Adoptive Therapy Using Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells to Manufacture T Cells Expressing CD19-Specific Chimeric Antigen Receptor

Abstract Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies. Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results: To date we have successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted. Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse. Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. Figure: Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Figure:. Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Disclosures No relevant conflicts of interest to declare.

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Adoptive Immunotherapy Following Umbilical Cord Blood Transplantation Using The Sleeping Beauty System and Artificial Antigen Presenting Cells To Generate Donor-Derived T Cells Expressing a CD19-Specific Chimeric Antigen Receptor

Blood ◽

10.1182/blood.v122.21.4208.4208 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4208-4208

Author(s):

Partow Kebriaei ◽

Helen Huls ◽

Harjeet Singh ◽

Simon Olivares ◽

Matthew Figliola ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Chimeric Antigen Receptor ◽

Umbilical Cord Blood ◽

Sleeping Beauty ◽

Car T Cells ◽

Artificial Antigen ◽

Car T ◽

Antigen Presenting

Abstract Background The ability to transplant across HLA disparities makes allogeneic umbilical cord blood (UCB) an attractive graft source for hematopoietic stem-cell transplantation (HSCT). Disease relapse remains a limitation, and adoptive transfer of tumor-specific T cells post UCB HSCT has not been feasible due to the functionally naïve CB T cells, and the small size as well as anonymity of the donor. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28) on UCB-derived T cells manufactured in compliance with current good manufacturing practice (cGMP). Methods After thawed UCB units are washed for clinical infusion 5% to 10% of cells are used to generate CAR+ T cells. The mononuclear cells are electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR are retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. Enrolled patients on our phase I trial receive two UCB units, thus two genetically modified T-cell products are made for each patient. We infuse thawed donor-derived CD19-specific CAR+ T cells from the dominant CB unit based on peripheral blood chimerism on days 40-100 post transplant in the adjuvant setting after double UCB HSCT Results To date we have successfully manufactured 8 products for 4 patients (ALL n=3, NHL=1) enrolled on trial. The median number of T cells in the starting CB aliquot was 8.6x106 (range, 2.5x106 to 54.8x106) with final modified T cell count at median 3x109 (range,1.7x108 to 4.1x1010) at time of cryopreservation days 28-32. In the final product, the median CD19-CAR+ cell purity by flow was 88% (range, 81.9% to 95.8%). The modified T cell product consisted of median 97.3% CD3+, 2.7 CD3-/CD56+ cells. All of the products exhibited CD19-specific killing by chromium assay as illustrated (Figure). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing. One patient with ALL has been infused to date with a T cell dose of 106T cells/m2 and no toxicity has been observed. The patient remains alive and in continued molecular remission at 111 days post HSCT. Conclusion We combined the SB system and aAPC-mediated propagation of T cells to successfully manufacture disease-specific T cells from small aliquots of UCB used to restore hematopoiesis. Importantly, this approach allows us to employ adoptive therapy to enhance the graft-versus-tumor effect in UCB HSCT as an approach to improve overall survival for these recipients. Accrual to the trial continues and updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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Clinical Development and Manufacture of Chimeric Antigen Receptor T cells and the Role of Leukapheresis

European Oncology & Haematology ◽

10.17925/eoh.2017.13.01.28 ◽

2017 ◽

Vol 13 (01) ◽

pp. 28 ◽

Cited By ~ 2

Author(s):

Andrew Fesnak ◽

Una O’Doherty ◽

◽

Keyword(s):

T Cells ◽

T Cell ◽

Chimeric Antigen Receptor ◽

Large Scale ◽

Mononuclear Cells ◽

Antigen Receptor ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T

Adoptive transfer of chimeric antigen receptor (CAR) T cells is a powerful targeted immunotherapeutic technique. CAR T cells are manufactured by harvesting mononuclear cells, typically via leukapheresis from a patient’s blood, then activating, modifying the T cells to express a transgene encoding a tumour-specific CAR, and infusing the CAR T cells into the patient. Gene transfer is achieved through the use of retroviral or lentiviral vectors, although non-viral delivery systems are being investigated. This article discusses the challenges associated with each stage of this process. Despite the need for a consistent end product, there is inherent variability in cellular material obtained from critically ill patients who have been exposed to cytotoxic therapy. It is important to carefully select target antigens to maximise effect and minimise toxicity. Various types of CAR T cell toxicity have been documented: this includes “on target, on tumour”, “on target, off tumour” and “off target” toxicity. A growing body of clinical evidence supports the efficacy and safety of CAR T cell therapy; CAR T cells targeting CD19 in B cell leukemias are the best-studied therapy to date. However, providing personalised therapy on a large scale remains challenging; a future aim is to produce a universal “off the shelf” CAR T cell.

