The International Congress of BMT 2017 In conjunction with 22 nd Annual Congress of KSBMT COI disclosure Name of author : Kyoo-Hyung Lee I have no personal or financial interests to declare. I have no financial support from an industry source at the current presentation.
NK Cell Therapy NK cell biology NK cell therapy University of Ulsan Asan Medical Center Kyoo-Hyung Lee
Human NK Cells Flow cytometry of PB lymphocytes from healthy subject NK-DC interaction and NK cell target recognition There are 10x10 9 lymphocytes in the blood Estimated 2x10 9 NK cells in the blood (3.3x10 7 /kg) (within 1 month after allogeneic HCT.) NK cells reconstitute most rapidly after allogeneic HCT when compared to CD8+ or CD4+ lymphocytes
Killer immunoglobulin receptors Inhibitory KIR2DL1 (CD158a) group 2 HLA-C KIR2DL2 (CD158b) group 1 HLA-C KIR2DL3 (CD158b) group 1 HLA-C KIR2DL5 unknown KIR3DL1 HLA-Bw4 KIR3DL2 HLA-A3, -A11 KIR3DL7 unknown C-type lectin receptors Inhibitory CD94/NKG2A/B HLA-E Activating CD94/NKG2C HLA-E CD94/NKG2E/H unknown Non-MHC class I specific Activating NKG2D MIC A, MIC B, ULBP-1, -2, -3 NKp46 unknown NKp44 unknown NKp30 unknown Natural cytotoxicity receptors Activating KIR2DS1 group 2 HLA-C KIR2DS2 group 1 HLA-C KIR2DL4 HLA-G KIR2DS4 unknown KIR2DS5 unknown KIR3DS1 unknown NK cell receptors (Farag S S et al. Blood 2002)
Donor-patient NK cell alloreactivity determined by HLA- C and B4 typing (Ligand mismatch) Pt A A B B C C Dr Dr HLA- Bw4 HLA- Bw4 HLA- C gr HLA- C gr HLA- C gr1 HLA- C gr2 HLA- Bw4 NK allo http://www.ebi.ac.uk/ipd/kir/ligand.html 1 206 207 2705 4601 102 102 101 803 pos neg 1 1 pos neg pos pos dnr 206 101 2705 5701 102 602 101 701 pos pos 1 2 2 201 3303 5401 5801 102 302 701 1201 neg pos 1 1 pos neg pos neg dnr 2602 3303 1501 5801 303 302 1406 1201 neg pos 1 1 Ligand HLA-C group 1 (Ser77, Asn80) HLA-C group 2 (Asn77, Lys80) HLA- Bw4 + + + Not found + + HLA-Bw4 HLA type of NK alloreactive donor + + HLA-C group 2 + + HLA-C group 1 + HLA-C group 2 and/or HLA-Bw4 + HLA-C group 1 and/or HLA-Bw4 Receptor KIR2DL2/3 KIR2DL1 KIR3DL1 HLA-C Gr1 HLA-C Gr2 KIR2DL2/3 KIR2DL1 Inhibited without ligand Allo-HCT HLA-C Gr1 HLA-C Gr2 KIR2DL2/3 KIR2DL1 Not inhibited HLA-B4 KIR3DL1 HLA-B4 KIR3DL1 Leukemia Patient NK cell Leukemia Donor NK cell
CD45+ leukemic cells Donor NK alloreactivity against host No Yes No of transplant 58 34 Donor displaying anti-recipient NK clones 1/58 34/34* Disease ALL AML 21 37 14 20 Transplant outcomes Rejection Acute GVHD grade II 16% 14% 0%* 0%* Bcr-Abl PCR results Relapse at 5 years ALL AML 90% 75% 85% 0%* Results of T cell depleted haploidentical-family HCT Survival of mice A single infusion of alloreactive human NK cells eradicated CML blast crises in NOD/SCID mouse. (Ruggeri et al, Science 2002)
MS-HCT U-HCT MSD-HCT (Hsu KC et al, Blood 2005) MUD-HCT (Hsu KC et al, BBMT 2006)
