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Introduction: Chimeric antigen receptor (CAR) T cells can mediate deep and durable responses in hematologic malignancies, however, achieving success in solid tumors has been so far limited largely by lack of suitable solid tumor-associated antigens and the immunosuppressive tumor microenvironment (TME). GPC3 is a surface antigen overexpressed in hepatocellular cancer (HCC) and virtually absent on healthy tissues. In this first-in-human (FIH) study, we investigated the feasibility, safety and initial anti- HCC efficacy of C-CAR031. C-CAR031 is an autologous, GPC3-directed armored CAR-T with affinity-tuned scFv to enhance the safety profile, and a 4-1BB and CD3ζ signaling domain. The C-CAR031 transgene includes a T2A viral self-cleaving peptide and a dominant negative TGF-β receptor II (TGFβRIIDN). The expression of TGFβRIIDN protects the cells against the immunosuppressive HCC TME and the T2A peptide allows for equimolar expression of the two transgene products. Methods: This FIH, open-label dose escalation trial employs an accelerated titration plus i3+3 design. Histologically confirmed GPC3+ advanced HCC patients (pts) who failed systemic treatments received a single-dose i.v. infusion of C-CAR031 following standard lymphodepletion. The primary objective was to assess the safety and tolerability. Adverse events (AEs) were graded using CTCAE 5.0, and cytokine release syndrome (CRS)/immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. Results: As of Dec. 31st 2022, 7 pts received two dose levels (DL1, n=1; DL2, n=6) of C-CAR031. The median number of prior lines of therapies was 4 (range 1-6). The median follow-up was 77 (40-213) days. Six pts with ≥28 days’ follow-up were eligible for safety evaluation. The only ≥Gr3 non-hematologic product-related AE observed was transient Gr3 AST elevation in two pts. Five of 6 pts experienced Gr1/2 CRS, with median time to onset and duration of 3 (range 2-7) and 4 (4-6) days. No DLT or ICANS was observed. Of the 5 pts evaluable for preliminary efficacy, 4 pts had unconfirmed PR, which are currently pending confirmation. AFP was also stabilized or reduced in all 4 patients with uPR. All 5 pts had reduction in tumor burden, with a median change of -31.2% (range -3.4- -60.6%)/-41.4% (-3.4- -56.6%) per RECIST1.1/mRECIST. C-CAR031showed a robust cellular kinetic profile. In DL2, the median Tmax, Cmax and AUC0-28Day were 15 days, 772,014 copies/μg gDNA and 7,747,054 days*copies/μg gDNA, respectively. CAR-T cells were detectable in blood of all pts in the last follow-up. Conclusions: In this FIH study, C-CAR031 is well tolerated and shows promising anti- tumor activity. Enrollment is ongoing to confirm initial results. Citation Format: Qi Zhang, Qihan Fu, Wanyue Cao, Xingyuan Xu, Ao Xia, Jiaqi Huang, Andy Zou, Judy Zhu, Fei Wang, Yi Hong, Hui Wan, Yihong Yao, Nina Chu, Ryan Gilbreth, Maria Letizia Giardino Torchia, John Stone, Attilio Bondanza, Benny Farsaci, Gordon Moody, Mark Cobbold, Tingbo Liang. First report of preliminary safety, efficacy, and pharmacokinetics of C-CAR031 (GPC3-specific TGFβRIIDN CAR-T) in patients with advanced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT097.

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes.

Chimeric antigen receptor (CAR) T cell expansion and persistence represent key factors to achieve complete responses and prevent relapses. These features are typical of early memory T cells, which can be highly enriched through optimized manufacturing protocols. Here, we investigated the efficacy and safety profiles of CAR T cell products generated from preselected naive/stem memory T cells (TN/SCM), as compared with unselected T cells (TBULK). Notwithstanding their reduced effector signature in vitro, limiting CAR TN/SCM doses showed superior antitumor activity and the unique ability to counteract leukemia rechallenge in hematopoietic stem/precursor cell-humanized mice, featuring increased expansion rates and persistence together with an ameliorated exhaustion and memory phenotype. Most relevantly, CAR TN/SCM proved to be intrinsically less prone to inducing severe cytokine release syndrome, independently of the costimulatory endodomain employed. This safer profile was associated with milder T cell activation, which translated into reduced monocyte activation and cytokine release. These data suggest that CAR TN/SCM are endowed with a wider therapeutic index compared with CAR TBULK.

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms., BRAF-MAPK activating mutations are reported in histiocytoses—hematological neoplasms with widespread pro-inflammatory myeloid cells. Here, the authors show that an activating mutant BRAF in haematopoietic stem and progenitor cells causes an oncogene-induced senescence response leading to myeloid restricted haematopoiesis, inflammation and histiocytosis.

