Ramireddy Bommireddy | Emory University (original) (raw)

Papers by Ramireddy Bommireddy

Research paper thumbnail of Generation of mice with a conditional allele for the transforming growth factor beta3 gene

Genesis, 2012

The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and sever... more The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGF b receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3 −/− mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3 flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3 flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3 cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3 −/− mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3 cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood. genesis 50:59-66, 2012.

Research paper thumbnail of Membrane-based immunotherapy with immune checkpoint blockade antibodies confers protection against metastatic breast cancer

Journal of Immunology, May 1, 2017

Objective The 4T1 triple negative breast cancer (TNBC) model resembles many of the hallmarks of a... more Objective The 4T1 triple negative breast cancer (TNBC) model resembles many of the hallmarks of advanced TNBC in humans. In this study we investigate if combining a membrane-based immunotherapy with immune checkpoint blockade antibodies in a metastatic tumor model could effectively generate protective immunity. This personalized strategy is applicable to indications such as TNBC that exhibit a high degree of heterogeneity and do not respond to approved cancer therapies. Methods 4T1 tumor tissue was harvested from BALB/c mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then incorporated with GPI-B7-1 and GPI-IL-12 by protein transfer and used for immunization in conjunction with immune checkpoint inhibitors. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis was assessed by clonogenic assay. Immune response was assessed by IFN-γ ELISPOT. Cell depletion studies were performed with anti-CD4 and anti-CD8 antibodies. Significance was determined using a student’s t test to compare metastasis and immune responses. Results TMV-based immunotherapy in combination with anti-CTLA-4 antibody significantly improved survival, reduced pulmonary metastasis, and increased tumor-specific CD8 T cell responses. Interestingly, either treatment alone failed to have a significant impact on survival, metastasis, or immune response. Conclusions These results strongly suggest that a TMV-based immunotherapy in combination with anti-CTLA-4 antibody generates effective antitumor immunity. Such a combinatorial approach could potentially translate into an effective treatment for patients with metastatic TNBC.

Research paper thumbnail of Abstract LB-203: Metformin enhances the efficacy of tumor membrane vesicle (TMV)-based vaccine immunotherapy in a murine model of breast

Cancer Research, Jul 1, 2019

Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000 new case... more Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000 new cases per year in the United States. Immunotherapy has emerged as a promising treatment of breast cancer, however response rates to therapy remain far from ideal. In this study, we investigated the combinatorial effect of tumor membrane vesicle (TMV)-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by maintaining CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while combination with metformin resulted in greater inhibition of primary growth. Importantly, TMV vaccination reduced the metastatic burden in the lungs while combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T cell infiltration to the tumor, while combination with metformin caused a decrease in the accumulation of T cells, despite having better outcomes. Further, we show that metformin is capable of reducing PD-L1 expression within the tumor microenvironment in both tumor cells as well as myeloid-derived suppressor cells, providing insight for the improvement of TMV-based immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley Huang, Janet Kim, Periasamy Selvaraj. Metformin enhances the efficacy of tumor membrane vesicle (TMV)-based vaccine immunotherapy in a murine model of breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-203.

Research paper thumbnail of Generation of mice with a conditional allele for transforming growth factor beta 1 gene

Genesis, Jun 1, 2009

Transforming growth factor β1 (TGFβ1) is a multifunctional growth factor involved in wound healin... more Transforming growth factor β1 (TGFβ1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer. Since the generation of Tgfb1 knockout mice, there has been extraordinary progress in understanding its physiological and pathophysiological function. Here, we report the generation of a conditional knockout allele for Tgfb1 in which its exon 6 is flanked with LoxP sites. As proof of principle, we crossed these mice to LckCre transgenic mice and specifically disrupted Tgfb1 in T cells. The results indicate that T-cell-produced TGFβ1 is required for normal in vivo regulation of peripheral T-cell activation, maintenance of T-cell homeostasis, and suppression of autoimmunity. Keywords transforming growth factorβ; conditional knockout mice Transforming growth factor betas (TGFβs) are regulatory cytokines that function in embryonic development and adults and in the pathogenesis of various diseases and cancer (Derynck and Akhurst, 2007; McLennan and Koishi, 2002; Sporn, 2006). In mammals, there are three TGFβ ligands which express differently in time and space (Azhar et al., 2003; Millan et al.,

