Presenter Disclosure: Renier J. Brentjens, MD, PhD. Nothing to disclose

Transcription

1 Presenter Disclosure: Renier J. Brentjens, MD, PhD Nothing to disclose

2 Adoptive Therapy of Cancer with T cells Genetically Targeted to Tumor Associated Antigens through the Introduction of Chimeric Antigen Receptors (CARs): Trafficking, Persistence, and Perseverance Renier Brentjens Leukemia Service Memorial Sloan Kettering Cancer Center New York, NY

3 Generation of a tumor targeted chimeric antigen receptor (CAR) α-taa mab TCR complex α-taa scfv CD8-ζ 5 LTR SD ψ SA α-tumor scfv V H V L CD8 ζ chain 3 LTR CAR retroviral vector

4 Generation of TAA-targeted T cells for treatment of Cancer TAA CAR αtaa scfv Native TCR 1. Construct a chimeric antigen receptor (CAR) CD8 CD3 ζ TAA CAR 3. Transduce and expand patient T cells ex vivo 4. Infuse transduced T cells to eradicate TAA + tumor cells 2. Subclone CAR gene into a retroviral vector (SFG) 5 LTR SD ψ SA αcd19 scfv V H V L SFG-CAR CD8 ζ chain 3 LTR TAA

5 Advantages of chimeric antigen receptors (CARs) HLA-independent antigen recognition CARs active in both CD4 + and CD8 + T cells Target antigens include proteins, carbohydrates and glycolipids Significant quantities of tumor specific T cells are rapidly generated Minimal risk of generating undesired autoimmunity or GvHD

6 What is the ideal TAA? Qualities of the ideal tumor antigen: Expression restricted to the tumor cell population alone Restricted expression to tumor and otherwise non-vital tissues Expressed by all tumor cells Expressed on the tumor cell surface The target antigen is required by the tumor cell for survival

7 CAR-targeted TAAs in clinical trials Jena et al Blood 2010

8 Expression of CD19 and other B cell markers on B lineage cells preb-all B cell lymphomas and leukemias myelomas Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Stem Cell pro B pre B immature B mature B plasma cell Y Y CD19 CD22 CD20

9 Development of CAR modified T cell cancer therapy CAR design CAR modified T cell trafficking CAR modified T cell persistence CAR modified T cell perseverance Lymphodepletion Early but promising clinical data

10 Expansion of CD19-targeted T cells with in vivo anti-tumor activity B A 1.E CAR Pre-expansion (Day 0) Post-expansion (Day 42) 5.4% 96% Cell Count 1.E E E E E Time (days) 3T3(19) IL15 3T3(19/7.1) IL15 3T3(19/7.1) IL2 IL15 alone C % Survival Raji tumor 19z1 Pz1 untreated P< Time (days) Brentjens et al Nat Med 2003

11 19z1 + T cells require in vivo costimulation NALM6 NALM6/CD % Survival z1 Pz1 P<0.001 % Survival z1 Pz1 P= Time (days) Time (days) Brentjens et al Nat Med 2003

12 T cell co-stimulation T cell activation and proliferation requires both signaling through the TCR (signal 1) and signaling through a co-stimulatory receptor (signal 2) (CD28, 4-1BB, OX-40) In the absence of co-stimulation (signal 2), the T cell will either become unresponsive (anergic) or undergo activation-induced cell death (AICD/apoptosis)

13 Evolution in CAR design

14 2nd generation CARs: in vitro Finney et al JI 1998 Imai et al Leukemia 2004 Maher et al Nat Biotech 2002

15 2nd generation CARs: in vivo 100% 80% Survival 60% 40% 20% 19z1 (n=10) 19-28z (n=16) Pz1 (n=8) P< % Days since tumor injection Brentjens et al Clin Can Res 2007

16 2nd generation CARs: in vivo Kowolik et al Can Res 2006

17 2nd generation CARs: in clinic Savoldo et al JCI 2011

18 3rd generation CARs Wang et al Hum Gen Ther 2007 Zhao et al JI 2009 Wilkie et al JI 2008

19 Development of CAR modified T cell cancer therapy CAR design CAR modified T cell trafficking CAR modified T cell persistence CAR modified T cell perseverance Lymphodepletion Early but promising clinical data

20 CAR modified T cell trafficking Santos et al Nat Med 2009

21 CAR modified T cell trafficking James et al Blood 2009

22 CD19-targeted T cell trafficking: Clinical Trials A T cell trafficking 48 hrs B T cell persistence 2 weeks T cell control Hilar LN CLL 7 19E3 ALL 1 19E3 Liver Bone Marrow Bone Marrow Brentjens et al submitted

23 CD19-targeted T cell trafficking: Clinical Trials Savoldo et al JCI 2011

24 Development of CAR modified T cell cancer therapy CAR design CAR modified T cell trafficking CAR modified T cell persistence CAR modified T cell perseverance Lymphodepletion Early but promising clinical data

25 Adoptive T cell therapy of cancer: Persistence is important Robbins et al JI 2004

26 Enhancing adoptively transferred T cell persistence: T cell phenotype Berger et al JCI 2008

27 Enhancing adoptively transferred T cell persistence: Virus specific CTLs Pule et al Nat Med 2008

28 Development of CAR modified T cell cancer therapy CAR design CAR modified T cell trafficking CAR modified T cell persistence CAR modified T cell perseverance Lymphodepletion Early but promising clinical data

