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Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

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.

Background MCLA-145 is a CD137 x PD-L1 bispecific antibody that releases PD-L1 mediated T-cell inhibition and activates and expands T cells through agonism of CD137. Immune checkpoint inhibitors (ICI) against PD-(L)1 have demonstrated anti-tumor efficacy in a fraction of patients across a broad range of cancers. CD137 (4-1BB, tumor necrosis factor receptor superfamily 9) is an inducible costimulatory receptor transiently expressed on T cells after TCR engagement. CD137 signaling is triggered by receptor clustering and leads to enhanced cytokine production; T cell proliferation, survival, and effector function; and immunological memory formation. Targeting of PD-L1 and CD137 with MCLA-145 may achieve synergistic activity by simultaneously blocking the inhibitory checkpoint PD-L1 and activating tumor specific T cells through co-stimulation. Methods We performed combinatorial functional screening of bispecific antibodies generated from high affinity inhibitory Fabs binding PD-L1 combined with a large and diverse panel of agonistic CD137 Fabs. Results MCLA-145 was selected based on its in vitro potency in multiple primary human immune cell assays. Further, it displays an ability to reverse T cell suppression mediated by M2 macrophages or Tregs. MCLA-145 binds to a unique epitope in the cysteine rich domain 2 of CD137 that overlaps with the CD137L binding region, and all potent bAbs in the screen were able to bind to this region. MCLA-145 drives activation of CD137 and the degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells. Using proximity ligation assays and confocal microscopy we demonstrated that MCLA-145 clusters CD137 on the surface of T cells resulting in internalization. The binding location of MCLA-145 on CD137 may be optimal for the formation of ‘immunological synapses’ with PD-L1 expressing antigen presenting cells or tumors resulting in the potent activation of tumor specific cytotoxic T cells. Conclusions These experiments demonstrate the dual anti-cancer activity of MCLA-145 in preclinical models: release of T-cell checkpoint inhibition through PD-L1; and activation and expansion of T cells through CD137, therefore overcoming T-cell exhaustion and increasing T-cell presence/activity (infiltration) in tumors. MCLA-145 is currently undergoing clinical development in an ongoing trial (NCT03922204). Ethics Approval Animal experiments were performed according to guidelines for animal care of the local Animal Experiments Committee; Use of human blood cells from healthy volunteers was approved by the blood bank’s Ethical Advisory Council.

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.

The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.

Background: In colorectal cancer (CRC) and other solid tumors, cancer stem cells (CSC) contribute to tumor progression and resistance to standard chemotherapies. The continuous regeneration of the colon is dependent on strict control of developmental (e.g. Wnt) and mitogenic (e.g. EGF) pathway signaling; dysregulation results in uncontrolled proliferation forming the basis of aggressive tumors with metastatic potential. Here we describe the generation of novel bispecific antibodies designed to target CSC through Wnt signaling receptors and block growth factor signaling. The Wnt targets LGR5, LGR4, ZNRF3 and RNF43 were selected since their expression is modulated in CSC populations. The GPCR family members LGR4/LGR5 are positive Wnt regulators and the transmembrane E3 ligases ZNRF3/RNF43 are negative Wnt regulators. The growth factor receptor EGFR is frequently (>70%) overexpressed in CRC and its blockade has demonstrated clinical benefit in a subgroup of patients. More recently, HER3 pathway activation has been implicated in resistance to EGFR-targeted therapies. Experimental procedures and results: Two parallel strategies were applied to generate panels of common light chain (cLC) Fab against LGR4, LGR5, ZNRF3 and RNF43. Humanized cLC mice (MeMo®) were immunized with recombinant protein or DNA, and materials harvested from these mice used to generate Fab regions against these antigens. The second approach utilized large and diverse synthetic cLC Fab-phagemid libraries. Combined, these methods resulted in ∼1500 unique antigen-specific Fab from which ∼300 were selected for further testing. Bispecific antibodies were produced in a human cLC IgG1 format using substitutions in the IgG Fc regions for coexpression of two different heavy chains resulting in the generation of large panels of pure and stable bispecific IgG suitable for screening. The Wnt target specific Fab were combined with a Tetanus toxoid-specific control Fab arm allowing for stringent ranking of these Wnt-specific panels in a monovalent format for specificity, affinity, stability, and ligand (R-Spondin3) blocking potency. Based on this characterization the 54 most promising Wnt targeting arms were combined with a panel of previously characterized EGFR and HER-3 specific Fab arms resulting in ∼ 500 different cLC bispecific IgG for functional testing. All bispecific IgG were screened for potency of growth inhibition of CSC using novel 3D high content imaging readouts on patient-derived CRC organoids. The organoids are cultured using growth factors that allow for the maintenance and proliferation of healthy and diseased stem cells and their offspring. Functional analysis revealed several bispecific antibodies that inhibited CRC organoid growth much more potently than comparator drugs such as cetuximab or erlotinib. Conclusion: Bispecific antibodies present a biological modality that result in unexpected functional activities by mechanisms possible unique to the architecture of these molecules. Identifying these unique properties requires the rapid generation and screening of large panels of bispecific IgG directly in the therapeutic format in relevant functional assays. Initial screening results of the bispecific antibodies targeting Wnt and HER family members supports the concept of CSC targeting and several leads are currently undergoing more extensive characterization. Citation Format: Berina Eppink, Rob Roovers, Bram Herpers, Wim de Lau, Carina Clements, Vanessa Zondag van der Zande, Abdul Basmeleh, Willem Bartelink, Marc van de Wetering, Robert Vries, Leo Price, John De Kruif, Mark Throsby. Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C21.