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Keywords:

  • gene therapy;
  • T cells;
  • Sleeping Beauty;
  • transposon;
  • transposase CD19;
  • adoptive immunotherapy

Summary

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing methods to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CARs) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR+ T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential.

This article is part of a series of reviews covering Adoptive Immunotherapy for Cancer appearing in Volume 257 of Immunological Reviews.

Genetic modification ex vivo to enhance in vivo therapeutic potential

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

Gene therapy has been successfully combined with T-cell therapy to generate potent immune cells that upon administration can sustain proliferation, home to sites of malignant disease, and recycle effector functions in the tumor microenvironment. This bench-to-bedside-to-bench circle of innovation is driven by an understanding of translating immunology into immunotherapy and harnessing vector systems for safe gene transfer. The stable integration of transgenes into T cells can be accomplished using viral and non-viral systems. Among the latter, the electro-transfer of DNA plasmids is appealing as investigators can use commercial electroporation devices and readily produce, or contract to have made, DNA plasmids suitable for ex vivo genetic manipulation. Up to now, the widespread adoption of electroporation of T cells to express transgene from an introduced DNA plasmid has been limited due to low frequency of integration events from an approach that had relied on illegitimate homologous recombination events. We have advanced the transposon/transposase system from Sleeping Beauty (SB) as an approach to improve the rate of transgene integration upon synchronous electro-transfer of DNA plasmids containing a SB transposon and encoding a SB transposase. This review summarizes this advance to gene therapy in the context of redirecting T-cell specificity.

Redirecting T-cell specificity for tumor via CARs

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

Cancer typically arises in the host with a healthy immune system due, in part, to tolerance of the T-cell receptor (TCR) to tumor-associated antigens (TAA). Circumventing tolerance to engender a desired immune response can be achieved by using the mouse to generate antibody against TAA found on the cell surface of human malignant cells. The antigen-binding region of a recovered mouse monoclonal antibody (mAb) can then be cloned and expressed as the scFv region imparting specificity to the prototypical chimeric antigen receptor (CAR). The complete CAR molecule consists of a scFv held in frame by an extracellular scaffold and fused via a transmembrane domain to one or more intracellular signaling domains. The CAR directly docks with TAA and can recognize tumor independent of human leukocyte antigen (HLA). Thus, this single-chain immunoreceptor can help broaden the application of adoptive immunotherapy as it avoids the need to pair TCR-mediated recognition of TAA-derived peptide antigen with the restricting HLA. The original first-generation CAR molecule was described by Zelig Eshhar in 1989 [1, 2] with a patent US 7,741,465 B1 apparently filed in 1993 and issued in 2010 stemming from this work. It has required years of investigation in not-for-profit academic centers to develop and implement the gene therapy tools, refine the CAR design, and codify an approach to production in compliance with current good manufacturing practice (cGMP) to enable T cells to be genetically modified to stably express CAR in a manner suitable for their human application.

The B-lineage antigens have been recognized as a ‘safe harbor’ for the development and implementation of novel clinical trials infusing T cells genetically modified to be specific for CD19. This TAA is present on the cell surface of most malignant B cells and thus a CAR designed to target CD19 has the potential to target a variety of hematologic malignancies. Similarly, as the distribution of CD19 is apparently confined to malignant and normal B cells, it is anticipated that there will be the potential for ‘on target’ adverse events due to the loss of the recipient's healthy CD19+ B cells and damage to humoral immune system. We and others have successfully shown that the T cells derived from peripheral blood can be genetically modified to express CD19-specific CARs [3, 4]. However, other sources of T cells and sub-populations of T cells may have clinical appeal as a cellular substrate for the stable expression of CARs. For example, the ability to undertake allogeneic hematopoietic stem-cell transplantation (HSCT) across traditional HLA barriers renders umbilical cord blood (UCB) an attractive source of T cells. In an effort to improve the graft-versus-tumor effect, we have demonstrated that CD19-specific CAR+ T cells can be manufactured from small amounts of UCB to provide recipients of allogeneic UCB transplantation (UCBT) with donor-derived tumor-specific T cells [5, 6]. Currently, studies infuse ‘bulk’ population of cells, therefore we and others seek to define and characterize the best cellular substrate for genetic modification and expression of CARs. Circulating T cells can be characterized by immunophenotype as naive (TN), central memory (TCM), and effector memory (TEM) cells. Naive-derived effector cells (TEFFN) are an attractive population of αβTCR+ T cells for adoptive immunotherapy, as they have conferred improved anti-tumor activity [7]. We determined the relative frequency of TN (CD62L+CD45RA+), TCM (CD62L+CD45RA), TEM (CD62LCD45RA), and TEMRA (CD62LCD45RA+) subsets in the peripheral blood (PB) of healthy donors and observed an abundance of circulating TN cells compared with TCM (P < 0.001), TEM (P < 0.05), or TEMRA (P < 0.001) cells, which supports circulating T cells as a rich source of naive T cells for expression of CARs [8]. Current clinical trials chiefly administer CAR+ on T cells co-expressing αβ TCRs, as this population dominates (95%) of T cells in PB. The remaining 1–5% includes T cells expressing γδ TCRs. This circulating T-cell sub-population are also attractive for adoptive immunotherapy, as they inherently recognize a broad range of tumor cells and potentially can be infused across HLA barriers without causing graft-versus-host-disease (GVHD). Therefore, we and others have expressed CD19-specific CARs in ζδTCR+ T cells [9, 10].

