Genetic Modification for Immune Evasion. Stephen Gottschalk. T-cell Therapy. in clinical studies BUT

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1 Genetic Modification for Immune Evasion Stephen Gottschalk T-cell Therapy T-cell Therapy has shown promise in clinical studies BUT The majority of administered T-cell products are sensitive to immune evasion mechanisms employed by tumors 1

2 Suboptimal T-cell products: sensitive to the immunosuppressive Tumor environment Genetic Modification of T cells Render T cells resistant to immune evasion mechanisms employed by tumors Improve T-cell function cytokines/cytokine receptors anti-apoptosis genes silencing negative regulators Antigen-specific T cells Chimeric antigen receptors (CAR) TCR 2

3 Tumor cells secrete factors to inhibit immune cells Expression of DN TGF- R Induction si RNA of FAS T-cell knock apoptosis down Dotti et al 25 FAS-L Clinical Study opened in 29 TGF- Bollard et al 22 Inhibition of T-cell effector function Expression of IL-12 IL-1 Immunoregulatory cytokines Wagner et al 24 Tumor cells have MHC processing defects & antigen expression is heterogeneous Expression of DN TGF- R Induction si RNA of FAS T-cell knock apoptosis down Dotti et al 25 FAS-L Clinical Study opened in 29 TGF- Bollard et al 22 Inhibition of T-cell effector function Expression of IL-12 IL-1 Immunoregulatory cytokines Wagner et al 24 3

4 CAR vs TCR T cells CAR T cells TCR T cells T-cell T-cell CAR vs TCR T cells FAS-L TGF- No Costimulatory Molecules IL-1 TCR T cells: incomplete T-cell activation CAR T cells: Addition of costimulatory signaling domains into CAR endodomain T-cell activation in the ABSENCE of costimulatory molecules 4

5 CAR T cells to overcome immune evasion Targeting cell surface antigens Targeting multiple antigens Targeting inhibitory stromal cells Rationale for immunotherapy for osteosarcoma Prognosis remains poor for patients with Metastatic disease (< 2% survival) Central tumors (< 35% survival) Relapsed disease Significant acute toxicities Long-term effects Disability after surgery 5

6 HER2 expression and prognosis Member of the EGFR family of receptors Expressed by up to 8% of primary osteosarcoma Important for malignant phenotype Worse overall survival rates HER2 negative HER2 positive (Gorlick et al. JCO 1999) 2 nd Generation HER2-specific CAR scfv GALV or RD114 pseudotyped retroviral particles CD28 zeta LTR LTR (Pule et al, Mol Thr 25; Ahmed et al, Mol Thr 29) 6

7 Generation of CART cells by retroviral transduction CD3/CD28 IL-2 Retroviral Transduction + Retronectin + IL-2 IL-2 Day Day 2 Day 3 Day freeze T cells Activated T cells donor A donor B 86.2% 83.% 86.2% 83.% donor C 87.8% 87.8% HER2-CAR T cells inhibit 2 ndary Sarcosphere formation Control + HER2-T cells + NT T cells Sarcosphere 143B MNNG/HOS 143B MNNG/HOS Monolayer (Rainusso et al; Cancer Gene Therapy 212) 7

8 Antitumor activity in metastatic Lung Model Wk 2 Control Wk 2 HER2-T cells (Ahmed et al. Mol Ther 29) Safety concerns with HER2-CAR T cells 6 patients have received conventional HER2-specific T cells 1-4x1 1 cells with no side effects 2 patients have received HER2-CAR T cells 1 st : 2x1 7 cells: no immediate toxicity 2 nd : 1x1 1 cells post nonmyeloablative conditioning: developed ARDS and died (Bernhard et al Cancer Immunol Imm 28, Disis et al JCO 29, Morgan et al Mol Thr 21) 8

9 T-cell Therapy for HER2-positive Osteosarcoma To determine the safety and antitumor activity of HER2-CAR T cells 9D Dose levels: l 1x11 4 to 1x1 1 8 cells/m 2 Characteristics of infused patients 9 females, 6 males Median age 17.3 (7 29) 13 OS, 1 EWS, 1 DSCRT Disease status 11 lung mets 1 lung and bone mets 1 lung and extraosseous mets 1 Liver mets All patients had failed multiple lines (>4) of salvage therapy 9

