Met Release Criteria Research Articles

Final results of a phase I trial of HERV-E TCR transduced T cells for the treatment of HLA-A*11 patients with metastatic clear cell renal cell carcinoma (mccRCC).

435 Background: Human endogenous retrovirus type E (HERV-E) is specifically expressed in ccRCC providing a safe target for T cell-based therapies. We investigated T cells transduced with a TCR targeting HERV-E (HERV-E T cells) for the treatment of mccRCC. Methods: This first-in-human study assessed the safety & efficacy of escalating doses of HERV-E T cells and manufacturing/clinical endpoints correlative analysis. HLA-A*11+ mccRCC patients (pts) were treated with a conditioning regimen, infusion of HERV-E T cells & IL-2. (NCT03354390). Results: Nineteen of185 pts tested were found to express HLA-A*11. 17 HLA-A*11 + pts enrolled on the study: 3 pts on each DL1-3 & 6 pts on DL4. 2 pts did not receive HERV-E T cells given disease progression during manufacturing period. Median age was 57 years. 86% received ≥ 3 prior systemic treatment (range 1-8). The manufacturing failure rate after first apheresis was 12% (n=2); both met target dose after second apheresis and repeat in vitro expansion. All HERV-E T cell products met release criteria for infusion including INF-γ production in response to HERV-E/HLA-A11-expressing tumor cells. Median HERV-E vector copy number (VCN) was 1.9. No dose-limiting toxicities (DLT), off-target toxicities or treatment-related deaths occurred. Pt#17 is on DLT monitoring period. 7 pts completed the planned 14 doses of IL-2 and all received at least 8 doses. Reasons for IL-2 discontinuation: hemodynamic (57%), cardiovascular (28%), pulmonary (28%), renal (14%) criteria & pts decision (14%). The best response was partial response in 7% & stable disease at least 8 weeks in 29% pts. HERV-E mRNA expression was detected in 5 primary & 9 metastatic specimens. HERV-E T cells were measurable in circulation post-dosing, with peak concentrations in the peripheral blood mononuclear compartment on day(D)+7. [DL1: 0.3 %, DL2: 1.2%, DL3: 0.5%, DL4: 12.3%]. Median HERV-E T cell VCN showed no correlation with HERV-E T cells peak concentration on D+4 & D+7. Conclusions: Proof of concept that HERV-T cells can induce tumor regression without evidence of causing off-target toxicities has been established by this trial. Infused HERV-E T cells were detectable transiently in vivo and induce effector cytokine production. Our initial results support the further development of HERV-E-directed therapies that focus on methods to improve in vivo persistence of TCR engineered T-cells and to target HERV-E antigens expressed on more commonly expressed HLA alleles.[Table: see text]

Prevention of CMV/EBV Reactivation By Double-Specific T Cells in Patients after Allogeneic Stem Cell Transplantation: Results from the Randomized Phase I/IIa Multivir-01 Study

Allogeneic stem cell transplantation (aSCT) is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population. Study Design: A randomized, controlled, phase I/IIa multi-center study was conducted in 5 centers in Germany between 2013 and 2018 (ClinicalTrials.gov Identifier: NCT02227641). The study aimed at prevention or pre-emptive treatment of reactivation of CMV and EBV post aSCT. Major inclusion criteria were male or female HLA matched (10/10) unrelated or related donor seropositive for CMV and EBV, patient age between 18 and 75, the use of PBSC as stem cell source and the presence of on of the following HLA alleles: A*01:01, A*02:01, B07:02, B*08:01, B*35:01, C*07:02. CMV/EBV specific T cells were generated from a fraction of the allogeneic stem cell graft by stimulation with peptide pools for both viruses developed at the University Hospital Erlangen. Three doses of 5x10E4 CD3+ T cells per kilogram body weight were applied with an interval of at least 30 days without dose escalation starting earliest at day 30 after aSCT. Observation period was 204 days after aSCT. Primary endpoint of the study was toxicity of the T-cell-product assessed as acute transfusion reaction and acute GvHD due to the infusion of activated T cells. Results: 33 patients were screened and 29 patients were randomized. 16 patients were randomized into the treatment group of who 3 patients crossed over to the control arm due to failure of manufacturing the product or due to not meeting release criteria. Four patients were excluded from the study before receiving the first dose due to transplant related toxicity or withdrawal of consent. In total 9 patients were treated with 25 doses of the T cell product (dose range 2.1-4.1x10E4/kg CD3+ T cells). The earliest infusion was on day 30 (range: 30-69) post aSCT. There was no statistical difference between the groups regarding disease, patient age, donor type, performance status and comorbidities. The median follow up for both groups was 930 days. None of the patients treated experienced any acute transfusion reaction. Two patients developed acute GvHD after the second infusion and did not receive the third dose. Overall there was no statistical significant difference regarding survival (A) (treatment: 55.9% vs. control 69.2%, p=0.44), transplant related mortality (B) (22.2% vs. 7.1%, p=0.28), relapse (C) (11.1% vs. 14.3%) and acute GvHD (D,E) (33.3% vs. 46.2%, p=0.44). Number of AE per patient (27.1±24.6 vs. 24.4±11.2, p=0.60) and severity of AE (1.60±0.39 vs. 1.64±0.24) were similar in both groups. The incidence of reactivation of CMV (44.4% vs. 64.3%, p=0.16) and EBV (77.8% vs. 44.4%, p=0.12) was not statistically different and not associated with the treatment as several reactivations of occurred before application of the first dose. Within the treatment group we observed in several cases substantial expansion of CMV and EBV specific T cells in the peripheral blood which could not attributed to the presence viral antigens in case of reactivation. Conclusion: The study met its primary endpoint as there was no toxicity of the T-cell product observed. The cumulative incidence of acute GvHD as the primary endpoint was not statistically different in both groups and there was no acute toxicity related to the product. The study suffered from slow recruitment due to the market approval of Letermovir and low patient numbers did not allow for conclusive analysis of secondary endpoints such as the use of antiviral therapy.