In addition to the constitution of the T cell, the design of the CAR impacts the therapeutic potential of the genetically modified product. Most CARs for oncology applications typically consist of a (i) scFv, comprising of VH and VL domains of a TAA-specific mAb, linked via (ii) flexible hinge sequence to the cell membrane by (iii) transmembrane sequence, fused to (iv) intracellular signaling domain (CD3-ζ from TCR complex) or co-expressed with costimulatory molecules. To provide a fully competent T-cell activation signal, minimally defined as TAA-dependent (i) proliferation, (ii) cytokine production, and (iii) serial killing, multiple investigators have advanced CAR structures to include one or more costimulatory signaling domains, such as derived from CD28, CD134, CD137, DAP10, DAP12, ICOS, Lck [11-15]. The clinical impact of such varied CAR designs to impact the ability of T cells to eliminate bulky disease, maintain remission, and prevent recurrence remains to be elucidated. For example, the CAR molecule that synchronously activates T cells to target relapsed hematologic disease may be different from the immunoreceptor species to address minimal residual disease or solid tumors.

Significant progress has been made toward the clinical application of CAR+ T cells especially to control B-cell malignancies. However, questions remain regarding the source of T cells and composition of the CAR, which may need to be personalized to meet the need of individual patients with regards to tumor type, burden, and biodistribution. Thus, while a CAR may be ‘universal’ in terms of the potential of the scFv to recognize one TAA present on a certain tumor type from multiple recipients, it is probable that this universality may not extend to other parts of the CAR molecule (e.g. the signaling moiety within the endodomain) and the cellular template on which the CAR is expressed. This is one of the reasons we implemented a new approach to human gene therapy based on non-viral gene transfer of the SB system recognizing that the approach could be rendered nimble and cost effective. DNA plasmids can be readily modified and produced for human application and this will enable investigators to have a clear line-of-sight between ideas tested in the laboratory and infusions of T cells that draw upon panels of CAR+ T cells.

Non-viral gene transfer

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

Non-viral gene transfer of CAR transgenes offers some advantages over viral-based systems to integrate transgenes into the T-cell genome, as DNA plasmids are comparatively inexpensive to produce, are non-immunogenic, and are not generally constrained by size of the cargo load (since there is no packaging needed to produce recombinant viral particles). However, the stable introduction of naked DNA plasmids has been a low-efficiency event subject to silencing of transgene expression and can be associated with genomic integration of multiple copies [16-18]. We have overcome these hurdles using SB-derived transposons which do not depend on illegitimate recombination to achieve stable integration. Originally described by Dr. Perry Hackett's Laboratory in 1996 [19], the SB transposon/transposase system [20] (Fig. 1) is derived from fish and has been adapted for gene therapy [21-23]. The SB system consists of a transposase enzyme which recognizes the inverted repeat containing direct repeated sequences (IR/DR) flanking the transgene (e.g. CAR) in a transposon. Recently, our group has employed the SB system to introduce CAR to redirect specificity of clinical-grade T cells [24, 25], as we have reviewed [26-29]. Concern of genotoxicity is mitigated as SB transposase-mediated insertion of the cargo load at TA dinucleotide basepairs and unlike other gene transfer systems, the SB transposition event does not have a preference for integration either into or proximal to, transcriptional units. Concerns for remobilization of transposon due to residual SB transposase activity are assuaged as the SB11 transposase is likely immunogenic and post-translational control of the transposase appears to be subject to a negative feedback loop by its degradation products [30, 31]. Furthermore, re-hopping of integrated SB transposon is unlikely as the human genome does not contain endogenous transposase capable of remobilizing introduced fish-derived SB genetic elements [29]. Thus, the SB system appears to have attractive features for human gene therapy and meets the need for an approach for non-viral gene transfer that might lower the cost and improve the speed to translate preclinical data into clinical trials.

image

Figure 1. Schematic of Sleeping Beauty (SB) system used to genetically modify a T cell to express a chimeric antigen receptor (CAR). The two plasmid SB system consists of a SB11 transposase (red) and CAR transposon (blue) simultaneously electro-transferred using a nucleofector device and solution. After translation, the SB11 enzyme (red circle) binds the inverted repeat sequences (shown as grey arrows) flanking the CAR transposon. Transposition occurs via a cut-and-paste mechanism to insert the CAR into TA sites in the T-cell genome (orange helix) resulting in stable CAR expression on the cell surface.

