Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease. Materials and methods: We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Results: Using HLA-A*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. Conclusion: PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.

Targeting the recurrent Rac1P29S neoepitope in melanoma with heterologous high-affinity T cell receptors.

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope FSGEYIPTV carries the Rac1P29S amino acid change caused by a c.85C>T missense mutation, which is the third most common hotspot mutation in melanoma. Here, we isolated and characterized TCRs to target this HLA-A*02:01-binding neoepitope by adoptive T cell therapy. Peptide immunization elicited immune responses in transgenic mice expressing a diverse human TCR repertoire restricted to HLA-A*02:01, which enabled isolation of high-affinity TCRs. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cells and we observed regression of Rac1P29S expressing tumors in vivo after adoptive T cell therapy (ATT). Here we found that a TCR raised against a heterologous mutation with higher peptide-MHC affinity (Rac2P29L) more efficiently targeted the common melanoma mutation Rac1P29S. Overall, our study provides evidence for the therapeutic potential of Rac1P29S-specific TCR-transduced T cells and reveal a novel strategy by generating more efficient TCRs by heterologous peptides.

H3.3K27M mutation is not a suitable target for immunotherapy in HLA-A2+ patients with diffuse midline glioma.

Diffuse midline glioma is the leading cause of solid cancer-related deaths in children with very limited treatment options. A majority of the tumors carry a point mutation in the histone 3 variant (H3.3) creating a potential HLA-A*02:01 binding epitope (H3.3K27M26-35). Here, we isolated an H3.3K27M-specific T cell receptor (TCR) from transgenic mice expressing a diverse human TCR repertoire. Despite a high functional avidity of H3.3K27M-specific T cells, we were not able to achieve recognition of cells naturally expressing the H3.3K27M mutation, even when overexpressed as a transgene. Similar results were obtained with T cells expressing the published TCR 1H5 against the same epitope. CRISPR/Cas9 editing was used to exclude interference by endogenous TCRs in donor T cells. Overall, our data provide strong evidence that the H3.3K27M mutation is not a suitable target for cancer immunotherapy, most likely due to insufficient epitope processing and/or amount to be recognized by HLA-A*02:01 restricted CD8+ T cells.

Isolation of Neoantigen-Specific Human T Cell Receptors from Different Human and Murine Repertoires.

(1) Background: Mutation-specific T cell receptor (TCR)-based adoptive T cell therapy represents a truly tumor-specific immunotherapeutic strategy. However, isolating neoepitope-specific TCRs remains a challenge. (2) Methods: We investigated, side by side, different TCR repertoires-patients' peripheral lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs), PBLs of healthy donors, and a humanized mouse model-to isolate neoepitope-specific TCRs against eight neoepitope candidates from a colon cancer and an ovarian cancer patient. Neoepitope candidates were used to stimulate T cells from different repertoires in vitro to generate neoepitope-specific T cells and isolate the specific TCRs. (3) Results: We isolated six TCRs from healthy donors, directed against four neoepitope candidates and one TCR from the murine T cell repertoire. Endogenous processing of one neoepitope, for which we isolated one TCR from both human and mouse-derived repertoires, could be shown. No neoepitope-specific TCR could be generated from the patients' own repertoire. (4) Conclusion: Our data indicate that successful isolation of neoepitope-specific TCRs depends on various factors such as the heathy donor's TCR repertoire or the presence of a tumor microenvironment allowing neoepitope-specific immune responses of the host. We show the advantage and feasibility of using healthy donor repertoires and humanized mouse TCR repertoires to generate mutation-specific TCRs with different specificities, especially in a setting when the availability of patient material is limited.

EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice.

Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often use high-avidity CD8+ T cells in adoptive-transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon noninflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen-mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibited enhanced tumor cell lysis. This property endowed Ebag9-/- mice with extended control of Tcl-1 oncogene-induced chronic lymphocytic leukemia progression. In Ebag9-/- mice, an expanded memory population was obtained for anti-HY and anti-SV-40 T antigen-specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9-/- T cells. In Ebag9-/- cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation and anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.

T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas.

