bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A20: A tumor-penetrating recombinant protein anti-EGFR-iRGD enhance efficacy of antigen-specific CTL in gastric cancer in vivo
Authors: Sha Huizi , Liu Baorui
Published: March 2017

Background: Strategies that enhance the function of T cells are critical for immunotherapy. Targeted delivery of T cells through BiTE (bispecific T-cell engager) platform to cancerous tissues shows potential in sparing unaffected tissues. However, it has been a major challenge for cells penetration in solid tumor tissues due to the complicated tumor microenvironment. Activated T cells expression integrin, which is the target of peptide RGD. Peptide iRGD (CRGDK/RGPD/EC) increased vascular and tissue permeability in a tumor-specific and neuropilin-1-dependent manner, allowing co-administered drugs to penetrate into extravascular tumor tissue. Recombinant protein anti-EGFR-iRGD was purified and examinated.

Methods: Recombinant protein anti-EGFR-iRGD consisting of an anti-EGFR VHH (the variable domain from the heavy chain of the antibody) fused to iRGD, a tumor-specific binding peptide with high permeability were expressed in E. coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid affinity chromatography. We use tumor cell lines and mice to analyze the targeting, penetrating and antitumor activity of antigen-secific T cells together with recombinant protein.

Results: We have successfully constructed a recombinant protein named anti-EGFR-iRGD, a dual targets of EGFR and integrin and high permeable protein. It could spread extensively throughout both the multicellular spheroids and the tumor mass. The recombinant protein anti-EGFR-iRGD could help more T cells infiltrating into tumor mass and also exhibited antitumor activity in tumor cell lines and mice.

Note: This abstract was not presented at the conference.

Conclusions: Our results provide impetus for further studies for potentially using iRGD based fusion protein anti-EGFR-iRGD with immune therapy regimens for enhancing therapy of gastric cancer patients.

Citation Format: Huizi Sha, Baorui Liu. A tumor-penetrating recombinant protein anti-EGFR-iRGD enhance efficacy of antigen-specific CTL in gastric cancer in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A20.
Abstract A35: Biomarker Strategy To Guide Clinical Development Of ImmTAC™ molecules , A Novel Class Of Bispecific T Cell Engaging Biologic Drugs
Authors: McAlpine Cheryl , Gonzalez Sandra Herrero , Bawden Lindsay , Krige David , Shingler William , Johnson Andy , Patel Sanjay , Parker Debbie , Coughlin Christina M. , Hassan Namir J. , Jakobsen Bent K.
Published: March 2017

IMCgp100, a first in class immunotherapy, is a T cell redirecting bispecific biologic comprising an affinity enhanced T-cell receptor specific for gp100 and an anti-CD3 scFV. Phase I/IIa data supported a favourable safety profile and durable responses in cutaneous and uveal melanoma (CM and UM) were observed. To complement the clinical studies we have developed a comprehensive biomarker strategy, which includes analysis of markers in both the tumour and periphery, to aid our understanding of pharmacodynamics, patient response and potential mechanisms of resistance. The data obtained provide evidence of the pharmacodynamic effects of chemokine/cytokine release, in both the tumor and periphery, and lymphocyte infiltration into tumors over 2 days post dose. Changes in the levels of certain chemokines following the first dose of IMCgp100 were associated with tumor shrinkage. In addition, biological differences in marker responses were also seen between CM and UM patients.

The biomarker strategy developed forms the basis for the on-going Ph II development of IMCgp100 in CM and UM and for other ImmTAC molecules, both as single agents and in combination, for the treatment of solid tumours.

Citation Format: Cheryl McAlpine, Sandra Herrero Gonzalez, Lindsay Bawden, David Krige, William Shingler, Andy Johnson, Sanjay Patel, Debbie Parker, Christina M. Coughlin, Namir J. Hassan, Bent K. Jakobsen. Biomarker Strategy To Guide Clinical Development Of ImmTAC™ molecules , A Novel Class Of Bispecific T Cell Engaging Biologic Drugs. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A35.
Abstract A38: Checkpoint blockade elicits unique T cell expansion to promote tumor regression
Authors: Crosby Erika J. , Wei Junping , Yang Xiao Yi , Lei Gangjun , Wang Tao , Liu Cong-Xiao , Agarwal Pankaj , Lyerly H. Kim , Hartman Zachary C.
Published: March 2017