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Human application of SB system

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

We have worked at MD Anderson Cancer Center (MDACC) to adapt the SB system to genetically modify T cells to express CD19-specific CAR in compliance with cGMP. Our first human trials infusing SB-modified CAR+ T cells, beginning in 2012 signify the fastest that a new gene therapy has been successfully brought from the bench to the bedside [32]. Our approach to manufacture was based on (i) the use of readily-available commercial device (e.g. Nucleofector from Lonza, http://www.lonza.com/) to synchronously electro-transfer two supercoiled DNA plasmids coding for SB transposon (the CAR) and SB transposase (SB11); (ii) commercial vendor to produce DNA plasmid (e.g. Waisman Biomanufacturing, http://gmpbio.org, and Aldevron, http://www.aldevron.com); and (iii) and engineered artificial antigen presenting cells (aAPC) to retrieve T cells stably expressing CAR integrants over culture time, with T cells lacking CAR unable to sustain proliferation and then dying by neglect. The criteria we use to release DNA plasmids for the electroporation in compliance with cGMP are shown in Table 1. The ability to selectively propagate T cells based upon activating CAR by TAA and costimulatory molecules co-expressed on designer (lethally irradiated) aAPC in the presence of soluble recombinant cytokines [4, 33] may allow us to test the hypothesis that those T cells genetically modified to numerically expand ex vivo may exhibit improved ability to proliferate and survive in vivo. Quiescent T cells (typically 1–2 × 107 mononuclear cells per cuvette) from PB and UCB are electroporated in the presence of supercoiled DNA plasmids coding for second-generation CD19-specific CAR [designated CD19RCD28 [34], that co-activates T cells via chimeric CD3-ζ and CD28] and SB11 transposase (transposon to transposase ratio of 3:1, 15:5 μg per cuvette) using the human T-cell kit and Nucleofector II electroporation device. The SB transposon is under control of human elongation factor-1α (EF-1α)/human T-cell leukemia virus hybrid composite promoter [35] and flanked by the pT [20] and pT2 [36] IR/DR, while SB11 [37] is under control of a transiently expressed CMV promoter [25]. We demonstrated a 60-fold improved integration efficiency of CAR transgene in SB-modified aAPC-propagated T cells, compared to electro-transfer of DNA without transposition [3]. Our typical clinical CAR+ T-cell products are characterized based on release criteria (Table 2) and in-process testing (Table 3) based upon published manufacturing and correlative data [24, 25, 38]. To date, we have opened four clinical trials at MDACC administering patient- and donor-derived CD19-specific CAR+ T cells after autologous and allogeneic HSCT, including UCBT, and after lymphodepleting chemotherapy (Table 4).

Table 1. Release criteria for manufacture of clinical grade DNA plasmids coding for SB system (transposon and transposase) used ex vivo to genetically modify T cells
TestSpecification
AppearanceClear colorless liquid
IdentityRestriction mapping and agarose gel electrophoresis agrees with predicted banding pattern
Sequencing
Plasmid concentration2.0 ± 0.2 mg/ml
1.8–2.0 (A260/A280)
>90% supercoiled form
Bacterial host-cell macromoleculesEndotoxin <50 EU/mg
Protein <0.3%
RNA <10%
SterilityNegative
Table 2. Criteria to release clinical grade CAR+ T cells after electroporation with SB-derived DNA plasmids and propagation on aAPCa/IL-2/IL-21
Testsb performed to generate the certificate of analysisSpecification
  1. a

    K562-derived aAPC (e.g. clone #4 that endogenously expresses CD32 and genetically modified to co-express CD19, CD64, CD86, CD137L, mIL15).

  2. b

    To establish chain-of-custody low resolution HLA class I may be confirmed with donor of T cells.

  3. c

    To assess presence of aAPC.

SterilityVisual inspection
No growth at 14 days (BD BACTEC)
PCR negative for Mycoplasma
Endotoxin LAL <5 EU/kg
Viability (Trypan blue exclusion)≥70%
Immunophenotyping (ungated)cCD32+7-AAD <5%
CD19+7-AAD <5%
Immunophenotyping (gated on lymphocytes)CD3+ ≥80%
CAR+ ≥10%
Autonomous growth≤10% cells viable at day 18 after withdrawal of cytokines and aAPCs
Table 3. In-process testing undertaken on CD19-specific CAR+ T cells cryopreserved for human administration
ParameterTestMethodReference to specialized methodologyNotes
  1. a

    This assay is also undertaken as part of release testing.

  2. b

    An assay is being developed to be compliant with guidance document from U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research; http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf.

