Crenolanib | DNA RNA synthesis Signal

CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+AML and act synergistically with the FLT3-inhibitor crenolanib

Hardikkumar Jetani, Irene Garcia-Cadenas, Thomas Nerreter, Simone Thomas, Julian Rydzek, Javier Briones Meijide, Halvard Bonig, Wolfgang Herr, Jordi Sierra, Hermann Einsele, Michael Hudecek

Cite this article as: Hardikkumar Jetani, Irene Garcia-Cadenas, Thomas Nerreter, Simone Thomas, Julian Rydzek, Javier Briones Meijide, Halvard Bonig, Wolfgang Herr, Jordi Sierra, Hermann Einsele and Michael Hudecek, CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+AML and act synergistically with the FLT3-inhibitor crenolanib, Leukemia _#####################_ doi:10.1038/s41375-018-0009-0

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Received 08 August 2017; accepted 07 December 2017; Author version _#####################_

1
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+ AML and act synergistically

with the FLT3-inhibitor crenolanib

Short title: FLT3-CAR T-cells targeting FLT3-ITD+ AML

6Hardikkumar Jetani1, Irene Garcia-Cadenas2, Thomas Nerreter1, Simone Thomas3, Julian Rydzek1, Javier

7Briones Meijide2, Halvard Bonig4,5, Wolfgang Herr3, Jordi Sierra2, Hermann Einsele1, and Michael

9
Hudecek1*

101Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany

112Hematology Department, Hospital de la Santa Creu i Sant Pau, Sant Pau and Jose Carreras Leukemia

12Research Institutes, Autonomous University of Barcelona, Spain

133Klinik und Poliklinik für Innere Medizin III, Universitätsklinikum Regensburg, Regensburg, Germany

144Institut für Transfusionsmedizin und Immunhämatologie, Goethe Universität Frankfurt, Frankfurt am

15Main, Germany

17
5Deutsches Rote Kreuz Blutspendedienst BaWüHe, Frankfurt, Germany

18*Correspondence should be addressed to:

19Dr. med. Michael Hudecek, Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II

20Oberdürrbacher Straße 6, 97080 Würzburg, Germany

22
Email: [email protected]; Phone: +49 931 201-40001; Fax: +49 931 201-640001

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

23Conflict-of-interest disclosure

24M.H. and H.J. are co-inventors on a patent related to the use of FLT3-CAR T-cells to treat AML filed by

25the University of Würzburg, Würzburg, Germany. M.H. is co-inventor on patents related to CAR-

26technologies filed by the Fred Hutchinson Cancer Research Center, Seattle, WA and the University of

28
Würzburg, Würzburg, Germany.

29Word count: 5567

30Number of Figures: 5

31Number of Tables: 0

33
This manuscript contains a supplement.

34Key words

35Cancer immunotherapy

36Chimeric antigen receptor (CAR)

37Acute myeloid leukemia

38FMS-like tyrosine kinase 3 (FLT3)

39FLT3-inhibitor

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

40Abstract

41FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem

42and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We

43engineered CD8+ and CD4+ T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and

44demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express

45either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment

46with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-

47ITD+ AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As

48anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt

49normal hematopoiesis in colony formation assays, suggesting that adoptive therapy with FLT3-CAR T-

50cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the

51hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with

52particular relevance in high-risk FLT3-ITD+ AML. Further, our data provide the first proof-of-concept

53that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified

54by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

55Introduction

56FMS-like tyrosine kinase 3 (FLT3) is a type I transmembrane protein that plays an essential role in normal

57hematopoiesis and is physiologically expressed on normal hematopoietic stem cells (HSCs), as well as

58lymphoid, myeloid and granulocyte/macrophage progenitor cells in humans1-4. In mature hematopoietic

59cells, FLT3-expression has been reported in subsets of dendritic cells and natural killer cells5-7. FLT3 is

60also uniformly present on malignant blasts in acute myeloid leukemia (AML), providing a target for

61antibody and cellular immunotherapy1,4,8-11. The antigen density of FLT3 protein on the cell surface of

62AML blasts is in the range of several hundreds to several thousand molecules per cell, which is optimal

63for recognition by engineered T-cells that are equipped with a synthetic chimeric antigen receptor

64(CAR)12,13.

65At the molecular level, FLT3 transcripts are universally detectable in AML blasts, with graded expression

66levels in distinct FAB (French-American-British) subtypes9,14. Higher FLT3 transcript levels correlate

67with higher leukocyte counts and higher degrees of bone marrow infiltration by leukemic cells,

68independent from the presence of FLT3 mutations11. FLT3 is important for survival and proliferation of

69AML blasts and of particular pathophysiologic relevance in AML cases that carry activating mutations in

70the FLT3 intracellular domain1,11. Of these, internal tandem duplications (ITDs) in the juxtamembrane

71domain and mutations in the intracellular tyrosine kinase domain (TKD) are the most common aberrations

72that collectively occur in approx. 30% of AML cases1,11,14,15. Both aberrations cause constitutive FLT3

73activation in a ligand-independent manner and act as gain-of-function ‘driver mutations’ that contribute to

74sustaining the malignant disease16,17. These attributes suggest FLT3-ITD+ AML is particularly susceptible

75and a preferred AML subset for anti-FLT3 immunotherapy because the risk to incur FLT3-/low antigen-loss

76variants is likely low. Indeed, the presence of an FLT3-ITD is associated with an inferior clinical outcome

77after induction/consolidation chemotherapy and allogeneic hematopoietic stem cell transplantation

78(HSCT), and defines a subset of high-risk AML patients that require novel, innovative treatment

79strategies18,19.