While PD-1 and CTLA-4 immune checkpoint antibodies have led to durable clinical activity in certain cancers, only a fraction of patients exhibit responses. In these responsive tumors, PD-1 and CTLA-4 antibodies are thought to interfere with tumor immunosuppression of T-cells; however, the exact mechanisms of action and potential synergism between these therapies remains unclear. As triple-negative breast cancers (TNBCs) are characterized by elevated expression of inflammatory and immunosuppressive molecules, as well as high levels of immune infiltrating T cells (TILs), we hypothesized that they would be susceptible to treatment with PD-1 and CTLA-4 antibodies. To test this hypothesis and further define the mechanisms of action of checkpoint blockade, we generated a model of murine TNBC (E0771) utilizing ovalbumin (OVA) as a defined antigen that is tumor-specific and recognizable by transgenic T cells (OT-I cells). Consistent with human TNBCs, E0771 tumors exhibit robust T cell infiltration, with >60% of CD4+ T cells being T-regulatory cells (Tregs), and tumor cells express high levels of PDL1. We found that despite the generation of systemic anti-tumor responses and the addition of OT-I cells, TNBC immunosuppression shielded tumors from immune mediated regression. We then tested the efficacy of anti-PD1 and anti-CTLA4 targeting antibodies to inhibit this tumor immunosuppression and demonstrate that they had an anti-tumor effect by blocking PD-1 signaling in the tumor microenvironment and reducing intratumoral Tregs, respectively. When combined, these distinct mechanisms of action led to regression of ~80% of tumors and were significantly associated with anti-tumor adaptive responses. T cell receptor (TCR) sequencing of TILS in treated mice demonstrated a hyperexpansion of several clones, while also a broadening of the total number of unique clones present. Surprisingly, we found that despite using a homogenous tumor model and adoptively transferring OT-1 cells, TCR sequencing revealed clonal populations that were almost entirely unique for each tumor, with the OTI TCR not representing an expanded clone. As such, our study demonstrates that dual CTLA-4 and PD-1 checkpoint blockade inhibits immunosuppression of T cells in the tumor microenvironment through different and complementary mechanisms to expand and broaden unique intrinsic T cell repertoires in the tumor.

Citation Format: Erika J. Crosby, Junping Wei, Xiao Yi Yang, Gangjun Lei, Tao Wang, Cong-Xiao Liu, Pankaj Agarwal, H. Kim Lyerly, Zachary C. Hartman. Checkpoint blockade elicits unique T cell expansion to promote tumor regression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A38.
Abstract A42: Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen
Authors: Rakhra Kavya , Zhu Eric F. , Abraham Wuhbet , Moynihan Kelly D. , Mehta Naveen , Wittrup Karl D. , Irvine Darrell J.
Published: March 2017

While cancer immunotherapies like checkpoint inhibitors have resulted in unprecedented clinical success, they only benefit a subset of patients. To improve therapeutic outcomes for greater numbers of patients, one strategy is to rationally combine different immunotherapy modalities. We recently demonstrated that attaching albumin-binding lipophilic tails to peptide antigens or molecular adjuvants (creating amphiphile vaccines) results in enhanced T-cell responses. Additionally, we observed significant tumor regression upon combining tumor-antigen targeting antibodies with extended half-life IL-2 (exPK-IL-2) in mouse models of melanoma and prostate cancer. In the present work, we combined both approaches to treat a spontaneous model of lung adenocarcinoma expressing carcinoembryonic antigen (CEA), an oncofetal protein expressed in some human lung cancers. KrasLSL-G12D/+;;p53fl/fl mice were crossed with transgenic mice expressing human-CEA to generate a CEA-tolerant background. Lung tumors were induced by infection with a lentivirus expressing Cre recombinase and human CEA. Due to the extended latency of tumor initiation in this model, we also generated a CEA-expressing cell line from these mice to test the efficacy of different combination immunotherapy regimens. We discovered that weekly treatments combining a CEA-targeting amphiphile-vaccine, exPK-IL-2, and an anti-CEA antibody with checkpoint inhibitors (anti-PD-1 and -CTLA4) resulted in sustained tumor regression in 50% of mice bearing established tumors. We are currently testing this combination immunotherapy on autochthonous lung tumors. Our results suggest that breaking tolerance to a tumor-associated self-antigen (CEA) and combining immunotherapies to recruit both innate and adaptive immune effectors can have a potent therapeutic effect in intractable tumors like lung cancer.

Citation Format: Kavya Rakhra, Eric F. Zhu, Wuhbet Abraham, Kelly D. Moynihan, Naveen Mehta, Karl D. Wittrup, Darrell J. Irvine. Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A42.
Abstract A50: Direct oncogene cancer killing and selective tumor Treg killing through the TNFR2 receptor via dominant antibody antagonists
Authors: Torrey Heather , Butterworth John , Mera Toshi , Okubo Yoshiaki , Wang Limei , Baum Danielle , Warden Sarah , Plager Sara , Huang Daniel , Vanamee Eva , Foster Rosemary , Faustman Denise
Published: March 2017

A major barrier to cancer immunotherapy is lack of selective inhibitors of the regulatory T cells (Tregs) of the cancer microenvironment and methods to directly kill tumors through novel surface oncogenes. Tumor necrosis factor receptor 2 (TNFR2) is a target protein with restricted expression on the most potent Tregs of the tumor infiltrate and on human tumors as a newly discovered human oncogene. We characterized the effect of TNFR2 antibody antagonists via TNFR2 in human samples from ovarian ascites compared to healthy controls. TNFR2 antagonists potently inhibited Treg proliferation with exponential potency and selectivity for the tumor microenvironment Tregs. Furthermore, common ovarian cancer cell lines such as OVCAR3 expressed the TNFR2 oncogene and were rapidly and completely killed by TNFR2 antagonistic antibodies. Dominant TNFR2 antagonists demonstrate tumor-specific Treg depletion. Blocking TNFR2 signaling with antagonist antibodies also creates a novel tool to possibly eliminate tumors expressing the TNFR2 oncogene and to more potently suppress Tregs of the tumor environment.