PhenotypeCell surface expression of CARaFlow cytometry [25, 61] Typically >80% T cells express CAR
Total CAR expressionWestern blotting [25] A 73 kDa chimeric CD3ζ band is seen for 2nd generation CAR CD19RCD28
PotencybSpecific lysisChromium Release Assay [25] CAR-dependent lysis of CD19+ target cells
Potential for persistenceMemory/Naïve phenotypeFlow Cytometry [25] Includes expression of CD28, CD62L, CCR7, CD45R0
Markers of exhaustionFlow Cytometry [25] Includes expression of PD1 and, CD57
Telomere lengthFlow-FISH [25] Not degraded compared with unmanipulated control T cells
SafetyCAR copy numberQ-PCR [25, 38] Typically one to two integrants per T-cell genome
Stable integration of DNA plasmid coding for SB11 by PCRPCR [25] Absence of PCR band by agarose gel electrophoresis
TCR repertoireFlow Cytometry for TCR Vβ and/or Direct TCR expression assay for TCR Vα & Vβ [25, 71] Absence of skewing (emergence of oligoclonality or monoclonality in pattern of TCR abundance)
KaryotypeG-banding [3] Normal
Table 4. Ongoing clinical trials at U.T. MD Anderson Cancer Center infusing CD19-specific CAR+ T cells electroporated with SB system and propagated on aAPC/IL-2/IL-21
Protocol titleIRB No.IND No.NIH-OBAClinicaltrials.gov
CD19-specific T-cell infusion in patients with B-lineage lymphoid malignancies after autologous hematopoietic stem cell transplantation2007–0635IND 141930804–922NCT00968760
CD19-specific T-cell infusion in patients with B-lineage lymphoid malignancies after allogeneic hematopoietic stem cell transplantation2009–0525IND 145770910–1003NCT01497184
Donor-derived, CD19-specific T-cell infusion in patients with B-lineage lymphoid malignancies after umbilical cord blood transplantation2010–0835IND 147391001–1022NCT01362452
Autologous CD19 specific T-cell infusion in patients with B-cell chronic lymphocytic leukemia (B-CLL)2011–1169IND 151801201–1142NCT01653717

Next generation SB systems

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

Efforts to improve the frequency of integration of SB system have been based upon systematically improving the enzymatic activity of the SB transposase with three muteins currently being employed and ranked here according to increasing ability to mediate transposition: SB10 [20], SB11[37], and SB100X [39]. We compared SB11 and SB100X to generate genetically modified T cells and observed increased enzymatic activity of SB100X, which resulted in a more efficient outgrowth of CD19-specific CAR+ T cells on aAPC, compared with SB11. However, the increased rate of transposition resulted in increased copy number of CAR integration events resulting in our need to reduce the amount of the associated DNA plasmid used to electroporate the T cells [40]. Thus, on balance there was little immediate advantage to alter our approach to manufacturing CD19-specific CAR+ T cells based on swapping SB11 for SB100X.

One potential limitation of the SB system is that the transposition rate may be inversely proportional to the size of the cargo. The efficiency of SB-mediated transposition appears to decrease when the expression cassette size is larger than 5 kb [41]. Other transposon systems such as piggyBac and Tol2 apparently tolerate larger transgenes than the SB system. We and others have successfully adapted the piggyBac system to generate CAR+ T cells [33, 42]. However, concerns remain that the piggyBac system has a pronounced preference for integration into transcriptional units compared with the SB system and that piggyBac transposase may liberate endogenous human sequences. The SB system has been modified by us to co-express more than one transgene. This can be achieved using bicistronic/multi-functional fusion SB DNA plasmids [43] or by the synchronous electro-transfer of two SB plasmids coding for different transposons that undergo transposition in the same T cell [44, 45] as mediated by electro-transfer of DNA (or mRNA) encoding for SB transposase. This has been employed by us to stably introduce not one but two transgenes at once (double transposition). The advantage of this approach is that the CAR can be combined with other molecules such as for non-invasive imaging, homing, or improving persistence.

Our approach to generating SB-modified T cells in compliance with cGMP is currently based of the electro-transfer of two DNA plasmids coding for the SB transposon and transposase in trans. We are adapting the electroporation conditions to eliminate the possibility that the introduced SB transposase can integrate into the T-cell genome. This is based on the co-electro-transfer of SB-derived DNA coding for the CAR and in vitro-transcribed mRNA coding for SB11. There is precedence for this approach [46], and our efforts are focused on bringing this to the GMP facility in support of next-generation and multi-center clinical trials. We are also developing an alternative based on a self-inactivating DNA construct, similar to piggyBac system [47] that eliminates expression of SB transposase placed in cis with SB transposon.

