PRECLINICAL CHARACTERIZATION AND MODELLING OF ALLOGENEIC Vg9Vd2 TAC T CELLS FOR THE TREATMENT OF

SOLID TUMORS

Stacey X. Xu, Suzanna L. Prosser, Ling Wang, Ritu R. Randhawa, Sailaja Pirati,

Deepika Bhemarasetty, Kyle MacDonald, Seung Mi Yoo, Miyoung E. Jung, Laurentia A. Gheorghiu, Chris L. Ayers,

Sadhak Sengupta, Christopher W. Helsen, and Andreas G. Bader

Triumvira Immunologics, 9433 Bee Caves Rd Building 1, Suite 240, Austin, TX 78733, USA (Headquarters),

270 Longwood Road South, Hamilton, Ontario L8P 0A6, Canada (Research Division)

ABSTRACT TAC SCIENCEA

CD69

Background

The T cell antigen coupler (TAC) is a novel, proprietary chimeric receptor that facilitates the redirection of Cells Antigen Binding + T cells to tumor cells and activates T cells by co-opting the endogenous T cell receptor complex with the Domain goal to elicit safe and durable anti-tumor responses. TAC01-HER2, a first-in-class, autologous TAC T cell %CD69 product targeting HER2 (ERBB2), has entered a phase I/II clinical trial in patients with HER2-positive solid tumors. Vγ9Vδ2 (γδ) T cells belong to a subset of T cells that recognize target cells in an HLA- CD3 e independent manner. Consequently, γδ T cells are not expected to cause GvHD and, thus, have the Binding Domain potential for allogeneic cell therapy applications. Here, we present preclinical data of an allogeneic Binder C D UCHT1 D A Full length T HER2-TAC γδ T cell product based on Vγ9Vδ2 T cells. B The membrane-bound TAC ...binds directly to the targeted Materials and Methods **IFN-**g receptor interacts directly with tumor antigen. Clustering of The potency and safety of HER2-TAC γδ T cells generated from multiple donors was evaluated using a C D T M Domain the TCR-CD3 epsilon domain TAC-TCR complexes leads to variety of in vitro and in vivo assays. Flow cytometric analysis was used to determine cellular phenotypes, Cells + Lck Interaction and... recruitment of kinases (Lck) via intracellular cytokines, CD69 upregulation, and T cell proliferation in response to target antigen. Site the cytoplasmic co-receptor okine Cytotoxicity was assessed via both luciferase-based killing and real-time microscopy-based co-culture domain and... assays. To assess HER2-TAC γδ T cell responses to HLA mismatches between unrelated donors, mixed %Cyt lymphocyte reactions (MLR) were performed using dendritic cells representing the major North American HLA subtypes. In vivo studies examined the anti-tumor effects of HER2-TAC γδ T cells against established Key features of TAC technology: HER2-expressing solid tumors. Binder UCHT1 C D A D Results Full length T

• TAC functions independently of HER2-TAC γδ T cells selectively reacted to HER2-expressing tumor cells in vitro, as measured by CD69 MHC Antigen negative Antigen positive T cell Activator upregulation, intracellular cytokine production, proliferation, and cytotoxicity. In contrast, HER2-TAC γδ T cells failed to show proliferative activity in MLR assays, indicating that HER2-TAC γδ T cells are likely- TAC activates T cells via the free of GvH reactivity. In addition, HER2-TAC γδ T cells showed strong anti-tumor efficacy in HER2-positive tumor xenograft models without signs of toxicity. Similar anti-tumor efficacy was observed in both NSG mice bearing the human IL-15 transgene (Tg-hIL-15) and NSG mice supplemented with

• TAC incorporates the co-receptor ... initiates T cell activation via This results in effective cell gd T cells were engineered using either the full length TAC, or exogenous IL-15. Comparison of serum IL-15 levels between Tg-hIL15 mice and NSG mice with cytokine and recruits the TCR complex, the endogenous CD3-TCR lysis of multiple tumor cells mutant TAC missing either the antigen-binding domain (DBinder) or supplementation revealed significantly lower levels in Tg-hIL15 mice, suggesting that Tg-hIL15 mice mimicking natural TCR activation complex. during multiple killing events. missing the CD3-binding domain (DUCHT1). Engineered T cells may be a more physiologically relevant model to study γδ T cells in vivo. were co-cultured with antigen-positive or -negative target cells, or Conclusions stimulated with a T cell activator as a positive control. A. Early HER2-targeted TAC γδ T cells display strong and specific activity against HER2-expressing tumor models activation as measured by CD69 upregulation. B. Intracellular in vitro and in vivo. This highlights the versatility of the TAC platform and its potential in the development cytokines. Watch a short animation to understand the TAC mechanism of an allogeneic product for therapeutic applications in solid tumors.