Citation Format: Heather Torrey, John Butterworth, Toshi Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Sarah Warden, Sara Plager, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise Faustman. Direct oncogene cancer killing and selective tumor Treg killing through the TNFR2 receptor via dominant antibody antagonists. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A50.
Abstract A53: Establishment of a brain tumor model with dysfunctional tumor immunity in mice as a platform for immunotherapeutic evaluation and PD-1 immunotherapy
Authors: Nakashima Hiroshi , Alayo Quazim , Penaloza Pablo , Barouch Dan , Chiocca Ennio Antonio
Published: March 2017

Several clinical trials are currently underway for patients with newly diagnosed and recurrent glioblastoma to reinvigorate tumor immunity using immune-checkpoint blockade. However, the CNS is immunologically unique in its maintenance of an immunosuppressive environment, to protect brain cells from autoimmunity and inflammation. Thus, immunotherapy of brain tumors requires novel regimens to overcome tumor immuno-suppression. Suitable preclinical animal models with exhausted tumor-specific immunity are currently not available. We thus hypothesized that a chronic LCMV (cLCMV) strain that induces T cell exhaustion in mice may be co-opted to model the impaired immunity that characterizes brain tumors. In this model, naïve and cLCMV-infected mice failed to reject a challenge with glioma in brain when murine glioma cells express one of LCMV antigens (VA-glioma) that are presented via MHC-I. In contrast, mice preimmunized with the acutely-controlled LCMV (aLCMV) strain did reject VA-glioma. In fact, in vitro co-culture of lymphocytes and glioma cells, and in vivo TIL analyses suggested that VA-glioma failed to induce CD8+ T cell responses in cLCMV-mice, in contrast to the robust CD8+ T cell response in aLCMV-mice. Furthermore, cLCMV-specific CD8 T cells lacked a central memory T cell phenotype (CD44+, CD62L-, CD127-) and expressed significantly high levels of PD-1. Thus, treatment with anti-PD1, but not isotype control antibody, successfully reinvigorated and restored antitumor immunity in cLCMV-mice that received VA-glioma cells. PD-1 blockade led to significantly improved survival. However, this treatment did not show a therapeutic effect in cLCMV-mice that received parental glioma cells that don't express the viral antigen. These data strongly suggest that PD-1 blockade is effective in brain tumors that induce impaired tumor-specific T cell immunity. In conclusion, this novel mouse model may be useful to screen for novel immune checkpoint inhibitors and combination regimes in the setting of a single dysfunctional CD8 T cell response.

Citation Format: Hiroshi Nakashima, Quazim Alayo, Pablo Penaloza, Dan Barouch, Ennio Antonio Chiocca. Establishment of a brain tumor model with dysfunctional tumor immunity in mice as a platform for immunotherapeutic evaluation and PD-1 immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A53.
Abstract A63: Macrophages expressing fibroblast activation protein-alpha are a tumor-educated polarization phenotype which facilitates metastasis
Authors: Koning Tamara de , Burchell Joy M. , Diebold Sandra S. , Arnold James N.
Published: March 2017

Tumor associated macrophages (TAMs) are one of the most abundant stromal cell types in cancer, and promote tumor progression by supporting angiogenesis, immunosuppression, and tumor cell migration. However, there is increasing evidence to suggest that the functions of these cells could be refined to specific subsets. A subset of TAMs identified by the cell surface marker fibroblast activation protein-alpha (FAP) has previously been demonstrated to facilitate immune suppression in an ectopic Lewis lung carcinoma model through its expression of the heme degrading enzyme heme oxygenase-1 (HO-1) (Arnold et al, 2014). Little is known about the origin of these cells, and whether they represent an autonomous population or a polarization phenotype. Here we show in orthotopic mammary 4T1 adenocarcinomas that FAP+ TAMs are a tumor-educated polarization phenotype which is functionally distinct from IL-4/-13 polarized alternatively activated macrophages. In contrast, IL-6 and integrin β1 signaling contribute to FAP+ TAM polarization. In addition to the previously described role of these cells in immune suppression we show that FAP+ TAMs facilitate pulmonary metastasis. FAP+ TAMs are the primary tumoral source of HO-1 and pharmacological inhibition of HO activity reduces pulmonary metastasis in both orthotopic 4T1 adenocarcinomas and aggressive autochthonous murine mammary adenocarcinomas (MMTV-PyMT). Using an in vitro transwell assay, we demonstrate that HO-1 activity by macrophages enhances the transendothelial migration of tumor cells. In vivo HO inhibition does not affect seeding of tumor cells in the lung once these cells have entered the circulation, suggesting that FAP+ TAMs facilitate the intravasation event. Together, these results demonstrate FAP+ TAMs to be a tumor-educated polarization phenotype which facilitates tumor cell metastasis by its expression of HO-1.