aAPCs to selectively propagate CAR+ T cells

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

Currently, our approach to manufacturing CD19-specific T cells is based on electro-transfer of DNA plasmids from the SB system and selective propagation of CAR+ T cells to clinically appealing numbers upon recursive (every 7–10 day) additions of γ-irradiated aAPCs in the presence of soluble recombinant IL-2 and IL-21 [4]. The immortalized K562 cells, a human erythroleukemic cell line derived from a patient with blast crisis [48], was selected as the source of aAPC, as these cells have been directly administered to humans [49, 50], can be readily genetically modified with lentivirus and SB systems, and express endogenous molecules [such as ICAM-1 and LFA-1 [51]] that can activate T cells (and NK cells). Furthermore, they lack of expression of HLA class I A and B alleles [they can express HLA class C molecules [52]] and all HLA class II alleles [53, 54] and thus are unlikely to propagate T cells with alloreactive potential. The K562-derived aAPC can be pre-irradiated (100 Gy) prior to thawing and co-culture with T cells and thus offers an attractive platform that avoids the need, inconvenience, and lot-to-lot variation associated with the use allogeneic peripheral blood mononuclear cells (PBMC) as irradiated (and sometimes pooled) feeder cells which are expensive and time-consuming to procure from volunteer donors. We initially showed that K562-derived aAPC co-expressing 4-1BBL and MICA [55] were more effective at numerically expanding CD19-specific CAR+ T cells compared with a rapid expansion protocol based on cross-linking CD3 (with mAb clone OKT3) in the presence of LCL and PBMC [56]. K562-derived aAPCs have further been modified by us and others to express a wide array of costimulatory molecules, including CD40, CD40L, CD70, CD80, CD83, CD86, CD137L, ICOSL, GITRL, CD134L, to facilitate proliferation of T cells [57, 58]. In collaboration with Carl June, we genetically modified CD32+ K562 to co-express CD64 (the high affinity Fc receptor, for loading mAb), CD86, CD137L (4-1BBL), and a membrane-bound IL-15 mutein (mIL15). A clone (number 4) of these K562-derived aAPC selectively propagates CAR+ T cells while preserving naïve and memory immunophenotypes [3, 4]. The aAPC clone #4 were produced for use in compliance with cGMP as a master cell bank (MCB, in collaboration with PACT under the auspices of NHLBI) and a derived working cell bank at MDACC in support of on-going clinical trials (Table 4). The testing to release the banks of clinical grade aAPCs are described in Table 5. Future developments include our plans to manufacture a MCB of one source of aAPC designed to activate and propagate T cells stably expressing CARs of any specificity.

Table 5. Criteria to produce a master cell bank of aAPCsa suitable for use in compliance with cGMP
ParameterTestb
  1. a

    K562-derived aAPC (e.g. clone #4 that endogenously expresses CD32 and genetically modified to co-express CD19 (the TAA), CD64, CD86, CD137L, mIL15).

  2. b

    Biologics safety testing are undertaken to ensure absence of contaminants. Some of these tests may be performed by contract services (e.g. BioReliance; http://www.bioreliance.com/default.aspx).

Endotoxin LAL<5 EU/mL
SterilityDirect inoculation method
VirusesReplication-competent lentivirus
In vitro and in vivo culturing
Bovine 9CFR in vitro assay
PCR for human viruses and Bovine polyoma virus
Quantitative product enhanced reverse transcriptase (Q-PERT) assay for retroviruses
MycoplasmaTest for presence of agar-cultivable and non-agar cultivable mycoplasma
IdentityTransmission electron microscopic examination
Isoenzyme Analysis
DNA fingerprinting
Immunophenotyping (gated flow cytometry)Viability ≥ 70%

Introduced cell surface antigen(s) ≥ 40%

CD32 ≥ 75%

Correlative studies to inform on therapeutic potential of infused CAR+ T cells

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

Clinical trials infusing SB-modified and aAPC-propagated T cells will reveal the feasibility and safety of this new approach to gene therapy. Since persistence of CAR+ T cells is likely proportional to the anti-tumor effect, we developed a mAb (clone 136.20.1) that binds the scFv region of CD19-specific CARs derived from mAb clone FMC63 [59, 60]. We demonstrated that clone 136.20.1 can detect (1:1000) CD19-specific CAR+ T cells [61], and this reagent has been successfully used by investigators to measure the abundance and biodistribution of CD19-specific CAR+ T cells after infusion [62, 63]. In some instances, particularly when the bioburden of circulating tumor cells is low, the persistence of infused T cells in peripheral blood may be below the limit of detection by flow cytometry and survival of the genetically modified T cells may be detected by quantitative PCR (Q-PCR) using CAR specific primers [38].

Measuring in vivo persistence provides one measure of success, but the infused T cells need to find and engage CAR molecules with TAA to eliminate tumor. Flow cytometry and Q-PCR on samples serially obtained from tissues is one approximation to the homing potential of infused CAR+ T cells. The SB system has been adapted to avoid the need for biopsy and sampling by co-expressing a reporter gene with CAR for non-invasive imaging. For example, a four-function reporter transgene can be introduced by ‘double transposition’ to achieve (i) in vitro selection under cytocidal concentration of hygromycin B, (ii) in vivo ganciclovir-dependent conditional ablation, (iii) in vivo bioluminescence imaging (BLI) simultaneous with (iv) positron emission tomography [43, 64]. Similarly, CD19-specific CAR+ T cells co-expressing a fusion gene of hygromycin phosphotransferase and thymidine kinase derived from herpes simplex virus-1 can be tracked in vivo in mice [64]. Developing methodologies capable of providing quantitative, non-invasive, longitudinal, and spatial in vivo information about the dynamic process of infused T cells will be critical to our understanding of adoptive immunotherapy in humans.

