The Microenvironment of Small Intestinal Neuroendocrine Tumours Contains Lymphocytes Capable of Recognition and Activation after Expansion

Traditionally, immune evasion and immunotherapy have been studied in cancers with a high mutational load such as melanoma or lung cancer. In contrast, small intestinal neuroendocrine tumours (SINETs) present a low frequency of somatic mutations and are described as genetically stable tumours, rendering immunotherapies largely unchartered waters for SINET patients. SINETs frequently metastasise to the regional lymph nodes and liver at the time of diagnosis, and no curative treatments are currently available for patients with disseminated disease. Here, we characterised the immune landscape of SINET and demonstrated that tumour-infiltrating lymphocytes (TILs) can be expanded and activated during autologous tumour challenge. The composition of lymphocyte subsets was determined by immunophenotyping of the SINET microenvironment in one hepatic and six lymph node metastases. TILs from these metastases were successfully grown out, enabling immunophenotyping and assessment of PD-1 expression. Expansion of the TILs and exposure to autologous tumour cells in vitro resulted in increased T lymphocyte degranulation. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.

P06.10 Short term inhibition of checkpoint proteins increases ex vivo expansion of tumour infiltrating lymphocytes in high grade serous ovarian cancer

BackgroundOvarian cancer is the most lethal gynaecological malignancy, accounting for approximately 185,000 deaths worldwide in 2018. The majority of patients will experience recurrence of disease. Therefore, there is an urgent need for the development of further therapies to improve patient survival. Tumour infiltrating lymphocyte (TIL) therapy has shown clear efficacy in immunogenic cancers, and TIL can be readily expanded ex vivo from samples of high grade serous ovarian cancer (HGSOC). Key indicators of effective TIL products for infusion are high TIL yield and functionality against autologous tumour. Blockade of checkpoint proteins is effective in increasing TIL yield and functional response from ovarian cancer TIL cultures. However, it is unknown whether blockade of other key checkpoints, including programmed death ligand-1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) increase TIL yield in ex vivo cultures from HGSOC samples.Materials and MethodsTIL cultures were generated from surgically resected HGSOC tumour samples and were incubated with CD3/CD28 Dynabeads. 3000IU/mL recombinant interleukin-2 (IL-2) was added on alternate days for 7 days before beads were removed. 1000IU/mL IL-2 was added on alternate days for a further 12 days of culture. In cohort 1, 10µg/mL αPD-1, αTIM-3 or αLAG-3 antibodies were added at initiation of TIL cultures only. In cohort 2, 10µg/mL αPD-1, αTIM-3 or αLAG-3 antibodies were added on alternate days until Day 19. Interferon gamma (IFN-γ) release in response to TIL co-culture with autologous tumour cultures was measured with a human IFN-γ ELISA kit. Data are presented as mean±SEM.ResultsAddition of checkpoint inhibitors at the initiation of HGSOC TIL culture in cohort 1 increased TIL expansion above untreated control in αPD-1 (1.20±0.04 fold, P<0.01, n=9) and αLAG-3 (1.31±0.08 fold, P<0.001, n=9) but not αTIM-3 treated cultures. However, intermittent dosing of HGSOC cultures in cohort 2 with either αPD-1, αTIM-3 or αLAG-3 antibodies did not increase TIL expansion above untreated cultures. In cohort 1, IFN-γ secretion was increased above untreated control in at least one culture treated with a checkpoint inhibitor in 5/7 patients. However, there was no overall fold change in IFN-γ secretion in either αPD-1, αTIM-3 or αLAG-3 treated cultures.ConclusionsThis data suggests that initial blockade of checkpoint proteins is effective in increasing the ex vivo expansion of TIL from HGSOC tumours, thus providing a method of improving the efficacy of TIL products in ovarian cancer patients.FundingGO was funded through a CRUK Manchester Centre Clinical Fellowship. PJ was in receipt of a bursary from the Emma Gyles Bursary Fund. The project was funded by TESARO Inc.Disclosure InformationC.A. Waddell: None. M.J. Price: None. P. Johnson: None. R.J. Edmondson: None. G.L. Owens None.

Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: A potential candidate molecule for immunotherapy in cervical cancer

Background Dendritic cell (DC)-based immunotherapy is capable of activating the immune system, and in particular tumour-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumour. However, major limitations are the availability of autologous tumour cells as antigenic source and the selection of antigen that may have potential to activate both CD8 + and CD4 + T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4 + and CD8 + T cell responses for future DCs-based vaccine trials in cervical cancer patients. Methods Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. Subsequently, the efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumour lysate (TLDCs), to induce CD4+, CD8 + T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. Results Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic CD4 + and CD8 + T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83 p = 0.4; for migration p = 0.2). In contrast, TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, but this was also not statistically significant (IL12p40, p = 0.06). We also found that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+Tregs in autologous co-cultures. Conclusions This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumour burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention. This strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer.

Complementary vaccination protocol with dendritic cells pulsed with autologous tumour lysate in patients with resected stage III or IV melanoma: protocol for a phase II randomised trial (ACDC Adjuvant Trial)

IntroductionSurgery is one of the treatments of choice for patients with a single metastasis from melanoma but is rarely curative. Such patients could potentially benefit from consolidation immunotherapy. Vaccination with dendritic cells (DCs) loaded with tumour antigens elicits a tumour-specific immune response. In our experience, patients who developed delayed type hypersensitivity (DTH) after DC vaccination showed a median overall survival (OS) of 22.9 monthsvs4.8 months for DTH-negative cases. A phase II randomised trial showed an advantage OS of a DC vaccine over a tumour cell-based vaccine (2-year OS 72% vs31%, respectively). Given that there is no standard therapy after surgical resection of single metastases, we planned a study to compare vaccination with DCs pulsed with autologous tumour lysate versus follow-up.Methods and analysisThis is a randomised phase II trial in patients with resected stage III/IV melanoma. Assuming a median relapse-free survival (RFS) of 7.0 months for the standard group and 11.7 months for the experimental arm (HR 0.60), with a two-sided tailed alpha of 0.10, 60 patients per arm must be recruited. An interim futility analysis will be performed at 18 months. The DC vaccine, produced in accordance with Good Manufacturing Practice guidelines, consists of autologous DCs loaded with autologous tumour lysate and injected intradermally near lymph nodes. Vaccine doses will be administered every 4 weeks for six vaccinations and will be followed by 3 million unit /day of interleukin-2 for 5 days. Tumour restaging, blood sampling for immunological biomarkers and DTH testing will be performed every 12 weeks.Ethics and disseminationThe protocol, informed consent and accompanying material given to patients were submitted by the investigator to the Ethics Committee for review. The local Ethics Committee and the Italian Medicines Agency approved the protocol (EudraCT code no.2014-005123-27). Results will be published in a peer-reviewed international scientific journal.Trial registration number2014-005123-27.