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Translational Implications for Off-the-shelf Immune Cells Expressing Chimeric Antigen Receptors Hiroki Torikai1 and Laurence JN Cooper1,2 1 Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; 2Ziopharm Oncology Inc., Boston, Massachusetts, USA

Chimeric antigen receptor (CAR) endows specificity to T-cells independent of human leukocyte antigen (HLA). This enables one immunoreceptor to directly target the same surface antigen on different subsets of tumor cells from multiple HLA-disparate recipients. Most approaches manufacture individualized CAR+T-cells from the recipient or HLA-compatible donor, which are revealing promising clinical results. This is the impetus to broaden the number of patients eligible to benefit from adoptive immunotherapy such as to infuse third-party donor derived CAR+T-cells. This will overcome issues associated with (i) time to manufacture T-cells, (ii) cost to generate one product for one patient, (iii) inability to generate a product from lymphopenic patients or patient’s immune cells fail to complete the manufacturing process, and (iv) heterogeneity of T-cell products produced for or from individual recipients. Establishing a biobank of allogeneic genetically modified immune cells from healthy third-party donors, which are cryopreserved and validated in advance of administration, will facilitate the centralizing manufacturing and widespread distribution of CAR+T-cells to multiple points-ofcare in a timely manner. To achieve this, it is necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to toxicity and rejection. We review the strategies to establish “off-theshelf” donor-derived biobanks for human application of CAR+T-cells as a drug. Received 3 March 2016; accepted 28 April 2016; advance online publication 14 June 2016. doi:10.1038/mt.2016.106

The chimeric antigen receptor (CAR) is an artificial immune receptor to redirect T-cell specificity to tumor-associated cell-surface molecules independent of HLA. The extracellular antigen-recognition domain of the prototypical CAR uses a single chain variable fragment from monoclonal antibody (mAb); however, this can be replaced with a receptor–ligand interaction of sufficient affinity, such as modified cytokine (e.g., IL-13) to target a cytokine receptor (e.g., IL-13Rα),1 a cell surface molecule (e.g., CD27) for targeting its ligand (e.g., CD70),2 or a pattern-recognition receptor (e.g., Dectin-1) for targeting foreign carbohydrates such as β-glucan on germinating Aspergillus.3 These binding motifs are typically fused to an extracellular stalk or varying lengths and composition, such as derived from the hinge with or without CH2–CH3 domains from IgG1 and IgG4 or hinge with extracellular domain of CD8α. The use of immunoglobulin regions raises the possibility that a CAR may bind to Fc receptors and unwanted elimination of infused T-cells which may be alleviated by introducing site-directed changes or eliminating CH2 region to reduce potential for such clearance.4–6 Recent evidence also suggests the importance of the stalk to impact the effector functioning of CAR+T-cells interrogating the binding motifs within tumor associated antigens (TAAs) proximal versus distal to the cell surface.6–9 CAR-dependent activation is dependent

on one or more intracellular signaling domains expressed in cis or trans. The signaling domain of CARs brought forward to clinical applications employed a single signaling molecule containing immunoreceptor tyrosin-based activation motifs (ITAMs) in cytoplasmic domain to mimic the T-cell receptor (TCR)/CD3 signal.10 These “first generation” CARs typically delivered an incomplete T-cell activation event which prompted embedding additional activation motifs within the CAR endodomain. Inclusion of costimulatory signaling, e.g., through CD28 (ref. 11), CD137 (refs. 12,13), OX40 (ref. 14), and CD27 domains,15 improved function of CAR+T-cells manifested by sustained persistence after adoptive transfer leading to improved therapeutic effect.16,17 In addition to “signal 1” delivered by phosphorylation of ITAMs and “signal 2” mediated by costimulatory molecules, T-cells typically require a third signal to achieve and perhaps sustain full activation. This third signal is mediated through the common γ-chain cytokine receptor and thus coordinated delivery or coexpression of certain cytokines can enhance CAR+T-cell functions, which may be especially useful for the application of genetically modified T-cells targeting solid tumors.18–20 Human applications of CAR+T-cells have shown promise in several early phase clinical trials, such as infusing targeting CD19

Correspondence: Hiroki Torikai, Division of Pediatrics, The University of Texas, MD Anderson Cancer Center, Unit 907, 1515 Holcombe Blvd., Houston, Texas 77030, USA. E-mail: [email protected] or Laurence JN Cooper, Division of Pediatrics, The University of Texas, MD Anderson Cancer Center, Unit 907, 1515 Holcombe Blvd., Houston, Texas 77030, USA. E-mail: [email protected]; [email protected]

