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Papers by Alan Epstein

Research paper thumbnail of Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

Cellular & Molecular Immunology, 2018

Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can... more Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40 − / − mice had significantly reduced numbers of CD25 − Foxp3 low tTreg precursors and CD25 + Foxp3 + mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.

Research paper thumbnail of Immunogenicity of Murine Solid Tumor Models as a Defining Feature of In Vivo Behavior and Response to Immunotherapy

Journal of Immunotherapy, 2013

Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify... more Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify novel targets for therapy. Two emerging uses of immune signatures are identification of likely responders to immunotherapy regimens among individuals with cancer or to understand the variable responses seen among subjects with cancer in immunotherapy trials. Here the immune profiles of six murine solid tumor models (CT26, 4T1, MAD109, RENCA, LLC, and B16) were correlated to tumor regression and survival in response to two immunotherapy regimens. Comprehensive profiles for each model were generated using quantitative RT-PCR, immunohistochemistry, and flow cytometry techniques, as well as functional studies of suppressor cell populations (Treg and MDSC), to analyze intratumoral and draining lymphoid tissues. Tumors stratified as highly or poorly immunogenic, with highly immunogenic tumors showing significantly greater presence of T-cell co-stimulatory molecules and immunosuppression in the tumor microenvironment. An absence of tumor-infiltrating CTL and mature DC was seen across all models. Delayed tumor growth and increased survival with suppressor cell inhibition and tumor-targeted chemokine +/− DC vaccine immunotherapy was associated with high tumor immunogenicity in these models. Tumor MHC class I expression correlated with overall tumor immunogenicity level and was a singular marker to predict immunotherapy response with these regimens. By using experimental tumor models as surrogates for human cancers, these studies demonstrate how select features of an immune profile may be utilized to identify patients most likely to respond to immunotherapy regimens.

Research paper thumbnail of Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling

Scientific reports, Jan 3, 2017

Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Tr... more Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset ...

Research paper thumbnail of Synthesis and preliminary evaluation of a new chelate N2S4 for use in labeling proteins with metallic radionuclides

International Journal of Radiation Applications and Instrumentation Part B Nuclear Medicine and Biology

The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodie... more The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodies for targeting tumor tissue in uivo depends upon the development and use of suitable bifunctional chelating agents. We have successfully synthesized a new N,S, (compound 4) chelate. This chelate forms a stable complex with 99mT~ and is capable of coupling to different proteins. The N,S, compound is quite stable if kept as a hydrochloride salt and is coupled to antibody under neutral conditions with better than 95% efficiency without the loss of immunoreactivity.

Research paper thumbnail of Molecular Cloning and Functional Expression of Contortrostatin, a Homodimeric Disintegrin from Southern Copperhead Snake Venom

Archives of Biochemistry and Biophysics, 2000

Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Th... more Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins αIIbβ3, α5β1, αvβ3, and αvβ5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part

Research paper thumbnail of Enhanced Tumor Uptake of Macromolecules Induced by a Novel Vasoactive Interleukin 2 Imrnunoconjugate1

Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer t... more Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer therapy. Recent studies have shown that antibody uptake in tumor is controlled in large part by the tumor blood flow and the vascular permeability of the tumor endothelium. We have hypothesized that these physiological properties of tumor vessels may be altered by pretreatment with vasoactive

Research paper thumbnail of MHC class I loss is a frequent mechanism of immune escape in papillary thyroid cancer that is reversed by interferon and selumetinib treatment in vitro

Clinical cancer research : an official journal of the American Association for Cancer Research, 2014

To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC e... more To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC expression and associated immune activation with current and experimental treatments for thyroid cancer using in vitro PTC cell lines. MHC class I expression and assessment of tumor-infiltrating leukocyte populations were evaluated by immunohistochemistry. PTC cell lines were analyzed for HLA-ABC expression by flow cytometry following tyrosine kinase inhibitor, IFNα or IFNγ, or radiation treatment. Functional changes in antigenicity were assessed by coculture of allogeneic donor peripheral blood leukocytes (PBL) with pretreated or untreated PTC cell lines and measurement of T-cell activation and cytokine production. Both MHC class I and β2-microglobulin expression was reduced or absent in 76% of PTC specimens and was associated with reduced tumor-infiltrating immune cells, including effector (CD3(+), CD8(+), CD16(+)) and suppressor (FoxP3(+)) populations. Treatment of PTC cell lines with ...

