Translational Implications for Off-the-shelf Immune Cells Expressing Chimeric Antigen Receptors

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Accepted Article Preview: Published ahead of advance online publication TRANSLATIONAL IMPLICATIONS FOR OFF-THE-SHELF IMMUNE CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTORS

Hiroki Torikai, and Laurence J.N. Cooper

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Cite this article as: Hiroki Torikai, and Laurence J.N. Cooper, TRANSLATIONAL IMPLICATIONS FOR OFF-THE-SHELF IMMUNE CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTORS, Molecular Therapy accepted article preview online 16 May 2016; doi:10.1038/mt.2016.106

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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 03 March 2016 ; accepted 28 April 2016 ; Accepted article preview online 16 May 2016

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Article title TRANSLATIONAL IMPLICATIONS FOR OFF-THE-SHELF IMMUNE CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTORS Short title: Off-the-shelf immune cells expressing CAR Authors: Hiroki Torikai1, and Laurence J.N. Cooper1,2 Author affiliation: Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX

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Ziopharm Oncology Inc. Boston, MA

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*Co-corresponding Authors:

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Laurence J.N. Cooper, M.D., Ph.D.

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Division of Pediatrics, The University of Texas MD Anderson Cancer Center

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Unit 907, 1515 Holcombe Blvd., Houston, TX 77030 Phone: (713) 563-3208; Fax: (713) 792-9832

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Hiroki Torikai, M.D.

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E-mail: [email protected] and [email protected]

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Division of Pediatrics, The University of Texas MD Anderson Cancer Center Unit 907, 1515 Holcombe Blvd., Houston, TX 77030 Phone: (713) 792-8195; Fax: (713) 792-9832 E-mail: [email protected]

Word count for text: 4481 words, Word count for abstract: 200 words, Figure count: 2 figures, Table Count: 4 tables, Reference count: 133 references

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ABSTRACT Chimeric antigen receptor (CAR) endows specificity to T-cells independent of 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

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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 bio-bank

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of allogeneic genetically modified immune-cells from healthy third-party donors, that are cryopreserved

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and validated in advance of administration, will facilitate the centralizing manufacturing and widespread

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distribution of CAR+T-cells to multiple points-of-care in a timely manner. To achieve this, it is

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necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to

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toxicity and rejection. We review the strategies to establish “off-the-shelf” donor-derived bio-banks for

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human application of CAR+T-cells as a drug.

KEY WORDS Off-the-shelf CAR+T-cell, genome editing, allogeneic immune reaction

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TEXT Introduction 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 antigenrecognition domain of the prototypical CAR uses a single chain variable fragment (scFv) 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

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receptor (e.g., Dectin-1) for targeting foreign carbohydrates such as β-glucan on germinating Aspergillus3. These binding motifs are typically fused to an extracellular stalk or varying lengths and

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composition, such as derived from the hinge with or without CH2-CH3 domains from IgG1 and IgG4 or

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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

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_ENREF_8. Recent evidence also suggests the importance of the stalk to impact the effector

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by introducing site-directed changes or eliminating CH2 region to reduce potential for such clearance4-

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functioning of CAR+T-cells interrogating the binding motifs within TAAs proximal versus distal to the cell surface7-10. 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 ITAM motifs in cytoplasmic domain to mimic the TCR/CD3 signal11. 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 CD2812, CD13713, 14, OX4015, and CD27 domains16, improved function of CAR+T-cells manifested by sustained persistence after adoptive transfer leading to improved therapeutic effect17, 18. In addition to “signal 1” delivered by phosphorylation of ITAMs and “signal 2” mediated by co-stimulatory molecules, T-cells typically require a third signal to achieve and perhaps

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sustain full activation. This third signal is mediated through the common γ-chain cytokine receptor and thus coordinated delivery or co-expression of certain cytokines can enhance CAR+T-cell functions, which may be especially useful for the application of genetically modified T-cells targeting solid tumors19-21. Human applications of CAR+T-cells have shown promise in several early phase clinical trials, such as infusing targeting CD19 on B-cell leukemias22-30 and lymphomas31-33 and targeting GD2 on neuroblastoma34_ENREF_32. Recently, several reviews have highlighted and summarized these clinical successes35-39. With these early successes, CAR+T-cell therapy is redeploying from academia to

