CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR sig- nalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8 + T cells to kill 4T1mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.Keywords: AICD r Anti-tumour immunity r CD5 r CD8 + T cell r Fas receptor Additional supporting information may be found online in the Supporting Information section at the end of the article. Faizah Alotaibi et al. Eur. J. Immunol. 2020. 50: 695-704 Abbreviations: AICD: activation-induce cell death · FasL: Fas ligand · FasR: Fas receptor · ICS: Intracellular cytokine staining · SRCR: scavenger receptor cysteine-rich · Treg: T regulatory
BackgroundCheckpoint inhibitors have changed the outcomes for patients with advanced melanoma. However, many patients still show primary resistance to single-agent therapy. Recently, the role of the gut microbiome in influencing antitumor immunity has been established. Currently, various methods of modifying the gut microbiome of cancer patients are being explored. We report the initial safety results of the first two patients treated on a phase I study combining Fecal Microbiota Transplantation (FMT) with single-agent anti-PD1 in treatment-naïve patients with advanced melanoma.MethodsTwo healthy donors were selected through our screening process and approximately 100 grams of fresh stool was processed and prepared for FMT as per our standardized protocol. FMT recipients were melanoma patients with unresectable or metastatic disease who were treatment naïve for their advanced disease. Bowel preparation was completed the day prior and FMT was performed using oral administration of approximately 40 capsules. Anti-PD1 was started at least 1 week after FMT to allow for microbiome engraftment. Blood and stool were analyzed at baseline (pre-FMT), before immunotherapy, and three weeks after it.ResultsPatient 1 was diagnosed with recurrent melanoma of the lower limb with multiple in-transit lesions refractory to control with surgery and a single intralesional injection of IL-2. Patient received stool from Donor 1 and did not experience any adverse effects from FMT. At the time of treatment #4, a solitary large cutaneous lesion stabilized but the patient experienced grade 1 diarrhoea, grade 2 nausea, and grade 2 fatigue, and grade 2 depression (NCI-CTCAE v5.0). Patient 2 was diagnosed with recurrent melanoma of the parotid gland with metastatic lesions in the lungs. Patient 2 received stool from Donor 2 and experienced only grade 1 flatus from FMT. At the time of treatment #3, the patient experienced grade 1 constipation. Both patients had a vigorous immune response to FMT measured by changes in the immune subpopulations in peripheral blood one week after FMT, including an increase in CD28+ CD8+ T cells and a decrease in PDL1+ CD3- cells. Following anti-PD1 therapy, both patients had an increase in CD39+ CD8+ T cell population. The PD1+ CD38+ CD8+ dysfunctional T cell levels decreased in both patients post-FMT and anti-PD1 therapy.ConclusionsFMT combined with anti-PD1 therapy in patients with advanced melanoma appears to be safe. A measurable immune response was observed one week after FMT in both patients. One patient experienced several grade 2 toxicities with stabilization of a large cutaneous lesion.AcknowledgementsThis study is funded by a grant from The Lotte & John Hecht Memorial Foundation and a grant from The Medical Oncology Research Funds (MORF) from Western University.Trial RegistrationNCT03772899Ethics ApprovalThe study was approved by Western University Institutution‘s Ethics Board, approval number 113131, date of approval March 15, 2019.
