Publications by authors named "Adrian P Gee"

86 Publications

T-Cell Therapy for Lymphoma Using Nonengineered Multiantigen-Targeted T Cells Is Safe and Produces Durable Clinical Effects.

J Clin Oncol 2021 Jan 28:JCO2002224. Epub 2021 Jan 28.

Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX.

Purpose: Patients with relapsed lymphomas often fail salvage therapies including high-dose chemotherapy and mono-antigen-specific T-cell therapies, highlighting the need for nontoxic, novel treatments. To that end, we clinically tested an autologous T-cell product that targets multiple tumor-associated antigens (TAAs) expressed by lymphomas with the intent of treating disease and preventing immune escape.

Patients And Methods: We expanded polyclonal T cells reactive to five TAAs: PRAME, SSX2, MAGEA4, SURVIVIN, and NY-ESO-1. Products were administered to 32 patients with Hodgkin lymphomas (n = 14) or non-Hodgkin lymphomas (n = 18) in a two-part phase I clinical trial, where the objective of the first phase was to establish the safety of targeting all five TAAs (fixed dose, 0.5 × 10 cells/m) simultaneously and the second stage was to establish the maximum tolerated dose. Patients had received a median of three prior lines of therapy and either were at high risk for relapse (adjuvant arm, n = 17) or had chemorefractory disease (n = 15) at enrollment.

Results: Infusions were safe with no dose-limiting toxicities observed in either the antigen- or dose-escalation phases. Although the maximum tolerated dose was not reached, the maximum tested dose at which efficacy was observed (two infusions, 2 × 10 cells/m) was determined as the recommended phase II dose. Of the patients with chemorefractory lymphomas, two (of seven) with Hodgkin lymphomas and four (of eight) with non-Hodgkin lymphomas achieved durable complete remissions (> 3 years).

Conclusion: T cells targeting five TAAs and administered at doses of up to two infusions of 2 × 10 cells/m are well-tolerated by patients with lymphoma both as adjuvant and to treat chemorefractory lymphoma. Preliminary indicators of antilymphoma activity were seen in the chemorefractory cohort across both antigen- and dose-escalation phases.
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http://dx.doi.org/10.1200/JCO.20.02224DOI Listing
January 2021

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

JACC CardioOncol 2020 Nov 22;2(4):581-595. Epub 2020 Dec 22.

Division of Cardiovascular Diseases, University of Kansas School of Medicine, Kansas City, Kansas, USA.

Background: Anthracycline-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.

Objectives: SENECA (Stem Cell Injection in Cancer Survivors) was a phase 1 study of allo-MSCs in AIC.

Methods: Cancer 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 × 10 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.

Results: A 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.

Conclusions: In 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.
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http://dx.doi.org/10.1016/j.jaccao.2020.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781291PMC
November 2020

Two Decades of Global Progress in Authorized Advanced Therapy Medicinal Products: An Emerging Revolution in Therapeutic Strategies.

Front Cell Dev Biol 2020 17;8:547653. Epub 2020 Dec 17.

Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.

The introduction of advanced therapy medicinal products (ATMPs) to the global pharma market has been revolutionizing the pharmaceutical industry and has opened new routes for treating various types of cancers and incurable diseases. In the past two decades, a noticeable part of clinical practices has been devoting progressively to these products. The first step to develop such an ATMP product is to be familiar with other approved products to obtain a general view about this industry trend. The present paper depicts an overall perspective of approved ATMPs in different countries, while reflecting the degree of their success in a clinical point of view and highlighting their main safety issues and also related market size as a whole. In this regard, published articles regarding safety, efficacy, and market size of approved ATMPs were reviewed using the search engines PubMed, Scopus, and Google Scholar. For some products which the related papers were not available, data on the relevant company website were referenced. In this descriptive study, we have introduced and classified approved cell, gene, and tissue engineering-based products by different regulatory agencies, along with their characteristics, manufacturer, indication, approval date, related regulatory agency, dosage, product description, price and published data about their safety and efficacy. In addition, to gain insights about the commercial situation of each product, we have gathered accessible sale reports and market size information that pertain to some of these products.
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http://dx.doi.org/10.3389/fcell.2020.547653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773756PMC
December 2020

Clinical effects of administering leukemia-specific donor T cells to patients with AML/MDS post-allogeneic transplant.

Blood 2020 Dec 3. Epub 2020 Dec 3.

Baylor College of Medicine, Houston, Texas, United States.