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industry40. Broadening the clinical experience of CAR+T-cell therapy is thus emerging as an important

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hurdle, especially for those tumor targets already associated with an anti-tumor effect. This underscores

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the need to expand the list of qualified TAAs that can be safely targeted by T cells though one or more

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engineered immunoreceptors41. 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

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recognition of more than one TAA,42, 43 or targeting intracellular proteins44, 45.

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Another limitation to the broad implementation of CAR+T-cell therapy resides with the

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production process to generate clinical-grade biologics. Currently, T-cells are genetically modified and propagated infusing either patient-derived T-cells often after lympho-depleting chemotherapy or donorderived T-cells administered in the context of hematopoietic stem-cell transplantation (HSCT). These Tcells 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 (GMP) for phase I and II trials on campuses that are in close proximity to clinical facilities to administer CAR+T-cells46. This leads to heterogeneity of manufacturing methods which 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 GMP facilities with infusion units can lead to

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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 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) cellbased biologic therapies in which immune cells can be manufactured ahead of need (in advance of

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consent) and infused on demand as required by the recipient, rather than when the biologic is produced.

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“Off-the-shelf” (OTS) CAR+T-cell therapy

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The current production and lot release (quality control) of clinical-grade genetically modified T-

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cells requires time in culture within GMP-compatible manufacturing facilities during which the intended

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recipient’s condition can deteriorate. Currently, the “distributed” approach to manufacturing by

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academic centers at multiple point-of-care has resulted in a portfolio of biologic products that differ in

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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 pre-deployment 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 multi-center trials powered for efficacy and to establish the maximum tolerated dose. The administration of a wellcharacterized product will then facilitate combination immunotherapies infusing OTS T-cells with other

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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-host-disease after infusion of OTS CAR+T-cells In the setting of an HLA-mismatch between donor and recipient, the frequency of T-cells

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specific for disparate HLA is estimated around 1 in 104 47, 48. In clinical trials, the number of administered CAR+T-cells is typically between 108 to 109 which could lead to the delivery 103 to 105 T-

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cells expressing αβTCR specific for allogeneic antigens and thus likely be sufficient to induce graft-

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versus-host-disease (GvHD). We describe five strategies to prevent this un-wanted activation through

1. Depletion of alloreactive T-cells

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endogenous TCR (Table 3).

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T-cells displaying αβTCRs play a central role inducing GvHD and therefore several strategies

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have been developed to ex vivo remove alloreactive T-cells such as contaminating the co-infusion of HLA-mismatched hematopoietic stem-cells (HSC) 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., CD2549-51, CD6952, and CD13753) upon co-culture with antigenpresenting cells (APC) derived from the intended recipient. Activated T-cells can be then be reduced using magnetic beads or immunotoxin conjugated to mAb52,49. Photo-depletion is an alternative approach for reduction of alloreactive T-cells based on the inability of activated T-cells to efflux of a phototoxic dye54. 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

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depletion cannot completely eliminate alloreactive T-cells. Moreover, the requirement to co-culture the CAR+T-cells with recipient’s cells reduces the speed and convenience associated with producing this OTS biologic. 2. Allo-anergization of T-cells We demonstrated that anergization of CAR+T-cells can be achieved in tissue culture by combining allo-stimulation with HLA-mismatched APC and concomitant blockade of CD28-mediated co-stimulation55. 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 induction of allo-

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anergization requires patient-derived stimulator cells which undermines its suitability for OTS CAR+T-

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cells.