Research paper thumbnail of Humanized mice challenged with TNBC tumor develop graft versus host disease and anemia limiting vaccine immunotherapy studies

Journal of Immunology, May 1, 2021

Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a... more Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a therapeutic cancer vaccine approach for triple negative breast cancer (TNBC). In this study, we investigated whether vaccine developed from a human TNBC cell line could effectively induce antitumor immunity, which control the growth of tumors in humanized mice implanted with the human TNBC cell line. Human CD34+ HSC implanted NSG-SGM3 from JAX labs and NOG-EXL mice from Taconic Biosciences used for the studies. Humanized NSG-SGM3 mice were vaccinated with 100 mg of the vaccine once every 2 weeks (3 doses). Control mice were injected with vehicle (PBS). One week after the last vaccination, mice were challenged with MDA-MB-231 TNBC cells subcutaneously in the hind flank. Body weight and tumor growth were monitored. All the mice in both groups have developed tumors, splenomegaly and metastatic lesions in the liver, lungs and spleen. However, all the mice also developed GvHD and need to be euthanized before completion of the study. We have also observed an allo-response (CD69+ T cells in control mice with MDA-MB-231 tumors) to the HLA-matched tumor cells but the tumors eventually developed into metastatic disease affecting multiple organs (liver, lungs and spleen) suggesting that the allo-response is not effective against the tumor. Since the tumor-bearing mice developed GvHD and becoming anemic even in a specific pathogen-free barrier facility, future studies will need to be tested in humanized mice which do not develop GvHD and live longer for carrying out vaccine immunotherapy studies. Funding: NIH/NCI R01 CA202763 (PS & CP).

Research paper thumbnail of Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Cancer Research, 2012

Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engine... more Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+...

Research paper thumbnail of HER-2+ breast cancer cells expressing GPI-anchored cytokines induce long lasting antitumor memory response

Journal of Immunology, May 1, 2017

Breast cancer is the leading cause of death among female cancer patients. Human epidermal growth ... more Breast cancer is the leading cause of death among female cancer patients. Human epidermal growth factor receptor 2 (HER-2) overexpression is associated with poor prognosis in 15–20% of breast cancer patients. Anti-HER-2 antibody (Herceptin) prolongs the survival of the HER-2+ breast cancer patients. However, resistance to Herceptin is a cause for relapse of aggressive metastatic cancer in these patients. Our earlier studies have demonstrated that PD-L1 blockade enhances the efficacy of HER-2+ breast cancer whole cell vaccine by increasing infiltration of T cells into the tumor. The goal of the present study is to determine the duration of protective anti-tumor memory response to HER-2+ breast cancer. Here, we demonstrate the beneficial effect of GPI-anchored cytokine molecules as adjuvants for generating long lasting memory response against HER-2+ as well as HER-2 negative breast cancer in syngeneic tumor models. Female BALB/c mice were challenged with D2F2/E2 (HER-2 transfected D2F2) cells or D2F2/E2 transfected with GPI-IL-12 or GPI-GM-CSF. While the wild-type challenged mice developed tumors, the mice challenged with transfected D2F2/E2 cells were protected. Protected mice were re-challenged with D2F2/E2 cells 3 months later and D2F2 cells 4 months later. All the mice challenged with D2F2 or D2F2/E2 were protected. We have observed strong antibody response against HER-2 and D2F2 cells in these mice. Our results show that long lasting protective anti-tumor memory response against D2F2 and D2F2/E2 is generated by vaccination with D2F2/E2 cells expressing GPI-anchored immunostimulatory molecules.

Research paper thumbnail of Hemagglutinin virus-like particles incorporated with membrane-bound cytokine adjuvants provide protection against homologous and heterologous influenza virus challenge in aged mice

Immunity & Ageing

Background Current influenza vaccines deliver satisfactory results in young people but are less e... more Background Current influenza vaccines deliver satisfactory results in young people but are less effective in the elderly. Development of vaccines for an ever-increasing aging population has been an arduous challenge due to immunosenescence that impairs the immune response in the aged, both quantitatively and qualitatively. Results To potentially enhance vaccine efficacy in the elderly, we investigated the immunogenicity and cross-protection of influenza hemagglutinin virus-like particles (HA-VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored cytokine-adjuvants (GPI-GM-CSF and GPI-IL-12) via protein transfer in aged mice. Lung viral replication against homologous and heterologous influenza viruses was significantly reduced in aged mice after vaccination with cytokine incorporated VLPs (HA-VLP-Cyt) in comparison to HA-VLP alone. Enhanced IFN-γ+CD4+ and IFN-γ+CD8+ T cell responses were also observed in aged mice immunized with HA-VLP-Cyt when compared to HA-VLP alone. C...