Case Summarized data of KIR genotyping from normal transplantation donors in Asan-KRIBB study (n=51) Donor Family relation Age KIR Genotyping Result (allele specifity) 2DL 2DS 3DL 1 2 3 4 5AB 5A 5B 1 2 3 4*001/ 002 4*003-007 5 1 2 3 3DS1 3DP 1*001/ 2DP1 002/00 1*003 4 1 심승 mother 64 O O O O O O O O O O O O O O 2 김춘 sister 49 O O O O O O O O O O 3 김수 daugth er 27 O O O O O O O O O O 4 김진 sister 25 O O O O O O O O O O O O O 5 윤지 mother 47 O O O O O O O O O 6 강모 father 48 O O O O O O O O O 7 이순 sister 45 O O O O O O O O O 8 윤진 brother 55 O O O O O O O O O O O O O 9 김미 mother 60 O O O O O O O O O O O 10 송희 son 31 O O O O O O O O O 11 진준 father 56 O O O O O O O O O O 12 조민 mother 46 O O O O O O O O O O O O O 13 이현 mother 53 O O O O O O O O O O 14 정교 brother 32 O O O O O O O O O 49 현민 sister 31 O O O O O O O O O O O O O 50 신수 sister 28 O O O O O O O O O 51 정정 mother 64 O O O O O O O O O KIR gene frequency(%) 100 12 96 100 43 27 6 37 12 18 86 39 24 98 100 98 31 100 6 96 Inhibitory Gene Frequency KIR2DL1 group 2 HLA-C (100%) KIR2DL2 group 1 HLA-C (12%) KIR2DL3 group 1 HLA-C (96%) KIR2DL5 unknown (6%-43%) KIR3DL1 HLA-Bw4 (98%) KIR3DL2 HLA-A3, -A11 (100%) KIR3DL7 unknown Activating Gene Frequency KIR2DS1 group 2 HLA-C (37%) KIR2DS2 group 1 HLA-C (12%) KIR2DS3 (18%) KIR2DL4 HLA-G (100%) KIR2DS4 unknown (39%-86%) KIR2DS5 unknown (24%) KIR3DS1 unknown (31%) KIR haplotype assignment At least one of KIR2DL5, 2DS1, 2DS2, 2DS3, 2DS5, or 3DS1 present > haplotype B/x Otherwise, haplotype A/A (Cooley S et al, Blood 2009) Activating KIR in KIR genotyping
KIR gene haplotype in MUD-HCT (Cooley S et al, Blood 2009)
1277 AML patients undergoing UD-HCT MUD-HCT (Venstrom JM et al. NEJM 2012)
SNP in inhibitory KIR, KIR2DL1 313 pediatric AML or ALL patients undergoing MS, UD, or haploidentical -HCT RR: homozygous for KIR2DL1-R 245 CC: homozygous for KIR2DL1-C 245 RC: heterozygous for KIR2DL1-R 245 /C 245 Donor inhibitory KIR2DL1 allele polymorphism in MSD, MUD, HFD HCT (Bari R et al. JCO 2013)
NKp30 isoforms in GIST (Delahaye NF, et al. Nature Med 2011)
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Lirilumab (BMS-986015) Fully human monoclonal antibody designed to block the interaction between KIR2DL-1,-2,-3 inhibitory receptors and their ligands.
LAK cell TIL cell CAR-T cell CIK cell NK cell Generation IL-2 IL-2 IL-2, IL-7, or IL-15 / anti- CD3 beads / OKT3 IFN-gamma, IL-2, OKT3, anti-cd3 monoclonal ab IL-15, IL-21 Phenotype CD3 + >95% CD8 + ~85% CD56 + ~30% CD3 + CD56 + ~30% CD56 + ~90% CD3 + CD56 + ~1% CD3 + CD56 - ~0.3% Auto vs allo Auto Auto Auto Auto Allo GVHD when used in allogeneic setting Prob yes Prob yes Prob yes Prob yes No Cell number ~10x10 10 3 10 8 T cells (1.5 10 7 transduced cells) ~1-10x10 10 ~1-5x10 10
Role of CART cell therapy in AML no replacement for allogeneic HCT!!! (Kenderian SS, et al, BBMT 2017)
NK cell therapeutics Donor source (allogeneic vs. autologous) Primary vs. chemotherapy-priming vs. post HCT NK cell acquisition by separation vs. ex vivo expansion Optimum dose? Is there MTD?
Patient A. Autologous NK cell therapy B. Primary adoptive NK cell therapy Patient tumor NK cells Donor NK cells C. Adoptive NK cell therapy based on HLA-mismatched HCT Patient tumor Donor Donor chimerism HCT Stem cells NK cells Alloreactivity against tumor without rejection from the host!!!