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on B-cell leukemias21–29 and lymphomas, 16,30,31 and targeting GD2 on neuroblastoma.32 Recently, several reviews have highlighted and summarized these clinical successes.33–37 With these early successes, CAR+T-cell therapy is redeploying from academia to industry.38 Broadening the clinical experience of CAR+T-cell therapy is thus emerging as an important hurdle, especially for those tumor targets already associated with an antitumor effect. This underscores the need to expand the list of qualified TAAs that can be safely targeted by T-cells though one or more engineered immunoreceptors.39 There are a variety of approaches to overcome this, such as to preidentify TAAs expressed solely on tumor cells and targeting aberrant cells based on combinatorial recognition of more than one TAA,40,41 or targeting intracellular proteins.42,43 Another limitation to the broad implementation of CAR+Tcell therapy resides with the production process to generate clinical-grade biologics. Currently, T-cells are genetically modified and propagated infusing either patient-derived T-cells often after lymphodepleting chemotherapy or donor-derived T-cells administered in the context of hematopoietic stem-cell transplantation (HSCT). These T-cells are produced for a given recipient on a case-by-case basis. This personalized approach to manufacturing reflects the approach pioneered by a subset of academic institutions that operate cell processing facilities in compliance with current good manufacturing practice for phase 1 and 2 trials on campuses that are in close proximity to clinical facilities to administer CAR+T-cells.44 This leads to heterogeneity of manufacturing methods that provides an intellectually fertile and competitive environment to determine facets of production that result in therapeutically appealing T-cells. However, the physical alignment of multiple geographically diverse good manufacturing practice facilities with infusion units can lead to duplication of efforts compounding the financial costs associated with a labor-intensive approach to manipulation and testing of T-cells (Figure 1). This in turn raises the cost of therapy and curtails the availability of CAR+T-cells, as these products are produced in a

a

b

Distributed manufacturing

Centralized manufacturing

limited number of production suites after the recipient has been identified. Moreover, the variability inherent to manufacturing CAR+T-cells from different patients and donors may confound an assessment of mechanisms associated with therapeutic success and complicate the implementation of combination therapies such as with other immune-based modalities. These challenges provide the impetus to develop and implement “off-the-shelf ” (OTS) cell-based biologic therapies in which immune cells can be manufactured ahead of need (in advance of consent) and infused on demand as required by the recipient, rather than when the biologic is produced.

OTS CAR+T-CELL THERAPY

The current production and lot release (quality control) of clinical-grade genetically modified T-cells requires time in culture within good manufacturing practice-compatible manufacturing facilities during which the intended recipient’s condition can deteriorate. Currently, the “distributed” approach to manufacturing by academic centers at multiple point-of-care has resulted in a portfolio of biologic products that differ in terms of quality and quantity (Table 1) which may impact the results of immunotherapy of CAR+T-cells. OTS immunotherapy may overcome these limitations as it lends itself to centralized manufacture of a well-characterized product. Our present definition of OTS CAR+T-cells is defined as a biologic that is pre-prepared in advance from one or more healthy unrelated donors, validated, and cryopreserved. Their manufacture can be readily undertaken in a centralized manufacturing facility for predeployment to treatment facilities and infused as needed rather than when the product is available (Figure 1). One or more biobanks of the OTS T-cell product(s) could then be administered into multiple recipients in multicenter trials powered for efficacy and to establish the maximum tolerated dose. The administration of a well-characterized product will then facilitate combination immunotherapies infusing OTS T-cells with other treatments (Table 2). To prepare the cells in advance, we propose the use of “universal” allogeneic donor-derived T-cells. These third party cells might be genetically modified, and as required genetically edited, to safely maintain CAR-mediated effector functions and sustain in vivo persistence by avoiding deleterious immune-mediated recognition by the recipient of allogeneic features on the product (Figure 2).

STRATEGIES TO AVOID GRAFT-VERSUS-HOSTDISEASE AFTER INFUSION OF OTS CAR+T-CELLS

In the setting of an HLA-mismatch between donor and recipient, the frequency of T-cells specific for disparate HLA is estimated ~1 in 104.45,46 In clinical trials, the number of administered Table 1 Heterogeneity in the biologic products as derived from different autologous or allogeneic donors

Number of copies of integrated chimeric antigen receptor Single point-of-care

Multiple points-of-care

Figure 1 Manufacturing of chimeric antigen receptor (CAR)+T-cells. (a) Current manufacturing of CAR+T-cells: CAR+T-cells are generated and infused into recipient in each facility. (b) Proposed manufacturing offthe-shelf (OTS) CAR+T-cells: CAR+T-cells are generated in a single facility and distributed to multiple points-of-care.

