Gianpietro Dotti Baylor College of Medicine, Houston TX, USA

Transcription

1 CARs in the clinic: first efficacy reports and concerns about safety Gianpietro Dotti Baylor College of Medicine, Houston TX, USA Adoptive Immunotherapy of Gene Modified T Cells T lymphocytes 1 Blood draw from patient or donor 3 Infusion 2 Genetic Modification Redirect tumor specificity 1

2 Redirect T-cell antigen specificity - Transgenic expression of T-cell receptors - Transgenic expression of Chimeric Antigen Receptors (CARs) Gene Transfer of CARs Monoclonal antibody TcR complex Antigen Linker Tumor scfv Spacer v T Cell CAR Intracytoplasmic Eshhar Z PNAS

3 Advantages of CAR-modified T cells - Virtually every surface antigen can be targeted - Non-processed molecules (including carbohydrates and glycolipids) can be targeted - Receptor specificity easily generated - No MHC restriction Limitations of CAR-modified T cells - Surface expression of the target antigen - Potential T cell interference if the antigen is shaded 3

4 Chimeric Antigen Receptors ScFv specificity PMSA HER-2 Folate-binding protein (FBP) Tumor target Prostate cancer Breast, lung, brain cancers Ovarian cancer CD3 CD19/CD2/CD23 Light chains of Igs GD2 CEA HD Lymphoma B cell lymphoid tumors B cell lymphoid tumors Neuroblastoma Melanoma Colon rectal carcinoma Generation of CAR-modified Activated T Lymphocytes (ATL) Step 2: CAR-gene transfer (DNA integration) PBMC PBMC ATL ATL PBMC ATL ATL Step1: Activation with OKT3 and IL-2 Step 3: Expansion with IL-2 Step 4: Sterility Phenotype Cytotoxicity 4

5 Expression of CARs by activated T lymphocytes 1 th generation CAR scfv v H v L 47% anti-igg1.ch2-ch3 counts chain anti-igg1.ch2ch3 Cytotoxicity of CD19 CAR + T cells Control T Cells CAR.CD19 + T cells % Specific Lysis JEKO-1 BJAB K562 % Specific Lysis JEKO-1 BJAB K562 Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio 4:1 2:1 :1 5:1 4:1 2:1 :1 5:1 Effector : Target Ratio JEKO-1 AND BJAB are CD19 + tumor cells Effector : Target Ratio 5

6 Cytotoxic activity of CAR-modified T cells is not MHC-restricted % of specific lysis Control T blasts CAR.CD19+ T blasts B-CLL autologous B-CLL allogeneic Clinical trials with CAR + T cells Antigen # of trials Tumor CD19 8 Hematological malignancies CD2 2 Hematological malignancies Kappa light chain 1 Hematological malignancies GD2 1 Neuroblastoma CEA 4 Adenocarcinoma PSMA 1 Prostate cancer CD171 1 Neuroblastoma FR 1 Ovarian cancer IL-13R 2 1 Gliobastoma HER2/neu 3 Osteosarcoma, lung cancer, other HER + tumors Jena B et al Blood 2 6

7 What are we learning from these trials? Does the CAR expression vector affect the outcome? Which co-stimulation is ideal for T cells? Which T cell subset should be used? What are the toxicities? Vectors for T cell CAR expression # of trials Retroviral vectors 17 Lentiviral vectors 1 Nonviral vector gene transfer - plasmids 5 - transposone/transposase Sleeping Beauty 1 7

8 Vectors for T cell CAR expression Vector Speed of manufacture Costs Retro/Lenti Short high Plasmids Long low Sleeping Beauty Intermediate low Generation of CAR-modified Activated T Lymphocytes (ATLs) Step 2: CAR-gene transfer (transposone/transposase) Step (plasmid (virus 2: CAR-gene mediated) transfer Step 4: PBMC Sterility PBMC ATL ATL PBMC Phenotype ATL ATL Cytotoxicity Step1: Activation with OKT3 and IL-2 Step 3: Step Drug Eexpansion 3: selection, with expansion IL-2 (± Expansion aapc?) with IL-2 with (± aapc?) IL-2 (± aapc?) 2-3 > 3-55 weeks 8