Research paper thumbnail of The magic of small-molecule drugs during ex vivo expansion in adoptive cell therapy

Frontiers in Immunology

In the past decades, advances in the use of adoptive cellular therapy to treat cancer have led to... more In the past decades, advances in the use of adoptive cellular therapy to treat cancer have led to unprecedented responses in patients with relapsed/refractory or late-stage malignancies. However, cellular exhaustion and senescence limit the efficacy of FDA-approved T-cell therapies in patients with hematologic malignancies and the widespread application of this approach in treating patients with solid tumors. Investigators are addressing the current obstacles by focusing on the manufacturing process of effector T cells, including engineering approaches and ex vivo expansion strategies to regulate T-cell differentiation. Here we reviewed the current small-molecule strategies to enhance T-cell expansion, persistence, and functionality during ex vivo manufacturing. We further discussed the synergistic benefits of the dual-targeting approaches and proposed novel vasoactive intestinal peptide receptor antagonists (VIPR-ANT) peptides as emerging candidates to enhance cell-based immunother...

Research paper thumbnail of Tumor membrane vesicle (TMV)-based cancer vaccine induces dendritic cell maturation and downstream T cell activation

Journal of Immunology, May 1, 2018

Research paper thumbnail of Efficacy of immunotherapy approaches in metastatic and non-metastatic lung cancer models

The Journal of Immunology

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all... more Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies in an aggressive, non-metastatic, immune checkpoint inhibitor resistant lung cancer model (LL/2) and a highly metastatic lung cancer (CMT-167) model. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then mixed with adjuvants and used for immunization in conjunction with immune checkpoint inhibitors. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of...

Research paper thumbnail of Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine generated CD8 T cells in a murine model of triple negative breast cancer

The Journal of Immunology

Immunotherapy has emerged as a promising treatment of breast cancer, however, response rates to t... more Immunotherapy has emerged as a promising treatment of breast cancer, however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by promoting CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Intere...

Research paper thumbnail of Tumor membrane vesicle-based vaccine immunotherapy inhibits tumor growth and metastasis of squamous cell carcinoma of head and neck in mice

The Journal of Immunology

Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% ... more Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine (TMV) immunotherapy in murine SCCHN models. The MOC1, MOC2 and SCC VII tumors grown subcutaneously in syngeneic mice were harvested to generate TMVs. TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules GPI-B7.1 and GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine after tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. The TMV vaccine inhi...

Research paper thumbnail of Tumor membrane-based vaccine immunotherapy in combination with anti-CTLA-4 antibody confers protection against immune checkpoint resistant murine triple-negative breast cancer

Human Vaccines & Immunotherapeutics, 2020

Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers an... more Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers and is associated with a worse clinical outcome. This poor clinical outcome is attributed to a lack of defined targets and patient-to-patient heterogeneity in target antigens and immune responses. To address such heterogeneity, we tested the efficacy of a personalized vaccination approach for the treatment of TNBC using the 4T1 murine TNBC model. We isolated tumor membrane vesicles (TMVs) from homogenized 4T1 tumor tissue and incorporated glycosyl phosphatidylinositol (GPI)-anchored forms of the immunostimulatory B7-1 (CD80) and IL-12 molecules onto these TMVs to make a TMV vaccine. Tumor-bearing mice were then administered with the TMV vaccine either alone or in combination with immune checkpoint inhibitors. We show that TMV-based vaccine immunotherapy in combination with anti-CTLA-4 mAb treatment upregulated immunomodulatory cytokines in the plasma, significantly improved survival, and reduced pulmonary metastasis in mice compared to either therapy alone. The depletion of CD8 + T cells, but not CD4 + T cells, resulted in the loss of efficacy. This suggests that the vaccine acts via tumor-specific CD8 + T cell immunity. These results suggest TMV vaccine immunotherapy as a potential enhancer of immune checkpoint inhibitor therapies for metastatic triple-negative breast cancer.

