Cell Transfer Immunotherapy Research Articles

Abstract IA14: Cell transfer immunotherapy targeting unique somatic mutations in cancer

Abstract Adoptive cell transfer (ACT) immunotherapy using T lymphocytes with antitumor activity is a living therapy that can be highly effective in patients with a variety of metastatic cancers. Naturally occurring tumor infiltrating lymphocytes (TIL) expanded in vitro and administered following a lymphodepleting preparative regimen mediated objective regression of metastatic melanoma in 55% of 194 patients including complete regressions in 24% of patients who remain ongoing disease-free 3 to 10 years later. To determine the antigens recognized by TIL we developed an approach based on deep exomic sequencing of the cancer and immunologic testing of TIL or peripheral lymphocytes to generate T-cells that recognized immunogenic mutations. TIL from 22 patients with metastatic melanoma recognized 54 random somatic mutations none of which were shared among different melanomas. We next extended these studies to patients with common epithelial cancers and showed that 81 of 99 (82%) patients with a variety human cancer types including esophageal, colorectal, bile duct, gastric, pancreatic, ovarian, cervical and lung cancer contained T-cells that recognized 197 neoantigens all of which were unique except for KRAS (2 patients). Targeting unique cancer mutations has extended the reach of ACT immunotherapy and was used to mediate objective regressions in selected patients with chemorefractory metastatic cancers of the bile duct, colon, cervix, and breast. In addition to the use of naturally occurring mutation-reactive cells, we have genetically engineered autologous lymphocytes to express shared antitumor T- cell receptors (TCR) or chimeric antigen receptors (CAR) for use in ACT immunotherapy that can mediate durable cancer regressions in heavily pretreated patients with refractory lymphomas, sarcomas and melanoma. Autologous T- cells can be used to provide a highly personalized immunotherapy for cancer patients refractory to conventional cancer treatments. Citation Format: Steven A. Rosenberg. Cell transfer immunotherapy targeting unique somatic mutations in cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA14.

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity.

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Abstract PR12: Epigenetic reprograming promotes an immunogenic ovarian tumor microenvironment and synergizes with adoptive transfer of engineered T cells expressing NY-ESO-1 specific T cell receptors

Abstract The ability of the immune system to recognize and reject tumors is dependent on several factors that include the expression of immunogenic target antigens on tumor cells, generation of high frequencies of tumor antigen-specific T cells with potent effector function, and capacity to overcome several mechanisms by which tumors escape immune attack. The immunosuppressive environment in the tumor bearing host and at the tumor site restricts persistence and function of antigen-specific T cells, and represent a major obstacle to a durable adoptive engineered TCR (T-cell receptor) cell transfer immunotherapy. DNA demethylation agents have been shown to change immune gene signatures of tumor cells and activate the expression of suppressed tumor antigens by relieving promotor hyper-methylation. In the current study, we aim to determine the immunomodulatory activity of demethylation agents on both ovarian cancer cells and immune cell components in tumor microenvironment, and identify strategies to enhance adoptive T-cell transfer (ACT) immunotherapy in ovarian cancer models. Using a preclinical xenograft model, NY-ESO-1 negative ovarian cancer, OVCAR3, we investigated therapeutic effects of three HLA-A*02 restricted clones of NY-ESO-1 specific CD8 TCR gene-engineered T cells in combination with the demethylation agents decitabine and SGI-110. Demethylation agent treatment followed by ACT elicited synergistic antitumor responses, ranging from inhibition to regression of tumor growth, with curative effect in some animals. In the NY-ESO-1 negative OVCAR3 model, demethylation agents not only induced expression of NY-ESO-1 tumor antigen and MHC I and II, rendering the tumor visible for recognition by CD8 T cells, but also dramatically promoted persistence and accumulation of adoptively transferred T cells at tumor site, as well as reduction of suppressive myeloid cells in the tumor. To further optimize the therapeutic effect of NY-ESO-1 specific CD8 T cells, we explored the activity of TCR gene-engineered NY-ESO-1 specific CD4 T cells that directly recognize cancer cells in a MHC II-restricted manner, in combination with decitabine. We also demonstrated that decitabine rendered the tumor visible for recognition by NY-ESO-1 specific CD4 T cells leading to significant tumor inhibition, and improved persistency of CD4 T cells at peripheral and tumor sites. To maximize curative effect and durability of the therapy, we are currently investigating the immunomodulatory role of decitabine in combination with both CD8 and CD4 T cells specific to NY-ESO-1 in ovarian cancer. Our findings provide insights into novel modes of action of demethylation agents, and indicate that, with optimized therapeutic strategies, demethylation agents can dramatically augment efficacy of adoptive T-cell transfer immunotherapy and counter the immunosuppressive mechanisms in ovarian cancer models. This abstract is also being presented as Poster B34. Citation Format: Li Shen, Bob McGray, Anthony Miliotto, Ariel Francois, Cheryl Eppolito, Junko Matsuzaki, Takemasa Tsuji, Richard Koya, Adekunle Odunsi. Epigenetic reprograming promotes an immunogenic ovarian tumor microenvironment and synergizes with adoptive transfer of engineered T cells expressing NY-ESO-1 specific T cell receptors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr PR12.

