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Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages., The anti-tumour effect of immune checkpoint inhibitors is potentiated by CD137 agonists in preclinical models, but translation of these results to the clinical practice is hampered by toxicity. Authors describe here a human CD137xPD-L1 bispecific antibody with improved anti-cancer activity whilst maintaining low toxicity in non-human primates.

CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. CD137 signaling requires receptor clustering normally facilitated by the trimeric CD137 ligand (CD137L). Alternatively, CD137 signaling can be triggered either directly by agonistic monoclonal antibodies (mAbs) or indirectly via crosslinking of CD137 binding mAbs by Fcγ receptors on neighboring cells. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist, monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. To address the issues of toxicity and efficacy a highly selective and potent CD137xPD-L1 bispecific antibody (bAb) was identified by applying an unbiased functional screening approach. Collections of common light chain Fabs recognizing CD137 and PD-L1 were produced based on antibody panels from immunized MeMo® mice. A large and diverse panel of CD137xPD-L1 bAbs was then produced by combining different CD137 and PD-L1 Fabs based on epitope and sequence diversity in the IgG1 Biclonics® format. The bAbs were screened for activity in reporter cell lines expressing the receptors. This unbiased combinatorial screening identified a CD137xPD-L1 bAb (MCLA-145) for which CD137 mediated activation is dependent on the presence of PD-L1 on a neighboring cell and, as such, the antibody acts in ‘trans’. Flow cytometry experiments demonstrated that MCLA-145 is fully cross-reactive to cynomolgus monkey CD137 and PD-L1. The CD137 Fab arm blocks the interaction of CD137 with CD137L as demonstrated in a competition assay by flow cytometry. The PD-L1 Fab arm blocks the interaction between PD-1 and PD-L1 as demonstrated in ELISA. Binding epitopes were mapped by shotgun mutagenesis using a flow-based screen. In addition, hydrogen-deuterium exchange experiments were performed to map the binding domain on CD137. Data show that MCLA-145 binds the ligand binding domain of CD137 domain (CRDII). The PD-L1 Fab arm binds PD-L1 in the PD-1 binding N-terminal V domain. Both epitope mapping data sets are consistent with the CD137 and PD-L1 ligand blocking activity of MCLA-145. Monovalent binding affinities were measured by surface plasma resonance (SPR) and radioactive iodine labeling and demonstrated affinities in the low nM (CD137) and subnanomolar (PD-L1) range. SPR experiments also confirmed that MCLA-145 was able to bind simultaneously to both CD137 and PD-L1 recombinant proteins. The unique binding properties of MCLA-145 may result in an increased therapeutic window by specifically activating CD137 expressing cells in the tumor niche where PD-L1 is expressed while simultaneously blocking inhibitory input from the PD-1/PD-L1 axis. Citation Format: Cecile A. Geuijen, Paul Tacken, Rinse Klooster, Horacio Nastri, Shaun Stewart, Jing Zhou, Steve Wang, Cheng-Yen Huang, Arjen Kramer, Linda Kaldenberg-Hendriks, John de Kruif, Renate den Blanken-Smit, Vanessa Zondag-van de Zande, Abdul Basmeleh, Willem Bartelink, Patrick Mayes, Gregory Hollis, Reid Huber, Mark Throsby. An unbiased screen identifies a CD137xPD-L1 bispecific IgG1 antibody with unique T cell activation and binding properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 541.

Lymphocyte activation gene 3 (LAG-3) is a cell surface receptor that negatively regulates antigen-specific T cell responses. LAG-3 expression is generally restricted to populations of recently activated and chronically stimulated exhausted T cells, and is often correlated with general T cell dysfunction across several human malignancies. Accordingly, the LAG-3 pathway has been identified as a potential barrier to productive tumor-specific T cell immunity generated by PD-1/PD-L1 blockade. The antitumor activity from targeting the LAG-3 pathway in preclinical models has provided further rationale for pharmacologic modulation of the LAG-3 axis in cancer patients. INCAGN02385 is an Fc-engineered IgG1κ antibody chosen for development based on its high-affinity binding to human LAG-3, cross-reactivity with cynomolgus monkey LAG-3, and ability to potently block LAG-3 binding with its MHC class II ligand. INCAGN02385 also enhances T cell responsiveness to TCR stimulation alone or in combination with PD-1/PD-L1 axis blockade. Evaluation of INCAGN02385 in cynomolgus monkeys was well-tolerated and demonstrated the expected pharmacokinetic profile. Altogether, these data support assessment of INCAGN02385 in patients with advanced or metastatic solid tumors. Citation Format: David Savitsky, Rebecca Ward, Christina Riordan, Cornelia Mundt, Shawn Jennings, Joe Connolly, Mark Findeis, Michele Sanicola, Dennis Underwood, Horacio Nastri, Peggy Scherle, Gregory Hollis, Reid Huber, Robert Stein, Marc van Dijk, Nicholas S. Wilson. INCAGN02385 is an antagonist antibody targeting the co-inhibitory receptor LAG-3 for the treatment of human malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3819.

The majority of immunotherapeutic agents developed thus far either attempt to stimulate a more productive anti-tumor immune response or to inhibit key proteins in the immunosuppressive tumor milieu. PD-1/PD-L1 axis blockade, CTLA-4 blockade and IDO1 inhibition are examples of the latter approach and have been utilized to reverse the suppressive tumor microenvironment, resulting in clinical benefit for cancer patients. Recent clinical and preclinical data have also demonstrated that combining these approaches results in enhanced therapeutic benefit. Notably, the IDO1-selective inhibitor epacadostat has been shown to increase the efficacy of two checkpoint inhibitors, the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody pembrolizumab, in patients with melanoma. Because both checkpoint receptors and IDO1 serve as negative regulators of the immune response, we also explored the ability of IDO1 inhibition to combine with agents that directly activate T cells through costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily. Rodent active surrogate agonist antibodies to 4-1BB, OX40 and GITR were tested with epacadostat in multiple preclinical models. In the B16-SIY melanoma model that does not express IDO1 in tumor cells, both epacadostat and anti-OX40 had little effect, but the combination resulted in enhanced efficacy. This was associated with increased infiltrates of CD8+ T cells and decreased numbers of FoxP3+ TILs. Increased numbers of SIY-reactive T cells were found in both the tumor and the TDLN post-therapy. In contrast, epacadostat did not provide any enhancement to the activity seen with 4-1BB. Clear combinatorial effects were seen with anti-GITR and epacadostat in the more inflamed, IDO1-expressing PAN02 pancreatic cancer model. These data suggest that IDO1 inhibition can be effective in combination with agents that agonize T cell costimulatory receptors as well as with agents that block coinhibitory receptors. Citation Format: Holly K. Koblish, Brendan Horton, Michael Hansbury, Sybil O'Connor, Kerri Lasky, Christina Stevens, Thomas Condamine, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Horacio Nastri, Gregory Hollis, Reid Huber, Thomas Gajewski, Peggy Scherle. Agonist antibodies targeting OX40 and GITR enhance the activity of the IDO1-selective inhibitor epacadostat in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2017-2618