Efficacy and Safety of a Third Generation CD20 CAR-T (MB-106) for Treatment of Relapsed/Refractory Indolent B-Cell Non-Hodgkin Lymphoma: Phase-1 Results from a Multicenter Trial

Background: Indolent B-cell non-Hodgkin lymphomas (NHL) remain incurable despite multiple therapeutic options, including chemoimmunotherapy, targeted therapies, bispecific antibodies, and CD19-targeted chimeric antigen receptor T (CAR-T) cells. MB-106, a fully human 3 rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains, had high overall response rate (100%) and complete response (CR) rate (91%) in follicular lymphoma (FL) patients at doses ≥ 3.3×10 6 cells/kg in a single institution trial (Shadman et al., 17-ICML, 2023). We seek to replicate the favorable efficacy and safety profile of MB-106 in a multicenter trial with centralized manufacturing. Methods: MB-106 is being evaluated in a Phase 1/2, open label, multicenter study utilizing a 3+3 dose-escalation design in R/R B-cell NHL patients with confirmed CD20 expression. Prior treatment with CD19-targeted CAR-T therapy is permitted. Following lymphodepletion (cyclophosphamide 300 mg/m 2/day and fludarabine 30 mg/m 2/day for 3 days), MB-106 is administered to patients with indolent B-cell NHL at one of two dose levels (DL): DL1, 3.3×10 6; and DL2, 1.0×10 7 cells/kg. MB-106 is infused in the outpatient setting if allowed by the institution, except for the first patient in each dose level cohort (overnight observation). Dose-limiting toxicities (DLT) are monitored through day 28. The treatment response is assessed on day 28 and on subsequent visits, and best overall response is reported here. CAR-T persistence is assessed by both flow cytometry and qPCR in peripheral blood mononuclear cells. Results: Three patients with indolent NHL (2 FL, 1 Waldenström macroglobulinemia [WM]) received MB-106 at DL1. In addition, one patient with hairy cell leukemia variant (HCL-v) received non-conforming material (drug product that failed to meet release criteria) at DL1, following FDA authorization. These four patients were heavily pre-treated with a median of 5.5 prior lines of treatment. High-risk features included progression of disease within 24 months of first-line treatment (POD24, n=1), prior autologous stem cell transplant (n=1) and prior CD19-targeted CAR-T therapy (n=1). No DLTs were observed at DL1. Three patients experienced Grade 1 cytokine release syndrome. There have been no occurrences of immune effector cell-associated neurotoxicity syndrome (ICANS). The 2 FL patients achieved CR by both PET-CT and bone marrow biopsy, one of whom had been previously treated with CD19-targeted CAR-T. The WM patient, who had nine prior treatments, including autologous stem cell transplant, and high disease burden, achieved very good partial response, with complete metabolic response by PET-CT, morphologic clearance of lymphoma in bone marrow, and resolution of the IgM paraprotein. The patient with HCL-v, who had been transfusion dependent, continues to have stable disease with decreased disease in the bone marrow, and achieved complete transfusion independence which is ongoing at 6 months. All patients displayed MB-106 expansion with peak levels between 7-14 days post-infusion. Persistence of CAR T-cells is ongoing at 6 months. Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in responses, including CRs, and CAR-T persistence in patients with R/R indolent NHL and was associated with favorable safety profile with no occurrence of Grade 3 or Grade 4 cytokine release syndrome and no ICANS of any grade. CRs have been observed in patients previously treated with CD19-targeted CAR-T in both this multicenter trial and the original single institution trial. Dosing is ongoing in the final dosing level (1.0×10 7 cells/kg) to establish the recommended Phase 2 dose for a planned pivotal trial in WM. It is anticipated that the complete data set from the Phase 1 indolent NHL arm will be presented.

Fully automated radiosynthesis of [68Ga]Ga-FAPI-46 with cyclotron produced gallium

BackgroundRadiopharmaceuticals capable of targeting the fibroblast activation protein have become widely utilized in the research realm as well as show great promise to be commercialized; with [68Ga]Ga-FAPI-46 being one of the most widely utilized. Until now the synthesis has relied on generator-produced gallium-68. Here we present a developed method to utilize liquid-target cyclotron-produced gallium-68 to prepare [68Ga]Ga-FAPI-46.ResultsA fully-automated manufacturing process for [68Ga]Ga-FAPI-46 was developed starting with the 68Zn[p,n]68Ga cyclotron bombardment to provide [68Ga]GaCl3, automated purification of the [68Ga]GaCl3, chelation with the precursor, and final formulation/purification. The activity levels produced were sufficient for multiple clinical research doses, and the final product met all release criteria. Furthermore, the process consistently provides < 2% of Ga-66 and Ga-67 at the 4-h expiry, meeting the Ph. Eur. standards.ConclusionsThe automated radiosynthesis on the GE FASTlab 2 module purifies the cyclotron output into [68Ga]GaCl3, performs the labeling, formulates the product, and sterilizes the product while transferring to the final vial. Production of > 40 mCi (> 1480 MBq) of [68Ga]Ga-FAPI-46 in excellent radiochemical yield was achieved with all batches meeting release criteria.

PO-04-065 THREE-DIMENSIONAL INTRACARDIAC ECHOCARDIOGRAPHY FOR LEFT ATRIAL APPENDAGE SIZING AND PERCUTANEOUS OCCLUSION GUIDANCE

Left atrial appendage (LAA) imaging is critical during percutaneous occlusion procedures. 3D-intracardiac echocardiography (ICE) features direct visualization of LAA from multiple cross-sectional planes at a time. To report procedural success of 3D-ICE-guided LAA occlusion and the correlation between pre-procedural transesophageal echocardiography (TEE) and intraprocedural 3D-ICE for LAA sizing. Among 274 patients undergoing LAAO with a Watchman FLX, periprocedural ICE guidance was achieved via a commercially available 2D-ICE catheter (220 patients) or a novel (NUVISION™) 3D-ICE one (54 patients). Technical success was defined as successful device implantation without periprocedural evidence of leaks≥3mm on color Doppler. Procedural success was a composite of technical success plus absence of procedure- or device-related major complications. Primary endpoint was a composite of procedural success and LAA sealing at follow-up TEE. Secondary endpoint was a composite of periprocedural device recapture/resizing plus presence of leaks≥3mm at follow-up TEE. We also compared mean and maximum mean and maximum landing zone (LZ) diameters obtained from same patient’s TEE and 3D-ICE; these measurements were used to assess the degree of correlation and agreement between the two imaging techniques. Procedural success was achieved in all 274 patients; all final devices met release criteria. The agreement between 3D-ICE-based device selection and final device size was 96.3% versus 79.1% with 2D-ICE (p=0.005). No major procedure- and device-related adverse events were documented The incidence of the primary endpoint was 98.1% with 3D-ICE and 97.3% with 2D-ICE (p=0.99). 2D-ICE patients had a trend towards a higher incidence of periprocedural device recapture/redeployment (31.5% vs 44.5%; p=0.09). The secondary endpoint occurred in 31.5% of 3D-ICE patients versus 45.9% of 2D-ICE ones (p=0.065). 3D-ICE measurements of maximum and mean LZ correlated highly with preprocedural TEE reference values [Pearson’s: 0.94; p<0.001; bias: -0.06 (-2.39, 2.27)] (Figure 1). ICE-guided LAAO showed a very high success, with no major adverse events. A very high level of agreement for LAA sizing was found between pre-procedural TEE and periprocedural 3D-ICE. 3D-ICE performed significantly better than 2D-ICE for FLX size selection and may provide better guidance during device deployment.

