Neoantigen landscape dynamics during human melanoma-T cell interactions.

Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance.

Potential use of lymph node-derived HPV-specific T cells for adoptive cell therapy of cervical cancer.

Adoptive transfer of tumor-specific T cells, expanded from tumor-infiltrating lymphocytes or from peripheral blood, is a promising immunotherapeutic approach for the treatment of cancer. Here, we studied whether the tumor-draining lymph nodes (TDLN) of patients with human papillomavirus (HPV)-induced cervical cancer can be used as a source for ACT. The objectives were to isolate lymph node mononuclear cells (LNMC) from TDLN and optimally expand HPV-specific CD4+ and CD8+ T cells under clinical grade conditions. TDLN were isolated from 11 patients with early-stage cervical cancer during radical surgery. Isolated lymphocytes were expanded in the presence of HPV16 E6 and E7 clinical grade synthetic long peptides and IL-2 for 22 days and then analyzed for HPV16 specificity by proliferation assay, multiparameter flow cytometry and cytokine analysis as well as for CD25 and FoxP3 expression. Stimulation of LNMC resulted in expansion of polyclonal HPV-specific T cells in all patients. On average a 36-fold expansion of a CD4+ and/or CD8+ HPV16-specific T cell population was observed, which maintained its capacity for secondary expansion. The T helper type 1 cytokine IFNγ was produced in all cell cultures and in some cases also the Th2 cytokines IL-10 and IL-5. The procedure was highly reproducible, as evidenced by complete repeats of the stimulation procedures under research and under full good manufacturing practice conditions. In conclusion, TDLN represent a rich source of polyclonal HPV16 E6- and E7-specific T cells, which can be expanded under clinical grade conditions for adoptive immunotherapy in patients with cervical cancer.

The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study.

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.

Timed adoptive T cell transfer during chemotherapy in patients with recurrent platinum-sensitive epithelial ovarian cancer.

BACKGROUND: The presence of T cells and suppressive myeloid cells in epithelial ovarian cancer (EOC) correlate with good and bad clinical outcome, respectively. This suggests that EOC may be sensitive to adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL), provided that immunosuppression by myeloid-derived suppressor cells and M2 macrophages is reduced. Platinum-based chemotherapy can alleviate such immunosuppression, potentially creating a window of opportunity for T cell-based immunotherapy. METHODS: We initiated a phase I/II trial (NCT04072263) in patients with recurrent platinum-sensitive EOC receiving TIL during platinum-based chemotherapy. TILs were administered 2 weeks after the second, third and fourth chemotherapy course. Patients were treated in two cohorts with or without interferon-α (IFNa), as conditioning and TIL support regimen. The primary endpoint was to evaluate the feasibility and safety according to CTCAE V.4.03 criteria and the clinical response and immune modulatory effects of this treatment were evaluated as secondary endpoints. RESULTS: Sixteen patients were enrolled. TIL could be successfully expanded for all patients. TIL treatment during chemotherapy without IFNa (n=13) was safe but the combination with IFNa added to the chemotherapy-induced toxicity with 2 out of 3 patients developing thrombocytopenia as dose-limiting toxicity. Fourteen patients completed treatment with a full TIL cycle and were further evaluated for clinical and immunological response. Platinum-based chemotherapy resulted in reduction of circulating myeloid cell numbers and IL-6 plasma levels, confirming its immunosuppression-alleviating effect. Three complete (CR), nine partial responses and two stable diseases were recorded, resulting in an objective response rate of 86% (Response Evaluation Criteria In Solid Tumors V.1.1). Interestingly, progression free survival that exceeded the previous platinum-free interval was detected in two patients, including an exceptionally long and ongoing CR in one patient that coincided with sustained alleviation of immune suppression. CONCLUSION: TIL therapy can be safely combined with platinum-based chemotherapy but not in combination with IFNa. The chemotherapy-mediated reduction in immunosuppression and the increase in platinum-free interval for two patients warrants further exploration of properly-timed TIL infusions during platinum-based chemotherapy, possibly further benefiting from IL-2 support, as a novel treatment option for EOC patients.

A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions.

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

Influenza matrix 1-specific human CD4+ FOXP3+ and FOXP3(-) regulatory T cells can be detected long after viral clearance.