MU-HCT MS-HCT Haplo-HCT CB-HCT US IBMTR data: D Souza A et al, BBMT 2017 in press
Clinical studies of systemic NK cell therapeutics (N=57) as of June 2017 (Clinicaltrial.com) 20 15 10 5 0 2001-2005 2006-2007 2008-2009 2010-2011 2012-2013 2014-2015 2016-2017 Study phase I 21 I-II 17 II 15 II with random control 4 Median patient number: 20 (range, 1-140)
Clinical studies of systemic NK cell therapeutics (N=57) as of June 2017 (Clinicaltrial.com) Dx AML 24 ALL 10 MDS 7 MM 6 NHL 2 Neuroblastoma 6 HCC 5 NK cell source Haplo donor 36 Haplo, KIR-mismatched 1 MS or MU donor 5 Autologous 9 CB derived 4 Deceased (Cadaveric) 1 Unclear 1 Sarcoma or melanoma 3 Breast ca 2 NSCLCa 2 GI malignancies 2
Clinical studies of systemic NK cell therapeutics (N=57) as of June 2017 (Clinicaltrial.com) Ex vivo treatment Separation only 20 Expansion 25 Expansion and gene modification unclear 9 3 HCT None 40 Haplo-HCT 6 Autologous HCT 5 Allo-HCT 3 CB-HCT 1 Unclear 2 Pre-NK conditioning Associated with HCT CTX / Fludara / IL-2 / cetuximab or rituximab or G-CSF or TBI or clofarabine 16 22 None 6 Lenalidomide based 3 Cetuximab 1 Bortezomib 1 Decitabine 1 Trastuzumab + IL-2 1 Hu3F8 + IL-2 1 Other chemo 5
동종자연살해세포서울아산병원 한국생명공학연구원공동연구요약 임상환자연구제목연구기간식약처허가번호발표단계수 1 차 반일치가족골수이식후발생할수있는암 / 백혈병의재발이나착상부전을예방하기위한공여자자연살해세포의투여 - 제 1상임상연구 2007.5-2 008.10 1 상 식품의약품안전청생물의약 품안전팀. 임상시험승인번 호 406 호. 2007.5.18 14 명 Bone Marrow Transplant 45:1038-46, 2010 2 차 혈액암또는고형종양환자에서 HLA-반일치가족공여자조혈세포이식후진행 / 재발한기저악성질환의치료를위한공여자자연살해세포투여 - 제 1-2상임상연구 2009.2-2 012.5 1-2 상 식품의약품안전청생물의약품정책과승인번호 80.2009.1.23 41명 (46명) Biol Blood Marrow Transplant 20:696-7 04, 2014 3 차 불응성급성백혈병에서반일치조혈세포이식후공여자유래자연살해세포투여 - 제1-2a상임상연구 2013.2-2 015.5 1-2a 상 식품의약품안전처접수번호 20120143817. 2013.1.18 51 명 Biol Blood Marrow Transplant 22: 2065-2076, 2016 고위험급성골수성백혈병및골수이형성 4 차 증후군에서반일치조혈세포이식단독또는이식후동종유래공여자자연살해세포투여의유효성과안전성평가를위한 2015.6- 현재 2b 상 식품의약품안전처접수번호 20140223597. 2015.5.13 96명 ( 예정 ) 진행중 (44 명등록 ) 무작위비교임상연구
반일치가족골수이식후발생할수있는암 / 백혈병재발이나착상부전을예방하기위한공여자자연살해세포의투여 제 1 상연구 <2007 년식약청임상시험계획승인번호 406 호 >
혈액암또는고형종양환자에서 HLA- 반일치가족공여자조혈세포이식후진행 / 재발한기저악성질환의치료를위한공여자자연살해세포투여 제 1-2 상연구 <2009.1.23 식약청임상시험계획승인번호생물의약품정책과 - 80> Days -8-7 -6-5 -4-3 -2-1 0 1 2-14 - 21 Patient conditioning HCT DNKI MNC MNC MNC Donor leukapheresis G-CSF BBMT Journal 2013 IF: 3.940
Cumulative incidence Event-free survival Overall survival Cumulative incidence Median F/U: 31.5 months (range, 16.6-53.0) 1.0 0.8 0.6 0.4 0.2 B ANC 500/µl Study (n=41) Control (n=31) Platelets 20,000/µl Study (n=41) Control (n=31) 1.0 0.8 0.6 0.4 0.2 C Acute GVHD, gr. 2-4 Study (n=41) Control (n=31) 1.0 0.8 0.6 0.4 0.2 D Chronic GVHD, mod-severe Study (n=41) Control (n=31) TRM Control (n=31) Study (n=41) 0.0 0 20 40 60 80 100 0.0 0 1 2 3 4 0.0 0 3 6 9 12 15 Days after HCT Months after HCT Months after HCT 1.0 0.8 E F G 0.6 0.4 0.2 0.0 0 12 24 36 48 60 Months after HCT Months after HCT Months after HCT Study refractory AML (n=29) Study refractory ALL/lymphoma (n=8) Control refractory AML (n=22) Control refractory ALL/lymphoma (n=9) Choi I and Lee K-H, Biol Blood Marrow Transplant (May 2014)