Immunological responses to adjuvant vaccination with combined CD1c+ myeloid and plasmacytoid dendritic cells in stage III melanoma patients

ABSTRACT We evaluated the immunological responses of lymph-node involved (stage III) melanoma patients to adjuvant dendritic cell vaccination with subsets of naturally occurring dendritic cells (nDCs). Fifteen patients with completely resected stage III melanoma were randomized to receive adjuvant dendritic cell vaccination with CD1c+ myeloid dendritic cells (cDC2s), plasmacytoid dendritic cells (pDCs) or the combination. Immunological response was the primary endpoint and secondary endpoints included safety and survival. In 80% of the patients, antigen-specific CD8+ T cells were detected in skin test-derived T cells and in 55% of patients, antigen-specific CD8+ T cells were detectable in peripheral blood. Functional interferon-γ-producing T cells were found in the skin test of 64% of the patients. Production of nDC vaccines meeting release criteria was feasible for all patients. Vaccination only induced grade 1–2 adverse events, mainly consisting of fatigue. In conclusion, adjuvant dendritic cell vaccination with cDC2s and/or pDCs is feasible, safe and induced immunological responses in the majority of stage III melanoma patients.

Polyclonal Regulatory T Cell Manufacturing Under cGMP: A Decade of Experience.

We report on manufacturing outcomes for 41 autologous polyclonal regulatory T cell (PolyTreg) products for 7 different Phase 1 clinical trials over a 10-year period (2011-2020). Data on patient characteristics, manufacturing parameters, and manufacturing outcomes were collected from manufacturing batch records and entered into a secure database. Overall, 88% (36/41) of PolyTreg products met release criteria and 83% (34/41) of products were successfully infused into patients. Of the 7 not infused, 5 failed release criteria, and 2 were not infused because the patient became ineligible due to a change in clinical status. The median fold expansion over the 14-day manufacturing process was 434.8 -fold (range 29.8-2,232), resulting in a median post-expansion cell count of 1,841 x 106 (range 56.9-16,179 x 106). The main correlate of post-expansion cell number was starting cell number, which positively correlates with absolute circulating Treg cell count. Other parameters, including date of PolyTreg production, patient sex, and patient age did not significantly correlate with fold expansion of Treg during product manufacturing. In conclusion, PolyTreg manufacturing outcomes are consistent across trials and dates of production.

BK virus-specific T cells for immunotherapy of progressive multifocal leukoencephalopathy: an open-label, single-cohort pilot study

Progressive multifocal leukoencephalopathy, a rare disease of the CNS caused by JC virus and occurring in immunosuppressed people, is typically fatal unless adaptive immunity is restored. JC virus is a member of the human polyomavirus family and is closely related to the BK virus. We hypothesised that use of partly HLA-matched donor-derived BK virus-specific T cells for immunotherapy in progressive multifocal leukoencephalopathy would be feasible and safe. We did an open-label, single-cohort pilot study in patients (aged 18 years or older) with clinically definite progressive multifocal leukoencephalopathy and disease progression in the previous month at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Overlapping peptide libraries derived from large T antigen and major capsid protein VP1 of BK virus with high sequence homology to JC virus counterparts were used to generate polyomavirus-specific T cells cross-recognising JC virus antigens. Polyomavirus-specific T cells were manufactured from peripheral blood mononuclear cells of first-degree relative donors aged 18 years or older. These cells were administered to patients by intravenous infusion at 1 × 106 polyomavirus-specific T cells per kg, followed by up to two additional infusions at 2 × 106 polyomavirus-specific T cells per kg. The primary endpoints were feasibility (no manufacturing failure based on meeting release criteria, achieving adequate numbers of cell product for clinical use, and showing measurable antiviral activity) and safety in all patients. The safety monitoring period was 28 days after each infusion. Patients were followed up with serial MRI for up to 12 months after the final infusion. This trial is registered at ClinicalTrials.gov, NCT02694783. Between April 7, 2016, and Oct 19, 2018, 26 patients were screened, of whom 12 were confirmed eligible and received treatment derived from 14 matched donors. All administered polyomavirus-specific T cells met the release criteria and recognised cognate antigens in vitro. 12 patients received at least one infusion, ten received at least two, and seven received a total of three infusions. The median on-study follow-up was 109·5 days (range 23-699). All infusions were tolerated well, and no serious treatment-related adverse events were observed. Seven patients survived progressive multifocal leukoencephalopathy for longer than 1 year after the first infusion, whereas five died of progressive multifocal leukoencephalopathy within 3 months. We showed that generation of polyomavirus-specific T cells from healthy related donors is feasible, and these cells can be safely used as an infusion for adoptive immunotherapy of progressive multifocal leukoencephalopathy. Although not powered to assess efficacy, our data provide additional support for this strategy as a potential life-saving therapy for some patients. Intramural Research Program of the National Institute of Neurological Disorders and Stroke of the NIH.