Transplacental trafficking of maternal and fetal cells during pregnancy establishes long-term reciprocal microchimerism in both mother and child. Consequently, the maternal immune system may become sensitized to paternal histocompatibility antigens. It has been hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect the outcome of hematopoietic stem cell transplantation (HSCT) when the mother serves as a donor for the child. In T cell-depleted HLA-haploidentical HSCT, maternal donors have been associated with improved transplantation outcomes. The present retrospective multicenter study, conducted on behalf of the Cellular Therapy and Immunobiology Working Party of the European Society of Blood and Marrow Transplantation, involved 409 patients (102 pediatric and 307 adult) with acute leukemia who underwent HLA-haploidentical HSCT. The goal of the study was to evaluate the role of maternal donors in a large cohort of haploidentical transplantation recipients. Transplantation from maternal donors was associated with a lower relapse incidence in T cell-depleted HSCTs (hazard ratio [HR], 2.13; 95% confidence interval [CI], 1.16 to 3.92; P = .018) as well as in a limited series of unmanipulated in vivo T cell-depleted HSCTs (HR, 4.15; 95% CI, 0.94 to 18.35; P= .06), along with better graft-versus-host disease/relapse-free survival (GRFS) in T cell-depleted HSCT (HR, 1.67; 95% CI, 1.02 to 2.73; P = .04). These results indicate that the mother is the preferred donor to provide better GRFS in T cell-depleted HLA-haploidentical HSCT for acute leukemia. (C) 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing., An immune suppressive tumor microenvironment (TME) is a limitation for immunotherapy. Here the authors show that, in a B cell acute lymphoblastic leukemia mouse model, gene-based delivery of IFNα reprograms the leukemia-induced immunosuppressive TME into immunostimulatory and enhances T-cell responses.