Activating NK Cell Ligands in Leukemia Leukemia Activating NK cell ligands Expression B-ALL MICA/B Negative ULBPs Mostly negative PVR and Nectin-2 Negative or low CD48 and NTBA Negative or low MDS MICA/B Positive in 30% of patients AML MICA/B Negative MICA/B Mostly positive PVR and Nectin-2 Positive ULBPs Negative or very low NCR ligands Negative or very low CML MICA/B Mostly positive B-CLL MICA Negative ULBP1, 2 and 4 Negative ULBP3 Low (Verheyden S et al, Leukemia 2007)
불응성급성백혈병에서반일치조혈세포이식후공여자유래자연살해세포투여 - 제 1-2a 상임상연구 < 식품의약품안전처접수번호 20120143817. 2013.1.18>
Other NK cell related variables, such as donor-patient ligand allo-reactivity, donor KIR typing (KIR2DS1 positivity, KIR3DS1 positivity, B/x vs A/A haplotype), and expressions of CD94, NKG2D, KIR2DL1, KIR2DL2/3, KIR3DL1, NKp46, NKp44 failed to predict CR or leukemia progression.
progression P=0.030 progression P=0.021 progression P=0.044 P=0.001 P=0.001
Cytokine release syndrome observed after early DNKI
B 10 599 Body weight (Kg) DNKI s D6 D9 D13 0.5 0.5 0.7 (x10 8 /kg) 588 577 566 555 544-20 -10 0 10 20 30 40 onset of fever 10 8 6 4 2 0 Total bilirubin (mg/dl) - - Creatinine (mg/dl) - - -20-10 0 10 20 30 40 1200 1000 800 600 400 200 0 ANC (/ul) Truncated at 1000-20 -10 0 10 20 30 40 Days after HCT UPN 1292: 28 yo Female with AML, recurrent then refractory Suppl Fig 1
<2015 년식약처임상시험계획승인번호 20140223597 호 ; 2015.5.13> 고위험급성골수성백혈병및골수이형성증후군에서반일치조혈세포이식단독또는이식후공 여자유래자연살해세포투여의유효성과안전성평가를위한무작위비교임상연구 무작위배정층화 불응성 AML 1차불응대재발후불응말초아세포 <5% 대 5% High-risk AML CR1 대상환자수 각군 48 명총 96 명 현재 40 명등록치료중 Intermediate -2/high-risk MDS
Summary 1. The role of NK cell biology in terms of donor selection in allogeneic HCT remains to be defined. 2. DNKI with or without HCT is safe. 3. MTD of DNKI remains to be defined. 4. DNKI more effective in AML than in Precursor B cell ALL. 5. Enhanced DNKI toxicity related to cytokine release was observed when given early after haploidentical HCT. 6. Enhanced anti-aml effect was observed with higher NKp30 expression of donor NK cells
Asan Medical Center Adult Allogeneic HCT Team Korea Research Institute of Bioscience and Biotechnology Team Kyoo-Hyung Lee, Je-Hwan Lee, Jung-Hee Lee, Han-Seung Park, Eun-Ji Choi Young-Shin Lee, Young-Ah Kang, Mijin Jeon, Miee Seol, Seunghyung Baek Inpyo Choi Suk-Ran Yoon Soo-Yeon Park Hanna Kim Sol-Ji Jung Korean Health Technology R&D Project, Ministry of Health and Welfare (A121934) R&D Convergence Program of National Research Council of Science & Technology (CRC-15-02-KRIBB) GRL project, Ministry of Education, Science and Technology (FGM1401223) KRIBB Research Initiative Program (KGM1211231)