9 Overview of Retroviral production Single cell clone of producer is expanded. Multiple aliquots are cryopreserved. Cells and supernatant are tested Cell Bank Testing Microbial Sterility Bacterial / fungal Sterility Mycoplasma testing Endotoxin Testing In vivo Assay for adventitious viral contaminants In Vitro assay for adventitious viral contaminants Mouse Antibody Production Test Thin section electron microscopy PCR testing for HIV, HTLV, HBV, HCV, HHV 6, HHV 7, CMV,. EBV, B19 Extended Mink PG4 S+L Focus Assay XC plaque assay Cell line species identification by isoenzyme electrophoresis Vector characterization by PCR or genomic Southern Functional testing on transduced primary T-cells Supernatant and cryopreserved cells for archiving. cell co-cultivation to detect GALV and ECO RCR Supernatant amplification to detect GALV and ECO RCR Overview of Retroviral production Single aliquot from cell bank is expanded to 8 CFs. 8X Supernatant is harvested, filtered, aliquoted and tested. End-of-production cells are then harvested and tested End-of-production cells are then harvested and tested 9

10 Overview of Retroviral production Overview of Retroviral production

11 What are we learning from these trials? Does the CAR expression vector affect the outcome? Which co-stimulation is ideal for T cells? Which T cell subset should be used? What are the toxicities? Second generation CAR to improve T cells activation Killing of tumor cells CAR T cell CD28 Tumor Tumor Tumor B7 Incomplete activation of Include costimulatory endodomains d in the CAR CAR scfv CD28 T cell IL2 T cell proliferation T cells Maher J et al Nat Biotechnol

12 Rational for exploring alternative costimulatory endodomains Early (CD28/PtdIns) costi. Late (4-1BB/TRAF) costi. Incorporation of co-stimulatory endodomains in CARs scfv-fc RI scfv 1 th generation CAR ScFv spacer Fc RI ScFv spacer Zeta 2 th generation CAR scfv-cd28 ScFv spacer CD28 Zeta scfv-4-1bb ScFv spacer 4-1BB Zeta scfv-cd28-ox4 scfv-cd28-4-1bb 3 th generation CAR ScFv spacer CD28 OX4 Zeta ScFv spacer CD28 4-1BB Zeta 12

13 CD28 induces T-cell expansion after antigen stimulation l counts x 6 Cell T blasts control T blasts CAR- T blasts CAR-CD Days of culture /ml/ 6 cells Pg IL-2 IFN- TNF- Vera et al, Blood 26;8(12):389-7 Are CAR-CD28 CD28 + T cells superior to CAR + T cells? Prepare two autologous activated T cell lines expressing CAR.CD19 or CAR.CD19-28 for each patient Infuse both T cell populations Track each T cell population in vivo Savoldo et al, JCI

14 Generation of CAR + T cells Peripheral blood drawn or patient pheresed PBMC activation on immobilized OKT3 + IL-2 Transduction with CAR.CD19-28 vector CAR.CD19-CD28 + T cells Expansion in IL-2 QA/QC testing and freezing Transduction with CAR.CD19 vector CAR.CD19 + T cells Expansion in IL-2 QA/QC testing and freezing Infuse Equal ex vivo killing by CAR.CD19 + and CAR.CD T cells pecific lysis % of s K562 HDLM-2 (CD19- target) Raji (CD19+ target) 51 Cr release assay 2:1 E:T ratio NT CAR.CD19 CAR.CD19-28 n=6 14