Relapse after allogeneic hematopoietic stem-cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusions of unselected donor lymphocytes (DLIs) are used to enhance the graft-versus-leukemia (GVL) effect, as treatment for relapsed disease. However, as the infused lymphocytes are not selected for leukemia-specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease(GVHD) due to the concurrent transfer of allo-reactive lymphocytes. Thus, to minimize GVHD and maximize GVL we selectively activated and expanded stem-cell donor-derived T cells that were reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, NY-ESO-1). Products were successfully generated from 29 HCT donors, and they demonstrated multi-leukemia antigen specificity (mLSTs). In contrast to DLIs, mLSTs selectively recognized and killed leukemia-antigen-pulsed cells with no activity against recipient-derived normal cells in vitro. We have now administered escalating doses of these mLSTs (0.5-10x107 cells/m2) to 25 trial enrollees with AML/MDS after HCT, 17 of whom were at high risk for relapse and 8 of whom had relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD up to a dose of 10x107 cells/m2. We observed anti-leukemia effects in vivo that translated into not yet reached median LFS and OS at 1.9 years of follow-up among survivors, evidence of sustained immune pressure and objective responses in the active disease cohort (1 CR and 1 PR). In conclusion, mLSTs are safe and promising for the prevention or treatment of AML/MDS following HCT.
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http://dx.doi.org/10.1182/blood.2020009471DOI Listing
December 2020

The safety and clinical effects of administering a multiantigen-targeted T cell therapy to patients with multiple myeloma.

Sci Transl Med 2020 07;12(554)

Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX 77030, USA.

Multiple myeloma (MM) is an almost always incurable malignancy of plasma cells. Despite the advent of new therapies, most patients eventually relapse or become treatment-refractory. Consequently, therapies with nonoverlapping mechanisms of action that are nontoxic and provide long-term benefit to patients with MM are greatly needed. To this end, we clinically tested an autologous multitumor-associated antigen (mTAA)-specific T cell product for the treatment of patients with high-risk, relapsed or refractory MM. In this study, we expanded polyclonal T cells from 23 patients with MM. T cells whose native T cell receptors were reactive toward five myeloma-expressed target TAAs (PRAME, SSX2, MAGEA4, Survivin, and NY-ESO-1) were enriched ex vivo. To date, we have administered escalating doses of these nonengineered mTAA-specific T cells (0.5 × 10 to 2 × 10 cells/m) to 21 patients with MM, 9 of whom were at high risk of relapse after a median of 3 lines of prior therapy and 12 with active, relapsed or refractory disease after a median of 3.5 prior lines. The cells were well tolerated, with only two transient, grade III infusion-related adverse events. Furthermore, patients with active relapsed or refractory myeloma enjoyed a longer than expected progression-free survival and responders included three patients who achieved objective responses concomitant with detection of functional TAA-reactive T cell clonotypes derived from the infused mTAA product.
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http://dx.doi.org/10.1126/scitranslmed.aaz3339DOI Listing
July 2020

Anti-CD30 CAR-T Cell Therapy in Relapsed and Refractory Hodgkin Lymphoma.

J Clin Oncol 2020 11 23;38(32):3794-3804. Epub 2020 Jul 23.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Purpose: Chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies has proved to be effective. We show how the same approach of CAR T cells specific for CD30 (CD30.CAR-Ts) can be used to treat Hodgkin lymphoma (HL).

Methods: We conducted 2 parallel phase I/II studies (ClinicalTrials.gov identifiers: NCT02690545 and NCT02917083) at 2 independent centers involving patients with relapsed or refractory HL and administered CD30.CAR-Ts after lymphodepletion with either bendamustine alone, bendamustine and fludarabine, or cyclophosphamide and fludarabine. The primary end point was safety.

Results: Forty-one patients received CD30.CAR-Ts. Treated patients had a median of 7 prior lines of therapy (range, 2-23), including brentuximab vedotin, checkpoint inhibitors, and autologous or allogeneic stem cell transplantation. The most common toxicities were grade 3 or higher hematologic adverse events. Cytokine release syndrome was observed in 10 patients, all of which were grade 1. No neurologic toxicity was observed. The overall response rate in the 32 patients with active disease who received fludarabine-based lymphodepletion was 72%, including 19 patients (59%) with complete response. With a median follow-up of 533 days, the 1-year progression-free survival and overall survival for all evaluable patients were 36% (95% CI, 21% to 51%) and 94% (95% CI, 79% to 99%), respectively. CAR-T cell expansion in vivo was cell dose dependent.

Conclusion: Heavily pretreated patients with relapsed or refractory HL who received fludarabine-based lymphodepletion followed by CD30.CAR-Ts had a high rate of durable responses with an excellent safety profile, highlighting the feasibility of extending CAR-T cell therapies beyond canonical B-cell malignancies.
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http://dx.doi.org/10.1200/JCO.20.01342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655020PMC
November 2020

T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells.

Clin Cancer Res 2019 12 26;25(24):7340-7350. Epub 2019 Sep 26.

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children's Hospital, Houston, Texas.

Purpose: Current protocols for CD19 chimeric antigen receptor-expressing T cells (CD19.CAR-T cells) require recipients to tolerate preinfusion cytoreductive chemotherapy, and the presence of sufficient target antigen on normal or malignant B cells.

