Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen
`receptor
`Bipulendu Jena, 1 Gianpietro Dotti, 2 and Laurence J. N. Cooper1
`'Division of Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston; and 2Center for Cell and Gene Therapy, Baylor College of Medicine,
`Houston, TX
`
`Infusions of antigen-specific T cells have
`yielded therapeutic responses in patients
`with pathogens and tumors. To broaden
`the clinical application of adoptive immu(cid:173)
`notherapy against malignancies, investi(cid:173)
`gators have developed robust systems
`for the genetic modification and character(cid:173)
`ization of T cells expressing introduced
`chimeric antigen receptors (CA Rs) to redi(cid:173)
`rect specificity. Human trials are under
`way in patients with aggressive malignan-
`
`cies to test the hypothesis that manipulat(cid:173)
`ing the recipient and reprogramming
`T cells before adoptive transfer may im(cid:173)
`prove their therapeutic effect. These ex(cid:173)
`amples of personalized medicine infuse
`T cells designed to meet patients' needs
`by redirecting their specificity to target
`molecular determinants on the underly(cid:173)
`ing malignancy. The generation of clinical
`grade CAR+ T cells is an example of
`bench-to-bedside translational science
`
`that has been accomplished using inves(cid:173)
`tigator-initiated trials operating largely
`without industry support. The next-gen(cid:173)
`eration trials will deliver designer T cells
`with improved homing, CAR-mediated
`signaling, and replicative potential, as
`investigators move from the bedside to
`the bench and back again. {Blood. 201 0;
`116(7):1035-1044)
`
`1 ntrod uction
`
`The systematic development and clinical application of genetically
`modified T cells is an example of how academic scientists working
`primarily at nonprofit medical centers are generating a new class of
`therapeutics. In this context, gene therapy has been used to
`overcome one of the major barriers to T-cell therapy of cancer,
`namely tolerance to desired target tumor-associated antigens (TAAs).
`This was achieved by the introduction of a chimeric antigen
`receptor (CAR) to redirect T-cell specificity to a TAA expressed on
`the cell surface. The prototypical CAR uses a mouse monoclonal
`antibody (mAb) that docks with a designated TAA and this binding
`event is reproduced by the CAR to trigger desired T-cell activation
`and effector functions. Multiple early-phase clinical trials are now
`under way or have been completed to evaluate the safety and
`feasibility of adoptive transfer of CAR+ T cells (Table 1). These
`pilot studies have revealed challenges in achieving reproducible
`therapeutic successes which may be solved by (1) reprogramming
`the T cells themselves for improved replicative potential, effector
`function, and in vivo persistence, (2) manipulating the recipient to
`improve TAA expression and survival of infused T cells, and
`(3) adapting the gene therapy platform to deliver (1) CARs capable
`of initiating an antigen-dependent fully-competent activation sig(cid:173)
`nal, and (2) transgenes to improve safety, persistence, homing, and
`effector functions within the tumor microenvironment. Academic
`investigators who work to both develop and deliver investigational
`biologic agents such as CAR+ T cells are poised to further tighten
`the pace of discovery between the bench and the bedside to
`improve the therapeutic potential of genetically modified T cells
`with redirected specificity. This review builds upon recent articles
`that describe the immunobiology of CAR+ T cells (Table 2) and we
`highlight how the CAR technology has been adapted to meet the
`challenges of infusing genetically modified T cells in medically
`fragile patients with aggressive malignancies and what new
`
`directions the field will need to embrace to undertake multicenter
`trials to prove their therapeutic efficacy.
`
`Redirecting T-cell specificity through the
`introduction of a CAR
`
`The generation of T bodies, or CARs, by Eshhar et al has been
`adapted by investigators as a tool to enable T cells, as well as other
`immune cells, to overcome mechanisms (eg, loss of human
`leukocyte antigen [HLA]1) by which tumors escape from immune
`surveillance of the patient's endogenous (unmanipulated) T-cell
`repertoire. 2•3 The specificity of a CAR is achieved by its exodomain
`which is typically derived from the antigen binding of a mAb
`linking the V Hand V L domains to construct a single-chain fragment
`variable (scFv) region. The exodomains of CARs have also been
`fashioned from ligands or peptides (eg, cytokines) to redirect
`specificity to receptors (eg, cytokine receptors), such as the
`IL-13Ru2-specific "zetakine."4 The exodomain is completed by
`the inclusion of a flexible (hinge) sequence, such as from CD8u or
`immunoglobulin sequence5·6 and via a transmembrane sequence,
`the exodomain is fused to 1 or more endodomain(s) which may
`include cytoplasmic domains from CD3-E, CD3--y, or CD3-1; from
`the T-cell receptor (TCR) complex or high-affinity receptor FcERI.7·9
`When CARs are expressed on the cell surface of genetically
`modified T cells, they redirect specificity to TAA (including TAA
`on tumor progenitor cells 10) independent of major histocompatibil(cid:173)
`ity complex (MHC). This direct binding of CAR to antigen ideally
`provides the genetically modified T cell with a fully-competent
`activation signal, minimally defined as CAR-dependent killing,
`proliferation, and cytokine production. Given that T cells targeting
`tumor through an endogenous ul3TCR (and even introduced TCR
`
`Submitted January 8, 2010; accepted April 26, 2010. Prepublished online as
`Blood First Edition paper, May 3, 201 O; DOI 10.1182/blood-2010-01-043737.
`
`© 201 O by The American Society of Hematology
`
`BLOOD, 19AUGUST 2010 • VOLUME 116, NUMBER 7
`
`1035
`
`Miltenyi Ex. 1031 Page 1
`
`

`

`1036
`
`JENA et al
`
`BLOOD, 19 AUGUST 2010 • VOLUME 116, NUMBER 7
`
`Table 1. Clinical trials in the United States infusing CAR+ T cells under IND
`Viral-
`specific Lympho-
`Teen
`depletion
`
`Tumor target
`
`Antigen
`
`CAR
`generation
`
`ClinicalTrial.gov
`identifier
`
`Enrolling
`
`SAE
`
`Gene transfer
`
`Kappa light chain B-NHL and B-CLL
`2 CD19
`Lymphoma/leukemia (B-NHL) and CLL
`3 CD19
`Advanced B-NHUCLL
`4 CD19
`Lymphoma and leukemia
`5 CD19
`ALL (post-HSCT)
`6 CD19
`Follicular NHL
`7 CD19
`CLL
`
`8 CD19
`9 CD19
`10 CD19
`11 CD19
`
`B-NHUleukemia
`B-cell leukemia, CLL and B-NHL
`B-ALL
`B-lymphoid malignancies
`
`No
`No
`Yes
`No
`Yes
`No
`No
`
`No
`No
`No
`No
`
`Yes
`No
`No
`Yes
`No
`Yes
`Yes/no
`
`Yes
`No
`Yes
`Yes
`
`First and second NCT00881920
`First and second NCT00586391
`First and second NCT00709033
`Second
`NCT00924326•
`Second
`NCT00840853
`First
`NCT00182650
`Second
`NCT00466531 •
`
`First and second NCT00891215
`Second
`NCT01087294
`Second
`NCT01044069
`Second
`NCT00968760
`
`Yes
`Yes
`Yes
`Yes
`Yes
`No
`Yes
`
`Yes
`Yes
`Yes
`No
`
`TBM
`TBM
`TBM
`TBM
`TBM
`No
`Yes
`(1 Death)
`TBM
`TBM
`TBM
`TBM
`
`Virus
`Virus
`Virus
`Virus
`Virus
`Electroporation
`Virus
`
`Virus
`Virus
`Virus
`Electroporation
`(SB system)
`Electroporation
`Electro po ration
`Virus
`Virus
`Virus
`Electroporation
`Virus
`Virus
`Virus
`Virus
`Electroporation
`Virus
`
`NCT00012207'
`NCT00621452
`NCT00085930•
`NCT00004178
`NCT00664 196
`NCT00006480
`NCT00019136
`NCT00429078
`NCT00673829
`NCT00673322
`NCT00730613
`NCT00924287
`
`No
`Yes
`Yes
`No
`Yes
`No
`No
`Yes
`Yes
`Yes
`No
`No
`
`12 CD20
`Relapsed/refractory B-NHL
`13 CD20
`Mantle cell lymphoma or indolent B-NHL
`14 GD2
`Neuroblastoma
`15 CEA
`Adenocarcinoma
`16 PSMA
`Prostate cancer
`17 CD171/L 1-CAM Neuroblastoma
`18 FR
`Ovarian epithelial cancer
`19 CEA
`Stomach carcinoma
`20 CEA
`Breast cancer
`21 CEA
`Colorectal carcinoma
`IL-13Ra2
`22
`Glioblastoma
`23 ERBB2
`Metastatic cancer
`(HER2/neu)
`24 HER2/neu
`25 HER2/neu
`
`Lung malignancy
`Advanced osteosarcoma
`
`No
`No
`Yes
`No
`No
`No
`No
`No
`No
`No
`No
`No
`
`Yes
`No
`
`Yes
`Yes
`Yes
`No
`Yes
`No
`No
`No
`No
`No
`NA
`Yes
`
`First
`Third
`First
`First
`First
`First
`First
`Second
`Second
`Second
`Second
`Third
`
`No
`TBM
`No
`No
`TBM
`No
`No
`TBM
`TBM
`TBM
`No
`Yes
`(1 Death)
`No
`Second
`NCT00889954
`Yes
`Virus
`TBM
`No
`Second
`NCT00902044
`Virus
`Yes
`TBM
`ALL indicates acute lymphoblastic leukemia; NHL, non-Hodgkin lymphoma; CLL, chronic lymphocytic leukemia; GD2, disialoganglioside; FR, alpha folate receptor; CEA,
`carcinoembryonic antigen; L 1-CAM, L 1 cell adhesion molecule; PSMA, prostate-specific membrane antigen; ERBB2, receptor tyrosine-protein kinase erbB-2; Her2, human
`epidermal growth factor receptor; HSCT, hematopoietic stem cell transplantation; NA, not available; SB, Sleeping Beauty; TBM, to be monitored; SAE, serious adverse event;
`and IND, investigational new drug.
`•studies have been described to demonstrate a CAR-mediated antitumor effect based on reduction in tumor size. Other trials demonstrated a biologic effect of CAR+ T
`cells based on reduction in biologic markers of tumor activity.
`
`Table 2. Published reviews since 2003 on immunobiology and
`clinical applications of CAR+ T cells
`References
`Authors
`
`Year published
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`
`Rossig and Brenner
`Willemsen et al
`Baxevanis and Papamichail
`Rossig and Brenner
`Rivie re et al
`Thistlethwaite et al
`Kershaw et al
`Dotti and Heslop
`Cooper et al
`Foster and Rooney
`Biagi etal
`Rossi et al
`Varela-Rohena et al
`Eshhar
`Marcu-Malina et al
`Berry et al
`Sadelain et al
`June et al
`Dotti et al
`Till and Press
`Vera et al
`Brenner and Heslop
`Westwood and Kershaw
`
`2003
`2003
`2004
`2004
`2004
`2005
`2005
`2005
`2005
`2006
`2007
`2007
`2008
`2008
`2009
`2009
`2009
`2009
`2009
`2009
`2009
`2010
`2010
`
`chains34) exhibit such a fully-competent activation signal, investi(cid:173)
`gators have iteratively designed and tested CARs, for example,
`with 1 or more activation motifs, to try and recapitulate the
`signaling event mediated by al3TCR chains. These changes to the
`CAR are enabled by the modular structure of a prototypical CAR
`and this has resulted in first-, second-, and third-generation CARs
`designed with 1, 2, or 3 signaling endodomains (Figure 1)
`including a variety of signaling motifs, such as chimeric CD28,
`CD134, CD137, Lek, ICOS, and DAPl0.6-35-37 A listing of such
`CARs is provided by Berry et al26 and Sadelain et al.27 While the
`optimal CAR design remains to be determined, at present it is
`believed that the first-generation technology, in which a CAR
`signals solely through immunoreceptor tyrosine-based activation
`motif (ITAM) domains on CD3-1;, is insufficient to sustain in vivo
`persistence of T cells. 38 This is supported by early clinical data
`which demonstrate
`that CD19-specific,39 CD20-specific,40
`GD2-specific (not Epstein-Barr virus [EBY] bispecific),41 and
`LI-CAM (cell adhesion molecule)-specific42 T cells had appar(cid:173)
`ently short-lived persistence in peripheral blood. The decision
`regarding which second- or even third-generation CAR design to
`use in clinical trials is predicated on the ability of a CAR to activate
`T cells for desired T-cell effector function, which at a minimum
`includes CAR-dependent killing. However, it is possible that
`next-generation CARs can be engineered to provide a supraphysi(cid:173)
`ologic activation signal which may be detrimental to continued
`T-cell survival and perhaps even the well-being of the recipient.43
`
`Miltenyi Ex. 1031 Page 2
`
`

`

`BLOOD, 19AUGUST2010 • VOLUME 116, NUMBER 7
`
`IMMUNOBIOLOGY OFT CELLS EXPRESSING CAR
`
`1037
`
`A
`
`Figure 1. Modular structure of prototypical CAR.
`CAR shown dimerized on the cell surface demonstrat(cid:173)
`ing the key extracellular (A-B) and intracellular (C-E)
`A domains. CARs may express 1, 2, or 3 signaling
`motifs within an endodomain to achieve a CAR(cid:173)
`dependent fully-<:ompetent T-<:ell activation signal.
`The modular structure of the CAR's domains, for
`example, the scFv (VL linked to VH) region (A) and the
`flexible hinge and spacer, for example, from lgG4
`hinge, CH2, and CH3 regions (8), allow investigators
`to change specificity through swapping of exodo(cid:173)
`mains and achieve altered function by varying trans(cid:173)
`membrane and intracellular signaling moieties (C-E).
`
`B
`
`1 s1 Generation CAR
`One T -cell signaling endodomain
`
`2 nd Generation CAR
`Two T-cell signaling endodomains
`
`3"' Generation CAR
`Three T-cell signaling endodomains
`
`Modifying the CAR to achieve a fully competent
`activation signal and reduce immunogenicity
`
`The inclusion of 1 or more T-cell costimulatory molecules within
`the CAR endodomain is in response to the appreciation that
`genetically modified and ex vivo-propagated T cells may have
`down-regulated expression of desired endogenous costimulatory
`molecules ( eg, CD28) or that the ligands for these receptors may be
`missing on tumor targets (eg, absence of CD80/CD86 on blasts
`from B-lineage acute lymphoblastic leukemia). By way of ex(cid:173)
`ample, chimeric CD28 and CD3-t38,44 CD137 and CD3-t23-36 or
`CD134 and CD3-t,45,46 have been incorporated into the design of
`second-generation CARs with the result that these CARs with
`multiple chimeric signaling motifs exhibited effector function
`stemming from a both a primary signal (eg, killing) and costirnula(cid:173)
`tory signal (eg, IL-2 production) after an extracellular recognition
`event.47·48 Animal studies have demonstrated that T cells express(cid:173)
`ing a second-generation CD19-specific36,38 and carcinoembryonic
`antigen-specific CAR49 exhibited an improved antitumor effect
`compared with T cells bearing a first-generation CAR. In addition
`to altering the endodomains, investigators have also made changes
`to the scFv to improve affinity based on selecting high-affinity
`binding variants from phage arrays.50,51 The approach to develop(cid:173)
`ing CAR+ T cells with a calibrated increase in functional affinity
`may be necessary to enable genetically modified T cells to target
`tumors with low levels of antigen expression or perhaps to target a
`cell-surface molecule in the presence of soluble antigen.52.53
`Because a CAR typically contains a murine scFv sequence which
`may be subject to immune recognition leading to deletion of
`infused T cells, investigators have developed humanized scFv
`regions to target carcinoembryonic antigen (CEA)54-55 and ERBB2
`
`(a member of epidermal growth factor receptor family).6 However,
`a benefit for using human or humanized scFv regions is yet to be
`established in the clinical setting.
`
`Imaging CAR+ T cells by positron emission
`tomography
`
`The ability to genetically modify T cells to redirect specificity
`provides investigators with a platform to express other transgenes
`such as for noninvasive imaging. Such temporal-spatial imaging is
`desired as a surrogate marker for a CAR-mediated antitumor effect
`and to serially determine number and localization of infused
`T cells. One imaging transgene coexpressed with CAR is thymi(cid:173)
`dine kinase (TK) and associated mutants from herpes simplex
`virus-I (HSV-1)56 which can be used to enzymatically trap
`radioactive substrates within the cytoplasm to image the locore(cid:173)
`gional biodistribution of T cells using positron emission tomogra(cid:173)
`phy.57-59 The expression ofTK also renders CAR+ T cells sensitive
`to conditional ablation using ganciclovir in a cell-cycle-dependent
`manner.60-62
`
`Gene transfer of CARs
`
`Approaches to the genetic manipulation of T cells for the introduc(cid:173)
`tion of CAR transgene use either viral-mediated transduction, or
`nonviral gene transfer of DNA plasmids or in vitro-transcribed
`mRNA species, The advantage of retrovirus63-65 or lentivi(cid:173)
`rus23·36·66·67 to modify populations of T cells lies in the efficiency of
`gene transfer, which shortens the time for culturing T cells to reach
`clinically-significant numbers (Figure 2), Gamma retroviruses, the
`
`Electroporation
`(Non-viral gene transfer of
`
`Numeric expansion using aAPC
`
`Harvest
`
`Figure 2. Timeline for in vitro gene transfer and propagation
`of CAR+ T cells. The electrotransfer of transposon/transposase
`systems has narrowed the gap between nonviral and viral-based
`gene transfer for the amount of time in tissue culture needed to
`generate a clinically sufficient number of genetically modified
`CAR+ T cells. Cells transduced with virus (blue text) are typically
`propagated for 3 weeks before infusion. 68,69 T cells that undergo
`nonviral gene transfer with the SB system (red text) can be
`typically harvested within 4 weeks.
`
`Woo~~ "f--s-y:_~_e_~_: _ _.__ _ _ _ _ _ _ ,__ _ _ _ _ _ __. _ _ __c:..:.;_ __ . . . , _ _ )
`I
`I
`Week4
`Week3
`Week2
`l
`J
`
`y
`Harvest
`
`Transduction
`(Viral-based gene transfer
`ofy-retrovirus)
`
`------------------
`
`Numeric expansion using 0KB and IL-2
`
`Miltenyi Ex. 1031 Page 3
`
`

`

`1038
`
`JENA et al
`
`Figure 3. Schematic of vector systems to express A
`CAR transgenes used in clinical trials. (A) Two SB
`DNA plasmids expressing a (CAR) transposon and a
`hyperactive transposase (eg, SB11 ). Transposition oc-
`curs at a TA dinucleotide sequence when the trans(cid:173)
`posase enzymatically acts on the internal repeat flank-
`ing the transposon. (B) A recombinant retroviral vector
`showing the long terminal repeats (LTR) containing the
`promoter flanking the CAR. SD and SA are the splice 8
`donor and splice acceptor sites, respectively, and tJ, is
`the viral packaging signal. (C) A self-inactivating recom(cid:173)
`binant lentiviral vector construct containing the LTR, tf,,
`SD and SA s~es, HIV Rev response element (ARE),
`HIV central polypurine tract (cPPT), CAR under control
`of an internal promoter, and the wood-chuck hepatitis
`virus posttranscriptional regulatory element (WPRE).
`
`... IR/OR
`
`Promoter
`
`Promoter
`
`BLOOD, 19AUGUST2010 • VOLUME 116, NUMBER 7
`
`Transposon ,
`
`CAR
`
`Poly A
`
`S'LTR
`
`CAR
`
`3'LTR
`
`C ,______sd ~l-0-c::$--Es:JC __ }- -{-J"_ -
`
`S'LTR
`
`'i' HIV RRE HIV cPPT
`
`Promoter
`
`CAR
`
`WPRE
`
`3'LTR
`
`most common vector system used to genetically modify clinical
`grade T cells (Figure 3), have introduced CAR into T cells with a
`proven ability to exert a therapeutic effect.41 Self-inactivating
`lentiviral vectors (Figure 3) hold particular appeal as they appar(cid:173)
`ently integrate into quiescent T cells.28 These recombinant viruses
`often have a high cost to manufacture at clinical grade in
`specialized facilities skilled in current good manufacturing practice
`(cGMP) which may preclude investigators from undertaking
`clinical trials. As an alternative to transduction, we and others have
`adapted electroporation as an approach to the nonviral gene transfer
`of DNA42,70,71 resulting in CAR+ T cells that have been attributed
`to have an antitumor effect in a clinical triaI.40 Previously, the
`electrotransfer and integration of naked plasmid DNA into T cells
`was considered inefficient because it depended on illegitimate
`recombination for stable genomic insertion of nonviral sequences.
`As a result, lengthy in vitro culturing times were required to select
`for T cells bearing stable integrants72•73 during which period the
`T cell became differentiated, and perhaps terminally differentiated,
`into effector cells which may have entered into replicative senes(cid:173)
`cence. This time in tissue culture can now be considerably
`shortened by using transposon/transposase systems such as Sleep(cid:173)
`ing Beauty (SB)7o,74,75 and piggyback16•11 to stably introduce CAR
`from DNA plasmids (Figure 3).71 •76•78· 84 We have shown that the SB
`system can be used to introduce CAR and other transgenes into
`human T cells with an approximately 60-fold improved integration
`efficiency compared with electrotransfer of DNA transposon
`plasmid without transposase74 and this provided the impetus to
`adapt the SB system for use in clinical trials. 85 After electropora(cid:173)
`tion, T-cell numbers from peripheral and umbilical cord blood can
`be rapidly increased in a CAR-dependent manner by recursive
`culture on -y-irradiated artificial antigen-presenting cells (aAPC)
`achieving clinically sufficient numbers of cells for infusion within
`3 to 4 weeks after electroporation (Figure 2). Given that T cells
`transduced with -y-retrovirus are typically cultured with OKT3 and
`IL-2 for approximately 3 weeks before infusion, the use of the SB
`system with aAPC does not appear to greatly lengthen the ex vivo
`culturing process.
`
`Improving the therapeutic potential of CAR+
`T cells
`
`T cells and polarized T-cell subsets, such as TH I and TH 17 cells,
`may be genetically modified to be specific for a catalog of
`
`cell-surface TAAs as described in recent reviews (Table 2). To
`enable these CAR+ T cells to achieve their full therapeutic
`potential in clinical trials, there are 3 major challenges to be
`overcome.
`
`Persistence
`
`The adoptively transferred CAR+ T cells must survive and perhaps
`also numerically expand to achieve a robust antitumor effect. One
`approach to improving persistence is to alter the host environment
`into which the T cells are infused. For example, rendering the
`recipient lymphopenic, or even aplastic by chemotherapy and/or
`radiation therapy, improves the persistence of adoptively trans(cid:173)
`ferred T cells (and natural killer [NK] cells).86·90 Presumably, the
`infused T cells proliferate in the lymphopenic recipient through
`homeostatic mechanisms mediated by the removal of regulatory/
`suppressor cells and the ready availability of previously scarce
`homeostatic cytokines. This approach likely improves in vivo
`persistence and thus efficacy of CAR+ T cells as a published trial
`and animal studies have hinted.40 However, combining chemo(cid:173)
`therapy and T-cell therapy might increase toxicity and compromise
`the interpretation of an antitumor effect for it may be difficult to
`definitively attribute efficacy to the infused CAR+ T cells in trials
`enrolling small numbers of patients who received concomitant
`chemotherapy. Trials infusing T cells expressing a first-generation
`CAR design (signaling solely through chimeric CD3-t endodo(cid:173)
`main) revealed that these T cells appear to have limited long-term
`persistence and one way of circumventing this limitation is to
`engineer CAR endodomains to deliver an activation signal for
`sustained proliferation. This has been achieved by the design of
`second-generation CARs that signal through 2 signaling domains
`and these are now in multiple clinical trials. Third-generation
`CARs are now being evaluated in humans in a few trials (Table 1)
`which are composed of 3 chimeric signaling moieties.6•35 Rather
`than modifying the CAR design to sustain proliferation, investiga(cid:173)
`tors have used signaling molecules alongside CARs to achieve
`improved persistence. For example, constitutively expressed recom(cid:173)
`binant CDS0 and CD137L alongside the CAR demonstrated
`coordinated signaling between these 2 introduced receptors with
`endogenous ligands CD28 and CD137 within the immunologic
`synapse on or between T cells, which could improve antitumor
`effects.91 CAR has been introduced into T cells expressing endoge(cid:173)
`nous al3TCR that recognize allo-92 or viral antigens.41 •93·95 Trigger(cid:173)
`ing such TCRs in vivo can be used to numerically expand T cells to
`
`Miltenyi Ex. 1031 Page 4
`
`

`

`BLOOD, 19AUGUST 2010 • VOLUME 116, NUMBER 7
`
`IMMUNOBIOLOGY OFT CELLS EXPRESSING CAR
`
`1039
`
`achieve an improved antitumor effect delivered by the CAR as was
`recently demonstrated by infusing bispecific T cells which recog(cid:173)
`nized EBY-specific antigens via the endogenous al3TCR and GD2
`on neuroblastoma cells by the introduced CAR.41 Because T cells
`may require exogenous cytokines to sustain in vivo persistence,96
`investigators have enforced expression of cytokines that signal
`through the common 'Y cytokine receptor chain. Animal experi(cid:173)
`ments97 as well as clinical experience98,99 have shown that long(cid:173)
`lived T cells are associated with expression of the IL-7Ra chain
`(CD127), but genetically modified and cultured T cells tend to
`down-regulate this receptor. Therefore, to enhance the ability of
`T cells to respond to IL-7, made available after inducing lymphope(cid:173)
`nia, 100·102 investigators have enforced the expression of IL-7Ra to
`demonstrate improved survival of EBY-specific T cells in an
`animal model103 or introduced a novel membrane-bound variant of
`IL-7 that when expressed on the cell surface improved persistence
`of CAR+ T cells. 104 Furthermore, overexpression of receptors for
`IL-2105 and IL-15 106 as well as enforced expression of the cytokines
`themselves107 have improved persistence ofT cells. 108·110 However,
`when this approach was tested in a clinical trial infusing tumor(cid:173)
`infiltrating lymphocytes (TIL) genetically modified to constitu(cid:173)
`tively secrete IL-2, the persistence of the adoptively transferred
`T cells was not improved compared with genetically unmodified
`TIL. 111 As constitutive expression of cytokines and/or cytokine
`receptors is tested in early clinical trials, investigators will need to
`consider coexpressing transgenes for conditional ablation of these
`genetically modified T cells to guard against aberrant proliferation
`as unopposed cytokine signaling may lead to aberrant T-cell
`growth. 109,112 The type of T cell into which the CAR is introduced
`may also impact persistence after adoptive immunotherapy. This
`has been demonstrated in the monkey by infusing autologous
`preselected central memory T cells which despite ex vivo numeric
`expansion retained superior in vivo persistence compared with
`adoptive transfer of differentiated effector T cells. 113 These observa(cid:173)
`tions have been expanded upon by Hinrichs et al who showed that
`an infusion of naive murine T cells was associated with improved
`T-cell persistence.114 These animal observations are likely to
`influence the design of trials infusing genetically modified T cells
`as investigators seek to introduce CARs into T cells that preserve
`the functional capacity of central memory or naive T cells.
`
`Homing
`
`an ability to overcome the adverse regulatory effects within the
`tumor microenvironment. For example, investigators have intro(cid:173)
`duced a dominant-negative receptor for TGFl3 to enable genetically
`modified T cells can resist the suppressive effects of this pleiotropic
`cytokine. 119 Recognizing that the tumor microenvironment con(cid:173)
`tains regulatory T cells (T,egs), the CAR signaling motif has been
`adapted to resist the suppressive effects of these cells by the
`expression of chimeric CD28. 120 The ability to engineer CAR+
`T cells to successfully function within tumor deposits remains
`relatively underexplored as most clinical trials to date have used
`CAR+ T cells with specificity for hematopoietic malignancies.
`However, trials have been published and are under way which
`seek to treat solid tumors 41•42·121 (Table 1) and systematic
`approaches to enabling infused T cells to effectively operate
`within a tumor microenvironment will be needed to reliably
`eliminate large tumor masses.
`
`Reprogramming T cells
`
`The ex vivo gene transfer and propagation (Figure 2) of T cells
`provides an opportunity to further manipulate T cells before
`infusion. The in vitro propagation of T cells provides investigators
`with an opportunity to numerically expand T cells from scant
`starting numbers, such as when T cells are genetically modified
`from umbilical cord blood with the intent to augment the graft(cid:173)
`versus-tumor-effect after allogeneic hematopoietic stem cell trans(cid:173)
`plantation (HSCT).61 However, the in vitro culturing process can
`also be adapted to modify T cells for desired effector function by
`the selective addition of a subset of soluble cytokines, for example,
`that bind via common 'Y chain receptor to the culture media during
`ex vivo culture. 122,123 In addition, to help maintain a desired T-cell
`phenotype after gene transfer, investigators have provided costimu(cid:173)
`latory signals by the addition of CD28-specific mAb in addition to
`OKT3, often using beads conjugated to these mAbs. As an
`alternative, we and others have used immortalized cells in tissue
`culture, such as 3T3 124·125 and K56274·126·128 which can be geneti(cid:173)
`cally modified to express desired T-cell costimulatory molecules
`and function when irradiated as aAPC.
`
`To act on and within the tumor, genetically modified T cells must
`home to the site(s) of malignancy. Migration may be compromised
`by the loss of desired chemokine receptors during genetic modifica(cid:173)
`tion and passage ex vivo, or may result from the selection ofT cells
`that are inherently unable to localize to certain tissues. Panels of
`tissue-specific homing receptors which are typically composed of
`integrins, chemokines, and chemokine receptors are associated
`with T-cell migration to anatomic sites of malignancy, and flow
`cytometry can therefore be used to describe the potential migration
`patterns of T cells before infusion. 115-116 It is unclear whether
`T cells capable of expressing a desired matrix of endogenous
`homing receptors can be genetically modified to express CAR.
`Therefore, investigators are manipulating the homing potential of
`T cells through the enforced expression of chemokine receptors
`such as CCR4. 117•118
`
`Overcoming mechanisms of resistance
`
`Once infused T cells persist and home, they must be able to execute
`and recycle their CAR-dependent effector function, which requires
`
`Expressing CARs in cells other than a(3 TCR+
`T cells
`
`Populations of hematopoietic cells other than T cells expressing
`al3TCR, such as NK cells, cytokine-induced killer (CIK) cells,
`monocytes, and neutrophils have been genetically modified to
`express CARs.27,28 Given the inherent lytic potential of NK cells,
`redirection of their specificity via a CAR is appealing. 129·133 CAR
`signaling endodomain(s) can be specifically adapted or chosen to
`enhance NK-cell signaling versus those that activate T cells. 134 One
`drawback to adoptive transfer of NK cells has been their limited
`survival in vivo after transfer. However, ex vivo NK-cell culturing
`using aAPC adapted from K562 may change this perception. 135-136
`Furthermore, the use of NK cells as a cellular platform for
`introducing CARs may be attractive after allogeneic HSCT as
`donor-derived NK cells do not appear to significantly contribute to
`graft-versus-host-disease (GYHD). 137-138 T cells expressing-y8TCR
`are another lymphocyte population with endogenous killing ability
`that has been modified to express CAR, and these cells can be
`
`Miltenyi Ex. 1031 Page 5
`
`

`

`1040
`
`JENAetal
`
`BLOOD, 19 AUGUST 2010 • VOLUME 116, NUMBER 7
`
`selectively proliferated using an aminobiphosphonate (Zoledr(cid:173)
`onate), 139 raising the possibility that a Food and Drug Administra(cid:173)
`tion (FDA)-approved phannacologic agent can be used to selec(cid:173)
`tively propagate these infused CAR+ T cells in vivo. Instead of
`genetically modifying mature lymphocytes to express CAR, inves(cid:173)
`tigators have introduced CAR into developing thymocytes140 and
`immunoreceptors into hematopoietic stem cells (HSC) 141·143 to
`avoid negative selection and improve immune-mediated surveil(cid:173)
`lance. The approach of genetically modifying lymphoid precursors
`has also been adapted by Zakrzewski et al to make "universal
`T cells" in which CAR+ HSC can be adoptively transferred across
`MHC barriers. 144 It is only a matter of time until embryonic stem
`cells and induced pluripotent stem cells are genetically modified to
`express CAR and differentiated into T cells with redirected and
`desired specificity. The question remains, however, whether these
`other populations of cells have a therapeutic advantage over
`genetic modification and infusion of o:~ + T cells that are geneti(cid:173)
`cally modified to express CAR.
`
`Safety of T cells expressing CAR
`
`There are 4 levels of concern associated with the infusion of
`genetically modified T cells. The first is whether the introduced
`genetic material can lead to genotoxicity. This remains a theoretical
`concern for genetically modified T cells, in contrast to HSC. 145 The
`stable expression of CAR requires the introduction of a promoter
`and the transgene cDNA which raises the possibility of insertional
`mutagenesis. However, to date, there have been no genotoxic
`events associated with serious adverse events attributed to geneti(cid:173)
`cally modified T