Publications by authors named "Michal J Besser"

60 Publications

Characteristics and risk factors of infections following CD28-based CD19 CAR-T cells.

Leuk Lymphoma 2021 Feb 10:1-14. Epub 2021 Feb 10.

Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

CAR T-cells are approved for the treatment of relapsed and refractory leukemia and lymphoma. Here, we studied the infectious complications in 88 patients treated with CD28-based CD19 CAR T-cells. Overall, 36 infections were documented in 24 patients within the first month after CAR T-cell infusion: Six events of bacteremia, sixteen focal bacterial infections, and fourteen systemic or localized viral infections. Seven patients had nine infectious episodes beyond the first 30 days of follow-up, including three events of bacteremia, three focal bacterial, two viral and one fungal infection. The presence of neutropenia, neutropenic fever and lack of response to treatment were associated with a higher rate of infections. Children had less severe infections than adults. In a multivariate analysis lack of response to treatment was the only significant risk factor. Overall, the incidence of bacterial infections following CAR T-cells is modest especially in children and in patients responding to therapy.
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http://dx.doi.org/10.1080/10428194.2021.1881506DOI Listing
February 2021

Genetic Modification of Tumor-Infiltrating Lymphocytes Retroviral Transduction.

Front Immunol 2020 7;11:584148. Epub 2021 Jan 7.

Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center, Ramat Gan, Israel.

Adoptive T cell therapy (ACT) holds great promise for cancer treatment. One approach, which has regained wide interest in recent years, employs antitumor T cells isolated from tumor lesions ("tumor-infiltrating lymphocytes" or TIL). It is now appreciated that a considerable proportion of anti-melanoma TIL recognize new HLA-binding peptides resulting from somatic mutations, which occurred during tumor progression. The clinical efficacy of TIL can potentially be improved their genetic modification, designed to enhance their survival, homing capacity, resistance to suppression, tumor killing ability and additional properties of clinical relevance. Successful implementation of such gene-based strategies critically depends on efficient and reproducible protocols for gene delivery into clinical TIL preparations. Here we describe an optimized protocol for the retroviral transduction of TIL. As the experimental system we employed anti-melanoma TIL cultures prepared from four patients, recombinant retrovirus encoding an anti-CD19 chimeric antigen receptor (CAR) as a model gene of interest and CD19+ and CD19- human cell lines serving as target cells. Transduction on day 7 of the rapid expansion protocol (REP) resulted in 69 ± 8% CAR positive TIL. Transduced, but not untransduced TIL, from the four patients responded robustly to CD19+, but not CD19- cell lines, as judged by substantial secretion of IFN-γ following co-culture. In light of the rekindled interest in antitumor TIL, this protocol can be incorporated into a broad range of gene-based approaches for improving the in-vivo survival and functionality of TIL in the clinical setting.
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http://dx.doi.org/10.3389/fimmu.2020.584148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817656PMC
January 2021

Immune imitation of tumor progression after anti-CD19 chimeric antigen receptor T cells treatment in aggressive B-cell lymphoma.

Bone Marrow Transplant 2020 Dec 3. Epub 2020 Dec 3.

Division of Hematology and Bone Marrow Transplantation, Sheba Medical Center, Ramat Gan, Israel.

We present three patients with aggressive non-Hodgkin's B-cell lymphoma (NHL) who received anti-CD19 chimeric antigen receptor T (CAR T) cells therapy after failure of several lines of chemotherapy that developed pseudo-progression. One-week clinical and radiological findings were consistent with tumor progression. Positron emission tomography-computed tomography (PET-CT) at 1 month post CAR T cells administration was consistent with treatment response. The rapid tumor growth and subsequent resolution are suggestive of tumor pseudo-progression mediated secondary to infiltration and immune activation of CAR T cells. Overall, 56 adult patients with NHL were enrolled in a phase 1b/2 in house clinical study with CD19 CAR T cells. Out of them 22/56 patients progressed as per PET-CT the 1 month post CAR T cells. In 14 patients, signs of progression started 7-10 days after CAR T cells infusion. In 11/14 patients, it was true progression, while in 3 it was pseudo-progression. Additional studies are warranted to describe the extent of this phenomenon and evaluate correlation with the CAR T activity and long-term disease control.
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http://dx.doi.org/10.1038/s41409-020-01156-yDOI Listing
December 2020

microRNA expression patterns in tumor infiltrating lymphocytes are strongly associated with response to adoptive cell transfer therapy.

Cancer Immunol Immunother 2020 Nov 17. Epub 2020 Nov 17.

Ella Lemelbaum Institute of Immuno-Oncology, Sheba Medical Center, Ramat Gan, 526260, Israel.

Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TILs) was previously shown to yield clinical response in metastatic melanoma patients as an advanced line. Unfortunately, there is no reliable marker for predicting who will benefit from the treatment. We analyzed TIL samples from the infusion bags used for treatment of 57 metastatic melanoma patients and compared their microRNA profiles. The discovery cohort included six responding patients and seven patients with progressive disease, as defined by RECIST1.1. High throughput analysis with NanoString nCounter demonstrated significantly higher levels of miR-34a-5p and miR-22-3p among TIL from non-responders. These results were validated in TIL infusion bag samples from an independent cohort of 44 patients, using qRT-PCR of the individual microRNAs. Using classification trees, a data-driven predictive model for response was built, based on the level of expression of these microRNAs. Patients that achieved stable disease were classified with responders, setting apart the patients with progressive disease. Moreover, the expression levels of miR-34a-5p in the infused TIL created distinct survival groups, which strongly supports its role as a potential biomarker for TIL-ACT therapy. Indeed, when tested against autologous melanoma cells, miR TIL cultures exhibited significantly higher cytotoxic activity than miR TIL cultures, and expressed features of terminally exhausted effectors. Finally, overexpression of miR-34a-5p or miR-22-3p in TIL inhibited their cytotoxic ability in vitro. Overall, we show that a two-microRNA signature correlates with failure of TIL-ACT therapy and survival in melanoma patients.
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http://dx.doi.org/10.1007/s00262-020-02782-7DOI Listing
November 2020

ADAR1 regulates melanoma cell invasiveness by controlling beta3-integrin via microRNA-30 family members.

Am J Cancer Res 2020 1;10(8):2677-2686. Epub 2020 Aug 1.

Ella Lemelbaum Institute for Immuno-Oncology Israel.

Melanoma cells utilize multiple mechanisms to exit the primary tumor mass, invade the surroundings and subsequently distant tissues. We have previously reported that the expression of the RNA editing enzyme ADAR1 (adenosine deaminase acting on RNA) is downregulated in metastatic melanoma, which facilitates proliferation and invasion. Here we show that ADAR1 controls melanoma invasiveness by regulating ITGB3 expression via miR-30a and miR-30d. ADAR1 overexpression or knockdown leads to an increase or decrease, respectively, in the expression of both microRNAs. The effect is independent of RNA-editing. Dual luciferase assays show that both microRNAs directly regulate the expression of the ITGB3 integrin. Overexpression of the miR-30a or miR-30d lead to a decrease in ITGB3 and a resultant decreased invasive and metastatic capacities. Neutralization of the endogenous miR-30a or miR-30d leads to the opposite effect. The microRNAs regulate ITGB3 levels probably through a post-transcriptional effect, as both mRNA and protein levels of ITGB3 are affected. These results further expand our knowledge on the ADAR1-ITGB3 network and its central role in acquisition of the invasive phenotype of metastatic melanoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471345PMC
August 2020

Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia.

Front Immunol 2020 2;11:1347. Epub 2020 Jul 2.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Autologous T cells engineered to express a chimeric antigen receptor (CAR) against the CD19 antigen are in the frontline of contemporary hemato-oncology therapies, leading to high remission rates in B-cell malignancies. Although effective, major obstacles involve the complex and costly individualized manufacturing process, and CD19 target antigen loss or modulation leading to resistant and relapse following CAR therapy. A potential solution for these limitations is the use of donor-derived γδT cells as a CAR backbone. γδT cells lack allogenecity and are safely used in haploidentical transplants. Moreover, γδT cells are known to mediate natural anti-tumor responses. Here, we describe a 14-day production process initiated from peripheral-blood mononuclear cells, leading to a median 185-fold expansion of γδ T cells with high purity (>98% CD3+ and >99% γδTCR+). CAR transduction efficacy of γδ T cells was equally high when compared to standard CAR-T cells (60.5 ± 13.2 and 65.3 ± 18.3%, respectively). CD19-directed γδCAR-T cells were effective against CD19+ cell lines and in vivo, showing cytokine production, direct target killing, and clearance of bone marrow leukemic cells in an NSG model. Multiple injections of γδCAR-T cells and priming of mice with zoledronate lead to enhanced tumor reduction . Unlike standard CD19 CAR-T cells, γδCAR-T cells were able to target CD19 antigen negative leukemia cells, an effect that was enhanced after priming the cells with zoledronate. In conclusion, γδCAR-T cell production is feasible and leads to highly pure and efficient effector cells. γδCAR-T cell may provide a promising platform in the allogeneic setting, and may target leukemic cells also after antigen loss.
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http://dx.doi.org/10.3389/fimmu.2020.01347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343910PMC
July 2020

Feasibility of leukapheresis for CAR T-cell production in heavily pre-treated pediatric patients.

Transfus Apher Sci 2020 Aug 25;59(4):102769. Epub 2020 Apr 25.

Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Electronic address:

Background: Autologous CD19 chimeric-antigen receptor (CAR) T-cells are an effective salvage therapy for patients with relapsed or refractory B cell malignancies. The essential first step in the production is the collection of mature lymphocytes through leukapheresis. It is a challenging procedure given the fact patients are heavily pretreated and the special considerations of pediatric apheresis.

Methods: We analyzed the data of leukapheresis outcome for CAR T production in a phase 1b/2 clinical trial enrolling 34 children, adolescents and young adults with relapsed or refractory B-cell malignancies.