Abstract CT031: GD2 CAR T cells mediate clinical activity and manageable toxicity in children and young adults with DIPG and H3K27M-mutated diffuse midline gliomas

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal central nervous system tumors. We previously discovered that the disialoganglioside GD2 is highly and homogenously expressed on H3K27M+ gliomas and demonstrated that GD2 CAR T cells are effective in preclinical models (Mount/Majzner et al., Nat Med, 2018). Methods: Four subjects (3 DIPG, 1 spinal cord DMG; 4-25 yr; 1M/3F) were enrolled at DL1. Three subjects with H3K27M+ DIPG received 1e6 autologous GD2 CAR T cells/kg intravenously (IV) on study. One patient, a 25 y/o with spinal cord DMG, developed rapidly progressive disease after enrollment, resulting in complete paraparesis that led to removal from the study prior to cell infusion; she was treated on a single patient eIND with the same treatment regimen as DL1. We utilized a retroviral vector expressing a 14g2a.4-1BB.z CAR construct and an inducible iCasp9 safety switch. Manufacturing was performed in the Miltenyi Prodigy on CD4/CD8 enriched apheresis product. CAR T cells were cultured in the presence of dasatinib to improve T cell fitness (Weber et al., Science, 2021). An Ommaya reservoir was placed in all patients for monitoring of intracranial pressure (ICP). Results: We generated GD2 CAR T cell products meeting release criteria for all four patients. All subjects received lymphodepletion with cyclophosphamide and fludarabine and remained inpatient for 14+ days after infusion. All patients developed cytokine release syndrome (Grade 1-3) manifested by fever, tachycardia and hypotension, beginning 6-7 days after infusion. Due to concern for tumoral edema and increased ICP, patients were managed with conservative fluid resuscitation, and early intervention with tocilizumab and anakinra +/- corticosteroids. Other toxicities included ICANS (Grade 1-2) and neurotoxicity mediated by inflammation in sites of disease which we have termed Tumor Inflammation-Associated Neurotoxicity (TIAN). TIAN most often manifested as worsening of existing deficits, but one patient developed symptoms of increased ICP which quickly resolved upon removal of CSF via the Ommaya. No evidence of on-target, off-tumor toxicity was observed in any patients. No dose-limiting toxicities occurred.CAR T cells trafficked to the CNS and were detected in both the CSF and peripheral blood. Inflammatory cytokines including IL-6 were elevated in the CSF and blood. 3/4 patients exhibited marked improvement or resolution of neurological deficits and some radiographic improvement. The patient treated on a single patient eIND exhibited a &amp;gt;90% reduction in her spinal cord DMG tumor volume at two months post-infusion. Durability of the therapeutic benefit remains to be determined. Conclusions: This is the first report of GD2 CAR T cell therapy for DIPG and spinal cord DMG. Toxicities are similar to other CAR T cells with additional, manageable complications due to inflammation at CNS sites of tumor. Treatment at DL1 demonstrated a tolerable safety profile and clear signs of T cell expansion and activity including clinical responses. This approach has the potential to transform therapy for patients with H3K27M+ DIPG/DMG. Further correlative studies, including single-cell RNAseq, longer-term outcomes and results from patients on subsequent dose levels will also be presented. Citation Format: Robbie G. Majzner, Sneha Ramakrishna, Aaron Mochizuki, Shabnum Patel, Harshini Chinnasamy, Kristen Yeom, Liora Schultz, Rebecca Richards, Cynthia Campen, Agnes Reschke, Jasia Mahdi, Angus Martin Shaw Toland, Christina Baggott, Sharon Mavroukakis, Emily Egeler, Jennifer Moon, Kayla Landrum, Courtney Erickson, Lindsey Rasmussen, Valentin Barsan, John S. Tamaresis, Anne Cunniffe Marcy, Michael Kunicki, Michelle Fujimoto, Zach Ehlinger, Sreevidya Kurra, Timothy Cornell, Sonia Partap, Paul Fisher, Gerald Grant, Hannes Vogel, Bita Sahaf, Kara Davis, Steven Feldman, Crystal L. Mackall, Michelle Monje. GD2 CAR T cells mediate clinical activity and manageable toxicity in children and young adults with DIPG and H3K27M-mutated diffuse midline gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT031.

Phase I Trial of Regulatory T-Cell Donor Lymphocyte Infusion Plus Daily Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Versus-Host Disease