Research paper thumbnail of Metformin reduces PD-L1 on tumor cells and enhances the anti-tumor immune response generated by vaccine immunotherapy

Journal for ImmunoTherapy of Cancer, 2021

BackgroundPD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumo... more BackgroundPD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumor immunity. Blockade of PD-L1/PD-1 has been a major improvement in the treatment of certain cancers, however, the response rate to checkpoint blockade remains low suggesting a need for new therapies. Metformin has emerged as a potential new drug for the treatment of cancer due to its effects on PD-L1 expression, T cell responses, and the immunosuppressive environment within tumors. While the benefits of metformin in combination with checkpoint blockade have been reported in animal models, little remains known about its effect on other types of immunotherapy.MethodsVaccine immunotherapy and metformin were administered to mice inoculated with tumors to investigate the effect of metformin and TMV vaccine on tumor growth, metastasis, PD-L1 expression, immune cell infiltration, and CD8 T cell phenotype. The effect of metformin on IFN-γ induced PD-L1 expression in tumor cells was assessed by f...

Research paper thumbnail of Improving T cell functionality for adoptive T cell therapy in metastatic colorectal cancer

Journal of Immunology, May 1, 2019

Research paper thumbnail of Calcineurin Aβ signalling is required for Treg-cell generation and prevention of T-cell activation and B-cell lymphomagenesis (100.13)

The Journal of Immunology

Introduction: Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved i... more Introduction: Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved in activation of NFAT. CN inhibitors such as cyclosporine A (CsA) and FK506 are potent inhibitors of T-cell responses and are commonly used to prevent graft rejection. Consistently, CNAβ-deficient mice exhibit defective thymocyte maturation in young mice. However, T cells undergo spontaneous activation in older Cnab-/- mice, and succumb to B-cell lymphomas. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines in culture supernatants are determined by ELISA, CBA and MSD methods. Cells are stained with CD4, CD25, CD62L and intracellular FOXP3. Results: We observed that FOXP3+ Treg cells are decreased while mature activated T cells (CD62L-CD44+) are increased in KO mice compared to control mice. While TGFβ1 induced the expression of FOXP3 in WT cultures, it failed to induce FOXP3 in KO cultures. Cytokine profiles revealed that CNAβ is...

Research paper thumbnail of Effect of Standard of Care Treatment on Tumor Membrane Vesicle Vaccine for Triple-Negative Breast Cancer

The Journal of Immunology

Metastatic triple-negative breast cancer (TNBC) has a poor prognosis primarily due to metastatic ... more Metastatic triple-negative breast cancer (TNBC) has a poor prognosis primarily due to metastatic potential and resistance to currently available therapies. Recent studies suggest that TNBC may be a viable target for immunotherapy due to the presence of high levels of infiltrating lymphocytes. However, many TNBC patients do not respond due to high intra-tumoral and inter-patient heterogeneity. Utilizing a novel protein transfer method, we generate a personalized vaccine based on tumor membrane vesicles (TMVs) derived from whole tumor tissues modified with glycolipid-anchored forms of the immunostimulatory molecules (GPI-ISMs) B7-1 and IL-12. In prior studies we observed that simultaneous delivery of TMVs along with biological adjuvants enhanced CD8 T cell immunity. Herein, we investigated the impact of standard-of-care (SOC) chemotherapy on the resulting quality of the TMV vaccine. Mice were injected with 4T1 TNBC cells subcutaneously and treated with three rounds of SOC drugs starti...

Research paper thumbnail of Immunotherapy with tumor membrane vesicle-based cancer vaccine inhibits metastatic tumor growth in lung cancer model

The Journal of Immunology

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all... more Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies lung cancer models LL/2 and CMT-167 models. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then modified with immunostimulatory molecules and used for immunization. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of CMT-167 was assessed by clonogenic assay. TMV vaccine alone, or in combination, anti-PD-1 or anti-CTLA-4 antibodies neither decreased...

Research paper thumbnail of Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor

PLOS ONE, 2021

Imaging techniques based on fluorescence and bioluminescence have been important tools in visuali... more Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cel...