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Donor-Derived CD19 CAR Cytokine Induced Killer (CIK) Cells Engineered with Sleeping Beauty Transposon for Relapsed B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Blood ◽

10.1182/blood-2019-125894 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 200-200 ◽

Cited By ~ 1

Author(s):

Chiara F Magnani ◽

Giuseppe Gaipa ◽

Daniela Belotti ◽

Giada Matera ◽

Sarah Tettamanti ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Sleeping Beauty ◽

Advisory Committees ◽

Cik Cells ◽

Car T Cells ◽

Car T ◽

Support Research

Background Immunotherapy using patient-derived CAR T cells has achieved complete remission and durable response in highly refractory populations. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Allogeneic Cytokine Induced Killer (CIK) cells, a T-cell population characterized by the enrichment of CD3+CD56+ cells, have demonstrated a high profile of safety in acute lymphoblastic leukemia (ALL) patients (Introna M et al. Biol Blood Marrow Transplant. 2017). CIK cells could be easily engineered by the non-viral Sleeping Beauty (SB) transposon for the clinical application (Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017). Methods CIK cells were generated from 50 ml of donor-derived peripheral blood (PB) by electroporation with the GMP-grade CD19.CAR/pTMNDU3 and pCMV-SB11 plasmids according to the method enclosed in the filed patent EP20140192371. After lymphodepletion with Fludarabine (30 mg/m2/day) x 4 days and Cyclophosphamide (300 mg/m2/day) x 2 days, CARCIK-CD19 were infused in pediatric and adult B-cell ALL (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The clinical trial follows a four-dose escalation scheme (1x106, 3x106, 7.5x106 and 15x106 transduced CAR+ T cells/kg) using the novel Bayesian Optimal Interval Design (BOIN). During the cell manufacturing period, bridging anti leukemic therapy from patient registration to the beginning of the lymphodepletion, was allowed. The primary endpoint was to define the Maximum Tolerated Dose (MTD) and a safety assessment. Key secondary endpoints included the assessment of complete hematologic response (CR), defined as < 5% bone marrow (BM) blasts, circulating blasts < 1%, no clinical evidence of extramedullary disease, as well as the characterization of CARCIK-CD19 persistence in PB and BM (NCT03389035). Results We manufactured eighteen batches by seeding a median of 126.8x106 allogeneicPBMCs. At the end of expansion, the mean harvesting was 6.46x109 nucleated cells (range 1.39 - 16.00x109). Manufactured cells were mostly CD3+ lymphocytes (mean 98.90% ±SE 0.30%). Of these, 43.57%±3.73% were CAR+, 47.07%±2.74% were CD56+, 80.44%±2.53% were CD8+. The quality requirements for batch release were met in 17 productions. As of the data cut-off date (July 19, 2019), 4 pediatric and 7 adult patients were infused with a single dose of CARCIK-CD19 (n=2 HLA identical sibling, n=4 MUD, n=5 haploidentical donor). The leukemic burden in the BM post lymphodepletion/pre-infusion ranged from 0% to 96%. CARCIK-CD19 were characterized by a high profile of safety in all treated patients. Toxicities reported were a grade I cytokine release syndrome and an infusion-related DMSO-associated seizure, with absence of dose-limiting toxicities, neurotoxicity and graft-versus-host disease (GvHD) in any of the treated patients. Four out of 5 patients, receiving the highest doses, achieved CR and CRi at day 28. The 3 patients in CR were also MRD- (by flow cytometry and RT-PCR) while the CRi was MRD+ and relapsed at day+49. Robust expansion was achieved in the majority of the patients as defined by detectable CAR T-cell detection (vector copy number VCN, range 4645-977992 transgene copies/ug) and flow (range 0.5-30%) in PB. The median time to peak engraftment was 14 days. The magnitude of expansion was correlated with the CD19+ burden in the BM at the time of the infusion (P value = 0.0006, R square 0.7469). CD8+ T cells represented the predominant CARCIK-CD19 T-cell subset (78.88%±5.33% d14 n=6) along with CD3+CD56+ CIK cells and CD4+ T cells to a lesser extent. The majority of CAR T cells had a central and effector memory phenotype. CAR T cells were measurable by VCN up to 6 months with a mean persistence of 70.5 ± 14.85 days (follow up ranging from 28 days to 1 year). No major difference was observed by integration analyses of the patients' PB and the cell products. The vector integration sites reflected the classical random distribution of SB without any tendency for gene dense regions. Conclusions Our ongoing phase I/II trial demonstrates that SB-engineered CARCIK-CD19 cells are able to expand and persist in pediatric and adult B-ALL patients relapsed after HSCT, with important implications for a non-viral technology. These encouraging results prompted us to expand our study. Disclosures Gritti: Autolus Ltd: Honoraria; Roche: Other: Not stated; Abbvie: Other: Not stated; Becton Dickinson: Other: Not stated. Rambaldi:Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Chimeric Antigen Receptor Transgenic, T Cell Receptor/CD3 Negative Monospecific T Cells Generated from Cord Blood CD34 Positive Cells