Each infusion product is a heterogeneous composition of genetically modified T cells, only some of which will persist and recycle effector function after adoptive transfer. The challenge to increase potency may be achieved by the a priori identification of subsets of CAR+ T cells that have superior anti-tumor activity for a given patient, tumor type, burden, and biodistribution. Standard immunologic assays report the overall functioning of populations of manufactured T cells, but cannot readily inform on the therapeutic potential of individual CAR+ T cells. To personalize CAR+ T-cell therapy, we have employed an integrated technology [65] that combines single-cell functional screening with systems-level molecular profiling to describe individual T cells linking their cytotoxicity, cytokine production, and molecular profile [66, 67]. It is expected that these data, compared before and after infusion, will help define desired T-cell populations that can be manufactured to meet the needs of a given patient.

Broadening the human application of CAR+ T cells

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

The SB and aAPC platforms can be readily used to genetically modify and propagate CAR+ T cells. This approach to manufacturing clinical grade T cells avoids the need, complexity, and cost of generating and releasing recombinant virus. Current practice is based on generating one population of CAR+ T cells to be infused into a given patient. This approach to manufacturing is costly, relies on both sufficient quality and quantity of host immune elements from which to harvest T cells, and takes time, during which the intended recipient's medical condition may deteriorate precluding infusion of the genetically modified T cells. Therefore, we have developed approaches whereby one (healthy) donor's T cells can be pre-prepared to express CAR and then infused on demand when needed, rather than when the T cells are available. The major impediment to the implementation of this embodiment of off-the-shelf therapy is the potential for adverse events stemming from the αβTCR on the infused CAR+ T cells recognizing major and minor histocompatibility antigens on normal cells in the recipient. This can be alleviated by eliminating alloreactive CAR+ T cells prior to infusion. One approach was based on allo-anergization of donor-derived T cells by in vitro induction of a hyporesponsive state which does not degrade signaling though introduced CAR, as achieved by allo-stimulation with concomitant blockade of CD28-mediated costimulation [68]. As an alternative, we have recently used engineered zinc finger nucleases (ZFNs) to target constant regions of the TCR α and β loci and thereby eliminate their expression. We combined the electro-transfer of SB-derived DNA plasmids to stably express CAR with electro-transfer of in vitro-transcribed mRNA coding for ZFNs which act in a hit-and-run fashion to introduce a desired double-strand break in the T-cell genome. The resultant CAR+TCRαβneg cells could not respond to TCR stimulation while maintained CD19-dependent effector function [69]. An application has completed review by NIH-OBA (#1307-1236) to undertake a clinical trial infusing CD19-specific T cells as an off-the-shelf therapy. Similarly, we have successfully used ZFNs to eliminate HLA-A expression in CAR+ T cells as an approach to prolonging the survival of infused off-the-shelf T cells by preventing their recognition by the recipient's immune system [70]. The ability to stably introduce transgenes using the SB system and eliminate undesired endogenous genes with artificial nucleases provides an experimental path to generating T cells with improved potency and broad application.

Summary

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References

The SB system provides investigators with a new approach to gene therapy. The initial clinical data are being obtained with CAR+ T cells targeting CD19 in patients with advanced B-cell malignancies. We find this approach to gene therapy to be nimble and cost-effective; for example it enables us to initiate next-generation clinical trials targeting ROR1 on CLL cells in attempt to spare normal B cells from CAR-mediated destruction (NIH-OBA #1210-1192) and test alternative CAR designs that activate T cells upon docking with CD19 via chimeric CD3-ζ and CD137 (NIH-OBA #1301-1203 and #1304-1225).