Low-dose daily Interleukin-2 (IL-2) therapy expands regulatory T cells (Treg) in vivo in patients with chronic graft-versus-host disease (cGvHD). Clinical improvement, primarily partial response, is seen in 50-60% of those treated. We hypothesized that infusion of fresh Tregs prior to low dose IL-2 may augment clinical and immune responses. In a Phase 1 study, we administered fresh Treg-enriched lymphocyte infusions (Treg-DLI) obtained from the original stem cell donor, without in vitro stimulation or expansion, followed by daily low-dose IL-2 as therapy for cGvHD. Methods: 24 recipients of 7-8/8 HLA-matched allogeneic hematopoietic stem cell transplants with steroid-refractory cGvHD on stable immune suppression received escalating doses Treg-DLI: 5 each at 0.1, 0.3, or 1x106 cells/kg plus an expansion cohort of 9 at 1x106/kg. Non-mobilized leukapheresis products were CD8/CD19 co-depleted, then positively selected for CD25+ Tregs via the CliniMACS system (Miltenyi). IL-2 was administered at 1x106 U/m2/day for 8 weeks after Treg-DLI infusion. Subjects: The first 22 subjects had a median age of 48 (range, 23-68), median of 3 cGvHD sites (range, 1-6), and median of 2 prior therapies in addition to steroids (range, 0 -5). Four had prior IL-2 exposure. Median time from cGvHD diagnosis to enrollment was 26 months (range, 7- 84). Manufacturing: All Treg-DLI products met release criteria. Median CD8 and CD19 log-depletions were 4.5 and 4.6, respectively. Released products contained a median of 92.6% CD4+CD25++ cells and 88.2% CD4+CD25+CD27low Tregs, a 24.8-fold enrichment (range, 12.7-43.4). At the highest targeted dose level, a median of 0.98x106 cells/kg were infused (range, 0.64-1.0). Safety: In the 21 subjects evaluable for safety, there were no infusional AEs, no DLTs, and no cases of relapse. 2 subjects underwent a 50% dose reduction of IL-2 for Grade 2 flu-like symptoms and injection site irritation, at provider discretion. Grade 3 AEs possibly related to therapy were dyspnea and edema in 1 subject. At median follow-up of 26 months, 17 subjects are alive. 2-year survival is 82% (95% CI, 54-95%). Response: At 8 weeks, 2 of 5 subjects in Cohort 1 (1 partial response (PR), 1 stable disease with minor response (SDMR)), 4 of 5 subjects in Cohort 2 (2 PR, 2 SDMR), and 8 of 11 subjects at the highest cell dose (1 complete response(CR), 3 PR, 4 SDMR) demonstrated clinical benefit. Response rate (CR+PR) was 33%; clinical benefit rate (CR+PR+SDMR) was 67%. 11 subjects opted to continue IL-2, for a median of 14.5 months (range, 3-39). Of 4 subjects previously on IL-2 alone, 2 were responders (CR, PR) and 1 each had mixed response and SDMR, respectively. Interestingly, 3 subjects with SDMR at 8 weeks manifested PR on extended IL-2. No impact of Treg-DLI dose was observed. At 6 months, median prednisone dose was 15mg (range, 5-40), down from 30mg daily at baseline (range, 5-80). Correlative Immunology: Treg counts and the ratio of Treg to conventional T cells (Tcon) rose over time. Higher Treg:Tcon ratios at baseline (p=0.08) and at 2 weeks into therapy (p=0.03) were associated with clinical response (Figure 1). Breadth of diversity within Treg T-cell receptor β (TCRβ) sequences (Shannon's entropy) and selective expansions of individual Treg clones (clonality) were low in cGvHD subjects at baseline, as compared to normal donors (p<0.01) and to Treg-DLI products (p<0.001). Treg TCRβ entropy increased in cGvHD subjects after 8 weeks of therapy (p<0.05), and resulting entropy was comparable to normal donors (n=16, Figure 2). This normalization was independent of clinical response and persisted through 6 months (n=8). Treg TCRβ clonality trended upwards at 8 weeks but did not reach levels seen in normal donors. Entropy and clonality of CD8 and Tcon TCRβ populations in cGvHD subjects were comparable to normal donors and did not change with therapy. Conclusions: GMP-grade manufacture ofCD25+ enriched Treg-DLI is feasible. In combination with low-dose IL-2, Treg-DLI is safe and has clinical efficacy in steroid-refractory cGvHD, including in those with inadequate responses to IL-2 alone. TCRβ population diversity specifically in Tregs is normalized with therapy. The incremental benefit of adding Treg-DLI to therapy with IL-2 alone on clinical responses, normalization of immunologic diversity, and functional suppressive activity, including at timepoints beyond 8 weeks, is the subject of ongoing analyses. [Display omitted] DisclosuresNikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Affimed: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Infinity: Consultancy; Otsuka: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Roche: Research Funding; Tensha: Research Funding; Pfizer: Consultancy, Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Koreth:Millennium Pharmaceuticals: Research Funding; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy.

Shortening the Time to Manufacture CAR+ T Cells with Sleeping Beauty System Supports T-Cell Engraftment and Anti-Tumor Effects in Patients with Refractory CD19+ Tumors

▪BACKGROUND:. We have previously reported the first human application of non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific chimeric antigen receptor (CAR) which resulted in survival benefits for recipients of autologous and allogeneic T cells delivered as adjuvant immunotherapy after hematopoietic stem cell transplantation (HSCT) (J Clin Invest. 2016 126(9):3363-76). This CAR (CD19RCD28) used a Fc-based extracellular stalk and T cells were generated after 28 days (4 “stimulations”) of recursive culture on activating and propagating cells (AaPC). To further improve persistence we modified this CAR construct and shortened the ex vivo time to produce clinical-grade T cells.METHODS: A new Phase 1 clinical trial (NCT02529813) at MD Anderson Cancer Center enrollspatients with a history of CD19+ lymphoid malignancy with active disease for dose escalation of SB-modified autologous T cells which were infused following lymphodepletion chemotherapy, starting at ≤105 (dose level, DL -1), >105 but ≤106 (dose level, DL 1) and then escalating in cohorts of 3 to the dose of >108 but ≤109 CD3+CAR+ cells/kg (DL 4). The CD19-specific CAR employed a revised stalk derived from CD8, and as per CD19RCD28, signaled through chimeric CD28 and CD3-z endodomains (designated CD19RCD8CD28). SB-derived DNA plasmids coding for CAR (transposon) and transposase were electro-transferred into peripheral blood mononuclear cells and subsequently co-cultured every ~7 days on irradiated K-562-derived CD19+ AaPC in the presence of IL-2 and IL-21 to selectively propagate CAR+ T cells. During dose escalation, T-cell manufacturing time was shortened by reducing the number of additions of AaPC from four to two stimulations. The final product met release criteria and was thawed prior to infusion.RESULTS: In this ongoing trial, 6 patients with median age 41 years (range 29-69 years) with active CD19+ lymphoid malignancy (CLL, n=1; ALL, n= 4; DLBCL, n=1) have been dosed so far (DL -1, n=1; DL 1, n=3; DL 2, n=2). All had refractory/relapsed disease with four having prior allogeneic HSCT. Median CAR expression upon infusion was 86% (range 19%-97%). No DLTs or CD19 related SAEs were observed. Only grade 1 cytokine release syndrome (CRS) was noted in 2 patients. The median follow-up for patients is 3 months (range 0.3-13.8 months). Anti-tumor effects were noted and infused T cells could be detected by flow cytometry and PCR (Figure).CONCLUSIONS: These initial data describing the infusion of SB-modified T cells with a revised extracellular domain and under reduced manufacturing time results in the detection of genetically modified T cells and anti-tumor effects in patients with relapsed/refractory ALL apparently without clinically-significant CRS. These data support advancing an approach for non-viral gene transfer to administer CAR+ T cells, and working toward even shorter manufacturing times.Figure: Persistence of circulating CD19-specific CAR+ T cells in patients after single infusion of SB-modified T cells, as determined by ddPCR. Circles represent a patient at given time point, short horizontal lines represent mean, and the continuous line connecting the means describes the trend. [Display omitted] DisclosuresHuls:Former employee of Intrexon: Employment. Smith:Ziopharm: Employment. De Groot:Ziopharm: Employment. Kantarjian:Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding. Cooper:Miltenyi Biotec: Honoraria; Ampliphi Biosciences: Equity Ownership; Procter & Gamble: Equity Ownership; Organovo Holdings: Equity Ownership; Targazyme, Inc.: Equity Ownership; Argos Therapeutics: Equity Ownership; Intrexon Corporation: Equity Ownership, Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties, Research Funding; Sangamo Biosciences: Patents & Royalties; Ziopharm: Employment, Equity Ownership, Research Funding; Ferring: Consultancy.