Blood ◽

10.1182/blood.v126.23.3087.3087 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3087-3087 ◽

Cited By ~ 1

Author(s):

Yasmine van Caeneghem ◽

Glenn Goetgeluk ◽

Karin Weening ◽

Greet Verstichel ◽

Sarah Bonte ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

T Cell Receptor ◽

First Generation ◽

Cell Receptor ◽

Car T Cells ◽

Car T

Abstract Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T cells can be generated in vitro that do not express an endogenous T cell receptor to avoid alloreactivity causing graft versus host reactions. We compared carcino-embryonic antigen (CEA)- specific CARs of the first generation (intracellular CD3ζ signaling chain), of the 2nd generation (intracellular CD3ζ and CD28 signaling chain) and of the 3rd generation (intracellular CD3ζ, CD28 and OX40 signaling chain). CD34+ progenitor cells were isolated from human cord blood or postnatal thymus and subsequently transduced with one of the three green fluorescent protein (GFP)-encoding CAR constructs. Transduced cells were subsequently co-cultured on OP9DL1 in the presence of stem cell factor, Flt3-ligand and interleukin-7. Unlike T cell receptor transduced precursors (1), expansion was not enhanced by transduction of the chimeric receptor. Expansion was highest with first generation CARs whereas second and third generation CARs displayed only restricted expansion. Similar to T cell receptor transduced progenitors, CAR transduced cells show an accelerated differentiation during co-culture compared to the non-transduced cells: first committed CD5+ CD7+ T precursors appear, then CD4+ CD8+ double positive cells (DP) and finally CD1- CD27- single positive or double negative (DN) mature T cells. In cultures transduced with 2nd and 3rd generation CARs, few transduced cells passed through the proliferative DP pathway but rather differentiated to mature CD1- CD27- non-proliferative DN cells without passing through the DP stage. This phenomenon is responsible for the limited expansion seen with precursors transgenic for 2nd or 3rd generation CARs. However, in all cultures CAR+ DP cells were generated and, as shown for TCR transgenic cells (1), we were able to induce CEA specific maturation after co-culturing these DP cells with a cell line expressing CEA or by antibody-induced cross-linking of the CAR, giving rise to CD1- CD27+ matured cells. The observations described above are compatible with data obtained in mice showing that strong T cell receptor (TCR) activation during thymocyte differentiation inhibits the generation of DP cells and induces maturation to DN cells. Both the spontaneously and induced mature CAR+ cells were TCR and CD3 negative, suggesting that the expression of a CAR in early T cell precursors shuts down rearrangements of the endogenous TCR chains. Moreover, these cells lack NK marker expression (CD56, NKG2D) and show expression of T cell markers (CD5, CD7, CD2), confirming their T cell nature. In conclusion, the CAR+ CD3/TCR negative cells are T cells as these are derived from T cell precursors (CD5+, DP cells) and express various membrane and nuclear T cell markers. Mature CD1- CD27- CAR+ cells can be expanded to large cell numbers using T cell expansion protocols. They displayed cytokine production specific for CEA+ tumor lines as well as specific cytotoxicity. Moreover, the 2nd and 3rd generation CAR expressing cells showed increased specific cytokine production when compared to the first generation CAR expressing cells. These results show that the cord blood-derived CAR+ cells have potent functional activity similar to peripheral blood derived CAR+ T cells. We believe that these in vitro generated CAR+ cells developed from HLA-matched cord blood progenitors may be ideal as an adjunct to cord blood transplantation. (1) Snauwaert et al, Leukemia, 2014 Disclosures No relevant conflicts of interest to declare.

