US 20190062735A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2019 / 0062735 A1 Welstead et al. ( 43 ) Pub. Date : Feb . 28 , 2019 ( 54 ) CRISPR - CPF1 -RELATED METHODS, (60 ) Provisional application No . 62/ 304 , 057, filed on Mar . COMPOSITIONS AND COMPONENTS FOR 4 , 2016 . CANCER IMMUNOTHERAPY ( 71 ) Applicant: EDITAS MEDICINE , INC ., Publication Classification CAMBRIDGE, MA (US ) (51 ) Int. CI. C12N 15 / 11 (2006 .01 ) ( 72 ) Inventors : Gordon Grant Welstead , Cambridge, C12N 9 /22 (2006 .01 ) MA (US ) ; Hariharan Jayaram , C12N 15 / 90 ( 2006 .01 ) Cambridge , MA (US ); Tongyao Wang , A61K 35 / 17 (2006 . 01) Malden , MA (US ) ; John Anthony (52 ) U . S . CI. Zuris , Cambridge, MA (US ) ; CPC .. .. C12N 15 / 11 ( 2013 .01 ) ; C12N 9 / 22 Christopher Borges, Reading , MA ( 2013 .01 ) ; C12N 15 / 907 (2013 . 01 ) ; C12N (US ) 2320 /32 (2013 . 01 ) ; C12N 2310 / 20 ( 2017 .05 ) ; (73 ) Assignee : EDITAS MEDICINE , INC . , C12N 2800 /80 (2013 . 01 ) ; A61K 35 / 17 CAMBRIDGE, MA (US ) (2013 .01 ) ( 21 ) Appl. No. : 16 /121 , 152 ( 22 ) Filed : Sep . 4 , 2018 (57 ) ABSTRACT Related U . S . Application Data CRISPR /Cpfl - related compositions and methods for treat (63 ) Continuation of application No . PCT/ US2017 / ment of cancer. 020598 , filed on Mar. 3 , 2017 . Specification includes a Sequence Listing . VIVUNIIKIZUIZIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Direct repeat domain Targeting domain Patent Application Publication Feb . 28 , 2019 Sheet 1 of 8 US 2019 /0062735 A1 Targetingdomain FIG.1 Directrepeatdomain Patent Application Publication Feb . 28 , 2019 Sheet 2 of 8 US 2019 / 0062735 A1 100 LadderLadder ????? ? GWED539GWED539 171 GWED540 GWED541 . 8888888888888 GWED543GWED543 GWED544 GWED545 FIG.2 GWED546GWED546 02 * * GWED547 Ladder cutsize: GWED539142,331 146,327GWED540 GWED541190,283 GWED543184,289 159,314GWED544 189,284GWED545 96,377GWED546 157GWED547,136 CrRNAID:Expected FulllengthPCRProduct=450bp Patent Application Publication Feb . 28 , 2019 Sheet 3 of 8 US 2019 /0062735 A1 GWED549 GWED548 GWED547 GWED546 GWED545 LLLLLLLLLLLZTULTITITZIZ GWED544 IDCrRNA FIG.3A WIMMINIMIZ GWED543 GWED541 GWED540 GWED539 & qur?ayaDIIIIIIIIIIIZZZZZZZZZZZZZZINITIZIE ControlH2O negative ß / TCRa % Patent Application Publication Feb . 28 , 2019 Sheet 4 of 8 US 2019 / 0062735 A1 Will GWED539 KOMEX 0.76% TTTTTTTTTTTTT GWED546 20.2% FIG.3B EN GWED545 . 16.5% E Cpf1Alone 0.8% ??????? TCRA/B CD4 Patent Application Publication Feb . 28 , 2019 Sheet 5 of 8 US 2019 / 0062735 A1 / / / www. / / / GWED549 GWED548 GWED547 .ww GWED546 GWED545 ** ** *** ** CrRNAID FIG.4 www GWED544 GWED543 GWED541 . NNN GWED540 / *** / . /* / GWED539 ControlH2O Lymphocytes " Live " % Patent Application Publication Feb . 28 , 2019 Sheet 6 of 8 US 2019 / 0062735 A1 GWED549 GWED546 GWED545 CrRNAID FIG.5 GWED541 Uncut NHEJ % Patent Application Publication Feb . 28 , 2019 Sheet 7 of 8 US 2019 /0062735 A1 GWED546 GWED545 CrRNAID FIG.6 Cpf1alone b / TCR0 + CD4 of Frequency Patent Application Publication Feb . 28 , 2019 Sheet 8 of 8 US 2019 /0062735 A1 NH2 ????? FIG.7 ARCASTRUCTURE H2CO—?0P???2 ?????3 CH? + z — NH2 US 2019 /0062735 A1 Feb . 28 , 2019 CRISPR -CPF1 -RELATED METHODS , domains to a cytoplasmic effector domain . The effector COMPOSITIONS AND COMPONENTS FOR domain is typically derived from the CD3 -zeta chain of the CANCER IMMUNOTHERAPY T cell co - receptor complex , and can also include domains derived from CD28 and / or CD137 receptor proteins . The CROSS REFERENCE TO RELATED CAR extra -cellular domain binds the tumor antigen in an APPLICATIONS MHC - independent manner leading to T cell activation and 0001 ] This application is a Continuation of International proliferation , culminating in cytotoxic anti - tumor activity as described for TCR engineered T cells . Patent Application No . PCT /US17 /20598 filed Mar. 3 , 2017 , [0007 ] To date , at least 15 different tumor antigens have which claims priority to U . S . Provisional Application No . been targeted in clinical trials of engineered T cells . In 62 / 304 ,057 , filed Mar. 4 , 2016 , the contents of each of which several trials , anti - tumor activity has been reported . The are incorporated by reference in their entireties herein , and greatest success has been achieved in hematologic malig priority to each of which is claimed . nancies. For example , adoptive transfer of CAR - T cells engineered to target the B cell antigen , CD19 , led to multiple SEQUENCE LISTING partial and complete responses in subjects with lymphoma, [ 0002 ] The specification further incorporates by reference acute lymphoblastic leukemia , acute lymphocytic leukemia the Sequence Listing submitted herewith via EFS on Sep . 