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      Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy

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          Abstract

          Background

          Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of TP53 gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape. Given the high mutation frequency of TP53 in TNBC cells, restoration of p53 function could be a potential strategy to overcome their resistance to anti-programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) therapy. Herein, we have assessed the use of Pos3Aa crystal-based platform to mediate the intracellular delivery of p53 protein to restore p53 activity in p53 null tumors and consequently augment anti-PD-1 activity.

          Methods

          The efficiency of Pos3Aa-p53 crystals in delivering p53 protein was evaluated using confocal imaging, immunofluorescence staining, flow cytometry and RNA-seq. The ability of Pos3Aa-p53 crystals to remodel tumor microenvironment was investigated by examining the markers of immunogenic cell death (ICD) and the expression of PD-L1, indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase 2 and type I interferon (IFN). Finally, both unilateral and bilateral 4T1 tumor mouse models were utilized to assess the efficacy of Pos3Aa-p53 crystal-mediated p53 restoration in enhancing the antitumor activity of ICIs. T cells in tumor tissues and spleens were analyzed, and the in vivo biosafety of the Pos3Aa-p53 crystal/anti-PD-1 antibody combination was also evaluated.

          Results

          Delivery of p53 protein into p53-null TNBC 4T1 cells via Pos3Aa-p53 crystals restored the p53 activity, and therefore led to the induction of ICD, activation of type I IFN signaling and upregulation of PD-L1 expression. Pos3Aa-p53 crystals significantly enhanced T cell infiltration and activation in 4T1 tumors, thereby sensitizing them to anti-PD-1 therapy. The combination of Pos3Aa-p53 crystals with anti-PD-1 antibody also induced a systemic antitumor immunity resulting in the inhibition of distal tumor growth with minimal toxicity.

          Conclusion

          This study validates that p53 restoration can be an effective approach to overcome ICI resistance and demonstrates that intracellular delivery of p53 protein can be an efficient, safe and potentially universal strategy to restore p53 activity in tumors carrying TP53 mutation.

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          Most cited references70

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          Cancer immunotherapy using checkpoint blockade

          The release of negative regulators of immune activation (immune checkpoints) that limit antitumor responses has resulted in unprecedented rates of long-lasting tumor responses in patients with a variety of cancers. This can be achieved by antibodies blocking the cytotoxic T lymphocyte antigen-4 (CTLA-4) or the programmed death-1 (PD-1) pathway, either alone or in combination. The main premise for inducing an immune response is the pre-existence of antitumor T cells that were limited by specific immune checkpoints. Most patients who have tumor responses maintain long lasting disease control, yet one third of patients relapse. Mechanisms of acquired resistance are currently poorly understood, but evidence points to alterations that converge on the antigen presentation and interferon gamma signaling pathways. New generation combinatorial therapies may overcome resistance mechanisms to immune checkpoint therapy.
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            PD-1 blockade induces responses by inhibiting adaptive immune resistance

            Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. 1–5 One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8 T-cells (termed adaptive immune resistance). 6,7 Here we show that pre-existing CD8 T-cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next generation sequencing for T-cell receptors (TCR). In serially sampled tumours, responding patients showed proliferation of intratumoural CD8+ T-cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8, PD1, and PD-L1 expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression following therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1 mediated adaptive immune resistance.
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              Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer

              We assessed the efficacy and safety of programmed cell death 1 (PD-1) inhibition with pembrolizumab in patients with advanced non-small-cell lung cancer enrolled in a phase 1 study. We also sought to define and validate an expression level of the PD-1 ligand 1 (PD-L1) that is associated with the likelihood of clinical benefit. We assigned 495 patients receiving pembrolizumab (at a dose of either 2 mg or 10 mg per kilogram of body weight every 3 weeks or 10 mg per kilogram every 2 weeks) to either a training group (182 patients) or a validation group (313 patients). We assessed PD-L1 expression in tumor samples using immunohistochemical analysis, with results reported as the percentage of neoplastic cells with staining for membranous PD-L1 (proportion score). Response was assessed every 9 weeks by central review. Common side effects that were attributed to pembrolizumab were fatigue, pruritus, and decreased appetite, with no clear difference according to dose or schedule. Among all the patients, the objective response rate was 19.4%, and the median duration of response was 12.5 months. The median duration of progression-free survival was 3.7 months, and the median duration of overall survival was 12.0 months. PD-L1 expression in at least 50% of tumor cells was selected as the cutoff from the training group. Among patients with a proportion score of at least 50% in the validation group, the response rate was 45.2%. Among all the patients with a proportion score of at least 50%, median progression-free survival was 6.3 months; median overall survival was not reached. Pembrolizumab had an acceptable side-effect profile and showed antitumor activity in patients with advanced non-small-cell lung cancer. PD-L1 expression in at least 50% of tumor cells correlated with improved efficacy of pembrolizumab. (Funded by Merck; KEYNOTE-001 ClinicalTrials.gov number, NCT01295827.).
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                Author and article information

                Journal
                J Immunother Cancer
                J Immunother Cancer
                jitc
                jitc
                Journal for Immunotherapy of Cancer
                BMJ Publishing Group (BMA House, Tavistock Square, London, WC1H 9JR )
                2051-1426
                2022
                14 September 2022
                : 10
                : 9
                Affiliations
                [1 ]departmentSchool of Life Sciences , The Chinese University of Hong Kong , Shatin, Hong Kong
                [2 ]departmentCenter of Novel Biomaterials , The Chinese University of Hong Kong , Shatin, Hong Kong
                Author notes
                [Correspondence to ] Professor Michael K Chan; michaelkchan88@ 123456cuhk.edu.hk ; Dr Marianne M Lee; mariannemmlee@ 123456cuhk.edu.hk
                Article
                jitc-2022-005068
                10.1136/jitc-2022-005068
                9476161
                36104100
                e8eab822-b5fc-46be-a913-bed56491c921
                © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

                This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

                Product
                Funding
                Funded by: Research Grants Council, University Grants Committee, Hong Kong;
                Award ID: AoE/P-705-16
                Funded by: Center of Novel Biomaterials, The Chinese University of Hong Kong;
                Categories
                Oncolytic and Local Immunotherapy
                1506
                2438
                Original research
                Custom metadata
                unlocked

                immunotherapy,drug therapy, combination,breast neoplasms

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