References

  1. Top of page
  2. Summary
  3. Genetic modification ex vivo to enhance in vivo therapeutic potential
  4. Redirecting T-cell specificity for tumor via CARs
  5. Non-viral gene transfer
  6. Human application of SB system
  7. Next generation SB systems
  8. aAPCs to selectively propagate CAR+ T cells
  9. Correlative studies to inform on therapeutic potential of infused CAR+ T cells
  10. Broadening the human application of CAR+ T cells
  11. Summary
  12. Acknowledgement
  13. References
  • 1
    Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA 1989;86:1002410028.
  • 2
    Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA 1993;90:720724.
  • 3
    Singh H, et al. Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res 2008;68:29612971.
  • 4
    Singh H, et al. Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies. Cancer Res 2011;71:35163527.
  • 5
    Serrano LM, et al. Differentiation of naive cord-blood T cells into CD19-specific cytolytic effectors for posttransplantation adoptive immunotherapy. Blood 2006;107:26432652.
  • 6
    Kelly SS, et al. Adoptive immunotherapy after umbilical cord blood (UCB) transplantation: manufacturing and analysis of CD19-specific UCB-derived T-cells from scant numbers of UCB mononuclear cells. Biol Blood Marrow Transplant 2010;16:S182S183.
  • 7
    Hinrichs CS, et al. Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proc Natl Acad Sci USA 2009;106:1746917474.
  • 8
    Singh H, et al. Naive CD19-specific T cells exhibit superior proliferation and potential for adoptive immunotherapy. Biol Blood Marrow Transplant 2012;18:S311S311.
  • 9
    Deniger DC, et al. Bispecific T-cells expressing polyclonal repertoire of endogenous gammadelta T-cell receptors and introduced CD19-specific chimeric antigen receptor. Mol Ther 2013;21:638647.
  • 10
    Rischer M, Pscherer S, Duwe S, Vormoor J, Jurgens H, Rossig C. Human gammadelta T cells as mediators of chimaeric-receptor redirected anti-tumour immunity. Br J Haematol 2004;126:583592.
  • 11
    Zhao Y, et al. A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. J Immunol 2009;183:55635574.
  • 12
    Sadelain M, Brentjens R, Riviere I. The promise and potential pitfalls of chimeric antigen receptors. Curr Opin Immunol 2009;21:215223.
  • 13
    Wang J, et al. Optimizing adoptive polyclonal T cell immunotherapy of lymphomas, using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains. Hum Gene Ther 2007;18:712725.
  • 14
    Yvon E, et al. Immunotherapy of metastatic melanoma using genetically engineered GD2-specific T cells. Clin Cancer Res 2009;15:58525860.
  • 15
    Singh H, et al. Third generation chimeric antigen receptors containing CD137 or CD134 signaling endodomains augment CD19-specific T-cell effector function. Blood 2009;114:15711572.
  • 16
    Bestor TH. Gene silencing as a threat to the success of gene therapy. J Clin Invest 2000;105:409411.
  • 17
    Henikoff S. Conspiracy of silence among repeated transgenes. BioEssays 1998;20:532535.
  • 18
    Selker EU. Gene silencing: repeats that count. Cell 1999;97:157160.
  • 19
    Ivics Z, Izsvak Z, Minter A, Hackett PB. Identification of functional domains and evolution of Tc1-like transposable elements. Proc Natl Acad Sci USA 1996;93:50085013.
  • 20
    Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 1997;91:501510.
  • 21
    Izsvak Z, Ivics Z. Sleeping beauty transposition: biology and applications for molecular therapy. Mol Ther 2004;9:147156.
  • 22
    Hackett PB, Ekker SC, Largaespada DA, McIvor RS. Sleeping beauty transposon-mediated gene therapy for prolonged expression. Adv Genet 2005;54:189232.
  • 23
    VandenDriessche T, Ivics Z, Izsvak Z, Chuah MK. Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells. Blood 2009;114:14611468.
  • 24
    Huls MH, et al. Clinical application of sleeping beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood. J Vis Exp 2013;e50070.
  • 25
    Singh H, et al. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using sleeping beauty system and artificial antigen presenting cells. PLoS ONE 2013;8:e64138.
  • 26
    Hackett PB, Largaespada DA, Cooper LJ. A transposon and transposase system for human application. Mol Ther 2010;18:674683.
  • 27
    Izsvak Z, Hackett PB, Cooper LJ, Ivics Z. Translating Sleeping Beauty transposition into cellular therapies: victories and challenges. BioEssays 2010;32:756767.
  • 28
    Hackett PB Jr., et al. Efficacy and safety of Sleeping Beauty transposon-mediated gene transfer in preclinical animal studies. Curr Gene Ther 2011;11:341349.
  • 29
    Hackett PB, Largaespada DA, Switzer KC, Cooper LJ. Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy. Transl Res 2013;161:265283.
  • 30
    Walisko O, et al. Transcriptional activities of the Sleeping Beauty transposon and shielding its genetic cargo with insulators. Mol Ther 2008;16:359369.
  • 31
    Schreifels J, et al. Autoregulation of transposition of Sleeping Beauty transposons by ubiquitous N-terminal, dominant negative peptides. Mol Ther 2007;15:S128S129.
  • 32
    Aronovich EL, McIvor RS, Hackett PB. The Sleeping Beauty transposon system: a non-viral vector for gene therapy. Hum Mol Genet 2011;20:R14R20.