Research paper thumbnail of Generation of mice with a conditional allele for the transforming growth factor beta3 gene

Genesis, 2012

The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and sever... more The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGF b receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3 −/− mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3 flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3 flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3 cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3 −/− mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3 cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood. genesis 50:59-66, 2012.

Research paper thumbnail of Membrane-based immunotherapy with immune checkpoint blockade antibodies confers protection against metastatic breast cancer

Journal of Immunology, May 1, 2017

Objective The 4T1 triple negative breast cancer (TNBC) model resembles many of the hallmarks of a... more Objective The 4T1 triple negative breast cancer (TNBC) model resembles many of the hallmarks of advanced TNBC in humans. In this study we investigate if combining a membrane-based immunotherapy with immune checkpoint blockade antibodies in a metastatic tumor model could effectively generate protective immunity. This personalized strategy is applicable to indications such as TNBC that exhibit a high degree of heterogeneity and do not respond to approved cancer therapies. Methods 4T1 tumor tissue was harvested from BALB/c mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then incorporated with GPI-B7-1 and GPI-IL-12 by protein transfer and used for immunization in conjunction with immune checkpoint inhibitors. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis was assessed by clonogenic assay. Immune response was assessed by IFN-γ ELISPOT. Cell depletion studies were performed with anti-CD4 and anti-CD8 antibodies. Significance was determined using a student’s t test to compare metastasis and immune responses. Results TMV-based immunotherapy in combination with anti-CTLA-4 antibody significantly improved survival, reduced pulmonary metastasis, and increased tumor-specific CD8 T cell responses. Interestingly, either treatment alone failed to have a significant impact on survival, metastasis, or immune response. Conclusions These results strongly suggest that a TMV-based immunotherapy in combination with anti-CTLA-4 antibody generates effective antitumor immunity. Such a combinatorial approach could potentially translate into an effective treatment for patients with metastatic TNBC.

Research paper thumbnail of Abstract LB-203: Metformin enhances the efficacy of tumor membrane vesicle (TMV)-based vaccine immunotherapy in a murine model of breast

Cancer Research, Jul 1, 2019

Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000 new case... more Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000 new cases per year in the United States. Immunotherapy has emerged as a promising treatment of breast cancer, however response rates to therapy remain far from ideal. In this study, we investigated the combinatorial effect of tumor membrane vesicle (TMV)-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by maintaining CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while combination with metformin resulted in greater inhibition of primary growth. Importantly, TMV vaccination reduced the metastatic burden in the lungs while combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T cell infiltration to the tumor, while combination with metformin caused a decrease in the accumulation of T cells, despite having better outcomes. Further, we show that metformin is capable of reducing PD-L1 expression within the tumor microenvironment in both tumor cells as well as myeloid-derived suppressor cells, providing insight for the improvement of TMV-based immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley Huang, Janet Kim, Periasamy Selvaraj. Metformin enhances the efficacy of tumor membrane vesicle (TMV)-based vaccine immunotherapy in a murine model of breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-203.

Research paper thumbnail of Generation of mice with a conditional allele for transforming growth factor beta 1 gene

Genesis, Jun 1, 2009

Transforming growth factor β1 (TGFβ1) is a multifunctional growth factor involved in wound healin... more Transforming growth factor β1 (TGFβ1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer. Since the generation of Tgfb1 knockout mice, there has been extraordinary progress in understanding its physiological and pathophysiological function. Here, we report the generation of a conditional knockout allele for Tgfb1 in which its exon 6 is flanked with LoxP sites. As proof of principle, we crossed these mice to LckCre transgenic mice and specifically disrupted Tgfb1 in T cells. The results indicate that T-cell-produced TGFβ1 is required for normal in vivo regulation of peripheral T-cell activation, maintenance of T-cell homeostasis, and suppression of autoimmunity. Keywords transforming growth factorβ; conditional knockout mice Transforming growth factor betas (TGFβs) are regulatory cytokines that function in embryonic development and adults and in the pathogenesis of various diseases and cancer (Derynck and Akhurst, 2007; McLennan and Koishi, 2002; Sporn, 2006). In mammals, there are three TGFβ ligands which express differently in time and space (Azhar et al., 2003; Millan et al.,