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Genomic sites of transgene insertion(s) T-cell immunophenotype TCR repertoire Effector function due to the phenotype, single nucleotide polymorphisms, etc.

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Table 2 Advantages associated with infusing off-the-shelf (OTS) chimeric antigen receptor (CAR)+T-cells manufactured from one or more third party donor(s) for administration and readministration into multiple unrelated recipients

Table 3 Potential strategies to generate off-the-shelf (OTS) cell products

Avoids difficulties in generating CAR T-cells from and for a recipient due to poor quality and/or quantity of T-cells Reduces the cost, time, and resources to manufacture CAR+T-cells that are needed for infusion into a single patient OTS CAR+T-cells can be pre-prepared for infusion as needed versus when available OTS CAR+T-cells can be predeployed at multiple points-of-care OTS CAR+T-cells have reduced heterogeneity compared with multiple patient-specific CAR+T-cells OTS CAR+T-cells can be produced using centralized manufacturing site(s) OTS CAR+T-cells reduce the barriers to undertaking phase 2b multicenter trials powered for efficacy OTS CAR+T-cells can be infused as part of multicomponent trials administering this biologic at predefined maximally tolerated dose

Eliminate alloreactive T-cells

+

Avoid GvHD   CD25, CD69, CD137 depletion, photodepletion Suppress TCR mediated activation   B7 specific mAb for blocking CD28 mediated costimulation Decrease the TCR repertoire   Memory T-cells T-cells expressing TCR with defined specificity   Virus or tumor specific T-cells, γ9δ2 T-cell Use cell types that are not expressing alloreactive immunoreceptors   γδ T-cells, NK-cells, NKT-cells Use precursor T-cells Conditional ablation

Allogeneic CAR+ T-cells

  HSV1-TK, iCasp9

Patient

Endogenous HLA class I & II

Limit

Eliminate of endogenous TCR T-cell

al urviv

long

  ZFN, TALEN, CRISPR/Cas9 and other gene editing technologies Avoid rejection

s -term

Suppress immune system in recipients Target antigen

Therapeutic effect CAR

  lymphodepletion (CY, Fludarabine, TBI, anti-CD52Ab) Modulation of immune system in recipients   FTY720

Tumor cells

Enforced expression of immune checkpoint molecules   PD-L1, CTLA4-Ig

Endogenous TCRαβ

HLA homozygous donor

GvH

D

Rec

ogn

ize no

n-se

lf H

Somatic cells

LA/

mH

Ag

Figure 2 Schematic presentation of potential issues in establishing off-the-shelf (OTS) chimeric antigen receptor (CAR)+T-cells from one or more third party donors. While allogeneic CAR+T-cells can destruct target tumor cells, they may also recognize patient’s somatic cells through endogenous TCR, which results in the deleterious graft-versushost disease (GvHD). We will need to avoid this allogeneic immune reaction induced by infused allogeneic CAR+T-cells. Further consideration will be needed to preclude recognition of infused CAR+T-cells by recipient’s immune system to sustain CAR+T-cells in vivo persistence.

CAR+T-cells is typically between 108 and 109 which could lead to the delivery 103–105 T-cells expressing αβTCR specific for allogeneic antigens and thus likely be sufficient to induce graft-versushost-disease (GvHD). We describe five strategies to prevent this unwanted activation through endogenous TCR (Table 3).

Depletion of alloreactive T-cells T-cells displaying αβTCRs play a central role inducing GvHD and therefore several strategies have been developed to ex vivo remove alloreactive T-cells such as contaminating the coinfusion of HLA-mismatched hematopoietic stem-cells (HSCs) to restore hematopoiesis without GvHD in the context of HSCT. These include the numeric depletion of T-cells that express one or more cell-surface markers consistent with activation (e.g., 1180

Gene editing of HLA   ZFN, TALEN, CRISPR/Cas9 and other gene editing technologies

CD25 (refs. 47–49), CD69 (ref. 50), and CD137 (ref. 51)) upon coculture with antigen-presenting cells (APCs) derived from the intended recipient. Activated T-cells can then be reduced using magnetic beads or immunotoxin conjugated to mAb.47,50 Photodepletion is an alternative approach for reduction of alloreactive T-cells based on the inability of activated T-cells to efflux of a phototoxic dye.52 Such methods have already tested in the clinic and significantly decreased the frequency of alloreactive T-cells while preserving viral- and tumor-specific T-cells which may benefit immune reconstitution in an immunocompromised recipient. However, strategies that rely on ex vivo depletion cannot completely eliminate alloreactive T-cells. Moreover, the requirement to coculture the CAR+T-cells with recipient’s cells reduces the speed and convenience associated with producing this OTS biologic.