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CAR T Cells Targeted with a High Affinity Scfv Against the HCMV Glycoprotein Gb As Adoptive T Cell Therapy after Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.5721.5721 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5721-5721 ◽

Cited By ~ 2

Author(s):

Renata Stripecke ◽

Laura Gerasch ◽

Sebastian Theobald ◽

Bala Sai Sundarasetty ◽

Maksim Mamonkin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Target Cells ◽

Hematopoietic Stem ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Infected Cells

Abstract Introduction: Reactivation of human cytomegalovirus (HCMV) in immune compromised patients after hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality, particularly after cord blood transplantation (CBT). Adoptive transfer of T cells expanded in vitro is currently used as therapy for drug-refractory HCMV disease. A major limitation of this approach is the requirement of HLA-restricted HCMV-specific memory T cells. An alternative approach exploring HLA-independent T cell recognition was sought. Because the HCMV envelope glycoprotein B (gB) is highly expressed during lytic infection and in latently infected cells, we hypothesized that T cells can be redirected to recognize and kill HCMV-specific cells by means of a gB-specific chimeric antigen receptor (CAR). We have synthesized and tested a gB-specific CAR derived from the SM5-1 monoclonal antibody which binds with high affinity (KD 5.7x1011) to a conserved antigenic and non-glycosylated domain of gB. Methods: We generated two codon-optimized SM5-derived scFvs (VH->VL and VL->VH) and fused with an existing CAR backbone containing an IgG Fc spacer and intracellular signaling domains. CARs containing either CD28.zeta or 4-1BB.zeta were synthesized and expressed in T cells following a standard retroviral transduction protocol yielding 80-90% transduction rate. Expression of the CARs on T cells was confirmed by flow cytometry using goat anti-human immunoglobulin reactive against the IgG Fc region. 293T cells co-expressing gB and dTomato were used for in vitro cytotoxicity assays. Results: T cells expressing gB-CAR/CD28.zeta were cytotoxic against gB+ target cells producing 90% killing of 293T/gB-dTom cells compared with control CD19 CAR/CD28.zeta cells at an effector-to-target ratio 3:1 for 48 h (parental 293T cells were not killed). The cytolytic activity correlated with expansion of CAR T cells and concomitant loss of gB-dTom expression in the remaining viable 293T cells. Sequential co-culture of these gB-CAR T cells with freshly seeded 293T/gB-dTom resulted into further elimination of target cells. We are currently evaluating the effects of different gB-CAR T cell designs in the killing of HCMV-infected cell lines and primary cells using HCMV laboratory strains expressing the GFP and Gaussia Luciferase reporter genes. Pilot experiments indicated that gB-CAR/CD28.zeta cells with the scFv in the VL->VH orientation resulted into more clustering and killing of HepG2 cells infected with HCMV-GFP after 24h of co-culture than a control CD19 CAR/CD28.zeta. Humanized mice transplanted with cord blood CD34+ stem cells and challenged with these HCMV laboratory strains will be used to evaluate the in vivo effectivity of cord blood-derived donor-matched gB-CAR-T cells to eliminate acute and latent HCMV infections. Conclusion: These studies explore a novel approach in preventing HCMV reactivation in immunosuppressed patients by redirecting T cells expressing a high-affinity gB-CAR to eliminate HCMV-infected cells in a TCR/MHC-independent manner. Disclosures No relevant conflicts of interest to declare.