Patients And Methods: We investigated whether additional stimulation of CD19.CAR-T cells through their native receptors can substitute for cytoreductive chemotherapy, inducing expansion and functional persistence of CD19.CAR-T even in patients in remission of B-cell acute lymphocytic leukemia. We infused a low dose of CD19.CAR-modified virus-specific T cells (CD19.CAR-VST) without prior cytoreductive chemotherapy into 8 patients after allogeneic stem cell transplant.

Results: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reactivation, up to 30,000-fold expansion of CD19.CAR-VSTs was observed, with depletion of CD19 B cells. Five patients remain in remission at 42-60+ months.

Conclusions: Dual T-cell receptor and CAR stimulation can thus potentiate effector cell expansion and CAR-target cell killing, even when infusing low numbers of effector cells without cytoreduction.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062259PMC
December 2019

"Mini" bank of only 8 donors supplies CMV-directed T cells to diverse recipients.

Blood Adv 2019 09;3(17):2571-2580

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children's Hospital, Houston, TX.

Cytomegalovirus (CMV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), and standard antiviral therapies are associated with significant side effects and development of drug-resistant mutants. Adoptively transferred donor-derived CMV-specific T cells (CMVSTs) can provide an alternative treatment modality with few side effects but are not widely available due to their patient-specific nature. Here we report the establishment and use of a bank of CMVSTs derived from just 8 CMV-seropositive donors, with HLA types representing the diverse US population, as an "off-the-shelf" therapy to treat drug-refractory infections. To date, we have screened 29 patients for study participation and identified a suitable line, with ≥2 of 8 shared HLA antigens, for 28 (96.6%) patients with a median of 4 shared HLA antigens. Of these, 10 patients with persistent/refractory CMV infections or disease were eligible for treatment; a single infusion of cells produced 3 partial responses and 7 complete responses, for a cumulative response rate of 100% (95% confidence interval, 69.2-100) with no graft-versus-host disease, graft failure, or cytokine release syndrome. Potential wider use of the tested CMVSTs across transplant centers is made more feasible by our ability to produce sufficient material to generate cells for >2000 infusions from a single donor collection. Our data indicate that a "mini" bank of CMVSTs prepared from just 8 well-chosen third-party donors can supply the majority of patients with an appropriately matched line that produces safe and effective anti-CMV activity post-HSCT.
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http://dx.doi.org/10.1182/bloodadvances.2019000371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737421PMC
September 2019

Safety and feasibility of virus-specific T cells derived from umbilical cord blood in cord blood transplant recipients.

Blood Adv 2019 07;3(14):2057-2068

Center for Cancer and Immunology Research, Children's National Health System and Department of Pediatrics, The George Washington University, Washington, DC.

Adoptive transfer of virus-specific T cells (VSTs) has been shown to be safe and effective in stem cell transplant recipients. However, the lack of virus-experienced T cells in donor cord blood (CB) has prevented the development of ex vivo expanded donor-derived VSTs for recipients of this stem cell source. Here we evaluated the feasibility and safety of ex vivo expansion of CB T cells from the 20% fraction of the CB unit in pediatric patients receiving a single CB transplant (CBT). In 2 clinical trials conducted at 2 separate sites, we manufactured CB-derived multivirus-specific T cells (CB-VSTs) targeting Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV) for 18 (86%) of 21 patients demonstrating feasibility. Manufacturing for 2 CB-VSTs failed to meet lot release because of insufficient cell recovery, and there was 1 sterility breach during separation of the frozen 20% fraction. Delayed engraftment was not observed in patients who received the remaining 80% fraction for the primary CBT. There was no grade 3 to 4 acute graft-versus-host disease (GVHD) associated with the infusion of CB-VSTs. None of the 7 patients who received CB-VSTs as prophylaxis developed end-organ disease from CMV, EBV, or adenovirus. In 7 patients receiving CB-VSTs for viral reactivation or infection, only 1 patient developed end-organ viral disease, which was in an immune privileged site (CMV retinitis) and occurred after steroid therapy for GVHD. Finally, we demonstrated the long-term persistence of adoptively transferred CB-VSTs using T-cell receptor-Vβ clonotype tracking, suggesting that CB-VSTs are a feasible addition to antiviral pharmacotherapy.
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http://dx.doi.org/10.1182/bloodadvances.2019000201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650740PMC
July 2019

In Vivo Fate and Activity of Second- versus Third-Generation CD19-Specific CAR-T Cells in B Cell Non-Hodgkin's Lymphomas.

Mol Ther 2018 12 13;26(12):2727-2737. Epub 2018 Sep 13.