Research paper thumbnail of The evaluation of 186Re-labeled antibodies using N2S4 chelate in vitro and in vivo using tumor-bearing nude mice

International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology, 1992

We have recently described a method for radiolabeling monoclonal antibodies, with metallic radion... more We have recently described a method for radiolabeling monoclonal antibodies, with metallic radionuclides using a new chelating agent N2S3. Using this chelate the monoclonal antibodies Lym-1 and B72.3 were labeled with 186Re and their biological integrity was evaluated in vitro and in vivo. 186Re-labeled antibodies using N2S4 methodology were found to be stable in human serum and retained their immunoreactivity. Intravenous administration of 0.5 mCi 186Re-labeled antibodies resulted in partial or complete regression of tumor tissue in mice.

Research paper thumbnail of Comparison of three different targeted tissue factor fusion proteins for inducing tumor vessel thrombosis

Cancer research, Jan 15, 2003

Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pat... more Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pathway of the blood coagulation cascade and normally released from damaged tissues. By substituting the attachment site with a tumor delivery agent, this potent thrombogenic protein in its truncated form (tTF) can be targeted to the tumor where it can initiate clotting, thereby occluding the tumor's blood supply and causing rapid tumor destruction. To test the therapeutic potential of this vascular targeting approach, three fusion proteins, chTNT-3/tTF, chTV-1/tTF, and RGD/tTF, which target DNA exposed in degenerative areas of tumors, fibronectin on the tumor vascular basement membrane, and alpha nu beta 3 on the luminal side of tumor vessels, respectively, were developed and tested for their antitumor effects. Antigen binding and clotting assays demonstrated that each of the fusion proteins retained their antigen binding and thrombogenic activities. In vivo studies in mice bearing est...

Research paper thumbnail of Synthesis and preliminary evaluation of a new chelate N2S4 for use in labeling proteins with metallic radionuclides

International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1991

The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodie... more The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodies for targeting tumor tissue in uivo depends upon the development and use of suitable bifunctional chelating agents. We have successfully synthesized a new N,S, (compound 4) chelate. This chelate forms a stable complex with 99mT~ and is capable of coupling to different proteins. The N,S, compound is quite stable if kept as a hydrochloride salt and is coupled to antibody under neutral conditions with better than 95% efficiency without the loss of immunoreactivity.

Research paper thumbnail of Monoclonal antibodies. Their promise for tumor diagnosis, staging, and therapy

Postgraduate medicine, 1985

Monoclonal antibodies will have utility as tools for diagnosis, staging, and therapy of malignant... more Monoclonal antibodies will have utility as tools for diagnosis, staging, and therapy of malignant disease. Investigators have produced monoclonal antibodies that are directed against tumor-associated antigens and have varying degrees of cross-reactivity against normal tissues. These reagents have homogeneous molecular structure, recognize specific antigenic sites, can be produced in mass quantities, and are easily purified. Clinical trials with monoclonal antibodies are in progress and suggest modest toxicity and potential therapeutic efficacy.

Research paper thumbnail of Evaluating Antibodies for Their Capacity to Induce Cell-mediated Lysis of Malignant B Cells1

Promising results from clinical trials have led to renewed interest in effector mechanisms operat... more Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells,

Research paper thumbnail of Soluble Fc Fusion Proteins for Biomedical Research

Methods in Molecular Biology, 2007

As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become va... more As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator's protein moiety of interest is genetically linked to the N-terminus of murine Fc and subsequently expressed in large quantity using a mammalian cell expression system. The resulting Fc fusion proteins are purified on a protein A column and may be stored for at least one year at -20 degrees C. The availability of easily purified, soluble Fc fusion proteins in such quantity can facilitate research in multiple fields of medicine and biotechnology.