80
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

FLT3 is being pursued as a target for tyrosine kinase inhibitors (TKIs) and numerous substances are at

81advanced stages of clinical development. However, the clinical efficacy of single agent therapy with type-

82II-inhibitors, e.g. midostaurin that target the inactive kinase conformation has been rather limited, owing

83at least in part to the development of resistance through novel mutations in the FLT3 intracellular domain,

84or FLT3 overexpression in AML blasts20-23. Crenolanib is a specific type-I-inhibitor that targets the active

85FLT3 kinase conformation and is effective against FLT3 with ITD and TKD mutations that confer

86resistance to midostaurin24,25. Crenolanib is also active against platelet-derived growth factor receptor

87alpha/beta and is being evaluated in patients with gastrointestinal stromal tumors and gliomas26,27. In

88AML, crenolanib is effective against AML with FLT3-ITD and TKD mutations28,29.

89FLT3 has also been pursued as a target for antibody immunotherapy, even though the antigen density of

90FLT3 on AML blasts is much lower compared to e.g. CD20 on lymphoma cells and not presumed to be

91optimal for inducing potent antibody-mediated effector functions12. A mouse anti-human FLT3

92monoclonal antibody (mAb) 4G8 has been shown to specifically bind to AML blasts and to a lesser extent

93to normal HSCs. After Fc-optimization, 4G8 conferred specific reactivity against AML blasts with high

94FLT3 antigen density in pre-clinical models12. Here, we engineered T-cells to express a FLT3-specific

95CAR with a targeting domain derived from the 4G8 mAb and analyze the antileukemia reactivity of

96FLT3-CAR T-cells against FLT3 wild-type (wt) and FLT3-ITD+ AML cells, alone and in combination

97with crenolanib. Further, we evaluate recognition of normal HSC as an anticipated side effect of

98effectively targeting FLT3 to instruct the clinical use of FLT3-CAR T-cells.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

99Materials and Methods

100Human subjects

101Peripheral blood was obtained from healthy donors and AML patients after written informed consent to

102participate in research protocols approved by the Institutional Review Boards of the University of

103

104
Würzburg and the University of Regensburg.

105CAR construction

106A codon optimized targeting domain comprising the VH and VL segments of the FLT3-specific 4G8

107mAb12 was synthesized (GeneArt ThermoFisher, Regensburg, Germany) and fused to a CAR backbone

108comprising a short IgG4-Fc hinge spacer, a CD28 transmembrane domain, CD28 or 4-1BB costimulatory

109moiety, and CD3z, in-frame with a T2A element and EGFRt transduction marker (Supplementary Figure

1101a)30-32. The entire transgene was encoded in a lentiviral vector epHIV7 and expressed under control of an

111EF1/HTLV hybrid promotor32. Similarly, targeting domains specific for CD19 (clone FMC63) and CD123

112(clone 32716) were used to generate CD19 and CD123-CARs with CD28 costimulatory moiety,

113

114
respectively30,31,33,34.

115In vivo experiments

116All experiments were approved by the competent Institutional Animal Care and Use Committees.

117NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (female, 6–8 week old) were purchased from Charles River

118(Sulzfeld, Germany) or bred in-house. Mice were inoculated with 1×106 ffluc_GFP+ MOLM-13 AML cells

119by tail vein injection on day 0 and randomly allocated to treatment and control groups. On day 7, mice

120received a single dose of 5×106 T-cells (i.e. 2.5×106 CD4+ and 2.5×106 CD8+ in 200µL of PBS/0.5% FCS)

121by tail vein injection. Crenolanib (Selleck Chemicals, Houston, TX) was formulated in 30% glycerol

122formal (SigmaAldrich, Munich, Germany) and administered intraperitoneally (i.p.) at a dose of 15mg/kg

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

123on Monday-Friday for 3 consecutive weeks. AML progression/regression was assessed by serial

124bioluminescence imaging following i.p. administration of D-luciferin substrate (0.3mg/g body weight)

125(Biosynth, Staad, Switzerland) using an IVIS Lumina imaging system (PerkinElmer, Waltham,

126Massachusetts). Data were analyzed using LivingImage software (PerkinElmer). In vivo models with

127

128
primary AML and normal HSCs are described in the supplement.

129Crenolanib-treatment of MOLM-13 AML cells

130MOLM-13 cells were maintained in RPMI-1640 medium, supplemented with 10% fetal calf serum, 2mM

131glutamine, 100U/mL penicillin/streptomycin, and 10nM crenolanib. Every 7 days, MOLM-13 cells were

132adjusted to 1×106/mL in fresh medium and 1mL of this cell suspension plated per well in 48-well plates

133(Costar, Corning, NJ). In some experiments, MOLM-13 cells were labelled with efluoro670 according to

134

135
the manufacturer’s instructions to assess proliferation by flow cytometry.

136Statistical analyses

137Statistical analyses were performed using Prism software v6.07 (GraphPad, San Diego, California).

138Unpaired Student’s t-tests were used to analyze data obtained in in vitro and in vivo experiments. Log-

139rank (Mantel-Cox) testing was performed to analyze differences in survival observed in in vivo

140experiments. Differences with a p value <.05 were considered statistically significant.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

141Results

142FLT3-CAR T-cells eliminate FLT3 wt and FLT3-ITD+ AML cells

143We prepared CD4+ and CD8+ FLT3-CAR-modified T-cell lines from healthy donors and AML patients

144(n=6). FLT3-CAR-expressing T-cells were enriched to >90% purity using the EGFRt selection marker

145prior to expansion and functional testing (Supplementary Figure 1a,b). First, we confirmed specific

146recognition of FLT3 surface protein by CD4+ and CD8+ FLT3-CAR T-cells using native K562

147(phenotype: FLT3-) and K562 target cells that had been transduced to stably express wt FLT3

148(K562/FLT3) (Supplementary Figure 2). Then, we included the AML cell lines THP-1 (FLT3 wt),

149MOLM-13 (FLT3-ITD+/-) and MV4;11 (FLT3-ITD+, loss of heterozygosity)35 into our analyses and

150confirmed specific high-level cytolytic activity of CD8+ FLT3-CAR T-cells against each of the cell lines
151at multiple effector to target cell ratios (E:T, range 10:1 – 2.5:1) (Figure 1a,b). Further, CD4+ and CD8+

152FLT3-CAR T-cells produced effector cytokines including IFN-γ and IL-2, and underwent productive

153proliferation after stimulation with each of the AML cell lines, whereas control T-cells derived from the

154same donors only showed background reactivity (Figure 1d,e; Supplementary Figure 3a,b). We found that

155our prototypic FLT3-CAR with CD28 costimulatory domain conferred higher levels of IL-2 production

156and superior proliferation in T-cells compared to a corresponding receptor with 4-1BB costimulatory

157domain and therefore used the FLT3-CAR/CD28 in all subsequent experiments (Supplementary Figure 4).

