Cell Therapy In Patients Research Articles

Safety and Efficacy of CD19 CAR T-Cells for Pediatric Relapsed Acute Lymphoblastic Leukemia with Active CNS Involvement

Background: CAR T cells targeting CD19 are approved for patients with relapsed or refractory acute lymphoblastic leukemia (ALL), failing two or more prior therapies. The central nervous system (CNS) is a known sanctuary site of ALL. Despite observations of CAR T-cells trafficking to the CNS in patients, most clinical trials excluded patients with active CNS leukemia, partially for concerns of neurotoxicity. Methods: We conducted an international retrospective study of patients who were referred to CAR T-cell therapy for relapsed ALL with CNS involvement. Results: We reviewed data of 27 patients (16 males and 11 females) from 8 centers who were treated with CD19 CAR T-cells for ALL relapse involving the CNS. Fifteen patients had a prior bone marrow transplantation, and in seven total body irradiation was given. Four additional patients received previous cranial radiation. At time of CAR T-cell order or clinical trial inclusion, ten patients had an isolated CNS relapse, and 17 had a combined CNS and marrow relapse. The median number of cells in the CSF of patients at enrollment was 65 (range 0-5670), and 16 of 21 patients who had imaging performed showed leukemia-associated changes in brain MRI. All patients received bridging chemotherapy between enrolment and lymphodepletion, including intrathecal chemotherapy alone or in combination with systemic chemotherapy and/or radiation. At lymphodepletion, 15 patients had cleared CNS disease while 12 had active CNS blasts or imaging lesions. Following lymphodepletion with fludarabine and cyclophosphamide, patients received 2nd-generation CAR T cells: 15 patients treated with a CD19-4-1BB CAR and 12 with a CD19-CD28 CAR. Cytokine release syndrome (CRS) occurred in 20 patients: 12 patients (100%) treated with CD19-CD28 CARs and 8 with CD19-4-1BB CARs (53%, p=0.006). Immune-effector cell neurotoxicity (ICANS) was diagnosed in 11 patients, 9 of 12 following CD28-based CARs, and 3 of 15 following 41BB-based CARs (p=0.004). Patients with active CNS-disease prior to lymphodepletion also had a higher rate of ICANS (75% vs 20%, p=0.004) but not of CRS. One patient died of grade 5 ICANS on day +7 following CD28-based CARs for an isolated CNS relapse. Twenty-four of 26 evaluable patients (92%) had a complete remission following CAR T-cells. Nine patients were further referred to an allogeneic transplant, all following CD28-based CARs. Overall, six patients had relapsed - in four relapse involved bone marrow, and in two the CNS. Conclusions: In this cohort of patients with active CNS disease treated with CD19 CAR T-cells, we report a high remission rate in previously heavily pre-treated patients. Toxicities are significant, and associated mostly with the costimulatory domain of the CAR (CD28>41BB) and the presence of active CNS disease at lymphodepletion. This may be informative when designing future clinical studies of CAR T cell therapy for patients with CNS manifestations of ALL. Disclosures Jacoby: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lonza: Membership on an entity's Board of Directors or advisory committees. Ghorashian:Novartis: Honoraria; UCLB: Patents & Royalties; Amgen: Honoraria. De Moerloose:Novartis: Consultancy. Rossig:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees; Genetech: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria. von Stackelberg:Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau. Bourquin:Servier: Other: Travel Support. OffLabel Disclosure: We describe the use of commercial and investigational CAR T-cell products

Enhancing the Therapeutic Efficacy of Bone Marrow-Derived Mononuclear Cells with Growth Factor-Expressing Mesenchymal Stem Cells for ALS in Mice.

