Antibody–drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. Significance: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.

MET, the cell-surface receptor for the hepatocyte growth factor/scatter factor, which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. BYON3521 is a novel site-specifically conjugated duocarmycin-based antibody–drug conjugate (ADC), comprising a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus MET. In vitro studies showed that BYON3521 internalizes efficiently upon MET binding and induces both target- and bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high MET–expressing cancer cell lines; in moderate and low MET–expressing cancer cell lines good potencies and partial efficacy were observed. In mouse xenograft models, BYON3521 showed significant antitumor activity upon single-dose administration in multiple non-MET–amplified tumor types with low, moderate, and high MET expression, including complete tumor remissions in models with moderate MET expression. In the repeat-dose Good Laboratory Practice (GLP) safety assessment in cynomolgus monkeys, BYON3521 was well tolerated and based on the observed toxicities and their reversibility, the highest non-severely toxic dose was set at 15 mg/kg. A human pharmacokinetics (PK) model was derived from the PK data from the cynomolgus safety assessments, and the minimal efficacious dose in humans is estimated to be in the range of 3 to 4 mg/kg. In all, our nonclinical data suggests that BYON3521 is a safe ADC with potential for clinical benefit in patients. A first-in-human dose-escalation study is currently ongoing to determine the maximum tolerated dose and recommended dose for expansion (NCT05323045).

C-Met, the cell-surface receptor for HGF/SF which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. However, the apparently delicate balance between anti-tumor activity and target mediated-toxicities proved to be quite a challenge. As a result, there are currently no c-Met targeting antibodies or antibody-drug conjugates (ADCs) on the market. We developed BYON3521, a novel site-specifically conjugated, duocarmycin-based ADC, comprising a humanized, cysteine-engineered IgG1 monoclonal antibody with low pM binding affinity towards both human and cynomolgus c-Met. In vitro studies showed that BYON3521 internalizes efficiently upon c-Met binding, and induces both target- as well as bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high c-Met-expressing cancer cell lines; in moderate and in low c-Met-expressing cancer cell lines partial efficacy was observed. PK/PD studies and a mouse clinical trial in patient-derived xenograft models showed significant anti-tumor activity of BYON3521 upon single dose administration in multiple tumor types. BYON3521 showed responses in non-MET-amplified tumors with low, moderate and high c-Met expression, with partial and complete tumor remission seen in several models with moderate c-Met expression. In the repeat-dose GLP safety assessment in cynomolgus monkeys BYON3521 was well tolerated. In all, BYON3521 was deemed a safe ADC with potential for clinical benefit in patients. Preparations are ongoing to start clinical Phase I in 2021. Citation Format: Patrick Groothuis, Danielle Jacobs, Inge Hermens, Desiree Damming, Ellen Mattaar, Myrthe Rouwette, Monique van der Vleuten, Aloys Sesink, Fred Dijcks, Ruud Coumans, Marion Blomenrohr, Ruud Ubink, Miranda van der Lee, Wim H.A. Dokter. BYON3521, a novel effective and safe c-Met targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 926.

Preclinical data have established CD47-SIRPα interactions as a myeloid immune checkpoint in cancer, which is corroborated by preliminary evidence from the first clinical studies with CD47 blockers. However, the ubiquitously expressed CD47 mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy. Here, we present preclinical results on a novel clinical candidate, BYON4228. BYON4228 is an antibody directed against SIRPα and recognizes both of the common allelic variants of human SIRPα which maximizes its potential clinical application in the broad human population. Notably, in contrast to several other anti-SIRPα antibodies in development, BYON4228 does not recognize the closely related T cell-expressed SIRPγ that has been reported to mediate interactions with CD47 as well, which are known to be instrumental in T cell extravasation and activation. BYON4228 binds to the N-terminal part of SIRPα and its epitope overlaps with the CD47-binding site. BYON4228 therefore prevents binding of CD47 to SIRPα and thus blocks inhibitory signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates both macrophage- and neutrophil-mediated elimination of hematologic and solid cancer cells in vitro in the presence of several different tumor targeting antibodies like trastuzumab, rituximab, daratumumab and cetuximab, illustrating the broad potential clinical benefit and application of BYON4228. Single intravenous infusion of up to 100 mg/kg BYON4228 to male and female cynomolgus monkeys was well tolerated and did not elicit any adverse effects. Collectively, this defines BYON4228 as a pan-allelic anti-SIRPα antibody without T cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2022. Citation Format: Mary van Helden, Seline Zwarthoff, Hugo Olsman, Marc Paradé, Ellen Mattaar, Karin de Laat, Imke Lodewijks, Paul Boersema, Lilian Driessen-Engels, Jorien Sanderink, Wendy Kappers, Diels van den Dobbelsteen, Ruud Ubink, Roel Arends, Gerard Rouwendal, Gijs Verheijden, Miranda van der Lee, Wim Dokter, Timo Van den Berg. BYON4228 is a pan-allelic SIRPα antibody that potentiates killing of antibody-opsonized tumor cells and lacks binding to T cells [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 5589.