Identification of clinically relevant T cell receptors for personalized T cell therapy using combinatorial algorithms.

A central challenge in developing personalized cancer cell immunotherapy is the identification of tumor-reactive T cell receptors (TCRs). By exploiting the distinct transcriptomic profile of tumor-reactive T cells relative to bystander cells, we build and benchmark TRTpred, an antigen-agnostic in silico predictor of tumor-reactive TCRs. We integrate TRTpred with an avidity predictor to derive a combinatorial algorithm of clinically relevant TCRs for personalized T cell therapy and benchmark it in patient-derived xenografts.

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5' cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.

Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma.

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

A phase 1 trial of adoptive transfer of vaccine-primed autologous circulating T cells in ovarian cancer.

We have previously shown that vaccination with tumor-pulsed dendritic cells amplifies neoantigen recognition in ovarian cancer. Here, in a phase 1 clinical study ( NCT01312376 /UPCC26810) including 19 patients, we show that such responses are further reinvigorated by subsequent adoptive transfer of vaccine-primed, ex vivo-expanded autologous peripheral blood T cells. The treatment is safe, and epitope spreading with novel neopeptide reactivities was observed after cell infusion in patients who experienced clinical benefit, suggesting reinvigoration of tumor-sculpting immunity.

Neoantigen-specific CD8 T cells with high structural avidity preferentially reside in and eliminate tumors.

The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. Here, we characterize the T cell receptor (TCR) functional (antigen sensitivity) and structural (monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy donors. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCR biophysicochemical properties, we derive and apply an in silico model predicting TCR structural avidity and validate the enrichment in high avidity T cells in patients' tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy.

Novel Immunotherapeutics: Perspectives on Checkpoints, Bispecifics, and Vaccines in Development.

Over the past decade, the advent of molecular techniques and deeper understanding of the tumor microenvironment (TME) have enabled the development of a multitude of immunotherapy targets and approaches. Despite the revolutionary advancement in immunotherapy, treatment resistance remains a challenge leading to decreased response rate in a significant proportion of patients. As such, there has recently been an evolving focus to enhance efficacy, durability, and toxicity profiles of immunotherapy. Although immune checkpoint inhibitors have revolutionized cancer treatment with many already-approved antibodies and several others in the pipeline, bispecific antibodies build on their success in an attempt to deliver an even more potent immune response against tumor cells. On the other hand, vaccines comprise the oldest and most versatile form of immunotherapy. Peptide and nucleic acid vaccines are relatively simple to manufacture compared with oncolytic virus-based vaccines, whereas the dendritic cell vaccines are the most complex, requiring autologous cell culture. Nevertheless, a crucial question in the development of cancer vaccines is the choice of antigen whereby shared and patient-private antigen approaches are currently being pursued. There is hope that cancer vaccines will join the repertoire of successful novel immunotherapeutics in the market. Better insights into the impact of immunotherapy on effector T cells and other immune cell populations in the TME shall be a major priority across the immune-oncology discipline and can help identify predictive biomarkers to evaluate response to treatment and identify patients who would most likely benefit from immunotherapy.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs.

T cell receptor (TCR) technologies, including repertoire analyses and T cell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from T cell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

Cancer vaccines based on whole-tumor lysate or neoepitopes with validated HLA binding outperform those with predicted HLA-binding affinity.

Antigen selection and prioritization represent crucial determinants of vaccines' efficacy. Here, we compare two personalized dendritic cell-based vaccination strategies using whole-tumor lysate or neoantigens. Data in mouse and in cancer patients demonstrate that peptide vaccines using neoantigens predicted on the sole basis of in silico peptide-major histocompatibility complex (MHC) binding affinity underperform relative to whole-tumor-lysate vaccines. In contrast, effective in vitro peptide-MHC binding affinity and peptide immunogenicity significantly improve the prioritization of tumor-rejecting neoepitopes and result in more efficacious vaccines.

Immunobiology of high-grade serous ovarian cancer: lessons for clinical translation.

Treatment of high-grade serous ovarian cancer (HGSOC) remains challenging. Although HGSOC can potentially be responsive to immunotherapy owing to endogenous immunity at the molecular or T cell level, immunotherapy for this disease has fallen short of expectations to date. This Review proposes a working classification for HGSOC based on the presence or absence of intraepithelial T cells, and elaborates the putative mechanisms that give rise to such immunophenotypes. These differences might explain the failures of existing immunotherapies, and suggest that rational therapeutic approaches tailored to each immunophenotype might meet with improved success. In T cell-inflamed tumours, treatment could focus on mobilizing pre-existing immunity and strengthening the activation of T cells embedded in intraepithelial tumour myeloid niches. Conversely, in immune-excluded and immune-desert tumours, treatment could focus on restoring inflammation by reprogramming myeloid cells, stromal cells and vascular epithelial cells. Poly(ADP-ribose) polymerase (PARP) inhibitors, low-dose radiotherapy, epigenetic drugs and anti-angiogenesis therapy are among the tools available to restore T cell infiltration in HGSOC tumours and could be implemented in combination with vaccines and redirected T cells.