Results: All patients underwent a single leukapheresis. Given a short production time for CAR T-cells, most patients received bridging therapy prior to apheresis. Leukapheresis was performed using peripheral venous access in the majority (82%) of patients, and the remainder required arterial line or central venous access. T-cell collection efficiency (CE) was variable with a median of 18%. No apheresis-related adverse events were noted, and all procedures were successful but two: one resulting in lower than target dose (1 × 10 CAR + cells/kg) and the other in failure of CAR T-cell production.

Conclusions: Collection of sufficient T-cells in heavily pretreated pediatric patients via a single apheresis procedure is feasible even with relatively low T-cell CE.
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http://dx.doi.org/10.1016/j.transci.2020.102769DOI Listing
August 2020

Comprehensive single institute experience with melanoma TIL: Long term clinical results, toxicity profile, and prognostic factors of response.

Mol Carcinog 2020 07 6;59(7):736-744. Epub 2020 Apr 6.

Ella Lemelbaum Institute for Immuno Oncology, Sheba Medical Center, Ramat Gan, Israel.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) mediates objective responses in 30% to 50% of patients with metastatic melanoma according to multiple, small phase 2 trials. Here we report the long-term clinical results, intent-to-treat analysis, predictors of response and toxicity profile in a large patient cohort. A total of 179 refractory melanoma patients were enrolled in the ACT trial. TIL were administered in combination with high-dose bolus interleukin-2 following preconditioning with cyclophosphamide and fludarabine. Patients were followed-up for a median of 7.2 years. A total of 107 (60%) of 179 enrolled patients were treated. The main reason for the drop out of the study was clinical deterioration. Of 103 evaluated patients, 29 patients (28%) achieved an objective response (OR), including complete remission (8%) or partial response (20%). Sixteen pateints exhibited stable disease. Predictors of response were performance status, time of TIL in culture and CD8 frequency in the infusion product. The absolute lymphocyte count 1 and 2 weeks after TIL infusion was the most predictive parameter of response. With a medium follow-up time of 7.2 years, OR patients reached a median overall survival (OS) of 58.45 months and a median progression-free survival (PFS) of 15.43 months, as compared with nonresponders, with 6.73 months OS and 2.60 months PFS. By 6 years, 50% of OR patients were alive and 43% had no documented progression. TIL ACT can yield durable objective responses, even as salvage therapy in highly advanced metastatic melanoma patients.
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http://dx.doi.org/10.1002/mc.23193DOI Listing
July 2020

Head-to-head comparison of in-house produced CD19 CAR-T cell in ALL and NHL patients.

J Immunother Cancer 2020 03;8(1)

Ella Lemelbaum Institute for Immuno Oncology, Sheba Medical Center, Tel Hashomer, Israel

Background: CD19 chimeric antigen receptor T (CAR-T) cells demonstrate remarkable remission rates in pediatric and adult patients with refractory or relapsed (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). In 2016, we initiated a clinical trial with in-house produced CD19 CAR-T cells with a CD28 co-stimulatory domain. We analyzed, for the first time, differences in production features and phenotype between ALL and NHL patients.

Methods: Non-cryopreserved CAR-T cells were produced from patients' peripheral blood mononuclear cells within 9 to 10 days. 93 patients with r/r ALL and NHL were enrolled under the same study. CAR-T cells of ALL and NHL patients were produced simultaneously, allowing the head-to-head comparison.

Results: All patients were heavily pretreated. Three patients dropped out from the study due to clinical deterioration (n=2) or production failure (n=1). Cells of ALL patients (n=37) expanded significantly better and contained more CAR-T cells than of NHL patients (n=53). Young age had a positive impact on the proliferation capacity. The infusion products from ALL patients contained significantly more naïve CAR-T cells and a significantly higher expression of the chemokine receptor CXCR3. PD-1, LAG-3, TIM-3, and CD28 were equally expressed. 100% of ALL patients and 94% of NHL patients received the target dose of 1×10e6 CAR-T/kg. The overall response rate was 84% (30/36) in ALL and 62% (32/52) in NHL. We further compared CAR-T cell infusion products to tumor infiltrating lymphocytes (TIL), another common type of T cell therapy, mainly clinically effective in solid tumors. CAR-T cells contained significantly more naïve T cells and central memory T cells and significantly less CCR5 compared to TIL infusion products.

Conclusions: The in-house production of CAR-T cells is highly efficient and fast. Clinical response rate is high. CAR-T cells can be successfully produced for 99% of patients in just 9 to 10 days. Cells derived from ALL patients demonstrate a higher proliferation rate and contain higher frequencies of CAR-T cells and naïve T cells than of NHL patients. In addition, understanding the differences between CAR-T and TIL infusion products, may provide an angle to develop CAR-T cells for the treatment of solid tumors in the future.

Trial Registration Number: ClinicalTrials.gov; CAR-T: NCT02772198, First posted: May 13, 2016; TIL: NCT00287131, First posted: February 6, 2006.
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http://dx.doi.org/10.1136/jitc-2019-000148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061891PMC
March 2020

Remission of acute myeloid leukemia with t(8;21) following CD19 CAR T-cells.