SummarySeveral treatments have been attempted in amyotrophic lateral sclerosis (ALS) animal models and patients. Recently, transplantation of bone marrow-derived mononuclear cells (MNCs) was investigated as a regenerative therapy for ALS, but satisfactory treatments remain to be established. To develop an effective treatment, we focused on mesenchymal stem cells (MSCs) expressing hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor using human artificial chromosome vector (HAC-MSCs). Here, we demonstrated the transplantation of MNCs with HAC-MSCs in ALS mice. As per our results, the progression of motor dysfunction was significantly delayed, and their survival was prolonged dramatically. Additional analysis revealed preservation of motor neurons, suppression of gliosis, engraftment of numerous MNCs, and elevated chemotaxis-related cytokines in the spinal cord of treated mice. Therefore, growth factor-expressing MSCs enhance the therapeutic effects of bone marrow-derived MNCs for ALS and have a high potential as a novel cell therapy for patients with ALS.

SAFEty and efficacy of autologous bone marrow – derived mononuclear stem cell therapy in patients with severe chronic ischemic Heart Failure – rationale and design

The main aim of this study is to explore the safety and efficacy of autologous bone marrow – derived mononuclear stem cell therapy in patients with severe chronic ischemic heart failure on short to medium-term (1-3 months). The SAFE-HF trial is a prospective, single center, two-arm, controlled trial with blind evaluation of endpoints. Target population will consist of patients with heart failure due to prior extensive myocardial infarction and left ventricle ejection fraction of less than 35% – namely the population with the highest risk of adverse events and the highest mortality. All patients will be treated with the state of the art reperfusion – primary percutaneous coronary intervention and also with guidelinebased optimal medical therapy. The stem cell injection will be performed 1 to 3 months after the acute event; evolution of stem cell treated patients will be compared with matched control–cases. Comprehensive assessment of outcomes will be done at 1 and 3 months follow-up; serum biomarkers, cardiac structural and physiological parameters, functional capacity and health-related quality of life will be evaluated. This pilot study possibly will serve as a foundation for designing future trials.

Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

Efficacy and Safety of Stem Cell Therapy for T1DM: An Updated Systematic Review and Meta-Analysis.

Background The long-term insulin therapy for type 1 diabetes mellitus (T1DM) fails to achieve optimal glycemic control and avoid adverse events simultaneously. Stem cells have unique immunomodulatory capacities and have been considered as a promising interventional strategy for T1DM. Stem cell therapy in T1DM has been tried in many studies. However, the results were controversial. We thus performed a meta-analysis to update the efficacy and safety of stem cell therapy in patients with T1DM. Methods We systematically searched the Medline, EMBASE, Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, Web of Science, Wan Fang Data, China National Knowledge Infrastructure, VIP database, and the Chinese Biomedical Literature Database (SinoMed) for relevant studies published before March 19, 2019. The outcomes included parameters for glycemic control (i.e., glycosylated hemoglobin (HbA1c) levels and insulin dosages), β cell function (i.e., fasting C-peptide levels and area-under-curve of C-peptide concentration (AUCC)), and relative risk of adverse events. Statistical analysis was conducted by using RevMan 5.3 and Stata 12.0. Results Five randomized controlled trials (RCTs) and eight nonrandomized concurrent control trials (NRCCTs) with a total of 396 individuals were finally included into the meta-analysis. Among RCTs, stem cell therapy could significantly reduce HbA1c levels (MD = −1.20, 95% CI -1.91 to -0.49, P = 0.0009) and increase fasting C-peptide levels (MD = 0.25, 95% CI 0.04 to 0.45, P = 0.02) and AUCC (SMD = 0.66, 95% CI 0.13 to 1.18, P = 0.01). Stem cell therapy could also reduce insulin dosages (SMD = −2.65, 95% CI -4.86 to -0.45, P = 0.02) at 6 months after treatment. NRCCTs also had consistent results. Furthermore, RCTs showed stem cell therapy did not increase relative risk of gastrointestinal symptom (RR = 0.69, 95% CI 0.14 to 3.28, P = 0.64) and infection (RR = 0.97, 95% CI 0.40 to 2.34, P = 0.95). However, NRCCTs showed stem cell therapy increased relative risk of gastrointestinal symptom (RR = 44.49, 95% CI 9.20 to 215.18, P < 0.00001). Conclusion Stem cell therapy for T1DM may improve glycemic control and β cell function without increasing the risk of serious adverse events. Stem cell therapy may also have a short-term (3-6 months) effect on reducing insulin dosages.

