Gene Modified T Cells Oncology Immunotherapeutics-I Laurence J.N. Cooper [email protected] 10-23-2014 10:30 am to 12:00 pm Thanks to Dr. George McNamara Disclosure. Dr. Laurence J. N. Cooper has been a consultant for American Stem Cells, Inc., GE Healthcare, Ferring Pharmaceuticals, Inc., and Bristol-Myers Squibb. Dr. Cooper has received multiple grants from foundations in the state of Texas, including CPRIT and UT STAR, and Federal to support research. Dr. Cooper has received honoraria and payment for the development of education presentations including service on speakers’ bureaus from Miltenyi Biotec. Dr. Cooper has received travel/accommodations expenses covered/reimbursed by Lonza. Broadening the clinical appeal of genetically modified T cells Tumor-associated antigens targeted by CARs Torikai H, Moyes JS, Cooper LJN 2014 engineering T cells to target tumor cells. In W. Cai (ed) Engineering in Translational Medicine, Springer-Verlag London 2014. Identify tumor-associated antigens Collaboration with Immatics and XPRESIDENT® Discovery Platform Targeting tumor-associated antigens Requires targeting self antigens? Targeting neoantigens possible? LB Alexandrov et al. Nature, 1-7 (2013) doi:10.1038/nature12477 • Boundaries on this slide are arbitrary • Immunogenic neoantigens may be identified by exome sequencing with sufficient probability only on mutation-rich tumors • Identifying immunogenic self antigens will require a different technology, e.g. mass spectrometry applicable to all HLA expressing tumors Broadening the clinical appeal of genetically modified T cells Approaches to gene transfer Viral • Retrovirus • Lentivirus • GOF Gain of function LOF Loss of function DGF Dominant gain of function DNF Dominant negative form Enhancer deleted, self inactivating (SIN) long terminal repeat lentivirus vectors. • Adenovirus • AAV • other Non-viral • DNA (electroporation or other) • Sleeping Beauty (SB11, SB100X) • piggyBac Cytoplasm Transposase mRNA mRNA • • Transient production Artificial nucleases, transposases, recombinases CAR Transposon Nucleus Genetic modification of T cells to redirect specificity Kershaw, Westwood, Darcy Nature Reviews Cancer 13, 525–541 (2013) Personalizing the therapy: Manipulating the T cell by harvesting and re-expressing melanoma-specific TCR 6 OCTOBER 2006 VOL 314 SCIENCE TCR gene therapy in patients with metastatic melanoma Target Antigen TCR Patient Response MART-1/A2 human 31 MART-1/A2 human high avidity gp100/A2 NYESO1/A2 CR PR Reference 4 4 Science 2006; 314:126-129 Blood. 2009;114:535-546 20 6 6 (3, 4, 9, 16+, 17+, 17+) Blood. 2009;114:535-546 mouse 16 3 1 (14+) 2 (4, 3) Blood. 2009;114:535-546 human 11 5 2 (22+, 20+) 3 (3, 8, 9+) J Clin Oncol 2011;29:917-924 Avoiding TCR miss-pairing Torikai, Moyes, Cooper 2014 Engineering T cells to target tumor cells. In W. Cai (ed) Engineering in Translational Medicine, Springer-Verlag London 2014. Zhang, Morgan Adv Drug Delivery Rev 2012 64: 756–762. Provasi et al., Nat Med. 2012 May;18(5):807-15. Torikai et al., Blood. 2012 Jun 14;119(24):5697-705. Govers et al 2014 J Immunol ε not CD3ζ CD3 (epsilon) (zeta) Identification of target antigens and TCRs for CD8+ T cells Siewert et al., Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides. Nat Med. 2012. Outcomes and toxicities infusing CD19specific CAR+ T cells Approaches to gene editing, genome engineering Nucleases CRISPR/Cas9 Meganucleases TALENs ZFNs site specific integration by HR, mutations by NHEJ. clustered regularly interspaced short palindromic repeat (RNA)/ Cas9 I-CreI meganucleases derived from self-splicing introns/inteins TAL Effector Nucleases