  • 33
    Manuri PV, et al. piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum Gene Ther 2010;21:427437.
  • 34
    Kowolik CM, et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res 2006;66:1099511004.
  • 35
    Kim DW, Uetsuki T, Kaziro Y, Yamaguchi N, Sugano S. Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene 1990;91:217223.
  • 36
    Cui Z, Geurts AM, Liu G, Kaufman CD, Hackett PB. Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon. J Mol Biol 2002;318:12211235.
  • 37
    Geurts AM, et al. Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther 2003;8:108117.
  • 38
    Maiti SN, et al. Sleeping beauty system to redirect T-cell specificity for human applications. J Immunother 2013;36:112123.
  • 39
    Mates L, et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 2009;41:753761.
  • 40
    Jin Z, et al. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor. Gene Ther 2011;18:849856.
  • 41
    Hackett PB. Integrating DNA vectors for gene therapy. Mol Ther 2007;15:1012.
  • 42
    Nakazawa Y, et al. PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol Ther 2011;19:21332143.
  • 43
    Bhatnagar P, et al. Imaging of genetically engineered T cells by PET using gold nanoparticles complexed to Copper-64. Integr Biol 2013;5:231238.
  • 44
    Hurton LV, et al. IL-7 as a membrane-bound molecule for the costimulation of tumor-specific T cells. Blood 2009;114:11831183.
  • 45
    Hurton LV, et al. Tethered IL-15 mutein on CD19-specific T cells sustains persistence when tumor antigen is low and can treat minimal residual disease. Mol Ther 2013;21:S237S237.
  • 46
    Peng PD, et al. Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity. Gene Ther 2009;16:10421049.
  • 47
    Marh J, et al. Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis. Proc Natl Acad Sci USA 2012;109:1918419189.
  • 48
    Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 1975;45:321334.
  • 49
    Nemunaitis J, et al. Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer. Cancer Gene Ther 2006;13:555562.
  • 50
    Smith BD, et al. K562/GM-CSF immunotherapy reduces tumor burden in chronic myeloid leukemia patients with residual disease on imatinib mesylate. Clin Cancer Res 2010;16:338347.
  • 51
    Maus MV, et al. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat Biotechnol 2002;20:143148.
  • 52
    Le Bouteiller P, et al. Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity. Proc Natl Acad Sci USA 2002;99:1696316968.
  • 53
    Hirano N, et al. Expression of costimulatory molecules in human leukemias. Leukemia 1996;10:11681176.
  • 54
    Papadimitriou L, Morianos I, Michailidou V, Dionyssopoulou E, Vassiliadis S, Athanassakis I. Characterization of intracellular HLA-DR, DM and DO profile in K562 and HL-60 leukemic cells. Mol Immunol 2008;45:39653973.
  • 55
    Numbenjapon T, et al. Characterization of an artificial antigen-presenting cell to propagate cytolytic CD19-specific T cells. Leukemia 2006;20:18891892.
  • 56
    Numbenjapon T, Serrano LM, Chang WC, Forman SJ, Jensen MC, Cooper LJ. Antigen-independent and antigen-dependent methods to numerically expand CD19-specific CD8(+) T cells. Exp Hematol 2007;35:10831090.
  • 57
    Suhoski MM, et al. Engineering artificial antigen-presenting cells to express a diverse array of costimulatory molecules. Mol Ther 2007;15:981988.
  • 58
    Zhang H, et al. 4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy. J Immunol 2007;179:49104918.
  • 59
    Zola H, MacArdle P, Bradford T, Weedon H, Yasui H, Kurosawa Y. Preparation and characterization of a chimeric CD19 monoclonal antibody. Immunol Cell Biol 1991;69:411422.
  • 60
    Nicholson IC, et al. Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma. Mol Immunol 1997;34:11571165.
  • 61
    Jena B, et al. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials. PLoS ONE 2013;8:e57838.
  • 62
    Kalos M, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73.
  • 63
    Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 2011;365:725733.
  • 64
    Singh H, et al. PET imaging of T cells derived from umbilical cord blood. Leukemia 2009;23:620622.
  • 65
    Varadarajan N, et al. A high-throughput single-cell analysis of human CD8(+) T cell functions reveals discordance for cytokine secretion and cytolysis. J Clin Invest 2011;121:43224331.
  • 66
    Singh H, Liadi I, Romain G, Varadarajan N, Cooper LJN. Single-cell imaging reveals that subsets of T cells expressing a CD19-specific chimeric antigen receptor differ in effector function. Mol Ther 2013;21:S151S151.
  • 67
    Liadi I, Roszik J, Romain G, Cooper LJ, Varadarajan N. Quantitative high-throughput single-cell cytotoxicity assay for T cells. J Vis Exp 2013;e50058.
  • 68
    Davies JK, et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res 2010;70:39153924.
  • 69
    Torikai H, et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood 2012;119:56975705.
  • 70
    Torikai H, et al. Towards eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 2013;122:13411349.
  • 71
    Zhang M, et al. A new approach to simultaneously quantify both TCR alpha- and beta-chain diversity after adoptive immunotherapy. Clin Cancer Res 2012;18:47334742.