Larger End-Diastolic Volume Associates With Response to Cell Therapy in Patients With Nonischemic Dilated Cardiomyopathy

ObjectiveTo investigate the association of left ventricular end-diastolic volume (LVEDV) and the response to cell therapy in patients with nonischemic dilated cardiomyopathy (NICM). Patients and MethodsFive-year registry data from 133 consecutive patients with NICM who underwent CD34+ cell treatment were analyzed. All patients received granulocyte-colony stimulating factor; CD34+ cells were collected by apheresis and delivered by transendocardial injections. Patients with baseline LVEDV less than 200 mL (group A; n=72) and patients with LVEDV 200 to 370 mL (group B; n=54) were included. Patients with LVEDV greater than 370 mL were excluded (n=7). Favorable ejection fraction response was pre-defined by improvement in left ventricular ejection fraction (LVEF) greater than or equal to 5% at 1 y post-cell therapy. ResultsAt baseline, groups A and B were comparable with regards to age (52±11 y in group A vs 53±10 y in group B; P=.95), sex (male: 79% vs 83%, respectively; P=.55), creatinine (1.07±0.28 mg/dL vs 1.03±0.21 mg/dL, respectively; P=.21), or N-terminal probrain natriuretic peptide (1454±1658 pg/mL vs 1589±1338 pg/mL, respectively; P=.80). Baseline LVEF was higher in group A (32.8±8.7%) than in group B (30.2±8.7%; P=.03). During follow-up, there were four deaths in group A (5.6%), and 2 in group B (3.7%, P=.63). At 1-year post-cell therapy, LVEDV decreased significantly in group B (-56±30 mL; P=.003), but not in group A (+12±97 mL; P=.13). On multivariate analysis, baseline LVEDV was an independent correlate of favorable response in LVEF to therapy (P=.02). ConclusionLarger LVEDV was associated with more pronounced increase in LVEF after transendocardial CD34+ cell therapy in NICM patients, informing target individuals with the highest likelihood of regenerative response. Trial Registrationclinicaltrials.gov identifier: NCT02445534

A Phase I Study of Allogeneic Mesenchymal Stem Cell Therapy in Patients with Heart Failure Secondary to Anthracycline-induced Cardiomyopathy: The Cctrn Stem Cell Injection in Cancer Survivors (Seneca) Trial

Background Anthracycline-induced cardiomyopathy (AIC) can be irreversible with a poor prognosis, and disproportionately affects women and young adults. Administration of allogeneic bone marrow-derived mesenchymal stromal cells (allo-MSCs) is a promising approach to heart failure treatment. Allo-MSCs are known to favorably affect fibrosis, apoptosis, and inflammation, which are key features of AIC. Methods This phase I study evaluated 37 adult cancer survivors (68% female) with left ventricular dysfunction secondary to chronic AIC. The study included a six-subject open-label, lead-in phase followed by 31 subjects randomized 1:1 to receive 1 × 108 allo-MSCs or vehicle via 20 transendocardial injections. Subjects were followed for 1 year to assess safety and feasibility (Fisher's test). Secondary efficacy measures included cardiac function/structure measured by magnetic resonance imaging (MRI), functional capacity, quality of life (MLHFQ), and biomarkers. These were assessed by ANCOVA adjusting for baseline values. Results Ninety-seven percent of subjects underwent successful study product injections, all allo-MSC-assigned subjects received the target dose. Follow-up visits were well attended (92%) with successful collection of endpoints in 94% at the 1-year visit. Although 58% of subjects had non-MRI compatible devices, MRI endpoints were successfully collected in 84% of subjects imaged at 1 year. No new tumors were reported. There were no significant differences between allo-MSC and vehicle groups with regard to clinical outcomes including major adverse cardiac events, other significant adverse events, and days alive and out of the hospital. Ten of the 13 efficacy measures numerically favored the allo-MSC group including a lower MLHFQ score (p = .048). Conclusion In this first-in-human study of cell therapy in patients with AIC, transendocardial administration of allo-MSCs appears safe and feasible and MRI was successfully performed in the majority of patients with devices. This study provides crucial information for designing Phase II trials aimed at assessing efficacy of cell therapy in patients with AIC.

