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WO 2016/174652 Al 3 November 2016 (03.11.2016) P O P C T

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WO 2016/174652 Al 3 November 2016 (03.11.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/174652 Al 3 November 2016 (03.11.2016) P O P C T (51) International Patent Classification: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, A61K 48/00 (2006.01) C07K 16/18 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C07K 14/705 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (21) International Application Number: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PCT/IL2015/050458 PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (22) International Filing Date: SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 30 April 2015 (30.04.2015) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: TECHNION RESEARCH & DEVELOP¬ TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, MENT FOUNDATION LIMITED [IL/IL]; Senate TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, House, Technion City, 3200004 Haifa (IL). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors: REITER, Yoram; 23 Ester Rabin street, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, 3491 784 Haifa (IL). OREN, Ravit; 22 Shalom Ash street, GW, KM, ML, MR, NE, SN, TD, TG). 6948322 Tel Aviv (IL). COHEN, Maya; Moshav Habon- im, Chof Hacarmel, 3084500 Moshav Habonim (IL). Published: — with international search report (Art. 21(3)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, — with sequence listing part of description (Rule 5.2(a)) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (54) Title: CHIMERIC ANTIGEN RECEPTORS AND METHODS OF THEIR USE (57) Abstract: Provided are chimeric antigen receptor (CAR) molecules comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein an affinity of said binding domain to said antigen is characterized by a K D higher than 150 n M. Also provided are isolated polynucleotides and nucleic acid constructs com prising same, cells transduced with same and methods of using same. CHIMERIC ANTIGEN RECEPTORS AND METHODS OF THEIR USE FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to chimeric antigen receptors, cells transduced with same and, more particularly, but not exclusively, to methods of using same for treating various pathologies, such as cancer and autoimmune diseases, as well as infection diseases caused by viral or bacterial infections. Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for hematological malignancies and solid cancers. This approach, in which tumor reactive T cells undergo ex-vivo expansion and are then infused back to patients, has proven to be effective in metastatic melanoma patients. However, the widespread use of this approach is limited by the need to isolate antigen-specific T lymphocytes for individual patients. To overcome this difficulty, the strategy of engineered T cells (CAR) has been developed, which involves genetic modifications based on 2 different approaches reviewed in Restifo, et al., 2012 (Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 12: 269-281). In the first approach, tumor targeting by T cells is achieved using a cloned β TCR which is introduced into the cells and enables specific MHC-restricted targeting of tumor cells. This approach has been proven effective in clinical trials in melanoma) and several groups are currently working to improve the expression of the exogenous TCR on the surface of T cells. The second strategy involves redirection of T cells based on antibody variable fragments (Fv). The availability of anti-tumor antibodies targeting a variety of tumors prompted the idea of incorporating the recognition domain of these antibodies in the form of single chain Fv (scFv) domain in a chimeric receptor construct (13). This chimeric antibody-based or antigen receptor (CAR) is based on linking the recognition elements of an antibody to signaling moieties for T cell activation, thereby redirecting T cells to a desired antigen in an either non-MHC restricted or an MHC restricted manner. In the non-MHC restricted manner, T cells are redirected independently of MHC and proved to be effective against tumor cells that lost their HLA expression due to tumor escape mechanisms (14). After several studies in the last decade that demonstrated the effectiveness of this approach in inducing tumor regression in mouse models, this approach is currently being evaluated in clinical trials (15-17). Furthermore, this strategy has shown dramatic responses in a pilot clinical trial in chronic lymphocytic leukemia (CLL) patients treated with CD19-specific CAR T cells (18). Targeting tumor-associated antigens with engineered T cells that express a specific CAR is an ideal approach combining the unique expression pattern of tumor associated antigens (TAAs) with potent killing mediated by the engineered effector T cell (19). For an MHC restricted manner, antibodies which recognize specific peptide/MHC complexes (termed "TCR like antibodies, or TCRLs) can be used (26). The present inventors and others have isolated antibodies which recognize HLA-A2 complexes bearing peptides derived from tumor and viral antigens by means of phage display and hybridoma strategies (27-30). These TCR-like antibodies, which exhibit both binding properties and kinetics of antibodies (e.g., high affinity), while mimicking the specificity of TCRs, are being used as a novel research tool to study antigen presentation and immunotherapy targets. Wilm's tumor suppressor gene 1 (WT1) is one of the most important TAAs classified by the National Cancer Institute (NCI). WT1, a zinc finger transcription factor, is highly expressed in many solid cancers and leukemia cells, but not in normal tissues (including hematopoetic progenitor and stem cells). Several studies have suggested that WT1 may have an essential role in leukemogenesis/tumorgenesis, and is required to maintain the transformed phenotype/function; therefore, tumor escape from immune surveillance as a result of down-regulation of WT1 expression is unlikely to occur, marking WT1 as an attractive and important target for immunotherapy. The WT1 Db126 (RMFPNAPYL; SEQ ID NO:l) peptide was identified by screening the WT1 amino acid sequence (GenBank Accession NO. EAW68225, Wilms tumor 1, isoform CRA_f; SEQ ID NO:3) for 9-mer peptides that include major anchor motifs for binding to HLA-A2 (24). In-vitro immunization elicits WT1 peptide-specific CTLs which mediate lysis of WTl -expressing tumor cells, indicating that this peptide constitutes a highly immunogenic epitope. The β genes of a TCR which recognizes HLA-A2-WT1 Db126 complexes were isolated using an allogeneic repertoire, and were introduced by retroviral transduction into human T cells (7). This TCR exhibited efficient and specific reactivity with HLA-A2-WT1 Db126 complexes, enabling specific cytolytic reactivity by CTLs expressing the TCR toward target cells (25)· CARs have been successfully used in the treatment of various leukemias, and they are currently being used in clinical trials also for various solid tumors, however, their use is limited by the number of cancer cells which are not recognized by T cells, mainly due to limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC) expression of cancer cells. Additional background art includes Chmielewski et al., 2013 (Antigen-specific T-cell activation independently of the MHC: chimeric antigen receptor-redirected T cells. Frontiers in Immunology, vol. 4, article 371), U.S. Patent Application No. 20130287748. SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a chimeric antigen receptor (CAR) molecule comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein an affinity of the binding domain to the antigen is characterized by a KD higher than 150 nM. According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the molecule of some embodiments of the invention. According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence encoding the molecule of some embodiments of the invention and a cis-acting regulatory element for directing transcription of the isolated polynucleotide in a host cell. According to an aspect of some embodiments of the present invention there is provided an isolated cell comprising the polynucleotide of claim of some embodiments of the invention or the nucleic acid construct of some embodiments of the invention. According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the CAR
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