CAR T cell immunotherapy for GBM: Bench to Bedside

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1 CAR T cell immunotherapy for GBM: Bench to Bedside Laura A Johnson, PhD Director, Solid Tumor Immunotherapy Laboratory Adj Asst Prof, Dept of Path & Lab Medicine Center for Cellular Immunotherapies University of Pennsylvania May 13 th, 2016

2 Adoptive T-cell Immunotherapy: Gene-modified patient T cells can destroy tumors 2

3 Gene-engineered anti-tumor receptors CAR = surface antigens TCR = intracellular antigens as peptide on MHC Chekmasova and Brentjens, Discovery Med

4 TCR-engineered anti MART1 therapy of melanoma can work (30% OR) ACT of patient TCR gene-engineered T cells can cause tumor regression in patients Johnson et al., Blood

5 TCR-engineered anti MART1 therapy of melanoma can work (30% OR) but at a cost! Unfortunately, they also recognize and destroy normal tissues bearing the same antigen Johnson et al., Blood

6 CD19 CARs treat patients with leukemia ~95%OR Chimeric Antigen Receptor Modified T Cells in Chronic Lymphoid Leukemia David L. Porter, M.D., Bruce L. Levine, Ph.D., Michael Kalos, Ph.D., Adam Bagg, M.D., and Carl H. June, M.D. N Engl J Med 2011; Chimeric Antigen Receptor Modified T Cells for Acute Lymphoid Leukemia Stephan A. Grupp, M.D., Ph.D., Michael Kalos, Ph.D., David Barrett, M.D., Ph.D., Richard Aplenc, M.D., Ph.D., David L. Porter, M.D., Susan R. Rheingold, M.D., David T. Teachey, M.D., Anne Chew, Ph.D., Bernd Hauck, Ph.D., J. Fraser Wright, Ph.D., Michael C. Milone, M.D., Ph.D., Bruce L. Levine, Ph.D., and Carl H. June, M.D. N Engl J Med 2013 Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia Shannon L. Maude, M.D., Ph.D., Noelle Frey, M.D., Pamela A. Shaw, Ph.D., Richard Aplenc, M.D., Ph.D., David M. Barrett, M.D., Ph.D., Nancy J. Bunin, M.D., Anne Chew, Ph.D., Vanessa E. Gonzalez, M.B.A., Zhaohui Zheng, M.S., Simon F. Lacey, Ph.D., Yolanda D. Mahnke, Ph.D., Jan J. Melenhorst, Ph.D., Susan R. Rheingold, M.D., Angela Shen, M.D., David T. Teachey, M.D., Bruce L. Levine, Ph.D., Carl H. June, M.D., David L. Porter, M.D., and Stephan A. Grupp, M.D., Ph.D. N Engl J Med 2014 See also Kochenderfer/Rosenberg (NCI) and Brentjens/Sadelaine (MSKCC) similar publications 6

7 CD19 CARs treat patients with leukemia ~95%OR 7

8 CD19 CARs treat patients with leukemia ~95%OR 8

9 Glioblastoma (GBM): Stage IV brain cancer Most common and most aggressive primary brain tumor Affects more patients annually than testicular cancer, multiple myeloma, and Hodgkin s disease Responsible for more deaths annually than melanoma Current treatment of resection, radiation, and chemotherapy is nonspecific and non-curative (life expectancy <15 months) One-third of GBM express a tumor-specific mutation in epidermal growth factor receptor, EGFRvIII 9

10 EGFRvIII CAR T cells destroy U87vIII glioma targets in vitro U87 (antigen -ve) glioma U87vIII (antigen +ve) glioma 10

11 What can we tell from xenogeneic models? U87vIII gliomas subcu or intracranial EGFRvIII CAR T-cells i.v. Tumor measurements -caliper (subcu) -luminescence (IVIS) -MRI Day 0 Day 7 Day 45+ NSG mouse (no T, B, NK cells, defective macrophages, DCs) 11

12 S.C. glioma + i.v. CAR T cell model in NSG mice: trafficking, anti-tumor, not correct anatomic site, no immune contribution, no safety data Tumor measurements Day 0 Day 7 Day 45 U87vIII gliomas subcu or intracranial NSG mouse CAR T-cells i.v. Tumor size (L x W x H) cm 3 ± SEM Tumor engraftment confirmed by luciferase 2173 CAR T cells n=10 3C10 CAR T cells n=10 CD19 T cells n=10 * ** ** + % Survival Time to endpoint 2173 CART 3C10 CART CART 19 n=10 n=10 n= tumor cell sq injection T cell iv injection ** p<0.005 Kruskal Wallis + all mice euthanized Days post-tumor injection p= Log rank Mantel Cox Johnson et al., Sci Transl Med

13 Orthotopic glioma + i.v. CAR T cell model in NSG mice: CNS organspecific trafficking, anti-tumor, but no immune contribution or safety data Day 18 after T cells CD4+ T cells CD8+ T cells Johnson et al., Sci Transl Med