Research paper thumbnail of Humanized mice challenged with TNBC tumor develop graft versus host disease and anemia limiting vaccine immunotherapy studies

Journal of Immunology, May 1, 2021

Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a... more Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a therapeutic cancer vaccine approach for triple negative breast cancer (TNBC). In this study, we investigated whether vaccine developed from a human TNBC cell line could effectively induce antitumor immunity, which control the growth of tumors in humanized mice implanted with the human TNBC cell line. Human CD34+ HSC implanted NSG-SGM3 from JAX labs and NOG-EXL mice from Taconic Biosciences used for the studies. Humanized NSG-SGM3 mice were vaccinated with 100 mg of the vaccine once every 2 weeks (3 doses). Control mice were injected with vehicle (PBS). One week after the last vaccination, mice were challenged with MDA-MB-231 TNBC cells subcutaneously in the hind flank. Body weight and tumor growth were monitored. All the mice in both groups have developed tumors, splenomegaly and metastatic lesions in the liver, lungs and spleen. However, all the mice also developed GvHD and need to be euthanized before completion of the study. We have also observed an allo-response (CD69+ T cells in control mice with MDA-MB-231 tumors) to the HLA-matched tumor cells but the tumors eventually developed into metastatic disease affecting multiple organs (liver, lungs and spleen) suggesting that the allo-response is not effective against the tumor. Since the tumor-bearing mice developed GvHD and becoming anemic even in a specific pathogen-free barrier facility, future studies will need to be tested in humanized mice which do not develop GvHD and live longer for carrying out vaccine immunotherapy studies. Funding: NIH/NCI R01 CA202763 (PS & CP).

Research paper thumbnail of Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Cancer Research, 2012

Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engine... more Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+...

Research paper thumbnail of HER-2+ breast cancer cells expressing GPI-anchored cytokines induce long lasting antitumor memory response

Journal of Immunology, May 1, 2017

Breast cancer is the leading cause of death among female cancer patients. Human epidermal growth ... more Breast cancer is the leading cause of death among female cancer patients. Human epidermal growth factor receptor 2 (HER-2) overexpression is associated with poor prognosis in 15–20% of breast cancer patients. Anti-HER-2 antibody (Herceptin) prolongs the survival of the HER-2+ breast cancer patients. However, resistance to Herceptin is a cause for relapse of aggressive metastatic cancer in these patients. Our earlier studies have demonstrated that PD-L1 blockade enhances the efficacy of HER-2+ breast cancer whole cell vaccine by increasing infiltration of T cells into the tumor. The goal of the present study is to determine the duration of protective anti-tumor memory response to HER-2+ breast cancer. Here, we demonstrate the beneficial effect of GPI-anchored cytokine molecules as adjuvants for generating long lasting memory response against HER-2+ as well as HER-2 negative breast cancer in syngeneic tumor models. Female BALB/c mice were challenged with D2F2/E2 (HER-2 transfected D2F2) cells or D2F2/E2 transfected with GPI-IL-12 or GPI-GM-CSF. While the wild-type challenged mice developed tumors, the mice challenged with transfected D2F2/E2 cells were protected. Protected mice were re-challenged with D2F2/E2 cells 3 months later and D2F2 cells 4 months later. All the mice challenged with D2F2 or D2F2/E2 were protected. We have observed strong antibody response against HER-2 and D2F2 cells in these mice. Our results show that long lasting protective anti-tumor memory response against D2F2 and D2F2/E2 is generated by vaccination with D2F2/E2 cells expressing GPI-anchored immunostimulatory molecules.

Research paper thumbnail of Hemagglutinin virus-like particles incorporated with membrane-bound cytokine adjuvants provide protection against homologous and heterologous influenza virus challenge in aged mice

Immunity & Ageing

Background Current influenza vaccines deliver satisfactory results in young people but are less e... more Background Current influenza vaccines deliver satisfactory results in young people but are less effective in the elderly. Development of vaccines for an ever-increasing aging population has been an arduous challenge due to immunosenescence that impairs the immune response in the aged, both quantitatively and qualitatively. Results To potentially enhance vaccine efficacy in the elderly, we investigated the immunogenicity and cross-protection of influenza hemagglutinin virus-like particles (HA-VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored cytokine-adjuvants (GPI-GM-CSF and GPI-IL-12) via protein transfer in aged mice. Lung viral replication against homologous and heterologous influenza viruses was significantly reduced in aged mice after vaccination with cytokine incorporated VLPs (HA-VLP-Cyt) in comparison to HA-VLP alone. Enhanced IFN-γ+CD4+ and IFN-γ+CD8+ T cell responses were also observed in aged mice immunized with HA-VLP-Cyt when compared to HA-VLP alone. C...