Research paper thumbnail of Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors

Immunotherapy, 2011

In this article, the role of chemokines and costimulatory molecules in the immunotherapy of exper... more In this article, the role of chemokines and costimulatory molecules in the immunotherapy of experimental murine solid tumors and immunotherapy used in ongoing clinical trials are presented. Chemokine networks regulate physiologic cell migration that may be disrupted to inhibit antitumor immune responses or co-opted to promote tumor growth and metastasis in cancer. Recent studies highlight the potential use of chemokines in cancer immunotherapy to improve innate and adaptive cell interactions and to recruit immune effector cells into the tumor microenvironment. Another critical component of antitumor immune responses is antigen priming and activation of effector cells. Reciprocal expression and binding of costimulatory molecules and their ligands by antigen-presenting cells and naive lymphocytes ensures robust expansion, activity and survival of tumor-specific effector cells in vivo. Immunotherapy approaches using agonist antibodies or fusion proteins of immunomodulatory molecules si...

Research paper thumbnail of Immunologic and Biochemical Analysis of TNT-1 and TNT-2 Monoclonal Antibody Binding to Histones

Hybridoma, 1993

Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and ... more Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and TNT-2, have been generated by immunizing mice with nuclear extracts from human lymphoma cells. The monoclonal antibodies were initially identified by indirect immunofluorescence on lymphoma cell lines and subsequently were found to stain the nucleus of all cell types from several species including plants by indirect immunofluorescence techniques. Immunoblot analysis demonstrated that TNT-1 bound to a protein of 22 kD and TNT-2 bound to two proteins of 15 and 22 kD, consistent with the known molecular weight of histones. To characterize their immunoreactivity, competition radioimmunoassays were performed using purified histone fractions HI, H2a, H2b and H3. By these assays, TNT-1 was found to bind to histone fraction HI and TNT-2 to an epitope common to histone fractions HI and H3. Histones are found in abundance in the nucleus of the cell and are known to play a major role in chromosome structure and gene expression. Upon cell death, histones remain tightly bound to DNA and consequently provide an abundant intracellular antigen that can be exploited in targeting necrotic cells, such as those found in tumors.

Research paper thumbnail of The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins

Planta, 2012

The spatial organisation of the splicing system in plant cells containing either reticular (Alliu... more The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei. Keywords Architecture of plant cell nucleus Á PANA antigen Á SnRNA Á Splicing Á SR proteins Abbreviations CB Cajal bodies DAPI 4,6-Diamidino-2-phenylindole PANA Proliferation-associated nuclear antigen SnRNP Small nuclear ribonucleoproteins SR Serine/arginine-rich proteins Electronic supplementary material The online version of this article (

Research paper thumbnail of Cytoreductive Chemotherapy Improves the Biodistribution of Antibodies Directed against Tumor Necrosis in Murine Solid Tumor Models

Molecular Cancer Therapeutics, 2013

Current strategies in cancer treatment employ combinations of different treatment modalities, whi... more Current strategies in cancer treatment employ combinations of different treatment modalities, which include chemotherapy, radiotherapy, immunotherapy, and surgery. Consistent with that approach, the present study demonstrates how chemotherapeutic agents can potentiate the delivery of radiolabeled, necrosis-targeting antibodies (chTNT-3, NHS76) to tumor. All chemotherapeutics in this study (5-fluorouracil, etoposide, vinblastine, paclitaxel, and doxorubicin) resulted in statistically significant increases in tumor uptake of radiolabeled antibodies and their F(ab')2 fragments compared to no pretreatment with chemotherapy. Labeled antibodies were administered at various time points following a single dose of chemotherapy in multiple tumor models, and the biodistribution of the antibodies was determined by measuring radioactivity in harvested tissues. MicroPET/CT was also done to demonstrate clinical relevancy of using chemotherapy pretreatment to increase antibody uptake. Results o...