158Because the FLT3-CAR binds to an epitope in the extracellular domain of FLT3, recognition of AML

159cells was independent from the mutation status of the intracellular tyrosine kinase domain, but rather

160correlated with the antigen density of FLT3 protein on the surface of target cells, as assessed by mean

161fluorescence intensity (MFI) (THP-1 ~ MOLM-13 > MV4;11) (Figure 1a).

162 We also confirmed potent activity of CD8+ and CD4+ FLT3-CAR T-cells against primary AML

163blasts (patients #1 and #2: FLT3-ITD+; patient #3: unknown) (Figure 1a,c; Supplementary Figure 3c,d). In

164particular, CD8+ FLT3-CAR T-cells conferred strong cytolytic activity, leading to eradication of >80%

165AML blasts within as short as 4 hours at an E:T ratio of 1:1 (Figure 1a,c). Notably, the antileukemia

166
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

activity of FLT3-CAR T-cells against primary AML blasts was equivalent to T-cells expressing a CAR

167

168

specific for the alternative AML antigen CD123 (Figure 1c; Supplementary Figure 3c,d).

169FLT3-CAR T-cells induce durable remission of AML in a xenograft model in vivo

170We performed experiments in xenograft AML models in immunodeficient NSG mice to analyze the

171function of FLT3-CAR T-cells in vivo. First, we inoculated mice with ffLuc_GFP-transduced MOLM-13

172AML cells that rapidly expanded to systemic leukemia in peripheral blood, and heavily infiltrated bone

173marrow and spleen (Figure 2a,b). Leukemia-bearing mice were treated with a single dose of 5×106 FLT3-

174CAR-modified or untransduced T-cells (CD4+:CD8+ ratio = 1:1), or received no treatment (Supplementary

175Figure 5a). We observed a strong antileukemia effect in all mice where FLT3-CAR T-cells engrafted. In

176these mice, FLT3-CAR T-cells could be readily detected in peripheral blood, increased in number during

177the antileukemia response, and were present in bone marrow and spleen at the end of the experiment,

178confirming persistence for >3 weeks after adoptive transfer (Figure 2b; Supplementary Figure 5b,c).

179Complete elimination of leukemia cells from peripheral blood occurred within 3 days after adoptive

180transfer (Figure 2b), and bioluminescence imaging confirmed strong systemic antileukemia activity

181(Figure 2a,c). Flow cytometric analyses in bone marrow and spleen confirmed that sustained complete

182remission of AML was accomplished after treatment with FLT3-CAR T-cells, which translated into

183superior overall survival compared with mice that received control T-cells and no treatment (p<.05)

184(Figure 2d, Supplementary Figure 6a,b). In all mice that responded to FLT3-CAR T-cell therapy, we

185observed recurrence of MOLM-13 cells in anatomical sanctuary sites (e.g. subcutaneous tissue and

186peritoneum) (Figure 2a). FLT3 was expressed at similar levels on native and recurring MOLM-13 cells.

187We could not detect infiltrating FLT3-CAR T-cells in specimen obtained from these sanctuary sites, even

188though they were present in peripheral blood and bone marrow (Supplementary Figure 5c, 7a,b).

189 Then, we inoculated NSG mice with primary AML blasts and confirmed development of

190leukemia within a 4-week engraftment period (Supplementary Figure 8a), consistent with prior work36.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

191Also in this model, FLT3-CAR T-cells could be detected at multiple time points following adoptive

192transfer, increased in number during the antileukemia response, and induced complete remission from

193AML in all treated mice (Supplementary Figure 8b-e). We did not observe recurrence of primary AML

194blasts in this model. In aggregate, these data show that FLT3-CAR T-cells confer potent antileukemia

195

196
activity against FLT3 wt and FLT3-ITD+ AML cell lines and primary AML cells in vitro and in vivo.

197Crenolanib induces increased FLT3 surface protein expression in FLT3-ITD+ AML cells

198We hypothesized that upregulation of FLT3 as a compensatory mechanism of AML blasts to counteract

199the effect of FLT3-inhibitors could be exploited to enhance the antileukemia efficacy of FLT3-CAR T-

200cells22,23. We cultured native MOLM-13 AML cells (MOLM-13Native) (FLT3-ITD+/-) in the presence of the

201FLT3-inhibitor crenolanib (MOLM-13Creno) using a 10nM dose, which is a clinically achievable serum

202level25,37. We analyzed FLT3-expression on MOLM-13Creno by flow cytometry after 7 days of exposure to

203the drug and observed significantly higher levels of FLT3 surface protein by MFI compared to MOLM-

20413Native cells (n=3 experiments, p<.05) (Figure 3a, Supplementary Figure 9a). Interestingly, withdrawal of

205crenolanib led to a decrease in FLT3-expression on MOLM-13 cells to baseline levels within 2 days, but

206increased again upon re-exposure to the drug (Figure 3b, Supplementary Figure 9b). After primary

207exposure to crenolanib, we observed a moderate cytotoxic effect and slower expansion of efluor670

208labeled MOLM-13Creno cells compared to MOLM-13Native cells for approx. 7 days (Supplementary Figure

2099a,c). However, despite continuous supplementation to the culture medium, the cytotoxic effect of

210crenolanib subsequently ceased and the expansion of MOLM-13Creno cells accelerated, suggesting they had

211acquired resistance to the drug.