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3. T-cells expressing αβTCR with limited or defined specificity

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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 naïve T-cells induced

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GvHD in a mouse model, whereas, administering memory T-cells did not56, 57_ENREF_29. This may be

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due to a difference in the αβTCR diversity between naïve and memory pools as revealed by Vβ CDR3

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spectratyping or sequencing58, 59. 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 GvHD60. This may have a human application as naïve T-cells can be depleted by recognition of CD45RA while preserving memory T-cells (and HSC)61. The therapeutic potential of adoptive immunotherapy appears to correlate with T-cells expressing a less-differentiated phenotype62 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 naïve population from allogeneic graft 63.

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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 so long as the restricting αβTCR fail 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 antigen64-67. 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

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endogenous TCR). An early-phase clinical trial has demonstrated that this can be accomplished in HLA-

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matched settings as donor-derived viral-specific CAR+T-cells were infused after allogeneic HSCT27.

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However, caution may be warranted when delivering antigen-specific T-cells to completely HLA-

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mismatched recipients as TCR-mediated recognition of target antigens is more promiscuous than

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anticipated68. Although GvHD has not been observed after infusing third party HLA-mismatched viral-

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substrate for OTS CAR+T-cells.

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specific T-cells69, further work is warranted before such antigen-specific T-cells can be used as a cellular

(i) γδT-cells

T-cells

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4. Effector cells other than peripheral blood-derived

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 γδ T-cells has been identified, the target antigens of most γδTCR are not well characterized and the impact of donorderived γδ T-cells in GvHD pathology is uncertain 70, 71,72, 73. We and others showed that ex vivo activated or expanded human γδ T-cell did not apparently cause xeno-GvHD74-77. Furthermore, the early engraftment of HLA-haploidentical γδ T-cells after infusion of HSC stripped of contaminating αβTCR did not lead to GvHD in humans78. The specificity of one subset of γδ T-cells expressing γ9δ2 TCR

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recognizes isopentenylpryophosphate (IPP) which can specifically propagated by co-culturing with clinical-grade aminobisphosphonate based upon the inhibition of cholesterol synthesis leading to the accumulation of IPP79. 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 trials80. The γ9δ2 T-cell may be combined with CAR to be an option for establishing OTS CAR+T-cell therapy81. 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

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appealing approach to generate OTS CAR+T-cells76. (ii) NKT-cells

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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

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reported after allogeneic HSCT while preserving a graft-versus-tumor (GVT) response82_ENREF_68.

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NKT-cell can also be expanded in vitro by aAPC83 and can be genetically modified to express CAR84.

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Thus, the limited TCR usage and emerging technology to obtain large numbers raises the possibility that

(iii) NK-cells

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NKT-cells can be used to generate OTS CAR+ immune 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 cytotoxicity85. An anti-tumor response attributed to NK-cells has been well documented in the context of HLA-mismatched HSCT86-89. 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 GvHD89, 90

. 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

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(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-1591 or IL-2192. Furthermore, the specificity of NK-cells can be directed through enforced expression of a CAR91. 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 clinicallysignificant GvHD93. 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.

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(iv) Precursor T-cells

T-cells can be generated in vitro by co-culturing CD34+ HSC on Notch-1 ligand-expressing

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mouse stroma cells (OP9/DLL-194 or OP9/DLL-495). This system generates precursor T-cells prior to

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thymic selection in vivo. 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-

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antigens were eliminated by host thymic selection96. This reveals an approach to administer OTS CAR+

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precursor T-cells generated in vitro by partially differentiating HSC with Notch-1 ligand97. As human

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HSC can apparently be safely genetically modified with lentivirus to express CAR98, 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-cells99. (v) Conditional ablation of T-cells Haploidentical T-cells have been genetically modified to express a suicide gene and infused to deliver a GvT-effect after HSCT with the expectation that GvHD can be controlled by eradicating infused T-cells using a pro-drug. The two suicide genes chiefly assessed in clinical trials are thymidine kinase (TK) from herpes simplex virus 1100 and induced caspase 9 (iCasp9)101. An advantage of TK is

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the ready availability of ganciclovir as a FDA-approved product100,

