Despite their favorable pharmacokinetic properties, singlechain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 ؋ 10 ؊9 to 1.5 ؋ 10Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 ؋ 10 ؊10 to <1.8 ؋ 10 ؊12 M. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86°C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.Because of their favorable pharmacokinetic properties, single-chain Fv (scFv) 3 antibody fragments represent an attractive format for therapeutic applications (1, 2). scFvs are often derived from monoclonal antibodies isolated from animal or human lymphocytes. As an alternative to hybridoma screening, in vitro display technologies, e.g. phage and ribosome display, enable the selection of high affinity-binding variable domains from natural or synthetic genetic libraries. Despite the successful use of in vitro randomization and selection systems, generation of antibodies by immunization and subsequent screening of full-size antibodies (e.g. hybridoma supernatants) includes conceptual advantages. For example, in contrast to in vitro display systems, in vivo methods are less prone to preferential selection of well expressed clones, which in many cases results in loss of potentially interesting antibodies. Moreover, in vivo methods are preferred in particular for addressing complex antigens, such as integral membrane proteins that are notoriously difficult to purify. However, reducing a full-length monoclonal antibody to the scFv format frequently is challenging particularly due to solubility and stability problems, which often impair expression and purification. Therefore, technologies to humanize and stabilize the scFv format following isolation of a monoclonal antibody remain critical for the generation of scFv therapeutics. Numerous approaches have been describ...

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Co-stimulatory 4–1BB receptors on tumor-infiltrating T cells are a compelling target for overcoming resistance to immune checkpoint inhibitors, but initial clinical studies of 4–1BB agonist mAbs were accompanied by liver toxicity. We sought to engineer a tri-specific antibody-based molecule that stimulates intratumoral 4–1BB and blocks PD-L1/PD-1 signaling without systemic toxicity and with clinically favorable pharmacokinetics. Recombinant fusion proteins were constructed using scMATCH3 technology and humanized antibody single-chain variable fragments against PD-L1, 4–1BB, and human serum albumin. Paratope affinities were optimized using single amino acid substitutions, leading to design of the drug candidate NM21-1480. Multiple in vitro experiments evaluated pharmacodynamic properties of NM21-1480, and syngeneic mouse tumor models assessed antitumor efficacy and safety of murine analogues. A GLP multiple-dose toxicology study evaluated its safety in non-human primates. NM21-1480 inhibited PD-L1/PD-1 signaling with a potency similar to avelumab, and it potently stimulated 4–1BB signaling only in the presence of PD-L1, while exhibiting an EC 50 that was largely independent of PD-L1 density. NM21-1480 exhibited high efficacy for co-activation of pre-stimulated T cells and dendritic cells. In xenograft models in syngeneic mice, NM21-1480 induced tumor regression and tumor infiltration of T cells without causing systemic T-cell activation. A GLP toxicology study revealed no evidence of liver toxicity at doses up to 140 mg/kg, and pharmacokinetic studies in non-human primates suggested a plasma half-life in humans of up to 2 weeks. NM21-1480 has the potential to overcome checkpoint resistance by co-activating tumor-infiltrating lymphocytes without liver toxicity.

Receptor tyrosine kinases (RTKs) play an important role in the control of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulated RTK signaling is critically involved in the development and progression of human cancer. Here, we present an assay for monitoring RTK activities in yeast, which provides an ideal heterologous cellular system to study these mammalian proteins in a null background environment. With our system, we have reconstituted aspects of the epidermal growth factor receptor (EGFR) signaling pathway as a model. Our approach is based on the Ras-recruitment system, in which membrane localization of a constitutively active human Ras achieved through protein-protein interactions can rescue growth of a temperature-sensitive yeast strain (cdc25-2). We show that co-expression of a dimerizing membrane-bound EGFR variant with specific adaptor proteins fused to the active Ras rescues growth of the cdc25-2 mutant yeast strain at the nonpermissive temperature. Using kinase-defective RTK mutants and selective EGFR kinase inhibitors, we demonstrate that growth rate of this yeast strain correlates with kinase activity of the EGFR derivatives. The RTK cellular assay presented here can be applied in high-throughput screens for selecting RTK-specific inhibitors that must be able to permeate the membrane and to function in an eukaryotic intrecellular environment.

