Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

[1]  E. Shpall,et al.  Infusing CD19-directed T cells to augment disease control in patients undergoing autologous hematopoietic stem-cell transplantation for advanced B-lymphoid malignancies. , 2012, Human gene therapy.

[2]  E. Shpall,et al.  Chimeric antibody receptors (CARs): driving T-cell specificity to enhance anti-tumor immunity. , 2012, Frontiers in bioscience.

[3]  E. Shpall,et al.  CARs: Driving T-cell specificity to enhance anti-tumor immunity , 2012 .

[4]  E. Kleinerman,et al.  Genetically modified T cells targeting interleukin-11 receptor α-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases. , 2012, Cancer research.

[5]  C. Rooney,et al.  PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. , 2011, Molecular therapy : the journal of the American Society of Gene Therapy.

[6]  Steven J. Kass,et al.  Efficacy and safety of Sleeping Beauty transposon-mediated gene transfer in preclinical animal studies. , 2011, Current gene therapy.

[7]  Z. Izsvák,et al.  The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor , 2011, Gene Therapy.

[8]  S. Rosenberg,et al.  Considerations for the clinical application of chimeric antigen receptor T cells: Observations from a Recombinant DNA Advisory Committee symposium held June 15, 2010 (Cancer Research (2011) 71, (3175-3181)) , 2011 .

[9]  S. Olivares,et al.  Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies. , 2011, Cancer research.

[10]  S. Rosenberg,et al.  Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. , 2011, Cancer research.

[11]  R. McIvor,et al.  The Sleeping Beauty transposon system: a non-viral vector for gene therapy. , 2011, Human molecular genetics.

[12]  S. Rosenberg,et al.  CARs on track in the clinic. , 2011, Molecular therapy : the journal of the American Society of Gene Therapy.

[13]  Z. Izsvák,et al.  Translating Sleeping Beauty transposition into cellular therapies: Victories and challenges , 2010, BioEssays : news and reviews in molecular, cellular and developmental biology.

[14]  G. Dotti,et al.  Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. , 2010, Blood.

[15]  E. Guinan,et al.  Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. , 2010, Cancer research.

[16]  D. Largaespada,et al.  A transposon and transposase system for human application. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[17]  P. Rao,et al.  piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. , 2010, Human gene therapy.

[18]  R. Lawrence.  Victories and challenges. , 2009, Breastfeeding medicine : the official journal of the Academy of Breastfeeding Medicine.

[19]  R. McIvor,et al.  DNA transposons for modification of human primary T lymphocytes. , 2009, Methods in molecular biology.

[20]  David A. Williams.  Sleeping beauty vector system moves toward human trials in the United States. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[21]  E. Shpall,et al.  System Sleeping Beauty the Redirecting Specificity of T-cell Populations for Cd19 Using Cited Articles Citing Articles E-mail Alerts Redirecting Specificity of T-cell Populations for Cd19 Using the Sleeping Beauty System , 2022 .

[22]  J. Wagner,et al.  Sleeping Beauty transposon-mediated engineering of human primary T cells for therapy of CD19+ lymphoid malignancies. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[23]  C. June,et al.  Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules. , 2007, Molecular therapy : the journal of the American Society of Gene Therapy.

[24]  David D. Smith,et al.  CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. , 2006, Cancer research.

[25]  Perry B. Hackett,et al.  Structure-based prediction of insertion-site preferences of transposons into chromosomes , 2006, Nucleic acids research.

[26]  S. Forman,et al.  Characterization of an artificial antigen-presenting cell to propagate cytolytic CD19-specific T cells , 2006, Leukemia.

[27]  Zoltán Ivics,et al.  Sleeping beauty transposition: biology and applications for molecular therapy. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[28]  R. Plasterk,et al.  Molecular Reconstruction of Sleeping Beauty , a Tc1-like Transposon from Fish, and Its Transposition in Human Cells , 1997, Cell.