102, 103

. Disadvantages are the

immunogenicity of viral-derived TK leading to recognition and elimination by the recipient104 and possibly the time course to destroy T-cells after delivery of ganciclovir. An alternative system uses a chemical dimerizer to cross-link iCasp9 leading to cell death. Since 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 published105, 106_ENREF_96 demonstrating resolution of GvHD after infusing haploidentical iCasp9+T-cells were rapidly eliminated by a single infusion of clinicalgrade dimerizer. The clinical appeal of OTS CAR+T-cells that co-express a suicide gene107 will depend

5. Elimination of expression of endogenous

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TCR

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on the kinetics of elimination and the associated potential for loss of the anti-tumor effect.

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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

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of the artificial nucleases108. Alternative artificial nucleases, such as transcription activator-like effector

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nuclease (TALEN)109, 110 and CRISPR/Cas9 (clustered regularly interspaced short palindromic

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repeat/CRISPR-associated proteins)111, 112 are recent technologies that may have translational appeal. We

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applied ZFNs to eliminate expression of endogenous αβTCR from CAR+T-cells thereby completely eliminating the possibility to induce GvHD113. Transient expression of a pair of ZFNs from electrotransferred in vitro-transcribed mRNA resulted in disruption of TCR expression on T-cells genetically modified using the Sleeping Beauty (SB) system to express a CD19-specific CAR. The TCRnegCAR+Tcells were readily enriched by magnetic depletion of the remaining CD3+ population using an approach amenable to clinical translation. Significantly, these genetically edited and modified T-cells could be numerically expanded on CD19+ aAPC. One concern regarding the human application of T-cells genetically edited with artificial nucleases is the occurrence of off-target introduction of double-stranded 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

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systems may improve off-target-cleavage and also the efficiency of disrupting αβTCR from T-cells114. Using these editing technologies, we have established that the manufacturing process can generate at least 3 x 109 TCRnegCAR+ OTS T-cells from one frozen cord blood unit, which typically contains about 8 x 107 CD3+ T-cells (unpublished data). We estimate that upon completing this propagation and given an infusion dose of 3.0 x 108 per patient29, 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 therapy62. Another round of ex vivo numeric expansion of TCRnegCAR+ OTS T-

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cells will typically increase the cell number 10- fold, which will result in sufficient product for injection

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leukapheresis product harvested from a healthy donor.

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into up to 100 patients. This number might be increased upon harvesting T-cells from a steady-state

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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

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infused. Yet, CAR+T-cells possess inherent advantages over mAbs due to their home to sites of

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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 immuno-monitoring to prevent recurrence. In the autologous setting, genetically modified T-cells can survive in human for over 10 years115. However, infusing allogeneic CAR+T-cells from just one unrelated and HLA-disparate donor could activate the recipients’ immune systems and be rejected before an anti-tumor effect is fully realized and prevent long-term immunoprotection. Thus, strategies to avoid clinically-deleterious immune-remediated rejection of OTS CAR+Tcells 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 3rd party OTS viral-specific T-cells, matched at only one HLA, are successfully infused to control EBV+

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lymphoproliferative disease after allogeneic HSCT67. 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. 1. 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 effect116, 117. In the context of infusion of allogeneic T-cells this immunosuppression may limit the emergence and/or potency of host-mediated immune response against HLAdisparate OTS CAR+T-cells. One approach to inducing immuno-suppression is based on using

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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 immune suppression by CD52-

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specific mAb118. This strategy may be used to sidestep clearance of infused OTS CAR+T-cells by an

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immunosuppressive agent while helping to avoid rejection by the recipient’s immune system. The

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degree of lymphodepletion achieved with more intensive regimens appears to correlate with improved

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persistence of infused T-cells and NK-cells119, 120. What is not known is whether the depth of such

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lymphodepletion will lead to sufficient suppression of the immune system to favorably prevent immune-

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mediated rejection of HLA-disparate immune cells. The length of time for the recipient to remain immunosuppressed to achieve a desired CAR-mediated anti-tumor 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, though follow-up is short121. 2. 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 Tcells from infiltrating into a transplanted allograft which would lead to rejection122, as well as preventing

