REGN2810 binds to PD-1 on engineered Jurkat cells and on activated human and cynomolgus monkey T cells.

Concentration-time profile of REGN2810 in serum following a single intravenous infusion to cynomolgus monkeys.

Supplementary Figure S2 shows binding of REGN3767 to T cells

REGN2810 binds to PD-1 on T cells in tumor-bearing human PD-1 knock-in mice.

Expression of human PD-1 on activated T cells from human PD-1 knock-in mice and expression of PD-L1 on MC38.Ova cells.

Supplementary figures S1-S6

SUPPLEMENTARY MATERIALS AND METHODS

Measuring binding kinetics of human and mouse PD-1/PD-L1 interactions using surface plasmon resonance.

Supplementary materials and methods

Supplementary Figure S3 shows blocking activity of anti-MHC class II antibody in in vitro assay

Supplementary Figure S1 shows the amino acid sequences of the heavy and light chains of REGN3767

Supplementary Figure S4 shows activity of REGN3767 in an engineered assay

Efficacy of immune checkpoint inhibitors in cancers can be limited by CD8 T cell dysfunction or HLA-I down-regulation. Tumor control mechanisms independent of CD8/HLA-I axis would overcome these limitations. Here, we report potent CD4 T cell-mediated tumor regression and memory responses in humanized immune system (HIS) mice implanted with HT-29 colorectal tumors. The regressing tumors showed increased CD4 cytotoxic T lymphocyte (CTL) infiltration and enhanced tumor HLA-II expression compared to progressing tumors. The intratumoral CD4 T cell subset associated with tumor regression expressed multiple cytotoxic markers and exhibited clonal expansion. Notably, tumor control was abrogated by depletion of CD4 but not CD8 T cells. CD4 T cells derived from tumor-regressing mice exhibited HLA-II-dependent and tumor-specific killing ex vivo. Taken together, our study demonstrates a critical role of human CD4 CTLs in mediating tumor clearance independent of CD8 T cells and provides a platform to study human anti-tumor immunity in vivo.

REGN2810 does not induce ADCC or CDC in either unstimulated or CD3/CD28 stimulated Jurkat cells.

Supplementary Figure S5 shows expression of human PD-1 and human LAG-3 in PD-1xLAG-3 mice

Cytokine release following human PBMC exposure to immobilized REGN2810, REGN2329 (TGN1412) and anti-CD3 (OKT3) antibodies.

Supplementary methods and figure legends

Background 4–1BB is a key costimulatory receptor that mediates robust and durable tumor control by providing a secondary signal to antigen-experienced effector T cells. First-generation 4–1BB agonistic antibodies activated 4–1BB in the presence of Fc receptors and showed signs of efficacy but triggered hepatotoxicity. The 4–1BB agonistic activity of CLN-418, a fully human bispecific antibody, is instead dependent on cross-linking by B7H4, a widely expressed tumor-associated antigen with high prevalence in solid tumors and minimal expression in normal tissues. Targeting B7H4, an immunosuppressive checkpoint that shows minimally overlapping expression with PD-L1, may potentially address patients resistant to PD1 blockade. Currently, CLN-418 is the only B7H4 x 4–1BB bispecific antibody in clinical development. Methods Expression of B7H4, 4–1BB and immune correlates were examined in PBMC, cancer cell lines and solid tumors from patients using bioinformatics analysis of public datasets. CLN-418 binding to B7H4 and 4–1BB was evaluated using surface plasmon resonance, and simultaneous CLN-418 binding to 4–1BB and B7H4-expressing cells was assessed by flow cytometry. B7H4-dependent agonistic activity of CLN-418 was demonstrated by co-incubation with engineered 4–1BB reporter or primary T cells in the presence of B7H4-expressing cancer cells. CLN-418 efficacy was examined in a MC38-hB7H4 syngeneic tumor model in 4–1BB-humanized mice, as well as a human xenograft model in human PBMC-engrafted mice. Results 4–1BB receptor expression was low or absent on resting PBMC but was induced on activated tumor antigen-specific T cells with levels increasing upon immune rechallenge. TCGA and CPTAC database mining revealed robust and consistent B7H4 RNA and protein expression across multiple solid cancers, including ovarian, endometrial and breast carcinoma. 4–1BB gene expression in solid tumors was associated with CD8 T cell gene signature. CLN-418 showed specific high-affinity binding to human B7H4 and 4–1BB proteins, as well as dual binding to 4–1BB and B7H4 expressing cells. CLN-418 binding to B7H4-positive cells promoted 4–1BB engagement and T cell activation, with costimulatory signal strength proportional to B7H4 levels. CLN-418 exhibited robust dose-dependent single agent efficacy against MC38-hB7H4 tumors, with 79% and 68% tumor growth inhibition at 3 and 0.5 mg/kg dose levels, respectively. CLN-418 monotherapy also controlled growth of a B7H4-positive human carcinoma in PBMC-engrafted mice. Conclusions B7H4 and 4–1BB expression in cancer cells, PBMC and solid tumors, together with robust B7H4-dependent single agent activity of CLN-418 in preclinical tumor models, support the development of CLN-418 in patients with difficult-to-treat solid cancers. A Phase 1 dose-escalation trial of CLN-418 is ongoing (NCT05306444). Ethics Approval The anti-tumor efficacy studies in mice were approved by the internal ethics board of the respective contract research organization (CRO)