15 CAR.CD19 + and CAR.CD T cells have comparable immunophenotypes CAR.CD19 CAR.CD19-28 positive cells % of CD8+ CD4+ CD45RA+ CD45RO+ CD62L+ CCR7+ n=6 -Three dose levels -Single dose Treatment plan - Modified continual reassessment method Dose level 1: 2x 7 cells/m 2 of each product Dose level 2: 1x 8 cells/m 2 of each product Dose level l 3: 2x 8 cells/m 2 of each product -Second infusion if stable disease/pr 15

16 Follow up studies - Q-PCR for transgene in PBMC at multiple times after infusion - CD19+ B-cells in peripheral blood - Imaging (PET or CT scan) at 6 weeks T cell infusions were well tolerated with no dose limited toxicity Monitoring expansion and persistence of each product PB DNA isolation primer probe R Q CAR.CD19 q-pcr assay CAR.CD19-28 ScFv spacer Zeta ScFv spacer CD28 Zeta 16

17 CAR.CD T cells have greater in vivo expansion and persistence Copy Numb bers x 3 ng of DNA 35 5 Pre Time post infusion CAR.CD19 signal in PBMC CAR.CD19.28 CD19 signal in PBMC 2w 4w Pre II 2w 4w 6w 3 mo 6 mo Pt #3, dose level 2 Critical points There is a consensus that CAR-modified T cells need the incorporation of costimulatory endodomains Open questions Which is the best costimulatory endodomain? Do we need 3 rd generation CARs early and late costimulation? Are preclinical models really predictive of efficacy and safety of 2 nd vs. 3 rd generation CARs? We are planning to carefully compare second vs. third generation in lymphoma patients 17

18 What can we learn from these trials? Does the CAR expression vector affect the outcome? What co-stimulation is ideal for T cells? Which T cell subset should be used? What are the toxicities? Which T cell subset? Polyclonal activated T lymphocytes y Virus-specific T cells Central memory T cells Natural Killer cells Gamma/delta T cells NKT cells 18

19 Multivirus-specific specific CTLs after HSCT- protective and efficacious in vivo Infused to 35 patients t HSCT no evidence of GvHD Cleared 9 of 9 EBV reactivations 7 of 8 CMV reactivations 8 of 8 adenovirus reactivations Nat Med. 26;12(): Critical problems: viral infections and relapse post-alternative donor SCT Relapse (39%) Viral infection (39%) Pulmonary toxicity (15%) Bacterial sepsis (7%) Alana Kennedy-Nasser, BBMT 28 19

20 Generation of CAR.CD19 + multivirus- specific CTLs Ad5f35CMVpp65 vector APC EBV-LCL +IL-2 +IL-2 +IL-2 PB Transduce CTLs with CAR.CD19-28 after third stimulation CAR + Multivirusspecific CTL Micklethwaite et al Blood 2; 17: CAR.CD19 Multi Virus-specific specific CTLs % 6.77% %.32% % 5.98% % Specific Lys sis (E:T 4:1) CAR.CD19 CMV pp % 2.49% %.3% 6% 3 CMV (YSE pent) Ad hexon 4 EBV LCL 1 61% 6.1% 2%.2% 1 2 Adeno 3 (TYF-pent) Irrel %.24% 1 Raji CD19+ 2 EBV 3 (RAK-pent) ALL 4 CAR.CD19+ CTL NT CTL HDLM CD19- K562 Micklethwaite et al Blood 2; 17:

21 Generation of CAR.CD19 + multivirus-specific specific CTLs Peripheral blood drawn multi-ctl generation Ad-pp65 DC generation LCL generation Transduction with CAR.CD19-28 retroviral vector after 3 rd stim Ad-pp65 CAR.CD multivirus CTLs Expansion with LCL and IL-2 QA/QC testing and freezing Infuse Pt 1: viral immune reconstitution and detection of CTLs CAR.CD19.28 signal in PBMC 15 CMV-pp65 responding cells 15 IFN SF FC/ 5 cells 5 5 Copies/ g DNA Pre 4wk 6wk Pre II 2wk 4wk 6wk 3 mo 4 mo No viral infections 21