Background: Despite unprecedented efficacy of existing CAR-T cell therapy, many pts fail to respond to the therapy, or relapse after initial response. YTB323 is an autologous CD19-directed CAR-T cell therapy utilizing the FMC63 domain for CD19 recognition and 4-1BB costimulatory domain. YTB323 is manufactured with an innovative simplified process, called T-Charge TM, which reduces the ex vivo culture time to about 24 hours and takes Methods: This Phase I, multicenter, dose-escalation study (NCT03960840) is evaluating safety and preliminary efficacy of YTB323 in pts with B-cell malignancies. Results presented here focus on the DLBCL cohort. Eligible pts are adults with measurable disease at enrollment, ECOG 0-1, and r/r DLBCL after ≥2 lines of prior therapies, including autologous hematopoietic stem cell transplantation (aHSCT). Pts received single-dose YTB323 at dose level 1 (DL1; 1-2.5×10 6 CAR+ cells), DL2 (5-12.5×10 6 CAR+ cells), or DL3 (25-40×10 6 CAR+ cells). Bridging therapy prior to YTB323 was optional. Primary endpoints are rate of dose-limiting toxicities (DLTs) in the first 28 d and safety to determine a recommended Phase II dose (RP2D). Secondary endpoints are cellular kinetics, overall response rate (ORR) by local investigator assessment, duration of response, and overall survival. Results: As of April 16, 2021, 15 pts with r/r DLBCL were infused with YTB323: 4 at DL1, 10 at DL2, and 1 at DL3 (Fig, n=14 for DL1 and DL2). Median age was 65 years; most (60%) received 2 prior lines of therapy and 4 (27%) had prior aHSCT. All adverse events were reported regardless of study drug relationship. Of the 15 pts evaluable for safety, 4 pts (27%) reported at least one Grade (Gr) 3 AE, 6 (40%) at least one Gr 4 AE, and 2 (13%) at least one Gr 5 AE. Most commonly reported Gr 3/4 AEs were thrombocytopenia (n=2, 13%), neutropenia (n=3, 20%), and decreased neutrophil count (n=3, 20%). Seven pts (47%) had neurological AEs, of which 2 events (13%) were considered serious-1 event each of Gr 3 peripheral neuropathy (unrelated) and Gr 2 seizure (immune effector cell-associated neurotoxicity syndrome Grade 3, related to treatment). Four pts (27%) experienced cytokine release syndrome (CRS), including 3 (20%) Gr 1/2 and 1 (7%) Gr 4 (Lee et al, 2014), which met DLT criteria. Tocilizumab and corticosteroids were administered for CRS management in 2 (13%) and 1 (7%) pts, respectively. There have been 4 deaths on trial, all unrelated to YTB323: 2 due to disease progression and 2 to sepsis (1 at DL1 and 1 at DL3). Median time to onset of CRS was 9 d (range, 9-9 d) for DL1 and 11 d (range, 8-17 d) for DL2. Preliminary dose-dependent response was observed. At DL1, 4 pts were evaluable for efficacy at Mo 3 and the ORR and CR rates were both 25% (95% CI, 0.6%-80.6%). At DL2, 8 pts were evaluable for efficacy at Mo 3, including 2 pts in CR prior to YTB323 infusion; ORR and CR rates were both 75% (95% CI, 34.9%-96.8%). Dose-dependent expansion (C max and AUC 0-28d) was observed following infusion with mean peak expansion (C max) at DL2 of 13.6% CAR+ in CD3+ cells or 32,100 copies/µg DNA. Although long-term persistence cannot be evaluated yet, of the 8 pts at DL2 with 3-mo follow-up, 3 had detectable CAR expression by flow cytometry (≥1%). Time of peak expansion (T max) coincided with peak cytokine levels (~16 d post infusion). The novel manufacturing methodology of YTB323 allowed preservation of CD4 and CD8 naive/T scm cells in the final product as ascertained by flow cytometry. Bulk and single-cell RNAseq analysis demonstrated that YTB323 retained a naive stem-like phenotype. Conclusions: YTB323 recruitment is ongoing at DL3; RP2D remains to be identified. At DL2, YTB323 showed promising efficacy and a favorable safety profile. Current data support continued development of YTB323 in r/r DLBCL pts. Clinical trial information: CYTB323A12101 Figure 1 Figure 1. Disclosures Flinn: ArQule: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Great Point Partners: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Gilead Sciences: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Constellation Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Teva: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Nurix Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Roche: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; AbbVie: Consultancy, Other: All Consultancy and Research Funding payments made to Sarah Cannon Research Institute, Research Funding; Infinity Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Yingli Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Forma Therapeutics: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Celgene: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Genentech: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Agios: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; AstraZeneca: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Acerta Pharma: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Forty Seven: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Karyopharm Therapeutics: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Incyte: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Unum Therapeutics: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; TG Therapeutics: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Takeda: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Loxo: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Pfizer: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Janssen: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; BeiGene: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Portola Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Merck: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Iksuda Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Juno Therapeutics: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Kite, a Gilead Company: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; MorphoSys: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Seagen: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Verastem: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; IGM Biosciences: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Rhizen Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Curis: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Novartis: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Calithera Biosciences: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Trillium Therapeutics: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Triphase Research & Development Corp.: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Century Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Hutchison MediPharma: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Vincerx Pharma: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Sarah Cannon Research Institute: Current Employment; Servier Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Yingli Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Seagen: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Servier Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Unum Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute, Research Funding; Johnson & Johnson: Current holder of individual stocks in a privately-held company; Seattle Genetics: Research Funding. Jaeger: BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Norvartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Shah: Epizyme: Consultancy; Legend: Consultancy; Incyte: Consultancy; Umoja: Consultancy; Lily: Consultancy, Honoraria, Research Funding; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Kite: Consultancy. Blaise: Jazz Pharmaceuticals: Honoraria. Boissel: Novartis: Consultancy, Honoraria, Research Funding; SANOFI: Honoraria; CELGENE: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Incyte: Honoraria; Servier: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; JAZZ Pharma: Honoraria, Research Funding; PFIZER: Consultancy, Honoraria. Bondanza: Novartis: Ended employment in the past 24 months, Patents & Royalties: Author of patent related to abstract; AstraZeneca: Current Employment. Lu: Novartis: Current Employment. Zhu: Novartis: Current equity holder in publicly-traded company; NIBR: Current Employment. Engels: Novartis: Current Employment, Current equity holder in publicly-traded company. Brogdon: Novartis Institutes for Biomedical Research: Current Employment. Mataraza: Novartis: Current Employment, Current holder of stock options in a privately-held company. Davis: Novartis: Current Employment, Current holder of stock options in a privately-held company. Marchal: Novartis: Current Employment. Mariconti: Novartis: Current Employment. Moschetta: Novartis: Current Employment. Parseval: Novartis: Current Employment. Barba: Gilead: Honoraria; Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Dickinson: Gilead Sciences: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, accommodation, expenses, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.