Abstract 963: Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity

Abstract Neoantigen-reactive T cells are considered to be crucial mediators for anti-tumor activity in adoptive T cell transfer (ACT) immunotherapy although the efficacy of ACT using in vitro expanded tumor-infiltrating T lymphocytes (TILs) has been limited. Since characterization of T cell receptor (TCR) repertoires of TILs before and after in vitro expansion has not well investigated, we in present study performed whole exome sequencing (WES) and transcriptome analysis, and selected candidate neoantigen epitopes to induce cytotoxic T lymphocytes (CTLs) in 20 patients with head and neck cancer (10 frozen tissues and 10 freshly resected tumors). 36 potential neoantigen peptides including missense somatic mutations were examined for induction of neoantigen-reactive cytotoxic T cells in vitro using patients' derived dendritic cells. We have so far confirmed three neoantigen-reactive T cells and obtained the sequence information of a pair of TCR alpha and beta chains, which were identical to those identified in resident dominant TILs in the original tumors. We cloned the TCR genes into T lymphocytes and constructed the neoantigen-reactive T-cell receptor-engineered (TCR-engineered) T cells, which showed HLA-restricted neoantigen-reactive cytotoxic activity. In addition, we compared the TCR composition of TILs before/after in vitro expansion in 10 fresh head and neck cancers, and found that TCR clonotypes in original TILs and in vitro expanded TILs are drastically different except for those between expanded CD8 cells and resident TILs in three tumors, which had unusually high mutational burden due to mutations in the mismatch repair genes (MSH2, MSH6) or a DNA polymerase (POLE) gene. Our data also suggest that some expanded TILs are likely to be cancer-specific T cells and may be good cell sources for ACT immunotherapy and that the mutational load might be a good biomarker to screen effective TIL treatment. We demonstrate establishment of an effective and rapid protocol to generate neoantigen-specific T cells and to identify neoantigen-specific TCRs for individual patients, which makes TCR-engineered T cells applicable for the clinical use. Citation Format: Lili Ren, Tatsuo Matsuda, Boya Deng, Kazuma Kiyotani, Taigo Kato, Jae-Hyun Park, Tanguy Seiwert, Everett Vokes, Nishant Agrawal, Yusuke Nakamura. Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 963.