Leukemia 2020 07 24;34(7):1939-1942. Epub 2020 Jan 24.

Division of Hematology and Bone Marrow Transplantation, Sheba Medical Center, Ramat Gan, Israel.

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http://dx.doi.org/10.1038/s41375-020-0719-yDOI Listing
July 2020

Treatment with anti CD19 chimeric antigen receptor T cells after antibody-based immunotherapy in adults with acute lymphoblastic leukemia.

Curr Res Transl Med 2020 01 25;68(1):17-22. Epub 2019 Dec 25.

Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Purpose Of The Study: The prognosis of patients with relapsed/refractory precursor B-acute lymphoblastic leukemia (ALL) is dismal. Antibody-based therapies, such as blinatumomab or inotuzumab ozogamycin (IO) have led to improved outcomes. The impact of prior immunotherapy on chimeric antigen receptor (CAR) T-Cell therapeutic efficacy and toxicity is unknown.

Methods: We describe a case series of ALL patients with prior exposure to blinatumomab or IO, who were treated with anti-CD19 CAR T cells with CD28 co-stimulatory domain (NCT02772198). We then review the literature on CAR-T post antibody-based therapy with either antibodies.

Results: Five adult patients with B-ALL were included. Three had active disease, and two were in morphological complete remission (CR) with minimal residual disease (MRD+). Therapy before CAR-T included blinatumomab (3/5 [60 %]) and IO (3/5 [60 %]), with one patient receiving both. One patient experienced severe cytokine release syndrome and central nervous system toxicity and subsequently died. At 28 days following treatment, two patients achieved CR with MRD negativity, and two had an MRD + CR. Two patients received allogeneic hematopoietic stem cell transplantation. At a median of 10 months (range, 5-26, three out of the four patients are still in CR, and one relapsed. The literature review identified a deficiency on data on the influence of blinatumumab and IO on outcomes post CAR-T therapy.

Conclusions: CD19 CAR T-cell therapy after treatment with blinatumomab and/or IO in patients with relapsed/refractory B-ALL is feasible and results in promising response rates in this case series. Future trails should specifically address outcomes in this population.
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http://dx.doi.org/10.1016/j.retram.2019.12.001DOI Listing
January 2020

Tumor-infiltrating lymphocytes from human prostate tumors reveal anti-tumor reactivity and potential for adoptive cell therapy.

Oncoimmunology 2019;8(12):e1672494. Epub 2019 Oct 11.

Ella Lemelbaum Institute for Immuno Oncology, Sheba Medical Center, Ramat Gan, Israel.

Advanced prostate cancer remains incurable and is the second leading cause of mortality in men. Immunotherapy based on the adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical results in patients with metastatic melanoma and lately also in other solid tumors. However, the ability to obtain TIL from patients with prostate cancer, considered poorly immunogenic, remains unknown. In this study, we investigate the feasibility of isolating and expanding TIL from primary prostate tumors. We collected tumor specimens from eight patients with diagnosed prostate adenocarcinoma undergoing radical prostatectomy and were able to successfully expand multiple autologous TIL cultures from all patients. Twenty-eight prostate-TIL cultures were further expanded using a standard rapid expansion procedure under Good Manufacturing Practice conditions. TIL cultures were phenotypically characterized for T cell subset composition, differentiation status and co-inhibitory/stimulatory markers such as PD-1, TIM-3, LAG-3, and CD28 and were found to have in general similarity to TIL obtained from patients with melanoma and lung carcinoma previously treated at our center. All analyzed TIL cultures were functional as determined by the capability to produce high level of IFNγ upon stimuli. Most importantly, co-culture assays of prostate-TIL with autologous tumors demonstrated anti-tumor reactivity. In conclusion, these findings demonstrate that functional and anti-tumor reactive TIL can be obtained, despite the immunosuppressive microenvironment of the cancer, thus this study supports the development of TIL therapy for prostate cancer patients.
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http://dx.doi.org/10.1080/2162402X.2019.1672494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844325PMC
October 2019

Tissue Harvesting for Adoptive Tumor Infiltrating Lymphocyte Therapy in Metastatic Melanoma.

Anticancer Res 2019 Sep;39(9):4995-5001

Ella Lemelbaum Institute for Immuno Oncology, Chaim Sheba Medical Center, Ramat Gan, Israel.

Background/aim: Adoptive transfer of tumor-infiltrating lymphocytes (TILs) combined with non-myeloablative chemotherapy (NMA) has been shown to prolong survival in patients with metastatic disease.

Materials And Methods: Tissue harvesting was performed form a variety of sites. TILs were isolated, expanded and infused with bolus high-dose IL-2.

Results: Between 2008 and 2018, 242 lesions were resected for TILs harvesting from a range of sites form 196 patients without mortality and with minimal morbidity. Of those harvested, 75 were unable to complete therapy because of clinical deterioration during the wait period. Of 121 evaluable treated patients, there was no effect of metastatic site biopsied on the mean fold TIL expansion. Those receiving prior ipilimumab had a higher TIL fold expansion but a lower TIL fold expansion than those exposed to anti-PD1 therapy.

