Abstract number: 893 A chimeric PD1-CD28 switch receptor enhances the activity of TRuC-T cells

Derrick P. McCarthy1, Mike Lofgren1, Shruti Datari1, Philippe Kieffer-Kwon1, Chris Rold1, Jian Ding1, Holly Horton1, Andrew Cornforth1, Robert Tighe1, Sebastian Kobold2, Robert Hofmeister1, Dario Gutierrez1. 1 TCR2 Therapeutics, Cambridge, MA, USA. 2Ludwig Maximilian University of Munich, Munich, Germany.

Abstract TRuC- Platform TC-210 Efficacy in Mesothelioma Xenograft Model A Chimeric PD-1 Receptor Cluster of differentiation 28 (CD28) and programmed death receptor 1 (PD-1) are members of the CD28 superfamily of co-receptors that have critical roles in the regulation of T cell-mediated immunity and inflammation. Upon ligand binding, CD28 signaling synergizes with T cell receptor (TCR) signaling to enhance T-cell activation through the PI3K-Akt PD- PD-L1 pathway, while PD-1 signaling upon binding to its ligands (PD-L1/L2) sequesters critical mediators of signaling from the TCR complex, thereby shunting T-cell activation and effector function. Thus, the expression of PD-L1/L2 in solid tumors Chimeric may pose a significant barrier to anti-tumor immunity and the efficacy of adoptive T cell therapies (ACT). We have PD-1 PD-1 recently described a novel class of engineered T cells that integrate a T cell receptor fusion construct (TRuC®) into the receptor receptor natural TCR complex, thereby reprogramming the specificity of the T cell to recognize tumor surface in a human leukocyte antigen (HLA)-independent fashion. TC-210 T cells expressing mesothelin (MSLN) specific TRuCs demonstrate CD28 robust anti-tumor immunity in preclinical models of mesothelioma, protecting mice from tumor re-challenge while intracellular inducing lower levels of inflammatory cytokine release when compared to a 2nd generation MSLN-targeted CAR T cell. domain Here, we show that co-expression of a PD-1:CD28 switch receptor comprising the PD-1 extracellular and transmembrane domains fused to the CD28 intracellular domain, enhances the activity of TC-210 T cells. When Ligation of PD-L1 by PD-1 Fusion of the CD28 compared to TC-210 expressing only the TRuC, co-expression of the PD1:CD28 receptor showed increased Phospho-Erk during TCR stimulation intracellular domain to the PD- mediates inhibition of T-cell 1 receptor can convert PD-1 signaling and maintained effector cytokine production in the presence of PD-L1. In a repetitive stimulation assay, TRuC function in the TME. signaling into a positive T cells bearing the PD-1:CD28 receptor demonstrated a competitive advantage in expansion and survival over time, and Functional persistence of TC-210 was tested in MSTO model by injecting new tumor cells to TC-210 treated NSG costimulatory signal. mice that had cleared the primary MSTO tumors (tumor-free for 30 days at the time of re-challenge). T cell dose: this enhanced fitness was dependent on the costimulatory domain of the chimeric PD-1 receptor. In vivo and further 2x106 TRuC+ cells. mechanistic studies are currently underway. Null TRuC-T cell phenotype The PD-1 switch enhances early TCR signaling and TH1 polarization A CD28 switch provides little enhancement to TRuC-T cell function A B A B C A NT TC-210 PD1TM PD1TMMutant PD1Trunc NT TC-210 CD28TM PD1TM 5 60 2.0×102 2.0×102 3×10 50 CD3+ T cells ) 2 ) 2 + + ) 1.5×10 1.5×10 CD3+ T cells + 40 2×105 +

30 1.0×102 2

1.0×10 (pg/mL) γ TRuC pERK 20 pERK 5 (% of CD3 1 1×10 -

1 1 IFN- (MFI of TRuC 5.0×10 (MFI of TRuC 5.0×10 PD 10 1 - TRuC NT TC-210 CD28TM PD1TM 0 PD NT TC-210 CD28TM PD1TM 0.0 0.0 0 TRuC MSTO MSTOMSLNMSTOMSLN-PD-L1 MSTO MSTOMSLNMSTOMSLN-PD-L1 MSTOMSLNMSTOMSLN-PD-L1 +αPD-1 At 10 minutes At 60 minutes TRuC+ T cells C D E F 5 4 3 4 C 1×10 1.5×104 8×10 6×10 2×10 B