AHER2-TAC γδ T cells NTD γδ T cellsB

T cells alone T cells + Target cells

C TAC

73.16% 26.83% GDT Donor #1

**94.29% 5.71%**s l l s GDT Donor #2 l e l e C GDT Donor #3+ C e ABT + n i k o NTD t D y C C GDT Donor #1 γδ % % GDT Donor #2 CR

0.00% 0.00% 0.00% 0.00% T GDT Donor #3 Count Normalized Division Index-10 -10 ABT TAC NTD TAC NTD IFN-g TNF-a CTV CD69

Stimulation with Target Antigen Elicits a Strong HER2-TAC gdT Cell Response

HER2-TAC gdT cells engineered from 3 healthy donors were tested in a range of in vitro assays. TAC01-HER2 ab T cells (ABT) served as a positive control.

Proliferation: **A.**4-day co-culture with HT1080 target cells. Early Activation: **B.**4-hour co-culture with N87 target cells. Intracellular Cytokines: **C.**4-hour co-culture with N87 target cells. All data shown as mean ± SD.

A B C

GDT #1 GDT #2 GDT #3 NTD )

Target alone HER2-TAC gd T Cells Eectively Kill Tumor Cells In 1:1 TAC HER2/eGFP Vitro GDT Donor #1 18-hour Cytotoxicity: A. Luciferase-based killing assay following co- GDT Donor #2 eLuc HER2/eGFP culture with HT1080 target cells. GDT Donor #3 NALM6 5-day Cytotoxicity: (B,C) HER2-TAC gd T cells were co-cultured with ABT C:NALM6DRAQ7HER2/eGFP 1:2.5NALM6 target cells at different E:T ratios. Tumor cell growth was A NTD (death dye) monitored by GFP fluorescence. Data is normalized to target cells alone**.**

% Cancer Cells % Cancer Cells

GDT Donor #1 T ar

B. Mean percentage tumor cell survival is shown ± SD. C. Example images of 5-day co-cultures. GDT Donor #2 get alone of 5-day co-cultures. GDT Donor #3 1:5 ABT E:T Ratio (T 1:1 1:5 1:1 5:1 1:10 1:2.5 1:100 1:1000

Tumor Spleen

A TAC gd T cells (Tg-NSG_hIL-15) NTD TAC gd T cells (NSG + IL-15) NT

0.74% 2.85% 1.87% 0.22%

Allele Frequency

) ) Tg-NSG_hIL-15 NSG + IL-15 DRA01:01;DRB101:01 6.99% ACT ACT DRA01:01;DRB107:01 11.83%

DRA01:01;DRB103:01 10.24%

-10 DRA01:01;DRB111:01 5.56% -10 olume (mm54.60% 41.81% 10.27% 87.65% olume (mm -10 CD56 -10-10-10 CD3

umor V umor V 0.32% 1.73% 0.57% 1.29% T T

% Stimulation

Add IL-15 3x per week

N87 N87

s.c. s.c. -10-10

ab-10-10 Days Post-ACT CR63.77% 34.18%-10 11.34% 86.79% TAC NTD TAC NTD TAC NTD T -10-10 HER2-TAC gd T Cells Demonstrate Ecacy in Mice ol #1 ol #2 Donor #1 Donor #2 Donor #3

B TCRgd

Tg-NSG_hIL-15 Expressing Human IL-15 Contr Contr NSG + IL-15

A. NSG and transgenic Tg-NSG_hIL-15 mice inoculated with N87 HER2-TAC gd T cells persist in vivo without expansion 6 tumor cells were treated with 12 x 10 HER2-TAC gd T cells or controls. MLR assay shows HER2-TAC gd T cells do not proliferate when 10 3 of natural killer and ab T cells NSG mice were supplemented with IL-15 (28 000 IU) starting 1 hour Tumor and spleen were isolated from a tumor-bearing transgenicstimulated with different HLA subtypes, indicating lack of 2 10 pre-ACT, then 3 times per week thereafter. B. IL-15 serum levels were measured in untreated, tumor-bearing NSG mice either 1 hour or 2 101 post-injection cells. Tissues were processed into single cell suspension and HER2-TAC gd T cells engineered from 3 healthy donors (GDT #1-3) were tested in a mixed 1 hr 48 hr days after a single IL-15 injection. Tg-NSG_hIL-15 mice were bled as positively-enriched for hCD45+ by magnetic beads. Populations were lymphocyte reaction (MLR) assay with dendritic cells derived from 5 different donors comparisons and not supplemented with any cytokines. gated on live cells and presence of natural killer (NK) cells was covering the most dominant North American HLA subtypes (inset table). assessed by CD56 and CD3 staining.

Summary

Ÿ HER2-TAC gd T cells are selectively activated in the presence of HER2-positive tumor Ÿ HER2-TAC gd T cells effectively eradicate HER2-expressing solid tumors

cells in vivo

Ÿ HER2-TAC gd T cells display strong cytotoxicity towards HER2-positive tumor cells in Ÿ HER2-TAC gd T cells lack activity in MLR assays, highlighting its potential as

co-culture assays an allogeneic cell therapy platform