Conclusion: Harvesting may be safely performed with successful TIL expansion from most sites. Prior check point inhibitory immunotherapy may potentially influence TIL fold expansion.
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http://dx.doi.org/10.21873/anticanres.13689DOI Listing
September 2019

Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.

Cell 2019 09 5;179(1):236-250.e18. Epub 2019 Sep 5.

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel-Aviv 6997801, Israel. Electronic address:

Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.
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http://dx.doi.org/10.1016/j.cell.2019.08.012DOI Listing
September 2019

Reduced CTL motility and activity in avascular tumor areas.

Cancer Immunol Immunother 2019 Aug 28;68(8):1287-1301. Epub 2019 Jun 28.

Department of Immunology, Weizmann Institute of Science, Wolfson Bldg., 234 Herzl St., 76100, Rehovot, Israel.

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.
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http://dx.doi.org/10.1007/s00262-019-02361-5DOI Listing
August 2019

Early and late hematologic toxicity following CD19 CAR-T cells.

Bone Marrow Transplant 2019 10 26;54(10):1643-1650. Epub 2019 Feb 26.

Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel.

Autologous T cells transduced with CD19-directed chimeric antigen receptors have recently been approved by several regulatory agencies for the treatment of relapsed and refractory leukemia and lymphoma, after demonstrating remarkable remission rate in advanced patients. The most common adverse events reported are cytokine-release syndrome (CRS), neurotoxicity, and hematologic toxicity. Here, we focus on early and late cytopenia occurring after CD19 CAR-T cells in 38 patients treated with CD19 CAR-T cells. Neutropenia, thrombocytopenia, and anemia occur frequently (94, 80, and 51%, respectively) after CAR-T cell infusion, and are associated with a biphasic nature, as in 93% of patients hematologic toxicity occurs after 21 days from cell infusion. Late hematologic toxicity was more common in patients with high grade CRS and in patients treated after a recent stem cell transplantation. Interestingly, since these events occur late after the lymphodepleting chemotherapy and after resolution of CRS, we found perturbations in SDF-1 levels to correlate with events of late neutropenia, likely associated with B-cell recovery.
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http://dx.doi.org/10.1038/s41409-019-0487-3DOI Listing
October 2019

Combined Expression of Genetic Adjuvants Via mRNA Electroporation Exerts Multiple Immunostimulatory Effects on Antitumor T Cells.

J Immunother 2019 Feb/Mar;42(2):43-50

Laboratory of Immunology, MIGAL, Galilee Research Institute, Kiryat Shmona.

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing antitumor TCRs or chimeric antigen receptors often yields a high rate of clinical response in several types of cancer. New approaches for enhancing the functional properties of antitumor T cells could improve the clinical outcome of these treatments. To this end, we created 3 classes of genes, each designed to operate autonomously upon expression in T cells. We recently reported on the enhancing effects of constitutively active toll-like receptor 4 (caTLR4), membrane (mem) interleukin-2, memIL-12, and memIL-15, and self-oligomerizing, constitutively active CD40 (caCD40). Here, we evaluated their combined effects on peripheral blood CD8 T cells and different antimelanoma TIL cultures following mRNA electroporation. Expression in CD8 T cells induced transient production of interferon-γ and prolonged and robust upregulation of CD25, CD69, 4-1BB, and OX40. The adjuvants enhanced cytolytic activity of TILs and production of interferon-γ and TNF-α in the presence of autologous, but not mismatched, melanoma for at least 3 days after electroporation. Expression of the 3 adjuvants in young TILs from different patients markedly increased the expression of CD25, OX40, 4-1BB, CD127, and CD28 and exhibited cooperative and, at times, synergistic effects. Furthermore, predefined mixtures of mRNA encoding these adjuvants markedly enhanced the specific antitumor response of selected TILs and killing of autologous melanoma cells by young TILs. Our findings suggest that combinations of these new genetic adjuvants can substantially improve the functional properties of antitumor T cells, offering a new tool of unique versatility in adoptive cell therapy.
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http://dx.doi.org/10.1097/CJI.0000000000000252DOI Listing
April 2020

Potent Activation of Human T Cells by mRNA Encoding Constitutively Active CD40.

J Immunol 2018 11 10;201(10):2959-2968. Epub 2018 Oct 10.

Laboratory of Immunology, MIGAL-Galilee Research Institute, Kiryat Shmona 11016, Israel;