The current clinical landscape of personalized cancer vaccines.

Due to the intrinsic genetic instability of tumor cells, aberrant and novel tumor antigens can be expressed and serve as potential targets for cancer immunotherapy. This intrinsic feature can be exploited by cancer immunotherapy, particularly with cancer vaccination. Personalized cancer vaccination strategy can be a potent approach to trigger a broad-based antitumor response that is both beneficial and relevant to individual cancer patients. Also, cancer vaccination strategy can be designed to help elicit immunological memory for long-lasting tumor control. In this review, we describe the different types of personalized cancer vaccines and summarize the completed and ongoing cancer vaccination clinical trials in the last 10 years (database from www.clinicaltrials.gov). We also discuss the pros and cons of using different tumor animal models, i.e. syngeneic models, patient-derived xenografts models and genetically engineered mouse models, as tools for investigating cancer vaccination strategies. Finally, we describe preclinical studies that seek to test new emerging vaccination strategies as well as improving existing methods.

TEM1-targeting PEGylated PLGA shikonin nanoformulation for immunomodulation and eradication of ovarian cancer.

Introduction: Tumor endothelial marker 1 (TEM1) is expressed by tumor vascular endothelial cells in various cancers. Methods: Here, we developed poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) PEGylated with polyethylene glycol (PEG) and functionalized with anti-TEM1 antibody fragment (78Fc) and loaded them with necroptosis-inducing agent shikonin (SHK) (78Fc-PLGA-SHK NPs). Results: The nanoformulation showed a smooth spherical shape (~120 nm; the ζ potential of -30 mV) with high drug entrapment and bioconjugation efficiencies (~92% and ~90%, respectively) and a sustained-release profile in serum. Having significant toxicity in vitro (e.g., MS1 and TC1 cells), the nanoformulation dramatically increased the cytotoxicity in the TC1 murine lung carcinoma subcutaneous and intravenous/metastatic models as aggressive tumor models. The injection of the 78Fc-PLGA-SHK NPs to the MS1-xenograft mice resulted in significantly higher accumulation and effects in the TEM1-positive tumor targets, while they were excreted via urine track without retaining in the liver/spleen. In the TC1 subcutaneous model, C57/BL6 mice treated with the 78Fc-PLGA-SHK NPs revealed a significant therapeutic effect. The mice, which were tumor-free after receiving the nanoformulation, were re-challenged with the TC1 cells to investigate the immune response. These animals became tumor-free a week after the injection of TC1 cells. Conclusion: Based on these findings, we propose the 78Fc-PLGA-SHK NPs as a highly effective immunostimulating nanomedicine against the TEM1-expressing cells for targeted therapy of solid tumors including ovarian cancer.

Sensitive identification of neoantigens and cognate TCRs in human solid tumors.

The identification of patient-specific tumor antigens is complicated by the low frequency of T cells specific for each tumor antigen. Here we describe NeoScreen, a method that enables the sensitive identification of rare tumor (neo)antigens and of cognate T cell receptors (TCRs) expressed by tumor-infiltrating lymphocytes. T cells transduced with tumor antigen-specific TCRs identified by NeoScreen mediate regression of established tumors in patient-derived xenograft mice.

Polymer Nanoparticle-Mediated Delivery of Oxidized Tumor Lysate-Based Cancer Vaccines.

Cancer vaccination is a powerful strategy to combat cancer. A very attractive approach to prime the immune system against cancer cells involves the use of tumor lysate as antigen source. The immunogenicity of tumor lysate can be further enhanced by treatment with hypochlorous acid. This study explores poly(lactic-co-glycolic acid) (PLGA) nanoparticles to enhance the delivery of oxidized tumor lysate to dendritic cells. Using human donor-derived dendritic cells, it is found that the use of PLGA nanoparticles enhances antigen uptake and dendritic cell maturation, as compared to the use of the free tumor lysate. The ability of the activated dendritic cells to stimulate autologous peripheral blood mononuclear cells (PBMCs) is assessed in vitro by coculturing PBMCs with A375 melanoma cells. Live cell imaging analysis of this experiment highlights the potential of nanoparticle-mediated dendritic-cell-based vaccination approaches. Finally, the efficacy of the PLGA nanoparticle formulation is evaluated in vivo in a therapeutic vaccination study using B16F10 tumor-bearing C57BL/6J mice. Animals that are challenged with the polymer nanoparticle-based oxidized tumor lysate formulation survive for up to 50 days, in contrast to a maximum of 41 days for the group that receives the corresponding free oxidized tumor lysate-based vaccine.

Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy.

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell-infiltrated tumors. SIGNIFICANCE: Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1.

Estimated Costs of the Ipilimumab-Nivolumab Therapy and Related Adverse Events in Metastatic Melanoma.