PURPOSE: Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy. EXPERIMENTAL DESIGN: Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intraventricular TCR-transgenic T-cell therapy was assessed in a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor-targeting guide RNAs. RESULTS: We identify recurrent capicua transcriptional repressor (CIC) inactivating hotspot mutations at position 215 CICR215W/Q as immunogenic MHC class II (MHCII)-restricted neoepitopes. Vaccination of MHC-humanized mice resulted in the generation of robust MHCII-restricted mutation-specific T-cell responses against CICR215W/Q. Adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert antitumor responses against CICR215W-expressing syngeneic gliomas. CONCLUSIONS: The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of human leukocyte antigen (HLA)-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T-cell adoptive therapy.

Lymphocyte access to lymphoma is impaired by high endothelial venule regression.

Blood endothelial cells display remarkable plasticity depending on the demands of a malignant microenvironment. While studies in solid tumors focus on their role in metabolic adaptations, formation of high endothelial venules (HEVs) in lymph nodes extends their role to the organization of immune cell interactions. As a response to lymphoma growth, blood vessel density increases; however, the fate of HEVs remains elusive. Here, we report that lymphoma causes severe HEV regression in mouse models that phenocopies aggressive human B cell lymphomas. HEV dedifferentiation occurrs as a consequence of a disrupted lymph-carrying conduit system. Mechanosensitive fibroblastic reticular cells then deregulate CCL21 migration paths, followed by deterioration of dendritic cell proximity to HEVs. Loss of this crosstalk deprives HEVs of lymphotoxin-ß-receptor (LTßR) signaling, which is indispensable for their differentiation and lymphocyte transmigration. Collectively, this study reveals a remodeling cascade of the lymph node microenvironment that is detrimental for immune cell trafficking in lymphoma.

In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo.

Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (TCR)-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm, TCRs were generated against putative KRASG12V- and RAC2P29L-derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope-specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRASG12V- and RAC2P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2P29L-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes.

Cooperation of genes in HPV16 E6/E7-dependent cervicovaginal carcinogenesis trackable by endoscopy and independent of exogenous estrogens or carcinogens.

Human papillomavirus (HPV) infection is necessary but insufficient for progression of epithelial cells from dysplasia to carcinoma-in situ (CIS) to invasive cancer. The combination of mutant cellular and viral oncogenes that regulate progression of cervical cancer (CC) remains unclear. Using combinations of HPV16 E6/E7 (E+), mutant Kras (mKras) (K+) and/or loss of Pten (P-/-), we generated autochthonous models of CC without exogenous estrogen, carcinogen or promoters. Furthermore, intravaginal instillation of adenoCre virus enabled focal activation of the oncogenes/inactivation of the tumor suppressor gene. In P+/+ mice, E6/E7 alone (P+/+E+K-) failed to cause premalignant changes, while mKras alone (P+/+E-K+) caused persistent mucosal abnormalities in about one-third of mice, but no cancers. To develop cancer, P+/+ mice needed both E6/E7 and mKras expression. Longitudinal endoscopies of P+/+E+K+ mice predicted carcinoma development by detection of mucosal lesions, found on an average of 23 weeks prior to death, unlike longitudinal quantitative PCRs of vaginal lavage samples from the same mice. Endoscopy revealed that individual mice differed widely in the time required for mucosal lesions to appear after adenoCre and in the time required for these lesions to progress to cancer. These cancers developed in the transition zone that extends, unlike in women, from the murine cervix to the distal vagina. The P-/-E+K+ genotype led to precipitous cancer development within a few weeks and E6/E7-independent cancer development occurred in the P-/-E-K+ genotype. In the P-/-E+K- genotype, mice only developed CIS. Thus, distinct combinations of viral and cellular oncogenes are involved in distinct steps in cervical carcinogenesis.

Identification and ranking of recurrent neo-epitopes in cancer.