Arnold, J.N., et al., Tumoral immune suppression by macrophages expressing fibroblast activation protein-α and heme oxygenase-1. Cancer Immunol Res, 2014. 2(2): p. 121-6

Citation Format: Tamara de Koning, Joy M. Burchell, Sandra S. Diebold, James N. Arnold. Macrophages expressing fibroblast activation protein-alpha are a tumor-educated polarization phenotype which facilitates metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A63.
Abstract A65: Mechanisms of melanoma cell resistance to phagocytosis
Authors: Anderson Katie L. , Snyder Kristin M. , Lins Debra , Weiskopf Kipp , Shimizu Yoji , Weissman Irving , Mescher Matthew , Modiano Jaime
Published: March 2017

Melanoma is notoriously resistant to apoptosis and to other cytolytic mechanisms used by immune cells; however, the mechanisms by which melanoma cells evade phagocytosis by the innate immune system are incompletely understood. Previous studies in our lab revealed that melanoma cells from human, mouse, and dog tumors display an evolutionarily conserved resistance to phagocytosis that was not observed in lymphoma cells or in other solid tumors from these three species. Blockade of CD47 to disable “don't eat me” signals and doxorubicin treatment to enhance the “eat me” signals calreticulin and phosphatidylserine lead to small increases in melanoma cell phagocytosis in vitro. However, this increase did not appear to be biologically relevant, as combination chemo-immunotherapy did not consistently promote enhanced activation of antigen-specific T cells in the tumor draining lymph node or reduce tumor burden in the B16 melanoma model. From these studies, we concluded that melanoma cells display an evolutionarily conserved resistance to phagocytosis, which cannot be fully mitigated by modulation of known pro- and anti-phagocytic pathways. Therefore, we hypothesize that melanoma cells possess an uncharacterized mechanism of resistance to phagocytosis, such as expression of a soluble or membrane bound “don't eat me” signal. To test for the presence of a soluble anti-phagocytic factor, we performed a series of assays in which human lymphoma (Raji) cells, shown to be readily phagocytosed by mouse macrophages, were placed in supernatant harvested from cultured human melanoma (M21) cells. Blockade of the known “don't eat me” signal CD47 was achieved using a SIRPα; mimotope, CV1-G4, or an isotype control. Lymphoma cell phagocytosis by mouse macrophages and response to CD47 blockade were unaffected by the presence of melanoma cell supernatant, suggesting that a secreted factor is not responsible for resistance. To test for the presence of an uncharacterized, membrane-bound “don't eat me” signal, we designed a siRNA panel containing 48 genes whose protein products are expressed on the cell surface of melanoma cells. In ongoing experiments, we are testing how knockdown of these proteins through RNA interference affects macrophage-mediated phagocytosis of melanoma cells and response to CD47 blockade.

Citation Format: Katie L. Anderson, Kristin M. Snyder, Debra Lins, Kipp Weiskopf, Yoji Shimizu, Irving Weissman, Matthew Mescher, Jaime Modiano. Mechanisms of melanoma cell resistance to phagocytosis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A65.
Abstract A68: Phosphodiesterase-5 inhibition modulates surgery-induced myeloid-derived suppressor cells to reduce postoperative metastatic disease
Authors: Tai Lee-Hwa , Alkayyal Almohanad A. , Baxter Katherine E. , Angka Leonard , Kennedy Mike , Souza Christiano Tanese de , Auer Rebecca C.
Published: March 2017

Purpose: Cancer surgery while necessary for primary tumor removal, has been shown to induce immune suppression and promote metastases in preclinical models and human cancer surgery patients. Activating the immune system and reversing immunosuppression have emerged as promising ways to treat cancer and they can be safely employed in the perioperative period. In this study, we evaluated the immunotherapeutic potential of phosphodiesterase-5 (PDE-5) inhibitors to target surgery-induced myeloid-derived suppressor cells (MDSC) and restore natural killer (NK) cell function in the clinically relevant perioperative period.

Experimental Design: Immunocompetent mice tumor models of major surgery were used to characterize the functional suppression of surgery-induced MDSC and to assess the in vivo efficacy of perioperative PDE5 inhibition. In cancer surgery patients with abdominal malignancies, we assessed NK cell function following co-culture with MDSC and PDE5 inhibition.

Results: Perioperative PDE5 inhibition reverses surgery-induced immunosuppression. In particular, sildenafil reduces surgery-derived granulocytic-MDSC (gMDSC) function through downregulation of arginase 1 (ARG1), IL4Rα; and reactive oxygen species (ROS) expression, enabling NK cell antitumor cytotoxicity and reducing postoperative disease recurrence. By removing surgery-derived immunosuppressive mechanisms on MDSC, sildenafil synergizes with the administration of perioperative influenza vaccination to reduce postoperative metastasis. Importantly, sildenafil reverses MDSC suppression in cancer surgery patients.

Conclusions: These findings demonstrate that PDE5 inhibitors reduce postoperative metastasis by their ability to inhibit surgery-induced MDSC. Further clinical studies are warranted to investigate the immunotherapeutic role of PDE5 inhibitors in combination with cancer surgery.

Note: This abstract was not presented at the conference.

Citation Format: Lee-Hwa Tai, Almohanad A. Alkayyal, Katherine E. Baxter, Leonard Angka, Mike Kennedy, Christiano Tanese de Souza, Rebecca C. Auer. Phosphodiesterase-5 inhibition modulates surgery-induced myeloid-derived suppressor cells to reduce postoperative metastatic disease. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A68.
Abstract A74: CAR-T cells harboring camelid single domain antibody as targeting agent to CEACAM6 antigen in pancreatic cancer
Authors: Wong Wah Yau , Tanha Jamshid , Krishnan Lakshmi , Tian Baomin , Kumar Praveen , Gaspar Kim , Demas Steve , Rohmann Sven , Chao Heman
Published: March 2017