New strategies for augmenting the actual performance of therapeutic T cells in vivo are needed for improving clinical outcome of adoptive cell therapy. Cumulative findings suggest that CD40 plays an intrinsic role in T cell costimulation. Recently, we demonstrated the ability of truncated, auto-oligomerizing CD40 derivatives to induce strong activation of APCs in a ligand-independent manner. We reasoned that constitutively active CD40 (caCD40) can similarly exert enhancing effects on human antitumor T cells. To test this assumption, we transfected human T cells with in vitro-transcribed caCD40 mRNA. In polyclonal T cells, caCD40 triggered IFN-γ secretion and upregulated CD25 and 4-1BB. In antimelanoma tumor-infiltrating lymphocytes (TILs), caCD40 induced massive production of IFN-γ, exerting a pronounced synergistic effect when coexpressed with constitutively active TLR4 devoid of its extracellular ligand binding. In unselected "young" TILs, caCD40 reproducibly increased surface expression of CD25, OX40, 4-1BB, CD127, and CD28. Three days post-mRNA electroporation of CD8 TILs, caCD40 elevated IFN-γ and TNF-α production and cytolytic activity in the presence of autologous but not HLA-I-mismatched melanoma. Enhanced killing of autologous melanoma by young TILs was observed 4 d posttransfection. These findings suggest that caCD40 can function as a potent T cell adjuvant and provide essential guidelines for similar manipulation of other key members of the TNFR family.
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http://dx.doi.org/10.4049/jimmunol.1701725DOI Listing
November 2018

Locally produced CD19 CAR T cells leading to clinical remissions in medullary and extramedullary relapsed acute lymphoblastic leukemia.

Am J Hematol 2018 12 26;93(12):1485-1492. Epub 2018 Sep 26.

Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Israel.

Autologous CD19 chimeric-antigen receptor (CAR) T cells demonstrated remarkable remission rates in relapsed and refractory acute lymphoblastic leukemia (R/R ALL). Here, we report results from a phase 1b/2 study of in-house produced CD19 CAR with a CD28 costimulatory domain. Twenty-one patients with R/R ALL were enrolled, and 20 infused. The median age was 11 years (range, 5-48). Patients had a median of 4 prior regimens, including blinatumomab in 6 and prior stem-cell transplantation in 10. In total 8 patients had extramedullary (EM) leukemic involvement, and prior to lymphodepletion and CAR 7 had active lesions, a group underrepresented in previous trials. In vivo expansion of CAR T cells was observed in 18 patients. In total 16 patients developed cytokine release syndrome, and 11 patients developed neurotoxicity, with no toxic deaths. All responding patients were referred to an allogeneic hematopoietic stem-cell transplantation. The remission rate was 90%, including resolution of all refractory EM sites. Four responding patients relapsed, 3 who had a PCR-MRD positive remission at 28 days following CAR-T cells and 1 patient 21 months after an MRD-negative response. The estimated 1-year event-free survival and overall survival are 73% and 90%, respectively. Patients with R/R EM ALL may also benefit from CAR-T cell therapy.
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http://dx.doi.org/10.1002/ajh.25274DOI Listing
December 2018

Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients.

Cancer Immunol Immunother 2018 08 29;67(8):1221-1230. Epub 2018 May 29.

Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center, 52621, Ramat Gan, Israel.

Adoptive cell therapy (ACT) of tumor infiltration lymphocytes (TIL) yields promising clinical results in metastatic melanoma patients, who failed standard treatments. Due to the fact that metastatic lung cancer has proven to be susceptible to immunotherapy and possesses a high mutation burden, which makes it responsive to T cell attack, we explored the feasibility of TIL ACT in non-small cell lung cancer (NSCLC) patients. Multiple TIL cultures were isolated from tumor specimens of five NSCLC patients undergoing thoracic surgery. We were able to successfully establish TIL cultures by various methods from all patients within an average of 14 days. Fifteen lung TIL cultures were further expanded to treatment levels under good manufacturing practice conditions and functionally and phenotypically characterized. Lung TIL expanded equally well as 103 melanoma TIL obtained from melanoma patients previously treated at our center, and had a similar phenotype regarding PD1, CD28, and 4-1BB expressions, but contained a higher percent of CD4 T cells. Lung carcinoma cell lines were established from three patients of which two possessed TIL cultures with specific in vitro anti-tumor reactivity. Here, we report the successful pre-clinical production of TIL for immunotherapy in the lung cancer setting, which may provide a new treatment modality for patients with metastatic NSCLC. The initiation of a clinical trial is planned for the near future.
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http://dx.doi.org/10.1007/s00262-018-2174-4DOI Listing
August 2018

Regulation of CEACAM1 Protein Expression by the Transcription Factor ETS-1 in BRAF-Mutant Human Metastatic Melanoma Cells.

Neoplasia 2018 04 17;20(4):401-409. Epub 2018 Mar 17.

Ella Lemelbaum Institute of Immuno-Oncology, Sheba Medical Center, Ramat-Gan, 5262620, Israel; Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; School of Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Electronic address:

BRAF becomes constitutively activated in 50% to 70% of melanoma cases. CEACAM1 has a dual role in melanoma, including facilitation of cell proliferation and suppression of infiltrating lymphocytes, which are consistent with its value as a marker for poor prognosis in melanoma patients. Here we show that BRAF melanoma cells treated with BRAF and MEK inhibitors (MAPKi) downregulate CEACAM1 mRNA and protein expression in a dose- and exposure time-dependent manners. Indeed, there is a significant correlation between the presence of BRAF and CEACAM1 expression in melanoma specimens obtained from 45 patients. Vemurafenib-resistant cell systems reactivate the MAPK pathway and restore basal CEACAM1 mRNA and protein levels. These combined results suggest transcriptional regulation. Indeed, luciferase reporting assays show that CEACAM1 promoter (CEACAM1p) activity is significantly reduced by MAPKi. Importantly, we show that the MAPK-driven CEACAM1p activity is mediated by ETS1, a major transcription factor and downstream effector of the MAPK pathway. Phosphorylation mutant ETS1 shows a dominant negative effect over CEACAM1 expression. The data are consistent with independent RNAseq data from serial biopsies of melanoma patients treated with BRAF inhibitors, which demonstrate similar CEACAM1 downregulation. Finally, we show that CEACAM1 downregulation by MAPKi renders the cells more sensitive to T-cell activation. These results provide a new view on a potential immunological mechanism of action of MAPKi in melanoma, as well as on the aggressive phenotype observed in drug-resistant cells.
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http://dx.doi.org/10.1016/j.neo.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909674PMC
April 2018

Histopathological expression analysis of intercellular adhesion molecule 1 (ICAM-1) along development and progression of human melanoma.

Oncotarget 2017 Nov 14;8(59):99580-99586. Epub 2017 Sep 14.

Ella Lemelbaum Institute of Immuno-Oncology, Ramat-Gan, Israel.

Intercellular adhesion molecule 1 (ICAM-1) protein is an important adhesion molecule that facilitates metastasis on one hand, and on the other hand supports the immunological synapse necessary for T-cell mediated elimination. The expression pattern of ICAM-1 in melanoma was studied more than two decades ago, mainly in cell lines or in unmatched melanoma specimens. By using real time PCR we could not demonstrate a clear difference in ICAM-1 mRNA levels between primary melanocytes and primary cultures of metastatic melanoma. However, immunohistochemistry staining of progression tissue microarray comprised of samples of different disease stages derived from different patients, demonstrated a dramatic ICAM-1 upregulation particularly upon the transition from primary tumor to lymph node metastasis. There was no significant difference between lymph node and distant metastases. Importantly, these results were confirmed in an independent tissue microarray comprised of patient-paired specimens from progressive stages of the patient's disease. These data indicate that ICAM-1 upregulation is required to initiate the lymphatic spread of melanoma (Stage III) but no further increase is associated with progression to remote organs (Stage IV).
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http://dx.doi.org/10.18632/oncotarget.20884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725116PMC
November 2017

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation.

Front Immunol 2017 10;8:1211. Epub 2017 Oct 10.

Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center, Tel Hashomer, Israel.

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT.
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http://dx.doi.org/10.3389/fimmu.2017.01211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641376PMC
October 2017

Adoptive Cell Therapy for Metastatic Melanoma.

Cancer J 2017 Jan/Feb;23(1):48-53

From the *Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center, Tel Hashomer; and †Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) is a powerful form of immunotherapy by inducing durable complete responses that significantly extend the survival of melanoma patients. Mutation-derived neoantigens were recently identified as key factors for tumor recognition and rejection by TILs. The isolation of T-cell receptor (TCR) genes directed against neoantigens and their retransduction into peripheral T cells may provide a new form of ACT.Genetic modifications of T cells with chimeric antigen receptors (CARs) have demonstrated remarkable clinical results in hematologic malignancies, but are so far less effective in solid tumors. Only very limited reports exist in melanoma. Progress in CAR T-cell engineering, including neutralization of inhibitory signals or additional safety switches, may open opportunities also in melanoma.We review clinical results and latest developments of adoptive therapies with TILs, T-cell receptor, and CAR-modified T cells and discuss future directions for the treatment of melanoma.
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http://dx.doi.org/10.1097/PPO.0000000000000240DOI Listing
August 2017

Metastatic Lung Lesions as a Preferred Resection Site for Immunotherapy With Tumor Infiltrating Lymphocytes.

J Immunother 2016 06;39(5):218-22

Departments of *Thoracic Surgery †Ella Lemelbaum Institute of Melanoma ‡General and Bariatric Surgery, Sheba Medical Center, Tel Hashomer §Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Adoptive cell therapy with tumor infiltrating lymphocytes (TIL) yields 50% response rates in metastatic melanoma and shows promising clinical results in other solid tumors. Autologous TIL cultures are isolated from resected tumor tissue, expanded ex vivo to large numbers and reinfused to the preconditioned patient. In this prospective study, we validate the origin of the tumor biopsy and its effect on T-cell function and clinical response. One hundred forty-four patients underwent surgery and 79 patients were treated with TIL adoptive cell therapy. Cultures from lung tissue were compared with other origins. The success rate of establishing TIL culture from lung tissue was significantly higher compared with nonlung tissue (94% vs. 72%, respectively, P≤0.003). Lung-derived TIL cultures gave rise to higher cell numbers (P≤0.011) and exhibited increased in vitro antitumor reactivity. The average fold expansion for lung-derived TIL during a rapid expansion procedure was 1349±557 compared with 1061±473 for nonlung TIL (P≤0.038). Patients treated with TIL cultures of lung origin (compared with nonlung) had prolonged median overall survival (29 vs. 9.5 mo; P≤0.065). Given the remarkable advancement in minimally invasive thoracic surgery and the results of this study, we suggest efforts should be taken to resect lung metastasis rather than other sites to generate TIL cultures for clinical use.
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http://dx.doi.org/10.1097/CJI.0000000000000124DOI Listing
June 2016

Membrane-attached Cytokines Expressed by mRNA Electroporation Act as Potent T-Cell Adjuvants.