The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.

Antagonistic molecules targeting the PD-1/PD-L1 axis have shown excellent activity in the clinic. However the majority of patients do not respond to the therapy due to multifaceted reasons implicating a non-effective activation of the immune system in those patients. The co-stimulatory molecule 4-1BB has been shown to be a key signalling component of T cells and the combination of 4-1BB activation and PD-1/PD-L1 antagonism has been shown to be highly active in preclinical models. Systemic application of first generation anti-4-1BB antibodies however have resulted in dose limiting hepatic toxicities. We have generated a novel 4-1BB/PD-L1/HSA trispecific MATCH3 immunomodulatory drug candidate (NM21-1480) that agonizes 4-1BB conditionally upon PD-L1 binding / blockade. Here we show the preclinical development data package for NM21-1480 demonstrating exquisite tumour-specific T cell activation associated with both 4-1BB agonism as well as PD-L1/PD-1 antagonism. We also demonstrate tumour specific localisation and accumulation of NM21-1480 in a mouse xenograft model. We report on the toxicity profile and pharmacokinetic properties of the molecule in non-human primates. We plan to initiate a first-in-human clinical study in the second half of 2020 to determine the safety, tolerability and first signs of clinical activity of the molecule. Citation Format: Daniel Snell, Tea Gunde, Stefan Warmuth, Peter Lichtlen, Julia Tietz, Matthias Brock, Alexandre Simonin, Christian Hess, Weinert Christopher, Robin Heiz, Naomi Flueckiger, Julia Zeberer, Dania Diem, Dana Mahler, Diego Morenzoni, Belinda Wickihalder, Simone Muntwiler, Antonia Poelderl, Benjamin Kuettner, Sandro Wagen, Sebastian Meyer, Timothy Egan, David Urech. Preclinical development and mechanism of action studies of NM21-1480, a PD-L1/4-1BB/HSA trispecific MATCH3 therapeutic clinical candidate [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 2276.

Antagonistic molecules targeting the PD-1/PD-L1 axis have shown excellent activity in the clinic. However, the majority of patients do not respond to the therapy due to multifaceted reasons implicating a non-effective activation of the immune system in those patients. The co-stimulatory molecule 4-1BB has been shown to be a key signalling component of T cells and the combination of 4-1BB activation and PD-1/PD-L1 antagonism has been shown to be highly active in preclinical models. Systemic application of first-generation anti-4-1BB antibodies however have resulted in dose limiting hepatic toxicities. We have generated and are currently clinically investigating a novel 4-1BB/PD-L1/HSA trispecific MATCH3 immunomodulatory drug candidate (NM21-1480) that agonizes 4-1BB conditionally upon PD-L1 binding and concomitantly blocks the interaction between PD-1 and PD-L1. Here we investigate the impact of dose dependency in vitro and the translation of this to in vivo studies to aid the selection of optimal doses for our ongoing clinical study of NM21-1480 in cancer patients. In human PD-L1 tumor bearing mice, triple knock-in for human PD-1, human PD-L1 and human 4-1BB, we have investigated the impact of dose titrations of NM21-1480. We observe a dose dependent effect of NM21-1480 on tumor growth, systemic exposure, tumor exposure and pharmacodynamic biomarkers of immune cell activation. We also demonstrate the formation of a memory response in treated mice through rechallenge of the mice with tumor. We also investigate the effect of combination therapy of NM21-1480 with immune-oncology targeted therapeutics both in vitro and in vivo. In particular, we demonstrate that the combination of NM21-1480 with anti-CD3 T cell engagers is highly effective in T cell activation and tumor control. These data highlight the potential of NM21-1480 for the treatment of cancer patients and enable greater understanding of the relationship between dose, tumor exposure, immune activation and tumor growth inhibition. Citation Format: Daniel Snell, Tea Gunde, Stefan Warmuth, Peter Lichtlen, Linlin Liu, Shu-wen Teng, Lan Zhang, ChaoHsuan Pan, Peiqi Li, Julia Tietz, Matthias Brock, Alexandre Simonim, Christian Hess, Christopher Weinert, Maria Johansson, Bithi Chatterjeee, Nicole Bassler, Niels Kirk, Catia Mendes, Robin Heiz, Naomi Flueckiger, Dania Diem, Dana Mahler, Belinda Wickihalder, Simone Muntwiler, Sandro Wagen, Elmar vom Baur, Archie N. Tse, David Urech. Dose selection investigations and combination strategies of NM21-1480, a PD-L1/4-1BB/HSA trispecific MATCH3 therapeutic clinical candidate [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 2870.

