METHODS OF CYTOTOXIC GENE THERAPY TO TREAT TUMORS

Abstract

A method is disclosed for decreasing or retarding an increase in the size of a localized or metastatic tumor by using a combination of an immune stimulating cytotoxic gene therapy and immune-checkpoint modulating agent, in conjunction with other therapies, including radiation therapy, chemotherapy, surgery, and immunotherapies.

Claims

1. A method of decreasing tumor burden or micrometastasis in a mammal, comprising: i.) administering a replication-incompetent adenoviral vector encoding thymidine kinase or cytosine deaminase to the mammal with a tumor intratumorally or to a tumor resection site in the mammal; ii.) administering a prodrug to the mammal orally or intravenously, the prodrug being activated by thymidine kinase or cytosine deaminase, the prodrug being ganciclovir, acyclovir, valacyclovir, valgancyclovir, famiciclovir, or an active analog thereof such that the prodrug is activated by the thymidine kinase; and iii.) administering a monoclonal antibody that recognizes a checkpoint protein which is PDL1, PDL2, CTLA4, TIM3, LAG3, B7-H4, CD80, CD86, BTLA, HVEM, KIR, or GALS to the mammal intravenously such that the antibody reduces the immune repressive response caused by the checkpoint protein, thereby allowing T cell activation, wherein the activated prodrug and enhanced T cell activation decrease tumor burden or decrease micrometastasis.

2-12. (canceled)

13. The method of claim 1, further comprising administering radiotherapy and/or chemotherapy to, and/or performing surgery on, the mammal before, during or following administrating the vector, prodrug, and monoclonal antibody.

14. The method of claim 1, wherein the replication-incompetent adenoviral vector encodes thymidine kinase.

15. The method of claim 1, wherein the replication-incompetent adenoviral vector encodes cytosine deaminase.

16. The method of claim 1, wherein the monoclonal antibody recognizes PDL1, CTLA4, or TIM3.

17. The method of claim 14, wherein the monoclonal antibody recognizes PDL1, CTLA4, or TIM3.

18. The method of claim 15, wherein the monoclonal antibody recognizes PDL1, CTLA4, or TIM3.

19. The method of claim 1, wherein the monoclonal antibody recognizes PDL1.

20. A method of reducing micrometastasis in a mammal with tumor resection, comprising: i.) administering a replication-incompetent adenoviral vector encoding thymidine kinase or cytosine deaminase to a tumor resection site in the mammal; ii.) administering a prodrug to the mammal orally or intravenously, the prodrug being activated by thymidine kinase or cytosine deaminase, the prodrug being ganciclovir, acyclovir, valacyclovir, valgancyclovir, famiciclovir, or an active analog thereof, such that the prodrug is activated by the thymidine kinase; and iii.) administering a monoclonal antibody that recognizes a checkpoint protein which is PDL1, PDL2, CTLA4, TIM3, LAG3, B7-H4, CD80, CD86, BTLA, HVEM, KIR, or GALS to the mammal intravenously such that the antibody reduces the immune repressive response caused by the checkpoint protein, thereby allowing enhanced T cell activation, wherein the activated prodrug and enhanced T cell activation reduce micrometastasis in the mammal.

21. The method of claim 20, further comprising administering radiotherapy and/or chemotherapy to, and/or performing surgery on, the mammal before, during or following administrating the vector, prodrug, and monoclonal antibody.

22. The method of claim 20, wherein the prodrug is activated by thymidine kinase.

23. The method of claim 20, wherein the prodrug is activated by cytosine deaminase.

24. The method of claim 22, wherein the monoclonal antibody recognizes PDL1, CTLA4, or TIM3.

25. The method of claim 23, wherein the monoclonal antibody recognizes PDL1, CTLA4, or TIM3.

26. The method of claim 20, wherein the monoclonal antibody recognizes PDL1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention is directed toward medicaments for inhibiting tumor growth and improving therapy in solid tumors and metastasis.

[0054] The invention provides a treatment of solid tumors comprising administering to a patient, an effective amount of an immune checkpoint inhibitor and an immune stimulating cytotoxic gene therapy. Tumor cells are transfected by direct injection of the gene delivery vehicle into accessible rumor lesions, followed by prodrug administration. The local reaction includes tumor cell necrosis, liberation of tumor antigens. Unexpectedly the immune stimulating cytotoxic gene therapy induces or increases the presence of immune checkpoint molecules, including PD-L1, PD-L2 within the tumor.