22 Pt 2: antitumor response and detection of CTLs D19+ lymphocytes % CD5+ C CAR.CD19.28 signal in PBMC 2 tumor Pre I 2wk 4wk 6wk Pre II 1wk Copies/ g DNA What can we learn from these trials? Does the CAR expression vector affect the outcome? What co-stimulation is ideal for T cells? Is host lymphodepletion necessary? Which T cell subset should be used? What are the toxicities? 22

23 Toxicities On target t toxicity it On target but out of organ toxicity Massive T cell expansion/cytokine storm On target toxicity (CD19-CAR) CAR) B cells pre vs 6 wks post Pt #1 <.1% Pt #2 <.1% Pt #3 4% vs 1% Pt #4 <.1% rs x 3 ng of DNA Copy Number CAR.CD19 signal in PBMC CAR.CD19-28 signal in PBMC CD2 + cells 8 Pre wk1 wk2 wk4 wk % of CD2 + cells Time post infusion 23

24 Toxicities On target t toxicity it On target but out of organ toxicity Her2 toxicity? Massive T cell expansion/cytokine storm 4-1BB costimulation toxicity? Do we need a suicide gene? Inducible Caspase-9 suicide gene Native Caspase-9 Inducible Caspase-9 CARD Apaf1 FKBP domain CID domain Chemical Inducer of Dimerization (AP2187) Apoptosis Apoptosis Straathof et al. Blood 25 24

25 Validation of the suicide gene in a Phase I clinical trial LTR icaspase9 2A CD19 LTR OKT3 rhil-2 rhil-2 Allodepleted T cells Day 4 Day 5 Day 6 Suicide gene modified allodepleted T cells Tey et al BBMT 27 Di Stasi et al NEJM 211 CD19 icasp9 Enrichment by Clinimacs Suicide gene modified T cells Suicide gene modified T cells after CD19 selection 25

26 CD3+CD19+CD45+ T Cell Depletion after CID Occurrence of Grade II GVHD CD3+/CD19+/CD45+ cells Bil Con (mg/dl) 4% 2.5 3% 2% % % Pre T day 7 cell post T infusion cell infus day 13 post T cell infus day 14 post T cell infus Bil con (mg/dl) CD19 Peripheral Blood 14d post T cell infusion CD3 9.6% 22% 2.9% T Cell Depletion after CID and skin GVD improvement 9.6% 22% 2.9% Pre CID infusion Pre AP193 2d Post AP193 CD19 CD3.2% 2% 2.7% 2.5% 2 days after CID infusion 26

27 Activation of the Caspase9 suicide gene eliminates CAR T cells in vivo LTR ic9 2A CAR LTR D1 D7 D14 CID D17 D21 T cell bioluminescence Hoyos et al. Leukemia 2 Conclusions Adoptive transfer of CAR modified T cells is a realistic therapeutic opportunity T cell costimulation is required Profound lymphodepletion may not be required Other T cell subsets should be explored in specific clinical contexts Toxicity remains a concern 27

28 Acknowledgements Malcolm Brenner Cliona Rooney Helen Heslop Barbara Savoldo Carlos Ramos Catherine Bollard Ann Leen Stephan Gottschalk Leonind Metelitsa Postdocs Russel Cruz Kenneth Micklethwaite Valentina Hoyos Clinical Research Bambi Grilley Alicia Brown Florence Noel Yu-Feng Lin Vicky Torrano Statistical analysis Hao Liu GMP Laboratory Adrian Gee Oumar Diouf Zhang Huimin Joyce Ku Weili Liu Pallavi Mahopatra Enli Liu Debbie Lyon Zhuyong Mei Funding: PO1, Leukemia and Lymphoma Society Specialized Center of Research, Leukemia and Lymphoma Society translational Research grants, Doris Duke Distinguished Clinical Scientist Award, Doris Duke Developmental Award, NIH NGVL, R1 from NCI, CLL Global Research Foundation, DOD, GCRC 28