BACKGROUND: Immune escape is one of the hallmarks of cancer and several new treatment approaches attempt to modulate and restore the immune system's capability to target cancer cells. At the heart of the immune recognition process lies antigen presentation from somatic mutations. These neo-epitopes are emerging as attractive targets for cancer immunotherapy and new strategies for rapid identification of relevant candidates have become a priority. METHODS: We carefully screen TCGA data sets for recurrent somatic amino acid exchanges and apply MHC class I binding predictions. RESULTS: We propose a method for in silico selection and prioritization of candidates which have a high potential for neo-antigen generation and are likely to appear in multiple patients. While the percentage of patients carrying a specific neo-epitope and HLA-type combination is relatively small, the sheer number of new patients leads to surprisingly high reoccurence numbers. We identify 769 epitopes which are expected to occur in 77629 patients per year. CONCLUSION: While our candidate list will definitely contain false positives, the results provide an objective order for wet-lab testing of reusable neo-epitopes. Thus recurrent neo-epitopes may be suitable to supplement existing personalized T cell treatment approaches with precision treatment options.

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation.

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed "memory inflation". Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation.

Oncogene-specific T cells fail to eradicate lymphoma-initiating B cells in mice.

To date, little is known about the interaction between (pre-)malignant B cells and T cells. We generated transgenic mice that allow B cell-specific induction of the oncogene SV40 large T-antigen (TAg) to analyze the role of oncogene-specific T cells during sporadic B-cell lymphoma development. Constitutive TAg expression in CD19-Cre × LoxP-Tag mice resulted in TAg-tolerant CD8+ T cells and development of B-cell lymphomas. In contrast, CD19-CreERT2 × LoxP-Tag mice retained TAg-competent CD8+ T cells at time of oncogene induction and TAg expression in few B cells of adult mice resulted in exceptionally rare lymphoma formation late in life. Increased lymphoma incidence in the absence of TAg-specific T cells suggested T cell-mediated inhibition of lymphoma progression. However, TAg-initiated B cells were not eliminated by T cells and detected long term. Our results demonstrate a failure of the immune system to eradicate lymphoma-initiating B cells, retaining the risk of lymphoma development.

Targeting Merkel Cell Carcinoma by Engineered T Cells Specific to T-Antigens of Merkel Cell Polyomavirus.

Purpose: The causative agent of most cases of Merkel cell carcinoma (MCC) has been identified as the Merkel cell polyomavirus (MCV). MCV-encoded T antigens (Tag) are essential not only for virus-mediated tumorigenesis but also for maintaining MCC cell lines in vitro MCV Tags are thus an appealing target for viral oncoprotein-directed T-cell therapy for MCC. With this study, we aimed to isolate and characterize Tag-specific T-cell receptors (TCR) for potential use in gene therapy clinical trials.Experimental Design: T-cell responses against MCV Tag epitopes were investigated by immunizing transgenic mice that express a diverse human TCR repertoire restricted to HLA-A2. Human lymphocytes genetically engineered to express Tag-specific TCRs were tested for specific reactivity against MCC cell lines. The therapeutic potential of Tag-specific TCR gene therapy was tested in a syngeneic cancer model.Results: We identified naturally processed epitopes of MCV Tags and isolated Tag-specific TCRs. T cells expressing these TCRs were activated by HLA-A2-positive cells loaded with cognate peptide or cells that stably expressed MCV Tags. We showed cytotoxic potential of T cells engineered to express these TCRs in vitro and demonstrated regression of established tumors in a mouse model upon TCR gene therapy.Conclusions: Our findings demonstrate that MCC cells can be targeted by MCV Tag-specific TCRs. Although recent findings suggest that approximately half of MCC patients benefit from PD-1 pathway blockade, additional patients may benefit if their endogenous T-cell response can be augmented by infusion of transgenic MCV-specific T cells such as those described here. Clin Cancer Res; 24(15); 3644-55. ©2018 AACR.

CD96 expression determines the inflammatory potential of IL-9-producing Th9 cells.

Recent findings demonstrated proinflammatory functions of interleukin (IL)-9-producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such as Cd40 ligand and Cd96, whereas expression of Cd25 and Cd83 was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9+ compared with IFN-γ+ T cells. Although Il9 was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96low cells. Transfer of CD96low Th9 cells into recombination activating gene 1-deficient (Rag1-/- ) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96high Th9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96high Th9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9-associated inflammations such as IBD.

ERAP1-Dependent Antigen Cross-Presentation Determines Efficacy of Adoptive T-cell Therapy in Mice.

