DuoBody -PD-L1 4-1BB (GEN1046) induces superior immune-cell activation, cytokine production and cytotoxicity by combining PD-L1 blockade with conditional 4-1BB co-stimulation Alexander Muik 1 , Isil Altintas 2 , Rachelle

MECHANISM OF ACTION OF

FC-SILENCED PD-L1 4-1BB BSABS

Fc-silenced PD-L1 4-1BB bsAbs are designed to re-activate anti-tumor immune responses by blockade of the PD-1:PD-L1 axis and conditional 4-1BB stimulation. PD-1:PD-L1 checkpoint blockade by the PD-L1-specific arm is constitutively active, whereas co-stimulatory activity mediated by the 4-1BB specific arm strictly depends

on cell-cell crosslinking by simultaneous binding to both 4-1BB and PD-L1. The resulting clustering of 4-1BB provides a local co-stimulatory signal to T cells, further enhancing its T-cell receptor (TCR)-mediated activity and leading to tumor destruction. PD-L1 4-1BB bsAbs are expected to support priming processes in lymphatic tissue, re-activate anti-tumor immune responses in tumor tissue and promote survival of

antigen-specific T cells.

combines checkpoint blockade and conditional 4-1BB agonist activity

Figure 1A. DuoBody-PD-L1 4-1BB was generated by controlled Fab-arm exchange of monoclonal

PD-L1 and 4-1BB antibodies using the DuoBody bispecific antibody platform.7-8 B. The effect of indicated antibodies on the interaction between PD-1 and its ligand PD-L1 was determined in a bioluminescent cell-based PD-1:PD-L1 Blockade Bioassay. C.

4-1BB- agonist activity of the indicated antibodies was determined in a modified luciferase-based

4-1BB-activation reporter assay with or without adherent human PD-L1+ ES-2 tumor cells.

induces expression of genes associated with enhanced immune responses

CD8+ T cells and CD14+ monocytes were isolated from PBMCs, and co-cultured in the presence of 10-90 ng/mL anti-CD3 and 3 g/mL anti-CD28, as well as 0.2 g/mL

DuoBody-PD-L1 4-1BB or atezolizumab. After 48 hours the cells were harvested and subjected to transcriptomic analysis by RNA-sequencing.

demonstrates enhanced proliferation and cytokine secretion of activated T cells in the presence of PD-L1+ cells in vitro

Antigen-specific T-cell proliferation assay: CD8+ T cells were electroporated with RNA encoding an

HLA-A2/CLDN6-specific TCR and PD-1 (A. 5 g; B. w/o, 2 or 10 g, as indicated), and labeled with CFSE,

co-cultured with autologous DCs electroporated with CLDN6-encoding RNA in presence of the indicated antibodies for 4 days. Polyclonal T-cell proliferation assay:

CFSE-labeled human PBMCs were stimulated with 0.1 g/mL anti-CD3 and incubated with the indicated antibodies for 4 days. CFSE dilution in CD8+ T cells

was analyzed by flow cytometry and the expansion index was calculated. C. Cell culture supernatants were collected 48 h after the start of the polyclonal T-cell proliferation assays, and cytokine

concentrations measured using the MSD V-PLEX Pro-inflammatory Panel 1 Human Kit. D: Naive CD8+ T cells were co-cultured with immature or LPS-matured (24 h prior to start of the co-culture) allogeneic dendritic cells in the presence of DuoBody-PD-L1 4-1BB or isotype control for 5 days. IFN- and TNF- concentrations

were determined in the culture supernatant.

demonstrates enhanced antigen-specific T-cell mediated cytotoxicity in vitro

PBMC-derived CD8+ T cells were electroporated with a CLDN6-specific TCR, and preactivated in co-culture with adherent MDA-MB-231 cells stably transduced with CLDN6, or co-cultured with untransduced parental cells. One day later, 4-1BB expression

in preactivated T cells was verified. The T cells were transferred to new co-cultures with CLDN6-transduced or untransduced

MDA-MD-231 cells as before, and incubated in the presence of the indicated antibodies. After 48 hours, T cell intracellular Granzyme B and

LAMP-1 expression was analyzed by flow cytometry (A-B). Separate co-cultures with the identical setup were

subjected to impedance analysis in 30-minute intervals over 5 days using an x CELLigence Real-Time Cell Analyzer (C). D. Naive CD8+ T cells

were co-cultured with LPS-matured (24 h prior to start of the co-culture) allogeneic dendritic cells in the presence of DuoBody-PD-L1 4-1BB or isotype control for 5 days, and GzmB concentrations were determined in the culture supernatant.

increases tumor infiltrating lymphocyte (TIL) expansion in human tumor tissue cultures ex vivo

Figure 5. A. Tumor tissue of a NSCLC patient was

resected, cut into pieces of 1-2 mm3, and cultured in the presence of IL-2 (10 U/mL) and the indicated antibodies.

On day 10, viable TIL were counted and the fold expansion of the indicated subpopulations was analyzed by flow cytometry. Data shown represents mean SD of n=5 individual wells. B. Clonotype repertoire analysis was performed on TCR

sequencing of the CDR3 region of the expanded TIL and the frozen tumor. The ten most frequent clones (1-10) for well 1 of DuoBody-PD-L1 4-1BB-treated cultures were used as reference and the frequency rank of these sequences in all other samples is visualized according to the

color scheme. About 200 sequences were obtained per sample. Each sample contained about 100,000 cells.