Allogeneic Mesenchymal Cell Therapy in Anthracycline-Induced Cardiomyopathy Heart Failure Patients: The CCTRN SENECA Trial

BackgroundAnthracycline-induced cardiomyopathy (AIC) may be irreversible with a poor prognosis, disproportionately affecting women and young adults. Administration of allogeneic bone marrow–derived mesenchymal stromal cells (allo-MSCs) is a promising approach to heart failure (HF) treatment. ObjectivesSENECA (Stem Cell Injection in Cancer Survivors) was a phase 1 study of allo-MSCs in AIC. MethodsCancer survivors with chronic AIC (mean age 56.6 years; 68% women; NT-proBNP 1,426 pg/ml; 6 enrolled in an open-label, lead-in phase and 31 subjects randomized 1:1) received 1 × 108 allo-MSCs or vehicle transendocardially. Primary objectives were safety and feasibility. Secondary efficacy measures included cardiac function and structure measured by cardiac magnetic resonance imaging (CMR), functional capacity, quality of life (Minnesota Living with Heart Failure Questionnaire), and biomarkers. ResultsA total of 97% of subjects underwent successful study product injections; all allo-MSC–assigned subjects received the target dose of cells. Follow-up visits were well-attended (92%) with successful collection of endpoints in 94% at the 1-year visit. Although 58% of subjects had non-CMR compatible devices, CMR endpoints were successfully collected in 84% of subjects imaged at 1 year. No new tumors were reported. There were no significant differences between allo-MSC and vehicle groups with regard to clinical outcomes. Secondary measures included 6-min walk test (p = 0.056) and Minnesota Living with Heart Failure Questionnaire score (p = 0.048), which tended to favor the allo-MSC group. ConclusionsIn this first-in-human study of cell therapy in patients with AIC, transendocardial administration of allo-MSCs appears safe and feasible, and CMR was successfully performed in the majority of the HF patients with devices. This study lays the groundwork for phase 2 trials aimed at assessing efficacy of cell therapy in patients with AIC. (Stem Cell Injection in Cancer Survivors [SENECA]; NCT02509156)

Autologous bone-marrow mononuclear stem cell therapy in patients with stroke: a meta-analysis of comparative studies

BackgroundThere is a need to promote recovery after stroke with novel therapeutic interventions. Of them, bone-marrow mononuclear cell (BM-MNC) therapy offers promising outcomes in preclinical and clinical models.AimsTo investigate the efficacy and safety of BM-MNCs versus traditional medical care of stroke patients.Summary of reviewA meta-analysis was conducted involving controlled prospective studies and randomized clinical trials (RCTs) which investigated the changes in the scores of neurological functions (the National Institutes of Health Stroke Scale [NIHSS]), the indices of functional recovery (the Barthel Index [BI] and the modified Rankin scale [mRS]) at 3 and 6 month post-transplantation. A total of nine studies (five RCTs) recruited 469 stroke patients (65.5% males, 49.25% received the intervention). There were no significant differences in NIHSS, BI, or mRS scores after 3 months of follow-up. However, the BI indices of BM-MNCs-receiving patients improved significantly after 6 months (standardized mean difference = 1.17, 95% confidence interval, 0.23 to 2.10, P = 0.01) as compared to traditional treatment. The risk of mortality and adverse events and the proportion of patients with favorable outcomes (mRS ≤ 3) were similar in both groups.ConclusionBoth the BM-MNCs and medical stroke treatment have similar outcomes in terms of safety and short-term efficacy, while the effect of therapy is significant only after 6 months. More well-designed, large sized RCTs are needed to confirm the efficacy of stem cell therapy over long periods of follow-up.

