Natural Kil er cel s

Transcription

1 NK Cells Evre Alici, MD, PhD Karoliska Istitutet, Departmet of Medicie, Divisio of Hematology Visitig Research Professor, Nova Southeaster Uiversity, FL, USA Erik G Svesso 25/11/2016 Name Surame 1

2 Natural Killer cells

3 Natural Killer Cells 3

4 What really goes o 4

5 NK cell abormalities Abormality Decreased cytotoxic activity of NK cells Defective expressio of activatig receptors Defective NK cell proliferatio NSC lug Ca HCC CRC H&N Ca Breast Ca Squamous cell Ca Brochogeic Ca HCC M. melaoma Real Ca Neuroblastoma Disease Cervical Ca Ovaria Ca AML ALL B-CLL CML MM AML MM Nasopharygeal Ca CML Icreased umber of CD56 bright NK cells H&N Ca Breast Ca Defective expressio of sigallig molecules Decreased NK cell couts Defective cytokie productio Cervical Ca CRC Ovaria Ca Nasopharygeal Ca Neuroblastoma AML ALL Prostate Ca AML CML CML ALL (Pediatric) CML

6 NK cell therapy overview Sutlu & Alici, JIM 2009

7 The expasio process Day 0 CellGro SCGM 5% Huma AB serum IL-2 (500 U/ml) OKT3 (10 g/ml) CD56 Detailed pheotypic characterizatio of NK cells Degraulatio ad cytotoxicity assays Day 0 Day 5 Day 10 Day 15 Day CD3 Day 20 Every 2-3 days Additioal medium with IL-2, without OKT3

8 cgmp certified expasio process

9 A phase-i cliical trial with expaded NK cells

10 Patiet follow-up

11 Multiple Myeloma The lifetime risk of gettig MM is 1 i 159 (0.63%). I 2015: 20,000 ew cases 11,000 me ad 9,000 wome 10,650 deaths 5-year survival rate: 35% 10-year survival rate: <2%

12 Natural Killer Cells i MM Various immue dysfuctios are observed i MM patiets Tumor-iduced immue dysfuctios regardig NK cells i MM: Icreased level of soluble IL-2 receptors High levels of M-compoet Defective expressio of activatig receptors Impaired NK cell cytotoxicity ad abormal NK cell couts Adoptive trasfer of IL-2 activated NK cells prologs survival i aimal models of MM 12

13 Expaded cells have cytotoxicity agaist autologous MM cells

14 Pheotypic chages Adhesio / Activatio Ihibitio Chemokie receptors N=7

15 Differet strategies for scalig up

16 NK cells from bioreactor expasios degraulate more efficietly

17 Pheotypic differeces Adhesio / Activatio Ihibitio Chemokie / cytokie receptors r 2 : p: r 2 : p: r 2 : p: MFI N=4 % Degraulatio

18 ACP-001 First-i-ma, Phase I/II Ope, sigle arm study Primary objective: Safety ad tolerability Secodary objective: Effect o serum Ig levels Iclusio: 20 MM patiets eligible for ASCT 3 escalatig ifusios/patiet (Weekly) 10 6, 5X10 7 ad 10 8 cells/kg Evaluatio: 4 weeks after ifusio, 6 moths follow up. 25/11/2016 Evre Alici 18

19 Daratumumab treatmet

20 Improvig letiviral ad retroviral gee delivery to NK cells

21 How could we help the viral gee delivery process?

22 Time dyamics of letiviral trasductio o NK cells N=4

23 Trasductio process does ot effect NK cell fuctio With BX795 Without BX795 SSC SSC SSC SSC FSC egfp FSC egfp CD CD CD CD CD107a CD107a CD107a CD107a

24 NK cell geetic modificatio Itracellular ati-viral defese mechaisms ca limit viral gee delivery i NK cells ad this ca be reduced by usig small molecule ihibitors Geomics & Bioiformatics Differetial gee expressio aalysis Virus +/- BX795 No Virus +/- BX795 Mock Virus +/- BX795 Sutlu 2012, Hum. Gee Ther.

25 A more targeted molecule: VyOxo

26 Ehacig letiviral gee delivery to NK cells: Vy-Oxo

27 NK-Tumor iteractios Geetic modificatio of NK cells from patiets with hematological maligacies Immuotherapy with geetically modified NK cells Icrease detectio of patiet-specific target ligads 27

28 LeGo-Vectors Gee of iterest Self- iactivatig-vector of the 3 rd geeratio replicatio icompetet ca ifect a broad group of cell types Backboe, packagig, evelope ad regulatio plasmid Pseudotyped with VSV-G HEK293FT as producer cell lie T R A N S L A T I N G R E S E A R C H I N T O H E A L T H

29 Geetically modified NK cells CD94 CD16a CD160 CD161 CD2 DNAM1 2B4 2B4T CD27 CRACC Siglec7 Siglec9 NKp46 NKp44 NKp30 NKG2A NKG2C NKG2D NKG2E NTBA NKp80 KIR2DL1 KIR2DL2 KIR2DL3 KIR2DL4 KIR2DL5 KIR3DL1 KIR3DL2 KIR3DL3

30 Protocol overview Activatig receptors Ihibitory receptors CD16 NKp44 DNAM1 KIR2DL1 KIR2DL4 KIR3DL2 CD160 CD27 NKp30 CD161 CRACC NKG2A CD2 NKG2C NKp46 2B4 NKp80 NKG2D &/ KIR2DL2 KIR2DL5 KIR3DL3 KIR2DL3 KIR3DL1 Siglec7 KIR2DL1 KIR2DL4 Siglec9 NKG2A Pheotypig Trasductio with differet vectors Gee of iterest IRES GFP Tumor cells magetic separatio 15PBMCs Pheotypig NK cell GFP Degraulatio Receptor 1 assays Receptor 2 GFP Viral vector degraulatio Trasduced NK cell CD107a UmodifiedNK CD107a cell No target Autologous PBMCs Autologous tumor 25 November

31 Goal: Patiet-specific data 31

32 A ew discovery: VyX 25/11/2016 Evre Alici 32

33 Vy-X effects i vitro Perfori Grazyme B Perfori (MFI) **** **** **** **** **** Grazyme B MFI **** **** **** **** **** 0 Medium VyX Cotrol A Cotrol B Il-2 +VyX Il-2 + Cotrol A IL-2+ Cotrol B 0 Medium NKx Cotrol Cotrol B Il-2 +NKx Il-2 + Cotrol A IL-2+ Cotrol B

34 16h Cyototoxicity assay 25/11/2016 Evre Alici 34

35 Direct VyX i vivo delivery 25/11/2016 Evre Alici 35

36 MM patiet Peripheral blood Boe marrow Aspiratio NK cell isolatio T cell isolatio CD138 + Plasma cell isolatio MSC isolatio Letivirus-based high-throughput expressio of NK cell receptors T cell expasio Degraulatio assays No target PBMCs MM cells Full geome sequecig mrna sequecig High-throughput drug screeig of MM cells co-cultured with cells Idetificatio of geetic predispositios to treatmet resistace to idetify optimum persoalized therapy for MM patiet

37 Ackowledgmets