4 , and B - cell acute lymphocytic leukemia . In contrast , trials 2018 . Pursuant to 37 C . F . R . § 1 .52 ( e ) (5 ) , the Sequence targeting other tumor types , especially solid tumors , includ Listing text file , identified as 0841770198SL2. txt , is 738 , ing renal cell carcinoma , neuroblastoma, colorectal cancer, 470 bytes and was created on Nov . 14 , 2018 . The entire breast cancer, ovarian cancer , melanoma, sarcoma and pros contents of the Sequence Listing are hereby incorporated by tate cancer , have been less successful. In many of these reference . The Sequence Listing does not extend beyond the trials , very few patients experienced objective responses . scope of the specification and thus does not contain new Thus , there is a need to improve the anti - tumor efficacy of matter . adoptively transferred engineered T cells . FIELD OF THE PRESENTLY DISCLOSED SUMMARY OF THE PRESENTLY DISCLOSED SUBJECT MATTER SUBJECT MATTER [0003 ] The invention relates to CRISPR / Cpf1 - related [ 0008 ] Methods and compositions disclosed herein pro methods, compositions and components for editing a target vide for the treatment of cancer using an immunotherapy nucleic acid sequence, or modulating expression of a target approach comprising administration of genetically engi nucleic acid sequence , and applications thereof in connec neered T cells or T cell precursors to a subject. An approach tion with cancer immunotherapy comprising adoptive trans to treat a subject suffering from cancer is to isolate T cells fer of engineered T cells or T cell precursors. from the subject, genetically modify them to target an antigen expressed by the cancer cells , then re - introduce them BACKGROUND into the subject ; a process referred to as adoptive T cell [ 0004 ] Adoptive transfer of genetically engineered T cells transfer. Methods to genetically modify T cells include has entered clinical testing as a cancer therapeutic modality . introduction of T cell receptor ( TCR ) or chimeric antigen Typically , the approach consists of the following steps : 1 ) receptor (CAR ) genes encoding trans -membrane TCR or obtaining leukocytes from the subject by apheresis ; 2 ) CAR proteins, respectively , which specifically recognize selecting / enriching for T cells ; 3 ) activating the T cells by particular cancer antigens . In certain embodiments , engage cytokine treatment; 4 ) introducing cloned T cell receptor ment of the tumor - expressed antigen with the antigen bind ( TCR ) genes or a chimeric antigen receptor (CAR ) gene by ing domain of the TCR or CAR protein initiates a signaling retroviral transduction , lentiviral transduction or electropo cascade leading to T cell activation , proliferation and , ulti ration ; 5 ) expanding the T cells by cytokine treatment ; 6 ) mately , destruction of the cancer cell via a cytotoxic immune conditioning the subject, usually by lymphodepletion ; and 7 ) response (Kershaw et al. , 2013 NatRevCancer 13 , 525 -541 ) . infusion of the engineered T cells into the subject. [0009 ] Adoptive T cell transfer utilizing genetically modi [ 0005 ] Sources of cloned TCR genes (TRAC and TRBC ) fied T cells has entered clinical testing as a therapeutic for include rare T cell populations isolated from individuals solid and hematologic malignancies. Results to date have with particular malignancies and T cell clones isolated from been mixed . In hematologic malignancies ( especially lym T cell receptor -humanized mice immunized with specific phoma, Chronic lymphocytic leukemia (CLL ) and Acute tumor antigens or tumor cells . Following adoptive transfer, lymphocytic leukemia ( ALL ) ) , the majority of patients in TCR - engineered T cells recognize their cognate antigen several Phase 1 and 2 trials exhibited at least a partial peptides presented by major histocompatibility complex response , with some exhibiting complete responses (MHC ) proteins on the tumor cell surface . Antigen engage (Kochenderfer , J . N . et al. , 2012 Blood 119 , 2709 -2720 ) . ment stimulates signal transduction pathways leading to T However, in most tumor types ( including melanoma, renal cell activation and proliferation . Stimulated T cells then cell carcinoma and colorectal cancer ) , fewer responses have mount a cytotoxic anti - tumor cell response , typically involv been observed ( Johnson , L . A . et al. , 2009 Blood 114 , ing a secreted complex comprising Granzyme B , perforin 535 - 546 ; Lamers , C . H . et al. , 2013 Mol. Ther . 21, 904 -912 ; and granulysin , inducing tumor cell apoptosis . Warren , R . S . et al ., 1998 Cancer Gene Ther. 5 , S1- S2 ) . [ 0006 ] Chimeric antigen receptor (CAR ) genes encode Thus , there exists a need to improve the efficacy of adoptive artificial T cell receptors comprising an extra - cellular tumor transfer of modified T cells in cancer treatment