Research paper thumbnail of Stepping Stones Toward Cancer Immunotherapy

Journal of Immunotherapy, 2005

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood ... more Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. The modulation of angiogenesis pathways using small interfering RNA (siRNA) inhibitors of gene expression has proven to be a powerful approach which would be applied for treatment of multiple cancers. Using a nanoparticle-mediated systemic delivery of siRNA with a dual targeting specificity to both neovasculature and VEGF receptor 2 (KDR), we were able to achieve anti-tumor efficacies in multiple tumor models. We further tested this potential anticancer drug candidate, ICS-283, in combination with Genentech's Avastin, a mAb against VEGF A in colorectal tumor cell DLD-1 xenograft model. The combined regimen resulted stronger anticancer efficacy than either of the component alone with significant evidence of anti-angiogenesis activities. We will report the latest results of this novel approach, further discuss the underlined mechanism action of this mAb-siRNA combination for potent anti-angiogenesis therapy and its clinical implication for cancer treatment. The dual-targeted therapeutics and the combination of siRNAs targeting multiple pro-angiogenesis factors have further demonstrated the advantages of this novel therapeutic modality. As with any emerging technologies, many issues related to siRNA therapeutics must be addressed diligently before the success of this novel approach can be achieved in clinics.

Research paper thumbnail of TNT-3/IL-4 and TNT-3/GM-CSF Fusion Proteins for the Promotion of Intratumoral DC Maturation and Initiation of Adaptive Tumor Immunity

Journal of Immunotherapy, 2005

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood ... more Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. The modulation of angiogenesis pathways using small interfering RNA (siRNA) inhibitors of gene expression has proven to be a powerful approach which would be applied for treatment of multiple cancers. Using a nanoparticle-mediated systemic delivery of siRNA with a dual targeting specificity to both neovasculature and VEGF receptor 2 (KDR), we were able to achieve anti-tumor efficacies in multiple tumor models. We further tested this potential anticancer drug candidate, ICS-283, in combination with Genentech's Avastin, a mAb against VEGF A in colorectal tumor cell DLD-1 xenograft model. The combined regimen resulted stronger anticancer efficacy than either of the component alone with significant evidence of anti-angiogenesis activities. We will report the latest results of this novel approach, further discuss the underlined mechanism action of this mAb-siRNA combination for potent anti-angiogenesis therapy and its clinical implication for cancer treatment. The dual-targeted therapeutics and the combination of siRNAs targeting multiple pro-angiogenesis factors have further demonstrated the advantages of this novel therapeutic modality. As with any emerging technologies, many issues related to siRNA therapeutics must be addressed diligently before the success of this novel approach can be achieved in clinics.

Research paper thumbnail of H60/TNT-3 Fusion Protein Activates NK Cells In Vitro and Improves Immunotherapeutic Outcome in Murine Syngeneic Tumor Models

Journal of Immunotherapy, 2006

H60 is a murine minor histocompatibility antigen that binds to NKG2D and activates an effector ph... more H60 is a murine minor histocompatibility antigen that binds to NKG2D and activates an effector phenotype in NK and T cells. In the present study, H60 was genetically fused to the tumor-targeting murine MAb TNT-3. The resultant fusion protein, named H60/TNT-3, was produced in NS0 cells and determined by ELISA to possess an H60 epitope. The K a of H60/TNT-3 (2.43 Â 10 9 M À 1) was nearly identical to that of the parental Ab (2.22 Â 10 9 M À 1), demonstrating that addition of the H60 moiety to the N-terminus of TNT-3 heavy chain did not affect antigen affinity. In vitro, H60/TNT-3 bound and activated murine NK cells, eliciting IFN-g production in a higher percentage of cells than the activating NKG2D Ab A10. In vivo, H60/TNT-3 possessed a half-life of approximately 12 hours and effectively targeted tumor tissue versus control organs, with nearly 2% injected dose per gram of tumor retained after 48 hours. Finally, H60/TNT-3 was tested for antitumor efficacy in BALB/c and C57BL/6 mice bearing subcutaneous syngeneic carcinomas. Tumor volume reduction was observed in both CT26 and Lewis Lung models (53% and 52%, respectively) relative to untreated control mice. Further, Lewis Lung carcinoma-bearing mice treated with H60/TNT-3 experienced a statistically significant survival advantage. Taken together, these data characterize a new immunotherapeutic MAb with antitumor efficacy that prolonged overall survival in a resistant solid tumor model.