Carcinoembryonic Antigen Related Cell Adhesion Molecule 6 (CEACAM6) is overexpressed in many types of human cancers such as breast, pancreatic, colorectal, and non-small-cell lung adenocarcinoma, and is an independent predictor of overall survival and disease free survival. Targeting this molecule by antibodies has slowed tumor progression in certain animal models. 2A3 is a camelid single domain antibody isolated from a whole cancer cell immunized llama library. The antibody binds specifically to the CEACAM6 antigen with high affinity (5nM as measured by SPR) and inhibits the proliferation of CEACAM6-expressing cancer cells in vitro. In this study, Chimeric-Antigen Receptor (CAR) T cells were engineered to target human CEACAM6 antigen by transducing the 2A3 antibody sequence to generate a modified chimeric CD28 signaling domain fused to chimeric CD3-zeta. Transduction efficiency and expression of 2A3 antibody were verified by flow cytometry. Co-incubation of CEACAM6-specific CAR-T (CEACAM6-CAR-T) cells with the CEACAM6-expressing pancreatic cell line BxPC-3 resulted in augmented cytotoxicity and cytokines (IL-2 and IFN-γ) release, suggesting potential anti-cancer activity of the CAR-T cells. Data from real-time cell analysis showed a significant increase in BxPC-3 cell cytotoxicity by CEACAM6-CAR-T cells, as compared to native T cells. However, CEACAM6-CAR-T cells showed much lower cytotoxic activity on negative control cell lines. The efficacy of CEACAM6-CAR-T cells in xenograft model was examined in vivo. BxPC-3 cells were inoculated subcutaneously into the hind flank of CIEA NOG mice. Three groups of mice then received intravenous injection of either PBS, native T cells, or CEACAM6-CAR-T cells, respectively, at day 1, 8, and 15. The data demonstrated very high efficacy of CEACAM6-CAR-T cells against the pancreatic cancer xenograft. CEACAM6-CAR-T cells significantly decreased the growth of the BxPC-3 xenograft as compared to that of native T cells (p-value=0.00025) and PBS (p-value=7.91x106). No toxic effect was observed based on body weight measurement. The results strongly support that CEACAM6-CAR-T cells can be used as an effective immunotherapy agent against CEACAM6-expressing cancers, and that camelid single domain antibodies can be easily adopted for CAR-T type therapies.

Citation Format: Wah Yau Wong, Jamshid Tanha, Lakshmi Krishnan, Baomin Tian, Praveen Kumar, Kim Gaspar, Steve Demas, Sven Rohmann, Heman Chao. CAR-T cells harboring camelid single domain antibody as targeting agent to CEACAM6 antigen in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A74.
Abstract A85: Investigating endogenous anti-tumor T cell responses in native tumor microenvironments
Authors: Joshi Nikhil , Jacks Tyler
Published: March 2017

Genetically-engineered mouse models (GEMMs) exist for most types of cancer in which autochthonous tumors develop into advanced disease in the presence of a fully functional immune system. These GEMMs would be ideal for investigating questions in cancer immunology, including those concerning the commonalities and differences between immune responses that target different cancers, and how the tumor microenvironments (TMEs) associated with different cancer types impact the functions of tumor-infiltrating immune cells. However, unlike human tumors, tumors in GEMMs have few somatic mutations and thus require exogenous neoantigens (neoAgs) to drive interactions with anti-tumor T cells. There are only a handful of mechanisms to program tumors to express neoAgs, and thus, GEMMs for most tumor types remain inaccessible for cancer immunology studies. Here, we describe the “iNversion INducible Joined neoAg” (NINJA) mouse, which can be used to program GEMM tumors with a common neoAg, and thus stimulate a common set of tumor-specific CD8 T cells. NINJA will be useful for addressing many key unanswered questions cancer immunology, including comparisons of immune responses across several cancer types and comparisons of anti-tumor T cell responses targeting de novo expressed neoAgs at different stages in tumor development. NINJA mice will also be invaluable as a preclinical platform for testing immunotherapeutics, and for investigating why certain immunotherapeutic regimens are more effective when treating some cancers and not others. NINJA is unique, because neoAgs are encoded within the genome, yet NINJA avoids central and peripheral tolerance, a common problem with other systems. This is achieved because the DNA sequences encoding the full-length neoAgs are discontinuous in the OFF state and, thus, neoAgs are silent. Induction of neoAgs is highly regulated, requiring a permanent inversion of a DNA element in NINJA, which allows production of the full-length NINJA neoAgs and GFP (a marker of neoAg expression). This inversion is regulated by Flippase (FlpO), which itself is highly regulated, requiring cells are exposed to Cre recombinase, and then doxycycline and tamoxifen. Therefore, NINJA is compatible with most Cre-inducible GEMMs of cancer. Activating NINJA with adenoviral infection results in a strong, NINJA-specific CD8 T cell response while, in contrast, activation in the setting of lung tumors leads to T cell dysfunction. Thus, NINJA is a powerful, highly versatile system for investigating T cell function in many settings, including cancer, immune tolerance, and autoimmunity.