Control and termination of infection with Influenza A virus is associated with increased IL-10 production in mouse models. Notably, IL-10 can be produced by Treg. Therefore, we investigated whether the population of IL-10-producing influenza-specific CD4(+) T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza-specific IL-10-producing T cells were detected in all human donors displaying influenza-specific immunity. Isolation of Matrix 1 protein-specific IL-10-producing T-cell clones revealed that a substantial proportion of these T-cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3(+) and FOXP3(-) CD4(+) Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus-infected cells. In vitro suppression was not mediated by IL-10 but involved interference with the IL-2 axis. The isolated Treg suppressed amongst others the IL-2 production of influenza-specific T-helper cells as well as partially prevented the upregulation of the high-affinity IL-2 receptor on CD8 effector cells. So far the induction of virus-specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus-specific Treg can also be induced by viruses that are rapidly cleared in humans.

Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha.

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38-474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success.

Low-dose interferon-alpha preconditioning and adoptive cell therapy in patients with metastatic melanoma refractory to standard (immune) therapies: a phase I/II study.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-reactive T cells has shown consistent clinical efficacy. We evaluated the response to ACT in combination with interferon alpha (IFNa) preconditioning in patients with stage IV metastatic melanoma, most of which were progressive on cytotoxic T-lymphocyte-associated protein 4 and/or programmed cell death protein 1 checkpoint blockade therapy. METHODS: Thirty-four patients were treated with ex vivo expanded tumor reactive T cells, derived from mixed lymphocyte autologous tumor cultures, or with autologous tumor-infiltrating lymphocytes and evaluated for clinical response. Clinical and immunological parameters associated with response were also evaluated. RESULTS: Best overall response defined as clinical benefit, comprising either complete response, partial response or stable disease >6 months, was observed in 29% of the patients. Forty-three per cent of the 14 immunotherapy-naïve patients and 20% of the 20 patients progressive on prior immunotherapy benefited from ACT. The overall survival (OS) was 90% versus 28.6% at 1 year and 46.7% versus 0% at 3 years follow-up, of responder and non-responder patients, respectively. Median OS was 36 versus 7 months, respectively. IFNa pretreatment resulted in leukopenia, neutropenia and lymphopenia, which was sustained during the treatment in clinical responders and associated with response. Differences in antigen specificity, but not in phenotype, cytokine profile or CD8+ T cell number of the ACT products correlated with clinical response. Cross-reactivity of the ACT products to one or more allogeneic human leukocyte antigen-matched melanoma cell lines was associated with short OS after treatment while the ACT products of very long-term survivors showed no cross-reactivity but recognized patient-specific neoantigens. CONCLUSION: This study demonstrates that ACT in combination with a mild IFNa preconditioning regimen can induce clinical benefit even in immunotherapy pretreated patients, although with lower success than in immunotherapy-naïve patients. ACT products comprising neoantigen reactivity may be more effective.

Phase I/II study protocol to assess safety and efficacy of adoptive cell therapy with anti-PD-1 plus low-dose pegylated-interferon-alpha in patients with metastatic melanoma refractory to standard of care treatments: the ACTME trial.

INTRODUCTION: Treatment with anti-PD-1 immunotherapy does not lead to long-lasting clinical responses in approximately 60% of patients with metastatic melanoma. These refractory patients, however, can still respond to treatment with tumour infiltrating lymphocytes (TIL) and interferon-alpha (IFNa). A combination of TIL, pegylated-interferon-alpha (PEG-IFNa) and anti-PD-1 is expected to provide a safe, feasible and effective therapy for patients with metastatic melanoma, who are refractory to standard of care treatment options. METHODS AND ANALYSIS: Patients are treated in two phases. In phase I, the safety of the combination TIL and anti-PD-1 is assessed (cohort 1) according to CTCAE 4.03 criteria. Subsequently, the safety of cotreatment with PEG-IFNa is tested in cohort 2. The efficacy will be evaluated in the second phase of the trial. Efficacy is evaluated according to RECIST 1.1 and immune-related response criteria. Clinical and immunological parameters will be evaluated for their relation with clinical responsiveness. ETHICS AND DISSEMINATION: Ethical approval of the trial was obtained from the Central Committee on Research Involving Human Subjects in the Netherlands. The trial results will be shared with the scientific community at (inter)national conferences and by publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT03638375; Pre-results.