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Successful Manufacturing of CAR T-Cells with Small Volume Peripheral Blood from Healthy Donors Using the Clinimacs Prodigy Device

Blood ◽

10.1182/blood-2020-136795 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Katie Palen ◽

Parameswaran Hari ◽

Nirav N. Shah ◽

Bryon Johnson

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Mononuclear Cells ◽

T Cell Therapy ◽

Cell Numbers ◽

Car T Cells ◽

Car T Cell ◽

Healthy Donors ◽

Car T

Introduction In recent years, CAR T-cell therapy has emerged as a potentially curative intent treatment for some patients with relapsed, refractory hematologic malignancies. Despite the exciting results, not all patients are able to receive CAR T-cells due to manufacturing failures. T-cells for CAR products are typically autologous and isolated from heavily pre-treated patients, which might account for some of the manufacturing failures and suboptimal clinical efficacy. T-cells collected either early into cancer diagnosis or prior to diagnosis may improve CAR T-cell expansion and limit manufacturing failure. We evaluated the feasibility of generating a CAR T-cell product manufactured from 50 ml of healthy donor blood. Methods Collaborators at Cell Vault collected 50 ml of whole blood from 3 healthy donors, isolated peripheral blood mononuclear cells (PBMCs), and cryopreserved the cells in cryovials at 5e6/vial (1.05-1.35e8 total cells). The vials were shipped to the Medical College of Wisconsin and stored frozen in liquid nitrogen until use. All PBMC vials for a given donor were thawed and pooled. Thawed PBMCs (0.93-1.17e8 cells) were loaded onto a CliniMACS Prodigy device, CD4 and CD8 T cells enriched by immunomagnetic sorting, and T cells placed in the culture chamber with IL-7, IL-15 and TransAct reagent to induce proliferation. On the second day of manufacturing, T cells were transduced with a lentiviral CAR vector encoding anti-CD19, 4-1BB and CD3z. Final CAR T-cell products for these pre-clinical studies were harvested on day 8 of manufacture. Results Starting enriched T-cell numbers from the 3 healthy donors ranged from 4.0-4.8e7 cells, the cells were 74-79% CD4/8+, and the average CD4/CD8 ratio was 1.4. On the day of CAR T harvest (day 8), total cells in the chamber had expanded to 3.6-4.6e9 cells (74-115 fold expansion), the cells were >99% CD3+, and the average CD4/CD8 ratio was 2.9 (Table 1). Final cell numbers were similar to what previously published CAR T manufacturing runs on the CliniMACS Prodigy (Zhu et al., Cytotherapy, 2018), that started with 1x108 enriched T-cells obtained from apheresed mononuclear cells. Cell surface CD19 CAR expression on the final cell products varied from 19.2-48.1%. While more than 50% of the starting T cells had a naïve (CD62L+ CD45RO-) phenotype, the final cell products contained greater than 80% central-memory (CD62L+ CD45RO+) T cells. Finally, the number of CD19 CAR T cells obtained from these pre-clinical manufacturing runs ranged from 7.82e8 to 2.21e9 cells. Conclusions 50 ml of cryopreserved PBMCs was adequate to manufacture clinically relevant CAR T-cell therapy doses from healthy donors not previously exposed to chemotherapy. Sufficient numbers of CAR T-cells were obtained to dose an 80 kg individual with at least 9e6 cells/kg which is greater than prescribed commercial doses of CD19 CAR T-cells. Further studies are indicated to determine if T-cells collected prior to disease modifying chemotherapies result in an improved product. These results demonstrate feasibility for generating CAR T cells from small volumes of whole blood collected at a time point before a cancer patient has been treated with multiple lines of therapy that could negatively impact starting T cell numbers and function. Disclosures Hari: GSK: Consultancy; Amgen: Consultancy; BMS: Consultancy; Takeda: Consultancy; Incyte Corporation: Consultancy; Janssen: Consultancy. Shah:TG Therapeutics: Consultancy; Celgene: Consultancy, Honoraria; Incyte: Consultancy; Kite Pharma: Consultancy, Honoraria; Cell Vault: Research Funding; Miltenyi Biotec: Honoraria, Research Funding; Lily: Consultancy, Honoraria; Verastim: Consultancy. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding.