Citation Format: Nikhil Joshi, Tyler Jacks. Investigating endogenous anti-tumor T cell responses in native tumor microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A85.
Abstract B01: Enhancing radiotherapy by targeting inflammatory monocytes
Authors: Connolly Kelli , Linehan David , Gerber Scott
Published: March 2017

Approximately 50% of cancer patients receive radiotherapy (RT) at some point throughout their treatment. Nevertheless, RT alone does not often result in cures, making it necessary to develop new ways to enhance this treatment modality. It is now understood that the efficacy of RT relies in large part on the anti-tumor immune response that it elicits. However, we believe that RT acts as a double-edged sword as it results in the infiltration of anti-tumor effector cells as well as myeloid cells associated with immunosuppression. One type of these myeloid cells, known as inflammatory monocytes (IM), are precursors for many immunosuppressive cells and have been shown to promote the progression of cancer in various models. However, the role these cells play in modulating the immune response following RT is unknown. To explore this, we focused on the specific chemokine receptors CCR2 and CCR5 and the primary ligands for these receptors, CCL2 and CCL5 respectively, which are present on IM and have been shown to play a role in the recruitment of myeloid cells into tissues and tumors alike. Here, we locally treat syngeneic tumors in C57BL/6 mice with 15Gy RT in order to study the role of CCL2+CCR5+ IM after RT. We show that RT upregulates the expression of intratumoral CCL2 and CCL5 by tumor cells. We also demonstrate that RT subsequently induces the increase of CCR2+ CCR5+ monocytes in circulation as well as their increased infiltration intratumorally. Therefore, we hypothesized that inhibiting the CCL2/CCR2 and CCL5/CCR5 axes and therefore blocking the infiltration of this potentially immunosuppressive cell population could improve the efficacy of RT. Administering a dual CCR2/CCR5 inhibitor (CCR2/CCR5i) daily beginning two days before RT, we demonstrate that inhibition of IM, both intratumorally and in circulation, results in smaller tumor burden and sustained tumor free survival in radioresponsive tumors. Furthermore, the anti-tumor effects of RT and the enhanced response afforded by CCR2/CCR5i is dependent on CD8+ T cells. These data suggest that inhibiting specific chemokine axes augments the anti-tumor effects of the adaptive arm of the immune system. Future studies will address the mechanism and clinical significance of targeting immunosuppressive myeloid cells as a means to enhance the effector functions of anti-tumor T-cells and further augment the efficacy of RT.

Citation Format: Kelli Connolly, David Linehan, Scott Gerber. Enhancing radiotherapy by targeting inflammatory monocytes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B01.
Abstract B08: Tumor-initiating cell associated macrophages play an essential role in tumorigenesis
Authors: Guo Xiaocan , Bin Zhao
Published: March 2017

Inflammation plays a critical role in different stages of tumor development such as initiation, progression, and metastasis. Macrophages are specifically recruited into human tumors and play a generally tumor-promoting role. However, whether macrophages are specifically recruited by tumor-initiating cells (TICs) and how they impact tumorigenesis in vivo have not been directly assessed. Here we show that single liver TICs induced by activation of the Hippo pathway effector YAP strongly recruits macrophages which we termed tumor-initiating cell associated macrophages (TICAMs). Eradication of TICAMs by repression of YAP-induced cytokine expression leads to clearance of TICs in a p53-dependent manner and completely eliminates tumorigenesis. Importantly, TICAMs are recruited to early human HCC lesions showing YAP activation. Our results demonstrate a new role of macrophages essential for tumorigenesis by associating with and promoting the survival of TICs from the single-cell stage. These findings suggest a unique opportunity for tumor prevention by blocking TICAMs.

Citation Format: Xiaocan Guo, Zhao Bin. Tumor-initiating cell associated macrophages play an essential role in tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B08.
Abstract B18: Noninvasive imaging of antitumor immune responses
Authors: Rashidian Mohammad , Dougan Michael , Ingram Jessica , Ploegh Hidde
Published: March 2017

By using the body's own immune response, immunotherapy may prove to be a turning point in the treatment of cancer. Predictors of a patient's response to immunotherapy remain to be defined, as actual responses are heterogeneous and difficult to study in real-time. Is it possible to predict the outcome of therapy for an individual patient? What is the behavior and what are the dynamics of immune cells in the tumor microenvironment? We need new methods to noninvasively explore the microenvironment of tumors and enumerate relevant cell types. Imaging the extent of infiltration of immune cells into tumors and identifying the relevant lymphocyte subsets is therefore essential. We developed single domain antibodies (VHHs, ~15 kDa) against a variety of lymphocyte surface antigens to image antitumor immune responses. VHHs were engineered to contain a sortase recognition tag, X-LPETGG, at their C-terminus to enable site-specific modification with tags of choice. We synthesized a substrate that contains both a click handle and a metal chelator, where the click handle can be used to attach a polymer, and the chelator is used to chelate a radiometal onto the VHH of interest. We thus produced VHHs equipped with a polyethyleneglycol (PEG) molecule to increase circulatory half-life and a chelator. C57/BL6 mice were injected with 89Zr-PEGylated VHHs, and images were acquired 24 h post injection. Images of the PEGylated-VHHs showed specific staining with low background in kidney and intestine. When we used 89Zr-labeled PEGylated-VHHs specific for CD8 or Class II MHC, secondary lymphoid structures, including the mesenteric lymph node, were clearly visible. Knock-out mice were used to establish specificity of PET imaging. Mice bearing panc02 tumors were imaged at 24 h p.i. and showed CD8 T cells infiltrating the tumor. We monitored the CD8-T cell response to immunotherapy in the B16 melanoma model by using the antiCD8 VHH. For animals that showed a response that led to regression of the tumor, the distribution of the signal was homogeneous throughout the tumor. It is thus possible to monitor antitumor immune responses. Noninvasive monitoring could therefore change how therapy is applied.

