SummaryAnti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes.

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

Regulatory T cells (Treg) play a major role in the suppression of protective anti-tumour T cell responses. In the CT26 BALB/c murine model of colorectal carcinoma, Tregs differentially suppress responses to two characterised CD8+ T epitopes, AH1 and GSW11, which results in an absence of detectable IFN-γ producing GSW11-specific T cells in the spleen and lymph nodes of tumour challenged mice. Activation of GSW11-specific T cells correlates with protection against tumour progression. Here we show that GSW11-specific T cells are in fact induced in Treg-replete, CT26-bearing mice, where they make up the majority of tumour infiltrating CD8+ lymphocytes, but exhibit an ‘exhausted’ phenotype. This dysfunctional phenotype is induced early in the anti-tumour response in tumours. Depletion of Tregs prior to tumour challenge correlates with an altered T cell receptor (TcR) repertoire. Moreover, the avidity of GSW11-specific TcRs that expanded in the absence of Tregs was significantly lower compared to TcRs of CD8+populations that were diminished in protective anti-tumour responses. This indicates that Tregs suppress the induction of protective anti-tumour T cell responses and may signify that low avidity T cells play an important role in this protection.

The pleiotropic TNF-α:TNFR axis plays a central role in the immune system. While the cellular expression of TNFR1 is broad, TNFR2 expression is mainly restricted to immune cells and especially high on T regs. The therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated but the mechanism-of-action (MoA) of these reagents remains unclear, with conflicting data reported by different investigators. To gain further insight, we identified and characterized a wide panel of human and mouse TNFR2-specific antibodies, generated from the n-CoDeR F.I.R.S.T™ phage display platform. Based on their ability to block TNF-α:TNFR2 binding and to agonize or antagonize TNFR2 signaling, we identified parallel human and mouse TNFR2-specific antibodies with activities ranging from agonist to antagonist, and from complete ligand blocking to non-blocking. Two antibody variants with distinctly opposing in vitro activities (complete ligand-blocking antagonist versus non-blocking agonist), were expressed in various IgG formats preferentially engaging activating FcγR (mIgG2a), inhibitory FcγR (mIgG1), or no FcγR (N297A Fc-mutated) and screened for in vivo antitumor activity. Both anti-TNFR2 antibody clones displayed anti-tumor efficacy but showed strikingly different FcγR-dependence for optimal antitumor activity. Further characterization demonstrated potent anti-tumor efficacy across several syngeneic in vivo cancer models (CT26, MC38 and B16), both as single agents, and when combined with anti-PD-1. In vivo mode-of-action studies indicated different initial events are evoked by the two antibodies but that they ultimately converge to elicit a similar immune modulation within the tumor that is associated with anti-tumor efficacy. The antagonist antibody caused early intra-tumoral T reg depletion, while the agonist caused dramatic CD8+ T cell increases. Over time, both antibodies induce an increase in effector T cells at the tumor site, improved CD8/T reg ratios, and tumor regression. In addition, the two antibodies similarly modulated the tumor myeloid content. Based on careful MoA-characterization, two human lead candidate anti-TNFR2 antibodies are being developed for treatment of solid cancer; BI-1808, a ligand-blocking T reg depleting antibody and BI-1910, a TNFR2 agonist. Citation Format: Linda Mårtensson, Kirstie Cleary, Monika Semmrich, Mathilda Kovacek, Petra Holmkvist, Caroline Svensson, Mimoza Demiri, Therese Blidberg, Ulla-Carin Thornberg, Vincentiu Pitic, Osman Dadas, Sean H. Lim, Stephen A. Beers, Mark S. Cragg, Björn Frendéus, Ingrid Teige. Targeting TNFR2 for cancer immunotherapy: Ligand blocking depletors versus receptor agonists [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 936.

The tumor necrosis factor superfamily (TNFSF) and their receptors (TNFRSF) are important regulators of the immune system, mediating proliferation, survival, differentiation, and function of immune cells. As a result, their targeting for immunotherapy is attractive, although to date, under-exploited. In this review we discuss the importance of co-stimulatory members of the TNFRSF in optimal immune response generation, the rationale behind targeting these receptors for immunotherapy, the success of targeting them in pre-clinical studies and the challenges in translating this success into the clinic. The efficacy and limitations of the currently available agents are discussed alongside the development of next generation immunostimulatory agents designed to overcome current issues, and capitalize on this receptor class to deliver potent, durable and safe drugs for patients.