An Fc-silenced bsAb targeting mouse

PD-L1 and 4-1BB (mbsAb-PD-L1 4-1BB) induces

tumor regression and increases the frequency of tumor-reactive T cells in CT26-tumor bearing mice

Figure 6. BALB/c mice bearing subcutaneous CT26 tumors

were treated after tumors reached a size 30 mm3. Antibody was dosed every 2-3 days for three weeks starting at day 11 (indicated by arrows). A. Individual tumor growth curves; CR: complete regression. B. Kaplan-Meier analysis of animal survival. C. On day 18, blood

was drawn and the relative frequency of CD8+ T cells specific for gp70, the immunodominant antigen expressed by CT26, was determined by flow cytometry. ****, p<0.0001, One-way ANOVA with Dunnett s multiple comparisons test.

shows superior efficacy compared to a combination of Fc-silenced PD-L1 or 4-1BB monovalent control

Figure 7. BALB/c mice bearing subcutaneous CT26 tumors

were treated after tumors reached a mean size of approximately 100 mm3. Antibody was dosed at 100 g per mouse every 4 days, for three weeks starting at day 20 (indicated by arrows).

A. Individual tumor growth curves, CR: complete regression. B. Kaplan-Meier analysis of animal survival. *, p<0.05; log-rank (Mantel-Cox) test.

shows superior efficacy compared to PD-L1 blockade

Figure 8. BALB/c mice bearing subcutaneous CT26 tumors

were treated after tumors reached a mean size of approximately 76 mm3. Antibody was dosed at 100 g per mouse every 3-4 days for three weeks starting

at day 14 (indicated by arrows). A. individual tumor growth curves, CR: complete regression. B. Kaplan-Meier analysis of animal survival. ***, p<0.001, log-rank

modulates tumor-draining lymph node and intratumoral immune-cell composition in favor of anti-tumor immunity

Figure 9. BALB/c mice bearing subcutaneous CT26 tumors

were treated with the indicated antibodies after tumors reached a median size of 38 mm3. Antibody was dosed at 100 g per mouse on days 12 and 17. Mice were sacrificed on day 18 after

inoculation for analysis. A. Flow cytometry analysis of tdLNs. B-C. tdLN cell suspensions were either left unstimulated (B) or restimulated with a gp70 peptide or CT26 cells

(C), and IFN- secretion was assessed by ELISpot. D. IFN- ELISpot using distant non-draining LN cell (ndLN) suspensions of mbsAb-PD-L1 4-1BB treated mice. E. Flow cytometry analysis of enzymatically dissociated tumors. Data from individual mice are shown, as well as group mean

( SD). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; One-way ANOVA with Dunnett s multiple comparisons test.

PD-(L)1 AND 4-1BB AS TARGETS

MOA AND PRECLINICAL DATA

Figure 1. GEN1046: IgG1 bispecific PD-L1 4-1BB antibody and mechanism of action

Figure 2. Trial design (dose escalation)

Patient characteristics and disposition

Data cut-off: August 31, 2020.

aCancer types occurring in <5 patients were categorized as Other .

ECOG, Eastern Cooperative Oncology Group; NSCLC, non-small cell lung cancer; PD-(L)1, programmed death (ligand) 1.

Data cut-off: August 31, 2020.

Adverse events graded according to National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v.5.0.

TRAE, treatment-related adverse event.

Data cut-off: August 31, 2020.

aOne patient in the 100-mg cohort died due to disease progression on study

bOne backfill patient in the 800-mg cohort did not complete the

C1D8 and C1D15 assessments. The patient elected not to attend follow-up visits to avoid contracting COVID-19.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; DLT, dose-limiting toxicity.

Presented at the Society for Immunotherapy of Cancer (SITC), November 11 14, 2020 Poster #412

Figure 1. Intratumoral mRNA-based cancer immunotherapy: Local approach with systemic antitumor effects

Table 1. Baseline characteristics of all treated patients

Table 2. Frequency of patients with a treatment emergent AEa related to SAR441000 by dose group and grade

Table 3. Frequency of patients with a treatment emergent AEa related to SAR441000 and/or cemiplimab by dose group and grade

Figure 2. Pharmacokinetic and

pharmacodynamic markers in monotherapy-Maximum fold change from baseline measured at any cycle in plasma for IL15 (a), IFNg (b), IP10 (c)

BCC, basal cell cancer; CSCC, cutaneous squamous cell cancer; MCC, Merkel cell carcinoma; TNBC, triple negative breast

A Personal Neoantigen Vaccine NEO-PV-01, in

Combination with Chemotherapy and Pembrolizumab, Induces Broad De Novo Immune

Responses in First-Line Non-Squamous NSCLC

Mark M. Awad1, David R. Spigel2, Edward B. Garon3, Saiama Waqar4, Aaron Lisberg3, Melissa Moles5, Jennifer Tepper5, April Lamb5, Amy Wanamaker5, Zak Khondker5, John Srouji5, Jesse Dong5, Asaf Poran5, Kristen Balogh5, Meghan Bushway5, Mark DeMario5, Lakshmi Srinivasan5, Richard B. Gaynor5, Ramaswamy

1Dana Farber Cancer Institute, Brigham and Women s Hospital, and Harvard Medical School, Boston, MA; 2The Sarah Cannon Research Institute, Tennessee Oncology, PLLC, Nashville, TN;

3Department of Medicine, University of California Los Angeles, Los Angeles, CA; 4Washington University School of Medicine, St Louis, MO; 5BioNTech US Inc., Cambridge, MA

Figure 1: Neoantigens Are Key Targets of Tumor-Directed T cell Responses

NT-002 is a Phase 1B trial of NEO-PV-01 in

combination with chemotherapy and pembrolizumab, in first-line therapy in patients with advanced or metastatic non-squamous NSCLC (NCT03380871). Target enrollment = 15 patients across 4 US sites.

Primary: To evaluate the safety of NEO-PV-01 with pembrolizumab