Citation Format: Mohammad Rashidian, Michael Dougan, Jessica Ingram, Hidde Ploegh. Noninvasive imaging of antitumor immune responses. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B18.
Abstract B21: Probing tumor heterogeneity and immune infiltration with Cyclic Immunofluorescence (CycIF), a robust, multiplexed imaging method
Authors: Lin Jia-Ren , Izar Benjamin , Hawthorne Sabrina , Nordberg Josh , Kaldjian Eric , Sorger Peter
Published: March 2017

Tumor microenvironment, defined by surrounding stromal/immune cells as well as blood vessels, plays an important role in disease progression and therapy resistance. Increasing understanding of the heterogeneity in both tumor and its microenvironment will be crucial to development more effective therapies. Recently, several studies employing state-of-the-art single-cell sequencing methods reveal enormous complexity in tumor microenvironment. However, the spatial information and cell-to-cell interaction could not be preserved in these dissociated cells. Immunofluorescence has been widely used in different fields of biological and medical research for decades. The ability to obtain in situ and single-cell information makes this technique particularly important in tumor biology. However, biochemical and optical constraints limit the number of signals that could be captured simultaneously within the same sample. We have developed the CycIF (Cyclic Immunofluorescence), an easy and low-cost method to increase the multiplexity of conventional immunofluorescence. The CycIF method has been first applied in pre-clinical drug discovery, cancer and stem cell biology with adherent cell cultures. In here, we modified the original CycIF method for IHC/IF on FFPE samples, and used that to probe tumor heterogeneity, microenvironment and immune infiltration in various types of tumors. Up to 30 different antigens/markers could be simultaneously detected in different tumor samples, and these makers represent a wide range of biological processes, including the key molecules for lymphocyte surface makers (CD45, CD4, CD8, CD20, CD11c), immune checkpoints (PD-1, PD-L1), stromal/EMT proteins (E-Cadherin, Vimentin), cell cycle regulators (CycD1, PCNA, Ki67, pRB, p21/CIP), signaling proteins (EGFR, pERK, pS6) and apoptosis mediators (p53, Bax, Bcl-2, Survivin). Our study not only provides the first detailed map of tumor and its immune microenvironment, but also illustrates a robust multiplexed imaging platform for probing tumor heterogeneity.

Citation Format: Jia-Ren Lin, Benjamin Izar, Sabrina Hawthorne, Josh Nordberg, Eric Kaldjian, Peter Sorger. Probing tumor heterogeneity and immune infiltration with Cyclic Immunofluorescence (CycIF), a robust, multiplexed imaging method. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B21.
Abstract B26: Immunomodulatory activity of Isatuximab
Authors: Srinivasan Srimathi S. , Wang Anlai , Song Zhili , Adrián Francisco
Published: March 2017

Isatuximab, previously known as SAR650984 is a naked chimeric IgG1 anti-CD38 monoclonal antibody. It is currently in clinical trials demonstrating single-agent activity in heavily pretreated relapsed/refractory multiple myeloma (RRMM). Isatuximab induces killing of CD38 positive tumor cells by triggering Fc dependent effector functions such as ADCC, ADCP and CDC, as well as Fc independent direct apoptotic effect. However, its effects in immune cells that express CD38 are emerging and are not yet well defined. The aim of this study was to investigate the effects of Isatuximab on immune effector cells, to better understand its anti-tumor mechanisms underlying the clinical benefit. Among immune sub-populations, Natural Killer (NK) cells and monocytes have the highest CD38 receptor density. Further, NK cells and monocytes play critical roles in immunity against cancer. We investigated Isatuximab's effect on these immune effector cells including their expansion, cytokine secretion and activation. Treatment (8 days at 1µg/ml) of 106 whole human peripheral blood mononuclear cells with Isatuximab induced expansion of NK and monocytes. In addition to enhance expansion, Isatuximab was able to induce the release of IFN-γ (~4000-7000 pg/ml) and TNF-α (~150-200 pg/ml) in NK cells in the absence of target cells. Incubation of Isatuximab (1µg/ml) for an hour also enhanced cytotoxic activity of human NK cells, both NK-92 cell line and isolated human NK cells, against CD38 negative K562 and JHH6 tumor cell lines. In the case of monocytes, Isatuximab activates phagocytosis by human monocytic cell line THP-1 and purified human monocytes. Furthermore, Isatuximab-activated NK cells can induce monocyte to M1 macrophage polarization through IFN-γ release in vitro. These findings collectively show evidence of Isatuximab being an immune-modulatory agent able to induce NK cell and monocyte activation. Further study is needed to determine whether Isatuximab's immunomodulatory activity could be utilized for the treatment of both CD38-positive and negative cancers by enhancing its anti-tumor function.

