(19) *EP002997132B1* (11) EP 2 997 132 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 5/0783 (2010.01) C07K 16/28 (2006.01) (2006.01) (2006.01) 12.08.2020 Bulletin 2020/33 C07K 14/735 C07K 14/725 (21) Application number: 14727626.5 (86) International application number: PCT/IB2014/061409 (22) Date of filing: 13.05.2014 (87) International publication number: WO 2014/184741 (20.11.2014 Gazette 2014/47) (54) METHODS FOR ENGINEERING ALLOGENEIC AND HIGHLY ACTIVE T CELL FOR IMMUNOTHERAPY VERFAHREN ZUM ENGINEERING ALLOGENER UND HOCHAKTIVER T-ZELLEN FÜR EINE IMMUNTHERAPIE PROCÉDÉ PERMETTANT D’OBTENIR PAR GÉNIE GÉNÉTIQUE DES LYMPHOCYTES T ALLOGÉNIQUES ET HAUTEMENT ACTIFS DESTINÉS À L’IMMUNOTHÉRAPIE (84) Designated Contracting States: (74) Representative: Santarelli AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 49, avenue des Champs-Elysées GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 75008 Paris (FR) PL PT RO RS SE SI SK SM TR (56) References cited: (30) Priority: 13.05.2013 US 201313892805 • MIZOGUCHI E ET AL: "Pathogenic role of IL-4, 13.05.2013 PCT/US2013/040755 but not IFN-gamma in colitis of TCRalpha 13.05.2013 PCT/US2013/040766 knockout mice", GASTROENTEROLOGY, 15.07.2013 US 201313942191 ELSEVIER, PHILADELPHIA, PA, vol. 114, 15 April 1998 (1998-04-15), page A1041, XP027469136, (43) Date of publication of application: ISSN: 0016-5085, DOI: 23.03.2016 Bulletin 2016/12 10.1016/S0016-5085(98)84235-2 [retrieved on 1998-04-15] (73) Proprietor: Cellectis • H. TORIKAI ET AL: "A foundation for universal 75013 Paris (FR) T-cell based immunotherapy: T cells engineered to express a CD19-specific (72) Inventors: chimeric-antigen-receptor and eliminate • GALETTO, Roman expression of endogenous TCR", BLOOD, vol. 75014 Paris (FR) 119, no. 24, 14 June 2012 (2012-06-14), pages • GOUBLE, Agnes 5697-5705, XP55071623, ISSN: 0006-4971, DOI: 75017 Paris (FR) 10.1182/blood-2012-01-405365 • GROSSE, Stephanie • ZACHARY A. COOPER ET AL: "Combining 77750 Saint Cyr Sur Morin (FR) checkpoint inhibitors and BRAF-targeted agents • SCHIFFER-MANNIOUI, Cécile against metastatic melanoma", 94350 Villiers sur Marne (FR) ONCOIMMUNOLOGY, vol. 2, no. 5, 1 May 2013 • POIROT, Laurent (2013-05-01), page e24320, XP055133315, ISSN: 75018 Paris (FR) 2162-4011, DOI: 10.4161/onci.24320 • SCHARENBERG, Andrew Seattle Washington 98177 (US) • SMITH, Julianne 92350 Le Plessis Robinson (FR) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 997 132 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 997 132 B1 • M. M. MAHFOUZ ET AL: "De novo-engineered • UTTENTHAL B J ET AL: "Challenges in T cell transcription activator-like effector (TALE) hybrid receptor gene therapy", JOURNAL OF GENE nuclease with novel DNA binding specificity MEDICINE, JOHN WILEY & SONS, INC, US, vol. creates double-strand breaks", PROCEEDINGS 14, no. 6, 1 June 2012 (2012-06-01), pages 386-399, OF THE NATIONAL ACADEMY OF SCIENCES, XP002760096, ISSN: 1099-498X, DOI: vol. 108, no. 6, 8 February 2011 (2011-02-08), 10.1002/JGM.2637 [retrieved on 2012-06-27] pages 2623-2628, XP055007615, ISSN: 0027-8424, • Michael S Magee ET AL: "Immunotherapeutic DOI: 10.1073/pnas.1019533108 strategies to target prognostic and predictive • PARTOW KEBRIAEI ET AL: "Infusing markers of cancer", Biomarkers in Medicine, vol. CD19-Directed T Cells to Augment Disease 7, no. 1, 1 February 2013 (2013-02-01), pages Control in Patients Undergoing Autologous 23-35, XP55486731, UK ISSN: 1752-0363, DOI: Hematopoietic Stem-Cell Transplantation for 10.2217/bmm.12.110 Advanced B-Lymphoid Malignancies", HUMAN GENE THERAPY, vol. 23, no. 5, 1 May 2012 (2012-05-01), pages 444-450, XP055130799, ISSN: 1043-0342, DOI: 10.1089/hum.2011.167 2 EP 2 997 132 B1 Description Field of the invention 5 [0001] The present invention relates to methods for developing engineered non-alloreactive T-cells for immunotherapy and more specifically to methods for modifying T-cells by inactivating both genes encoding T-cell receptor and at least one immune checkpoint gene to unleash the potential of immune response. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture 10 of primary cells. The invention also relates to further attributes, which can be brought into such engineered T cells, such as preTCRα ("pTalpha") and functional derivatives thereof, Chimeric Antigen Receptor (CAR), multichain CAR and their use thereof to enhance the efficiency of immunotherapy. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections. 15 Background of the invention [0002] Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et 20 al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma. [0003] Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a 25 targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect 30 T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010). 35 [0004] Present CAR architectures are built on a design in which all relevant domains are contained within a single polypeptide. This design necessitates serial appending of signaling domains, thus necessitating moving some domains from their natural juxtamembrane positions. Thus, architectures in which ligands and signaling domains are separate may allow for improved function of costimulatory domains placed on different chains in their normal juxtamembrane positions, rather than appended together with some domains positioned distal from the plasma membrane. A natural 40 receptor, the high affinity receptor for IgE (FcεRI) would afford such architecture. FcεRI present on mast cells and basophils binds IgE with high affinity. FcεRI is a tetrameric receptor complex consisting of ligand binding alpha subunit, a beta subunit and a homodimer of two signal-transducing gamma subunits (Metzger, Alcaraz et al. 1986). FcεRI alpha domain consists of an extracellular domain containing two Ig-like domains that bind IgE, a transmembrane domain and a short cytoplasmic tail. Beta subunit contains four transmembrane segments separating amino and carboxy terminal 45 cytoplasmic tails. The gamma chain consists essentially of a transmembrane region and cytoplasmic tail containing one immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995). The zeta chain of the TCR complex is closely related to the gamma chain and can substitute for the gamma chain of FcεRI (Howard, Rodewald et al. 1990). H. TORIKAI ET AL, BLOOD, 2012, 119: 5697-5705, discloses elimination of endogenous TCR in CAR Tcells. [0005] The current protocol for treatment of patients using adoptive immunotherapy is based on autologous cell transfer. 50 In this approach, T lymphocytes are recovered from patients, genetically modified or selected ex vivo, cultivated in vitro in order to amplify the number of cells if necessary and finally infused into the patient. In addition to lymphocyte infusion, the host may be manipulated in other ways that support the engraftment of the T cells or their participation in an immune response, for example pre-conditioning (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2). Each patient receives an individually fabricated treatment, using the patient’s own lymphocytes (i.e. an 55 autologous therapy). Autologous therapies face substantial technical and logistic hurdles to practical application, their generation requires expensive dedicated facilities and expert personnel, they must be generated in a short time following a patient’s diagnosis, and in many cases, pretreatment of the patient has resulted in degraded immune function, such that the patient’s lymphocytes may be poorly functional and present in very low numbers. Because of these hurdles, 3 EP 2 997 132 B1 each patient’s autologous cell preparation is effectively a new product, resulting in substantial variations in efficacy and safety.