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital and Texas Children's Hospital, Houston, TX 77030, USA; Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Second-generation (2G) chimeric antigen receptors (CARs) targeting CD19 are highly active against B cell malignancies, but it is unknown whether any of the costimulatory domains incorporated in the CAR have superior activity to others. Because CD28 and 4-1BB signaling activate different pathways, combining them in a single third-generation (3G) CAR may overcome the limitations of each individual costimulatory domain. We designed a clinical trial in which two autologous CD19-specific CAR-transduced T cell products (CD19.CARTs), 2G (with CD28 only) and 3G (CD28 and 4-1BB), were infused simultaneously in 16 patients with relapsed or refractory non-Hodgkin's lymphoma. 3G CD19.CARTs had superior expansion and longer persistence than 2G CD19.CARTs. This difference was most striking in the five patients with low disease burden and few circulating normal B cells, in whom 2G CD19.CARTs had limited expansion and persistence and correspondingly reduced area under the curve. Of the 11 patients with measurable disease, three achieved complete responses and three had partial responses. Cytokine release syndrome occurred in six patients but was mild, and no patient required anti-IL-6 therapy. Hence, 3G CD19.CARTs combining 4-1BB with CD28 produce superior CART expansion and may be of particular value when treating low disease burden in patients whose normal B cells are depleted by prior therapy.
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http://dx.doi.org/10.1016/j.ymthe.2018.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277484PMC
December 2018

EBV/LMP-specific T cells maintain remissions of T- and B-cell EBV lymphomas after allogeneic bone marrow transplantation.

Blood 2018 11 27;132(22):2351-2361. Epub 2018 Sep 27.

Center for Cancer and Immunology Research, Children's National Health System and George Washington University, Washington, DC.

Autologous T cells targeting Epstein-Barr virus (EBV) latent membrane proteins (LMPs) have shown safety and efficacy in the treatment of patients with type 2 latency EBV-associated lymphomas for whom standard therapies have failed, including high-dose chemotherapy followed by autologous stem-cell rescue. However, the safety and efficacy of allogeneic donor-derived LMP-specific T cells (LMP-Ts) have not been established for patients who have undergone allogeneic hematopoietic stem-cell transplantation (HSCT). Therefore, we evaluated the safety and efficacy of donor-derived LMP-Ts in 26 patients who had undergone allogeneic HSCT for EBV-associated natural killer/T-cell or B-cell lymphomas. Seven patients received LMP-Ts as therapy for active disease, and 19 were treated with adjuvant therapy for high-risk disease. There were no immediate infusion-related toxicities, and only 1 dose-limiting toxicity potentially related to T-cell infusion was seen. The 2-year overall survival (OS) was 68%. Additionally, patients who received T-cell therapy while in complete remission after allogeneic HSCT had a 78% OS at 2 years. Patients treated for B-cell disease (n = 10) had a 2-year OS of 80%. Patients with T-cell disease had a 2-year OS of 60%, which suggests an improvement compared with published posttransplantation 2-year OS rates of 30% to 50%. Hence, this study shows that donor-derived LMP-Ts are a safe and effective therapy to prevent relapse after transplantation in patients with B cell- or T cell-derived EBV-associated lymphoma or lymphoproliferative disorder and supports the infusion of LMP-Ts as adjuvant therapy to improve outcomes in the posttransplantation setting. These trials were registered at www.clinicaltrials.gov as #NCT00062868 and #NCT01956084.
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http://dx.doi.org/10.1182/blood-2018-07-863654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265652PMC
November 2018

Regulation of Regenerative Medicine Products.

Authors:
Adrian P Gee

Adv Exp Med Biol 2018 ;1098:189-198

cGMP Facilities, Center for Cell & Gene Therapy, Baylor College of Medicine, Houston, TX, USA.

Cellular therapies have moved to the forefront based upon promising results from clinical trials using both chimeric antigen receptor T lymphocytes to treat leukemia and other cell types to restore structure and function to tissues that have been damaged by disease or physical injury. The pace at which these treatments have evolved has posed a regulatory challenge to agencies, such as the Food and Drug Administration (FDA). This chapter describes how a specific regulatory strategy was developed and how it has evolved in response to the demand for these new therapies.
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http://dx.doi.org/10.1007/978-3-319-97421-7_10DOI Listing
February 2019

Rationale and Design of the SENECA (StEm cell iNjECtion in cAncer survivors) Trial.

Am Heart J 2018 07 4;201:54-62. Epub 2018 Apr 4.

University of Kansas School of Medicine, Kansas City, Kansas.

Objectives: SENECA (StEm cell iNjECtion in cAncer survivors) is a phase I, randomized, double-blind, placebo-controlled study to evaluate the safety and feasibility of delivering allogeneic mesenchymal stromal cells (allo-MSCs) transendocardially in subjects with anthracycline-induced cardiomyopathy (AIC).

Background: AIC is an incurable and often fatal syndrome, with a prognosis worse than that of ischemic or nonischemic cardiomyopathy. Recently, cell therapy with MSCs has emerged as a promising new approach to repair damaged myocardium.