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rejection of administered allogeneic CAR+T-cells123, and preventing GvHD in the context of allogeneic HSCT in mice124. The mechanism of suppressing GvHD includes not only inhibition of egress, but apparently also induction of apoptosis of alloreactive T-cells in lymph nodes (LNs)124. It is likely that continuous exposure to FTY720 will be needed to maintain a beneficial effect125, which may adversely affect immune cells other than alloreactive T-cells. Furthermore, since normal B-cells reside within LNs, this might induce 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

the potential benefit on survival of infused OTS T-cells.

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3. Enforced expression of immune checkpoint molecules

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disparate HLA on infused OTS cells. However, safeguarding the specificity of the CAR may preclude

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Blockade of immune checkpoints can be used to activate resident tumor-specific T-cells to

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achieve clearance of tumors. Enforced expression of PD-L1 and CTLA4-Ig in human embryonic stem

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cells has successfully suppressed rejection of derived allogeneic cells in mice reconstituted with

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elements of the human immune system126. Furthermore, allogeneic pancreatic cells differentiated from

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human embryonic stem cells can engraft in humanized mice127. 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. 4. HLA homozygous donors One 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 un-related patient is low as even millions registered with the National Marrow Donor Program cannot provide coverage for the entire U.S. population128. One 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

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73% of the Japanese population with HLA-matched 3rd party cells and these donors can be found by screening 37,000 Japanese129. In the United Kingdom, 50 donors homozygous for the most prevalent HLA alleles can provide approximately 80% of the population with HLA-matched allogeneic cells130. Therefore, the establishment of a biobank that is homozygous at HLA loci is anticipated to generate OTS CAR+T-cells that are HLA-matched with multiple recipients. In addition to the matching of HLAA/-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

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importance of minor histocompatibility antigen(s) in the mis-matched setting needs to be addressed. One potential source for obtaining HLA homozygous donor derived T-cells would be frozen cord blood units

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in the public cord blood bank, as HLA information is usually available in those cord blood units and

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these cells are ready to use.

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5. Genetic editing of HLA

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We have developed an approach to bio-engineering T-cells to limit the number of HLA-

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homozygous donors needed to match to an HLA-diverse population. Indeed, designer ZFNs have been

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used to eliminate expression of HLA-A from CAR+T-cells and also TCRnegCAR+T-cells131. One area of possible concern is deleterious NK-cell mediated attack upon recognition of infused HLAnegT-cells. However, this may have limited clinical impact as we have demonstrated that the enforced expression of non-classical non-polymorphous HLA molecules, such as HLA-E or HLA-G, can suppress lysis of genetically edited T-cells by NK-cells circulating in peripheral blood131. Another strategy to avoid NKcell attack may to enforce expression of Siglec-7 and -9 ligands on HLAnull cells132.

Conclusion Autologous CAR+T-cells have demonstrated dramatic anti-tumor responses especially in patients with B-cell tumors. The human applications of OTS CAR+T-cells currently serves as a type of cellular

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

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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 editing133. 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,

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in the absence of suitable animal models, the anti-tumor activity associated with successive advances in OTS therapies will need to be revealed in iterative clinical trials.

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In aggregate, targeting of T-cells to antigens independent of HLA provides optimism that

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CAR+T-cells can be rendered as a “drug”. At this time, OTS approaches belong within a constellation of

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other immunotherapies such as the adoptive transfer of autologous T cells. For example, during the time

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that patient-derived CAR+T-cells are prepared the recipient could receive an OTS cellular therapeutic.

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The clinical evaluation of OTS cells for cancer can be contemplated as a modality to provide disease

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control (transient remission) or long-term remission. This distinction will help calibrate the emphasis on bio-engineering methodologies 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.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

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ACKNOWLEDGEMENT We thank Dr. Judy Moyes for assistance with editing.