8×104

) 4 + 6×10 4 3 1×104 1.0×10 4×10 6×104 CD4

4 (pg/mL) (pg/mL) CD8 4×10 NT TC-210 CD28TM PD1TM γ 4 α 4×10 5.0×103 2×103 5×103 IFN- IL-2 (pg/mL)

(MFI of TRuC 4 + + 2×10 TNF- 2×104 TRuC CD4 T GM-CSF (pg/mL) TRuC Expression cells 0 0.0 0 0 0 NT TC-210 CD28TM PD1TM MSLN MSLN-PD-L1 MSTO MSTOMSLN MSTOMSLN-PD-L1 MSTOMSLNMSTOMSLN-PD-L1 +αPD-1 MSTOMSLNMSTOMSLN-PD-L1 +αPD-1 MSTO MSTO +αPD-1 D At 10 minutes D G H I E

CD45RA 1×104 8×103 3×102 3 2×102 3×101 CCR7 NT TC-210 CD28TM PD1TM 8×10 3 8×10 6×103

) 3 + + + 6×10 2×102 TRuC CD8 T 1×102 2×101 6×103 cells 3 (pg/mL) 3 4×10

4×10 α 3 4×10 2 1 1 1×10 5×10 1×10 IL-2 (pg/mL) 3 IL-4 (pg/mL) 2×10 TNF-

3 IL-10 (pg/mL) IL-17A (pg/mL) IL-17A (MFI of TRuC 2×10 3 PD-1 Expression 2×10

0 0 CD45RA 0 0 0 0 MSTO MSTOMSLN MSTOMSLN-PD-L1 MSTO MSTOMSLN MSTOMSLN-PD-L1 CCR7 NT TC-210 CD28TM PD1TM MSTOMSLNMSTOMSLN-PD-L1 +αPD-1 MSTOMSLNMSTOMSLN-PD-L1 +αPD-1 MSTOMSLNMSTOMSLN-PD-L1 +αPD-1

Day 10 TRuC-T cell characterization. (A) Primary T cells were transduced 24 hours after activation with a lentiviral construct containing the MH1 anti- Day 10 TRuC-T cell receptor signaling and polarization. (A-B) TruC-T cells were thawed and co-cultured at a 3:1 ratio with the low-antigen expressing cell Importance of the intracellular costimulatory domain of the chimeric PD-1 receptor. (A) TRuC-T cell characterization of transduction efficiency mesothelin TRuC (TC-210), or with either of two bicistronic constructs containing the anti-mesothelin TRuC followed by a sequence encoding the line MSTO-211H (MSTO), MSTO overexpressing mesothelin (MSTOMSLN), or MSTOMSLN overexpressing PD-L1 (MSTOMSLN-PD-L1). Cultures were harvested at by lentiviral constructs encoding anti-mesothelin TRuC (TC-210), the anti-mesothelin TRuC plus PD1TM, and a PD1TM receptor with null CD28 extracellular PD-1 domain and the intracellular signaling domain of CD28. The transmembrane domain of either CD28 (CD28TM) or PD-1 (PD1TM) was the indicated timepoints, fixed and permeabilized in methanol before staining with antibodies. Graphs depict the mean fluorescence intensity (MFI) of ITAMs (PD1TMMutant) or a truncated version of the PD-1 receptor lacking an intracellular domain (PD1Trunc). TRuC-T cells were stained for the MH1 used to stabilize expression of the chimeric receptor at the cell membrane. At day 10 of expansion, T cells where characterized for co-expression of PD-1 phosphorylated ERK (pERK) at 10 minutes (D) and at 60 minutes (E) of incubation. Plotted data represent the mean ± SEM of two independent experiments TRuC receptor and PD-1 prior to normalizing for transduction efficiency with expanded non-transduced T cells. (B) T cells were incubated at a 3:1 and the TRuC receptor, CD4 and CD8, and memory phenotype (CD45RA versus CCR7). Data shown are from a single experiment representing 3 normal with two separate donors. Data were analyzed for statistical significance by two-way ANOVA.(C-I) TRuC T cells were co-cultured with MSTO, MSTOMSLN, ratio with the tumor cell lines for 10 minutes and then fixed and stained for p-ERK. Data are from two experiments where mean and SEM are shown. donors. (B) Percent transduction of CD3+ T cells as measured by TRuC receptor expression on Day 10 of expansion following activation and transduction MSTOMSLN-PD-L1 and MSTOMSLN-PD-L1 plus a monoclonal anti-PD-1 antibody (+αPD-1) at a 1:1 effector to tumor ratio for 24 hours. At the end of incubation, the (C-E) T cells normalized for transduction efficiency were plated with tumor cells at a a 1:1 ratio and incubated for 72 hours. At 72 hours, supernatants (C) Mean florescence intensity (MFI) of TRuC receptor expression on TRuC+ T cells. (D) MFI of PD-1 expression on TRuC+ T cells.. culture supernatants were harvested for cytokine analysis by MSD ELISA. Results shown are from 2 donors and are plotted as mean (± SEM). Data were were collected from the cultures and analyzed by MSD for IFN-γ (C), IL-2 (D), and TNF-α (E). Data shown are from a single donor representing 2 analyzed for statistical significance by two-way ANOVA.. independent experiments. The chimeric PD-1 receptor confers a competitive advantage to TRuC-T cells in a repetitive stimulation assay Conclusions +Tumor In agreement with previously published reports1,2, the expression of a chimeric PD-1:CD28 receptor A 1 The chimeric PD-1 receptor confers enhanced fitness during 40 B 4 C 4×103 D 4×10 5×10 repeated stimulation challenge. (A) Day 10 TRuC-T cells were significantly enhanced the function of our anti-mesothelin TRuC-T cell when cultured in the presence of cancer 3 +Tumor 3×10 normalized for transduction efficiency to 20% and cultured with MSLN- 4×104 1 cell lines expressing PD-L1 by augmenting TCR signaling and effector cytokine production. 30 2×103 3×10 bearing tumor cells at a 1:20 effector to tumor ratio. On day 4, day 8 and