Methods: The study population is 36 cancer survivors with a diagnosis of AIC, left ventricular (LV) ejection fraction ≤40%, and symptoms of heart failure (NYHA class II-III) on optimally-tolerated medical therapy. Subjects must be clinically free of cancer for at least two years with a ≤ 30% estimated five-year risk of recurrence. The first six subjects participated in an open-label, lead-in phase and received 100 million allo-MSCs; the remaining 30 will be randomized 1:1 to receive allo-MSCs or vehicle via 20 transendocardial injections. Efficacy measures (obtained at baseline, 6 months, and 12 months) include MRI evaluation of LV function, LV volumes, fibrosis, and scar burden; assessment of exercise tolerance (six-minute walk test) and quality of life (Minnesota Living with Heart Failure Questionnaire); clinical outcomes (MACE and cumulative days alive and out of hospital); and biomarkers of heart failure (NT-proBNP).

Conclusions: This is the first clinical trial using direct cardiac injection of cells for the treatment of AIC. If administration of allo-MSCs is found feasible and safe, SENECA will pave the way for larger phase II/III studies with therapeutic efficacy as the primary outcome.
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http://dx.doi.org/10.1016/j.ahj.2018.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282462PMC
July 2018

GMP CAR-T cell production.

Authors:
Adrian P Gee

Best Pract Res Clin Haematol 2018 06 2;31(2):126-134. Epub 2018 Feb 2.

cGMP Facilities, Center for Cell & Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

The clinical success achieved using CD19-directed CAR-T cells has stimulated many academic institutions to explore the feasibility of manufacturing these, and other CAR-T cells, in-house. This article reviews the issues that must be addressed in order to achieve this goal. It includes the manufacturing infrastructure, the regulatory environment, practical aspects of production, and the costs involved.
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http://dx.doi.org/10.1016/j.beha.2018.01.002DOI Listing
June 2018

Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma.

J Clin Oncol 2018 04 9;36(11):1128-1139. Epub 2018 Jan 9.

Catherine M. Bollard, Tamara Tripic, Gianpietro Dotti, Stephen Gottschalk, Vicky Torrano, Olga Dakhova, George Carrum, Carlos A. Ramos, Adrian P. Gee, Malcolm K. Brenner, Helen E. Heslop, and Cliona M. Rooney, Houston Methodist Hospital, and Texas Children's Hospital; Catherine M. Bollard, Gianpietro Dotti, Stephen Gottschalk, George Carrum, Carlos A. Ramos, Hao Liu, Meng-Fen Wu, Andrea N. Marcogliese, Adrian P. Gee, Malcolm K. Brenner, Helen E. Heslop, and Cliona M. Rooney, Baylor College of Medicine, Houston, TX; Catherine M. Bollard, Conrad Russell Cruz, and Cecilia Barese, Children's National Health System, Washington, DC; Youli Zu and Daniel Y. Lee, Weill Medical College of Cornell University; and Owen O'Connor, Columbia University College of Physicians and Surgeons, The New York Presbyterian Hospital, New York, NY.

Purpose Transforming growth factor-β (TGF-β) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-β receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 10 and 1.5 × 10 cells/m of DNRII-expressing T cells with specificity for the Epstein Barr virus-derived tumor antigens, latent membrane protein (LMP)-1 and LMP-2 (DNRII-LSTs). Lymphodepleting chemotherapy was not used before infusion. Results DNRII-LSTs were resistant to otherwise inhibitory concentrations of TGF-β in vitro and retained their tumor antigen-specific activity. After infusion, the signal from transgenic T cells in peripheral blood increased up to 100-fold as measured by quantitative polymerase chain reaction for the transgene, with a corresponding increase in the frequency of functional LMP-specific T cells. Expansion was not associated with any acute or long-term toxicity. DNRII-LSTs persisted for up to ≥ 4 years. Four of the seven evaluable patients with active disease achieved clinical responses that were complete and ongoing in two patients at > 4 years, including in one patient who achieved only a partial response to unmodified tumor-directed T cells. Conclusion TGF-β-resistant tumor-specific T cells safely expand and persist in patients with Hodgkin lymphoma without lymphodepleting chemotherapy before infusion. DNRII-LSTs can induce complete responses even in patients with resistant disease. Expression of DNRII may be useful for the many other tumors that exploit this potent immune evasion mechanism.
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http://dx.doi.org/10.1200/JCO.2017.74.3179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891126PMC
April 2018

Absence of Replication-Competent Retrovirus in Vectors, T Cell Products, and Patient Follow-Up Samples.

Mol Ther 2018 01 9;26(1):6-7. Epub 2017 Dec 9.

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital and Texas Children's Hospital, Houston, TX, USA. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2017.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763145PMC
January 2018

TIME Trial: Effect of Timing of Stem Cell Delivery Following ST-Elevation Myocardial Infarction on the Recovery of Global and Regional Left Ventricular Function: Final 2-Year Analysis.