212 An increase in FLT3-expression upon exposure to crenolanib was also observed with MV4;11

213AML cells (FLT3-ITD+), but did not occur in several cell lines expressing wt FLT3, i.e. THP-1 AML

214cells, JeKo-1 mantle cell lymphoma, and K562 erythro-myeloid leukemia, suggesting upregulation of

215FLT3-expression in response to crenolanib treatment specifically occurred in FLT3-ITD+ AML cells

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

216(Figure 3a, Supplementary Figure 9d). In contrast to FLT3, CD33 and CD123-expression on both

217

218
MOLM-13 and MV4;11 was not affected by crenolanib and did not increase (Supplementary Figure 9e).

219Higher FLT3-expression on crenolanib-treated MOLM-13 AML cells leads to enhanced antileukemia

220reactivity of FLT3-CAR T-cells in vitro

221We sought to determine whether the higher antigen density of FLT3 on MOLM-13Creno would enhance

222recognition by FLT3-CAR T-cells. Our earlier data showed rapid modulation of FLT3-expression upon

223exposure to and withdrawal of crenolanib (Figure 3a,b), suggesting maximum reactivity of FLT3-CAR T-

224cells against MOLM-13Creno would be accomplished in the presence of the drug. It is known that TKI may

225interfere with T-cell activation and function38,39, and we therefore confirmed that crenolanib per se did not

226affect the effector function of FLT3-CAR T-cells (Supplementary Figure 10). In subsequent functional

227analyses, we observed superior cytolytic activity of CD8+ FLT3-CAR T-cells against MOLM-13Creno

228compared to MOLM-13Native cells (p<.05), enhanced production of IFN-γ and IL-2, as well as enhanced

229proliferation of FLT3-CAR T-cells after stimulation with MOLM-13Creno compared to MOLM-13Native

230(Figure 3c). In contrast to FLT3-CAR T-cells, the antileukemia reactivity of CD123-CAR T-cells against

231MOLM-13Creno and MOLM-13Native was not significantly different (Figure 3c).

232 We confirmed that upregulation of FLT3 after treatment with crenolanib also occurred on primary

233FLT3-ITD+ AML blasts and led to increased cytolysis by FLT3-CAR T-cells (Supplementary Figure 11).

234Collectively, these data show that treatment with crenolanib leads to increased expression of FLT3

235

236
specifically in FLT3-ITD+ AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells.

237FLT3-CAR T-cells and the FLT3-inhibitor crenolanib act synergistically in mediating regression of

238AML in vivo

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

239This encouraged us to examine the antileukemia effect of FLT3-CAR T-cells in combination with

240crenolanib in the MOLM-13/NSG xenograft model. Mice were inoculated with MOLM-13Native AML cells

241on day 0, and on day 7 were treated with either FLT3-CAR T-cells alone, crenolanib alone, the

242combination treatment with FLT3-CAR T-cells and crenolanib, or were left untreated. We observed potent

243antileukemia efficacy in mice receiving the combination treatment with FLT3-CAR T-cells and crenolanib

244(Figure 4a). There was superior engraftment and in vivo expansion of FLT3-CAR T-cells (Figure 4b); a

245higher overall response rate (combination: n=8/8, 100% vs. FLT3-CAR T-cells alone n=6/8, 75% vs.

246crenolanib alone n=0/8, 0% vs. no treatment n=0/8, 0%); faster and deeper remissions as assessed by

247bioluminescence imaging (Figure 4a,c); as well as improved overall survival of mice receiving the FLT3-

248CAR T-cell and crenolanib combination, compared to monotherapy with either FLT3-CAR T-cells or

249crenolanib alone, or no treatment (p<.05) (Supplementary Figure 12a). Crenolanib monotherapy had only a

250minuscule antileukemia effect and MOLM-13 cells that we recovered from peripheral blood and bone

251marrow at the experiment endpoint had uniformly and strongly upregulated FLT3, consistent with our

252earlier observation in vitro (Figure 4d). At the experiment endpoint, peripheral blood, bone marrow and

253spleen in mice treated with the FLT3-CAR T-cell/crenolanib combination and FLT3-CAR T-cell alone

254were completely free from MOLM-13 AML cells, whereas mice receiving crenolanib monotherapy and

255untreated mice showed a high degree of infiltration with MOLM-13 cells (Figure 4e; Supplementary Figure

25612b). Also with the FLT3-CAR T-cell/crenolanib combination treatment, mice experienced recurrences of

257FLT3+ MOLM-13 cells in anatomical sanctuary sites (Supplementary Figure 12c). Collectively, our data

258show that FLT3-CAR T-cells and crenolanib can be used synergistically in combination therapy to confer a

259

260
potent antileukemia effect against FLT3-ITD+ AML cells in vitro and in vivo.

261FLT3-CAR T-cells recognize normal HSCs and compromise hematopoiesis in colony formation assays

262We sought to determine the collateral damage of FLT3-CAR T-cells on normal hematopoietic stem and

263progenitor cells. We confirmed expression of FLT3 on normal GM-CSF-mobilized peripheral blood

264
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

CD34+CD38- HSCs and CD34+CD38+ progenitor cells, although the antigen density of FLT3 was lower

265compared to AML cells by MFI (Figure 5a, Supplementary Figure 13a). We performed a flow

266cytometry-based cytotoxicity assay in vitro and found that FLT3-CAR T-cells lysed approx. 50% and 80%

267of normal HSCs within 4 and 24 hours, respectively (E:T ratio = 5:1) (Figure 5b). As a reference, we

268included T-cells expressing a CD123-specific CAR, that has been reported to completely eliminate normal

269HSCs and induce myeloablation34. CD123-CAR T-cells exerted a faster and stronger cytolytic effect on

270normal HSCs compared to FLT3-CAR T-cells and lysed >95% of HSCs within 24 hours (Figure 5b).