[0055] The increased levels of immune checkpoint molecules in the tumor resulting from immune stimulating cytotoxic gene therapy enhances efficacy of immune checkpoint inhibitors such as antibodies that recognize CTLA4, PD-1, PD-L1, or PD-L2. Immune checkpoint inhibitors such as antibodies that recognize CTLA4, PD-1, PD-L1, or PD-L2 enhance the efficacy of immune stimulating cytotoxic gene therapy.

[0056] The use of cytotoxic gene therapy in combination with an immune checkpoint inhibitor is intended to enhance T-cell activation through different mechanisms, respectively augmenting T-cell activation and augmenting dendritic cell-mediated tumor antigen presentation (Kaufman et al., Ann Surg Oncol., 17(3):718-730, 2010) following the release of tumor antigens by the cytotoxic activity and antagonizing immune tolerance by blocking inhibitory signals mediated by a immune checkpoint inhibitor, such as CTLA-4 or PD-1 on T lymphocytes (Kapadia and Fong, J Olin Oncol., 23:8926-8928, 2005).

[0057] The result is an enhanced anti-tumor effect against both the injected tumor and non-injected tumors, including metastases. The combination of provides a cumulative effect that is greater than the expected additive effect for the agents individually.

[0058] The invention is intended to enhance the local anti-tumor response to tumor antigens following the cytotoxic activity in tumors, leading to a greater systemic protective effect. Therefore, the combination therapy may result in enhanced destruction of injected tumors as well as uninjected/distant tumors, including micrometastatic disease to improve the rate of overall tumor response and duration of response. Overall, these effects may contribute to an improvement in overall survival, particularly when compared to treatment using either agent alone.

[0059] Without wishing to be bound by any theory of the invention, in one embodiment the combination of a cytotoxic gene therapy and an immune checkpoint inhibitor may increase the frequency or intensity of tumor-specific T cell responses in treated patients as compared to either agent alone

[0060] In another embodiment of this invention the combination of a cytotoxic gene therapy and an immune checkpoint inhibitor may result in reduction in cancer recurrences in treated patients, as compared to either agent alone.

[0061] In yet another embodiment of this invention the combination of a cytotoxic gene therapy and an immune checkpoint inhibitor may result in reduction of the presence or appearance of metastases or micro metastases in treated cancer patients, as compared to either agent alone.

[0062] In another embodiment of this invention the combination of a cytotoxic gene therapy and an immune checkpoint inhibitor may result improved overall survival of treated cancer patients, as compared to either agent alone.

[0063] As used herein, the term “immune checkpoint inhibitor” refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD 80 and CD86; and PD-1 with its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer 12: 252-264, 2012). These proteins are responsible for co-stimulatory or inhibitory interactions of T-cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0064] CTLA-4 is an immune checkpoint molecule that down-regulates pathways of T-cell activation. CTLA-4 is a negative regulator of T-cell activation. Blockade of CTLA-4 has been shown to augment T-cell activation and proliferation. The combination of the cytotoxic gene therapy and the anti-CTLA-4 antibody is intended to enhance T-cell activation through two different mechanisms in order to augment the anti-tumor immune response to tumor antigen released following the cytotoxic tumor lysis of the cytotoxic gene therapy in the tumor. Therefore, the combination of the cytotoxic gene therapy and the anti-CTLA-4 antibody may enhance the destruction of the injected and un-injected/distal tumors, improve overall tumor response, and extend overall survival, in particular where the extension of overall survival is compared to that obtained using an anti-CTLA-4 antibody alone.

[0065] PD-1 is an immune checkpoint molecule that down-regulates pathways of T-cell activation. PD-1 binds to PD-L1 and PD-L2. PD-1 is a negative regulator of T-cell activation. Blockade of PD-1 PD-L1/PD-L2 interactions have been shown to augment T-cell activation and proliferation. The combination of the cytotoxic gene therapy and the antibody against PD-1, PD-L1, or PL-L2 is intended to enhance T-cell activation through two different mechanisms in order to augment the anti-tumor immune response to tumor antigen released following the cytotoxic tumor lysis of the cytotoxic gene therapy in the tumor. Therefore, the combination of the cytotoxic gene therapy and the anti-PD-1 antibody may enhance the destruction of the injected and un-injected/distal tumors, improve overall tumor response, and extend overall survival, in particular where the extension of overall survival is compared to that obtained using an anti-PD-1 antibody alone.