Evaluation of Tumor Vaccine Generation in a Phase II Multicenter Trial of Single Autologous Hematopoietic Cell Transplant (AutoHCT)Followed By Lenalidomide Maintenance for Multiple Myeloma (MM) with or without Vaccination with Dendritic Cell/ Myeloma Fusions (DC/MM fusion vaccine): Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 1401

BMT CTN 1401 is an academically led randomized study of a personalized cancer vaccine in which the combination of DC/MM fusion vaccine and lenalidomide maintenance after autoHCT is compared with maintenance alone as upfront treatment of MM (NCT#02728102). Assessment of the percentage of patients achieving CR and therapy-induced changes in MM-specific immunity are the primary clinical and immunologic endpoints, respectively. The protocol represents a transformative open source approach to cell therapy in MM and required collection of tumor cells prior to treatment initiation and vaccine production at day 60 after autoHCT. The process featured a multi-step approach of procedural standardization, training, and centralized verification of manufactured product/release criteria. Here we present initial analysis of accrual and vaccine manufacturing. Enrollment of 203 patients completed within 26 months with 140 patients randomized. Drop-out rate from enrollment to randomization was 31% (expected 30%). Sixty-eight patients were randomized to the DC/MM fusion vaccine arm, with 63 having products successfully manufactured and approved for release at 14 manufacturing sites. Vaccine products for 5 patients were not approved for release for the following reasons: final TC/DC viability did not meet release criteria (n = 3), product failed to meet sterility release criteria (n = 1), and disease progression prior to manufacture (n = 1). Among 63 participants who received vaccine, mean DC viability and CD86 expression was 79.3% (Standard Deviation = 11.3) and 80.6% (SD = 12.9), respectively. Mean viability and percent cells co-expressing DC and tumor antigens was 78.6% (SD = 13.0) and 47.9% (SD = 11.4), respectively. The process devised for BMT CTN 1401 led to rapid patient accrual and successful point-of-care DC/MM fusion vaccine generation. Feasibility of serial sample collection and performance of immunologic correlates were demonstrated. This multi-center collaboration highlights the development of a personalized tumor vaccine platform that serves as a model for future immunotherapy trials in MM.

GMP-Compliant Manufacturing of NKG2D CAR Memory T Cells Using CliniMACS Prodigy.

Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA− memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA+ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 108 CD45RA− cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.

Identifying and Managing Sources of Variability in Cell Therapy Manufacturing and Clinical Trials

Identifying and managing cell therapy variability can be a significant challenge for a company seeking to commercialize a new product. Failure to address this issue can lead to negative consequences such as delayed approval due to unsuccessful clinical investigations, or failed product lots that do not meet release criteria. Allogeneic cell therapies can be particularly prone to variability challenges due to the use of variable input material. In order to support the manufacturers of cell therapies, the FDA has identified two primary regulatory pathways (351 vs 361) that reflect the relative risk of the product. In this review, we will discuss criteria that separate the two potential regulatory pathways for cell therapy products in the USA. Also, we will discuss what aspects of manufacturing and clinical trial execution might introduce undesired variability that can derail the path towards licensure and commercialization, along with tools to minimize these potential sources of variability. ClinicalTrials.gov Identifier: NCT03347708 and NCT03955315Lay SummaryTherapies that utilize live cells as the active ingredient, known as cell therapies, are a promising approach to treating many diseases that cannot be addressed with traditional medicines. However, with this great potential comes specific challenges associated with cell therapy, including identifying the appropriate regulatory approval pathway, manufacturing it in a reproducible way, and successfully executing clinical trials. In this review, we describe potential sources of variability that can negatively impact the translation of a cell therapy, and ways to minimize those risks.

Results of point-of-care manufacturing of bispecific chimeric antigen receptor (CAR) LV20.19 CAR-TCELLS in a phase i study for relapsed/refractory (R/R), non-hodgkin lymphoma (NHL)

Background & Aim Current approved CAR-T cell manufacturing employs off-site production, a labor-intensive process that requires several weeks and involves shipping back and forth to the production site. This production model may limit availability and accessibility to CAR-T cells and delay treatment for patient (pt) s with rapidly progressive disease. To address these limitations, we utilized the Miltenyi CliniMACS Prodigy, a GMP-compliant device, to produce CAR-T cells on-site for a first-in-human bi-specific CD19/20 (LV20.19) CAR-T Phase I trial. Methods, Results & Conclusion CAR-T cells were manufactured within our Cell Therapy Laboratory, an ISO7 facility. Pt's mononuclear cell apheresis was collected and loaded onto the Prodigy to isolate CD4/CD8 T cells. The T cells were suspended in TexMACS supplemented with human AB serum and IL-2, activated with TransACT, and then transduced by LV20.19 the next day. Culture wash and feedings were done during the production process, and the final cell product was harvested at day 14. The process was automated with limited machine/operator interactions. CAR-T expression was assessed by protein L staining. CAR-T cells were either administered fresh or cryopreserved for later infusion. Dose was escalated in a 3+3 manner from 2.5e+5 cells/kg to a target dose of 2.5e+6 cells/kg. CAR-T cells have been successfully generated for 11/11 enrolled pts thus far with no manufacture failures, and all products met release criteria. Median T cell recovery from the apheresis products was 59.5% with a median CD3+ purity of 97.2% (Figure 1). Final product CAR expression was at a median of 17.6%. A median of 4.7e+8 CAR T cells was obtained at harvest, exceeding the required target dose. CAR-T cells contained both CD4 and CD8 T cells with higher CD4 content. The majority of CAR-T cells showed an effector-memory phenotype. CAR-T cells were able to kill CD19+/20+ target cells and produced IFN-gin response to antigens. Among the 11 treated pts 8 received fresh CAR-T cells and 3 received CAR-T cells after cryopreservation. As no DLTs were observed at the 3 dose levels, a dose of 2.5e+6 cells/kg has been selected for further expansion. Clinical response shows 6 pts have achieved a CR at Day +28 with a duration of response between 2 to 14 months. In summary, initial results demonstrated feasibility for point-of-care manufacturing of bispecific LV20.19CAR-T cells with safe and promising efficacy in treating heavily pretreated pts with R/R B-cell NHL, which warrants further investigations.

