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Presented at the 56th American Society of Clinical Oncology (ASCO) Virtual Annual Meeting, May 29–31, 2020, Chicago, IL, United States CD56 bright /CD16 bright FC21-NK-cell adoptive immunotherapy in patients with concurrent CNS disease and relapsed or refractory (R/R) AML LUCIA SILLA, 1 ALESSANDRA PAZ, 2 NELSON HAMERSCHLAK, 3 ROSANE BITTENCOURT, 2 DEAN A LEE 4 1 Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; 2 Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil; 3 Hospital Albert Einstein, São Paulo, SP, Brazil; 4 Nationwide Children’s Hospital, Columbus, OH, USA [email protected] • Patients with relapsed or refractory acute myeloid leukemia (R/R AML) and concurrent central nervous system (CNS) disease rarely respond to chemotherapy and have a dismal prognosis 1 • Adoptive immunotherapy with haploidentical ex vivo-activated/expanded natural killer (NK) cells shows promise in refractory AML 2–4 • However, it is unknown whether the blood–brain barrier (BBB) represents an obstacle to this approach in patients with R/R AML and CNS disease • Double-bright (CD56 bright /CD16 bright ; DB) NK cells are generated from peripheral blood using transfected feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and 4-1BB ligand (FC21) 5 • FC21-derived NK cells present a unique DB phenotype and express hyperfunctional characteristics with high levels of cytolytic activity 5 • In a phase I study (NCT02809092), we investigated multiple doses of DB FC21- NK cells following standard fludarabine, cytarabine, and granulocyte-colony stimulating factor (FLAG) induction in patients with R/R AML INTRODUCTION • To investigate preliminary safety and efficacy data in a subgroup of patients with R/R AML and concurrent CNS disease from this phase I study OBJECTIVE METHODS • This open-label, phase I study was performed in patients treated at a single center in Brazil • Eligible patients were ≥2 years old, with R/R AML; a Karnofsky or Lansky performance status score of ≥70; and adequate renal, liver, and pulmonary function • Eligible donors were a human leukocyte antigen-haploidentical relative selected for best NK alloreactivity • Cytogenetic risk was determined according to European LeukemiaNet (ELN) guidelines 6 • FC21 cells were expanded from donors on FC21 feeder cells as previously described 5 • The treatment schema is shown in Figure 1 Figure 1: Treatment schema Day -30 Cryopreservaon -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 (Connues thrice weekly for 6 IV infusions in total) G-CSF 5 µg/kg daily from day -7 unl post-nadir ANC ≥1000 Fludarabine (30 mg/m 2 daily × 5) Cytarabine (2 g/m 2 daily × 5, 4 hours aſter fludarabine) Donor blood obtained FC21-NK infusion Treatment day NK-cell expansion on FC21 × 14 days* FC21-NK infusions were performed 2–14 days after FLAG chemotherapy. *Up to 28 days for some cultures. ANC, absolute neutrophil count; G-CSF, granulocyte-colony stimulating factor. • This post hoc analysis assessed the following outcomes in a subgroup of patients with concurrent CNS disease: – Overall response; assessed at day 28 using the International Working Group (IWG) response criteria 7 – CNS responses; assessed at day 30 using the Response Assessment in Neuro-Oncology (RANO) criteria 8 – Adverse events (AEs); reported from day 0 up to day 56 and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4 • Gene expression profiling was performed by next-generation sequencing on mRNA libraries (TruSeq RNA Sample Preparation Kit. Illumina Inc., San Diego, CA) made from total RNA extracted from NK cells prior to and after mbIL-21 expansion Patient characteristics • Of 13 patients treated with FLAG chemotherapy followed by FC21-NK, 4 patients had concurrent CNS disease and were included in this analysis • Characteristics of patients with CNS disease are summarized in Table 1 – All 4 patients had received a prior stem cell transplant and were heavily pretreated, and 1 patient had primary refractory disease – Two patients were treated under compassionate use Table 1: Patient characteristics Characteristic Patient 1 Patient 4 Patient 5* Patient 6* Sex Female Male Male Female Age, years 