Citation Format: Srimathi S. Srinivasan, Anlai Wang, Zhili Song, Francisco Adrián. Immunomodulatory activity of Isatuximab. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B26.
Abstract B28: Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective FcγR-engagement
Authors: Dahan Rony , Barnhart Bryan C. , Korman Alan J. , Ravetch Jeffrey V.
Published: March 2017

The use of immunomodulatory mAbs to stimulate effective therapeutic anticancer immunity has shown clinical successes for the treatment of an increasing number of cancer types. An approach that is currently under clinical development uses agonistic anti-CD40 mAbs to induce APC activation, thereby harnessing the potency of the immune response to eradicate tumors. While engagement of the inhibitory Fcγ Receptor(FγγR)IIB is required for the in vivo agonistic activity of anti-mouse CD40 mAbs, a similar requirement for human mAbs has been disputed based on in vitro studies and in vivo studies performed either in wild-type or mouse FcγR knockout strains and thus fails to address the unique binding affinities and tissue distribution of human FcγRs. To overcome these limitations we generated and used unique mouse model fully humanized for its FcγRs and CD40, and revealed that FcγRIIB engagement is also essential for the activity of the human CD40 agonistic mAbs, while engagement of the activating FcγRIIA inhibits their activity. By engineering Fc variants of human mAbs with selective enhancement to FcγRIIB but not to FcγRIIA, significant improvement of antitumor immunity was observed in multiple tumor models. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using pre-clinical model that accurately reflects the unique affinities and cellular expression of human FcRs, thus providing a development approach for therapeutic human antibodies based on their requirement for selective FcγR interactions. Here we describe the selection of optimized next-generation Fc-engineered agonistic CD40 mAbs.

Citation Format: Rony Dahan, Bryan C. Barnhart, Alan J. Korman, Jeffrey V. Ravetch. Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective FcγR-engagement. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B28.
Abstract B71: Circulating PD-1+CD8+T cells reflect response to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer patients
Authors: Lee Sung Yong , Lee Sungjong , Oh Jee Youn , Min Kyung Hoon , Hur Gyu Young , Shim Jae Jeong , Kang Kyung Ho
Published: March 2017

The programmed death-1 (PD-1)/programmed cell death-1 ligand (PD-L1) pathway plays a crucial role in tumor evasion. Alteration in Epidermal Growth Factor Receptor (EGFR) and KRAS are oncogenic drivers in lung cancer and activation of this pathway is a critical event during lung tumorigenesis. However, how this oncogenic signaling influences immunity in the tumor microenvironment is just beginning to be understood. According to other report, expression of mutant EGFR in bronchial epithelial cells induces PD-L1 and PD-L1 expression is reduced by EGFR inhibitors in lung cancer cell lines with activated EGFR mutation. Thus, we evaluated the levels of PD-1+ T cells in peripheral blood according to the response of EGFR Tyrosine Kinase Inhibitors (TKIs) in Non-small Cell Lung Cancer (NSCLC) patients. Blood samples of NSCLC patients were obtained at baseline and after EGFR TKI treatment. Flow cytometry was used to detect PD-1 expressing CD4 or CD8 T cells. Total 53 patients were enrolled in the study. 17 patients had EGFR mutation and treated with EGFR TKIs. The expression of PD-1+CD8+ T cells in peripheral blood are increased according to their clinical stage. The expression levels of PD-1+CD8+ T cells significantly decreased after EGFR TKIs treatment (pretreatment vs. post-treatment 0.93±1.25% vs. 0.43±0.59%, P=0.006). Post/pre EGFR TKIs treatment ratio of PD-1+CD8+ T cells was lower in partial response group than stable disease group (0.19±0.17 vs. 0.9±0.68, P=0.017). So this result showed that PD-1+CD8+ T cells was more decreased in good responder group. EGFR mutant lung tumor inhibited antitumor immunity by activating PD-1 and PD-1 expression was reduced by EGFR TKI, which was proportional to treatment response. In conclusion, the change of PD-1+CD8+ T cell in peripheral blood may be a surrogate marker to predict EGFR TKIs treatment response.

Citation Format: Sung Yong Lee, Sungjong Lee, Jee Youn Oh, Kyung Hoon Min, Gyu Young Hur, Jae Jeong Shim, Kyung Ho Kang. Circulating PD-1+CD8+T cells reflect response to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B71.
Abstract B74: Development of Salmonella-based vaccine against mesothelioma
Authors: Xu Chen
Published: March 2017

Purpose: Salmonella-based vaccine can provide protection against different kinds of cancer, whether used alone or used as a vehicle to deliver heterologous proteins. In this study, the functionality of a live Salmonella-p24 vaccine as a prophylactic anti-tumor vaccine against murine mesothelioma was analyzed.

Experimental Design: Malignant murine mesothelioma cell was engineered to express GAG, an HIV capsid protein, regarding as an artificial tumor associated antigen. Attenuated Salmonella enterica serovar Typhimurium aroA strain was modifed to encode p24, derived from GAG. BalB/C mice were orally immunized with Salmonella-p24 vaccine thrice at 2-week interval. P24-specific humoral and cellular immune responses were examined by ELISA and ELISPOT, respectively. Protection against the challenge with GAG-expressing mesothelioma was also evaluated after immunization.

Results: P24 antigen was successfully expressed in the cytoplasm of an attenuated Salmonella enterica serovar thyphimurium aroA strain. Immunization of wild-type BalB/C by oral gavage could elicit specific anti-p24 humoral and cellular immune response. In vivo protection studies revealed that over 80% of the treated mice remained free of GAG-mesothelioma after challenge, whereas all mice in control group developed tumor growth.

Conclusions: This p24-Salmonella vaccine could efficiently prevent GAG-expressing mesothelioma tumor growth in mice.

Citation Format: Chen Xu. Development of Salmonella-based vaccine against mesothelioma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B74.