Research paper thumbnail of Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

Cellular & Molecular Immunology, 2018

Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can... more Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40 − / − mice had significantly reduced numbers of CD25 − Foxp3 low tTreg precursors and CD25 + Foxp3 + mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.

Research paper thumbnail of Immunogenicity of Murine Solid Tumor Models as a Defining Feature of In Vivo Behavior and Response to Immunotherapy

Journal of Immunotherapy, 2013

Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify... more Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify novel targets for therapy. Two emerging uses of immune signatures are identification of likely responders to immunotherapy regimens among individuals with cancer or to understand the variable responses seen among subjects with cancer in immunotherapy trials. Here the immune profiles of six murine solid tumor models (CT26, 4T1, MAD109, RENCA, LLC, and B16) were correlated to tumor regression and survival in response to two immunotherapy regimens. Comprehensive profiles for each model were generated using quantitative RT-PCR, immunohistochemistry, and flow cytometry techniques, as well as functional studies of suppressor cell populations (Treg and MDSC), to analyze intratumoral and draining lymphoid tissues. Tumors stratified as highly or poorly immunogenic, with highly immunogenic tumors showing significantly greater presence of T-cell co-stimulatory molecules and immunosuppression in the tumor microenvironment. An absence of tumor-infiltrating CTL and mature DC was seen across all models. Delayed tumor growth and increased survival with suppressor cell inhibition and tumor-targeted chemokine +/− DC vaccine immunotherapy was associated with high tumor immunogenicity in these models. Tumor MHC class I expression correlated with overall tumor immunogenicity level and was a singular marker to predict immunotherapy response with these regimens. By using experimental tumor models as surrogates for human cancers, these studies demonstrate how select features of an immune profile may be utilized to identify patients most likely to respond to immunotherapy regimens.

Research paper thumbnail of Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling

Scientific reports, Jan 3, 2017

Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Tr... more Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset ...

Research paper thumbnail of Synthesis and preliminary evaluation of a new chelate N2S4 for use in labeling proteins with metallic radionuclides

International Journal of Radiation Applications and Instrumentation Part B Nuclear Medicine and Biology

The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodie... more The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodies for targeting tumor tissue in uivo depends upon the development and use of suitable bifunctional chelating agents. We have successfully synthesized a new N,S, (compound 4) chelate. This chelate forms a stable complex with 99mT~ and is capable of coupling to different proteins. The N,S, compound is quite stable if kept as a hydrochloride salt and is coupled to antibody under neutral conditions with better than 95% efficiency without the loss of immunoreactivity.

Research paper thumbnail of Molecular Cloning and Functional Expression of Contortrostatin, a Homodimeric Disintegrin from Southern Copperhead Snake Venom

Archives of Biochemistry and Biophysics, 2000

Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Th... more Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins αIIbβ3, α5β1, αvβ3, and αvβ5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part

Research paper thumbnail of Enhanced Tumor Uptake of Macromolecules Induced by a Novel Vasoactive Interleukin 2 Imrnunoconjugate1

Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer t... more Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer therapy. Recent studies have shown that antibody uptake in tumor is controlled in large part by the tumor blood flow and the vascular permeability of the tumor endothelium. We have hypothesized that these physiological properties of tumor vessels may be altered by pretreatment with vasoactive

Research paper thumbnail of MHC class I loss is a frequent mechanism of immune escape in papillary thyroid cancer that is reversed by interferon and selumetinib treatment in vitro

Clinical cancer research : an official journal of the American Association for Cancer Research, 2014

To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC e... more To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC expression and associated immune activation with current and experimental treatments for thyroid cancer using in vitro PTC cell lines. MHC class I expression and assessment of tumor-infiltrating leukocyte populations were evaluated by immunohistochemistry. PTC cell lines were analyzed for HLA-ABC expression by flow cytometry following tyrosine kinase inhibitor, IFNα or IFNγ, or radiation treatment. Functional changes in antigenicity were assessed by coculture of allogeneic donor peripheral blood leukocytes (PBL) with pretreated or untreated PTC cell lines and measurement of T-cell activation and cytokine production. Both MHC class I and β2-microglobulin expression was reduced or absent in 76% of PTC specimens and was associated with reduced tumor-infiltrating immune cells, including effector (CD3(+), CD8(+), CD16(+)) and suppressor (FoxP3(+)) populations. Treatment of PTC cell lines with ...