Alloanergization of T-cells We demonstrated that anergization of CAR+T-cells can be achieved in tissue culture by combining allostimulation with HLA-mismatched APC and concomitant blockade of CD28mediated costimulation.53 This resulted in the reduction of recognition of disparate HLA by third-party T-cells mediated by αβTCR while preserving CAR-mediated effector function. The www.moleculartherapy.org  vol. 24 no. 7 jul. 2016

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induction of alloanergization requires patient-derived stimulator cells that undermines its suitability for OTS CAR+T-cells.

T-cells expressing αβ TCR with limited or defined specificity One strategy to reduce TCR diversity and thus potential of alloreactivity is to employ T-cells from memory pools as a cellular template for introduction of CAR. Injecting naive T-cells induced GvHD in a mouse model, whereas administering memory T-cells did not.54,55 This may be due to a difference in the αβTCR diversity between naive and memory pools as revealed by Vβ CDR3 spectratyping or sequencing.56,57 There may also be a functional advantage as mouse memory T-cells could respond to alloantigen, but could not maintain a proliferative response which thus blunted GvHD.58 This may have a human application as naive T-cells can be depleted by recognition of CD45RA while preserving memory T-cells (and HSC).59 The therapeutic potential of adoptive immunotherapy appears to correlate with T-cells expressing a less-differentiated phenotype60 and the sustained numeric expansion of a T-cell subset derived from memory pools to achieve a sizeable biobank may undermine this approach to OTS CAR+T-cell therapy. Enthusiasm for their clinical translation is also undermined by a recent report that failed to show a reduction of acute GvHD using the strategy to deplete naive population from allogeneic graft.61 Using the T-cells expressing a defined antigen specificity can curtail the TCR diversity. Adoptive T-cell therapy against a defined peptide/HLA complex should not cause GvHD as long as restricting αβTCR fails to recognize allogeneic antigens. This is the premise behind clinical trials infusing virus-specific T-cells isolated and expanded from third-party donors to successfully treat opportunistic viral diseases in immunocompromised hosts based on matching at least one HLA allele between donor and recipient that expresses an immunodominant antigen.62–65 Administering T-cells expressing αβTCR with defined specificity may be advantageous, as after genetic modification, they will have dual specificity against cell surface TAA (through CAR) and peptide/HLA complex (through endogenous TCR). An early-phase clinical trial has demonstrated that this can be accomplished in HLA-matched settings as donor-derived viral-specific CAR+Tcells were infused after allogeneic HSCT.26 However, caution may be warranted when delivering antigen-specific T-cells to completely HLA-mismatched recipients as TCR-mediated recognition of target antigens is more promiscuous than anticipated.66 Although GvHD has not been observed after infusing third party HLA-mismatched viral-specific T-cells,67 further work is warranted before such antigen-specific T-cells can be used as a cellular substrate for OTS CAR+T-cells. Effector cells other than peripheral blood-derived αβ T-cells γδ T-cells. Compared to αβTCR, the diversity of γδTCR chain usage is limited which hints that this T-cell population may be less prone to alloreactivity. Although the specificity of some specific γδT-cells has been identified, the target antigens of most γδTCR are not well characterized and the impact of donor-derived γδ T-cells in GvHD pathology is uncertain.68–71 We and othMolecular Therapy  vol. 24 no. 7 jul. 2016