AUTHORSHIP Author contribution: H.T. and L.J.N.C. wrote the paper. Conflict of Interest: Some of the technology described in this presentation was advanced through research conducted at the MD Anderson Cancer Center under the direction of Laurence J.N. Cooper. In January 2015, the technology was licensed for commercial application to ZIOPHARM Oncology, Inc.,

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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, Dr. Cooper was appointed as the Chief

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Executive Officer at ZIOPHARM. Dr. Cooper is now a Visiting Scientist at MD Anderson.

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FIGURE LEGENDS Figure 1. Manufacturing of 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 OTS CAR+T-cells: CAR+T-cells are generated in a single facility and distributed to multiple points-of-care.

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Figure 2. Schematic presentation of potential issues in establishing OTS CAR+T-cells from one or

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more third party donors.

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While allogeneic CAR+T-cells can destruct target tumor cells, they may also recognize patient’s somatic

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cells through endogenous TCR, which results in the deleterious GvHD. We will need to avoid this

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allogeneic immune reaction induced by infused allogeneic CAR+T-cells. Further consideration will be

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needed to preclude recognition of infused CAR+T-cells by recipient’s immune system to sustain

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CAR+T-cells in vivo persistence.

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Table 1: Heterogeneity in the biologic products as derived from different autologous or allogeneic donors

Number of copies of integrated CAR Genomic sites of transgene insertion(s) T-cell immuno-phenotype TCR repertoire

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Effector function due to the phenotype, single nucleotide polymorphisms (SNPs), etc…

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Table 2: Advantages associated with infusing OTS CAR+T-cells manufactured from one or more 3rd party donor(s) for administration and re-administration into multiple unrelated recipients. 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 pre-deployed at multiple points-of-care

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OTS CAR+T-cells have reduced heterogeneity compared with multiple patient-specific

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CAR+T-cells

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OTS CAR+T-cells can be produced using centralized manufacturing site(s) OTS CAR+T-cells reduce the barriers to undertaking Phase IIb multi-center trials powered

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for efficacy

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OTS CAR+T-cells can be infused as part of multi-component trials administering this

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biologic at predefined maximally tolerated dose

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Table3: Potential strategies to generate OTS cell products Avoid GvHD Eliminate alloreactive T-cells CD25, CD69, CD137 depletion, photo-depletion Suppress TCR mediated activation B7 specific mAb for blocking CD28 mediated co-stimulation 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

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γδ T-cells, NK-cells, NKT-cells Use precursor T-cells

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Conditional ablation

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HSV1-TK, iCasp9 Eliminate of endogenous TCR

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

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Avoid rejection

Suppress immune system in recipients

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lymphodepletion (CY, Fludarabine, TBI, anti-CD52Ab)

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FTY720

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Modulation of immune system in recipients

Enforced expression of immune checkpoint molecules PD-L1, CTLA4-Ig HLA homozygous donor Gene editing of HLA ZFN, TALEN, CRISPR/Cas9 and other gene editing technologies

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Table 4: Questions to improve the therapeutic potential and acceptance of infused 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 anti-tumor response? What lympho-depleting and/or immune-suppressive regimens are needed to prevent immunemediated rejection and deleterious clearance leading to loss of anti-tumor 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 anti-tumor response both be measured/optimized?

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Will scale up and manufacturing of large bio-banks impact the anti-tumor activity and how will

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this be a prior assessed?

Should investigators and recipients expect (temporary) disease control rather than (sustained)

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anti-tumor effects?

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What clinical context is best suited for initial clinical testing given the variabilities of tumor (i)

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types, (ii) burdens, and (iii) distributions?

What clinical context is best suited for initial clinical testing given the variabilities of each

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recipient’s (i) genetic background, (ii) body mass, and (iii) prior therapies?

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How to address the targeting of solid tumors?

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Should Phase I trials anticipate that recipients receive more the one infusion?

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Are T cells the preferred cellular substrate for an OTS biologic/product?

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Figure 1

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Figure 2

© 2016 The American Society of Gene & Cell Therapy. All rights reserved