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Genomic Analyses of SLAMF7 CAR-T Cells Manufactured by Sleeping Beauty Transposon Gene Transfer for Immunotherapy of Multiple Myeloma

10.1101/675009 ◽

2019 ◽

Author(s):

Csaba Miskey ◽

Maximilian Amberger ◽

Michael Reiser ◽

Sabrina Prommersberger ◽

Julia Beckmann ◽

...

Keyword(s):

Clinical Trial ◽

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Gene Transfer ◽

Copy Number ◽

Sleeping Beauty ◽

Vector System ◽

Car T Cells ◽

Car T

ABSTRACTWidespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a ‘safe’ integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.In the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).Here we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.DisclosuresThis project is receiving funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).

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IL-7 as a Membrane-Bound Molecule for the Costimulation of Tumor-Specific T Cells.

Blood ◽

10.1182/blood.v114.22.3035.3035 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3035-3035 ◽

Cited By ~ 2

Author(s):

Lenka V. Hurton ◽

Harjeet Singh ◽

Simon Olivares ◽

Sonny O. Ang ◽

Susan Staba Kelly ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Sleeping Beauty ◽

Specific Lysis ◽

Car T Cells ◽

Car T ◽

Membrane Bound

Abstract Abstract 3035 Poster Board II-1011 Redirecting specificity to a selected cell surface tumor-associated antigen can be accomplished by the genetic modification of T cells to express a chimeric antigen receptor (CAR). Despite systematic modifications to the CAR endodomains to provide T cells with competent signaling, CAR-dependent T-cell activation may remain incomplete resulting in inferior in vivo persistence leading to a suboptimal therapeutic response. To improve T-cell survival and therefore an anti-tumor response, investigators have co-infused soluble recombinant cytokines such as IL-2 and IL-7. IL-7 is a homeostatic cytokine for T cells and supports survival of memory T cells. To provide IL-7 mediated signaling to T cells in the tumor microenvironment and thus enhance the proliferation and survival of CAR+ T cells, we constructed a version of IL-7 as a novel membrane-bound molecule (mIL7) designed to stimulate T cells in cis and trans. The mIL7 construct was electro-transferred with a CD19-specific CAR (on day 0) into primary T cells via multiple transposition of Sleeping Beauty DNA plasmids. These genetically modified T cells could be numerically expanded ex vivo without additional soluble cytokine supplementation on CD19+ artificial antigen presenting cells (aAPC) derived from K562. This resulted in the preferential outgrowth of T cells expressing both mIL7 and CAR (Figure A and B) while CAR+ T cells receiving no soluble cytokine supplementation did not sustain proliferation (Figure B). These mIL7+CAR+ T cells exhibited redirected specific lysis of CD19+ tumor targets. Significantly, the kinetics of propagation of mIL7+CAR+ T cells in the absence of exogenous cytokine was comparable to CAR+ T cells that were numerically expanded in the presence of soluble IL-2. Signaling by IL-7 receptor signal induction in mIL7+CAR+ T cells appeared comparable to signaling by soluble IL-7 in CAR+ T cells, as assessed by phosphorylation of signal transducer and activator of transcription 5 (STAT5). These data demonstrate that mIL7 can be expressed by CAR+ tumor-redirected T cells to enhance their proliferation without the need for additional cytokine support. These results have implications for the design of clinical trials to evaluate whether mIL7+CAR+ T cells can exhibit enhanced persistence and thus improved therapeutic potential. Disclosures No relevant conflicts of interest to declare.

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Combination Immunotherapy with NY-ESO-1-Specific CAR+ T Cells with T-Cell Vaccine Improves Anti-Myeloma Effect

Blood ◽

10.1182/blood.v128.22.3366.3366 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3366-3366 ◽

Cited By ~ 7

Author(s):

Krina Patel ◽

Simon Olivares ◽

Harjeet Singh ◽

Lenka V. Hurton ◽

Mary Helen Huls ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Sleeping Beauty ◽