Background: Tisagenlecleucel (tisa-cel), an autologous anti-CD19 chimeric antigen receptor (CAR)-T cell therapy, has demonstrated durable responses and a manageable safety profile in adult patients (pts) with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). It has previously been suggested that prior therapy with ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, may improve tisa-cel manufacturing, in vivo cellular kinetics, and antitumor efficacy (Fraietta et al. Blood. 2016). Moreover, since BTK signaling is involved in direct pro-inflammatory polarization of macrophages, as well as indirectly by T cells, it is hypothesized that ibrutinib may mitigate CAR-T cell-related toxicities such as cytokine release syndrome (CRS) and neurological events (NE). We report the initial results from a Phase Ib, multicenter, open-label trial evaluating the safety and tolerability of tisa-cel in combination with ibrutinib in adult pts with r/r DLBCL. Methods: Adult pts with r/r DLBCL who received >2 prior lines of systemic therapy, including pts who progressed after or were ineligible for autologous stem cell transplant, were enrolled. The study design has 2 nonrandomized arms. In Arm 1, pts received ibrutinib 560 mg/d for ~4 weeks prior to leukapheresis; in Arm 2, pts were exposed to ibrutinib after leukapheresis. In both arms, ibrutinib was continued throughout lymphodepleting chemotherapy, tisa-cel infusion, and post infusion for up to 24 months. Lymphodepleting chemotherapy, ending at least 2 days before tisa-cel infusion, was either fludarabine (25 mg/m2) and cyclophosphamide (250 mg/m2) daily for 3 days or bendamustine (90 mg/m2) daily for 2 days. Pts received a single infusion of tisa-cel (target dose: 0.6-6.0×108 viable CAR+ T cells). Primary endpoints are incidence and severity of adverse events and ibrutinib dose interruptions/modifications. Secondary endpoints include best overall response (BOR) by Lugano criteria and cellular kinetics of tisa-cel. Results: As of June 9, 2020, 10 pts have been treated and observed through at least the Day 28 assessment: 4 in Arm 1 and 6 in Arm 2. Median age was 59 (range, 32-67) in Arm 1 and 64 (range, 58-76) in Arm 2. Median number of prior therapies was 3.5 (range, 2-5) in Arm 1 and 2 (range, 2-3) in Arm 2. Three of 10 pts (Arm 1, n=1; Arm 2, n=2) had an activated B-cell-like subtype of DLBCL. Six of 10 pts (Arm 1, n=1; Arm 2, n=5) had grade 1 CRS (by Lee scale) and 1 pt had NE (Arm 2, grade 1 by ASTCT criteria; Table). One pt in Arm 2 had grade 3 neutropenia lasting >28 days post tisa-cel infusion. No other pts had grade 3 or 4 neutropenia or thrombocytopenia lasting >28 days. No major bleeding events were observed. Ibrutinib-related bradycardia and atrial fibrillation (both grade 2) were each observed in 1 pt in Arm 1; supraventricular tachycardia (grade 1) related to tisa-cel was observed in 1 pt in Arm 2. No pt required tocilizumab or ICU admission. As of data cutoff, BOR in Arm 1 was complete response (CR) in 2 pts and partial response (PR) in 2 pts, with no relapses. BOR in Arm 2 was CR in 2 pts, PR in 1 pt, and progressive disease in 3 pts (Table). CAR-T cell expansion in vivo by qPCR was in line with data from the pivotal JULIET trial, except for 1 pt in Arm 2 whose transgene levels were below the limit of quantification at all points in time and who progressed at Day 28. Median viability of the leukapheresis material was 96.80% (range, 88.8-97.3) in Arm 1 and 90.95% (range, 88.1-94.7) in Arm 2. A naïve/stem cell-like central memory phenotype (CD45RA+/CCR7+) was observed in 24.05% (median; range, 15.9-37.0) of CD8+ T cells in the leukapheresis material for Arm 1 and in 8.12% (median; range, 1.3-20.4) for Arm 2 (Fig.1A). Fig.1B shows total CAR+ manufactured cells in each arm. The median dose of the final product was 3.9×108 CAR+ T cells in Arm 1 (range, 3.4-4.6×108 CAR+ T cells; median viability 92.25%) and 1.7×108 CAR+ T cells in Arm 2 (range, 1.2-3.0×108 CAR+ T cells; median viability 85.8%; Fig.1C). IFNγ secretion of tisa-cel in vitro in response to CD19+ target cells was similar between the 2 arms, whereas median normalized IL-2 responses were 23.1 fg/CAR+ cell in Arm 1 (range, 16.7-43.8) and 1.1 fg/CAR+ cell in Arm 2 (range, 0-17.3). Conclusions: These results support the feasibility of administering ibrutinib to pts with DLBCL throughout tisa-cel therapy. When given before apheresis, ibrutinib may improve CAR-T cell manufacturing, although further studies are needed to confirm this finding. Disclosures Chavez: AstraZeneca: Speakers Bureau; Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding; Bayer: Consultancy; BeiGene: Speakers Bureau; Karyopharm: Consultancy; Genentech: Speakers Bureau; AbbVie: Consultancy; Epizyme: Speakers Bureau; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Speakers Bureau; Verastem: Consultancy; Pfizer: Consultancy. Locke:Kite, a Gilead Company: Consultancy, Research Funding; Calibr: Consultancy; Celgene/Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; GammaDelta Therapeutics: Consultancy; Cellular Biomedicine Group: Other: Consultancy with grant options; Allogene: Consultancy; Wugen: Consultancy. Simon:Novartis: Current Employment. Lewandowski:Novartis Institutes for BioMedical Research: Current Employment. Awasthi:Novartis Institutes for BioMedical Research: Current Employment. Engels:Novartis Institutes for BioMedical Research: Current Employment. Georgala:Novartis Pharmaceuticals Corporation: Current Employment. Bondanza:Novartis Institutes for BioMedical Research: Current Employment. Schuster:AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria; Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding.