Nanotechnology‐Based CAR‐T Strategies for Improving Efficacy and Safety of Tumor Immunotherapy

AbstractTherapeutic responses to chimeric antigen receptor (CAR) T cell therapy in patients with limited treatment options have been appealing in several clinical trials. However, the efficacy of CAR‐T therapy has been challenged by several obstacles when treating patients with solid tumors, such as severe toxicities, restricted access to tumor sites, suboptimal therapeutic persistence, and manufacturing issues. Nanotechnology has the advantages of protecting CAR‐T cells from being suppressed by tumor microenvironment (TME) and favorably adapting immune‐modulating drugs’ pharmacokinetics by modifying their spatiotemporal release profiles. Loaded with nanoparticles and packed onto CAR‐T cells, immune‐modulating drugs can be delivered to the tumor site and lymph node more efficiently, stimulating the expansion and activity of CAR‐T cells. To protect normal tissues from the nonspecific toxicity of the activated CAR‐T cells, formulations are optimized toward tumor targeting delivery of nanotechnology. This review summarizes the nanotechnology strategies to improve the safety and efficacy of CAR‐T therapy. In addition, the unsolved problems existing in the clinical application of CAR‐T therapy are focused on, where study and exploration by the way of nanotechnology is needed.

BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

CAR T cell therapies for patients with multiple myeloma.

Despite several therapeutic advances over the past decade, multiple myeloma (MM) remains largely incurable, indicating a need for new treatment approaches. Chimeric antigen receptor (CAR) T cell therapy works by mechanisms distinct from those of other MM therapies and involves the modification of patient or donor T cells to target specific cell-surface antigens. B cell maturation antigen (BCMA) is expressed only on plasma cells, a small subset of B cells and MM cells, which makes it a suitable target antigen for such therapies. At the time of writing, data from >20 clinical trials involving anti-BCMA CAR T cells have demonstrated that patients with relapsed and/or refractory MM can achieve objective responses. These early investigations have been instrumental in demonstrating short-term safety and efficacy; however, most patients do not have disease remission lasting >18 months. Attempts to reduce or delay the onset of relapsed disease are underway and include identifying additional CAR T cell target antigens and methods of enhancing BCMA expression on MM cells. Engineering CAR T cells to enhance both the activity and safety of treatment continues to be a promising avenue for improvement. In this Review we summarize data from clinical trials that have been carried out to date, describe novel antigens that could be targeted in the future, and highlight potential future innovations that could enhance the efficacy and/or reduce the toxicities associated with CAR T cell therapies.

Thioredoxin activity confers resistance against oxidative stress in tumor-infiltrating NK cells.

To improve the clinical outcome of adoptive NK cell therapy in patients with solid tumors, NK cells need to persist within the tumor microenvironment (TME) in which the abundance of ROS could dampen antitumor immune responses. In the present study, we demonstrated that IL-15-primed NK cells acquired resistance against oxidative stress through the thioredoxin system activated by mTOR. Mechanistically, the activation of thioredoxin showed dependence on localization of thioredoxin-interacting protein. We show that NK cells residing in the tumor core expressed higher thiol densities that could aid in protecting other lymphocytes against ROS within the TME. Furthermore, the prognostic value of IL15 and the NK cell gene signature in tumors may be influenced by tobacco smoking history in patients with non-small-cell lung cancer (NSCLC). Collectively, the levels of reducing antioxidants in NK cells may not only predict better tumor penetrance but potentially even the immune therapy response.