CD3-based T cell engagers are highly potent therapeutic molecules which enable T cell-mediated cytotoxic activity toward cells expressing selected tumor-associated antigens. Alongside a highly potent anti-tumor activity, is the risk of on-target off-tumor side effects due to low levels of expression of the target antigen in normal tissue. We have sought to overcome this issue with the design and generation of a target-density-dependent activation mechanism. We have generated reduced-affinity antibody fragments to the tumor associated antigen mesothelin and constructed a multi-domain MATCH4 molecule encompassing bivalent mesothelin binding domains, a CD3 binding domain, and a human serum albumin (HSA)-binding domain for half-life extension. Here we report the design of the MATCH4 molecule and the preclinical activity of the molecule in vitro and in vivo. We also report the biochemical characteristics of the therapeutic candidate molecule showing its highly favorable properties for clinical development. We demonstrate that the bivalent mesothelin T cell engager has increased in vitro potency in T cell activation and tumor cell killing in the presence of high mesothelin expressing cells, when compared to a higher-affinity monovalent counterpart. We also demonstrate that the activity on low mesothelin expressing cells, such as healthy mesothelial cells, is reduced for the bivalent molecule compared to the higher affinity monovalent molecule. Due to the shedding of mesothelin from the surface of cancer cells and the high circulating levels of soluble mesothelin in patient sera, we also demonstrate that the bivalent molecule is still highly potent in cytotoxic activity in the presence of concentrations of soluble mesothelin up to 500 ng/ml. We also demonstrate dose dependent anti-tumor activity in in vivo efficacy studies in PBMC-reconstituted mice, and combination therapeutic activity with an anti-PD-L1, anti-4-1BB bispecific molecule (NM21-1480). Collectively, these data demonstrate an increased selectivity to mesothelin-overexpressing cells by this novel MATCH4 reduced affinity bivalent T cell engager. These data indicate the potential of this molecule to increase the therapeutic window by reducing safety concerns on normal tissue where mesothelin expression is low, and yet promote cytotoxicity on mesothelin over-expressing cancer cells. Citation Format: Bithi Chatterjee, Christian Hess, Daniel Snell, Tea Gunde, Stefan Warmuth, Alexandre Simonin, Matthias Brock, Fabio Spiga, Maria Johansson, Christopher Weinert, Julia Tietz, Niels Kirk, Nicole Bassler, Dana Mahler, Dania Diem, Alessandra Carrella, Noreen Giezendanner, Alessandra Alberti, Giorgio Gambino, Belinda Wickihalder, Bettina Bommer, Simone Muntwiler, Yasemin Yaman, Naomi Flueckiger, Robin Heiz, Sandro Wagen, David Urech. NM28-2746, a reduced affinity bivalent mesothelin-binding MATCH4 T cell engager, with half-life extension, increases selectivity for killing of mesothelin-overexpressing 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 2871.