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ers showed that ex vivo activated or expanded human γδ T-cell did not apparently cause xeno-GvHD.72–75 Furthermore, the early engraftment of HLA-haploidentical γδ T-cells after infusion of HSC stripped of contaminating αβTCR did not lead to GvHD in humans.76 The specificity of one subset of γδ T-cells expressing γ9δ2 TCR recognizes isopentenylpryophosphate (IPP) which can specifically propagated by coculturing with clinical-grade aminobisphosphonate based on the inhibition of cholesterol synthesis leading to the accumulation of IPP.77 The γ9δ2 T-cell subset can recognize several kinds of tumor cells although to date there has been only a marginal beneficial effect of this subpopulation in clinical trials.78 The γ9δ2 T-cell may be combined with CAR to be an option for establishing OTS CAR+T-cell therapy.79 However, stimulation(s) with aminobisphosphonate may lead to terminal differentiation which could curtail in vivo persistence. Thus, methodologies to propagate γδ T-cells, including those with polyclonal TCR repertoire, such as using K562-derived artificial antigen presenting cells (aAPC) may be an appealing approach to generate OTS CAR+T-cells.74 NKT-cells. The NKT-cell is a relatively uncommon circulating immune cell that expresses a unique TCR species (Vα24Jα18 in humans). An inverse correlation of recovery of NKT-cells and GvHD has been reported after allogeneic HSCT while preserving a graft-versus-tumor response.80 NKT-cell can also be expanded in vitro by aAPC81 and can be genetically modified to express CAR.82 Thus, the limited TCR usage and emerging technology to obtain large numbers raises the possibility that NKT-cells can be used to generate OTS CAR+ immune cells. NK-cells. These cells from the innate immune system express a constellation of inhibitory and activation receptors which in aggregate determine the potential for NK-cell mediated cytotoxicity.83 An antitumor response attributed to NK-cells has been well documented in the context of HLA-mismatched HSCT.84–87 To broaden this clinical effect, haploidentical NK-cells have been isolated and infused to treat relapse of acute myelogenous leukemia after lympho-depleting chemotherapy apparently without causing GvHD.87,88 Despite success in leukemia, currently the evidence that NK-cells can target solid tumor cells in humans is limited. However, adoptive NK-cell therapy may be advanced through genetic manipulation and/or combining with immune modulators (e.g., lenalidomide and cytokines) and targeting molecules (e.g., therapeutic mAbs participating in antibody-dependent cell-mediated cytotoxicity). Ex vivo numeric expansion of NK-cells can be achieved with recursive additions of γ-irradiated K-562-derived aAPC coexpressing 4-1BBL and membrane bound IL-15 (ref. 89) or IL-21 (ref. 90). Furthermore, the specificity of NK-cells can be directed through enforced expression of a CAR.89 These observations lay the foundation for deploying NK-cells as OTS CAR+ immunotherapy. However, a surprising clinical result infusing ex vivo expanded HLA-matched unrelated donor NK-cells revealed an unexpectedly high rate of clinically significant GvHD.91 Thus, until additional clinical experience is forthcoming and the mechanism for this adverse event is understood, caution is warranted using allogeneic NK-cells as OTS therapy. 1181

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Precursor T-cells. T-cells can be generated in vitro by coculturing CD34+ HSC on Notch-1 ligand-expressing mouse stroma cells (OP9/DLL-1 (ref. 92) or OP9/DLL-4 (ref. 93)). This system generates precursor T-cells prior to thymic selection in vivo. Major histocompatibility antigen (MHC)-mismatched precursors have been infused into mice with normal thymic function and did not cause GvHD presumably because T-cells expressing TCRs that recognize self-antigens were eliminated by host thymic selection.94 This reveals an approach to administer OTS CAR+ precursor T-cells generated in vitro by partially differentiating HSC with Notch-1 ligand.95 As human HSC can apparently be safely genetically modified with lentivirus to express CAR,96 this approach to OTS immunotherapy may be rendered suitable for human applications. A drawback includes the scale up of ex vivo programming T-cells from HSC to sufficient numbers for biobanking. Furthermore, this strategy might not be suitable for recipients with an immediate need to control of underlying tumor burden due to the time needed in vivo for precursor CAR+T-cells to differentiate into effector T-cells.97 Conditional ablation of T-cells. Haploidentical T-cells have been genetically modified to express a suicide gene and infused to deliver a graft-versus-tumor-effect after HSCT with the expectation that GvHD can be controlled by eradicating infused T-cells using a prodrug. The two suicide genes chiefly assessed in clinical trials are thymidine kinase (TK) from herpes simplex virus 1 (ref. 98) and induced caspase 9 (iCasp9).99 An advantage of TK is the ready availability of ganciclovir as a FDA-approved product.98,100,101 Disadvantages are the immunogenicity of viral-derived TK leading to recognition and elimination by the recipient102 and possibly the time course to destroy T-cells after delivery of ganciclovir. An alternative system uses a chemical dimerizer to crosslink iCasp9 leading to cell death. Because iCasp9 is derived from the human gene, there is likely reduced potential to elicit immune-mediated recognition and thus deletion. The clinical application of iCasp9 has been published103,104 demonstrating resolution of GvHD after infusing haploidentical iCasp9+T-cells were rapidly eliminated by a single infusion of clinical-grade dimerizer. The clinical appeal of OTS CAR+T-cells that coexpress a suicide gene105 will depend on the kinetics of elimination and the associated potential for loss of the antitumor effect.