Background The thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients’ survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined. Methods We performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets. Results We show in vitro that IL-1α and IL-1β released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1β secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients. Conclusions Our findings indicate that tumor released IL-1α and IL-1β and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0521-4) contains supplementary material, which is available to authorized users.

The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (TSCM) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1+Eomes+T-bet−) BM-TSCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease., Allogeneic hematopoietic cell transplantation is the standard treatment of acute myeloid leukemia, but many patients relapse. Here the authors show increased markers of exhaustion and cancer antigen specificity within bone marrow-residing memory T cells precede and potentially predict the relapse.

The array of cellular players involved in the biology of HSCT clearly extends beyond HSC themselves and, in the case of transplantation from allogeneic sources, importantly includes cells of the innate and adaptive immune system. Historically, the discovery of the HLA system and the functional characterization of the different immune cell types had a transformational impact on our current understanding of the pathobiological sequelae of allo-HSCT (rejection, GVHD, the GVL effect). This body of knowledge coupled to the most recent exploit of biotechnology nowadays allows us to design strategies for in vivo stimulation or adoptive transfer of specific immune cell types with the potential to dramatically improve transplantation outcome.

Over the last 60 years, more than a million patients received hematopoietic cell transplantation. Having incorporated multiple changes in clinical practices, it remains a complex procedure facing a dual challenge: cure of the underlying disease and prevention of relapse while controlling potentially severe complications. Improved understanding of underlying biological processes resulted in the design of innovative therapies engineered from defined cell populations and testing of these therapies as addition or substitution at virtually every step of the procedure. This review provides an overview of these developments, many of them now applied outside the historical field of hematopoietic cell transplantation.

The cellular basis of cancer immune surveillance, already hypothesized in ancient times, was only demonstrated with the advent of HSCT. Indeed, the discovery of the nature of GVHD and its antileukemic effects (Weiden et al.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p

In the clinic, chimeric antigen receptor-modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell-mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies.

CD1c self-reactive T cells recognize a novel class of self-lipids that are accumulated on leukemia cells., T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c+ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c+ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.

Neuromyelitis optica is a rare neurological autoimmune disorder characterized by a poor prognosis. Immunosuppression can halt disease progression, but some patients are refractory to multiple treatments, experiencing frequent relapses with accumulating disability. Here we report on durable clinical remissions after allogeneic hematopoietic stem cell transplantation in 2 patients suffering from severe forms of the disease. Immunological data evidenced disappearance of the pathogenic antibodies and regeneration of a naive immune system of donor origin. These findings correlated with evident clinical and radiological improvement in both patients, warranting extended clinical trials to investigate this promising therapeutic option.

NA A major hurdle to the field of allogeneic hematopoietic stem cell transplantation (HSCT) is the lack of validated biomarkers of protective immunity against opportunistic infections, including CMV reactivation syndromes. This issue is particularly relevant to alternative-donor settings, for example, HLA-haploidentical HSCT (haplo-HSCT) and cord blood transplantation, where aggressive GvHD prophylaxis results in a profound and long-lasting state of immune incompetence. Despite the introduction of routine post-transplant viral monitoring and preemptive antiviral therapy, CMV reactivation syndromes remain a major cause of transplant related mortality. Accordingly, serological evidence of prior CMV infection is still one of the main negative prognostic factors in HSCT. Different studies have demonstrated that a faster recovery of CMV-specific T-cell responses associates with a reduced CMV reactivation incidence in HSCT from HLA-identical sibling and matched unrelated donors. Given its clinical and biological peculiarities, it is currently unknown whether these observations are directly translatable to the haplo-HSCT setting. In this study, while aiming to follow the recovery of CMV-specific T-cell responses after haplo-HSCT, we unexpectedly found that protective levels of CMV immunity were associated with a reduced incidence of severe infections overall.

Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM. ""Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem\\\/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3\\\/CD28 beads and interleukin (IL)-7\\\/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.""