Blockade of programmed cell death protein 1 (PD-1) is approved for treatment of multiple human cancers and is the focus of multiple studies due to its key role in T cell function. However, only patients with ‘'hot'' tumors (i.e. those with increased numbers of tumor-infiltrating CD8+ T cells) respond well to the blockade. This suggests that new strategies to increase infiltration of CD8+ T cells into tumors will increase the number of tumor types and patients benefiting from anti-PD-1 therapy. CD5, a member of the scavenger receptor cysteine-rich (SRCR) superfamily, is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signaling and impair cytotoxic T lymphocyte (CTL) activation. CD5 knockout mice have increased anti-tumor immunity and reduced homograft tumor growth: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumor response and increase tumor-infiltrating CD8+ T cells. We report that in vivo administration of anti-CD5 blocking MAb treatment increased primary T cell activation in response to 4T1 tumor cell homografts and ex vivo activation as measured by increased CD69, Fas, Fas ligand, IFNγ, PD-1, and apoptosis. Further, anti-CD5 treatment enhanced the capacity of primary T cells to kill 4T1 tumor cells ex vivo. Mice receiving anti-CD5 and anti-PD-1 in combination exhibited increased overall survival compared to mice treated with either agent alone. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumor immunity and PD-1 blockade treatment. Citation Format: Faizah Alotaibi, Mateusz Rytelewski, Rene Figueredo, Ronak Zareardalan1, Saman Maleki Vareki, Xiufen Zheng, Meng Zhang, Peter Ferguson, Mikal El-Hajjar, Yousef Najajreh, Wei-ping Min, James Koropatnick. Functional blocking of CD5 on T cells to enhance the efficacy of therapeutic PD-1 blockade in treatment of mouse 4T1 breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 917.
BackgroundDespite rigorous multimodal therapy, recurrence is common among high-risk neuroblastoma patients. Neuroblastoma is a poorly immunogenic tumor with low tumor mutational burden (TMB). Currently, immunotherapy with immune checkpoint inhibitors (ICIs) are not approved for neuroblastoma. Novel strategies to sensitize neuroblastoma to ICIs are urgently needed. We have induced mismatch repair (MMR) deficiency in mouse neuro-2a tumors and show that these tumors become highly immunogenic and responsive to anti-CTLA4 but not anti-PD1 therapy.MethodsThe MMR gene Mlh1 was knocked out of neuro-2a and B16F10 cells, and clones were selected. MMR-deficient (dMMR) and -proficient (pMMR) neuroblastoma tumors were grown in immunocompetent and immunodeficient animals. Tumors were harvested, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Neuro-2a tumor-bearing mice were treated with anti-PD1, anti-CTLA4, or a combination of both antibodies. NK cells were depleted in mice treated with anti-CTLA4 using the anti-asialo GM1 antibody to examine their role in the efficacy of anti-CTLA4 treatment. dMMR B16F10 melanoma tumor-bearing mice were treated with anti-PD1 followed by the analysis of TILs. Publicly available TARGET and TCGA databases were mined to examine the effect of T-cell infiltration on neuroblastoma and melanoma patient‘s survival, respectively.ResultsWe show that high-risk neuroblastoma and melanoma patients with tumors containing high levels of T-cell and memory T-cell-related genes have improved survival. Additionally, inducing MMR deficiency in neuro-2a cells renders these tumors immunogenic, with high T-cell infiltration, and inhibits tumor growth in mice in an immune-dependent manner. We also show that dMMR neuroblastoma tumors are highly sensitive to anti-CTLA4 but not anti-PD1 treatment. Anti-CTLA4 therapy cured most tumor-bearing mice, inducing immune memory, epitope spreading, and increased tumor-specific T-cells in cured animals. Notably, the effect of anti-CTLA4 therapy was independent of NK cells. In neuroblastoma tumors with induced MMR deficiency, anti-PD1 treatment antagonized anti-CTLA4 by upregulating inhibitory molecules on T-cells and increasing the level of dysfunctional TILs. Interestingly, anti-PD1 therapy was effective against high TMB-background melanoma tumors with induced MMR deficiency. Our data suggests that this effect relied on lowering T-cell inhibitory molecules rather than increasing T-cell infiltration into tumors.ConclusionsInducing MMR deficiency in low and high TMB-background tumors leads to distinct responses to anti-PD1 therapy, with low levels of T-cell exhaustion being a positive indicator of response. Anti-CTLA4 therapy in combination with induced MMR deficiency is a novel strategy for treating low TMB-background high-risk neuroblastoma.Ethics ApprovalThis study was approved by the Animal Care Committee at Western University; approval number 2017-030.
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