) 3 + +Tumor 4 1×10 day 12 post initiation, cultures were harvested, counted, and stained for 3×10 1 20 (pg/mL) 2×10 analysis by flow cytometry. The data plotted represent the mean (± SEM) 1 α (pg/mL) 2×10 γ 4 fold expansion of TRuC T cells over time in two combined experiments This enhanced responsiveness of TRuC T cells bearing the chimeric receptor may be partially attributed to a (TRuC 2×10 MSLN 3 IL-2 (pg/mL) 1 using separate donors. Closed symbols represent cultures of MSTO TNF- 10 IFN- 1×10 small role of the PD-1 switch receptor functioning as a decoy PD-1 ; however, the major contribution of the PD- Fold Expansion 1 1×104 1×10 while open symbols represent cultures of MSTOMSLN-PD-L1. Arrows indicate days on which T cells were challenged with additional tumor cell 1 switch receptor was in providing costimulation to TRuC-T cells encountering PD-L1 as evidenced by the 0 0 0 0 targets. Data were analyzed for statistical significance by two-way enhanced proliferation and survival that it conferred in a repetitive stimulation assay. Day 0 Day 4 Day 8 Day 12 1st Stim 2nd Stim 3rd Stim 1st Stim 2nd Stim 3rd Stim 1st Stim 2nd Stim 3rd Stim ANOVA. (B-D) Supernatants were harvested from MSTOMSLN-PD-L1 cultures 72 hours after each challenge (1st stim, 2nd stim, and 3rd stim) and analyzed by MSD ELISA for (B) IFN-γ, (C) IL-2 and (D) TNF-α. (E) TC-210 contracts following repeated challenge with PD-L1 expressing Expression of a chimeric PD-1:CD28 receptor represents a significant enhancement to our TRuC-T Mutant Trunc E NT TC-210 PD1TM PD1TM PD1 F Day 4 Day 8 Day 12 targets. FACs plots of PD-1 and TRuC receptor expression on viable cells to counteract the PD-L1/PD-L2-mediated inhibition of T-cell function in solid tumors. CD45+CD3+ T cells from MSTOMSLN-PD-L1 cultures at day 12 of culture. (F) TRuC T cell proliferation as measured by CellTraceViolet (CTV) labeling References CD3+ T cells on day 4, day 8, and day 12 of cultures repeatedly challenged with MSTOMSLN-PD-L1 targets. T cells were gated on viable CD45+CD3+TRuC+ 1. Kobold S, et al. Impact of a new fusion receptor on PD-1 mediated immunosuppression in adoptive T cell cells. therapy. J Natl Canc Inst 2015. 2. Liu X, et al. A chimeric switch receptor targeting PD-1 augments the efficacy of second-generation CAR T 1 - cells in advanced solid tumors. Cancer Res 2016. PD Day 12 TRuC Expression TRuC CTV 3. Cherkassky L, et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. JCI 2016.