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WO 2019/014581 Al 17 January 2019 (17.01.2019) W !P O PCT

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WO 2019/014581 Al 17 January 2019 (17.01.2019) W !P O PCT (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 2019/014581 Al 17 January 2019 (17.01.2019) W !P O PCT (51) International Patent Classification: HOSPITAL, INC. [US/US]; 75 Francis Street, Boston, C12Q 1/6886 (2018.01) C12N 5/0783 (2010.01) Massachusetts 021 15 (US). CI2Q 1/00 (2006.01) (72) Inventors; and (21) International Application Number: (71) Applicants: KUCHROO, Vijay K. [US/US]; c/o 75 PCT/US20 18/042069 Francis Street, Boston, Massachusetts 021 15 (US). AN¬ DERSON, Ana C. [US/US]; c/o 75 Francis Street, Boston, (22) International Filing Date: Massachusetts 021 15 (US). KURTULUS, Sema [US/US]; 13 July 2018 (13.07.2018) c/o 75 Francis Street, Boston, Massachusetts 021 15 (US). (25) Filing Language: English MADI, Asaf [US/US]; c/o 75 Francis Street, Boston, Mass achusetts 021 15 (US). (26) Publication Language: English (72) Inventor: REGEV, Aviv; c/o 415 Main Street, Cambridge, (30) Priority Data: Massachusetts 02142 (US). 62/532,556 14 July 2017 (14.07.2017) 62/636,637 28 February 2018 (28.02.2018) (74) Agent: SCHER, Michael B. et al; Johnson, Marcou & Isaacs, LLC, P.O. Box 691, Hoschton, Georgia 30548 (US). (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; 415 Main Street, Cambridge, Massachusetts 02142 (US). (81) Designated States (unless otherwise indicated, for every MASSACHUSETTS INSTITUTE OF TECHNOLOGY kind of national protection available): AE, AG, AL, AM, [US/US]; 77 Massachusetts Avenue, Cambridge, Massa AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, chusetts 02139 (US). THE BRIGHAM AND WOMEN'S CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (54) Title: METHODS AND COMPOSITIONS FOR MODULATING CYTOTOXIC LYMPHOCYTE ACTIVITY PD-r CD8+ FIG. 2 H CD6 artit/- CX C 1" CX3CR1 2 1.8 PD-1 CD8 j 79.4 ! KLRG1 - 00 (57) Abstract: The subject matter disclosed herein is generally directed to novel CD8 tumor infiltrating lymphocyte (TIL) subtypes associated with response to immunotherapy treatment. Specifically, the subtypes are associated with checkpoint blockade therapy. © Moreover, the subject matter disclosed herein is generally directed to methods and compositions for use of the subtypes. Also, disclosed herein are gene signatures and markers associated with the subtypes and use of said signatures and markers. Further disclosed are thera peutic methods of using said gene signatures and immune cell subtypes. Further disclosed are pharmaceutical compositions comprising o populations of CD8+TILs enriched for a specific subtype. o [Continued on nextpage] WO 2019/014581 Al llll II II 11III III III I i I II il II I II DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, 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, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: METHODS AND COMPOSITIONS FOR MODULATING CYTOTOXIC LYMPHOCYTE ACTIVITY CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Nos. 62/532,556, filed July 14, 2017 and 62/636,637, filed February 28, 2018. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under Grant Nos. CA1 87975, AI073748 and NS045937 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The subject matter disclosed herein is generally directed to CD8+ tumor infiltrating lymphocyte subtypes associated with response to immunotherapy treatment. Moreover, the subject matter disclosed herein is generally directed to detecting, isolating and using said subtypes. BACKGROUND [0004] The CD8+ T cell response within the tumor microenvironment (TME) is functionally (Sakuishi et al, 2010; Williams et al., 2017; Woo et al, 2012; Xu et al., 2015) and transcriptionally (Singer et al., 2016; Tirosh et al., 2016; Zheng et al., 2017) heterogeneous. At one end of the functional spectrum are CD8+ tumor-infiltrating lymphocytes (TILs) that lack the expression of co-inhibitory or immune checkpoint receptors (eg. CTLA-4 and PD-1) and exhibit effector potential, while at the opposite end are CD8+ TILs that co-express multiple checkpoint receptors and exhibit an "exhausted" or dysfunctional phenotype. Checkpoint blockade immunotherapy, using antibodies against co-inhibitory receptors, unleashes a potent effector CD8+ T cell response resulting in anti-tumor immunity and durable clinical responses. However, it is not clear which CD8+ T cell