271Next, we performed in vitro colony formation assays from residual HSCs at the end of the 24-hour co-

272culture with FLT3-CAR T-cells. After 14 days, we only detected a small number of erythroid colonies,

273whereas formation of myeloid colonies was completely abrogated (Figure 5c). To corroborate our data, we

274administered normal HSCs to NSG-3GS mice and after 8-week engraftment, treated them with FLT3-

275CAR T-cells. We found that also in vivo, normal HSCs and progenitor cells were depleted from bone

276marrow after treatment with FLT3-CAR T-cells, similar to mice that received CD123-CAR T-cells

277(Supplementary Figure 13b-d). To assess the influence of crenolanib on normal HSCs, we analyzed FLT3

278expression which, consistent with their wt FLT3 genotype, did not increase; and assessed viability, which

279in contrast to MOLM-13 AML cells, did not decrease within 7 days of exposure to 10nM of the drug

280(Supplementary Figure 14; Supplementary Figure 9c).

281 Collectively, these data show that normal HSCs are recognized and eliminated by FLT3-CAR T-

282cells. These data suggest that the clinical use of FLT3-CAR T-cells against AML would be restricted to a

283defined window of time prior to allogeneic HSCT that allows subsequent CAR T-cell depletion and

284reconstitution of the hematopoietic system.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

285Discussion

286We are pursuing FLT3 as a novel target antigen for CAR T-cells in AML. Clinical trials with CD19-CAR-

287modified T-cells in acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma have provided

288evidence for the curative potential of CAR T-cells in hematologic malignancies. However, they also

289exposed several challenges that affect safety and limit efficacy of CAR T-cell therapy, including on-target

290recognition and long-term elimination of non-tumor cells that express the targeted antigen40,41. We

291detected FLT3 surface protein on normal HSCs, consistent with previous work that demonstrated uniform

292expression of FLT3 on human hematopoietic stem and progenitor cells obtained from bone marrow and

293cord blood of healthy donors1,2,4. Our data also confirm the previous notion that FLT3 density is lower on

294normal HSCs compared to AML blasts by flow cytometry (MFI)12. Even though the precise antigen

295density on target cells that is required to induce CAR T-cell triggering is unknown, it is presumed that this

296threshold is in the order of (few) hundreds of molecules per cell13, which is the range that has been

297estimated for FLT3-expression on HSCs12. Indeed, our data show that FLT3-CAR T-cells eliminate the

298majority of HSCs within a 24-hour co-culture assay, leading to qualitatively and quantitatively impaired

299hematopoiesis in colony formation assays in vitro. Overall, recognition of normal HSCs by FLT3-CAR T-

300cells is an anticipated finding, in line with the previous demonstration that mAb 4G8 – from which we

301derived the targeting domain of our FLT3-CAR – shows uniform binding to normal HSCs12. Our data

302suggest that the clinical use of FLT3-CAR T-cells may be limited to a defined therapeutic window of time

303and restricted to a clinical context that permits subsequent reconstitution of the hematopoietic system.

304Such a window of opportunity is provided in the context of allogeneic HSCT with adoptive transfer of

305FLT3-CAR T-cells prior to HSCT to reduce leukemia burden and/or induce minimal residual disease

306(MRD)-negativity, followed by FLT3-CAR T-cell depletion and engraftment of donor-derived normal

307HSCs. This strategy requires the ability to rapidly and completely remove CAR T-cells to protect

308incoming normal HSCs. The FLT3-CAR T-cells employed in our study are equipped with an EGFRt

309depletion marker. We have recently shown in immunocompetent mice that administration of an anti-

310EGFR mAb can mediate CAR T-cell depletion and reversal of CAR T-cell induced systemic toxicity42. An

311
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

alternative strategy is inclusion of the iCasp9 suicide gene that has been demonstrated to accomplish

312(near)-complete removal of CAR T-cells in the order of minutes to few hours following administration of

313an inducer drug43. Operational models of myeloablative CAR T-cell therapy have been established44 and

314will aid in defining the optimal timing of FLT3-CAR T-cell administration and elimination, and

315subsequent reconstitution with normal HSCs.

316 It is uncertain, whether a small proportion of FLT3low HSCs may escape elimination by FLT3-

317CAR T-cells, and whether this diminished pool of HSCs would be capable of replenishing a quantitatively

318and qualitatively normal hematopoietic system. A recent study even suggested that FLT3-CAR T-cells

319would not deplete HSCs and preserve HSC differentiation in vivo45. However, the only experiment to

320substantiate this statement had been performed in immunodeficient NSG-3GS mice that received

321simultaneous injections of human HSCs and FLT3-CAR T-cells, and it is unclear whether this application

322mode actually leads to any interactions of HSC and FLT3-CAR T-cells, especially in the bone marrow.

323Rather, FLT3-CAR T-cells should have been administered after HSC engraftment and hematopoietic

324differentiation is established, which takes several weeks in this model45. Indeed, our data in the NSG-

3253GS/HSC model show that normal HSCs are eliminated after adoptive transfer of FLT3-CAR T-cells.

326Several alternative CAR target antigens are being pursued in AML, including CD33 and CD123 that have

327advanced to clinical evaluation, but share with FLT3 the challenge of being expressed on normal

328HSCs34,46. CD123-CAR T-cells have been shown to induce myeloablation in vivo due to recognition of

329normal HSCs, consistent with our data that showed rapid and complete elimination of normal HSCs by

330CD123-CAR T-cells in our co-culture assay in vitro and in HSC-engrafted NSG-3GS mice in vivo34.