[0066] Within another aspect of the present invention, GDV constructs resulting in the expression of the thymidine kinase, are administered to a solid tumor in a patient at various doses In the case of AdV-tk the expected range is between 10.sup.4 and 10.sup.15 vector particles (vp). Titres of adenoviral vectors used in the clinic typically range between 10.sup.8 vp/ml and 10.sup.13 vp/ml. Patients can be injected with 0.3 ml to 500 ml of vector with single or repeated doses. The tumor lesion may vary from 1 to 20 cm in size, for example, in the case of soft tissue sarcoma, multiple myeoloma, or head and neck squamous cell carcinoma. The dose can be administered in a single injection or in multiple injections within the same tumor site over a time period. Alternatively, a dose consisting of up to 500 mls per day, can be administered over a time period of 5 days in order to establish one course. Patients can receive as many courses as necessary in order to establish a response without proving toxic. Courses can be given, for example, weekly or every other week over months or over years.

[0067] As noted above, pharmaceutical compositions are described comprising a GDV carrying the thymidine kinase vector construct, in combination with a pharmaceutically acceptable carrier or diluent (see Nyberg-Hoffman and Aguilar-Cordova, Nature Medicine, April 1999). The composition may be prepared either as a liquid solution, or as a solid form (e.g., lyophilized), which is suspended in a solution prior to administration. In addition, the composition may be prepared with suitable carriers or diluents for surface administration, injection, oral, or rectal administration.

[0068] Pharmaceutically acceptable carriers or diluents are nontoxic to recipients at the dosages and concentrations employed. Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH or a pH for vector stability (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, sucrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin.

[0069] Various methods may be utilized within the context of the present invention in order to directly administer the vector construct to the tumor, including direct intra-lesional injection, intra-cavital, iv administration or topical delivery. For example, within one embodiment a lesion may be located, and the vector injected once or several times in several different locations within the body of the tumor. Alternatively, arteries or blood vessels, which serve a tumor, may be identified and the vector injected into such blood vessel, in order to deliver the vector directly into the tumor. Within another embodiment, a tumor that has a necrotic center may be aspirated, and the vector injected directly into the now empty center of the tumor. Within yet another embodiment, the vector construct may be directly administered to the surface of the tumor, for example, by application of a topical pharmaceutical composition containing the vector construct, or preferably, a recombinant viral vector carrying the vector construct. Vector particles may be administered either directly (e.g., intravenously, intramuscularly, intraperitoneally, subcutaneously, orally, rectally, intraocularly, intranasally, intravesically, during surgical intervention) to the site of a tumor lesion, or the vector construct may be delivered after formulation by various physical methods such as lipofection (Feigner et al., PNAS 84:_7413-7417, 1989), direct DNA injection (Fung et al., PNAS 80:_353-357, 1983; Seeger et al., PNAS 81:_5849-5852; Acsadi et al., Nature 352:_815-818, 1991); microprojectile bombardment (Williams et al., PNAS 88:_2726-2730, 1991); liposomes of several types (see, e.g., Wang et al., PNAS 84:_7851-7855, 1987); CaPO4 (Dubensky et al., PNAS 81:_7529-7533, 1984); DNA ligand (Wu et al., J. Biol. Chem. 264: 16985-16987, 1989); administration of nucleic acids alone (WO 90/11092); or administration of DNA linked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3:_147-154, 1992); via polycation compounds such as polylysine, utilizing receptor specific ligands; as well as with psoralen inactivated viruses such as Sendai or Adenovirus, by electroporation or by pressure-mediated delivery. In addition vector particles or formulated construct may either be administered by direct injection to the desired site or by other clinically acceptable means such as by various forms of catheter that can be introduced into the patient with minimal discomfort, followed by injection or release of the vector in conjunction with operations made possible by the catheter, such as multiple injection, introduction of radioactive seeds, tissue disruption and other means known to those skilled in the art.

[0070] Vector particles and formulated vector constructs may be administered to a wide variety of tissue and/or cell types where lesions may exist, including for example, the brain and/or spinal cord, bone marrow, eyes, the liver, nose, throat and lung, heart and blood vessels, spleen, skin, circulation, muscles, prostate, breast, pancreas, kidney, cervix, and other organs.

Evaluation of Patients with Cancer

[0071] Every patient with any form of tumor is followed and staged in different ways depending on the tools and methods available and their accumulated track record in allowing reliable evaluation and effective disease management (for example see Medical Oncology: Basic Principles and Clinical Management of Cancer by Paul Calabresi, Philip S. Schein McGraw-Hill 1993, and Cancer, Principles and Practice of Oncology 5th edition, Vincent T. DeVita, Jr., Samuel Hellman and Steven A. Rosenberg Eds. Lippincott-Raven 1997)). For example, testicular cancer is followed with Beta HCG, AFP, and LDH, colon cancer is often followed with CEA levels, prostate cancer is followed with PSA levels, and ovarian cancer is followed with CA-125 levels. Tumors of the Head and Neck area, of the upper air and digestive tract are often followed by visual inspection as they are in an easy location to visualize. This is also the case for cervical cancer. Areas that are not easily visualized or palpated are evaluated with CT scans or MRI scans. MRI scans are extremely helpful in the evaluation of tumors of the neuraxis and the central nervous system. PET Scan is not routinely utilized to follow cancer. In the proceeding paragraphs prostate cancer is used as an example of the staging and treatment decisions of a solid tumor disease.

[0072] In one aspect, the technology provides methods for determining the efficacy cytoxic GDV and checkpoint inhibitor combination therapy for killing neoplastic cells and inducing a systemic immune response. There are many instances where it might be desirable to determine the efficacy of such a combination therapy. For example, it may be desirable to evaluate efficacy during the development of a new combination therapy. It may also be desirable to evaluate efficacy of a previously developed therapies to, for example, evaluate additional properties such as shelf life, production methods, etc.

[0073] In some embodiments, the methods involve measuring the efficacy of the combination therapy in vivo. For example, the tumor may be examined using classical imaging techniques (e.g., CT and PET) before and after treatment to determine the effects of the combination therapy.

[0074] In some embodiments, the invention will be used in combination with an adjuvant. In one embodiment, the adjuvant comprising a cytokine for enhancements of innate and adaptive immunity. The cytokine can be administered as a cytokine containing formulation or a GDV that will result in the expression of a cytokine upon administration to the patient.

[0075] In some embodiments, the invention involves contacting a cancer cell with the cytotoxic GDV and determining the viability of the cancer cell. Cell viability may be evaluated by anyone of a number of methods known in the art. For example, the viability may be evaluated in a cell counting assay, a replication labeling assay, a cell membrane integrity assay, a cellular ATP-based viability assay, a mitochondrial reductase activity assay, a caspase activity assay, an Annexin V staining assay, a DNA content assay, a DNA degradation assay, and a nuclear fragmentation assay. It is understood that assays of cell viability are capable of detecting cell killing (i.e., cell death). Cell death may be, for example, cytolytic, apoptotic, or necrotic.

[0076] Other exemplary assays of cell viability include BrdU, EdU, or H3-Thymidine incorporation assays; DNA content assays using a nucleic acid dye, such as Hoechst Dye, DAPI, Actinomycin D, 7-aminoactinomycin D or Propidium Iodide; Cellular metabolism assays such as AlamarBlue, MTT, XTT, and CeliTitre Glo; Nuclear Fragmentation Assays; Cytoplasmic Histone Associated DNA Fragmentation Assay; PARP Cleavage Assay; TUNEL staining; and Annexin staining. Still other assays will be apparent to one of ordinary skill in the art.

[0077] The cancer cells used in the efficacy evaluation methods may be any of the cancer cell lines disclosed herein and/or known in the art. In certain cases, it is desirable that the cancer cell is of a specific type. For example, it is particularly desirable that the cancer cell is a pancreatic cell when the condition to be treated by the oncolytic virus under evaluation is a pancreatic cancer.

[0078] In some cases, evaluation methods involve determining the expression of a cancer cell marker (e.g., at least one) in the cancer cell. Any appropriate cancer cell biomarker may be used. The cancer cell biomarkers can be evaluated by any appropriate method known in the art. For example, immunoblotting, immunohistochemistry, immunocytochemistry, ELISA, radioimmunoassays, proteomics methods, such as mass spectroscopy or antibody arrays may be used. In some embodiments, high-content imaging or Fluorescence-activated cell sorting (FACS) of cells may be used. Other exemplary methods will be apparent to the skilled artisan.