T regulatory cells (Tregs) play a key role in modulating T cell responses. Clinical trials showed that Tregs modulate graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity (graft-versus-leukemia [GvL]) is largely unknown. Enforced interleukin-10 (IL-10) expression converts human CD4+ T cells into T regulatory type 1 (Tr1)-like (CD4IL-10) cells that suppress effector T cells in vitro and xenoGvHD in humanized mouse models. In the present study, we show that CD4IL-10 cells mediate anti-leukemic effects in vitro and in vivo in a human leukocyte antigen (HLA) class I-dependent but antigen-independent manner. The cytotoxicity mediated by CD4IL-10 cells is granzyme B (GzB) dependent, is specific for CD13+ target cells, and requires CD54 and CD112 expression on primary leukemic target blasts. CD4IL-10 cells adoptively transferred in humanized mouse models directly mediate anti-tumor and anti-leukemic effects. In addition, when co-transferred with peripheral blood mononuclear cells (PBMCs), CD4IL-10 cells contribute to the GvL activity but suppress xenoGvHD mediated by the PBMCs. These findings provide for the first time a strong rationale for CD4IL-10 cell immunotherapy to prevent GvHD and promote GvL in allo-HSCT for myeloid malignancies., Graphical Abstract, Tr1 cells are generated by overexpressing IL-10 in CD4+ T cells (CD4IL-10). In this issue of Molecular Therapy, Locafaro et al. (2017) show that CD4IL-10 cells kill myeloid leukemia in an HLA class I-dependent mechanism, mediate anti-tumor and anti-leukemic effects, and contribute to GvL while preventing GvHD in humanized mice.

Chimeric antigen receptor (CAR) T cell therapy, together with checkpoint inhibition, has been celebrated as a breakthrough technology due to the substantial benefit observed in clinical trials with patients suffering from relapsed or refractory B‐cell malignancies. In this review, we provide a comprehensive overview of the clinical trials performed so far worldwide and analyze parameters such as targeted antigen and indication, CAR molecular design, CAR T cell manufacturing, anti‐tumor activities, and related toxicities. More than 200 CAR T cell clinical trials have been initiated so far, most of which aim to treat lymphoma or leukemia patients using CD19‐specific CARs. An increasing number of studies address solid tumors as well. Notably, not all clinical trials conducted so far have shown promising results. Indeed, in a few patients CAR T cell therapy resulted in severe adverse events with fatal outcome. Of note, less than 10% of the ongoing CAR T cell clinical trials are performed in Europe. Taking lead from our analysis, we discuss the problems and general hurdles preventing efficient clinical development of CAR T cells as well as opportunities, with a special focus on the European stage.

T regulatory cells (Tregs) play a key role in modulating T cell responses. Clinical trials showed that Tregs modulate graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity (graft-versus-leukemia [GvL]) is largely unknown. Enforced interleukin-10 (IL-10) expression converts human CD4+ T cells into T regulatory type 1 (Tr1)-like (CD4IL-10) cells that suppress effector T cells in vitro and xenoGvHD in humanized mouse models. In the present study, we show that CD4IL-10 cells mediate anti-leukemic effects in vitro and in vivo in a human leukocyte antigen (HLA) class I-dependent but antigen-independent manner. The cytotoxicity mediated by CD4IL-10 cells is granzyme B (GzB) dependent, is specific for CD13+ target cells, and requires CD54 and CD112 expression on primary leukemic target blasts. CD4IL-10 cells adoptively transferred in humanized mouse models directly mediate anti-tumor and anti-leukemic effects. In addition, when co-transferred with peripheral blood mononuclear cells (PBMCs), CD4IL-10 cells contribute to the GvL activity but suppress xenoGvHD mediated by the PBMCs. These findings provide for the first time a strong rationale for CD4IL-10 cell immunotherapy to prevent GvHD and promote GvL in allo-HSCT for myeloid malignancies. Tr1 cells are generated by overexpressing IL-10 in CD4+ T cells (CD4IL-10). In this issue of Molecular Therapy, Locafaro et al. (2017) show that CD4IL-10 cells kill myeloid leukemia in an HLA class I-dependent mechanism, mediate anti-tumor and anti-leukemic effects, and contribute to GvL while preventing GvHD in humanized mice.

Infection-related mortality (IRM) is a substantial component of nonrelapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). No scores have been developed to predict IRM before transplantation. Pretransplantation clinical and biochemical data were collected from a study cohort of 607 adult patients undergoing allo-HSCT between January 2009 and February 2017. In a training set of 273 patients, multivariate analysis revealed that age >60 years (P = .003), cytomegalovirus host/donor serostatus different from negative/negative (P