331 Another observation from the clinical use of CD19-CAR T-cells in ALL is relapse with leukemia

332cell variants that have lost CD19-expression under therapeutic pressure either through lymphoid to

333myeloid trans differentiation; alternative splicing, resulting in loss of the epitope targeted by the CAR; or

334selection of pre-existing antigen-negative leukemia cell clones47-49. These data underscore the need to

335select CAR target antigens that are uniformly expressed and ideally of pathophysiologic relevance for

336leukemia cells. Encouragingly, FLT3 is uniformly expressed in AML blasts, including leukemia

337
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

stem/initiating cells (LSC/LIC), suggesting the potential to confer a curative treatment for AML with

338FLT3-CAR T-cell therapy14,17. There is no clinical experience with FLT3 as an immune target to aid in

339assessing the risk that mutations in the extracellular FLT3 domain may arise during therapy that delete the

340epitope recognized by our FLT3-CAR. We observed disease recurrence in anatomical niches in our

341xenograft model with MOLM-13 AML cells. We confirmed that FLT3-expression and the epitope

342recognized by the FLT3-CAR had been retained on recurring MOLM-13 cells, but could not detect FLT3-

343CAR T-cells in sanctuary lesions. This is in line with the prior notion that human T-cells are not readily

344able to migrate through murine endothelial and epithelial barriers50.

345 There is increasing clinical data from the use of small molecule FLT3-inhibitors20-22 and

346intriguingly, an observation that has been made with the FLT3-inhibitor lestaurtinib is upregulation of

347FLT3-expression in AML blasts after repeated exposure to this drug22. The conceptual appeal of using

348FLT3-inhibitors and FLT3-CAR T-cells in combination is that AML blasts that acquire resistance to

349FLT3-inhibitors and upregulate FLT3, expose themselves to recognition and elimination by FLT3-CAR

350T-cells. Indeed, our data show strong upregulation of FLT3 in FLT3-ITD+ AML cells after treatment with

351the FLT3-inhibitor crenolanib, and enhanced antileukemia reactivity of FLT3-CAR T-cells against

352crenolanib treated FLT3-ITD+ AML cells in vitro and in vivo. We show that upregulation of FLT3 occurs

353specifically in FLT3-ITD-mutated AML cells, supporting the critical role of FLT3-ITD in AML

354pathogenesis. These data demonstrate for the first time that CAR T-cell immunotherapy and small

355molecule inhibition can be used synergistically in a hematologic malignancy, and provide proof-of-

356concept with the FLT3-CAR T-cell/crenolanib combination. Experiments that evaluate synergy between

357FLT3-CAR T-cells and other FLT3-inhibitors are ongoing. Upregulation of FLT3 has been previously

358reported in FLT3-ITD+ MOLM-13 AML cells that acquired resistance to the FLT3-inhibitor

359midostaurin23, providing another attractive combination partner for FLT3-CAR T-cells. Clonal

360heterogeneity is a challenge for treating AML, and even though FLT3 is uniformly expressed on AML

361blasts, there is a potential risk that FLT3-/low AML relapse may develop from FLT3 wt subclones even in

362patients that are classified as FLT3-ITD+. We did not observe FLT3-/low AML escape variants in our in

363
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

vivo model with primary AML blasts. However, it has been demonstrated that FLT3-ITD+ AML subclones

364show preferential engraftment in NSG mice compared to FLT3 wt subclones and therefore, this model

365may underestimate this risk51.

366 It has recently been shown that FLT3 acts as a cytoprotective kinase in cardiomyocytes52. It is

367unknown whether FLT3 surface expression on cardiomyocytes is sufficient for CAR recognition and

368hence, particular caution must be taken when clinically translating FLT3-CAR T-cell therapy, especially

369in combination with FLT3-inhibitors. We did not detect FLT3 upregulation after crenolanib treatment in

370AML and non-AML cells that express wt FLT3 (including HSCs), suggesting the concomitant use of

371crenolanib enhances selectivity of FLT3-CAR T-cells for FLT3-ITD+ AML blasts compared to HSC and

372non-AML cells.

373 Our data suggest that FLT3-ITD+ AML cases are particularly susceptible and have a high

374likelihood to benefit from FLT3-CAR T-cell therapy. In particular, the risk for encountering FLT3 loss

375under therapeutic pressure with FLT3-CAR T-cells is likely lower in FLT3-ITD+ compared to FLT3 wt

376cases, and may be further lowered through concomitant treatment with crenolanib and other FLT3-

377inhibitors. Further, our data suggest that FLT3 in FLT3-ITD+ AML cases is a preferred CAR target,

378advantageous to the alternative antigens CD33 and CD123, receptors for sialic acid and interleukin-3

379respectively, that are of less pathophysiologic relevance in AML compared to FLT3. Clinical trials have

380already demonstrated that rapid antigen downregulation occurs in AML blasts after treatment with anti-

381CD33 and anti-CD123 mAbs53,54. FLT3-expression has also been demonstrated in pre-B-ALL and T-ALL,

382mixed-lineage leukemia and myelodysplastic syndrome1,4,8, expanding the spectrum of hematologic

383diseases amenable to adoptive immunotherapy with T-cells expressing our novel FLT3-CAR.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

384Acknowledgements

385We thank Silke Frenz and Elke Spirk for their expertise in performing the mouse experiments. H.J. was

386supported by a grant from the German Excellence Initiative awarded to the Graduate School of Life

387Sciences (GSLS), University of Würzburg. I.G.G. was supported by a grant from Fundación Alfonso

388Martin Escudero, Spain. M.H. is a member of the Young Scholar Program (Junges Kolleg) and

389

390
Extraordinary Member of the Bavarian Academy of Sciences (Bayerische Akademie der Wissenschaften).

391Author contributions

392H.J. designed and performed experiments, analyzed data and wrote the manuscript. I.G.G., T.N., S.T. and

393J.R. designed and performed experiments and analyzed data. W.H., J.B.M and J.S. analyzed data. H.B.

394provided biologic material and analyzed data. M.H. and H.E. designed experiments, analyzed data, wrote

395the manuscript and supervised the project.

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

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607Figure legends

608Figure 1. FLT3-CAR T-cells recognize and eliminate FLT3 wt and FLT3-ITD+ AML cell lines and

609primary AML blasts in vitro. (a) Flow cytometric analysis of FLT3-expression on AML cell lines

610(MOLM-13, THP-1, MV4;11) and primary AML blasts from n=3 patients. Histograms show staining with

611anti-FLT3 mAb (4G8) (solid line) and isotype control antibody (zebra line). Inset numbers state the

612absolute difference in mean fluorescence intensity obtained by staining with anti-FLT3 mAb and isotype.