10 Phenotype of GMP grade HER2-CAR T-cells Frequency of CD4 & CD8 T-cell subsets Frequency of CD3+CD45RO+ CD45RO Effector & Central memory T cells Characterization of GMP grade HER2-CAR T-cells from OS patients 1 8 Transduction Efficiency P< Cytotoxicity NT T cells HER2-CAR T cells P<.1 P<.1 tive (%) Posi Lys sis (%) 4 2 n=9 NT T cells HER2-CAR T cells K562 MDA NCI-H1299 LM7 HER2 - HER2 + 1

11 No increase in pro-inflammatory cytokines post infusion DL1 DL1 DL1 DL2 DL2 DL3 DL3 DL5 DL5 DL5 conc (pg/ml) GMCSF IFN IL TNF Pre 3h Wk1 Wk2 Wk4 wk6 Pre 3h Wk1 Wk2 Wk4 wk6 Pre 3h Wk1 Wk2 Wk4 wk6 Pre 3h Wk1 Wk2 Wk4 wk6 post Infusion post Infusion post Infusion post Infusion In vivo persistence of adoptively transferred HER2-CAR T cells Dose Level 6 (3x1 6 cells/m 2 ) Dose Level 7 (1x1 7 cells/m 2 ) Dose Level 8 (3x1 7 cells/m 2 ) Tumor biopsy post infusion 11

12 Clinical Outcome 4 patients with stable disease 1 patient for 6 weeks (then had tumor resected) 1 patient for 12 weeks (then had tumor resected) 1 patient for 4 months 1 patient for 5½ months (ongoing) 11 patients with progressive disease Conclusions: T-cell Therapy for sarcoma HER2-CAR T cells Have antitumor activity in preclinical models Adoptive transfer of HER2-CAR T cells in humans Evaluated cells doses safe (up to 3x1 7 /m 2 ) Limited T-cell persistence Plan Clinical: Escalate HER2-CAR T-cell dose to 1x1 8 /m 2 Preclinical: Improve CAR; better preclinical models 12

13 CAR T cells to overcome immune evasion Targeting cell surface antigens Targeting multiple antigens Targeting inhibitory stromal cells HER2 as an Immunotherapy target for GBM ~ 7% of GBM positive HER2 HER2 promotes malignant phenotype expressed in CD133+ glioma initiating cell population IHC CD133 HER2 CD133 ve CD133 +ve CD133-PE HER2-PE 13

14 HER2-specific T cells induce regression of autologous GBM day Tumor NT HER2 only T cells T cells Su urvival Probability HER2-T cells NT T cells Tumor only Survival Time (days) (Ahmed et al, Clin Can Res 21) CMV as an Immunotherapy target for GBM Majority of GBMs are positive for CMV IE1 and pp65 IE1 pp65 Phase II adjuvant pp65/dc vaccine study: Safe Induction of pp65-specific T-cell responses Prolonged survival in comparison to historic controls 14

15 GBM patients have decreased T-cell responses to pp65 SFC per 2x1 5 PBMC CMV.pp65 CMV.IE1 p=.22 6 p=.31 GBM Healthy Donor per 2x1 5 PBMC SFC 4 2 GBM Healthy Donor (Ghazi et al, JIT 212) Generation of CMV-specific CTL using Ad5f35 Vectors Ad5f35 CMVpp65 LCL PBMC + IL-2 O/N Adherence CMVspecific CTL (Leen et al, Nat Med 26) 15

16 Generation of CMV-specific T cells from GBM patients 6 ) T-cell number (x1 6 SFC per 1x1 5 cells 1 Expansion n= Week Elispot Assay pp65 Control Lys sis (%) Cytotoxic Activity CMV(+) auto GBM CMV(+) mismatched GBM 1 CMV(-) auto GBM CMV(-) mismatched GBM :1 2:1 1:1 4:1 2:1 1:1 T cell : target ratio 1 Generation of HER2-CAR CMV-specific CTL Ad5f35 CMVpp65 LCL PBMC + IL-2 O/N Adherence HER2-CAR retrovirus HER2-CAR CMV-CTL 16