Research paper thumbnail of The evaluation of 186Re-labeled antibodies using N2S4 chelate in vitro and in vivo using tumor-bearing nude mice

International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology, 1992

We have recently described a method for radiolabeling monoclonal antibodies, with metallic radion... more We have recently described a method for radiolabeling monoclonal antibodies, with metallic radionuclides using a new chelating agent N2S3. Using this chelate the monoclonal antibodies Lym-1 and B72.3 were labeled with 186Re and their biological integrity was evaluated in vitro and in vivo. 186Re-labeled antibodies using N2S4 methodology were found to be stable in human serum and retained their immunoreactivity. Intravenous administration of 0.5 mCi 186Re-labeled antibodies resulted in partial or complete regression of tumor tissue in mice.

Research paper thumbnail of Comparison of three different targeted tissue factor fusion proteins for inducing tumor vessel thrombosis

Cancer research, Jan 15, 2003

Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pat... more Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pathway of the blood coagulation cascade and normally released from damaged tissues. By substituting the attachment site with a tumor delivery agent, this potent thrombogenic protein in its truncated form (tTF) can be targeted to the tumor where it can initiate clotting, thereby occluding the tumor's blood supply and causing rapid tumor destruction. To test the therapeutic potential of this vascular targeting approach, three fusion proteins, chTNT-3/tTF, chTV-1/tTF, and RGD/tTF, which target DNA exposed in degenerative areas of tumors, fibronectin on the tumor vascular basement membrane, and alpha nu beta 3 on the luminal side of tumor vessels, respectively, were developed and tested for their antitumor effects. Antigen binding and clotting assays demonstrated that each of the fusion proteins retained their antigen binding and thrombogenic activities. In vivo studies in mice bearing est...

Research paper thumbnail of Synthesis and preliminary evaluation of a new chelate N2S4 for use in labeling proteins with metallic radionuclides

International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1991

The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodie... more The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodies for targeting tumor tissue in uivo depends upon the development and use of suitable bifunctional chelating agents. We have successfully synthesized a new N,S, (compound 4) chelate. This chelate forms a stable complex with 99mT~ and is capable of coupling to different proteins. The N,S, compound is quite stable if kept as a hydrochloride salt and is coupled to antibody under neutral conditions with better than 95% efficiency without the loss of immunoreactivity.

Research paper thumbnail of Monoclonal antibodies. Their promise for tumor diagnosis, staging, and therapy

Postgraduate medicine, 1985

Monoclonal antibodies will have utility as tools for diagnosis, staging, and therapy of malignant... more Monoclonal antibodies will have utility as tools for diagnosis, staging, and therapy of malignant disease. Investigators have produced monoclonal antibodies that are directed against tumor-associated antigens and have varying degrees of cross-reactivity against normal tissues. These reagents have homogeneous molecular structure, recognize specific antigenic sites, can be produced in mass quantities, and are easily purified. Clinical trials with monoclonal antibodies are in progress and suggest modest toxicity and potential therapeutic efficacy.

Research paper thumbnail of Evaluating Antibodies for Their Capacity to Induce Cell-mediated Lysis of Malignant B Cells1

Promising results from clinical trials have led to renewed interest in effector mechanisms operat... more Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells,

Research paper thumbnail of Soluble Fc Fusion Proteins for Biomedical Research

Methods in Molecular Biology, 2007

As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become va... more As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator's protein moiety of interest is genetically linked to the N-terminus of murine Fc and subsequently expressed in large quantity using a mammalian cell expression system. The resulting Fc fusion proteins are purified on a protein A column and may be stored for at least one year at -20 degrees C. The availability of easily purified, soluble Fc fusion proteins in such quantity can facilitate research in multiple fields of medicine and biotechnology.