Human umbilical cord-derived mesenchymal stem cell therapy in patients with COVID-19: a phase 1 clinical trial

No effective drug treatments are available for coronavirus disease 2019 (COVID-19). Host-directed therapies targeting the underlying aberrant immune responses leading to pulmonary tissue damage, death, or long-term functional disability in survivors require clinical evaluation. We performed a parallel assigned controlled, non-randomized, phase 1 clinical trial to evaluate the safety of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) infusions in the treatment of patients with moderate and severe COVID-19 pulmonary disease. The study enrolled 18 hospitalized patients with COVID-19 (n = 9 for each group). The treatment group received three cycles of intravenous infusion of UC-MSCs (3 × 107 cells per infusion) on days 0, 3, and 6. Both groups received standard COVID-treatment regimens. Adverse events, duration of clinical symptoms, laboratory parameters, length of hospitalization, serial chest computed tomography (CT) images, the PaO2/FiO2 ratio, dynamics of cytokines, and IgG and IgM anti-SARS-CoV-2 antibodies were analyzed. No serious UC-MSCs infusion-associated adverse events were observed. Two patients receiving UC-MSCs developed transient facial flushing and fever, and one patient developed transient hypoxia at 12 h post UC-MSCs transfusion. Mechanical ventilation was required in one patient in the treatment group compared with four in the control group. All patients recovered and were discharged. Our data show that intravenous UC-MSCs infusion in patients with moderate and severe COVID-19 is safe and well tolerated. Phase 2/3 randomized, controlled, double-blinded trials with long-term follow-up are needed to evaluate the therapeutic use of UC-MSCs to reduce deaths and improve long-term treatment outcomes in patients with serious COVID-19.

Safety and Efficacy of Stem Cell Therapy in Patients With Ischemic Stroke.

Stem cell therapy is emerging as a promising treatment strategy to treat patients with stroke. While there are established modes of treatment for stroke patients such as thrombolysis and endovascular intervention, most of the stroke patients frequently end up with major residual deficits or even death. The use of stem cells to treat stroke has been found to be beneficial in the animal models but strict evidence for the same in humans is still lacking. We reviewed 13 clinical trials of stem cell therapy in stroke patients conducted between 2014 and 2020 based on the search using the database PubMed, and the clinical trial registry (www.clinicaltrials.gov). We aimed to assess the safety and efficacy of stem cell treatment in stroke patients who participated in the trials. Quality assessment of the clinical trials revealed a sub-optimal score. We found mixed results regarding the efficacy of stem cells in the treatment of ischemic stroke although we could not do a quantitative analysis of the effect outcomes. Assessment for safety revealed promising results as there were only minor side effects related to cell therapy. Although stem cell therapy seems to be a promising strategy to treat stroke patients in the future, we concluded that the field needs more evidence regarding the safety and efficacy of the use of stem cells in stroke patients before we use them in the clinic.

Abstract 2192: Non-viral genome engineering method allows highly efficient, single-step removal and precise insertion of multiple large genes