48 31 22 1.6 CNS involvement Mycetoma ‡ Bone and nerve root Uncus/brain stem Chloromas Cytogenetic risk ‡ Favorable Favorable Adverse Intermediate Chromosomal aberrations inv(16) (p13;q22) t(8;21) (q22;q22) t(6;9)(p22;q34) DEK/NUP214 Normal Prior treatments, n 6 4 7 5 Prior SCT Autologous Allogeneic Allogeneic Haploidentical Relapse, n or refractory 4 2 3 Refractory * Patient treated under compassionate use; † Diagnosed during treatment. SCT, stem cell transplant; ‡ Patients were wild-type for FLT-3 internal tandem repeat mutations. Treatment exposure and safety • Treatment exposure is shown in Table 2; all patients completed ≥6 IV infusions of FC21-NK Table 2: Treatment exposure Characteristic Patient 1* Patient 4 Patient 5 Patient 6 Cell dose per infusion 1 × 10 6 /kg 5.5 × 10 6 /kg 8.3 × 10 6 /kg 6.6 × 10 6 /kg Number of infusions 11 6 6 6 *Patient 1 completed 2 FC21-NK treatments: the first treatment comprised 6 infusions of FC21-NK only; the second comprised 5 infusions of FC21-NK following FLAG chemotherapy (6th infusion discarded due to contamination of the infusion sample). • All patients experienced grade 4 febrile neutropenia attributed to hematologic toxicity secondary to FLAG • AEs considered to be related to treatment are shown in Table 3; all were manageable • No FC21-NK cell infusion-related toxicities or cytokine storm syndrome were reported • Patient 5 experienced graft-versus-host disease (GVHD): – Grade 1 acute GVHD occurred 2 days after the last FC21-NK infusion and was managed with tacrolimus and high-dose steroids – Re-activation of chronic liver GVHD was suspected at day 38; this resolved after removal of hepatotoxic medications including tacrolimus Table 3: Adverse events considered to be related to FC21-NK cell treatment Adverse event Grade Description Patient 1* Worsening of pulmonary symptoms 3 Pre-existing invasive pulmonary aspergillosis, managed with voriconazole Probable CNS aspergillosis 2 Resolved without medication CNS hypertension 2 Transient, secondary to CNS inflammation Patient 4 Anemia 4 Resolved with high-dose dexamethasone Patient 5 CNS hypertension 4 Transient, secondary to CNS inflammation. Resolved with high dose dexamethasone *AEs experienced by patient 1 occurred during the first treatment. Treatment outcome • All four patients showed a response (Table 4) Table 4: Outcome after treatment with FC21-NK following FLAG chemotherapy Characteristic Patient 1 Patient 4 Patient 5 Patient 6 Response CR CRi CR PR Duration of response*, days 151 31 168 90 Time to patient death † , days 344 176 505 224 *Time from first reporting of response to relapse; † Time from start of the protocol (from the first FC21-NK infusion in the case of patient 1 who did not receive FLAG in her first treatment). Responses were durable, lasting up to 5.5 months (Figure 2) • CNS responses as indicated by cellular infiltration or the resolution of CNS lesions, was observed in all four patients with nervous system lesions (including probable aspergilloma), shown in Figure 3: – Complete resolution of CNS lesions were documented in patients 1 and 5, and patient 1 also experienced no relapse of pulmonary or CNS aspergillosis – Almost complete resolution of bone and nerve root leukemic infiltration was observed in patient 4, and patient 6 experienced a 50% reduction of CNS chloromas Figure 2: Swimmer plot showing the course of patients with R/R AML and CNS involvement 0 90 180 270 360 450 PR PD PD/Other Tx CR Protocol CRi R D 1 D 4 R R 5 6 Days R D R relapse Death D # Paent Swimmer plot of patient courses, indicating duration of protocol therapy, remission, relapse or persistent disease, additional therapy, and death. CR complete response; CRi, complete response with incomplete hematologic recovery; D, death; PD, progressive disease; PR, partial response; R, relapse; Tx, treatment. Figure 3: CNS responses in patients treated with FC21-NK after FLAG chemotherapy Head MRI of paent #1, showing parenchymal mycetoma obtained (leſt), and resoluon at day 28 (right). Spine MRI of paent #4 showing sacral leukemic infiltraon with soſt ssue and probable cauda equina involvement (leſt) and resoluon at day 28 (right). T1, axial, and