populations change in response to checkpoint blockade therapy. One possibility is that checkpoint blockade acts directly on dysfunctional T cells that express checkpoint receptors, thereby re-invigorating them. Conversely, checkpoint blockade may indirectly generate an environment that promotes optimal differentiation of T cell precursors into effector cells. Understanding how the functional spectrum of CD8+ TILs changes upon checkpoint blockade immunotherapy could provide information to improve current strategies for harnessing the anti-tumor CD8+ T cell response and could lead to the identification of biomarkers to track responses to therapies. [0005] Antibodies that block the activity of checkpoint receptors, including CTLA-4, PD- 1, Tim-3, Lag-3, and TIGIT, either alone or in combination, have been associated with improved effector CD8+ T cell responses in multiple pre-clinical cancer models (Johnston et al., 2014; Ngiow et al, 2011; Sakuishi et al, 2010; Woo et al, 2012). Similarly, blockade of CTLA-4 and PD-1 in patients (Brahmer et al., 2012; Hodi et al., 2010; Schadendorf et al, 2015; Topalian et al, 2012; Wolchok et al., 2017) has shown increased frequencies of proliferating T cells, often with specificity for tumor antigens, as well as increased CD8+ T cell effector function (Ayers et al, 2017; Das et al, 2015; Gubin et al, 2014; Huang et al., 2017; Kamphorst et al, 2017; Kvistborg et al, 2014; van Rooij et al, 2013; Yuan et al, 2008). Accordingly, the success of checkpoint receptor blockade has been attributed to the binding of blocking antibodies to checkpoint receptors expressed on dysfunctional CD8+ T cells and restoring effector function in these cells. [0006] A recent study suggests that PD-1 blockade acts on a distinct subset of PD-1 + precursors in the setting of chronic viral infection (Im et al., 2016; Utzschneider et al, 2016). However, several studies have indicated that although PD-1 pathway blockade can re- invigorate the effector functions of PD-1 +CD8+ T cells, this effect was transient as these cells had limited memory potential due to their acquisition of a stable epigenetic state that cannot be modified by therapy (Ghoneim et al, 2017; Pauken et al, 2016; Philip et al, 2017; Scott- Browne et al, 2016; Sen et al, 2016). These observations raise the important question of the origin and phenotype of the effector T cells that arise after checkpoint blockade therapy and are responsible for the therapeutic effect. Thus, there is a need to better understand tumor immunity and response to immunotherapy. [0007] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY [0008] The mechanisms underlying how checkpoint blockade therapy alters the functional spectrum of CD8+ tumor-infiltrating lymphocytes (TILs) is poorly understood. Applicants have examined the RNA profiles from bulk and single-cell CD8+ tumor-infiltrating lymphocytes (TILs) following Tim-3/PD-l blockade. Surprisingly, there were significantly higher transcriptional changes in Tim-3 PD- compared to Tim-3+PD-l +CD8+ TILs, leading to the identification of three novel precursor PD- populations that separately have features of naive, effector, and memory-precursor-like CD8+ T cells. Following Tim-3/PD-l blockade, the proportion of memory-precursor-like and effector-like TIL subsets increases relative to the naive-like subset. Applicants further identified Tcf7 as a regulator of the memory-precursor- like subset and show that different immunotherapies fail in its absence. The memory-precursor- and effector-like subsets contain tumor-antigen specific cells and expand following multiple checkpoint blockade therapies in different cancers. The memory-precursor-like subset shares features with CD8+ T cells that are associated with response to checkpoint blockade in patients and is compromised in the absence of Tcf7, which Applicants show is requisite for the efficacy of diverse immunotherapies. The findings uncover previously unappreciated changes in PD- precursor populations within CD8+ TILs, providing critical insight into development of the effector CD8+ T cell response after immunotherapy.. [0009] It is an objective of the present invention to identify CD8+ TIL subtypes responsive to checkpoint blockade therapy. It is another objective of the present invention to detect gene signatures and biomarkers specific to the CD8+ TIL subtypes, whereby cells may be detected and isolated.
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