Cytotoxic T lymphocytes can reject established tumors if their target peptide is efficiently presented by MHC class I molecules (pMHC-I) on the surface of cancerous cells. Therapeutic success upon adoptive T-cell transfer (ATT), however, requires additional cross-presentation of the same pMHC-I on noncancerous cells. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that customizes the N-terminus of proteasome-generated peptides so they can be loaded onto MHC-I molecules in the endoplasmic reticulum (ER). We show here that ERAP1 is critically involved in the process of tumor rejection and assumes a dual role by independently operating on both sides. Direct presentation of two MHC-I-restricted epitopes of a cancer-driving transplantation rejection antigen through ERAP1 moderately affected tumor rejection by adoptively transferred T-cell receptor gene-modified T cells in each case. ERAP1 expression by antigen cross-presenting cells of the ATT recipients was critical for expansion of therapeutic monospecific T cells and correlated with tumor rejection. Specifically, lack of ERAP1 expression in the ATT recipient's noncancerous cells enabled progression of pMHC-I-positive, IFNγ-responsive tumors, despite the presence of antigen-specific functional cytotoxic T lymphocytes. These data reveal a decisive role for ERAP1 in T-cell-mediated tumor rejection and will enhance the choice of MHC-I-restricted epitopes targeted by adoptive T-cell transfer.Significance: This study demonstrates a role of ERAP1 in the efficacy of adoptive T-cell transfer and has potential to improve personalized T-cell therapy for solid tumors. Cancer Res; 78(12); 3243-54. ©2018 AACR.

Clinical translation and regulatory aspects of CAR/TCR-based adoptive cell therapies-the German Cancer Consortium approach.

Adoptive transfer of T cells genetically modified by TCRs or CARs represents a highly attractive novel therapeutic strategy to treat malignant diseases. Various approaches for the development of such gene therapy medicinal products (GTMPs) have been initiated by scientists in recent years. To date, however, the number of clinical trials commenced in Germany and Europe is still low. Several hurdles may contribute to the delay in clinical translation of these therapeutic innovations including the significant complexity of manufacture and non-clinical testing of these novel medicinal products, the limited knowledge about the intricate regulatory requirements of the academic developers as well as limitations of funds for clinical testing. A suitable good manufacturing practice (GMP) environment is a key prerequisite and platform for the development, validation, and manufacture of such cell-based therapies, but may also represent a bottleneck for clinical translation. The German Cancer Consortium (DKTK) and the Paul-Ehrlich-Institut (PEI) have initiated joint efforts of researchers and regulators to facilitate and advance early phase, academia-driven clinical trials. Starting with a workshop held in 2016, stakeholders from academia and regulatory authorities in Germany have entered into continuing discussions on a diversity of scientific, manufacturing, and regulatory aspects, as well as the benefits and risks of clinical application of CAR/TCR-based cell therapies. This review summarizes the current state of discussions of this cooperative approach providing a basis for further policy-making and suitable modification of processes.

Oleate but not stearate induces the regulatory phenotype of myeloid suppressor cells.

Tumor infiltrating myeloid cells play contradictory roles in the tumor development. Dendritic cells and classical activated macrophages support anti-tumor immune activity via antigen presentation and induction of pro-inflammatory immune responses. Myeloid suppressor cells (MSCs), for instance myeloid derived suppressor cells (MDSCs) or tumor associated macrophages play a critical role in tumor growth. Here, treatment with sodium oleate, an unsaturated fatty acid, induced a regulatory phenotype in the myeloid suppressor cell line MSC-2 and resulted in an increased suppression of activated T cells, paralleled by increased intracellular lipid droplets formation. Furthermore, sodium oleate potentiated nitric oxide (NO) production in MSC-2, thereby increasing their suppressive capacity. In primary polarized bone marrow cells, sodium oleate (C18:1) and linoleate (C18:2), but not stearate (C18:0) were identified as potent FFA to induce a regulatory phenotype. This effect was abrogated in MSC-2 as well as primary cells by specific inhibition of droplets formation while the inhibition of de novo FFA synthesis proved ineffective, suggesting a critical role for exogenous FFA in the functional induction of MSCs. Taken together our data introduce a new unsaturated fatty acid-dependent pathway shaping the functional phenotype of MSCs, facilitating the tumor escape from the immune system.

Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR.