CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

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1 The Americn Society of Gene & Cell Therpy originl rticle CD123-Engger T Cells s Novel Immunotherpeutic for Acute Myeloid Leukemi Chllice L Bonifnt 1,2,3, Arpd Szoor 1,2,3, Dvid Torres 1,2,3, Nicholos Joseph 1,2,3, Mirey Pulin Velsquez 1,2,3, Kot Iwhori 1,2,3, Amos Gikwd 3,4, Phuong Nguyen 1,2,3, Croline Arer 1,2,5, Xio-Tong Song 1,4, Michele Redell 2,3 nd Stephen Gottschlk 1,2,3,4 1 Center for Cell nd Gene Therpy, Texs Children s Hospitl, Houston Methodist Hospitl, Bylor College of Medicine, Houston, Texs, USA; 2 Texs Children s Cncer nd Hemtology Center, Texs Children s Hospitl, Bylor College of Medicine, Houston, Texs, USA; 3 Deprtment of Peditrics, Bylor College of Medicine, Houston, Texs, USA; 4 Deprtment of Pthology nd Immunology, Bylor College of Medicine, Houston, Texs, USA; 5 Deprtment of Medicine, Bylor College of Medicine, Houston, Texs, USA Immunotherpy with CD123-specific T-cell engger protei or with T cells expressing CD123-specific chimeric ntigen receptors is ctively eing pursued for cute myeloid leukemi. T cells secreting ispecific engger molecules (ENG-T cells) my present promising lterntive to these pproches. To evlute therpeutic potentil, we generted T cells to secrete CD123/CD3- ispecific engger molecules. T cells recognized primry cute myeloid leukemi (AML) cells nd cell lines in n ntigen-dependent mnner s judged y cytokine production nd/or tumor killing, nd redirected ystnder T cells to AML cells. Infusion of T cells resulted in regression of AML in xenogrft models conferring significnt survivl dvntge of treted mice in comprison to mice tht received control T cells. At high effector to trget rtios, T cells recognized norml hemtopoietic stem nd progenitor cells (HSPCs) with preferentil recognition of HSPCs from cord lood compred to one mrrow. We therefore introduced the CD2 suicide gene tht cn e trgeted in vivo with rituxim into T cells. The expression of CD2 did not diminish the nti-aml ctivity of T cells, ut llowed for rituxim-medited ENG-T cell elimintion. Thus, ENG-T cells coexpressing CD2 suicide nd CD123 engger molecules my present promising immunotherpeutic pproch for AML. Received 4 Novemer 215; ccepted 1 June 216; dvnce online puliction 12 July 216. doi:1.138/mt INTRODUCTION The outcome for peditric nd dult ptients with cute myeloid leukemi (AML) remi poor, prticulrly in those with high risk or relpsed disese. 1 3 Additionlly, current tretment protocols hevily rely on chemotherpeutic gents whose use commonly leds to serious cute nd long-term toxicities. Given this, there is need to develop novel trgeted therpies tht improve outcomes nd reduce tretment-relted complictio of current therpies. The ex vivo preprtion of ntigen-specific T cells followed y their doptive trfer is one ttrctive strtegy to improve outcomes for hemtologicl mlignncies, since T-cell killing does not rely on the rodly cytotoxic mechnisms of conventionl therpies. 4 7 Indeed the doptive trfer of T cells tht re geneticlly modified with CD19-specific chimeric ntigen receptors (CARs) hs resulted in impressive clinicl respoes; especilly in ptients with cute lympholstic leukemi However, for AML, there hs een limited success. Lewis Y (LeY)-specific CAR T cells hve een tested so fr in one clinicl study without roust respoe. 16 In ddition, CD33-specific CAR T cells were evluted in single ptient with limited success. 17 Severl groups hve explored interleukin-3 receptor lph (IL3Rα, CD123)-specific CAR T cells for AML in preclinicl models, nd while these cells hd potent ntitumor ctivity, one group demotrted tht norml hemtopoietic stem nd progenitor cells (HSPCs) re lso eliminted We nd others hve developed n lterntive strtegy to generte tumor-specific T cells y genetic modifiction with diodies, 23 or secretle, ispecific T-cell engger molecules, which coist of two single chin vrile frgments (scfvs) specific for tumorssocited ntigen nd CD3ε (ENG-T cells). 24 These T cells not only recognize nd kill tumor cells in tumor-ssocited ntigendependent mnner, ut lso hve the unique ility to redirect ystnder T cells to tumor cells. 24 Coistent synthesis of enggers y doptively trferred T cells should e superior to the direct infusion of the recominnt ispecific ntiody, ecuse these typiclly hve short hlf-lives nd do not ccumulte t tumor sites. Here, we report the development of T cells nd demotrte tht these ENG-T cells recognize nd kill CD123- positive trget cells in vitro, redirect ystnder T cells, nd hve potent ntitumor ctivity in vivo. Since T cells recognized norml HSPCs t high effector to trget rtios, we further geneticlly modified T cells to lso include the CD2 suicide gene (CD2. T cells) CD2. T cells hd equivlent effector function in comprison to CD123- ENG T cells nd were redily killed in the presence of rituxim Correspondence: Stephen Gottschlk, Center for Cell nd Gene Therpy, Texs Children s Hospitl, Houston Methodist Hospitl, Bylor College of Medicine, 112 Btes Street, Suite 177, Houston, Texs 773, USA. E-mil: smgottsc@txch.org Moleculr Therpy vol. 24 no. 9, sep

2 CD123-Engger T Cells for the Tretment of AML The Americn Society of Gene & Cell Therpy nd complement mking them promising T-cell therpy cndidte for future clinicl development. RESULTS Genertion of T cells T cells were generted y trduction with n RD114-pseudotyped retrovirl vector encoding ispecific CD123-CD3 engger molecule, n internl riosoml entry site (IRES), nd mornge (Figure 1). Trduction of T cells ws confirmed y fluorescence-ctivted cell sorting (FACS) nlysis for mornge expression. Men trduction efficiency ws 79.2% (rnge: %; n = 14; Figure 1,c). Phenotypic nlysis of trduced T cells reveled mixture of CD4- nd CD8-positive T cells, with reproducile percentges of nive, centrl memory, nd effector memory cell popultio (Supplementry Figure S1, n = 5). Trduction of cells nd expression of did not lter the T-cell phenotype in comprison to nontrduced (NT) T cells ctivted nd expnded in prllel. secretion nd inding to oth trduced nd NT T cells ws confirmed y FACS nlysis using n nti-mouse F( ) 2 (Figure 1d). To quntify protein in cell culture medi, we developed n enzyme-linked immunosorent ssy (ELISA) using recominnt CD123 T-cell ENG protein s stndrd (Supplementry Figure S2). CD123 T-cell ENG protein ws redily detected in medium conditioned y T cells (men: 7.5 µg/ml, 95% CI: µg/ml) in contrst to medium conditioned y T cells expressing CD19 T-cell ENG protein ( T cells; men: 9.8 ng/ml, 95% CI: 26.6 ng/ml) confirming specificity of the developed ssy (Figure 1e). T cells recognize nd kill CD123-positive AML cells in vitro To demotrte the specificity of T cells git tumor cells expressing the ntigen trget, we used pnel of CD123-positive (K562-CD123, KG1, MV-4 11, MOLM-13) nd CD123-negtive (K562) cell lines (Supplementry Figure S3). We performed coculture ssys with these tumor cells nd ENG T cells trgeting CD123 or n irrelevnt ntigen not expressed on cell trgets (CD19). T cells secreted significnt mounts of IFNγ nd IL-2 when exposed to cells expressing CD123 (Figure 2,). In contrst, coculture with CD123-negtive cells did not induce cytokine production. Similrly, T cells did not produce cytokine when cocultured with trget cells (Figure 2,). To further confirm specificity, we ssessed the killing potentil of T cells git CD123-positive trget cells in 4-hour chromium relese cytotoxicity ssy. CD123- ENG T cells effectively lysed AML cells expressing CD123, ut did not kill the CD123-negtive K562 cell line (Figure 2c). CD19- ENG T cells did not hve ny cytolytic ctivity git the tested cell lines, confirming specificity. T cells redirect ystnder T cells to CD123-positive AML cells in vitro To exmine the potentil of ENG T cells to ctivte ystnder cells in vitro, we performed trwell ssys with iert wells tht llow the diffusion of molecules ut not the migrtion of cells. ENG T cells were plted in the iert well nd KG1 trget cells with NT T cells in the ottom well. Only T cells, in contrst to NT T cells nd T cells, were le to induce significnt trget killing y NT T cells (Figure 3). No tumor cell killing ws oserved in wells tht contined only trget cells without T cells, indicting tht engger protein lone is inherently nontoxic. We confirmed the ility of T cells to redirect T cells tht hd not een expnded ex vivo y repeting the experiment with peripherl lood mononucler cells (PBMCs) s source of ystnder T cells in the ottom well (Figure 3). T cells kill primry AML lsts in vitro To further ssess the potentil trltionl impct of CD123- ENG T cells s tretment for AML ptients, we tested their cytolytic ctivity git primry AML lsts. Peditric AML smples were collected, nd first evluted for CD123 expression (Figure 4). The lst percentge recorded y our clinicl hemtopthology lortory nd further descried in the methods section ws AML#1: 33%, AML#2: 9%, nd AML#3: 65%. Primry smples were then treted with CD123- or T cells t n E:T rtio of 1:1 or 1:1 prior to plting in semisolid medi with growth fctors present. Untreted smples served s controls. After 1 14 dys of culture, cells were counted. At oth E:T rtios tested T cells induced significnt AML lst killing in comprison to T cells (Figure 4, P <.5). T cells hve nti-aml ctivity in vivo We evluted the nti-aml ctivity of T cells in our MOLM-13 AML NSG xenogrft model. Post sulethl irrdition, mice were injected with MOLM-13.GFP.ffLuc cells nd on dy 7 nd 14 received n intrvenous injection of or T cells. Untreted mice served s controls. Within 4 dys post the first T-cell injection mice treted with CD123- ENG T cells hd significnt decline in their ioluminescence signl (P <.1) with susequent leukemi erdiction in ll treted mice (Figure 5,d). In contrst, untreted mice nd mice treted with T cells hd significnt increse (P <.2) in their ioluminescence signl (Figure 5,c) nd died of progressive disese y dy 2 (Figure 5e). While none of the T-cell treted mice developed recurrent leukemi, 4/5 mice died 29 ( 2), 46, nd 67 dys post-tumor cell injection. The cuse of deth ws xenogeneic grft versus host disese (GVHD) s commonly reported with doptive trfer of humn T cells in the NSG murine model. 28,29 We confirmed this for 1/4 mice y demotrting humn T-cell infiltrtion throughout the liver (Supplementry Figure S4A). To ssess the contriution of trget ntigen-specific CD123- ENG T-cell stimultion in inducing GVHD, MOLM-13 ering nd control mice were injected on dy 7 with T cells tht were lso geneticlly modified to express GFP.ffLuc. While in control mice there ws rpid, significnt decline (P <.5) of infused T cells, no significnt decline of T cells ws oserved for 5 dys postinfusion in MOLM-13 ering mice (Figure 6,). Mice were euthnized on dy 6 to determine the presence of humn T cells (humn CD3 nd CD45 positive) nd expressing humn T cells (mornge positive) in spleen nd one mrrow. The verge frequency of humn T cells ws 1.1% (rnge: %) in splee nd.3% (rnge: vol. 24 no. 9 sep. 216

3 The Americn Society of Gene & Cell Therpy CD123-Engger T Cells for the Tretment of AML LTR CD123-CD3 IRES mo LTR c NT T cells.8% ENG T cells 9.4% Positive (%) mo e (µg/ml) NT T cells T cells *** d NT T cells T cells T cells T cells nti-f( ) 2 mo nti-f( ) 2 Figure 1 Genertion of T cells. () Schemtic of retrovirl vector encoding nd mornge. (,c) Representtive FACS digrm nd summry dt ( T cells (n = 14), NT T cells (n = 6) of mornge expression post-trduction. (d) A mouse F( ) 2 ntiody ws used to detect cell surfce-ound CD123 T-cell ENG protein. mornge-positive nd -negtive T cells stined positive (filled curve) for CD123 T-cell ENG in contrst to smples tht were stined with isotype lone (open curve). NT T cells cultured without T cells did not stin positive with the mouse F( ) 2 ntiody, confirming specificity. (e) Detection of CD123 T-cell ENG protein in medi of nd T cells fter 24 hours of culture (n = 4, performed in triplictes, ox grph, whiskers: min, mx, versus T cells P <.1). IFN-γ (pg/ml) 18, 16, 14, 4, 2, *** * ** * IL-2 (pg/ml) 15, 1, 5, *** *** *** ** c Lysis (%) ** * ** K562 K562-CD123 MV-4-11 MOLM-13 KG1 K562 K562-CD123 MV-4-11 MOLM-13 KG1 K562 K562-CD123 MV-4-11 KG1 Figure 2 T cells recognize nd kill CD123-positive cute myeloid leukemi cells. (, ) or T cells were cocultured with CD123-positive (K562-CD123, MV-4-11, MOLM-13, KG1) or -negtive (K562) cell lines. After 24 hours, () IFNγ or () IL-2 ws determined y ELISA (n = 3 4, ssy performed in duplictes; versus : *P <.5, **P <.1, ***P <.1). (C) Cytotoxicity ssys were performed using or T cells s effectors nd CD123-positive (K562-CD123, MV-4-11, KG1) or -negtive (K562) cell lines s trgets t E:T rtio of 1:1 (men + SD; n = 4; ssy ws performed in triplictes, *P <.2, **P <.2). Moleculr Therpy vol. 24 no. 9 sep

4 CD123-Engger T Cells for the Tretment of AML The Americn Society of Gene & Cell Therpy.27.37%) in one mrrows with no significnt difference etween control nd MOLM-13 ering mice (Figure 6c). In the infused T-cell product 5.7% of cells were mornge positive. On verge 56.3% (rnge: %) of humn T cells from splee nd 57.3% (rnge: %) of humn T cells from one mrrows were positive for mornge ( T cells) with no significnt difference etween control nd MOLM- 13 ering mice, nd no significnt difference to the infused T-cell product (Figure 6d). Residul MOLM-13 cells were not detectle in splee nd one mrrows of mice tht hd een injected with MOLM-13 s judged y FACS nlysis of CD123 (dt not shown). Toxicity of T cells git hemtopoietic stem nd progenitor cells Given the reported low level of expression of CD123 on HSPCs, we sought to determine if T cells recognize this cell popultion. 3,31 We used cord lood or one mrrow s source of HSPCs. Smples were treted with CD123- or T cells t E:T rtios of 5:1, 1:1, or 1:1 prior to plting in semisolid medi with growth fctors present. Untreted smples served s controls. After 1 14 dys, colony forming unit (CFUs) were counted. At n E:T rtio of 5:1, significnt toxicity ws oserved (Figure 7,). At n E:T rtio of 1:1 or 1:1, toxicity ws dependent on the stem cell source with umilicl cord- derived HSPCs eing more seitive to T cells thn one mrrow-derived HSPCs. T cells expressing the CD2 suicide gene retin their nti-aml ctivity nd re eliminted in the presence of rituxim nd complement While T cells hd potent nti-aml ctivity, they induced killing of norml HSPCs t high E:T rtios, indicting tht T cells should lso express suicide gene to llow their selective depletion in the event of unwnted side effects. We focused on trgenic expression of CD2, which llows the depletion of T cells y the FDA-pproved CD2 monoclonl ntiody rituxim vi complement-medited or ntiody-dependent cell-medited cytotoxicity. To coexpress CD2 nd CD123 T-cell ENG molecules in T cells, we generted retrovirl vector encoding CD2, 2A sequence, nd our (CD2.CD123- ENG; Supplementry Figure S5A). CD2.CD123 ENG T cells were generted y retrovirl trduction nd men of 6.3% (rnge: %, n = 8, Supplementry Figure S5B) of T cells were geneticlly modified s judged y FACS nlysis for CD2. There ws no chnge in the percentge of CD2-positive T cells erly (dy 3 5) versus lte (dy 13 15) post-trduction, indicting tht trgene expression is stle over time (Supplementry Figure S5C). We oserved no difference in the cytolytic ctivity etween T cells nd CD2. T cells (Figure 8, P =.45). In vivo, in the MOLM-13 AML NSG xenogrft model, single dose of CD2. T cells hd similr ntitumor ctivity (Figure 8) s T cells in the previously performed experiment (Figure 5d). To evlute the functionlity of the CD2 suicide gene, we performed cytotoxicity ssy with 51Cr-leled NT, CD123- ENG, or CD2. T cells in the presence of rituxim nd/or complement. While complement or rituxim lone Numer of live MOLM-13 cells T cells in iert well NT-T cells in ottom well Numer of live MOLM-13 cells T cells in iert well PBMC in ottom well Figure 3 T cells ctivte ystnder T cells nd freshly isolted peripherl lood mononucler cells (PBMCs) git CD123- positive trget cells. () MOLM-13.GFP.ffLuc cells were plted in the ottom well with or without NT T cells. Control (NT, CD19- ENG) or T cells were plted in the iert well t the indicted T-cell dose. Following 24-hour incution, vile MOLM-13.GFP. ffluc cells were quntified y luciferse ssy (n = 3; ssy ws performed in duplictes; for : 1 6 ENG-T cells in iert, 1 6 NT T cells in ottom well versus 1 6 ENG-T cells in iert, no NT T cells in ottom well: P <.5, 1 5 ENG-T cells in iert, 1 6 NT T cells in ottom well versus 1 6 ENG-T cells in iert, no NT T cells in ottom well: P <.5). () MOLM-13.GFP.ffLuc cells were plted in the ottom well with or without PBMCs utologous NT,, or CD123- ENG T cells were plted in the iert well. Following 24-hour incution, vile MOLM-13.GFP.ffLuc cells were quntified y luciferse ssy (n=3; ssy ws performed in triplictes; for : PBMCs versus no PBMCs: P <.2). induced <2% killing of CD2. T cells, ~6% killing ws oserved in the presence of rituxim nd complement in 4-hour chromium relese ssy (Figure 8c). Since the men trduction efficiency of CD2. T-cell lines ws 6.3% (rnge: %) in the performed ssys, these results demotrte >95% killing of trduced T cells in the presence of rituxim nd complement. These findings were confirmed in 4-hour chromium relese ssy using CD2-selected CD2. T cells (Supplementry Figure S6A,B). In contrst, rituxim nd complement hd no cytolytic ctivity git NT or T cells. To determine the durility of rituxim/complement-medited killing of CD2. T cells in cell culture ssy, CD2. T cells were treted with rituxim/complement for two hours, NT P <.5 P <.5 P <.2 NT vol. 24 no. 9 sep. 216

5 The Americn Society of Gene & Cell Therpy CD123-Engger T Cells for the Tretment of AML 1 4 AML #1 AML #2 AML # k K 2K 15K 1K k % of control cells * * 1:1 1:1 ENG-T cell: Leukemi cell rtio SSC 5K 5k 1k 15k 2k 25k FSC CD CD CD123 Figure 4 Primry AML cells re killed y T cells in vitro. () FACS nlysis of peditric primry cute myeloid leukemi (AML) smples for CD123 expression. AML lsts,(cd33+, CD3-, CD19- (dt not shown for CD19)) were nlyzed for CD123 expression (filled curve: isotype control; open curve: CD123 MA). () Primry AML smples were treted with or T cells t E:T rtios of 1:1 nd 1:1 for 6 hours. Following coculture, cells were plted in MethoCult medi nd incuted for 1 14 dys. Finl cell counts re displyed s percentge of control; n = 3; versus T cells: *P <.5. wshed, nd the surviving T cells cultured for 7 dys. Greter thn 9% of CD2. T cells were killed s judged y FACS nlysis (Figure 8d, n = 5, P <.1). In contrst, no significnt killing of CD2-positive cells ws oserved in the presence of rituxim or complement lone, when compred to untreted cells. Concurrent with FACS nlysis, stndrd chromium relese cytotoxicity ssy ws performed using KG1 s trgets nd untreted or rituxim/complement-treted CD2. T-cell lines s effectors, one week fter tretment. T-cell lines served s negtive, nd CD123- ENG T-cell lines s positive controls. While untreted CD2. T-cell lines redily killed KG1 cells, rituxim/ complement-treted CD2. T-cell lines did not, indicting successful depletion of CD2. T cells (Figure 8e). Hving estlished the functionlity of the CD2 suicide gene in vitro, we ssessed the ility of rituxim to deplete CD2. T cells in vivo. Post sulethl irrdition, mice were injected with MOLM-13 cells, nd on dy 7 received CD2. T cells tht were lso geneticlly modified with GFP.ffLuc, nd selected for CD2 expression (Supplementry Figure S6A). Strting dy 3 post-t-cell infusion, mice received intrperitonel injection (IP) of 25 µg rituxim for 3 coecutive dys. Rituxim dministrtion resulted in greter thn 9% decrese in the ioluminescence signl, demotrting the effectiveness of rituxim to destroy CD2. T cells in vivo. (Figure 8f, rituxim versus no tretment: P <.5 strting dy 2 post-first dose of rituxim). DISCUSSION Herein, we descrie the genertion of T cells nd demotrte tht these cells recognize nd kill CD123-positive trget cells in n ntigen-dependent mnner, redirect ystnder T cells to CD123-positive trget cells, nd hve potent ntitumor ctivity in vivo. We lso oserved killing of HSPCs t high effector to trget rtios, nd therefore comined the expression of molecules with CD2 suicide gene in T cells. CD2. T cells retined their ntileukemi ctivity nd could e redily depleted in the presence of rituxim nd complement. T-cell immunotherpy s less toxic, precision therpy for the tretment of leukemi is therpeutic pproch tht hs enjoyed recent populrity due to the success of CD19-CAR T cells dministered for refrctory cute lympholstic leukemi In contrst to CAR T-cell therpy for cute lympholstic leukemi, CAR T-cells trgeting AML hve not een exteively tested in the clinic. However, severl ntige re currently eing ctively explored in preclinicl studies including CD33, CD44v6, CD123, nd LeY ,32 36 While ll of these ntige hve een trgeted with CAR T cells, none hs een trgeted with ENG T cells, which re new clss of T cells with the unique ility to redirect ystnder T cells to tumor cells in n ntigen dependent fshion. 24 T cells not only killed AML cell lines ut lso primry AML lsts in vitro, confirming studies y others tht CD123-positive AML cells cn e trgeted with geneticlly modified T cells While ll previous studies used T cells expressing Moleculr Therpy vol. 24 no. 9 sep

6 CD123-Engger T Cells for the Tretment of AML The Americn Society of Gene & Cell Therpy Untreted Dys post-tumor injection Untreted c Rdince Rdince d Dys post-tumor injection 1 11 e Dys post-tumor injection Rdince Percent survivl NT Dys post-tumor injection Dys post-tumor injection Figure 5 T cells hve potent nti-cute myeloid leukemi ctivity in vivo. Antitumor ctivity of T cells in MOLM-13 leukemi NSG xenogrft model. MOLM-13.GFP.ffLuc-ering mice received n i.v. dose of (n = 5) or T cells (n = 5) on dy 7 nd 14 post-tumor cell injection. Untreted nimls served s controls (n = 1). Tumor growth ws followed y ioluminescence imging. () Representtive imges of nimls (dy post-tumor cell injection is shown in the upper right corner of imges). ( d) Quntittive ioluminescence imging results (dotted lines: individul mice; solid lines: medin; rdince=photo/sec/cm 2 /sr). Strting dy 3 post-first T-cell injection for CD123- ENG versus T cells, nd T cells versus untreted: P <.1). (e) Kpln-Meier survivl curve (control versus T cells: P =.4; control versus T cells: NS; versus T cells: P =.15). CD123-CARs, which cnnot ctivte ystnder T cells git AML cells, we show here tht ENG T cells redily redirect unmodified ystnder T cells to CD123-positive trget cells. Since the efficcy of CAR T-cell therpy relies on significnt in vivo expion of T cells, the oserved ystnder effect might llow for disese control with limited in vivo expion of doptively trferred T cells. We hve shown in vivo tht the short-term persistence of T cells is significntly impcted y the presence of the ntigen trget. In our model system, the rtio of expressing to unmodified humn T cells is mintined, suggesting equl stimultion of nd unmodified cells. This indictes tht ENG T cells hve the cpility of vol. 24 no. 9 sep. 216

7 The Americn Society of Gene & Cell Therpy CD123-Engger T Cells for the Tretment of AML T cells T cells + MOLM MOLM-13 Dys post-t-cell injection Rdince * ** ** ** ** Dy post-t-cell injection 6 c 5 d 1 CD3+ CD45+ (%) MOLM-13 mornge+ % (of CD3+ CD45+) MOLM-13 Spleen Bone mrrow Spleen Bone mrrow Figure 6 Antigen-specific persistence of T cells in vivo. MOLM-13 ering or control mice were injected I.V. with mornge T cells tht were lso geneticlly modified to express GFP.ffLuc (n = 5 per group). () Imges of individul mice. () Quntittive ioluminescence imging results (rdince = photo/sec/cm 2 /sr, men nd SD is plotted, *P <.1, **P <.1). (c, d) On dy 6 post-t-cell injection, mice were euthnized nd splee nd one mrrows (oth femurs) of ll (5) cute myeloid leukemi (AML)-ering mice nd of two control mice tht received T cells were processed for FACS nlysis. (c) Percentge of humn T cells (CD45-positive, CD3-positive) gted on live cells (AML-ering mice versus control mice: P = NS). (d) mornge-positive cells (%) gted on humn T cells (CD45-positive, CD3-positive); dotted line: mornge-positive cells (%) in infused T cells (AML- ering mice versus control mice: P = NS; % mornge positive cells in infused T cells versus % mornge-positive T cells on dy 6 postinfusion: P = NS). ctivting 1% of infused T cells, potentilly overcoming lower trduction efficiencies of ptients T cells. We oserved xenogenic GVHD in treted mice. In our in vivo T-cell persistence studies we only oserved significnt difference in the frequency of T cells etween MOLM-13 ering mice nd control mice for the first 5 dys fter injection. Thus GVHD in our model is most likely due to nopecific xenogenic T-cell stimultion t lter time points post-infusion s oserved y others. 28,29 molecules induced significnt IL-2 production in n ntigen-dependent mnner without hving costimultory domin. While AML lsts do not express stndrd costimultory molecules such s CD8, 37 they express for exmple NKG2D lignds, 38 which cn provide costimultion 39 explining the oserved IL-2 production. One potentil dvntge of ENG T cells versus the infusion of ispecific ntiodies is tht T cells cn trffic to tumor sites nd produce ENG molecules loclly. We could not demotrte this dvntge in the descried systemic AML model, ut hve shown ENG T-cell homing to tumor sites in locl B-cell mlignncy model. 4 Bispecific ntiodies such s lintumom tht coist of 2 scfvs hve hlf-life of 2 hours in vivo. 41 Since hlf-life depends on moleculr weight, we expect tht scfv-sed ENG molecules secreted from T cells will hve similr hlf-life. CD123 is expressed t low levels on plsmcytoid dendritic cells, sophils, monocytes, endothelil cells, nd HSPCs. 3,42,43 Moleculr Therpy vol. 24 no. 9 sep

8 CD123-Engger T Cells for the Tretment of AML The Americn Society of Gene & Cell Therpy 12 P =.1 5 P <.1 P <.1 P <.2 Numer of colonies Numer of colonies :1 1:1 1:1 5:1 1:1 1:1 ENG-T cell: BMMC rtio ENG-T cell: CBMC rtio Figure 7 T cells recognize HSPCs t high effector to trget rtios. Bone mrrow mononucler cells (BMMCs) or cord lood mononucler cells (CBMCs) were cultured of E:T rtios of 5:1, 1:1, nd 1:1 with or T cells for 6 hours. Cells were plted in MethoCult medi nd CFUs were counted fter 1 14 dys. Box grph, whiskers: min, mx. () BMMCs: n = 4; versus T cells: 5:1 P =.1, 1:1 P =, nd 1:1 P =. () CBMCs: n = 4; versus CD19- ENG T cells: 5:1 P <.1, 1:1 P <.1, nd 1:1 P <.2. Controversy exists in regrds to the recognition of norml HSPCs y CD123-CAR T cells. While one group demotrted tht the infusion of CD123-CAR T cells inhiited the engrftment of fetl liver- or one mrrow-derived HSPCs in NSG mice, 21,22 two groups reported limited toxicity of CD123-CAR T cells Limited toxicity hs lso een oserved for CD123/CD3-specific dul ffinity retrgeting recominnt protein. In non-humn primte model, CD123/CD3-specific dul ffinity retrgeting induced cytokine relese syndrome (CRS) with the first infusion nd trient decrese in hemtocrit fter multiple infusio. 44 Here we show tht the toxicity of T cells depends on the used E:T rtio s well s the source of the HSPCs in CFU ssys. We did not differentite etween linege-specific CFUs since we wnted to ssess the glol toxicity on norml HSPCs of T cells. We re plnning to determine the effects on linege-specific CFUs in the future. We oserved tht HSPCs derived from cord lood were more seitive to T-cell medited killing thn HSPCs derived from one mrrow. Others hve reported tht the percentge of HSPCs expressing CD123 s well s the expressed level of CD123 is higher in cord lood thn one mrrow derived HSPCs, which supports our findings. 45 While high rtios cused significnt killing of HSPCs, limited killing ws oserved t low rtios t which AML lsts were still killed. These results suggest tht there my e therpeutic window, which would llow the killing of AML lsts, while spring norml HSPCs. Since it is impossile to predict the effective E:T rtio in vivo, we resoned tht introducing suicide gene into T cells would e dvisle for future clinicl development. We first evluted the inducile cspse 9 suicide gene in T cells, ut oserved seline toxicity (Supplementry Figure S7). We next focused on expressing CD2 in T cells in comintion with rituxim dministrtion s suicide gene pproch, since ntiodies cn trvel freely through the sinusoidl clefts tht re present in one mrrow, 46 the site of potentil HSPC toxicity. CD2. T cells hd stle CD2 expression, comprle ntitumor ctivity in vitro nd in vivo, nd were redily eliminted y rituxim in the presence of complement in vitro. We lso showed tht rituxim dministrtion in vivo resulted in greter thn 9% depletion of CD2./GFP.ffLuc T cells s judged y ioluminescence imging in MOLM-13-ering NSG mice. Of note, NSG mice hve deficient complement pthwys. 47 Thus the oserved in vivo T-cell killing in these mice relies minly on FC-receptor medited killing. In hum, which hve n intct complement pthwy s well s FC-receptor medited killing, we expect rituxim to even e more effective in depleting CD2- positive T cells. Thus, our in vitro nd in vivo results indicte tht CD2 expression in comintion with rituxim dministrtion is suitle suicide gene method to eliminte T cells. Nevertheless, to move our pproch to clinicl ppliction, we propose to perform first-in-humn studies in the pretrplnt setting. We im to use CD2. T cells to induce complete remission in refrctory AML ptients who hve n identified stem cell donor, efore proceeding to n llogeneic stem cell trplnt. Our pproch is comprle to the strtegy tken y other investigtors who hve open clinicl studies with CD123-CAR T cells (NCT , NCT ), nd is expected to mitigte the risk of long-term plsi nd grft filure from ll CD123-trgeted T-cell therpies, including T cells. In summry, our study demotrtes tht CD2. T cells hve potent ntitumor ctivity, redirect ystnder T cells to CD123-positive AML, nd re redily eliminted y rituxim/ complement. Thus, CD2. T cells my e promising lterntive to current CD123-trgeted immunotherpy pproches tht either rely on the continuous infusion of recominnt protein or the doptive trfer of CAR T cells. MATERIALS AND METHODS Cells nd culture conditio. 293T, K562, KG1, nd MV-4-11 cell lines were purchsed from the Americn Type Culture Collection (ATCC, Mnsss, VA) nd cultured in Dulecco's Modified Egle Medium (DMEM, ThermoScientific, Wlthm, MA) supplemented with 2 mmol/l Glutmx (Invitrogen, Crlsd, CA) nd 1% Fetl Bovine Serum (FBS, ThermoScientific (293T)) or Roswell Prk Memoril Ititute (RPMI, ThermoScientific), supplemented with 2 mmol/l Glutmx nd 1% (K562) or 2% (MV-4 11) FBS or scove's Modified Dulecco's Medium (IMDM, ThermoScientific) supplemented with 2 mmol/l Glutmx nd 2% Fetl Bovine Serum (FBS) FBS (KG1). MOLM-13 cell line ws purchsed from the Leiniz Ititute (DSMZ, Germn Collection of Microognisms nd Cell Cultures, Bruchweig, Germny) nd cultured in RPMI supplemented vol. 24 no. 9 sep. 216

9 The Americn Society of Gene & Cell Therpy CD123-Engger T Cells for the Tretment of AML Lysis (%) CD2. Rdince CD :1 2:1 1:1 5:1 Effector: trget rtio Dys post-tumor injection c d P <.1 8 P =.3 1 Lysis (%) NT CD2. CD2(+) cell deth (%) Complement Rituxim Complement Rituxim e 1 f 1 9 Lysis (%) CD2. Rdince Control Rituxn 2 CD2. (Rituxn + Comp) 1 6 4:1 2:1 1:1 5:1 Effector: trget rtio Dys post-t-cell injection Figure 8 CD2 gene-modified T cells retin their effector function git CD123-positive cute myeloid leukemi nd re effectively eliminted y rituxim. () Cytotoxicity ssys were performed using, CD2., or T cells s effectors nd KG1 s CD123-positive trget. (n = 3; ssy ws performed in triplictes, P =.45, versus CD2. T cells). () MOLM-13. GFP.ffLuc-ering mice received n i.v. dose of CD2. T cells on dy 7 post-tumor cell injection. Quntittive ioluminescence imging results (dotted lines: individul mice; solid line: medin; rdince = photo/sec/cm 2 /sr). (c) CD2.,, or NT T cells were leled with 51Chromium nd treted with rituxim nd/or complement in stndrd cytotoxicity ssy. (n = 4; ssy ws performed in triplicte; for CD2. T cells: untreted versus rituxim nd complement: P =.3). (d, e) CD2. T cells were treted with rituxim, complement, or rituxim nd complement, nd cultured for 7 dys. (D) FACS nlysis for CD2-positive cells. Dt is presented s percent cell deth of CD2-positive cells 7 dys post-tretment (n = 5; untreted, rituxim treted, complement treted vs rituxim nd complement treted cells: P <.1). (e) Cytotoxicity ssys using untreted,, CD2., or rituxim nd complement treted CD2. T-cell lines s effectors nd KG1 cells s trgets. n = 3; ssy performed in triplictes; untreted versus treted CD2. T cells: P <.1; untreted CD2. T cells versus T cells: P <.1; treted CD2. T cells versus T cells: P = for ll E:T rtios tested. (f) Mice engrfted with MOLM-13 were treted with 3x1 6 CD2./GFP.ffLuc T cells on Dy 7 postleukemi injection. On Dys 3 5 post-t-cell injection mice (n = 4) received 25 µg rituxim IP. Untreted mice (n = 3) served s controls. Quntittive ioluminescence imging results (rdince = photo/sec/cm 2 /sr, men nd SD is plotted; P <.5 strting 2 dys post-first dose of rituxim). with 2 mmol/l Glutmx nd 1% FBS. All cells were mintined in humidified tmosphere contining 5% CO 2 t 37 C. K562 cells expressing CD123 (K562.CD123) were generted y trducing K562 cells with lentivirl vector encoding CD123 nd green fluorescent protein/puromycin resistnce gene (GFPpuro; pcdh.cmv.cd123.ef1.gfppuro). MOLM-13 cells expressing n enhnced GFP firefly luciferse fusion gene (MOLM-13. GFP.ffLuc) were generted y trducing MOLM-13 cells with retrovirl vector encoding GFP.ffLuc. 48 GFP-positive cells were sorted nd mintined in RPMI supplemented with 2 mmol/l Glutmx nd 1% FBS. Luciferse expression ws confirmed using D-luciferin. Moleculr Therpy vol. 24 no. 9 sep

10 CD123-Engger T Cells for the Tretment of AML The Americn Society of Gene & Cell Therpy Flow cytometry. Fluorochrome conjugted isotype controls, nti-cd123, nti-cd3, nti-cd4, nti-cd8, nti-cd33, nti-cd34, nti-cd45, nti- CD45RA, nd nti-ccr7 were purchsed from BD Biosciences (Sn Jose, CA). CD123-specific scfvs were detected with n AlexFluor 647-conjugted got nti-mouse F( ) 2 (Jckson Immunoreserch, West Grove, PA). Expression of mornge ws detected y FACS. Anlysis ws performed on t lest 2, cells per smple using FACSCliur itrument nd Cell Quest softwre (BD Biosciences). Cotruction of retrovirl vectors. The cotruction of the retrovirl vector encoding the CD19-specific engger molecule hs een previously reported. 24 The CD123-specific engger molecule contining the immunogloulin hevy-chin leder peptide, the CD123-specific scfv (26292), 48 short serine-glycine linker, nd CD3-specific scfv derived from OKT3 ws synthesized y ThermoFisher Scientific (Grnd Islnd, NY), nd sucloned into psfg-ires-mornge. The retrovirl vector encoding full length CD2, 2A sequence, nd the CD123 engger molecule ws generted y sulconing minigene encoding CD2.2A. (ThermoFisher Scientific) into psfg retrovirl vector. RD114-pseudotyped retrovirl prticles were generted s previously descried. 5 pcdh.cmv.hcd123.ef1.gfppuro ws generted y cloning codon optimized cdna (ThermoFisher Scientific) of humn CD123 into the multiple cloning site (MCS) of pcdh.cmv.mcs. EF1.GFPpuro. VSVG-pseudotyped lentivirl prticles were generted ccording to the mnufcturer s itructio (System Biosciences, Inc., Mountin View, CA). Genertion of Engger-T cells. PBMCs were otined from helthy donors under Bylor College of Medicine Ititutionl Review Bord (IRB)- pproved protocol, fter informed coent ws otined in ccordnce to the Declrtion of Helsinki. Cells were ctivted y stimultion on OKT3 (CRL-81, ATCC) nd nti-cd28 (Becton Dickion, Mountin View, CA) coted non-tissue culture-treted 24-well pltes. Recominnt humn interleukin (IL)-7 (1 ng/µl, R&D Systems, Minnepolis, MN) nd IL-15 (5 ng/µl, R&D Systems) were dded to cultures on dy 2, nd the following dy cells were trduced with retrovirl prticles immoilized on RetroNectin (Clontech Lorotories, Mountin View, CA). T cells were mintined nd expnded in the presence of IL-7 nd IL-15. Cells were nlyzed for expression of mornge y FACS 5 to 7 dys post-trduction. ELISA ssy. Humn recominnt CD123 (R&D Systems) ws plted t 1 ng/well on 96-well non- tissue culture treted plte. Medi from ENG-T cells (CD123 nd control: CD19) ws plted nd llowed to incute for 1 hour t room temperture (RT). Got nti-mouse F( ) (Jckson Immunoreserch) ws dded, nd incuted t RT for 1 hour. The plte ws wshed, nd secondry nti-got Horserdish Peroxidse (HRP) ntiody (Jckson Immunoreserch) ws dded. After 1 hour incution t RT, the plte ws wshed nd developing gent ws dded (tetrmethylenzidine (TMB) sustrte, Sigm-Aldrich). Asornce ws red t 45 nm. A stndrd curve ws generted using recominnt CD123 T-cell ENG protein (custom synthesis, ThermoFisher Scientific; Supplementry Figure S2). Cytokine secretion ssy. ENG-T cells were plted with trget cells t 2:1 rtio. Following 24 hours of culture, superntnt ws hrvested nd nlyzed for the presence of interferon (IFN) γ or IL-2 using ELISA kits (R&D systems) ccording to the mnufcturer s itruction. Chromium relese ssy. Trget cells were leled with.1 mci 51 Chromium(Cr) nd incuted with nontrduced (NT) or ENG-T cells t 4:1, 2:1, 1:1, nd 5:1 effector to trget (E:T) rtios for 4 hours. Trgets in medi lone or 1% Triton X-1 were used for spontneous nd mximum 51 Cr relese, respectively. Superntnts were collected nd rdioctivity mesured on gmm counter. Men percentge of specific lysis of triplicte smples ws clculted s 1* (experimentl relese spontneous relese)/(mximum relese spontneous relese). Trwell ssy. MOLM-13.GFP.ffLuc cells were plted in the ottom well of 24-well tissue culture plte, nd NT T cells, PBMCs, or medi were dded. Medi, NT T cells, or ENG-T cells were dded to the iert well (.4 µm pore, Corning, Corning, NY). After 24 hours, vile tumor cells were quntified y luciferse ssy. Primry leukemi smple cytotoxicity ssy. Peditric AML smples were otined under Bylor College of Medicine IRB pproved protocol, fter informed coent ws otined in ccordnce to the Declrtion of Helsinki. The clinicl flow lortory uses multi-prmetric flow cytometric ssy with set of 33 ntiodies. The lst popultion of ech smple ws essentilly identified using CD45 nd side sctter, with lmost ll myeloid clones expressing dim to moderte CD45 compred to norml cell popultio (lymphocytes nd monocytes). The lst percentge in ech specimen ws clculted y gting on CD45/SSC-A long-with one or more distinct immunophenotypes expressed on the lsts. The lst percentge in the specime used in this study rnged from 33 9%. Cells were incuted with medi lone or ENG-T cells t indicted E:T rtios for 6 hours, then plted in MethoCult (Clssic) medi (Stemcell Technologies, Vncouver, BC, Cnd) per mnufcturer s itruction. After 1 14 dys, medi ws resuspended with dilution into RPMI nd cells were mnully counted with hemocytometer nd trypn lue exclusion. CFU ssys. Bone mrrow nd cord lood units were otined under Bylor College of Medicine IRB pproved protocol in ccordnce to the Declrtion of Helsinki. Bone mrrow or cord lood mononucler cells (BMMCs, CBMCs) were isolted y stndrd deity grdient centrifugtion nd cyropreserved. For CFU ssys, CBMCs or BMMCs were incuted with medi lone or ENG-T cells t indicted E:T rtios for 6 hours, then plted in MethoCult (Clssic) medi (Stemcell Technologies, Vncouver, BC, Cnd) per mnufcturer s itruction. After 1 14 dys, colonies were counted. Complement-dependent cytotoxicity ssy. T cells trduced with CD2. were lelled with.1 mci 51 Cr nd then treted with 1 µg/ml rituxim (Roche, Sn Frncisco, CA), 1% y rit complement (Cedrlne Ls, Burlington, NC), or 1 µg/ml rituxim nd 1% y rit complement. Cells were incuted for 4 hours. Trgets in medi lone or 1% Triton X-1 were used for spontneous nd mximum 51 Cr relese, respectively. Superntnts were collected nd rdioctivity mesured on gmm counter. Men percentge of specific lysis of triplicte smples ws clculted s 1* (experimentl relese spontneous relese)/(mximum relese spontneous relese). FACS-sed cytotoxicity ssy. CD2. T cells were treted with 1 µg/ml rituxim, 1% y rit complement, or 1 µg/ml rituxim nd 1% y rit complement nd incuted for 2 hours. Following incution, the remining T cells were expnded for one week in IL-7 nd IL-15 efore determining the presence of CD2-positive cells y FACS nlysis. Xenogrft NSG model. All niml experiments were performed on protocol pproved y the Bylor College of Medicine Ititutionl Animl Cre nd Use Committee in ccordnce to the Americn Assocition for Lortory Animl Science. NSG mice (NOD.Cg- Prkdcscid/ Il2rgtm1Wjl/SzJ, Br Hror, ME) were su-lethlly irrdited with 2 cgy 24 hours prior to til vein injection with 5x1 4 MOLM-13.GFP.ffLuc cells. Mice were treted with ENG-T cells intrvenously t dy 7 or t dys 7 nd 14 dys fter MOLM-13.GFP.ffLuc injection. Untreted mice served s controls. In experiments designed to trck T-cell expion nd persistence in vivo, T cells were modified with GFP.ffLuc. Unmodified MOLM-13 AML cells were used s descried. To test the ility of rituxim to deplete T cells in vivo, mice were su-lethlly irrdited with 2 cgy 24 hours prior to til vein injection with MOLM- 13 cells. Mice received on dy 7 post-molm-13 injection CD vol. 24 no. 9 sep. 216

11 The Americn Society of Gene & Cell Therpy CD123-Engger T Cells for the Tretment of AML -T cells. CD2. T cells lso expressed GFP.ffLuc. On dy 3 post-t-cell injection, mice received 25 µg of rituxim IP for 3 coecutive dys. Untreted mice served s controls. Bioluminescent imging ws performed on n IVIS system (IVIS, Xenogen, Almed, CA) s previously descried. 51 Euthnsi ws performed t prior determined time points or when nimls met euthnsi criteri in ccordnce with Bylor College of Medicine s Center for Comprtive Medicine. Sttisticl nlysis. GrphPd Prism 5 softwre (GrphPd softwre) ws used for sttisticl nlysis. Mesurement dt were presented s men ± stndrd error. For comprison etween two groups, two-tiled t-test ws used. For compriso of three or more groups, the vlues were nlyzed y one-wy nlysis of vrince with Bonferroni s post-test. For the mouse experiments, survivl, determined from the time of tumor cell injection, ws nlyzed y the Kpln Meier method nd y the log-rnk test. SUPPLEMENTARY MATERIAL Figure S1. T cells exhiit similr T-cell phenotype to non-trduced, ex-vivo expnded T cells. Figure S2. Stndrd curve of developed ELISA to detect CD123 T-cell ENG protein. Figure S3. Myeloid leukemi cell lines express CD123. Figure S4. Humn T-cell infiltrtion into liver of NSG mice treted with T cells. Figure S5. Genertion of CD2. T cells. Figure S6. Selection nd in vitro elimintion of CD2. T cells. Figure S7. Expression of inducile cspse 9 (ic9) in CD123 ENG T cells is toxic. ACKNOWLEDGMENTS We thnk Clion Rooney, Mlcolm Brenner, nd Helen Heslop for helpful discussio nd dvice. This work ws supported y The Leukemi & Lymphom Society, Ldies Leukemi Legue, Alex s Lemonde Stnd Foundtion for Childhood Cncer, When Everyone Survives Foundtion, nd philnthropic gift to fund AML reserch t the Center for Cell nd Gene Therpy. C.L.B. ws supported y NIH grnt 5T32HL M.P.V. is St. Bldrick s Foundtion fellow. C.A. is n ASH Reserch Scholr. The Center for Cell nd Gene Therpy hd reserch collortion with Celgene nd Blueird Bio. K.I., M.P.V., C.A., nd S.G. hve ptent pplictio in the field of T cell nd genemodified T-cell therpy for cncer. AUTHOR CONTRIBUTION C.L.B. nd S.G. designed the study. C.L.B., A.S., D.T., N.J., M.P.V., K.I., A.G., P.N., nd C.A. performed experiments. All uthors contriuted to dt nlysis nd mnuscript preprtion. References 1. Löwenerg, B, Downing, JR nd Burnett, A (1999). Acute myeloid leukemi. N Engl J Med 341: Woods, WG (26). Curing childhood cute myeloid leukemi (AML) t the hlf-wy point: promises to keep nd miles to go efore we sleep. Peditr Blood Cncer 46: Gormn, MF, Ji, L, Ko, RH, Brnette, P, Bostrom, B, Hutchion, R et l. (21). Outcome for children treted for relpsed or refrctory cute myelogenous leukemi (raml): Therpeutic Advnces in Childhood Leukemi (TACL) Coortium study. Peditr Blood Cncer 55: Mnzo, T, Heslop, HE nd Rooney, CM (215). Antigen-specific T cell therpies for cncer. Hum Mol Genet 24(R1): R67 R Hinrichs, CS nd Rosenerg, SA (214). Exploiting the curtive potentil of doptive T-cell therpy for cncer. Immunol Rev 257: Themeli, M, Rivière, I nd Sdelin, M (215). New cell sources for T cell engineering nd doptive immunotherpy. Cell Stem Cell 16: June, CH, Mus, MV, Ples, G, Johon, LA, Zho, Y, Levine, BL et l. (214). Engineered T cells for cncer therpy. Cncer Immunol Immunother 63: Klos, M, Levine, BL, Porter, DL, Ktz, S, Grupp, SA, Bgg, A et l. (211). T cells with chimeric ntigen receptors hve potent ntitumor effects nd cn estlish memory in ptients with dvnced leukemi. Sci Trl Med 3: 95r Kochenderfer, JN, Dudley, ME, Feldmn, SA, Wilson, WH, Spner, DE, Mric, I et l. (212). B-cell depletion nd remissio of mlignncy long with cytokine-ssocited toxicity in clinicl tril of nti-cd19 chimeric-ntigen-receptor-trduced T cells. Blood 119: Kochenderfer, JN, Dudley, ME, Crpenter, RO, Kssim, SH, Rose, JJ, Telford, WG et l. (213). Donor-derived CD19-trgeted T cells cuse regression of mlignncy persisting fter llogeneic hemtopoietic stem cell trplnttion. Blood 122: Brentje, RJ, Dvil, ML, Riviere, I, Prk, J, Wng, X, Cowell, LG et l. (213). CD19- trgeted T cells rpidly induce moleculr remissio in dults with chemotherpyrefrctory cute lympholstic leukemi. Sci Trl Med 5: 177r Cruz, CR, Micklethwite, KP, Svoldo, B, Rmos, CA, Lm, S, Ku, S et l. (213). Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell mlignncies relpsed fter llogeneic stem cell trplnt: phse 1 study. Blood 122: Mude, SL, Frey, N, Shw, PA, Aplenc, R, Brrett, DM, Bunin, NJ et l. (214). Chimeric ntigen receptor T cells for sustined remissio in leukemi. N Engl J Med 371: Kochenderfer, JN, Dudley, ME, Kssim, SH, Somerville, RP, Crpenter, RO, Stetler- Steveon, M et l. (215). Chemotherpy-refrctory diffuse lrge B-cell lymphom nd indolent B-cell mlignncies cn e effectively treted with utologous T cells expressing n nti-cd19 chimeric ntigen receptor. J Clin Oncol 33: Lee, DW, Kochenderfer, JN, Stetler-Steveon, M, Cui, YK, Delrook, C, Feldmn, SA et l. (215). T cells expressing CD19 chimeric ntigen receptors for cute lympholstic leukemi in children nd young dults: phse 1 dose-escltion tril. Lncet 385: Ritchie, DS, Neeson, PJ, Khot, A, Peinert, S, Ti, T, Tinton, K et l. (213). Persistence nd efficcy of second genertion CAR T cell git the LeY ntigen in cute myeloid leukemi. Mol Ther 21: Wng, QS, Wng, Y, Lv, HY, Hn, QW, Fn, H, Guo, B et l. (215). Tretment of CD33-directed chimeric ntigen receptor-modified T cells in one ptient with relpsed nd refrctory cute myeloid leukemi. Mol Ther 23: Mrdiros, A, Dos Sntos, C, McDonld, T, Brown, CE, Wng, X, Budde, LE et l. (213). T cells expressing CD123-specific chimeric ntigen receptors exhiit specific cytolytic effector functio nd ntitumor effects git humn cute myeloid leukemi. Blood 122: Tettmnti, S, Mrin, V, Pizzitol, I, Mgnni, CF, Giordno Attinese, GM, Criioli, E et l. (213). Trgeting of cute myeloid leukemi y cytokine-induced killer cells redirected with novel CD123-specific chimeric ntigen receptor. Br J Hemtol 161: Pizzitol, I, Anjos-Afoo, F, Rouult-Pierre, K, Lssilly, F, Tettmnti, S, Spinelli, O et l. (214). Chimeric ntigen receptors git CD33/CD123 ntige efficiently trget primry cute myeloid leukemi cells in vivo. Leukemi 28: Gill, S, Tsin, SK, Ruell, M, Shestov, O, Li, Y, Porter, DL et l. (214). Preclinicl trgeting of humn cute myeloid leukemi nd myeloltion using chimeric ntigen receptor-modified T cells. Blood 123: Kenderin, SS, Ruell, M, Shestov, O, Klichiky, M, Aikw, V, Morrissette, JJ et l. (215). CD33-specific chimeric ntigen receptor T cells exhiit potent preclinicl ctivity git humn cute myeloid leukemi. Leukemi 29: Compte, M, Blnco, B, Serrno, F, Cuest, AM, Snz, L, Bernd, A et l. (27). Inhiition of tumor growth in vivo y in situ secretion of ispecific nti-cea x nti-cd3 diodies from lentivirlly trduced humn lymphocytes. Cncer Gene Ther 14: Iwhori, K, Kkrl, S, Velsquez, MP, Yu, F, Yi, Z, Gerken, C et l. (215). Engger T cells: new clss of ntigen-specific T cells tht redirect ystnder T cells. Mol Ther 23: Intron, M, Brui, AM, Bmcioni, F, Csti, C, Gip, G, Borleri, G et l. (2). Genetic modifiction of humn T cells with CD2: strtegy to purify nd lyse trduced cells with nti-cd2 ntiodies. Hum Gene Ther 11: Serfini, M, Mngnini, M, Borleri, G, Bonmino, M, Imerti, L, Biondi, A et l. (24). Chrcteriztion of CD2-trduced T lymphocytes s n lterntive suicide gene therpy pproch for the tretment of grft-versus-host disese. Hum Gene Ther 15: Griffioen, M, vn Egmond, EH, Kester, MG, Willemze, R, Flkenurg, JH nd Heemskerk, MH (29). Retrovirl trfer of humn CD2 s suicide gene for doptive T-cell therpy. Hemtologic 94: King, MA, Covssin, L, Brehm, MA, Rcki, W, Person, T, Leif, J et l. (29). Humn peripherl lood leucocyte non-oese dietic-severe comined immunodeficiency interleukin-2 receptor gmm chin gene mouse model of xenogeneic grft-versushost-like disese nd the role of host mjor histocomptiility complex. Clin Exp Immunol 157: Ali, N, Flutter, B, Snchez Rodriguez, R, Shrif-Pghleh, E, Brer, LD, Lomrdi, G et l. (212). Xenogeneic grft-versus-host-disese in NOD-scid IL-2Rγnull mice disply T-effector memory phenotype. PLoS One 7: e Muñoz, L, Nomdedéu, JF, López, O, Crnicer, MJ, Bellido, M, Aventín, A et l. (21). Interleukin-3 receptor lph chin (CD123) is widely expressed in hemtologic mlignncies. Hemtologic 86: Mnz, MG, Miymoto, T, Akshi, K nd Weissmn, IL (22). Prospective isoltion of humn clonogenic common myeloid progenitors. Proc Ntl Acd Sci USA 99: Peinert, S, Prince, HM, Guru, PM, Kershw, MH, Smyth, MJ, Trpni, JA et l. (21). Gene-modified T cells s immunotherpy for multiple myelom nd cute myeloid leukemi expressing the Lewis Y ntigen. Gene Ther 17: Csucci, M, Nicolis di Roilnt, B, Flcone, L, Cmis, B, Norelli, M, Genovese, P et l. (213). CD44v6-trgeted T cells medite potent ntitumor effects git cute myeloid leukemi nd multiple myelom. Blood 122: Test, U, Pelosi, E nd Frnkel, A (214). CD 123 is memrne iomrker nd therpeutic trget in hemtologic mlignncies. Biomrk Res 2: O Her, C, Heier, JF, Schuert, I, Fey, G nd Geiger, TL (215). Anti-CD33 chimeric ntigen receptor trgeting of cute myeloid leukemi. Hemtologic 1: Buckley, SA nd Wlter, RB (215). Updte on ntigen-specific immunotherpy of cute myeloid leukemi. Curr Hemtol Mlig Rep 1: Chn, L, Hrdwick, N, Drling, D, Gle-Luri, J, Gäken, J, Devereux, S et l. (25). IL-2/B7.1 (CD8) fusgene trduction of AML lsts y self-inctivting lentivirl Moleculr Therpy vol. 24 no. 9 sep