Abstract 225: Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis

Abstract Tubo-ovarian high-grade serous cancer (HGSC) is the most common histotype of epithelial ovarian cancer (70%-80%) and the leading cause of death from gynecological malignancy. Novel monoclonal antibody and adoptive cell transfer immunotherapies have found success in cancer in the past five years; however success in HGSC treatment has been elusive thus far. We have recently shown that CD8+ T-lymphocytes infiltrating the tumor proper (TILs) associate in a dose response manner with longer patient survival time regardless of age, stage or residual disease. As understanding host factors associated with this clinically relevant tumor phenotype will contribute to the development of targeted therapies, we sought to identify germline genetic factors associated with CD8+ TIL levels in HGSC. We performed a germline genome-wide association study (GWAS) of CD8+ TILs using 1204 HGSC patients of European ancestry from twelve studies with centralized genotyping, immunohistochemistry, and harmonization of clinical data. Germline genotypes were assayed using the Illumina Infinium OncoArray Beadchip and were imputed to over 11.4 million variants based on the 1000 Genomes Project reference panel. CD8 immunohistochemistry was performed on tissue microarrays using the Leica Bond Rx stainer and scored into four levels of CD8+ TILs per x400 magnification of a 0.55-mm diameter field for each tumor hotspot: none (no CD8+ TILs), low (1-2 CD8+ TILs), moderate (3-19 CD8+ TILs), high (20 or more CD8+ TILs). GWAS used multinomial regression modelling of these four CD8+ TIL levels adjusted for study site, age, and European ancestry principal components. On the average, CD8+ TIL levels were 18%, 19%, 44% and 18% for none, low, moderate and high levels, respectively. As expected, CD8+ TIL levels associated with overall survival time (p < 2.6x10-8), and CD8+ TIL levels and age at diagnosis were similarly distributed across study sites. We identified a region on chromosome 22 with multiple, correlated variants (MAF > 20%) associated with CD8+ TIL levels at p-value < 2.0x10-5. The most statistically significant variant was an indel (rs557925408) located in the an intron of the MYO18B gene (Myosin XVIIIB, p-value < 8.7x10-6). Each copy of this variant associated with a reduced extent of CD8+ TIL infiltration (OR low v none = 0.92; OR moderate v none = 0.61). An independent set of 1200 OncoArray genotyped HGSC cases are undergoing CD8+ TIL scoring in December 2017 and will be included in final genome-wide analysis. This will provide improved statistical power and enable consideration of additional genetic models (e.g., linear trend tests). Comprehensive integrative analysis of germline, tumor, and clinical features provides a model for clinical molecular epidemiology studies and will be key to increasing our understanding of this important HGSC immunophenotype. Citation Format: Yanina Natanzon, Martin Köbel, Stacey J. Winham, Sebastian M. Armasu, Bryan M. McCauley, Robert A. Vierkant, Julie M. Cunningham, David Bowtell, Ian G. Campbell, Jenny Chang-Claude, Anna deFazio, Peter A. Fasching, Mark T. Goodman, Beth Y. Karlan, Francesmary Modugno, Kirsten B. Moysich, Roberta B. Ness, Weiva Sieh, Paul D. Pharoah, Susan J. Ramus, Ellen L. Goode. Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 225.

Reenergized Adoptive Cell Transfer Immunotherapy

Immunotherapy, in treating solid tumors, has seen increased efficacy due to the development of novel strategies of implementation. One innovative strategy, in development by Dr. Weiguo Cui and his colleagues, combines adoptive cell transfer (ACT) with pathogen‐based vaccine techniques in what is called Reenergized ACT (ReACT). Tumor specific CD8 T‐Cells that already have a tumor specific T‐Cell Receptor (TCR), are genetically modified outside the body to express a second TCR that will recognize a bacterial antigen. These newly created, dual specific T cells, when introduced to mice in combination with intratumoral injection of bacterial vaccine, show a significant decrease in tumor volume than their mono‐specific T‐Cell counterparts. The Marquette High SMART (Students Modeling a Research Topic) Team used 3D printing technology, in partnership with the Milwaukee School of Engineering Center for Biomolecular Modeling, to model the TCR complex in order to study its structure and function to better understand ReACT. TCR is a heterodimeric protein consisting of highly variable alpha and beta chains. Both chains are composed of a variable region and a constant region, both of which form antiparallel beta sheets. The variable region binds to the MHC complex whereas the constant region is near the cell membrane of the T‐cell. The constant domains of the TCR consists of cysteine residues that form disulfide bonds, which form a link between the two chains. Further research into immunotherapy, TCR structure and function, and the implementation of Reenergized ACT has major promise in increasing the efficacy of treating disease in human beings now and in the future to come.Support or Funding InformationWeiguo Cui, MD, PhD at the Blood Research Institute, BloodCenter of Wisconsin & Medical College of WisconsinDiane Muzenmaier at the Milwaukee School of Engineering Center for Biomolecular ModelingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

Intra-tumoral delivery of CXCL11 via a vaccinia virus, but not by modified T cells, enhances the efficacy of adoptive T cell therapy and vaccines

ABSTRACTT cell trafficking into tumors depends on a “match” between chemokine receptors on effector cells (e.g., CXCR3 and CCR5) and tumor-secreted chemokines. There is often a chemokine/chemokine receptor “mismatch”, with tumors producing minute amounts of chemokines, resulting in inefficient targeting of effectors to tumors. We aimed to alter tumors to produce higher levels of CXCL11, a CXCR3 ligand, to attract more effector cells following immunotherapy. Mice bearing established subcutaneous tumors were studied. In our first approach, we used modified chimeric antigen receptor (CAR)-transduced human T cells to deliver CXCL11 (CAR/CXCL11) into tumors. In our second approach, we intravenously (iv) administered a modified oncolytic vaccinia virus (VV) engineered to produce CXCL11 (VV.CXCL11). The effect of these treatments on T cell trafficking into the tumors and anti-tumor efficacy after subsequent CAR T cell injections or anti-tumor vaccines was determined. CAR/CXCL11 and VV.CXCL11 significantly increased CXCL11 protein levels within tumors. For CAR/CXCL11, injection of a subsequent dose of CAR T cells did not result in increased intra-tumoral trafficking, and appeared to decrease the function of the injected CAR T cells. In contrast, VV.CXCL11 increased the number of total and antigen-specific T cells within tumors after CAR T cell injection or vaccination and significantly enhanced anti-tumor efficacy. Both approaches were successful in increasing CXCL11 levels within the tumors; however, only the vaccinia approach was successful in recruiting T cells and augmenting anti-tumor efficacy. VV.CXCL11 should be considered as a potential approach to augment adoptive T cell transfer or vaccine immunotherapy.

Abstract KN03: Keynote Lecture: Cell transfer immunotherapy targeting unique somatic mutations in cancer

Abstract Adoptive cell transfer (ACT) immunotherapy using T lymphocytes with antitumor activity is a living therapy that can be highly effective in patients with a variety of metastatic cancers. Naturally occurring tumor-infiltrating lymphocytes (TIL) expanded in vitro and administered following a lymphodepleting preparative regimen mediated objective regression of metastatic melanoma in 56% of 194 patients, including complete regressions in 20% of patients who remain ongoing disease-free 3 to 10 years later. To determine the antigens recognized by TIL we developed an approach based on deep exomic sequencing of the cancer and immunologic testing of TIL or peripheral lymphocytes to generate T cells that recognized immunogenic mutations. TIL from 34 patients with metastatic melanoma recognized 78 random somatic mutations, none of which were shared among different melanomas. We next extended these studies to patients with common epithelial cancers and showed that 64 of 84 (70%) patients with a variety of human cancer types, including esophageal, colorectal, bile duct, gastric, pancreatic, ovarian, cervical, and lung cancer, contained T cells that recognized 142 neoantigens, all of which were unique except for KRAS (3 patients) and p53 (4 patients). Targeting unique cancer mutations has extended the reach of ACT immunotherapy and was used to mediate objective regressions in selected patients with chemorefractory metastatic cancers of the bile duct, colon, cervix, and breast. In addition to the use of naturally occurring mutation-reactive cells, we have genetically engineered autologous lymphocytes to express antitumor T-cell receptors (TCR) or chimeric antigen receptors (CAR) for use in ACT immunotherapy that can mediate durable cancer regressions in heavily pretreated patients with refractory lymphomas, sarcomas, and melanoma. Autologous T-cells can be used to provide a highly personalized immunotherapy for cancer patients refractory to conventional cancer treatments. Citation Format: Steven A. Rosenberg. Keynote Lecture: Cell transfer immunotherapy targeting unique somatic mutations in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr KN03.

Imaging T Cell Dynamics and Function Using PET and Human Nuclear Reporter Genes.

Adoptive cell transfer immunotherapy has demonstrated significant promise in the treatment of cancer, with long-term, durable responses. T cells expressing T cell receptors (TCRs) that recognize tumor antigens, or engineered with chimeric antigen receptors (CARs) can recognize and eliminate tumor cells even in advanced disease. Positron emission tomography (PET) imaging with nuclear reporter genes, a noninvasive method to track and monitor function of engineered cells in vivo, allows quantitative, longitudinal monitoring of these cells, including their expansion/contraction, migration, retention at target and off-target sites, and biological state. As an additional advantage, some reporter genes also exhibit "suicide potential" permitting the safe elimination of adoptively transferred T cells in instances of adverse reaction to therapy. Here, we describe the production of human nuclear reporter gene-expressing T cells and noninvasive PET imaging to monitor their cell fate in vivo.

14PD - Expansion of tumor specific tumor-infiltrating lymphocytes (TIL) from sarcoma and the potential benefit of anti-CD137 stimulation: A prerequisite for adoptive cell transfer (ACT) immunotherapy

14PD - Expansion of tumor specific tumor-infiltrating lymphocytes (TIL) from sarcoma and the potential benefit of anti-CD137 stimulation: A prerequisite for adoptive cell transfer (ACT) immunotherapy

Identification of essential genes for cancer immunotherapy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

Enhanced Therapeutic Efficacy and Memory of Tumor-Specific CD8 T Cells by Ex Vivo PI3K-δ Inhibition.