Combined ipilimumab and nivolumab significantly improve outcomes in metastatic melanoma patients but bear an important financial impact on the healthcare system. Here, we analyze the treatment costs, focusing on irAE. We conducted a retrospective analysis of 62 melanoma patients treated with ipilimumab-nivolumab at the Lausanne University Hospital between 1 June 2016 and 31 August 2019. The frequency of irAEs and outcomes were evaluated. All melanoma-specific costs were analyzed from the first ipilimumab-nivolumab dose until the therapy given subsequently or death. A total of 54/62 (87%) patients presented at least one irAE, and 31/62 (50%) presented a grade 3-4 irAE. The majority of patients who had a complete response 12/14 (86%) and 21/28 (75%) of overall responders presented a grade 3-4 toxicity, and there were no responses in patients without toxicity. Toxicity costs represented only 3% of the total expenses per patient. The most significant contributions were medication costs (44%) and disease costs (39%), mainly disease-related hospitalization costs, not toxicity-related. Patients with a complete response had the lowest global median cost per week of follow up (EUR 2425) and patients who had progressive disease (PD), the highest one (EUR 8325). Except for one patient who had a Grade 5 toxicity (EUR 6043/week), we observe that less severe toxicity grades (EUR 9383/week for Grade 1), or even the absence of toxicity (EUR 9922/week), are associated with higher median costs per week (vs. EUR 3266/week for Grade 4 and EUR 2850/week for Grade 3). The cost of toxicities was unexpectedly low compared to the total costs, especially medication costs. Patients with higher toxicity grades had better outcomes and lower total costs due to treatment discontinuation.

A Personalized Neoantigen Vaccine in Combination with Platinum-Based Chemotherapy Induces a T-Cell Response Coinciding with a Complete Response in Endometrial Carcinoma.

Endometrial cancer (EC) is a common gynecological malignancy and the fourth most common malignancy in European and North American women. Amongst EC, the advanced serous, p53-mutated, and pMMR subtypes have the highest risk of relapse despite optimal standard of care therapy. At present, there is no standard of care maintenance treatment to prevent relapse among these high-risk patients. Vaccines are a form of immunotherapy that can potentially increase the immunogenicity of pMMR, serous, and p53-mutated tumors to render them responsive to check point inhibitor-based immunotherapy. We demonstrate, for the first time, the feasibility of generating a personalized dendritic cell vaccine pulsed with peptide neoantigens in a patient with pMMR, p53-mutated, and serous endometrial adenocarcinoma (SEC). The personalized vaccine was administered in combination with systemic chemotherapy to treat an inoperable metastatic recurrence. This treatment association demonstrated the safety and immunogenicity of the personalized dendritic cell vaccine. Interestingly, a complete oncological response was obtained with respect to both radiological assessment and the tumor marker CA-125.

Vaccines as Priming Tools for T Cell Therapy for Epithelial Cancers.

Impressive progress has recently been made in the field of cancer immunotherapy with the adoptive transfer of T cells, a successful personalized strategy, and checkpoint inhibitors (CPI) having extended the survival of numerous patients. However, not all patients have been able to benefit from these innovations. A key determinant of the responsiveness to cancer immunotherapies is the presence of T cells within the tumors. These tumor-infiltrating lymphocytes (TILs) are crucial in controlling tumor growth and their activity is being potentiated by immunotherapies. Although some epithelial cancers are associated with spontaneous T-cell and B-cell responses, which makes them good candidates for immunotherapies, it remains to create strategies that would promote lymphocyte infiltration and enable sustained immune responses in immune-resistant tumors. Therapeutic cancer vaccines hold the potential of being able to render "cold", poorly infiltrated tumors into "hot" tumors that would be receptive to cellular immunotherapies. In this review, we elaborate on the obstacles that need to be overcome and the strategies that are being explored to that end, including various types of antigen repertoires and different vaccine platforms and combinations with other available treatments.

Myeloid antigen-presenting cell niches sustain antitumor T cells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

Integrating Cancer Vaccines in the Standard-of-Care of Ovarian Cancer: Translating Preclinical Models to Human.

As the majority of ovarian cancer (OC) patients are diagnosed with metastatic disease, less than 40% will survive past 5 years after diagnosis. OC is characterized by a succession of remissions and recurrences. The most promising time point for immunotherapeutic interventions in OC is following debulking surgery. Accumulating evidence shows that T cells are important in OC; thus, cancer vaccines capable of eliciting antitumor T cells will be effective in OC treatment. In this review, we discuss different cancer vaccines and propose strategies for their incorporation into the OC standard-of-care regimens. Using the murine ID8 ovarian tumor model, we provide evidence that a cancer vaccine can be effectively combined with OC standard-of-care to achieve greater overall efficacy. We demonstrate several important similarities between the ID8 model and OC patients, in terms of response to immunotherapies, and the ID8 model can be an important tool for evaluating combinatorial regimens and clinical trial designs in OC. Other emerging models, including patient-derived xenograft and genetically engineered mouse models, are continuing to improve and can be useful for evaluating cancer vaccination therapies in the near future. Here, we provide a comprehensive review of the completed and current clinical trials evaluating cancer vaccines in OC.