Discovery and characterization of novel TNFR2 antibodies to modulate T cell activities

in immunosuppressive environment Presented at the AACR

in immunosuppressive environment

Shuo Wei, Ross Fulton, Guang Yang, Juying Li, Qian Zhang, Matthieu Delince, Greg Rose, Charina Ortega, Pascaline Mary, He Zhou, Andreas Raue, Dean Lee, Roshan Kumar, Stephanie Beq, Jennifer Watkins‐Yoon, Nicola Beltraminelli, Francisco Adrian, Liang Schweizer

Summary

Tumor necrosis factor (TNFR2) TNFR1 has recently emerged a promising therapeutic target for receptor‐2 immuno‐oncology. and TNFR2 areasreceptors for tumor necrosis factor (TNFa). However, unlike TNFR1, TNFR2 is expressed exclusively on immune cells. TNFR2 is a potent co‐stimulatory molecule expressed on the surface of CD8 and CD4 T cells in the tumor micro‐environment. An agonistic antibody against TNFR2 has the potential to further enhance effector T cell functions and their anti‐tumor response. We applied our single‐cell platform CelliGO™ for antibody discovery and discovered a panel of antibodies binding TNFR2 on distinct epitopes (HFB3‐18:CRD1, HFB3‐1:CRD2, HFB3‐14:CRD3).

The selected antibodies bind to TNFR2 without competing with its ligand TNFα, stimulate activated CD4 and CD8 T‐cells and enhance their proliferation in‐vitro. Interestingly, HFB3‐18 requires cross‐linking of Fc receptors for its activity while HFB3‐1 and HFB3‐14 do not. The most potent antibody, HFB3‐1, was humanized and the resulting humanized, HFB200301, was tested in human TNFR2 knock‐in mice bearing syngeneic subcutaneous MC38 tumors. HFB200301 displays potent anti‐tumor activity and combination with PD1 resulted in enhanced in this model01,indicatingthatTNFR2co‐stimulationandPD1 blockade could lead to blockade a synergistic anti‐tumor immunesurvival response. HFB2003 was further evaluated in non‐human primate toxicity model. When administrated to adult cynomolgus monkeys up to 150mg/kg, HFB3‐200301 displays favorable pharmacokinetic agent in Oncology.and safety profiles. All together these data support the development of HFB200301 as a novel therapeutic

TNFR2 expression in TILs

C

Figure 1. Single cell analysis of TNFR2 expression . (A) Expression of TNFR2 and other immuno‐stimulating genes, OX40, 4‐1BB and GITR. TNFR2 is expressed in most CD8 T cells and a large proportion of CD4 T cells, including Tregs, and monocytes. (B) scRNA‐sequence analysis on multiple cancer types indicates TNFR2 expression mainly on Treg, CD4 and CD8 cells. BCC, basal cell carcinoma; SCC, sarcoma cell carcinoma; HCC, hepatocellular carcinoma; NSCLC, non small cell lung cancer; CRC, colorectal cancer; RCC, renal cell carcinoma. (C) Expression of TNFR2 on exhausted CD8 cells aligns with those from other immune T cell targets, such as PD‐1, TIM‐3, CTLA‐4 and 4‐1BB, in the sample of SCC.References: Breast cancer (Azizi et al., 2018 Cell); BCC/SCC (Yost et al.,2019 Nat. Med.); NSCLC (Zilionis et al., 2019 Immunity); Melanoma (Schelker et al.,2017 Nat. Comm.); HCC (Qiming et al., 2019 Cell); CRC/RCC (Wu et al., 2020 Nature)

Discovery of anti‐TNFR2 antibodies

Figure 2. Discovery of anti‐TNFR2 antibodies using HiFiBiO CelliGO™ platform. Splenocytes from C57BL/6 mice immunized with human recombinant TNFR2 ECD–Fc fusion protein were isolated, B‐cell enriched and subjected to CelliGO™ droplet‐based single cell screening for TNFR2 binding. The screening resulted in 17 potent TNFR2 binders with diverse CDRs. Color‐labeled HFB3‐1, ‐14 and ‐18 were selected based on their different epitopes on TNFR2.

Results

Epitope mapping, binding and crossreactivity of anti‐TNFR2 antibodies

A A A B B C

Figure 3. In vitro binding characterization of HFB3 antibodies. (A) Diverse epitopes identified by HDX‐MS. (B) Kd values of HFB3 antibodies to rhTNFR2‐His. (C‐D) Cellular binding of humanized HFB3 antibodies to human TNFR2. (E‐G) ELISA assay demonstrates the binding EC₅₀ of humanized HFB3 antibodies to recombinant human and cynomolgus TNFR2 range from a sub‐ to single digit‐nM, without recognizing TNFR1. All data represented as mean and SD (N=3).