14 EGFRvIII copy number per cell Alex Cogdill, Chong Xu 14

15 EGFRvIII copy number per cell EGFRwt copy number per cell Alex Cogdill, Chong Xu 15

16 Ex-vivo human cell studies: h2173 CART-EGFRvIII do not cross react in vitro with normal cells, EGFRwt CAR do: some safety data 35 %CD3+TNFa EGFRwt 2173 UTD CD34+ HCM Kerat HMSC HNP1 HOB HREpC U87 U87vIII NS 2173 = EGFRvIII CAR EGFRwt = EGFR CAR UTD = untransduced T cells CD34+ = enriched hematopoetic stem cells HCM = cardiac myocytes Kerat = keratinocytes HMSC = mesenchymal stem cells %CD3+IL EGFRwt 2173 UTD HNP1 = neural progenitor cells HOB = osteoblasts HREpC = renal epithelial cells U87 = glioblastoma NS = no stimulation 0 CD34+ HCM Kerat HMSC HNP1 HOB HREpC U87 U87vIII NS 16

17 Ex-vivo human cell studies: EGFRvIII CARs do not cross react in vitro with normal cells, but EGFRwt CAR do: some safety data U87 targets cardiac myocyte targets mesenchymal stem cell targets % lysis UTD EGFRwt 2173 % lysis UTD EGFRwt 2173 % lysis UTD EGFRwt to 1 10 o 1 5 to to to to to to 1 10 o 1 5 to to to to to to 1 10 o 1 5 to to to to to E:T ratio E:T ratio E:T ratio U87vIII targets renal cell targets keratinocyte targets 25 UTD 25 UTD 25 UTD % lysis EGFRwt 2173 % lysis EGFRwt 2173 % lysis EGFRwt to 1 10 o 1 5 to to to to to to 1 10 o 1 5 to to to to to to 1 10 o 1 5 to to to to to E:T ratio E:T ratio E:T ratio 17

18 Xenograft tissue transplant: EGFRvIII CARs do not cause toxicity in vivo in human-skin grafted NSG mice, but EGFRwt CAR do: some safety data Day 0 Day 30 Day 45 Human skin graft CAR T-cells i.v. NSG mouse Johnson et al., Sci Transl Med

19 Xenograft tissue transplant: EGFRvIII CARs do not cause toxicity in vivo in human-skin grafted NSG mice, but EGFRwt CAR do: some safety data Tumor measurements Day 0 Day 30 Day 45 Human skin graft NSG mouse CAR T-cells i.v. CAR T cell infiltration in skin grafts (day 15 after CAR T cells) Johnson et al., Sci Transl Med

20 What more can we learn from Syngeneic mouse models? Has intact immune system, tumor microenvironment Mouse CD28, 4-1BB and CD3z replaced in CAR to make mcar CD8TM, CD28, 4-1BB, CD3ζ Ψ LTR 139 scfv LTR sd sa Inject sma560viii mouse glioma tumors i.c. or sub cu 5 Gy Inject mcar transduced mouse T-cells i.v. Day 0 Day 3 Measure s.c. tumors Plot i.c. survival curves Day 60 VmDk (immune intact) Sampson et al., Clin Cancer Res

21 EGFRvIII mcars treat S.C. and IC tumors in vivo: organ-specific trafficking, antitumor, impact of endogenous immune cells, impact of endogenous cytokines A 300 D0 5e5 sma560viii subcu D3 5e6 mcar T cells IV Tumor size (mm 2 ) Untreated 5Gy TBI Vector control 5Gy TBI EGFRvIII CAR 5Gy TBI *5/8 complete regression B Days 100 D0 5e4 sma560viii IC D3 mcar T cells IV Percent survival (%) x10 6 Vector Control 5Gy TBI 0.7x10 5 EGFRvIII CAR 5Gy TBI 3.5x10 6 EGFRvIII CAR 5Gy TBI 1.0x10 7 EGFRvIII CAR 5Gy TBI P < Days 60 Sampson et al., Clin Cancer Res

22 Syngeneic mice: Look for de novo immune response by antigen-loss tumor rechallenge Previously cured mice or naïve animals were rechallenged with 5e5 sma560 parental (non-egfrviii) tumors subcutaneously: All rejected tumors Sampson et al., Clin Cancer Res

23 Need fully immune intact, syngeneic system to completely evaluate immunotherapy impact Johnson et al., OncoImmunol

24 UPENN Clinical trial: NCT Based upon these results: University of Pennsylvania has now opened a clinical trial to treat patients with EGFRvIII positive GBM with the humanized 2173 EGFRvIII CAR T cells (NCT ), PI Dr. Don O Rourke. 24

25 CCI Carl June Anne Chew Dana Hamill Marcela Maus Gabi Plesa Regina Young UPENN Don O Rourke John Seykora Benny Nace Nadia Dahmane Maria Lage-Martinez Rob Bailey Jay Dorsey Human Immunology Core Small Animal Imaging Facility Stem Cell & Xenograft Core Acknowledgements Johnson Lab Alina Boesteanu Alexandria Cogdill Danielle Cook Bevin McGettigan-Croce Reiss Reid Jesse Rodriguez Chong Xu Yibo Yin CCI Collaborators Steve Albelda Gregory Beatty Saar Gill Mike Milone Avery Posey John Scholler Yangbing Zhao Novartis Jen Brogdon Boris Engels Andreas Loew Reshma Singh Pramod Thekkat James Sutton (CA) 25