Abstract Recent advances in gene editing have enabled the targeted engineering of primary cells to insert entire transgenes without the use of viral vectors. Using these methods, novel genes can be inserted in a seamless manner into the specified genomic locus, realizing the goal of precise targeted genomic modifications. Non-viral modification of primary cells holds potential to significantly reduce costs and time needed for the generation of therapeutic cell products. It has also allowed the generation of personalized cell therapies involving patient-specific manufacturing of DNA constructs, such as for the developed of patient-derived, neoepitope-specific TCR-T cells. Proof-of-concept experiments have shown that non-viral genome engineering methods can integrate small genetic elements such as GFP, while the delivery of larger therapeutically relevant payloads at high efficiency has previously been challenging. Here we describe a proprietary single-step, DNA-mediated method developed for seamlessly engineering fully natural neoepitope-specific TCRs (neoTCR) into the endogenous TCR locus of primary human T cells at high efficiencies (i.e. &amp;gt;50% gene editing efficiency). This method allows the delivery of the two genes comprising the neoTCR without the need for selection necessitated by less efficient approaches. To further evaluate this approach, fresh human donor T cells were engineered to express the patient-specific neoTCR plus two additional gene products, CD8α and CD8β (i.e. precise genome engineering of four ectopic genes). The data shows that these modifications were made at high efficiency, resulting in fully functional CD8 and CD4 T cells. Surface expression of CD8 coreceptor together with the neoE-targeted TCR increased T cell signaling sensitivity of the engineered neoTCR-T cells by 10-100 fold. The potential for off-target cleavage or unexpected genomic outcomes was assessed using multiple methods, including a newly developed primary T cell GUIDE-seq assay. Despite multi-locus editing, no evidence of off-target insertion or unexpected genomic rearrangements were observed. In summary, these results demonstrate the applicability of a single step, highly efficient method for manufacturing fresh human T cells into neoTCR-T cell therapies, engineered with multiple functionalities. This proprietary precision genome engineering technology supports the on-going Phase 1 clinical trial of personalized autologous, NeoTCR-P1 engineered T cell therapies for patients with solid tumors (NCT03970382). Citation Format: Kyle Jacoby, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Alex Franzusoff, Stefanie Mandl. Non-viral genome engineering method allows highly efficient, single-step removal and precise insertion of multiple large genes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2192.

Abstract 3253: A high-throughput platform to produce neoE-HLA libraries for capturing neoE-specific T cells from the peripheral blood of patients with solid tumors

Abstract A critical step in the development of personalized neoTCR-T cell therapies is operational selection of tumor-exclusive neoantigen peptide targets (neoepitopes or neoE) presented by each person's own HLA proteins (neoE-HLA targets). We have developed a high-throughput, automated process for the cloning, expression and purification of soluble proteins comprising neoE peptide with beta2m and the HLA heavy chain fused into a single polypeptide (neoE-HLA proteins). Of the 13 thousand HLA alleles in the human population, with 6 HLA alleles expressed in each person, our HLA catalog enables rapid production of neoE-HLA protein candidate libraries, representing &amp;gt;4 of 6 HLAs for ≥95% of all patients, regardless of ethnicity. We apply our proprietary bioinformatics pipeline to predict and prioritize tumor-exclusive neoE-HLA candidates for each patient. We then generate neoE-HLA libraries in single production runs for up to several hundred neoE-HLA candidates per patient. These soluble neoE-HLA proteins are then assembled into barcoded snare multimers. These barcoded snare libraries are used to interrogate matched PBMCs from that patient to capture rare (&amp;gt;1 per 106) to abundant antigen-experienced CD8 T cells which specifically bind the cognate neoE-HLA tumor targets, using the imPACT Isolation Technology®. This platform is capable of producing barcoded snares predicted to represent high affinity (≤500 nM) and low affinity (&amp;gt;500nM) neoE binding to their cognate HLAs. These capabilities permit deep and broad interrogation of the tumor-targeted neoE-specific T cell repertoire in the blood of patients with solid cancers. Data shown here demonstrate that &amp;gt;85% of patients with different cancer types, indeed, harbor a surprising repertoire of tumor-targeted neoE-specific T cells. These antigen-experienced T cells reveal the pre-existing immune response to each person's cancer, and how that repertoire evolves in longitudinal analyses of patients that respond/do not respond to their cancer therapies. In summary, PACTs proprietary neoE-HLA prediction pipeline and the proprietary high-throughput neoE-HLA protein production platform enables the discovery of tumor-targeted, neoE-specific T cells from patients. We leverage the neoTCRs cloned from these captured T cells to engineer fresh T cells from the patient into a full dose of tumor-targeted personalized neoTCR-T cells (NeoTCR-P1 T cell therapy). This fully personalized NeoTCR-P1 cell therapy is manufactured in fully enclosed, automatable systems for each individual patient with solid tumors. These breakthrough technologies support the on-going Phase 1 clinical trial of personalized engineered autologous NeoTCR-P1 T cell therapies for patients with six different solid tumor types (NCT03970382). Citation Format: Olivier Dalmas, Zheng Pan, Christine Shieh, Allison Xu, Jason Kwa, Katharine Heeringa, Yan Ma, John Collins, Duo An, Songming Peng, Alex Franzusoff. A high-throughput platform to produce neoE-HLA libraries for capturing neoE-specific T cells from the peripheral blood of patients with solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3253.