Binding and functional effect of anti‐TNFR2 antibodies on CD4 and CD8 T cells

A B C

TNFRSF9 (4‐1BB) TNFRSF18 (GITR)

NFκB signal activation

CD4 D CD8

CTV‐tracked proliferation

E CD4 CD8

Figure 4. In vitro functional characterization of HFB3 antibodies. (A) Epitope binning shows HFB3 antibodies do not compete with ligand TNF‐α. (B) Humanized HFB3 antibodies preferentially binds to TCR‐primed CD4 and CD8 T cells after CD3/28 co‐stimulation. (C,D) Co‐stimulation of isolated conventional CD4 and CD8 T cells with HFB3 antibodies triggers TNFR2 downstream NFκB signaling, measured as gene expression upregulation of NFKB2 and RELB. Treatment of T cells with HFB3 antibodies in the presence of TNF‐α enhances NFκB signaling responses (green bars). (E,F) Co‐stimulation of isolated CD3 T cells with humanized HFB3 antibodies triggers cell proliferation and cell activation of CD4 and CD8 T cells. Cell proliferation is monitored by with cell trace violet (CTV), while cell activation is detected by changes of CD25 positive population in CD4 or CD8 T cells. Treatment of HFB3 antibodies in the presence of TNF‐α enhances cell proliferation and cell activation of CD4 and CD8 T cells. All data denoted as mean and SD (N=3) or representative data from 3 independent experiments. In addition, HFB3 antibodies have no/minimal ADCC effect comparable to isotype (data not shown).

Results (cont.)

Pharmacokinetic and antitumor activity of HFB200301 (HFB3‐1hz6‐hG1) in mice

Figure 5. In vivo evaluation of HFB3 antibodies. (A) Pharmacokinetic profile of development candidate HFB3‐1hz6‐hG1. (B,C) Efficacy and survival studies in MC38 tumor model in hTNFR2 knock‐in (TNFR2 KI) mice (n=8/group). Tumor growth inhibitions (TGI) is statistically significant for both 3 and 10 mg/kg HFB3‐ 1hz6‐hG1, anti‐PD‐1 (RMP‐14), and HFB3‐1hz6‐hG1/PD‐1 combination groups, that result in extended lifespan of the HFB3‐treated animals. Particularly, HFB3‐1hz6/PD‐1 combination results in increased survival than PD‐1 alone. HFB3‐1hz6‐hG1 and anti‐PD‐1 antibodies were injected 7 x 10 mg/kg or 4 x 10 mg/kg (vertical arrows) respectively, every 3 days intraperitoneally from day 0. Tumor inoculation was at day ‐7. Data are analyzed using ANOVA comparing treatment groups to isotype control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Exploratory toxicological evaluation of HFB200301 in NHPs

A B C D

Figure 6. Exploratory toxicological evaluation of HFB3‐1hz6‐hG1 in NHPs.(A) Scheme of experimental design. Two cynomolgus monkeys per group were injected with a single dose of 15 mg/kg (low), 50 mg/kg (medium) and 150 mg/kg(high) of HFB3‐1hz6‐hG1, after which plasma was collected at different time points until 336h (day 14). (B) Toxicokinetic analysis of HFB3‐1hz6‐hG1. (C) No elevation of cytokines was observed at the 15, 50 or 150 mg/kg of HFB3‐ 1hz6‐hG1 in comparison to reported data (dotted lines) from CD3xCD20 bispecific IgG at ≤ 3 mg/kg (D) Cell count analysis of HFB3‐1hz6‐hG1. No abnormality was found in cell count examination, as compared to historical data range from normal monkeys indicated by the blue and red lines.

Drug Intelligent Science (DIS™)

• Target discovery benefiting from patient single‐cell analysis

DIS approach captures single‐cell biology from target to patients

• High‐quality and differentiated antibody leads empowered by single B cell analysis

• Innovative lead optimization in picoliter droplets and rapid process optimization

• Identification of predictive biomarkers at single‐cell level to select responsive patient populations

CONCLUSION

We have discovered a number of anti‐TNRFR2 antibodies capable of co‐stimulating CD4 and CD8 T cells and selected a candidate, HFB200301, for further development. Highlight of HFB200301:

• First‐in‐class Immuno‐stimulatory humanized anti‐TNFR2 antibody

• Exhibit potent anti‐tumor activity in MC38 syngeneic model, alone and in combination with anti‐PD‐1

• Co‐stimulate cell proliferation and activation on CD4 and CD8 cells

• Specifically binds to human TNFR2 with sub‐nanomolar affinity

• Cross‐reactive with monkey TNFR2 ortholog

• Favorable acute toxicity profile to cynomolgus monkeys

• Favorable developability and pharmacokinetic profiles