A Novel Low Affinity CD19CAR Results in Durable Disease Remissions and Prolonged CAR T Cell Persistence without Severe CRS or Neurotoxicity in Patients with Paediatric ALL

Introduction:Published studies of CD19 CAR T cells have shown unprecedented response rates in ALL but with a 23-27% incidence of severe Cytokine Release Syndrome (CRS) and 27-50% incidence of severe neurotoxicity which may limit broader application. We developed a novel second generation CD19CAR (CAT-41BBz CAR) with a lower affinity and faster off-rate but equivalent on-rate than the FMC63-41BBz CAR (Kd 116 nM vs 0.9 nM, T1/2 10s vs 1260s) utilised in CTL019 currently under consideration by the FDA. Pre-clinical studies indicated T-cells transduced with CAT-41BBz mediate enhanced tumor clearance and show increased expansion in an NSG-NALM6 stress test model (Kramer et al., submitted). We here report interim results from a multi-centre, Phase I clinical study of autologous CAT-41BBz CAR T cells as therapy for high risk/relapsed paediatric ALL, CARPALL (NCT02443831) demonstrating efficacy with an excellent safety profile.Methods:Autologous T cells were activated with anti-CD3/CD28 beads, transduced with a SIN lentiviral vector encoding CAT-41BBz CAR and expanded for 4 days prior to magnetic bead removal and cryopreservation. Transduction efficiency was assessed using an anti-idiotype antibody. Serum levels of cytokines associated with CRS were measured using cytometric bead array.All patients received lymphodepletion with fludarabine 150 mg/m2 + cyclophosphamide 1.5g/m2 followed by a single infusion of CAR T cells at a dose of 1x106 CAR+ T cells. Patients were monitored for the presence of CAR T cells in the blood by flow cytometry and by qPCR for the 41BBz junctional region, as well as circulating B cell count monthly for 6 months and then 6 weekly to 1 year. Disease status was assessed in the bone marrow morphologically, by IgH qPCR, as well as by flow cytometric assessment of MRD at the same time-points to establish durability of responses as a stand-alone therapy. The primary end-points were incidence of grade 3-5 toxicity related to CAR T cells within 30 days and the proportion of patients achieving molecular remission.Results:We have enrolled 10 patients and treated 8 to date. Six of 8 had relapsed post myeloablative SCT. The median disease burden prior to lymphodepletion was 9% blasts (ranging from molecular CR to 74% blasts, Table 1). It was possible to generate a product meeting release criteria in all but 1 patient (90% feasibility). Median transduction efficiency was 18.1% (range 6.7 to 76.3%). All treated patients received the anticipated dose of 1x106 CAR T cells/kg.Cytokine release syndrome occurred in all patients (grade 1 n=4, grade 2 n=4), but to date none have developed ≥ grade 3 CRS, required ICU admission or therapy with Tocilizumab. CRS was associated with modest elevations of IL-6, IFN-γ and IL-10 and resolved spontaneously in all. Grade 2 neurotoxicity was observed in 3 patients and resolved spontaneously, but no severe (≥grade 3) neurotoxicity was seen. Five patients had prolonged grade 4 neutropenia lasting > 30 days but this resolved in all by 2 months. Only 1 patient experienced significant infective complications in the context of pre-existing poor marrow reserve following allogeneic SCT.6/7 (86%) evaluable patients achieved molecular remission at a median of 30 days post infusion (range 30-60 days, Table 1). One patient did not respond and died of CD19+ disease progression. At a median follow-up of 5.9 months (range 28-328 days), 4/7 evaluable patients remain in flow MRD negative remission of whom 3 show no evidence of molecular MRD at 1, 7.5 and 9 months. Two patients relapsed with CD19- disease at 3 and 4 months post infusion: 1 of these remains alive with disease at 11 months and the other died of disease progression.Reflecting our pre-clinical data with CAT-41BBz CAR, we have seen excellent CAR T cell expansion (median 65459 copies/µg DNA at 1 month, range 609 to 230112) and persistence at up to 11 months post-infusion (Figure 1). All 7 evaluable patients have ongoing CAR T cell persistence detectable by both flow and qPCR as well as ongoing B cell aplasia at last follow-up.Conclusions:These interim results with a novel low affinity CD19 CAR show similar remission rates to those reported by US studies in paediatric ALL with an improved safety profile. No severe (grade ≥3) CRS or neurotoxicity has occurred to date despite high tumour burden in 4 patients. Excellent CAR T cell expansion has been documented, as well as long duration of CAR T cell persistence and associated B cell aplasia. [Display omitted] DisclosuresGhorashian:UCL: Patents & Royalties: UCL Business. Kramer:UCL: Patents & Royalties: UCL Business. Lucchini:Alexion: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL: Patents & Royalties: UCL Business.

Manufacture of Autologous CD34+ Selected Grafts in the NIAID-Sponsored HALT-MS and SCOT Multicenter Clinical Trials for Autoimmune Diseases

To ensure comparable grafts for autologous hematopoietic cell transplantation (HCT) in the National Institute of Allergy and Infectious Diseases–sponsored Investigational New Drug protocols for multiple sclerosis (HALT-MS) and systemic sclerosis (SCOT), a Drug Master File approach to control manufacture was implemented, including a common Master Production Batch Record and site-specific standard operating procedures with “Critical Elements.” We assessed comparability of flow cytometry and controlled rate cryopreservation among sites and stability of cryopreserved grafts using hematopoietic progenitor cells (HPCs) from healthy donors. Hematopoietic Progenitor Cells, Apheresis-CD34+ Enriched, for Autologous Use (Auto-CD34+HPC) graft specifications included ≥70% viable CD34+ cells before cryopreservation. For the 2 protocols, 110 apheresis collections were performed; 121 lots of Auto-CD34+HPC were cryopreserved, and 107 of these (88.4%) met release criteria. Grafts were infused at a median of 25 days (range, 17 to 68) post-apheresis for HALT-MS (n = 24), and 25 days (range, 14 to 78) for SCOT (n = 33). Subjects received precryopreservation doses of a median 5.1 × 106 viable CD34+ cells/kg (range, 3.9 to 12.8) for HALT-MS and 5.6 × 106 viable CD34+ cells/kg (range, 2.6 to 10.2) for SCOT. Recovery of granulocytes occurred at a median of 11 days (range, 9 to 15) post-HCT for HALT-MS and 10 days (range, 8 to 12) for SCOT, independent of CD34+ cell dose. Subjects received their last platelet transfusion at a median of 9 days (range, 6 to 16) for HALT-MS and 8 days (range, 6 to 23) for SCOT; higher CD34+/kg doses were associated with faster platelet recovery. Stability testing of cryopreserved healthy donor CD34+ HPCs over 6 months of vapor phase liquid nitrogen storage demonstrated consistent 69% to 73% recovery of viable CD34+ cells. Manufacturing of Auto-CD34+HPC for the HALT-MS and SCOT protocols was comparable across all sites and supportive for timely recovery of granulocytes and platelets.