Research paper thumbnail of Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors

Immunotherapy, 2011

In this article, the role of chemokines and costimulatory molecules in the immunotherapy of exper... more In this article, the role of chemokines and costimulatory molecules in the immunotherapy of experimental murine solid tumors and immunotherapy used in ongoing clinical trials are presented. Chemokine networks regulate physiologic cell migration that may be disrupted to inhibit antitumor immune responses or co-opted to promote tumor growth and metastasis in cancer. Recent studies highlight the potential use of chemokines in cancer immunotherapy to improve innate and adaptive cell interactions and to recruit immune effector cells into the tumor microenvironment. Another critical component of antitumor immune responses is antigen priming and activation of effector cells. Reciprocal expression and binding of costimulatory molecules and their ligands by antigen-presenting cells and naive lymphocytes ensures robust expansion, activity and survival of tumor-specific effector cells in vivo. Immunotherapy approaches using agonist antibodies or fusion proteins of immunomodulatory molecules si...

Research paper thumbnail of Immunologic and Biochemical Analysis of TNT-1 and TNT-2 Monoclonal Antibody Binding to Histones

Hybridoma, 1993

Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and ... more Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and TNT-2, have been generated by immunizing mice with nuclear extracts from human lymphoma cells. The monoclonal antibodies were initially identified by indirect immunofluorescence on lymphoma cell lines and subsequently were found to stain the nucleus of all cell types from several species including plants by indirect immunofluorescence techniques. Immunoblot analysis demonstrated that TNT-1 bound to a protein of 22 kD and TNT-2 bound to two proteins of 15 and 22 kD, consistent with the known molecular weight of histones. To characterize their immunoreactivity, competition radioimmunoassays were performed using purified histone fractions HI, H2a, H2b and H3. By these assays, TNT-1 was found to bind to histone fraction HI and TNT-2 to an epitope common to histone fractions HI and H3. Histones are found in abundance in the nucleus of the cell and are known to play a major role in chromosome structure and gene expression. Upon cell death, histones remain tightly bound to DNA and consequently provide an abundant intracellular antigen that can be exploited in targeting necrotic cells, such as those found in tumors.

Research paper thumbnail of The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins

Planta, 2012

The spatial organisation of the splicing system in plant cells containing either reticular (Alliu... more The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei. Keywords Architecture of plant cell nucleus Á PANA antigen Á SnRNA Á Splicing Á SR proteins Abbreviations CB Cajal bodies DAPI 4,6-Diamidino-2-phenylindole PANA Proliferation-associated nuclear antigen SnRNP Small nuclear ribonucleoproteins SR Serine/arginine-rich proteins Electronic supplementary material The online version of this article (

Research paper thumbnail of Cytoreductive Chemotherapy Improves the Biodistribution of Antibodies Directed against Tumor Necrosis in Murine Solid Tumor Models

Molecular Cancer Therapeutics, 2013

Current strategies in cancer treatment employ combinations of different treatment modalities, whi... more Current strategies in cancer treatment employ combinations of different treatment modalities, which include chemotherapy, radiotherapy, immunotherapy, and surgery. Consistent with that approach, the present study demonstrates how chemotherapeutic agents can potentiate the delivery of radiolabeled, necrosis-targeting antibodies (chTNT-3, NHS76) to tumor. All chemotherapeutics in this study (5-fluorouracil, etoposide, vinblastine, paclitaxel, and doxorubicin) resulted in statistically significant increases in tumor uptake of radiolabeled antibodies and their F(ab')2 fragments compared to no pretreatment with chemotherapy. Labeled antibodies were administered at various time points following a single dose of chemotherapy in multiple tumor models, and the biodistribution of the antibodies was determined by measuring radioactivity in harvested tissues. MicroPET/CT was also done to demonstrate clinical relevancy of using chemotherapy pretreatment to increase antibody uptake. Results o...