Circ Res 2018 02 5;122(3):479-488. Epub 2017 Dec 5.

From the Department of Cardiology, Minneapolis Heart Institute Foundation, Abbott Northwestern Hospital, MN (J.H.T., T.D.H.); Department of Medicine, University of Minnesota School of Medicine, Minneapolis (J.H.T., G.R.); Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA (T.D.H.); Department of Medicine, College of Medicine, University of Florida, Gainesville (C.J.P., J.R.F.); Stem Cell Center (J.T.W., E.C.P.), and Regenerative Medicine Research (D.A.T.), Texas Heart Institute, CHI St. Luke's Health Baylor College of Medicine Medical Center, Houston; Franciscan Saint Francis Health, Indianapolis, IN (A.C.); Department of Cardiovascular Medicine, Stanford University School of Medicine, CA (P.C.Y.); Department of Cardiology, Wake Forest University School of Medicine, Winston-Salem, NC (D.X.M.Z.); Department of Cardiovascular Medicine, Cleveland Clinic Foundation, OH (S.G.E.); Summa Health Heart and Vascular Institute, Akron, OH (M.S.P.); Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN (A.K.H.); Metropolitan Heart and Vascular Institute, Mercy Hospital, Coon Rapids, MN (J.C.C.); United Heart and Vascular Clinic (K.W.B.); Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX (A.P.G.); Coordinating Center for Clinical Trials, UTHealth School of Public Health, Houston, TX (L.M.); National Heart Lung, and Blood Institute, Bethesda, MD (R.F.E.); and University of Kansas School of Medicine (R.D.S.).

Rationale: The TIME trial (Timing in Myocardial Infarction Evaluation) was the first cell therapy trial sufficiently powered to determine if timing of cell delivery after ST-segment-elevation myocardial infarction affects recovery of left ventricular (LV) function.

Objective: To report the 2-year clinical and cardiac magnetic resonance imaging results and their modification by microvascular obstruction.

Methods And Results: TIME was a randomized, double-blind, placebo-controlled trial comparing 150 million bone marrow mononuclear cells versus placebo in 120 patients with anterior ST-segment-elevation myocardial infarctions resulting in LV dysfunction. Primary end points included changes in global (LV ejection fraction) and regional (infarct and border zone) function. Secondary end points included changes in LV volumes, infarct size, and major adverse cardiac events. Here, we analyzed the continued trajectory of these measures out to 2 years and the influence of microvascular obstruction present at baseline on these long-term outcomes. At 2 years (n=85), LV ejection fraction was similar in the bone marrow mononuclear cells (48.7%) and placebo groups (51.6%) with no difference in regional LV function. Infarct size and LV mass decreased ≥30% in each group at 6 months and declined gradually to 2 years. LV volumes increased ≈10% at 6 months and remained stable to 2 years. Microvascular obstruction was present in 48 patients at baseline and was associated with significantly larger infarct size (56.5 versus 36.2 g), greater adverse LV remodeling, and marked reduction in LV ejection fraction recovery (0.2% versus 6.2%).

Conclusions: In one of the longest serial cardiac magnetic resonance imaging analyses of patients with large anterior ST-segment-elevation myocardial infarctions, bone marrow mononuclear cells administration did not improve recovery of LV function over 2 years. Microvascular obstruction was associated with reduced recovery of LV function, greater adverse LV remodeling, and more device implantations. The use of cardiac magnetic resonance imaging leads to greater dropout of patients over time because of device implantation in patients with more severe LV dysfunction resulting in overestimation of clinical stability of the cohort.

Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00684021.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.311466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805626PMC
February 2018

Clinical and immunological responses after CD30-specific chimeric antigen receptor-redirected lymphocytes.

J Clin Invest 2017 Sep 14;127(9):3462-3471. Epub 2017 Aug 14.

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital and Texas Children's Hospital, Houston, Texas, USA.

Background: Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity.

Methods: We conducted a phase I dose escalation study in which 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-Ts) encoding the CD28 costimulatory endodomain. Three dose levels, from 0.2 × 108 to 2 × 108 CD30.CAR-Ts/m2, were infused without a conditioning regimen. All other therapy for malignancy was discontinued at least 4 weeks before CD30.CAR-T infusion. Seven patients had previously experienced disease progression while being treated with brentuximab.

Results: No toxicities attributable to CD30.CAR-Ts were observed. Of 7 patients with relapsed HL, 1 entered complete response (CR) lasting more than 2.5 years after the second infusion of CD30.CAR-Ts, 1 remained in continued CR for almost 2 years, and 3 had transient stable disease. Of 2 patients with ALCL, 1 had a CR that persisted 9 months after the fourth infusion of CD30.CAR-Ts. CD30.CAR-T expansion in peripheral blood peaked 1 week after infusion, and CD30.CAR-Ts remained detectable for over 6 weeks. Although CD30 may also be expressed by normal activated T cells, no patients developed impaired virus-specific immunity.