[0079] Typically, the methods for determining the efficacy of cytotoxic GDV for killing cancer cells are carried out in vitro under standard cell culture conditions. However, the methods are not so limited. The methods may involve growing cancer cells and optionally control cells, which mayor may not be cancer cells. The cells may be grown in single well or multi-well format (e.g., 6, 12, 24, 96, 384, or 1536 well format). Thus, in some cases the assays may be adapted to a high-throughput format.

Example 1

AdV-tk Mediated Gene Therapy in Combination with Checkpoint Inhibitors for the Treatment of Glioma

[0080] One application of this invention would be in the treatments of malignant gliomas patients where a craniotomy with resection of the tumor or tumors will be performed. At the neurosurgeon's discretion, this may involve stereotactic methods and/ or intraoperative navigational guidance and/or intraoperative MRI or other radiologic guidance. Tumor resection may be partial or complete. After the tumor resection has been completed, freehand injections of between 10-1000 microliters of the AdV-tk virus will be performed by the neurosurgeon in the wall of the resection cavity at a number of sites ranging from 1 to 50 to a total volume injected of between 100 microliters and 5000 microliters. The total dose of the AdV-tk may range between 1×10.sup.8 to 1×10.sup.12 vector particles. After the injections are completed, the remainder of the operation will consist of routine wound closure.

[0081] After completion of surgery, the patient will receive prodrug. In this example, Valacyclovir treatment will begin 1-3 days after vector administration at a dose of 2 grams orally three times a day for 14 days. Certain patients, such as those with impaired renal function, may receive a modified dose schedule such as 1.5 grams orally three times a day, or 1.5 grams twice a day. Alternatively, if a patient is unable to take the oral prodrug for any reason, intravenous acyclovir at 10 mg/kg tid may be substituted.

[0082] In this example, administration of an immune checkpoint inhibitor antibody such as pembrolizumab (anti-PD-1) will then be initiated within 1-21 days after surgery. Intravenous dosing will be approximately 1-4 mg/kg once every two or three weeks.

[0083] Clinical patient outcomes will be monitored using standard methodology, including tumor progression, quality of life, blood chemistry, immune system status, general wellness, and survival.

[0084] In this example, the patients receiving the combination treatment will have improved outcomes when compared to patents with similar disease characteristics that receive current standard of care or either single agent alone. Improvements in outcomes may include improved survival time post-treatment, increased time to disease recurrence, and better quality of life.

Example 2

[0085] AdV-tk Mediated Gene Therapy in Combination with Checkpoint Inhibitors for the Treatment of Pancreatic Adenocarcinoma

[0086] Another application of this invention would be in the treatment of patients with pancreatic adenocarcinoma. In this example, two courses of AdV-tk+prodrug will be delivered. Prior to the first injection, pathologic diagnosis of pancreatic adenocarcinoma will be made. For the injections that performed before surgery or injections to patents not receiving surgery, between 3×10.sup.10 and 1×10.sup.12 vector particles of AdV-tk will be delivered by Endoscopic Ultrasound or CT-guided injection into the pancreas in a total volume in the range of 0.5 mls to 4 mls. For patients receiving surgery, freehand injections, each of a volume of between 10 and 1000 microliters of the AdV-tk vector formulation, will be performed by the surgeon into each of between and 2-20 sites into the soft tissue of the retroperitoneum or to any residual macroscopic tumor. The total dose of the AdV-tk will be between 3×10.sup.10 and 1×10.sup.12 vector particles in a total volume in the range of 1 ml to 5 ml. The sites will be at least 1 cm apart and will be selected by the surgeon to avoid injections into vessels, bowel or other critical structures.

[0087] After the completion of each course, the patient will receive prodrug. In this example, Valacyclovir treatment will begin 1-3 days after vector administration at a dose of 2 grams orally three times a day for 14 days. Certain patients, such as those with impaired renal function, may receive a modified dose schedule such as 1.5 grams orally three times a day, or 1.5 grams twice a day. Alternatively, if a patient is unable to take the oral prodrug for any reason, intravenous acyclovir at 10 mg/kg tid may be substituted.

[0088] In this example, administration of an immune checkpoint inhibitor antibody such as Ipilimumab (anti-CTLA-4) will then be initiated within 1-21 days after surgery. Intravenous dosing will be approximately 1-4 mg/kg once every two or three weeks.

[0089] Clinical patient outcomes will be monitored using standard methodology, including tumor progression, quality of life, blood chemistry, immune system status, general wellness, and survival.