Research paper thumbnail of Stepping Stones Toward Cancer Immunotherapy

Journal of Immunotherapy, 2005

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood ... more Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. The modulation of angiogenesis pathways using small interfering RNA (siRNA) inhibitors of gene expression has proven to be a powerful approach which would be applied for treatment of multiple cancers. Using a nanoparticle-mediated systemic delivery of siRNA with a dual targeting specificity to both neovasculature and VEGF receptor 2 (KDR), we were able to achieve anti-tumor efficacies in multiple tumor models. We further tested this potential anticancer drug candidate, ICS-283, in combination with Genentech's Avastin, a mAb against VEGF A in colorectal tumor cell DLD-1 xenograft model. The combined regimen resulted stronger anticancer efficacy than either of the component alone with significant evidence of anti-angiogenesis activities. We will report the latest results of this novel approach, further discuss the underlined mechanism action of this mAb-siRNA combination for potent anti-angiogenesis therapy and its clinical implication for cancer treatment. The dual-targeted therapeutics and the combination of siRNAs targeting multiple pro-angiogenesis factors have further demonstrated the advantages of this novel therapeutic modality. As with any emerging technologies, many issues related to siRNA therapeutics must be addressed diligently before the success of this novel approach can be achieved in clinics.

Research paper thumbnail of TNT-3/IL-4 and TNT-3/GM-CSF Fusion Proteins for the Promotion of Intratumoral DC Maturation and Initiation of Adaptive Tumor Immunity

Journal of Immunotherapy, 2005

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood ... more Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. The modulation of angiogenesis pathways using small interfering RNA (siRNA) inhibitors of gene expression has proven to be a powerful approach which would be applied for treatment of multiple cancers. Using a nanoparticle-mediated systemic delivery of siRNA with a dual targeting specificity to both neovasculature and VEGF receptor 2 (KDR), we were able to achieve anti-tumor efficacies in multiple tumor models. We further tested this potential anticancer drug candidate, ICS-283, in combination with Genentech's Avastin, a mAb against VEGF A in colorectal tumor cell DLD-1 xenograft model. The combined regimen resulted stronger anticancer efficacy than either of the component alone with significant evidence of anti-angiogenesis activities. We will report the latest results of this novel approach, further discuss the underlined mechanism action of this mAb-siRNA combination for potent anti-angiogenesis therapy and its clinical implication for cancer treatment. The dual-targeted therapeutics and the combination of siRNAs targeting multiple pro-angiogenesis factors have further demonstrated the advantages of this novel therapeutic modality. As with any emerging technologies, many issues related to siRNA therapeutics must be addressed diligently before the success of this novel approach can be achieved in clinics.

Research paper thumbnail of H60/TNT-3 Fusion Protein Activates NK Cells In Vitro and Improves Immunotherapeutic Outcome in Murine Syngeneic Tumor Models

Journal of Immunotherapy, 2006

H60 is a murine minor histocompatibility antigen that binds to NKG2D and activates an effector ph... more H60 is a murine minor histocompatibility antigen that binds to NKG2D and activates an effector phenotype in NK and T cells. In the present study, H60 was genetically fused to the tumor-targeting murine MAb TNT-3. The resultant fusion protein, named H60/TNT-3, was produced in NS0 cells and determined by ELISA to possess an H60 epitope. The K a of H60/TNT-3 (2.43 Â 10 9 M À 1) was nearly identical to that of the parental Ab (2.22 Â 10 9 M À 1), demonstrating that addition of the H60 moiety to the N-terminus of TNT-3 heavy chain did not affect antigen affinity. In vitro, H60/TNT-3 bound and activated murine NK cells, eliciting IFN-g production in a higher percentage of cells than the activating NKG2D Ab A10. In vivo, H60/TNT-3 possessed a half-life of approximately 12 hours and effectively targeted tumor tissue versus control organs, with nearly 2% injected dose per gram of tumor retained after 48 hours. Finally, H60/TNT-3 was tested for antitumor efficacy in BALB/c and C57BL/6 mice bearing subcutaneous syngeneic carcinomas. Tumor volume reduction was observed in both CT26 and Lewis Lung models (53% and 52%, respectively) relative to untreated control mice. Further, Lewis Lung carcinoma-bearing mice treated with H60/TNT-3 experienced a statistically significant survival advantage. Taken together, these data characterize a new immunotherapeutic MAb with antitumor efficacy that prolonged overall survival in a resistant solid tumor model.