Conclusion: CD30.CAR-Ts are safe and can lead to clinical responses in patients with HL and ALCL, indicating that further assessment of this therapy is warranted.

Trial Registration: ClinicalTrials.gov NCT01316146.

Funding: National Cancer Institute (3P50CA126752, R01CA131027 and P30CA125123), National Heart, Lung, and Blood Institute (R01HL114564), and Leukemia and Lymphoma Society (LLSTR 6227-08).
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http://dx.doi.org/10.1172/JCI94306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669573PMC
September 2017

Off-the-Shelf Virus-Specific T Cells to Treat BK Virus, Human Herpesvirus 6, Cytomegalovirus, Epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell Transplantation.

J Clin Oncol 2017 Nov 7;35(31):3547-3557. Epub 2017 Aug 7.

Ifigeneia Tzannou, Anastasia Papadopoulou, Swati Naik, Kathryn Leung, Caridad A. Martinez, Carlos A. Ramos, George Carrum, Ghadir Sasa, Premal Lulla, Ayumi Watanabe, Manik Kuvalekar, Adrian P. Gee, Bambi J. Grilley, Robert A. Krance, Stephen Gottschalk, Malcolm K. Brenner, Cliona M. Rooney, Helen E. Heslop, Ann M. Leen, and Bilal Omer, Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital, and Houston Methodist Hospital; Meng-Fen Wu and Hao Liu, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX.

Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.
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http://dx.doi.org/10.1200/JCO.2017.73.0655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662844PMC
November 2017

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

Sci Rep 2017 04 27;7:46731. Epub 2017 Apr 27.

Cell Processing Section, Department of Transfusion Medicine, Clinical Center; National Institutes of Health, Bethesda, Maryland, USA.

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.
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http://dx.doi.org/10.1038/srep46731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406832PMC
April 2017

Evaluation of Cell Therapy on Exercise Performance and Limb Perfusion in Peripheral Artery Disease: The CCTRN PACE Trial (Patients With Intermittent Claudication Injected With ALDH Bright Cells).

Circulation 2017 Apr 16;135(15):1417-1428. Epub 2017 Feb 16.

From Texas Heart Institute (E.C.P., J.T.W., A.G., D.A.T., M.R., B.C., N.P.), CHI St. Luke's Health (R.M.), Houston; Indiana University School of Medicine, Indianapolis (M.P.M., K.L.M., P.G.); University of Louisville, KY (R.B., J.L., S.W.); Stanford University School of Medicine, CA (P.C.Y., N.J.L., R.L.D., F.K., E.G.R., M.R.S.); Minneapolis Heart Institute Foundation at Abbott Northwestern Hospital, MN (J.A., J.H.T., J.G.); Cedars-Sinai Heart Institute, Los Angeles, CA (T.D.H.); University of Florida School of Medicine, Gainesville (C.J.P., R.D.A., S.B., D.L., E.H.); University of Minnesota Medical School, Minneapolis (G.R., B.B.-D., E.C., A.T.H.); University of Miami Miller School of Medicine, FL (O.V., J.M.H., I.H.S., D.D.); Orlando Health, FL (V.S.K.); University of Colorado School of Medicine and CPC Clinical Research, Aurora (W.R.H.); Johns Hopkins University, Baltimore, MD (B.A.- V., J.A.L.); Baylor College of Medicine, Houston, TX (A.P.G., A.G.D., J. Bloom, S.R.); University of Texas School of Public Health, Houston (J. Bettencourt, D.L., L.P., L.S., M.C., S.L.S., R.W.V., L.M.); National Institutes of Health, National Heart, Lung, and Blood Institute, Bethesda, MD (R.F.E.); and University of Kansas School of Medicine, Kansas City (R.D.S.).

Background: Atherosclerotic peripheral artery disease affects 8% to 12% of Americans >65 years of age and is associated with a major decline in functional status, increased myocardial infarction and stroke rates, and increased risk of ischemic amputation. Current treatment strategies for claudication have limitations. PACE (Patients With Intermittent Claudication Injected With ALDH Bright Cells) is a National Heart, Lung, and Blood Institute-sponsored, randomized, double-blind, placebo-controlled, phase 2 exploratory clinical trial designed to assess the safety and efficacy of autologous bone marrow-derived aldehyde dehydrogenase bright (ALDHbr) cells in patients with peripheral artery disease and to explore associated claudication physiological mechanisms.

Methods: All participants, randomized 1:1 to receive ALDHbr cells or placebo, underwent bone marrow aspiration and isolation of ALDHbr cells, followed by 10 injections into the thigh and calf of the index leg. The coprimary end points were change from baseline to 6 months in peak walking time (PWT), collateral count, peak hyperemic popliteal flow, and capillary perfusion measured by magnetic resonance imaging, as well as safety.