[0090] In this example, the patients receiving the combination treatment will have improved outcomes when compared to patents with similar disease characteristics that receive current standard of care or either single agent alone. Improvements in outcomes may include improved survival time post-treatment, increased time to disease recurrence, and better quality of life.

Example 3

AdV-tk Mediated Gene Therapy in Combination with Checkpoint Inhibitors for the Treatment of Malignant Pleural Effusion

[0091] Yet another application of this invention would be in the treatment of patients with malignant pleural effusions. Eligible patients will be seen by the interventional pulmonologists for placement of pleural catheter as per current standard of care. The cytotoxic gene therapy, such as AdV-tk vector in this case, will be delivered to the pleural space through the pleurex catheter after drainage of any fluids. For AdV-tk cytotoxic gene therapy, between 1×10.sup.12 and 1×10.sup.13 vector particles will be delivered in a total volume in the range of 10 mls and 50 mls. After a period of time within the range of 5 minutes and 60 minutes, the pleural cavity will be drained. A second infusion may be administered between 1-5 days after the first infusion.

[0092] After completion of infusions, the patient will receive prodrug. In this example, Valacyclovir treatment will begin 1-3 days after vector administration at a dose of 2 grams orally three times a day for 14 days. Certain patients, such as those with impaired renal function, may receive a modified dose schedule such as 1.5 grams orally three times a day, or 1.5 grams twice a day. Alternatively, if a patient is unable to take the oral prodrug for any reason, intravenous acyclovir at 10 mg/kg tid may be substituted.

[0093] In this example, administration of an immune checkpoint inhibitor antibody such as lambrolizumab (anti-PD-1) will then be initiated within 1-21 days after surgery. Intravenous dosing will be approximately 1-4 mg/kg once every two or three weeks.

[0094] Clinical patient outcomes will be monitored using standard methodology, including tumor progression, quality of life, blood chemistry, immune system status, general wellness, and survival.

[0095] In this example, the patients receiving the combination treatment will have improved outcomes when compared to patents with similar disease characteristics that receive current standard of care or either single agent alone. Improvements in outcomes may include improved survival time post-treatment, increased time to disease recurrence, and better quality of life.

FIG. 1 Induction of PD-L1 in Human Glioma Cells Response to Cytotoxic Gene Therapy

[0096] FIG. 1 shows a time course of PD-L1/PD-L2 expression levels in human glioma (U251) cells in culture after treatment with cytotoxic gene therapy. The effect of AdV-tk/prodrug cytotoxicity on the expression of immune checkpoint proteins (PD-1, PD-L1, PD-L2, CTLA-4, CD80, CD86) was tested by quantitative PCR. A substantial induction of gene expression relative to GAPDH mRNA observed with each checkpoint protein. The induction expression indicates that the AdV-tk/prodrug induced immunogenicity would be potentiated by inhibition of checkpoint blockade.

FIG. 2 Induction of PD-L1 in Glioma Cells Response to Cytotoxic Gene Therapy

[0097] FIG. 2 shows a time course of PD-L1/PD-L2 expression levels in murine glioma cells (GL-261) in culture after treatment with cytotoxic gene therapy. The effect of AdV-tk/prodrug cytotoxicity on the expression of immune checkpoint ligands (PD-L1, PD-L2) was tested by quantitative PCR. A substantial induction of gene expression relative to GAPDH mRNA observed with each PD-L1 and PD-L2. The induction of PD-L1/PD-L2 expression indicates that the AdV-tk induced immunogenicity would be potentiated by inhibition of checkpoint blockade.

FIG. 3 Induction of PD-L1 Expression in Tumor Tissue from Pancreatic Cancer Patients in Response to AdV-tk/Prodrug

[0098] PD-L1 expression was characterized in resected tumors after AdV-tk/prodrug treatment and compared to pre-treatment fine needle aspirate samples from a study of AdV-tk in pancreatic cancer. All samples analyzed had an average fold increase of 21.66 (range 6.00-74.85, p=0.0021) in CD8+ T cell infiltrate; with CD4+ infiltrates not significantly altered. PDL1 expression levels were increased in 5 of 7 samples analyzed. These data further support the combined use of AdV-tk immune-stimulation with ICI of the PD1/PD-L1 axis. Two examples are shown: before (A,C) and after (B,D) AdV-tk injection and 14 days of valacyclovir; Paraffin sections from pretreatment fine needle aspirate (A and C) or post-treatment surgical resection (B and D) were stained with the anti-PD-L1 antibody.