Elimination of expression of endogenous αβTCR Artificial nucleases are harnessed ex vivo as genome-editing tools to permanently disrupt the expression of endogenous genes. Zinc finger nucleases (ZFNs) are currently the most clinically mature of the artificial nucleases.106 Alternative artificial nucleases, such as transcription activator-like effector nuclease (TALEN)107,108 and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins)109,110 are recent technologies that may have translational appeal. We applied ZFNs to eliminate expression of endogenous αβTCR from CAR+T-cells thereby completely eliminating the possibility to induce GvHD.111 Transient expression of a pair of ZFNs from electro-transferred in vitro-transcribed mRNA resulted in disruption of TCR expression on T-cells genetically modified using the Sleeping Beauty system to express a CD19-specific CAR. The TCRnegCAR+T-cells were readily enriched by magnetic depletion of the remaining CD3+ 1182

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population using an approach amenable to clinical translation. Significantly, these genetically edited and modified T-cells could be numerically expanded on CD19+ aAPC. A concern regarding the human application of T-cells genetically edited with artificial nucleases is the occurrence of off-target introduction of doublestranded breaks and the potential for translocation. These must be avoided to exclude genotoxicity and potentially detrimental effects arising after administration of TCRnegCAR+ OTS T-cells. TALEN or CRISPR/Cas9 systems may improve off-target-­cleavage and also the efficiency of disrupting αβTCR from T-cells.112 Using these editing technologies, we have established that the manufacturing process can generate at least 3 × 109 TCRnegCAR+ OTS T-cells from one frozen cord blood unit, which typically contains about 8 × 107 CD3+ T-cells (unpublished data). We estimate that upon completing this propagation and given an infusion dose of 3.0 × 108 per patient,28 one can prepare CAR+ OTS T-cells for up to 10 patients within 4 weeks from a single cord blood unit although recurrent stimulation may lead to T-cells with more differentiated phenotype and shortened telomeres that thus may not be suitable for effective adoptive CAR T-cell therapy.60 Another round of ex vivo numeric expansion of TCRnegCAR+ OTS T-cells will typically increase the cell number 10-fold, which will result in sufficient product for injection up to 100 patients. This number might be increased upon harvesting T-cells from a steady-state leukapheresis product harvested from a healthy donor.

STRATEGIES TO SUSTAIN PERSISTENCE OF OTS CAR+T-CELLS IN VIVO

Monoclonal antibodies have found favor as an OTS therapy as they can be readily deployed and simply infused. Yet, CAR+Tcells possess inherent advantages over mAbs due to their home to sites of malignancy, migrate through tissues despite elevated interstitial pressures, increase in numbers, and serially kill target tumor cells. Furthermore, infused CAR+T-cells may survive over the long term and play an unassisted role in immunomonitoring to prevent recurrence. In the autologous setting, genetically modified T-cells can survive in human for over 10 years.113 However, infusing allogeneic CAR+T-cells from just one unrelated and HLA-disparate donor could activate the recipient’s immune systems and be rejected before an antitumor effect is fully realized and prevent long-term immunoprotection. Thus, strategies to avoid clinically deleterious immune-remediated rejection of OTS CAR+T-cells are needed. In some instances, when the recipient is heavily immunosuppressed, the impact of immune-mediated rejection may not compromise the therapeutic effect such as revealed when third party OTS viral-specific T-cells, matched at only one HLA, are successfully infused to control EBV+ lymphoproliferative disease after allogeneic HSCT.65 If an HLA-restricted rejection response does arise, then OTS T-cells might be generated as biobanks from more than one donor that are HLA distinct from each other and thus be accessed for the recipient to receive more than one infusion.