Research paper thumbnail of The magic of small-molecule drugs during ex vivo expansion in adoptive cell therapy

Frontiers in Immunology

In the past decades, advances in the use of adoptive cellular therapy to treat cancer have led to... more In the past decades, advances in the use of adoptive cellular therapy to treat cancer have led to unprecedented responses in patients with relapsed/refractory or late-stage malignancies. However, cellular exhaustion and senescence limit the efficacy of FDA-approved T-cell therapies in patients with hematologic malignancies and the widespread application of this approach in treating patients with solid tumors. Investigators are addressing the current obstacles by focusing on the manufacturing process of effector T cells, including engineering approaches and ex vivo expansion strategies to regulate T-cell differentiation. Here we reviewed the current small-molecule strategies to enhance T-cell expansion, persistence, and functionality during ex vivo manufacturing. We further discussed the synergistic benefits of the dual-targeting approaches and proposed novel vasoactive intestinal peptide receptor antagonists (VIPR-ANT) peptides as emerging candidates to enhance cell-based immunother...

Research paper thumbnail of Tumor membrane vesicle (TMV)-based cancer vaccine induces dendritic cell maturation and downstream T cell activation

Journal of Immunology, May 1, 2018

Research paper thumbnail of Efficacy of immunotherapy approaches in metastatic and non-metastatic lung cancer models

The Journal of Immunology

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all... more Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies in an aggressive, non-metastatic, immune checkpoint inhibitor resistant lung cancer model (LL/2) and a highly metastatic lung cancer (CMT-167) model. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then mixed with adjuvants and used for immunization in conjunction with immune checkpoint inhibitors. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of...

Research paper thumbnail of Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine generated CD8 T cells in a murine model of triple negative breast cancer

The Journal of Immunology

Immunotherapy has emerged as a promising treatment of breast cancer, however, response rates to t... more Immunotherapy has emerged as a promising treatment of breast cancer, however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by promoting CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Intere...

Research paper thumbnail of Tumor membrane vesicle-based vaccine immunotherapy inhibits tumor growth and metastasis of squamous cell carcinoma of head and neck in mice

The Journal of Immunology

Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% ... more Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine (TMV) immunotherapy in murine SCCHN models. The MOC1, MOC2 and SCC VII tumors grown subcutaneously in syngeneic mice were harvested to generate TMVs. TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules GPI-B7.1 and GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine after tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. The TMV vaccine inhi...

Research paper thumbnail of Tumor membrane-based vaccine immunotherapy in combination with anti-CTLA-4 antibody confers protection against immune checkpoint resistant murine triple-negative breast cancer

Human Vaccines & Immunotherapeutics, 2020

Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers an... more Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers and is associated with a worse clinical outcome. This poor clinical outcome is attributed to a lack of defined targets and patient-to-patient heterogeneity in target antigens and immune responses. To address such heterogeneity, we tested the efficacy of a personalized vaccination approach for the treatment of TNBC using the 4T1 murine TNBC model. We isolated tumor membrane vesicles (TMVs) from homogenized 4T1 tumor tissue and incorporated glycosyl phosphatidylinositol (GPI)-anchored forms of the immunostimulatory B7-1 (CD80) and IL-12 molecules onto these TMVs to make a TMV vaccine. Tumor-bearing mice were then administered with the TMV vaccine either alone or in combination with immune checkpoint inhibitors. We show that TMV-based vaccine immunotherapy in combination with anti-CTLA-4 mAb treatment upregulated immunomodulatory cytokines in the plasma, significantly improved survival, and reduced pulmonary metastasis in mice compared to either therapy alone. The depletion of CD8 + T cells, but not CD4 + T cells, resulted in the loss of efficacy. This suggests that the vaccine acts via tumor-specific CD8 + T cell immunity. These results suggest TMV vaccine immunotherapy as a potential enhancer of immune checkpoint inhibitor therapies for metastatic triple-negative breast cancer.