613(b) Specific cytolytic activity of CD8+ FLT3-CAR T-cells, CD19-CAR T-cells and untransduced T-cells

614(UTD) against AML cell lines in a bioluminescence-based assay (4-hour incubation). Assay was

615performed in triplicate wells with 5,000 target cells/well. Values are presented as mean + s.d. (c) Specific

616cytolytic activity against primary AML blasts analyzed in a flow cytometry-based assay (4-hour

617incubation). Assay was performed in triplicate wells with 10,000 target cells/well. Counting beads were

618used to quantitate the number of residual live target cells at the end of co-culture. Of note, AML blasts

619from patient #3 were CD19+ by flow cytometry. (d) ELISA to detect IFN-γ and IL-2 in supernatant

620obtained from 24-hour co-cultures of CD4+ and CD8+ FLT3-CAR T-cells with MOLM-13 target cells. T-

621cells (50,000/well) and target cells (25,000/well) were seeded in triplicate wells. Values are presented as

622mean ± s.d. (e) Proliferation of FLT3-CAR T-cells examined by CFSE dye dilution after 72 hours of co-

623culture with MOLM-13 target cells. Assay was performed in triplicate wells with 50,000 T-cells/well and

62425,000 target cells/well. Histograms show proliferation of live (7-AAD-) T-cells. No exogenous cytokines

625were added. Data shown in b, d, e are representative for results obtained with FLT3-CAR and control T-

626

627
cell lines prepared from at least three donors.

628
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

Figure 2. FLT3-CAR T-cells confer potent antileukemia activity in a xenograft model of AML in

629immunodeficient mice in vivo. NSG mice were inoculated with 1×106 MOLM-13 AML cells

630(ffluc+GFP+) and on day 7, were treated with 5×106 CAR-modified or untransduced (UTD) T-cells

631(CD4+:CD8+ ratio = 1:1) or were left untreated. (a) Serial bioluminesence (BL) imaging to assess

632leukemia progression and regression. Note the scale indicating upper and lower BL thresholds at each

633analysis time point (right). (b) Flow cytometric anaysis of peripheral blood on day 10 and 14 after

634leukemia inoculation. Data show the frequency of leukemia cells (GFP+/FLT3+) and transferred T-cells

635(CD45+/CD3+) as percentage of live (7-AAD-) cells. (c) Waterfall plot showing the Δ (increase/decrease)

636in absolute BL values obtained from individual mice between day 7 and day 14 after tumor inoculation.

637BL values were obtained as photon/sec/cm2/sr in regions of interest encompassing the entire body of each

638mouse. (d) Flow cytometric anaysis of peripheral blood (PB), spleen (Sp) and bone marrow (BM) at the

639experiment endpoint in each mouse. Diagrams show the frequency of leukemia cells (GFP+/FLT3+) as

640percentage of live (7-AAD-) cells. *p<.05 (Student's t-test). Data shown are representative for results

641

642
obtained in independent experiments with FLT3-CAR T-cell from n=3 donors.

643Figure 3. Crenolanib treatment leads to enhanced FLT3-expression on FLT3-ITD+ AML cell lines

644and enhances recognition by FLT3-CAR T-cells in vitro. (a) Flow cytometric analysis of FLT3-

645expression on cell lines that had been cultured in the absence or presence of 10nM crenolanib for 7 days.

646Histograms show staining with anti-FLT3 mAb (4G8) (gray) compared to isotype (black). Inset numbers

647state the absolute difference in MFI of treated - non-treated cells. (b) Histograms show FLT3-expression

648on MOLM-13 cells after 7-day culture in presence of 10nM crenolanib (exposure), 2 days after

649subsequently withdrawing the drug (withdrawal), and 7 days afer re-exposure to 10nM crenolanib (re-

650exposure). (c) Recognition of MOLM-13Creno and MOLM-13Native AML cells by FLT3-CAR T-cells.

651Assays with MOLM-13Creno were performed in medium containing 10nM crenolanib. Cytolytic activity in

652a bioluminescence-based assay (4-hour incubation at a 10:1 E:T ratio with 5,000 target cells/well) (Left

653diagram). IFN-γ and IL-2 ELISA (24-hour incubation at a 4:1 E:T ratio with 50,000 T-cells/well) (middle

Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

654diagrams). Proliferation of CD4+ FLT3-CAR and CD123-CAR T-cells assessed by CFSE dye dilution

655(72-hour co-culture of 50,000 T-cells with 12,500 target cells/well) (right diagrams). Numbers below

656histograms indicate the number of cell divisions the proliferating subset underwent, and the fraction (%)

657of T-cells in each gate that underwent ≥4/3/2/1 cell divisions. Data shown in c are representative for

658results obtained in independent experiments with FLT3-CAR T-cells prepared from n=2 donors.

659

660
**p<.005, ***p<.0005 (Student’s t-test).

661Figure 4. FLT3-CAR T-cells and the FLT3-inhibitor crenolanib act synergistically in mediating

662regression of AML in vivo. NSG mice were inoculated with 1x106 MOLM-13 cells (ffluc+GFP+) and on

663day 7 were treated with 5x106 FLT3-CAR T-cells alone (CD4+:CD8+ ratio = 1:1), crenolanib alone

664(15mg/kg body weight as i.p. injection), or both (combination), or were left untreated. The first dose of

665crenolanib was given on day 7 and mice received 15 doses (Monday-Friday) for 3 consecutive weeks. (a)

666Serial bioluminesence (BL) imaging to assess leukemia progression/regression in each treatment group.