Zinc Finger Nucleases Recombinases / Transposases Adenovirus Cre SB11, SB100X piggyBac Adenovirus delivery of TALENs+DNA with protein capped ends (Holkers 2014) Cre recombinase Sleeping Beauty transposase (2 of various versions), random AT sites piggyBac transposases (various versions), random AATT sites Activation sgRNA:dCas9-TF TALE ZFP-TF Synthetic guide RNA:disabled Cas9 nuclease-Transcription Factor Transcription Activator like-Effector Zinc Finger Protein-Transcription Factor Alternative functional domains include: Paired Nickases (increase selectivity for double stranded breaks), recombinases, repressors, epigenomic modifiers (DNA methyltransferases, histone acetylases or deacetylases, etc), fluorescent proteins (SUNtags, TALEcolor, TALE-Lights). NHEJ: Non-homologous end joining (& micro-homologous recombination). HR: Homologous recombination. Select companies in this field are highlighted later. Artificial nucleases Hard Clinical Easy Research http://www.toolgen.com/html/eng/technology/engineered_nucleases.php Cellular substrates Delete TILs, CAR or Hypoxia High adenosine, H2O2, lactate, PGE2, TGFβ Arg, Trp depletion Tumor Adenosine A2AR (drug or delete gene) BCM: “Rewire”: TGFβRex-TLR4in co-stimulation Arginase and iNOS deplete Arg, iNOS generates H2O2 Adapted from: Gattinoni, Klebanoff, Restifo 2012 Nat Rev Cancer 12: 671-684 CAR designs Jena et al. Curr Hematol Malig Rep. 2014 Mar;9(1):50-6. CAR design scFv • affinity • Other binders … ex. D-CAR based on Dectin-1 (Kumarasen et al. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10660-5. ) Stalk • Which stalk … Ig vs. CD8 vs. short • Length of hinge+ stalk for proximal vs. distal epitopes • Reduce FcR binding Signaling domains • Signal 1, 2, 3 Combinatorial CARs 2nd: inhibitory costimulation Abate-Daga, et al., Oncoimmunology. 2014 Jun 18;3:e29194. modulatory ligands-> signal(s) K = kinase docking sites P = phosphorylation sites Other sites possible (ITAMs, ITIMS …) Conditional ablation of T cells Casucci, Bondanza 2011 J of Cancer HSV-tk: phosphorylates pro-drug ganciclovir, that is then incorporated into DNA. HSV-tk can be immunogenic Approaches to propagation of genetically modified T cells • CD3 with CD28 beads (Novartis) • CD3 and CD28 beads (TransACT, Miltenyi Biotec) • Activating and propagating cells (AaPC), • many variations … different outputs Huls et al., J Vis Exp. 2013 Feb 1;(72):e50070. doi: 10.3791/50070. Forget et al. 2014 J Immunother 37: 448-460. T-cell subpopulations for genetic engineering Gattinoni, Klebanoff, Restifo 2012 Nat Rev Cancer 12: 671-684. Lymphocyte populations for engineering CD16 T cells Deniger, … Cooper 2014 Clin Cancer Res T cells NK cells NK cells* NKT cells * Kudo, … Campana 2014 Cancer Res Chu, ... Yu, Hofmeister 2014 Leukemia Denman, ... Cooper, Lee 2012 PLoS One Heczey, ... Metelitsa 2014 Blood mbIL-21 AaPC Broadening the clinical appeal of genetically modified T cells Approaches to manufacture and distribution of engineered T cells T cells are rendered as a drug Insert CAR or TCR Eliminating TCR on CAR+ T cells Patient Gene insertion: Retrovirus/lentivirus Sleeping Beauty mRNA CD19 Intended response CAR HLAs B-cell leukemia/lymphoma TCRαβ HLAs Artificial nuclease Blood. 2013 Aug 22;122(8):1341-9 Blood. 