Inhibition of specific Akt isoforms in CD8+ T cells promotes favored differentiation into memory versus effector cells, the former of which are superior in mediating antitumor immunity. In this study, we investigated the role of upstream PI3K isoforms in CD8+ T-cell differentiation and assessed the potential use of PI3K isoform-specific inhibitors to favorably condition CD8+ T cells for adoptive cell therapy. The phenotype and proliferative ability of tumor antigen-specific CD8+ T cells was assessed in the presence of PI3K-α, -β, or -δ inhibitors. Inhibition of PI3K-δ, but not PI3K-α or PI3K-β, delayed terminal differentiation of CD8+ T cells and maintained the memory phenotype, thus enhancing their proliferative ability and survival while maintaining their cytokine and granzyme B production ability. This effect was preserved in vivo after ex vivo PI3K-δ inhibition in CD8+ T cells destined for adoptive transfer, enhancing their survival and also the antitumor therapeutic activity of a tumor-specific peptide vaccine. Our results outline a mechanism by which inhibitions of a single PI3K isoform can enhance the proliferative potential, function, and survival of CD8+ T cells, with potential clinical implications for adoptive cell transfer and vaccine-based immunotherapies. Cancer Res; 77(15); 4135-45. ©2017 AACR.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

Human T memory stem (TSCM) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long‐lived T‐cell memory and are thus considered an attractive population to be used in adoptive transfer‐based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T‐cell receptor stimulation curbs human effector CD8+ T‐cell differentiation and allows the generation of CD45RO–CD45RA+CCR7+CD27+CD95+ ‐phenotype cells from highly purified naïve T‐cell precursors, resembling naturally‐occurring human TSCM. These cells proliferate extensively in vitro and in vivo, express low amounts of effector‐associated genes and transcription factors and undergo considerable self‐renewal in response to IL‐15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly‐activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long‐lived memory T cells with potential application in adoptive cell transfer immunotherapy.

Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells.

The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens.

Abstract 1949: Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma

Abstract The aim of this study is to investigate the potential of Hypoxanthine phosphoribosyltransferase (HPRT) as a surface biomarker and target for future immunotherapies in Burkitt’s B-cell Lymphoma. B-cell malignancies are the most common type of childhood cancer. Development of immunotherapies could improve current treatment. HPRT is a cytosolic transferase involved in nucleotide production via the purine salvage pathway. Altered expression of TK1, a salvage pathway enzyme, is an indicator of prognosis and diagnosis in multiple cancer types due to active proliferation of cells and the resulting elevated nucleotide demand. It has previously been reported that some salvage pathway enzymes are found on the surface of Burkitt’s Lymphoma cells. HPRT presence on the surface of this B-cell lymphoma subtype could provide a target for adoptive cell transfer and other immunotherapies. The potential surface presentation of HPRT was assessed using flow cytometry, scanning electron microscopy (SEM), and cytoplasmic staining on both healthy lymphocytes and Burkitt’s Lymphoma cells (Raji). Flow cytometry experiments with HPRT antibodies and fluorescent-labeled secondary antibodies show that Raji cells exhibit an 81.4% (p-value .0001) positive fluorescence shift when compared to IgG controls (1.5%). Healthy lymphocytes had a fluorescence shift of 2.38% (p-value .9787). The presence of HPRT on the surface of both Raji cells and healthy lymphocytes was further confirmed using gold-labeled antibodies. Utilizing a scanning electron microscope, the presence of the protein on the surface was evaluated and quantified via increases in gold weight percentage of the sample. When treated with antibodies against HPRT, there was a significant increase in gold binding along with an increase in gold weight percentage. These results suggest a direct relationship between HPRT and the surface of Burkitt’s lymphoma cells, indicating HPRT as a potential target for future immunotherapeutic treatment in Burkitt’s B-cell lymphoma pediatric patients. Citation Format: Michelle H. Townsend, John Ellis Lattin, Michael D. Anderson, Abigail Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, Kim L. O'Neill. Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2017-1949

Abstract 4932: Oxygen sensing by T cells establishes an immunologically favorable metastatic niche

Abstract Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: David Clever, Nicholas Restifo. Oxygen sensing by T cells establishes an immunologically favorable metastatic niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4932. doi:10.1158/1538-7445.AM2017-4932

Tumor-penetrating peptide iRGD conjugation effectively potentiates intratumor T cell infiltration.