J Immunother 2016 Feb-Mar;39(2):60-70

*Laboratory of Immunology, MIGAL-Galilee Research Institute, Kiryat Shmona †Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem ‡Ella Lemelbaum Institute for Melanoma, Sheba Medical Center, Ramat Gan §Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv ∥Tel-Hai College, Upper Galilee, Israel.

Proinflammatory cytokines are widely explored in different adoptive cell therapy protocols for enhancing survival and function of the transferred T cells, but their systemic administration is often associated with severe toxicity which limits their clinical use. To confine cytokine availability to the therapeutic T cells, we expressed 3 key cytokines, IL-2, IL-12, and IL-15, as integral T-cell membrane proteins. To prevent permanent activation of growth signaling pathways, we delivered these genes to T cells through mRNA electroporation. The engineered cytokines could be detected on the surface of mRNA-transfected cells and binding to their cell-surface receptors mainly occurred in cis. The 3 human cytokines supported the ex vivo growth of activated human CD8 and CD4 T cells for at least 6 days posttransfection, comparably to high-dose soluble IL-2. Similarly, membrane IL-2, membrane IL-12, and, to a lesser extent, membrane IL-15, were comparable with their soluble counterparts in supporting proliferation of splenic mouse CD8 T cells. Following electroporation of human CD8 T cells and antimelanoma tumor-infiltrating lymphocytes, membrane cytokines synergized with constitutively active toll-like receptor 4 in inducing interferon-γ secretion. Efficient cooperation with TLR4 was also evident in the upregulation of the activation molecules CD25, CD69, CD137 (4-1BB), and CD134 (OX40). Taken together, membrane cytokines expressed through mRNA transfection emerge as effective tools for enhancing T-cell proliferation and function and may have potential use in adoptive T-cell therapy.
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http://dx.doi.org/10.1097/CJI.0000000000000109DOI Listing
November 2016

Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens.

Oncotarget 2016 Feb;7(5):5110-7

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.
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http://dx.doi.org/10.18632/oncotarget.6960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868674PMC
February 2016

Predictors of tumor-infiltrating lymphocyte efficacy in melanoma.

Immunotherapy 2016 10;8(1):35-43. Epub 2015 Dec 10.

The Ella Lemelbaum Institute for Melanoma, Sheba Medical Center, 52621 Ramat-Gan, Israel.

In the past decades, the increasing knowledge in cellular immunology and tumor-host immune interactions, led to the development of immunotherapy approaches. Immunotherapy, based on adoptive cell transfer of ex vivo activated and expanded tumor-infiltrating T lymphocytes (TILs), has shown promising clinical results in patients with metastatic melanoma. TIL therapy yields response rates of around 50% and significant survival benefit in refractory melanoma patients, even after failing other immunotherapies, such as checkpoint inhibitors or cytokine-based therapy. Identifying predictors of TIL efficacy and detection of TIL subsets with specific reactivity against the patient's tumor might be an important milestone toward further improvement of clinical responses and prolonged survival.
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http://dx.doi.org/10.2217/imt.15.99DOI Listing
June 2016

Tumor-Infiltrating Lymphocytes: Clinical Experience.

Cancer J 2015 Nov-Dec;21(6):465-9

From the *Ella Lemelbaum Institute for Melanoma, Sheba Medical Center, Tel Hashomer; and †Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) in combination with lymphodepletion and high-dose interleukin 2 has shown reproducible objective response rates of approximately 50% in patients with highly advanced, refractory metastatic melanoma. As confirmed by different clinical centers, TIL ACT can yield durable responses especially in patients with complete regression, who are mostly disease-free many years after treatment, suggesting the possibility of cure. Most TIL ACT trials have been conducted as salvage therapy for patients with multiple metastases, frequently in visceral organs and even brain, and who failed numerous treatments, including checkpoint inhibitors, which underlines the value of the treatment. Recent developments in the identification and selection of tumor-specific T-cell populations have facilitated the implementation of TIL ACT also in nonmelanoma malignancies. We summarize the clinical experience of TIL ACT in melanoma, briefly discuss new directions for further improvement of this promising therapy, and present the latest clinical results in nonmelanoma cancers.
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http://dx.doi.org/10.1097/PPO.0000000000000154DOI Listing
September 2016

A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme.

Oncotarget 2015 Oct;6(30):28999-9015

Ella Lemelbaum Institute of Melanoma, Sheba Medical Center, Israel.

The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance. Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity. Here we investigate the role of ADAR1 in melanoma immune resistance.Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism. We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance. ADAR1 thus has a novel, pivotal, role in cancer immune resistance. Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab.These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment.
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http://dx.doi.org/10.18632/oncotarget.4905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745707PMC
October 2015