Stem Cell Therapy for Chronic and Advanced Heart Failure.

The purpose of this review is to discuss recent advances in the field of cell therapy in patients with heart failure with reduced ejection fraction (HFrEF) of ischemic (iCMP) and nonischemic (dCMP) etiology, heart failure with preserved ejection fraction (HFpEF), and in advanced heart failure patients undergoing mechanical circulatory support (LVAD). In HFrEF patients (iCMP and dCMP cohorts), autologous and/or allogeneic cell therapy was shown to improve myocardial performance, patients' functional capacity, and neurohumoral activation. In HFpEF patient population, the concept of cell therapy in novel and remains largely unexplored. However, initial data are very encouraging and suggest at least a similar benefit in improvements of myocardial performance (also diastolic function of the left ventricle), exercise capacity, and neurohumoral activation. Recently, cell therapy was explored in the sickest population of advanced heart failure patients undergoing LVAD support also showing a potential benefit in promoting myocardial reverse remodeling and recovery. In the past decade, several cell therapy-based clinical trials showed promising results in various chronic and advanced heart failure patient cohorts. Future cell treatment strategies should aim for more personalized therapeutic approaches by defining optimal stem cell type or their combination, dose, and delivery method for an individual patient adjusted for patient's age and stage/duration of heart failure.

The efficacy and safety of CAR-T cell therapy in patients with refractory ALL and concomitant HBV infection.

The efficacy and safety of CAR-T cell therapy in patients with refractory ALL and concomitant HBV infection.

Reduced graphene oxide facilitates biocompatibility of alginate for cardiac repair

The benefits of combined cell/material therapy appear promising for myocardial infarction treatment. The safety of alginate, along with its excellent biocompatibility and biodegradability, has been extensively investigated for cardiac tissue engineering. Among graphene-based nanomaterials, reduced graphene oxide has been considered as a promising candidate for cardiac treatment due to its unique physicochemical properties. In this study, the reduced graphene oxide incorporation effect within alginate hydrogels was investigated for cardiac repair application. Reduced graphene oxide reinforced alginate properties, resulting in an increase in gel stiffness. The cytocompatibility of the hydrogels prepared with human bone marrow–derived mesenchymal stem cells was assessed by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. Following reduced graphene oxide addition, alginate-reduced graphene oxide retained significantly higher cell viability compared to that of alginate and cells cultured on tissue culture plates. Acridine orange/propidium iodide staining was also used to identify both viable and necrotic human bone marrow–derived mesenchymal stem cells within the prepared hydrogels. After a 72-h culture, the percentage of viable cells was twice as much as those cultured on either alginate or tissue culture plate, reaching approximately 80%. Quantitative reverse transcription polymerase chain reaction analysis was performed to assess gene expression of neonatal rat cardiac cells encapsulated on hydrogels for TrpT-2, Conx43, and Actn4 after 7 days. The expression of all genes in alginate-reduced graphene oxide increased significantly compared to that in alginate or tissue culture plate. The results obtained confirmed that the presence of reduced graphene oxide, as an electro-active moiety within alginate, could tune the physicochemical properties of this material, providing a desirable electroactive hydrogel for stem cell therapy in patients with ischemic heart disease.