Detection and functional analysis of CD8+ T cells specific for PRAME: a target for T-cell therapy.

PURPOSE: Preferentially expressed antigen on melanomas (PRAME) is an interesting antigen for T-cell therapy because it is frequently expressed in melanomas (95%) and other tumor types. Moreover, due to its role in oncogenic transformation, PRAME-negative tumor cells are not expected to easily arise and escape from T-cell immunity. The purpose of this study is to investigate the usefulness of PRAME as target for anticancer T-cell therapies. EXPERIMENTAL DESIGN: HLA-A*0201-subtyped healthy individuals and advanced melanoma patients were screened for CD8+ T cells directed against previously identified HLA-A*0201-binding PRAME peptides by IFN-gamma enzyme-linked immunosorbent spot assays and tetramer staining. PRAME-specific T-cell clones were isolated and tested for recognition of melanoma and acute lymphoid leukemia (ALL) cell lines. PRAME mRNA expression was determined by quantitative real-time reverse transcription-PCR. RESULTS: In 30% to 40% of healthy individuals and patients, PRA(100-108)-specific CD8+ T cells were detected both after in vitro stimulation and directly ex vivo after isolation by magnetic microbeads. Although CD45RA- memory PRA(100-108)-specific T cells were found in some individuals, the majority of PRA(100-108)-tetramer+ T cells expressed CD45RA, suggesting a naive phenotype. PRA(100-108)-tetramer+ T-cell clones were shown to recognize and lyse HLA-A*0201+ and PRAME+ melanoma but not ALL cell lines. Quantitative real-time reverse transcription-PCR showed significantly lower PRAME mRNA levels in ALL than in melanoma cell lines, suggesting that PRAME expression in ALL is below the recognition threshold of our PRA(100-108)-tetramer+ T cells. CONCLUSION: These data support the usefulness of PRAME and in particular the PRA(100-108) epitope as target for T-cell therapy of PRAME-overexpressing cancers.

Induction of CAMEL/NY-ESO-ORF2-specific CD8+ T cells upon stimulation with dendritic cells infected with a modified Ad5 vector expressing a chimeric Ad5/35 fiber.

Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8(+) T cells specific for the known HLA-A(*)0201-binding CAMEL/NY-ESO-ORF2(1-11) epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8(+) T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF2(46-54) epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8(+) T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8(+) T cell responses in vivo.

Identification of multiple HLA-DR-restricted epitopes of the tumor-associated antigen CAMEL by CD4+ Th1/Th2 lymphocytes.

CD4(+) Th cells play an important role in the induction and maintenance of adequate CD8(+) T cell-mediated antitumor responses. Therefore, identification of MHC class II-restricted tumor antigenic epitopes is of major importance for the development of effective immunotherapies with synthetic peptides. CAMEL and NY-ESO-ORF2 are tumor Ags translated in an alternative open reading frame from the highly homologous LAGE-1 and NY-ESO-1 genes, respectively. In this study, we investigated whether CD4(+) T cell responses could be induced in vitro by autologous, mature dendritic cells pulsed with recombinant CAMEL protein. The data show efficient induction of CAMEL-specific CD4(+) T cells with mixed Th1/Th2 phenotype in two healthy donors. Isolation of CD4(+) T cell clones from the T cell cultures of both donors led to the identification of four naturally processed HLA-DR-binding CAMEL epitopes: CAMEL(1-20), CAMEL(14-33), CAMEL(46-65), and CAMEL(81-102). Two peptides (CAMEL(1-20) and CAMEL(14-33)) also contain previously identified HLA class I-binding CD8(+) T cell epitopes shared by CAMEL and NY-ESO-ORF2 and are therefore interesting tools to explore for immunotherapy. Furthermore, two CD4(+) T cell clones that recognized the CAMEL(14-33) peptide with similar affinities were shown to differ in recognition of tumor cells. These CD4(+) T cell clones recognized the same minimal epitope and expressed similar levels of adhesion, costimulatory, and inhibitory molecules. TCR analysis demonstrated that these clones expressed identical TCR beta-chains, but different complementarity-determining region 3 loops of the TCR alpha-chains. Introduction of the TCRs into proper recipient cells should reveal whether the different complementarity-determining region 3 alpha loops are important for tumor cell recognition.