Results: A total of 82 patients with claudication and infrainguinal peripheral artery disease were randomized at 9 sites, of whom 78 had analyzable data (57 male, 21 female patients; mean age, 66±9 years). The mean±SEM differences in the change over 6 months between study groups for PWT (0.9±0.8 minutes; 95% confidence interval [CI] -0.6 to 2.5; =0.238), collateral count (0.9±0.6 arteries; 95% CI, -0.2 to 2.1; P=0.116), peak hyperemic popliteal flow (0.0±0.4 mL/s; 95% CI, -0.8 to 0.8; =0.978), and capillary perfusion (-0.2±0.6%; 95% CI, -1.3 to 0.9; P=0.752) were not significant. In addition, there were no significant differences for the secondary end points, including quality-of-life measures. There were no adverse safety outcomes. Correlative relationships between magnetic resonance imaging measures and PWT were not significant. A post hoc exploratory analysis suggested that ALDHbr cell administration might be associated with an increase in the number of collateral arteries (1.5±0.7; 95% CI, 0.1-2.9; =0.047) in participants with completely occluded femoral arteries.

Conclusions: ALDHbr cell administration did not improve PWT or magnetic resonance outcomes, and the changes in PWT were not associated with the anatomic or physiological magnetic resonance imaging end points. Future peripheral artery disease cell therapy investigational trial design may be informed by new anatomic and perfusion insights.

Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01774097.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.025707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388585PMC
April 2017

Clinical responses with T lymphocytes targeting malignancy-associated κ light chains.

J Clin Invest 2016 07 6;126(7):2588-96. Epub 2016 Jun 6.

Background: Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan-B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment.

Methods: We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used.

Results: κ.CART expansion peaked 1-2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2-17 months. No toxicities attributable to κ.CARTs were observed.

Conclusion: κ.CART infusion is feasible and safe and can lead to complete clinical responses.

Trial Registration: ClinicalTrials.gov NCT00881920.

Funding: National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018.
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http://dx.doi.org/10.1172/JCI86000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922690PMC
July 2016

Large-Scale Culture and Genetic Modification of Human Natural Killer Cells for Cellular Therapy.

Methods Mol Biol 2016 ;1441:195-202

Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital, Houston Methodist Hospital, 1102 Bates St, Houston, TX, 77030, USA.

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.
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http://dx.doi.org/10.1007/978-1-4939-3684-7_16DOI Listing
December 2017

International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials.

Cytotherapy 2016 Feb 23;18(2):151-9. Epub 2015 Dec 23.

UMR5273 STROMALab CNRS/EFS/UPS-INSERM U1031, Toulouse, France.

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection.
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http://dx.doi.org/10.1016/j.jcyt.2015.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745114PMC
February 2016

Quantitative activation suppression assay to evaluate human bone marrow-derived mesenchymal stromal cell potency.

Cytotherapy 2015 Dec 28;17(12):1675-86. Epub 2015 Sep 28.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.

Background Aims: With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs).

Methods: Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression.

Results: The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression.

Conclusions: This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay.
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http://dx.doi.org/10.1016/j.jcyt.2015.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655179PMC
December 2015

Quantitative activation suppression assay to evaluate human bone marrow-derived mesenchymal stromal cell potency.

Cytotherapy 2015 Dec 28;17(12):1675-86. Epub 2015 Sep 28.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.

Background Aims: With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs).

Methods: Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression.

Results: The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression.

Conclusions: This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay.
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http://dx.doi.org/10.1016/j.jcyt.2015.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655179PMC
December 2015

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

J Immunother Cancer 2015 18;3. Epub 2015 Feb 18.

Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.

Background: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2).

Results: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro.

Conclusions: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).
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http://dx.doi.org/10.1186/s40425-015-0049-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346112PMC
March 2015

A reproducible immunopotency assay to measure mesenchymal stromal cell-mediated T-cell suppression.

Cytotherapy 2015 Feb 21;17(2):140-51. Epub 2014 Nov 21.

Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA; University of Wisconsin Carbone Cancer Center, Madison, Wisconsin, USA. Electronic address:

Background Aims: The T-cell suppressive property of bone marrow-derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression.

Methods: At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation. We measured MSC-induced suppression of CD4+ T-cell proliferation at various effector-to-target cell ratios with the use of defined peripheral blood mononuclear cells and in parallel compared with a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product.

Results: Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T-cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T-cell proliferation, with immunopotency assay values ranging from 27% to 88%. However, MSC suppression did not correlate with human leukocyte antigen-DR expression.

Conclusions: We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy.
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http://dx.doi.org/10.1016/j.jcyt.2014.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297551PMC
February 2015

CD34 measurement: setting standards.

Authors:
Adrian P Gee

Cytotherapy 2014 Nov;16(11):1451-1452

Center for Cell & Gene Therapy, Baylor College of Medicine, Houston, Texas, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jcyt.2014.09.001DOI Listing
November 2014