Iranian homograft tissue processing.

Tissue transplantation is a life-enhancing therapeutic modality for damaged or non-functioning tissues. In most cases, there is no alternative other than human tissue as replacement, and taking into account the ever-increasing demand for tissue grafts, it makes sense to set up an establishment in charge of human tissue procurement to meet local needs. A quality assurance system, clearly defined standards, and regular audits complement the infrastructure which make this activity feasible. The process of tissue procurement consists of donor identification, consent, tissue recovery, donor screening and testing, tissue processing, preservation, packaging, labeling, terminal sterilization, storage and distribution. The transplantation of homograft heart valves remains controversial, due to the availability of prosthetic and bioprosthetic alternatives. The limited durability of homografts has not yet outweighed the advantages which this graft offers. Adherence to regulations and regularly revised guidelines improve long-term efficacy and minimizes complications or malfunction. Furthermore, the lower price of homograft heart valves and the removal of the need for a lifetime of anticoagulation therapy are noteworthy advantages of this replacement. In our practice, the proportion of homograft heart valves meeting release criteria and successfully implanted grafts were 83% and 95%, respectively.

THE IMPACT-CABG TRIAL: A CANADIAN RCT OF CD133+ STEM CELL THERAPY FOR ISCHEMIC CARDIOMYOPATHY

BACKGROUND: The IMPACT-CABG trial (NCT01033617) is a Canadian multicenter randomized, prospective, doubleblinded study investigating the feasibility, safety and efficacy of intramyocardial injections of autologous CD133+ stem cells in patients with chronic ischemic cardiomyopathy and impaired LV dysfunction (LVEF 25-45%) undergoing isolated CABG surgery. The primary safety endpoint was freedom from major adverse cardiac event (MACE) at time of injection and 6 months follow-up, including freedom from cardiac death, myocardial infarction (MI), repeat revascularization or severe arrhythmias. The secondary efficacy endpoint was regional myocardial perfusion and function assessed by magnetic resonance imaging (MRI). Herein we are reporting the safety results. METHODS: After an initial 5 (Montreal) or 2 (Toronto) openlabel patients who received stem cells, patients were then randomized in a 1:1 (Toronto1⁄418) or 2:1 (Montreal1⁄415) ratio to receive either stem cells or placebo. All forty patients (20 each in Montreal and Toronto) with ischemic cardiomyopathy and clinical indications for CABG provided informed consent and were assessed with preoperative stress echo and perfusion MRI. All patients had 100-150 cc of bone marrow aspirated from the iliac crest under local anaesthesia and light sedation on the morning of surgery. Stem cells were isolated using the CliniMACS CD133+ Reagent System (Miltenyi Biotec), which utilizes superparamagnetic beads composed of iron-dextran particles conjugated to monoclonal antibodies. On the afternoon following cell preparation, patients underwent conventional CABG surgery under cardiopulmonary bypass. Following the completion of the distal coronary anastomoses, the cell preparation containing up to 10 million cells or placebo was injected (10-15 injection sites) into the infarct and border zone as identified by MRI. RESULTS: Patient demographics, intraoperative data and hospital outcomes are presented in Table 1 for all 40 patients. Stem cell isolation procedures yielded cell preparations meeting release criteria in all patients randomized to cell delivery. There was no in-hospital mortality. No patient had a perioperative MI, 4 had renal failure, one had a wound infection and one had a seizure. During the 6-month followup period, one patient died from a pulmonary embolus, and no other MACE. CONCLUSION: This work represents the first Canadian experience with CD133+ stem cells for the treatment of chronic ischemic cardiomyopathy. There were no protocol-related complications, demonstrating that same-day isolation and delivery of stem cells in conjunction with CABG is safe and feasible. Cell therapy may become a valuable adjunct treatment for myocardial repair in heart failure patients in whom conventional therapies for ventricular dysfunction are limited.

Safety and feasibility for pediatric cardiac regeneration using epicardial delivery of autologous umbilical cord blood-derived mononuclear cells established in a porcine model system.

Congenital heart diseases (CHDs) requiring surgical palliation mandate new treatment strategies to optimize long-term outcomes. Despite the mounting evidence of cardiac regeneration, there are no long-term safety studies of autologous cell-based transplantation in the pediatric setting. We aimed to establish a porcine pipeline to evaluate the feasibility and long-term safety of autologous umbilical cord blood mononuclear cells (UCB-MNCs) transplanted into the right ventricle (RV) of juvenile porcine hearts. Piglets were born by caesarean section to enable UCB collection. Upon meeting release criteria, 12 animals were randomized in a double-blinded fashion prior to surgical delivery of test article (n=6) or placebo (n=6). The UCB-MNC (3×10(6) cells per kilogram) or control (dimethyl sulfoxide, 10%) products were injected intramyocardially into the RV under direct visualization. The cohorts were monitored for 3 months after product delivery with assessments of cardiac performance, rhythm, and serial cardiac biochemical markers, followed by terminal necropsy. No mortalities were associated with intramyocardial delivery of UCB-MNCs or placebo. Two animals from the placebo group developed local skin infection after surgery that responded to antibiotic treatment. Electrophysiological assessments revealed no arrhythmias in either group throughout the 3-month study. Two animals in the cell-therapy group had transient, subclinical dysrhythmia in the perioperative period, likely because of an exaggerated response to anesthesia. Overall, this study demonstrated that autologous UCB-MNCs can be safely collected and surgically delivered in a pediatric setting. The safety profile establishes the foundation for cell-based therapy directed at the RV of juvenile hearts and aims to accelerate cell-based therapies toward clinical trials for CHD.