2012 Jun 14;119(24):5697-705 Normal cells Cellectis/Pfizer Summary Inter- and intra-tumor heterogeneity T cells are precision tools CAR TCR++ T cells CAR TCR+ T cells CAR TCR+ T cells CAR TCR+ T cells CAR TCR+ T cells Infuse T cells with one or more specificity Personalized for the patient and the disease “N=1” trial paradigm 27 Power-law curve The teaching of Beyoncé Number of purchases Brick & Mortar Cost of distribution iTunes Number of artists Power-law curve The new industrialization of T cells Number of patients Traditional Med Centers Cost of distribution Immuno-oncology at Med Centers 1 Number of trials The N=1 approach has been shown for non-genetically modified T cells Evidence of tumor regression after treatment with a highly pure population of V22+ ERBB2IP mutation–reactive CD4+ T cells. COMMERCIALIZATION OF T-CELL THERAPIES Cell Immunotherapy of Cancer – Deals 2012-2014 Institution Amount Year Emphasis Adaptimmune $104M 2014 Series A financing, enhanced TCR(s) to cancer specific antigens (NY-ESO-1, …) Amgen-Micromet $1.16B 2012 Acquisition. BiTES, including Blinatumomab (CD3&CD19 bispecific antibody) $42M 2013 Dendritic cell personalized kidney cancer vaccine AGS-003 $225M* 2013 PD-1 inhibitor AMP-514; preclinical B7 pathway molecules. * up to $275M milestones. 2014 Research – Moon Shot Program’s Immunotherapy platform $69M* 2014 CAR T-cells, iCasp-9 inducible suicide gene switch (2014 series C $55M; 2014 series B $14M) $50M 2014 Bellicum worldwide exclusive license to Dimerizer (ex. AP1903) for use in human cell therapies for all diseases. $225M* 2013 Chimeric antigen receptor (CAR) T-cells; *up to $225M per product $50M* 2014 Two immune checkpoint pathways. * up to $300M milestones 2014 Nivolumab (anti-PD-1) FDA approval (Ipilimumab, anti-CTLA, was approved in 2011) $15 2012 CPRIT Commercialization award. EBV+ tumor cells antigen specific T-cells in collaboration with BCM. $335M* 2012 OX40L mimicking mAbs (co-stimulation) 2014 Research – Moon Shot Program’s Immunotherapy platform $47M 2013 Series A funding. Founders: Allison, Gajewski, Pardoll, Sharma, Weiner $300M 2013 Chimeric antigen receptor (CAR) T-cells (MSKCC, FHCRC CAR T-cells) Lion Biotechnologies $23M 2013 Tumor infiltrating lymphocytes (TILs, Rosenberg-NCI). SAB: Hwu, Radvanyi, Yee. Kite Pharma-NCI/NIH $128M 2014 Rosenberg CAR T-cell therapies (CART’s) Merck-Ablynx $20M* 2014 Immune checkpoint targeting nanobodies. * plus up to $2.3B royalties Merck-Pfizer 2014 Collaboration: PD-1 inhibitor pembrolizumab (MK-3475); TKI Inlyta; 4-1BB agonist Merck-Incyte 2014 Collaboration: PD-1 inhibitor pembrolizumab (MK-3475) plus INCB24360 IDO inhibitor Novartis-CoStim 2014 Acquisition. Co-stimulatory pathways pipeline $43M 2012 Novartis bought cell manufacturing plant (i.e. for UPenn CAR T-cells) Novartis-UPenn $20M* 2012 Chimeric antigen receptor (CAR) T-cells. * plus milestones and royalties. Pfizer-Cellectis $110M 2014 CAR T-cells, TALEN genome engineering. * plus up to $2.9B milestones Pfizer-MDA 2014 Research – Moon Shot Program’s Immunotherapy platform Pierre Fabre-Aurigene 2014 AUNP-12, 29 amino acid peptide based PD-1 inhibitor (license) Argos Therapeutics AstroZeneca/MedImmune-Amplimmune AstroZeneca/MedImmune-MDA Bellicum Pharmaceuticals Bellicum Pharmaceuticals-Ariad Bluebird Bio-Celgene Bristol-Myers Squibb-Five Prime Ther. Bristol-Myers Squibb Cell Medica GlaxoSmithKline-MDA Johnson&Johnson/Janssen Biotech-MDA Jounce Therapeutics Juno Therapeutics Novartis-Dendreon Roche-Immatics $17M* 2013 Access to Immatics proprietary TUMAP peptide vaccines. * plus up to $1B research funding and milestones. Roche-Inovia $10M* 2013 Two of Inovia’s preclinical therapies. * Plus up to $412M in funding and potential milestones. $12M 2014 Series A funding. Founder: Dario Campana. T-cells with CD16 FcReceptors to bind mAbs. UNUM Therapeutics LC 10/22/2014. Data from Sheridan 2013, 2014 Nat Biotechnol; Herper 2014 Forbes; MDA Strategic Alliances web page; Companies Internet web sites; current news.