// Sara Sandri 1,* , Francesco De Sanctis 1,* , Alessia Lamolinara 2 , Federico Boschi 3 , Ornella Poffe 1 , Rosalinda Trovato 1 , Alessandra Fiore 1 , Sara Sartori 1 , Andrea Sbarbati 4 , Attilio Bondanza 5 , Simone Cesaro 6 , Mauro Krampera 7 , Maria T. Scupoli 7,8 , Michael I. Nishimura 9 , Manuela Iezzi 2 , Silvia Sartoris 1 , Vincenzo Bronte 1 and Stefano Ugel 1 1 Department of Medicine, University of Verona, Section of Immunology, Verona, Italy 2 Department of Medicine and Aging Science, Center of Excellence on Aging and Translational Medicine (CeSi-Met), G. D’Annunzio University, Chieti-Pescara, Italy 3 Department of Computer Science, University of Verona, Verona, Italy 4 Department of Neurological and Movement Sciences, University of Verona, Verona, Italy 5 Innovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Vita-Salute San Raffaele University, Milano, Italy 6 Department of Pediatric Haematology Oncology, University of Verona, Verona, Italy 7 Department of Medicine, University of Verona, Section of Haematology, Verona, Italy 8 University of Verona, Interdepartmental Laboratory for Medical Research (LURM), Verona, Italy 9 Department of Surgery, Loyola University Medical Center, Maywood, IL, United States * These authors have contributed equally to this work Correspondence to: Stefano Ugel, email: // Keywords : acute myeloid leukaemia (AML), B-cell acute lymphoblastic leukaemia (B-ALL), telomerase (TERT), TCR-redirected T-cells, adoptive cell therapy (ACT) Received : November 08, 2016 Accepted : May 12, 2017 Published : May 23, 2017 Abstract Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment.

Hematopoietic stem cell transplantation (HSCT) from an allogeneic donor is an effective form of cancer immunotherapy, especially for acute leukemias. HSCT is however frequently complicated by the occurrence of graft-versus-host disease (GVHD). Immunocompromised mice infused with human T cells often develop a clinical syndrome resembling human GVHD (xenogeneic or X-GVHD). Herein, we describe a method for inducing X-GVHD in a highly reproducible manner. Given the human nature of immune effectors, this xenogeneic model can be routinely adopted for screening the efficacy of new treatments for GVHD.

The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+-cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31(+) recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution. (Blood. 2012;120(9):1820-1830) The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially-incompatible Hematopoietic Stem Cell Transplantation (HSCT). In the TK007 clinical trial 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the Herpes Simplex Virus Thymidine Kinase suicide gene (TK(pos) cells). After a first wave of circulating TK(pos) cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naïve lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK(pos) cell engraftment. Accordingly, after the infusions we documented an increase in circulating T cell Receptor Excision Circles and CD31+ recent thymic emigrants, and a substantial expansion of the active thymic tissue at chest tomography scans. Interestingly, a peak in the serum level of interleukin-7 was observed after each infusion of TK(pos) cells, anticipating the appearance of newly generated T cells. Taken together, our data show that the infusion of genetically modified donor T cells after transplantation can drive the recovery of thymic activity in adults, leading to immune reconstitution.

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR beta- and alpha-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy—that is, the genetic induction of a conditional suicide phenotype into donor T cells—allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk+ T cells contained HA-1– and H-Y–specific CD8+ cytotoxic T cells (CTL) precursors. Thymidine kinase–positive HA-1– and H-Y–specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Chimeric antigen receptors (CARs) are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb) with the signal transduction machinery of the T-cell receptor (TCR). The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs) allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT), wherein allorecognition causes both the graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD). Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

Background. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML); nonetheless, relapse remains the major cause of death after such therapeutic option. Patients and Methods . We investigated the expression of Inhibitory Receptors (IR; i.e. PD-1, CTLA-4, TIM-3, LAG-3 and KLRG1) on different T-cell subsets infiltrating the bone marrow (BM) of 8 healthy donors (HD) and 32 allogeneic HSCT recipients diagnosed with Acute Myeloid Leukemia, collected at relapse (median 251 days) or at complete remission (CR) 1 year after HSCT. Inclusion criteria were: a diagnosis of acute myeloid leukemia or myelodysplastic syndrome, a relapse-free survival of at least 4 months after allogenein HSCT, absence of active GvHD, CMV infections or other complications at the time of sampling. Samples were analysed by multi-parametric flow cytometry for the expression of inhibitory receptors on T-cell subsets and the results were validated with BH-SNE, an unbiased dimensionality reduction algorithm. We exploited HLA-mimicking fluorescent molecules loaded with a specific epitope to screen anti-tumour and anti-viral T cells whereas the T-cell receptor repertoire was assessed by TRAC and TRBC RNA sequencing and the relative frequency of each T-cell receptor calculated. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified. Results. After Haploidentical-HSCT PD-1, CTLA-4, 2B4 and Tim-3 were expressed at higher percentage when compared to HD, independently from the clinical outcome. In contrast, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To gain insights on the inhibited T-cell subpopulation identified in the BM of relapsing patients, we separately profiled the different T-cell memory subsets: in both HD and CR patients the IR expression was confined to effector memory and effectors whereas at relapse PD-1, 2B4, KLRG1 and Tim-3 were also expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p Interestingly, the TCR repertoire of BM-infiltrating T cells at relapse displayed a restricted clonality, suggesting that immune inhibitory signals are active on discrete and specific T-cell clones. To gain further insights on such clones, we assessed the IR expression profile on CD8 T cells specific for viral (CMV) and tumor-associated antigens (including peptides from WT1, EZH2 and PRAME). We observed a higher IR expression and co-expression on tumor-specific T cells when compared to viral-specific CD8 cells, particularly in case of patients who experienced post-transplant relapse. In accordance, IRpos sorted T cells harvested from relapsing patients showed a restricted TCR repertoire and, when challenged with autologous leukemic blasts, proved enriched in leukemic specificities as shown by higher expression of the activation marker HLA-DR (p Conclusion. These results highlight a wide, yet reversible, immunological dysfunction likely mediated by AML blasts in the BM of patients relapsing after allogeneic HSCT, that is particularly evident on memory T cells specific for tumor antigens. This suggest and open new therapeutic opportunities for AML. Figure. Figure. Disclosures Bondanza: Novartis: Employment. Vago:GENDX: Research Funding; Moderna TX: Research Funding. Bonini:Intellia Therapeutics: Research Funding.