29 The hostile tumor microenvironment The tumor microenvironment contains multiple inhibitory factors designed to potentially suppress effector T cells. CD4 + CD25 hi FoxP3 + regulatory T cells (Tregs) MDSCs TAMs Expression of inhibitory ligands by tumor (PD-L1) Tumor secretion of T cell suppressive cytokines (TGF-β and IL-10)

30 Transduction of Tregs A FL4-H: CD25 APC Tregs % of Max FL1-H: CD4 FITC FL2-H: foxp3 PE Untransduced Transduced B Foxp3 FL4-H: Foxp3 APC FL4-H: Foxp3 APC FL2-H: 12d11 PE FL2-H: 12d11 PE 19z1 CAR Lee et al Can Res 2011

31 19z1 + Tregs abrogate anti-tumor efficacy of 19z1 + effector T cells A % Survival p < * 1928z Teff alone (n=10) 1928z Teff + 19z1 Treg (n=10) 19z1 Treg alone (n=10) Pz1 Teff alone (n=10) B % Survival p < p = :16 Tregs (n=10) 1:8 Tregs (n=10) 1928z Teff alone 1:1 Tregs (n=10) 1:4 Tregs (n=10) Pz1 Teff alone (n=4) Time (d) Time (d) Lee et al Can Res 2011

32 Development of CAR modified T cell cancer therapy CAR design CAR modified T cell trafficking CAR modified T cell persistence CAR modified T cell perseverance Lymphodepletion Early but promising clinical data

33 Lymphodepletion: Mechanism(s) Promotion of homeostatic proliferation Depletion of cytokine sinks by reduction of endogenous lymphocytes Depletion of endogenous inhibitory CD4 + T regulatory cells (Tregs) Depletion of endogenous inhibitory myeloid derived suppressor cells (MDSCs) Decreased competition for access to antigen presenting cells such as dendritic cells (DCs) Translocation from the gut of bacterial products including LPS and other Toll-like receptor agonists (TLRs) induces activation and maturation of DCs and heighten levels of systemic inflammatory cytokines (IL- 12p70) Gattinoni et al Nat Rev Imm (2006), Paulos et al JCI (2007)

34 Lymphodepletion in the clinical setting Treatment of patients with metastatic melanoma using ex vivo expanded TILs Improved response rates (RR) with enhanced lymphodepleting regimens Non-myeloablative: 49% RR Ablative (200cGy TBI): 52% RR Ablative (1,200cGy TBI): 72% RR Dudley et al JCO (2008)

35 Syngeneic EL4(hCD19) tumor model EL4(hCD19) IV injection 53% Assess T cell eradication of tumor mcd19 -/- hcd19 +/- Assess T cell homing to tumor IV injection Assess longterm survival of T cells Assess T cell proliferation in vivo Harvest splenocytes Assess memory T cell response to rechallenge with tumor Assess the efficacy of suicide vectors Determine the side effects of therapy Retroviral transduction with chimeric receptor mcd19 -/- hcd19 +/-

36 hcd19 hcd19 Persistent B cell aplasias induced by lymphodepletion and 19z1 + T cells i.p. cyclophosphamide %hcd19+ cells i.v. T cells cyclophosphamide alone cyclophosphamide+19z1 T cells cyclophosphamide+pz1 T cells 19z1 T cells alone 1Élive cells FL2-H: hcd19 PE % FL1-H: FL1-Height 6Élive cells FL2-H: hcd % Time (Weeks) FL1-H: mcd19

37 Lymphodepletion enhances anti-tumor efficacy of CD19 targeted T cells 100 Percent Survival Cytoxan + 19z1 No Cytoxan + 19z1 Cytoxan Pz Time (d) Days since tumor cell injection

38 Lymphodepletion enhances anti-tumor efficacy of CD19 targeted T cells Cheadle et al J Immunother 2009 Kochenderfer et al Blood 2010

39 Development of CAR modified T cell cancer therapy CAR design CAR modified T cell trafficking CAR modified T cell persistence CAR modified T cell perseverance Lymphodepletion Early but promising clinical data

40 Clinical responses following infusion with CD19 targeted T cells Kochenderfer et al Blood 2010

41 Clinical responses following infusion with CD19 targeted T cells Pretreatment 1 month following treatment 3 months following treatment Brentjens et al submitted

42 Clinical responses following infusion with CD19 targeted T cells ALL#1 CD3+ and CD19+ cell count 9.0E E E+05 + CD3 + CD19 6.0E E E E+05 cell count/m 2.0E E E+00 Pre-CX Pre-Infusion Post inf +1 hr (Day 0) Post inf +24hrs (Day 1) Post inf 2 +1 hr (Day 1) Day 2 Day 4 Week 1 Week 2 Week 3 Week 4 Week 5 Brentjens et al submitted

43 Conclusions Adoptive CAR modified T cell therapy of cancer holds great promise based on both pre-clinical and clinical studies, however potentially significant obstacles remain: Unforeseen toxicities (Lamers et al JCO 2006, Morgan et al Mol Ther 2010, Brentjens et al Mol Ther 2010) Limited in vivo persistence of modified T cells Limited in vivo perseverance of modified T cells in the setting of a hostile tumor microenvironment Immunogenicity of CAR modified T cells (Lamers et al Blood 2011) The need for multicenter clinical trials CAR design Gene transfer technology Ex vivo T cell expansion Target patient population Funding