The pleiotropic TNF-α:TNFR axis plays a central role in immune regulation. While the cellular expression of TNFR1 is broad, TNFR2 expression is mainly restricted to immune cells. The therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated and to gain further insight, we generated a wide panel of TNFR2-specific antibodies using the n-CoDeR F.I.R.S.T™ target and antibody discovery platforms. We identified antibodies against mouse or human TNFR2 that could block TNF-α binding and showed potent anti-tumor activity in several immune-competent models, both as single agent and in combination with anti-PD1. The mechanism-of-action was shown to be FcγR dependent and likely mediated through a combination of intra-tumor T reg depletion, CD8+ T cell expansion and modulation of innate immune cells e.g. neutrophils and myeloid cells. Toxicological studies of the human lead-candidate BI-1808 in non-human primates (NHP) demonstrated very good tolerability at doses up to 200 mg/kg, and in vitro studies with human cells showed no signs of harmful cytokine release. Furthermore, in vivo studies using immune competent mouse cancer experimental models showed a clear relationship between dose, receptor occupancy (RO) and efficacy. In both murine models and in the NHPs, soluble TNFR2 was clearly modulated by the treatment and closely correlated with RO. In addition, several changes in the immune cells (e.g. T cells and neutrophils) in the blood compartment were observed. Since January 2021, BI-1808 is evaluated in an ongoing Phase I/IIa trial. Similar to the preclinical studies, correlations between dose, RO and soluble TNFR2 has been clearly observed in the patients. In addition, and consistent with TNF-α:TNFR biology being conserved across the species, modulations of T cell and neutrophil numbers are also paralleled between the patients, NHPs and mice, increasing the likelihood of successful clinical translation of our findings. Citation Format: Linda Mårtensson, Kirsty Cleary, Petra Holmkvist, Mathilda Kovacek, Carolin Svensson, Monika Semmrich, Therese Blidberg, Mimoza Demiri, Osman Dadas, Marie Borggren, Vici Pitic, Sean H. Lim, Stephen A. Beers, Susanne Gertsson, Kristoffer S. Rohrberg, Ingrid Karlsson, Andres McAllister, Mark S. Cragg, Björn Frendeus, Ingrid Teige. BI-1808 - a first in class ligand-blocking αTNFR2 antibody for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4156.

Agonistic antibodies directed to immunostimulatory receptors are a currently untapped source for immunotherapy. Whereas checkpoint blockers have translated into the clinic, the rules for agonistic antibodies have been more difficult to discern and these reagents await further optimization. Here we discuss the salient properties of monoclonal antibodies (mAb) required to strongly agonize these receptors and discuss potential strategies for the future. We show that immunostimulatory mAb (ISA) can agonize key stimulatory receptors on the cell surface—many of which are from the TNFR family—and highlight the importance of isotype. Using CD40 mAb as a paradigm, we show that receptor clustering is key for ISA responses—probably mimicking ligand. This clustering can be achieved via Fc gamma receptor (FcgR) engagement or independently with the hIgG2(B) isotype. For CD40 this is highly epitope dependent, showing that epitope, isotype, and domain location all interact to drive mAb agonism. Finally, we show that isotype engineering can overcome “weak” epitopes. This can be completely independent of FcgR interaction in the case of hIgG2B, which may be of use in humans where the FcgR profile within the tumor microenvironment may vary. Citation Format: Xiaojie Yu, Anne White, Martin Glennie, Ivo Tews, Hayden Fisher, Chris Orr, Ruth French, Stephen Beers, Aymen Al'Shamkhani, Osman Dadas, Ali Roghanian, Jane Willoughby, Mark S. Cragg. Optimizing immunostimulatory antibodies for cancer immunotherapy [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA03.

29Regulatory T cells (Treg) play a major role in the suppression of protective anti-30 tumour T cell responses. In the CT26 BALB/c murine model of colorectal carcinoma, 31Tregs differentially suppress responses to two characterised CD8+ T epitopes, AH1 32 and GSW11, which results in an absence of detectable IFN-γ producing GSW11-33 specific T cells in the spleen and lymph nodes of tumour challenged mice. Activation 34 of GSW11-specific T cells correlates with protection against tumour growth. Here we 35show that GSW11-specific T cells are in fact induced in Treg-replete, CT26-bearing 36 mice, where they make up the majority of tumour infiltrating CD8+ lymphocytes, but 37 exhibit a dysfunctional 'exhausted' phenotype. This dysfunctional phenotype is 38 induced early in the anti-tumour response in draining lymph nodes, spleens and 39 tumours and is significantly more pronounced in GSW11-specific T cells compared to 40 other tumour-specific T cell responses. Depletion of Tregs prior to tumour challenge 41 significantly reduces the induction of exhaustion in GSW11-specific T cells and 42 correlates with altered T cell receptor (TcR) usage. Moreover, the avidity of GSW11-43 specific TcRs that expanded in the absence of Tregs was significantly lower compared 44