Background: The adoptive transfer of CAR-T cells have shown impressive results against B-cell malignancies, but still limited efficacy against solid tumors. The discovery of the key factors regulating the activity of CAR-T cells is required to improve their antitumor potency and modulate toxicities. Since solid tumors display a wide range of glycosylation alterations, including increased N-glycan branching, we hypothesized that peptidic epitopes may be masked by glycans from CAR-T cell targeting, especially in richly glycosylated proteins. Results: To investigate if sugar chains may be sterically hulking for CAR-T cell targeting, we generated N-glycosylation-defective pancreatic tumor cell lines. This aim has been achieved by knocking-out the expression of the glycosyltransferase Mgat5, a key enzyme involved in the process of N-glycan branching, using the CRISPR-Cas9 technology. As model antigens for CAR targeting, we focused on CD44v6 and CEACAM-5 (CEA) since they are both heavily glycosylated proteins over-expressed on a wide variety of solid tumors, including pancreatic adenocarcinoma. Strikingly, the impairment of N-glycosylation resulted in a dramatic increase of tumor targeting by both CD44v6 (4-fold, p Conclusions: Our results indicate that i) the glycosylation status of tumor cells regulates the efficacy of CAR-T cells, especially when targeting highly glycosylated antigens, and ii) combining CAR-T cells with the de-glycosylation agent 2DG, which preferentially accumulates in tumor masses, may pave the way for a successful immunotherapy against solid tumors. Disclosures Bonini: Intellia Therapeutics: Research Funding. Bondanza:Novartis: Employment.

Background: Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. Methods: We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. Results: In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donor's haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. Conclusions: After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor's antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse. N Engl J Med 2009;361:478-88. RI Cosentino, Cristian/F-2604-2010 Background: Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. Methods: We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. Results: In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donor's haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. Conclusions: After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor's antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse. N Engl J Med 2009;361:478-88. ACKGROUND: Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. METHODS: We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. RESULTS: In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donor's haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. CONCLUSIONS: After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor's antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse.

Carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections are an emerging cause of death after hematopoietic stem cell transplantation (HSCT). In allogeneic transplants, mortality rate may rise up to 60%. We retrospectively evaluated 540 patients receiving a transplant from an auto- or an allogeneic source between January 2011 and October 2015. After an Institutional increase in the prevalence of KPC-Kp bloodstream infections (BSI) in June 2012, from July 2012, 366 consecutive patients received the following preventive measures: (i) weekly rectal swabs for surveillance; (ii) contact precautions in carriers (iii) early-targeted therapy in neutropenic febrile carriers. Molecular typing identified KPC-Kp clone ST512 as the main clone responsible for colonization, BSI and outbreaks. After the introduction of these preventive measures, the cumulative incidence of KPC-Kp BSI (P=0.01) and septic shocks (P=0.01) at 1 year after HSCT was significantly reduced. KPC-Kp infection-mortality dropped from 62.5% (pre-intervention) to 16.6% (post-intervention). Day 100 transplant-related mortality and KPC-Kp infection-related mortality after allogeneic HSCT were reduced from 22% to 10% (P=0.001) and from 4% to 1% (P=0.04), respectively. None of the pre-HSCT carriers was excluded from transplant. These results suggest that active surveillance, contact precautions and early-targeted therapies, may efficiently control KPC-Kp spread and related mortality even after allogeneic HSCT.

Hematopoietic stem cell transplantation (HSCT) from an allogeneic donor is an effective form of cancer immunotherapy, especially for acute leukemias. HSCT is however frequently complicated by the occurrence of graft-versus-host disease (GVHD). Immunocompromised mice infused with human T cells often develop a clinical syndrome resembling human GVHD (xenogeneic or X-GVHD). Herein, we describe a method for inducing X-GVHD in a highly reproducible manner. Given the human nature of immune effectors, this xenogeneic model can be routinely adopted for screening the efficacy of new treatments for GVHD.