Suppression of immune system in recipients The depletion of resident immune cells before adoptive transfer of T-cells is generally accepted to enhance the therapeutic effect.114,115 In the context of infusion of allogeneic T-cells this www.moleculartherapy.org  vol. 24 no. 7 jul. 2016

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Table 4 Questions to improve the therapeutic potential and acceptance of infused off-the-shelf (OTS) T-cells in human trials for oncology

What degree of HLA matching is needed between a donor and multiple recipients to prevent immune-mediated rejection and deleterious clearance leading to loss of antitumor response? What lymphodepleting and/or immunosuppressive regimens are needed to prevent immune-mediated rejection and deleterious clearance leading to loss of antitumor response? What defines the ideal donor and do these criteria differ based on tumor burden, type, and recipient? How will donor-to-donor variation and the potential to impact the antitumor response both be measured/optimized? Will scale up and manufacturing of large biobanks impact the antitumor activity and how will this be a prior assessed? Should investigators and recipients expect (temporary) disease control rather than (sustained) antitumor effects? What clinical context is best suited for initial clinical testing given the variabilities of tumor (i) types, (ii) burdens, and (iii) distributions? What clinical context is best suited for initial clinical testing given the variabilities of each recipient’s (i) genetic background, (ii) body mass, and (iii) prior therapies? How to address the targeting of solid tumors? Should phase 1 trials anticipate that recipients receive more the one infusion? Are T-cells the preferred cellular substrate for an OTS biologic/product?

immunosuppression may limit the emergence and/or potency of host-mediated immune response against HLA-disparate OTS CAR+T-cells. An approach to inducing immunosuppression is based on using chemotherapy (e.g., cyclophosphamide and fludarabine) to lympho-deplete the recipient. For example, the elimination of CD52 from CAR T-cells by TALEN may avoid immunosuppression by CD52-specific mAb.116 This strategy may be used to sidestep clearance of infused OTS CAR+T-cells by an immunosuppressive agent while helping to avoid rejection by the recipient’s immune system. The degree of lymphodepletion achieved with more intensive regimens appears to correlate with improved persistence of infused T-cells and NK-cells.117,118 What is not known is whether the depth of such lymphodepletion will lead to sufficient suppression of the immune system to favorably prevent immune-mediated rejection of HLA-disparate immune cells. The length of time for the recipient to remain immunosuppressed to achieve a desired CAR-mediated antitumor response remains to be determined and during this period the patient is at risk from opportunistic infection. . The human application of OTS T-cells may be useful when combined with allogeneic HSCT and the resultant immunocompromised clinical state. Indeed, an apparently successful infusion of OTS CAR+T-cell therapy using this strategy was recently announced in one recipient, although follow-up is short.119

Modulation of immune system in recipients Small molecules, such as sphingosine-1 receptor agonist, FTY720, may be used as modulators of immune system function rather causing immune-mediated suppression. Thus, FTY720 can preclude T-cells from infiltrating into a transplanted allograft which would lead to rejection,120 as well as preventing rejection of administered allogeneic CAR+T-cells,121 and preventing GvHD in the context of allogeneic HSCT in mice.122 The mechanism of suppressing GvHD includes not only inhibition of egress, but also apparently induction of apoptosis of alloreactive T-cells in lymph nodes.122 It is likely that continuous exposure to FTY720 will be needed to maintain a beneficial effect,123 which may adversely affect immune cells other than alloreactive T-cells. Furthermore, since normal B-cells reside within lymph nodes, this might induce Molecular Therapy  vol. 24 no. 7 jul. 2016

the unwanted apoptosis of CD19-specific OTS CAR+T-cells residing in such lymphoid spaces. Thus, FTY720 may prevent adverse events arising from donor-derived OTS CAR+T-cells recognizing recipient alloantigens as well as limiting host-derived T-cells deleteriously recognizing disparate HLA on infused OTS cells. However, safeguarding the specificity of the CAR may preclude the potential benefit on survival of infused OTS T-cells.

Enforced expression of immune checkpoint molecules Blockade of immune checkpoints can be used to activate resident tumor-specific T-cells to achieve clearance of tumors. Enforced expression of PD-L1 and CTLA4-Ig in human embryonic stem cells has successfully suppressed rejection of derived allogeneic cells in mice reconstituted with elements of the human immune system.124 Furthermore, allogeneic pancreatic cells differentiated from human embryonic stem cells can engraft in humanized mice.125 However, the expression of PD-L1 and CTLA4-Ig on CAR+T-cells will presumably result in suppression of effector functions, and thus this approach may not be suitable for OTS T-cell therapy. HLA homozygous donors An approach to minimize the rejection of introduced allogeneic cells is to match HLA type between one or more recipients and one or more donors. However, the probability to find a suitable HLA-matched donor for an unrelated patient is low as even millions registered with the National Marrow Donor Program cannot provide coverage for the entire US population.126 An approach to decrease the number of donors is to use individuals that are homozygous at one or more HLA alleles. For instance, 50 unique donors with HLA homozygous at HLA-A/B/DRB1 are calculated to provide 73% of the Japanese population with HLA-matched third party cells and these donors can be found by screening 37,000 Japanese.127 In the United Kingdom, 50 donors homozygous for the most prevalent HLA alleles can provide ~80% of the population with HLA-matched allogeneic cells.128 Therefore, the establishment of a biobank that is homozygous at HLA loci is anticipated to generate OTS CAR+T-cells that are HLAmatched with multiple recipients. In addition to the matching 1183