diffusion-weighted head MRI of paent #6 showing chloromas non-responsive to prior treatment (top row), and 2 months aſter treatment showing resoluon (boom row). Head MRI axial FLAIR of paent #5 showing signal intensity consistent with refractory AML in both unci and brainstem (leſt), five days post last NK cell infusion showing extension of signal to anteromedial temporal lobes and opc chiasm, opc nerves, and ventral striatum (not shown) consistent with inflammatory response (middle), and 6 months post treatment showing normalizaon of the signal in unci with evoluon to cavitary lesions bordered by low signal rim (consistent with hemosiderin; right). A C B D Gene expression profiles in FC21-NK cells • Expression profiles of genes related to adhesion and cell trafficking are shown in Figure 4 • Notably, CNS and inflammation-homing addressins ALCAM, 9 CCR2, CCR5 and CXCR3, 10,11 ICAM-2, 12 ITGB2 13 (CD18/LFA-1), and NCAM1 (CD56) were all significantly increased with FC21-NK Figure 4: Gene expression profiling in FC21-NK 10 8 6 4 2 0 -2 -4 Fold change (log2) Relave expression -6 -8 ALCAM CCR1 CCR2 CCR5 CCR7 CCRL2 CXCR1 CXCR2 CXCR3 CXCR4 CXCR6 ICAM2 ICAM3 ITGA1 ITGA2 ITGA3 ITGA4 ITGA6 ITGA9 ITGAD ITGB2 GB3BP ITGB7 L1CAM NCAM1 ECTIN1 ECTIN3 SMAGP B1BP1 100 1000 10000 LCAM CCR1 CCR2 CCR5 CCR7 CCRL2 CXCR1 CXCR2 CXCR3 CXCR4 CXCR6 CAM2 CAM3 ITGA1 ITGA2 ITGA3 ITGA4 ITGA6 ITGA9 TGAD B1BP1 ITGB2 GB3BP ITGB7 1CAM CAM1 CTIN1 CTIN3 MAGP B A A Relative mRNA expression levels. B Fold change in mRNA expression of mbIL-21-expanded FC21-NK cells compared with freshly isolated peripheral blood NK cells. Shown are genes related to cell adhesion and trafficking with DESeqScore ≥ 1.5 or ≤ -1.5. Figures show mean ± standard deviation.. • Multiple IV infusions of ex vivo-expanded FC21-NK cells were well tolerated and demonstrated unprecedented CNS responses in patients with R/R AML • These data demonstrate the potential of FC21-NK to traverse the BBB and mediate therapeutic anti-leukemic effect • Receptors for CNS homing that are upregulated on FC21-NK cells may influence their migration into the CNS • The role of ex vivo-expanded FC21-NK cells for use in therapeutic applications for CNS malignancies warrants further investigation CONCLUSIONS REFERENCES 1. Rozovski U, et al. Leuk Lymphoma 2015;56:1392–7; 2. Miller JS, et al. Blood 2005;105:3051–7; 3. Romee R, et al. Sci Transl Med 2016;8:357ra123; 4. Vela M, et al. Cancer Lett 2018;422:107–17; 5. Denman CJ, et al. PLoS One 2012;7:e30264; 6. Döhner H, et al. Blood 2017;129:424–47; 7. Cheson BD, et al. J Clin Oncol 2003;21:4642–9; 8. Chukwueke UN and Wen PY. CNS Oncol 2019;8:CNS28; 9. Cayrol R, et al. Nat Immunol 2008;9:137–45; 10. Keawvichit R, et al. Immunology 2018;153:455–65. 11. Sørensen TL, et al. J Clin Invest 1999;103:807–15. 12. Haghayegh Jahromi N, et al. Front Immunol 2020;10:3056. 13. Schwab N, Schneider-Hohendorf T, Wiendl H. Oncotarget 2015;6:17863–4. DISCLOSURES Silla: Nothing to disclose Paz: Nothing to disclose Hamerschlak: Nothing to disclose Bittencourt: Nothing to disclose Lee: CytoSen Therapeutics, leadership role & consultancy; Kiadis Pharma, stock ownership, consultancy, research funding, & patents/ royalties; Caribou Biosciences, consultancy; Courier Therapeutics, consultancy ACKNOWLEDGMENTS This study was supported by a grant to LS from a Consortium formed by the MCTI/CNPQ/MS-SCTIE - DECIT # 401193/2013-6; MS/DECIT/MCTI/ FINEP # 01.08.0630.01; MEC/CAPES/PRODOC # 23038.004681/2014- 17; CGSAU 2012 (APQ) # 404896/2012-0; GESCON # 375/2013; MS/ FNS/MCTI/CNPQ # 455405/2014-0; MEC/MCTI/CAPES/CNPQ/FAPS # 401086/2014-3; FIPE – HCPA # 10-0457, which supported the installation of a GMP facility at our Institution, acquisition of related equipment and material, validation of NK cell manufacturing processes, and this clinical trial. Hospitalization costs for patients were supported by the Brazilian National System of Health Care (SUS – Sistema Unico de Saude) or by private health insurance (some had support through UNIMED). Paul Hoban, a medical writer supported by funding from Kiadis Pharma, provided drafts and editorial assistance to the authors during preparation of this poster. RESULTS Abstract 3025