17 A Characterization of HER2-CAR CMV-specific T cells HER2-CAR expression Cytotoxic Activity SFC per 1x1 5 cells NLV (CMV) CD8 HER2-CAR Elispot Assay CMV-CTL 51.9% HER2-CAR CMV-CTL Lysis s (%) HER2(+) autologous GBM HER2(-) MDA-MB-468 GBM # GBM #4 4:1 2:1 1:1 4:1 2:1 1:1 T cell : target ratio pp65 neg Co pos Co Characteristics & Outcome of infused patients 4 patients with recurrent GBM Cell dose: 1x1 6 /m 2 Outcome: 2 progressive disease 1 stable disease for 4½ months 1 partial response 17

18 Clinical PREINFUSIONresponse post T-cell infusion Pre CTL 6 weeks 4 months Conclusions: T-cell Therapy for GBM HER2 and CMV antigens are expressed in GBM Generation of clinical grade HER2/CMV-specific T cells is feasible Clinical Trial: Safe data so far encouraging Plan: Escalate HER2/CMV-specific T-cell dose to 1x1 8 /m 2 18

19 CAR T cells to overcome immune evasion Targeting cell surface antigens Targeting multiple antigens Targeting inhibitory stromal cells CAFs play critical role in tumorgenesis Immunosuppression TGF- IL-1 TGF- CXCL14 FAS-L Tumor cells CAFs FAP VEGF Promote Tumor growth VEGF MMPs 19

20 Characterization of FAP-CAR T cells FAP-expression in cancer cell lines FAP-CAR T cells recognize FAP+ targets: cytokine secretion 25 NT-T cells FAP-T cells 2 p<.5 p<.5 Hs894 SENMA IFN (pg/ml) C t score C666.1 PL45 U87 A549 LCL A549.hFAP A549.mFAP A549 A549.mFAP A549.hFAP FAP-CAR T cells recognize FAP+ targets: cytolytic activity % Lysis (1:1 E:T Ratio) NT-T cells FAP-T cells p<.5 p<.5 A549 A549.mFAP A549.hFAP FAP-CAR T cells have antitumor activity in vivo In vivo induction of murine FAP Murine FAP fold expression Days post tumor injection FAP-CAR T cells target FAP+ cells in vivo Murine FAP fold expression p<.5 Normal NT-T cells FAP-T cells Pretreatment Day 5 Day 15 Day 25 Percent survival FAP-CAR T cells have antitumor activity in vivo Untreated NT-T cells FAP-T cells NT (n=9) FAP (n=8) Untreated (n=9) p< Days post tumor injection 2

21 Targeting CAFs induces expression of pro inflammatory genes NTC3 NTC2 NTC1 NTC4 NTC5 NTC6 FAP5 FAP4 FAP1 FAP2 FAP3 FAP6 Pathway analysis - enrichment of genes involved in chemokine & cytokine-cytokine receptor interaction toll-like receptor pathway natural killer cell mediated cytotoxicity Jak-STAT pathway B-cell receptor signaling g antigen processing and presentation pathway complement and coagulation cascades Combining FAP-CAR T cells with tumorspecific T cells enhances antitumor effects Pretreatment Untreated FAP-T cells EphA2-T cells FAP- T cells + EphA2-T cells Day 11 Day 23 Day 33 21

22 Conclusions: Targeting tumor stroma CAFs contribute to immunosuppressive tumor environment Targeting CAFs results in antitumor effects and induces expression of pro-inflammatory genes Targeting CAFs and tumor cells results in enhanced antitumor effects Conclusions Tumors pursue a gamut of immune evasion mechanisms BUT G ti difi ti h ld th Genetic modifications holds the promise to engineer T cells that are resistant to these ploys 22

23 Acknowledgements Go Lab Sunitha Kakarla Claudia Gerken Johanna Yi Mamta Kalra Melinda D Souza Simone Krebs Tania Rodriguez Cruz Kato Iwahori David Torres LaTerrica Williams Chris DeRenzo Paulina Velasquez GMP Facility Adrian Gee Zhuyong Mei Oumar Diouf Humin Zhang Vita Brawley CAGT Nabil Ahmed Gianpietro Dotti Malcolm Brenner Helen Heslop Cliona Rooney Clinical Research Bambi Grilley Bridget Medina Catherine Perera Enli Liu Tara Gray Collaborators HER2-CAR Winfried Wels Osteosarcoma patients Pete Anderson, Lisa Wang Osteosarcoma stem cells Nino Rainusso, Jeff Rosen GBM Robert Grossman, Murali Chintagumpala, JackSu Pathology John Hicks, Suzanne Powell 23