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of HLA-A/-B/-DRB1, we may need to explore the importance of matching other HLA loci (HLA-C/-DPB1) in OTS T-cell therapeutic settings, as these molecules have been identified as the important alleles related to allogeneic immune responses in allogeneic HSCT setting. Furthermore, the potential clinical importance of minor histocompatibility antigen(s) in the mismatched setting needs to be addressed. A potential source for obtaining HLA homozygous donor derived T-cells would be frozen cord blood units in the public cord blood bank, as HLA information is usually available in those cord blood units and these cells are ready to use.

Genetic editing of HLA We have developed an approach to bioengineering T-cells to limit the number of HLA-homozygous donors needed to match to an HLA-diverse population. Indeed, designer ZFNs have been used to eliminate expression of HLA-A from CAR+T-cells and also TCRnegCAR+T-cells.129 An area of possible concern is deleterious NK-cell mediated attack upon recognition of infused HLAnegTcells. However, this may have limited clinical impact as we have demonstrated that the enforced expression of nonclassical nonpolymorphous HLA molecules, such as HLA-E or HLA-G, can suppress lysis of genetically edited T-cells by NK-cells circulating in peripheral blood.129 Another strategy to avoid NK-cell attack may be to enforce expression of Siglec-7 and -9 ligands on HLAnull cells.130

CONCLUSION

Autologous CAR+T-cells have demonstrated dramatic antitumor responses especially in patients with B-cell tumors. The human applications of OTS CAR+T-cells currently serve as a type of cellular template for testing CAR species that can recognize hematologic tumors. However, these clinical data also reveal challenges (Table 4) to widen their human application within adoptive immunotherapy. For example, we recognize that the initial embodiments of genetically reprogrammed T-cells sourced from a limited number of third party HLA-mismatched donors may be rejected. This will likely be lessened after immune suppression and/or elimination of one or more HLA through genetic editing.131 Even so, the presence of minor histocompatibility antigens may still engender an unwanted response in the recipient leading to immune-mediated clearance. The impact of this rejection vector might be blunted by sequentially infusing products derived from more than one donor to achieve a therapeutic response. Thus, in the absence of suitable animal models, the antitumor activity associated with successive advances in OTS therapies will need to be revealed in iterative clinical trials. In aggregate, targeting of T-cells to antigens independent of HLA provides optimism that CAR+T-cells can be rendered as a “drug”. At this time, OTS approaches belong within a constellation of other immunotherapies such as the adoptive transfer of autologous T-cells. For example, during the time that patientderived CAR+T-cells are prepared the recipient could receive an OTS cellular therapeutic. The clinical evaluation of OTS cells for cancer can be contemplated as a modality to provide disease control (transient remission) or long-term remission. This distinction will help calibrate the emphasis on bioengineering methodologies 1184

to improve in vivo persistence. The former, while still clinically meaningful, implies that survival of the infused product may be compromised, whereas striving for long-term remission implies that the OTS product will not be subject to immune-mediated rejection and is HLA compatible with recipient. The decision to infuse autologous or allogeneic CAR+T-cells as monotherapies or in combinations will evolve as the technologies associated with each cellular product is advanced to meet the dual needs of safely and completely eliminating tumor. Overall, the benefits of immediacy, logistics, and trial design governing OTS immunotherapies, such as with CAR+T-cells, justify the development of a path to their human applications. ACKNOWLEDGMENTS The authors thank Judy Moyes for assistance with editing. Some of the technology described in this article was advanced through research conducted at the MD Anderson Cancer Center under the direction of L.J.N.C. In January 2015, the technology was licensed for commercial application to ZIOPHARM Oncology, Inc., and Intrexon Corporation in exchange for equity interests in each of these companies for which both authors are entitled to receive a portion. On May 7, 2015, Cooper was appointed as the Chief Executive Officer at ZIOPHARM. Cooper is now a Visiting Scientist at MD Anderson Cancer Center.

AUTHOR CONTRIBUTIONS H.T. and L.J.N.C. wrote the article.

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