Employment Equity ◽

Effector Cells ◽

T Effector Cells ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Abstract Adoptive transfer of T cells expressing chimeric antigen receptor (CAR) has demonstrated clinical effectiveness in early phase clinical trials, with persistence of effector cells typically leading to improved outcomes. Most CARs directly dock with cell-surface antigens, but this limits the number of tumor-derived targets. Thus, we have adapted two technologies to target intracellular antigens and improve survival of infused T cells. This was accomplished by expressing a CAR on T effector cells that functions as a mimetic of T-cell receptor (TCR) to recognize NY-ESO-1 in the context of HLA A2 and adapting HLA-A2+ T cells to serve as antigen presenting cells (T-APC) by expressing NY-ESO-1 antigen. NY-ESO-1 is a desirable target for T-cell therapy of high risk multiple myeloma (MM) with efficacy in trials infusing T cells expressing TCR recognizing this antigen. We hypothesized combined immunotherapy with NY-ESO-1-specific CAR+ T cells and an NY-ESO-1+ T-APC vaccine will lead to enhanced anti-myeloma efficacy due to improved persistence of the CAR+ T effector cells. An NY-ESO-1-specific CAR and control TCR were expressed on primary T cells using the Sleeping Beauty (SB) transposon/transposase system. T-APC was generated by electro-transfer of DNA plasmids from SB system coding for NY-ESO-1 and membrane-bound IL-15 (mbIL15). The tethered cytokine functions as co-stimulatory molecule to improve the potency of the vaccine. In vitro studies confirmed the NY-ESO-1-specific CAR+ (and TCR+) T cells could be numerically expanded upon co-culture with T-APC. A mouse model of NY-ESO-1+HLA-A2+(CD19neg) multiple myeloma was used to compare tumor growth for CAR+ T effector cells with and without T-APC. The NY-ESO-1-specific CAR+ T effector cells displayed anti-tumor effect that was superior to control mice without T cells and mice receiving CD19-specific control CAR+ T cells. Mice receiving both NY-ESO-1-specific CAR+T effector cells and T-APC exhibited further improvement in anti-myeloma activity. This group demonstrated superior persistence of T effector cells with recovered cells exhibiting a memory phenotype. In summary, T cells can target intracellular NY-ESO-1 using a TCR mimetic CAR. Improved anti-tumor effect attributed to better persistence can be achieved by co-infusion of T-APC vaccine. These data provide the foundation to assess T cells targeting NY-ESO-1 in a clinical trial. Disclosures Patel: Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Olivares:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Singh:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Hurton:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Cooper:City of Hope: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.

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CD19 CAR-T cell treatment conferred sustained remission in B-ALL patients with minimal residual disease

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-02941-4 ◽

2021 ◽

Author(s):

Wenyi Lu ◽

Yunxiong Wei ◽

Yaqing Cao ◽

Xia Xiao ◽

Qing Li ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Minimal Residual Disease ◽

Residual Disease ◽

Sustained Remission ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Minimal Residual ◽

Active Disease

AbstractThe persistence or recurrence of minimal residual disease (MRD) after chemotherapy predicts relapse of B-cell acute lymphoblastic leukemia (B-ALL). CD19-directed chimeric antigen receptor T (CD19 CAR-T) cells have shown promising responses in B-ALL. However, their role in chemotherapy-refractory MRD-positive B-ALL remains unclear. Here we aimed to assess the effectiveness and safety of CD19 CAR-T cells in MRD-positive B-ALL patients. From January 2018, a total of 14 MRD-positive B-ALL patients received one or more infusions of autogenous CD19 CAR-T cells. Among them, 12 patients achieved MRD-negative remission after one cycle of CAR-T infusion. At a median follow-up time of 647 days (range 172–945 days), the 2-year event-free survival rate in MRD-positive patients was 61.2% ± 14.0% and the 2-year overall survival was 78.6 ± 11.0%, which were significantly higher than patients with active disease (blasts ≥ 5% or with extramedullary disease). Moreover, patients with MRD had a lower grade of cytokine release syndrome (CRS) than patients with active disease. However, the peak expansion of CAR-T cells in MRD positive patients showed no statistical difference compared to patients with active disease. Five patients received two or more CAR-T cell infusions and these patients showed a decreased peak expansion of CAR-T cell in subsequent infusions. In conclusion, pre-emptive CD19 CAR-T cell treatment is an effective and safe approach and may confer sustained remission in B-ALL patients with chemotherapy-refractory MRD. The trials were registered at www.chictr.org.cn as ChiCTR-ONN-16009862 (November 14, 2016) and ChiCTR1800015164 (March 11, 2018).

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