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

Preclinical development of tumor-infiltrating lymphocyte (TIL) based adoptive cell transfer (ACT) immunotherapy for patients with sarcoma and the potential benefit of anti-CD137 stimulation.

e14545 Background: Tumor specific TILs can be in vitro expanded and have the ability to induce complete and durable tumor regression in some patients following ACT. In this preclinical study we investigated the feasibility of expanding TILs from sarcomas, as well as performing functional in vitro analyses on these. Methods: Fresh tumor samples from sarcoma patients were obtained, and TILs were isolated and expanded in growth medium containing IL-2. In a sub study, we investigated the effect of adding an agonistic CD137 antibody (Urelumab, BMS) and/or an anti-CD3 antibody (OKT3) to the medium. Phenotype and functional analyses was performed using flow cytometry and IFNγ-Elispot. Results: Tumor samples from 30 patients with various types of sarcomas were obtained, and we were able to expand a minimum of 40 million TILs from 27 of these. Mean expansion times were 32 days (14 - 61). 87,7 % (36,4 – 99,1) of these cells were CD3+, and of these, 66,7 % (16,3 – 99,1) were CD4+, and 21,8 % (0,1 – 50,6) were CD8+. A sub study revealed that adding anti-CD137 and/or OKT3 increased total yield of TILs; anti-CD137 skewed the phenotype significantly towards more CD8+ TILs and in some cases NK cells and γδ T cells. TILs from 8 of 17 tested tumor samples from four different sarcoma subtypes (undifferentiated pleomorphic sarcoma, myxofibrosarcoma, myxoid liposarcoma and osteosarcoma) demonstrated reactivity against autologous tumor cells using IFNγ-Elispot (40 – 500 spots per 100.000 TILs). In TILs stimulated with anti-CD137 the reactivity increased. Conclusions: We were able to expand TILs from 90 % of the acquired tumor samples to numbers needed for possible future clinical ACT implementation. Expanded TILs were a mix of CD4+ and CD8+ with CD4+ being predominant. Approximately half of the TIL cultures showed some degree of in vitro tumor reactivity as determined by Elispot. Early analyses suggest that addition of anti-CD137 could influence expansion time, phenotype, and functional capacity of the expanded TILs. Based on these results, we conclude that it is feasible to translate TIL based ACT into clinical testing in sarcoma patients.

Isolation of T-Cell Receptors Specifically Reactive with Mutated Tumor-Associated Antigens from Tumor-Infiltrating Lymphocytes Based on CD137 Expression

Purpose: The adoptive transfer of lymphocytes genetically modified to express tumor reactive T-cell receptors (TCR) can mediate tumor regression. Some tumor-infiltrating lymphocytes (TIL) recognize somatic mutations expressed only in the patient's tumors, and evidence suggests that clinically effective TILs target tumor-specific neoantigens. Here we attempted to isolate neoantigen-reactive TCRs as a prelude to the treatment of patients with autologous T cells genetically modified to express such TCRs.Experimental Design: Mutations expressed by tumors were identified using whole-exome and RNA sequencing. Tandem minigene (TMG) constructs encoding 12-24 mutated gene products were synthesized, each encoding the mutated amino acid flanked by 12 amino acids of the normal protein sequence. TILs were cultured with autologous dendritic cells (DC) transfected with in vitro transcribed (IVT) mRNAs encoding TMGs and were evaluated for IFNγ secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+ CD8+ T cells and expanded in vitro Dominant TCR α and β chains were identified in the enriched populations using a combination of 5' rapid amplification of cDNA ends, deep sequencing of genomic DNA, PairSeq analysis, and single-cell RT-PCR analysis. Human PBL retrovirally transduced to express the TCRs were evaluated for recognition of relevant neoantigens.Results: We identified 27 TCRs from 6 patients that recognized 14 neoantigens expressed by autologous tumor cells.Conclusions: This strategy provides the means to generate T cells expressing neoantigen-reactive TCRs for use in future adoptive cell transfer immunotherapy trials for patients with cancer. Clin Cancer Res; 23(10); 2491-505. ©2016 AACR.