667(b) Percentage of live (7-AAD-) T-cells (CD45+CD3+) in peripheral blood (on day 11 and 15 after tumor

668inoculation, i.e. on day 4 and day 8 after T-cell transfer) of mice treated with FLT3-CAR T-cells alone or

669in combination with crenolanib. **p<.005 (Student’s t-test) (c) Water fall plot showing the difference in

670absolute BL values obtained from individual mice between day 7 and day 14 after tumor inoculation. Data

671shown are combined from two independent experiments with T-cells prepared from different donors. (d)

672FLT3-expression on MOLM-13 cells obtained from bone marrow of untreated and crenolanib only mice

673(after 5 doses of crenolanib). Data were analyzed using Student’s t-test (*p<.05) (e) Diagrams show the

674frequency of leukemia cells (GFP+/CD45+) as percentage of live (7-AAD-) cells. *p<.05, **p<.005

675

676
(Student's t-test).

677Figure 5. Recognition of human HSCs by FLT3-CAR T-cells leads to compromised normal
678hematopoiesis. (a) Flow cytometric analysis of FLT3-expression on GM-CSF-mobilized CD34+CD38-

679
Jetani et al. FLT3-CAR T-cells targeting FLT3-ITD+ AML

HSCs and CD34+CD38+ progenitors from n=5 healthy donors. Histograms show staining with anti-FLT3

680mAb (4G8) (solid line) and isotype control antibody (zebra line). (b) Percentage of live HSCs (7-AAD-)

681cells after 4-hour (upper diagram) and 24-hour (lower diagram) co-incubation with CD8+ FLT3-CAR T-

682cells, CD123-CAR T-cells or untransduced T-cells. Assay was performed in triplicate wells with 5,000

683target cells/well. Counting beads were used to quantitate the number of residual live HSCs at the end of

684co-culture. Data shown are mean ± s.d. from n=3 independent experiments with T cells and HSCs

685obtained from different donors. *p<.05, **p< .005 (Student's t-test). (c) Colony formation assay

686performed with residual live HSCs after 24 hours of co-incubation with CD8+ FLT3-CAR, CD123-CAR

687or control T-cells. Diagram shows the absolute number of colonies (mean ± s.d.) per 55 mm plate as

688determined by microscopy on day 14. Data shown are representative for results obtained in independent

689experiments with T-cells from n=3 donors. GEMM (Granulocyte/erythroid/macrophage/megakaryocyte);

690GM (Granulocyte/macrophage); CFU-E (Colony forming unit-erythroid); CFU-M (Colony forming unit-

691Macrophage); CFU-G (Colony forming unit-Granulocyte).

Figure 1:

MOLM-13
DMFI=4341

FLT3

THP-1
DMFI=3088

MV4;11
DMFI=855

Pt#1
DMFI=810

Pt#2
DMFI=644

Pt#3
DMFI=703

b c
MOLM-13 THP-1 MV4;11 Pt#1 Pt#2 Pt#3

d
100
80
60
40
20
0
10:1 5:12.5:1
E:T ratio

2.0 CD4+

1.5

1.0

0.5

0.0
100
80
60
40
20
0
10:1 5:12.5:1

0.6

0.4

0.2

0.0
100
80
60
40
20
0
10:1 5:1 2,5:1

CD8+

UTD
FLT3 CAR CD19 CAR
100
80
60
40
20
0
20:1 10:1 1:1

e
100
80
60
40
20
0
20:1 10:1 1:1

CD4+

CD19
CAR
FLT3
CAR
UTD
100
80
60
40
20
0
20:1 10:1 1:1

CD8+

UTD
FLT3 CAR CD19 CAR CD123 CAR

Figure 2:

d14

d18

Untreated

UTD

FLT3 CAR

1.5

1.0

0.5

6
b

x107

x108

ns *

0.8

0.6

0.4

0.2

0.0

d10

d14

d31

d
Untreated UTDFLT3 CAR

BM
UntreatedUTDFLT3 CARUntreatedUTDFLT3 CAR

Sp PB

1.0´ 10 10

5.0´ 10 09

-2.0´ 10 8

Untreated UTD
FLT3 CAR

10
5
0
ns

0
ns
*

Figure 3:

MOLM-13 MV4;11 THP-1
b

DMFI= 2822

Iso
DMFI=677
DMFI=61

Re-exposure Withdrawal Exposure Isotype

60
50
0

**
FLT3

IFN-g

ns
***

0.3

0.2

0.1

0.0

IL-2
ns

**
FLT3

FLT3 CAR

MOLM-13Creno MOLM-13Native No target

CD123 CAR

CAR CAR UTD CAR CAR UTD CAR CAR CFSE

Cell divisions 4 3 2 1 4 3 2 1
MOLM-13Native 10.5 19.3 24.9 27.8 6.5 12.8 15.6 27.9
MOLM-13Creno 23.9 31.4 24.4 12.4 8.6 15.2 19.0 31.9

Figure 4:

a b
Untreated Crenolanib FLT3 CAR FLT3 CAR
only only +crenolanib **

d14

1
0 x107 5
5

3 x108

1
1
1.5

1.0

0.5

0.0

d10 d15
FLT3 CAR
only FLT3 CAR +
crenolanib

d18 0.8

0.6
x109 8 *

d24
0.4

0.6

0.2
6
4
Untreated Crenolanib
only

2
0
c e
1´ 10 8 BM Sp PB

5´ 10 7 2´ 10 6
1´ 10 6
0

-2´ 10 6
-4´ 10 6
-6´ 10 6
80

5
4
3
2
1
0
ns

**
5 ns 4
3
2
1
0

Figure 5:
a b

HSC
Avg. DMFI
= 211
HSCP
Avg. DMFI
= 260

Donor

120

4 h

5
100 *

4
3

2
1 Isotype
60
40
20
*

c
FLT3 0

1:1 2:1 5:1

GEMM GM BFU-E CFU-M CFU-G

Untreated UTD
FLT3 CAR CD123 CAR

Crenolanib
120
100
80
60
40
20
0

24 h

1:1 2:1 5:1
E:T ratio

UTD
FLT3 CAR CD123 CAR