Research paper thumbnail of Metformin reduces PD-L1 on tumor cells and enhances the anti-tumor immune response generated by vaccine immunotherapy

Journal for ImmunoTherapy of Cancer, 2021

BackgroundPD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumo... more BackgroundPD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumor immunity. Blockade of PD-L1/PD-1 has been a major improvement in the treatment of certain cancers, however, the response rate to checkpoint blockade remains low suggesting a need for new therapies. Metformin has emerged as a potential new drug for the treatment of cancer due to its effects on PD-L1 expression, T cell responses, and the immunosuppressive environment within tumors. While the benefits of metformin in combination with checkpoint blockade have been reported in animal models, little remains known about its effect on other types of immunotherapy.MethodsVaccine immunotherapy and metformin were administered to mice inoculated with tumors to investigate the effect of metformin and TMV vaccine on tumor growth, metastasis, PD-L1 expression, immune cell infiltration, and CD8 T cell phenotype. The effect of metformin on IFN-γ induced PD-L1 expression in tumor cells was assessed by f...

Research paper thumbnail of Improving T cell functionality for adoptive T cell therapy in metastatic colorectal cancer

Journal of Immunology, May 1, 2019

Research paper thumbnail of Calcineurin Aβ signalling is required for Treg-cell generation and prevention of T-cell activation and B-cell lymphomagenesis (100.13)

The Journal of Immunology

Introduction: Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved i... more Introduction: Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved in activation of NFAT. CN inhibitors such as cyclosporine A (CsA) and FK506 are potent inhibitors of T-cell responses and are commonly used to prevent graft rejection. Consistently, CNAβ-deficient mice exhibit defective thymocyte maturation in young mice. However, T cells undergo spontaneous activation in older Cnab-/- mice, and succumb to B-cell lymphomas. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines in culture supernatants are determined by ELISA, CBA and MSD methods. Cells are stained with CD4, CD25, CD62L and intracellular FOXP3. Results: We observed that FOXP3+ Treg cells are decreased while mature activated T cells (CD62L-CD44+) are increased in KO mice compared to control mice. While TGFβ1 induced the expression of FOXP3 in WT cultures, it failed to induce FOXP3 in KO cultures. Cytokine profiles revealed that CNAβ is...

Research paper thumbnail of Effect of Standard of Care Treatment on Tumor Membrane Vesicle Vaccine for Triple-Negative Breast Cancer

The Journal of Immunology

Metastatic triple-negative breast cancer (TNBC) has a poor prognosis primarily due to metastatic ... more Metastatic triple-negative breast cancer (TNBC) has a poor prognosis primarily due to metastatic potential and resistance to currently available therapies. Recent studies suggest that TNBC may be a viable target for immunotherapy due to the presence of high levels of infiltrating lymphocytes. However, many TNBC patients do not respond due to high intra-tumoral and inter-patient heterogeneity. Utilizing a novel protein transfer method, we generate a personalized vaccine based on tumor membrane vesicles (TMVs) derived from whole tumor tissues modified with glycolipid-anchored forms of the immunostimulatory molecules (GPI-ISMs) B7-1 and IL-12. In prior studies we observed that simultaneous delivery of TMVs along with biological adjuvants enhanced CD8 T cell immunity. Herein, we investigated the impact of standard-of-care (SOC) chemotherapy on the resulting quality of the TMV vaccine. Mice were injected with 4T1 TNBC cells subcutaneously and treated with three rounds of SOC drugs starti...

Research paper thumbnail of Immunotherapy with tumor membrane vesicle-based cancer vaccine inhibits metastatic tumor growth in lung cancer model

The Journal of Immunology

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all... more Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies lung cancer models LL/2 and CMT-167 models. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then modified with immunostimulatory molecules and used for immunization. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of CMT-167 was assessed by clonogenic assay. TMV vaccine alone, or in combination, anti-PD-1 or anti-CTLA-4 antibodies neither decreased...

Research paper thumbnail of Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor

PLOS ONE, 2021

Imaging techniques based on fluorescence and bioluminescence have been important tools in visuali... more Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cel...