The cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride in dependence of sodium and potassium. The proteins share a conserved structural scaffold that consists of a transmembrane transport domain followed by a cytoplasmic regulatory domain. We have determined the X-ray structure of the C-terminal domain of the archaea Methanosarcina acetivorans. The structure shows a novel fold of a regulatory domain that is distantly related to universal stress proteins. The protein forms dimers in solution, which is consistent with the proposed dimeric organization of eukaryotic CCC transporters. The dimer interface observed in different crystal forms is unusual because the buried area is relatively small and hydrophilic. By using a biochemical approach we show that this interaction is preserved in solution and in the context of the full-length transporter. Our studies reveal structural insight into the CCC family and establish the oligomeric organization of this important class of transport proteins.

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.

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.

Targeting PD-L1-overexpressing cells with therapeutic antibodies is a clinically validated strategy for the treatment of multiple solid tumors. In order to increase efficacy, PD-1/PD-L1 blocking agents are currently being tested in combination with additional immune checkpoint modulators (ICMs). However, such combination therapies are associated with considerable treatment-related adverse events, resulting in a narrow therapeutic window and thereby limiting treatment efficacy. To maximize potency and improve the safety of ICM combination approaches, we sought to design a multi-specific molecule bearing two ICM domains that depletes PD-L1-overexpressing cancer cells via selective recruitment and stimulation of tumor-reactive effector T cells in the tumor microenvironment. The molecule's design consists of three monovalent antibody variable-domain fragments (Fvs) specific for PD-L1, human serum albumin and a T cell costimulatory receptor fused in a single-chain (PD-L1/HSA/costim tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that agonism of the costimulatory receptor on effector cells can only arise when the molecule concomitantly binds to PD-L1 on the surface of target cells. The addition of a half-life-extending anti-SA domain, meanwhile, not only enables convenient dosing but also should promote delivery of the molecule to tumor microenvironments. Here, we demonstrate the successful production of a novel multi-specific antibody that potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of cells that overexpress PD-L1, as confirmed using a transgenic Jurkat reporter T cell-line in co-cultures with HT29 and HCC827 cancer cell-lines. We show that the molecule elicits such costimulation considerably more selectively and potently than a first-generation, clinical-stage, anti-costim IgG. Furthermore, the tri-specific is incapable of stimulating T cells in the absence of primary T cell activation. Finally, in an HCC827 xenograft model in hPBMC-supplemented NOG mice, the tri-specific slowed tumor growth and enhanced intratumoral CD8+ T cell activation to a greater extent than monospecific IgG variants of the anti-PD-L1 and anti-costimulatory receptor domains. These data support the hypothesis that broadening the therapeutic window for a promising, clinically validated ICM-based treatment strategy can be achieved by combining multiple ICM domains in an Fc-less, monovalent multi-specific that selectively forms immunological synapses between cancer cells and activated effector cells. Citation Format: Tea Gunde, Sebastian Meyer, Stefan Warmuth, Alexandre Simonin, Christian Hess, Matthias Brock, Timothy Egan, David M. Urech. A novel multi-specific antibody targeting PD-L1-overexpressing cancers that redirects and stimulates antigen-committed CD8+ T cells through concomitant engagement of a T cell costimulatory receptor [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 3818.

BackgroundThe effective treatment of solid tumors remains an unmet medical need. Several concepts exist to treat malignancies, including antibody-drug or -immunotoxin conjugates, immune checkpoint inhibition, CAR- T cells, as well as bispecific T cell engagers. CD3-based T cell engagers are highly potent therapeutic molecules with T cell cytotoxicity activities in the picomolar range. Alongside this highly potent anti-tumor activity is the risk of on-target off-tumor effects due to low levels of expression of the target antigen in normal tissue, as has been observed for the tumor-associated antigen mesothelin (MSLN).MethodsLow-affinity antibody fragments to the tumor-associated antigen MSLN were generated, and a multispecific MATCH4 molecule was constructed containing bivalent low-affinity MSLN binding domains, a CD3 binding domain, and a serum albumin-binding domain for half-life extension. This molecule was tested in a cytotoxicity assay using human PBMCs co-cultured with H226 or MeT-5A cells, which express high or low levels of MSLN, respectively. The MeT-5A line, derived from mesothelial cells in the pleural fluid of non-cancerous individuals, represents normal MSLN-expressing cells. Soluble MSLN was added to determine effects on cytotoxicity. In vivo xenograft mouse studies were conducted using a tumor cell/PBMC co-implantation model, followed by regular dosing with molecules of interest.ResultsHere we report the design and the promising preclinical activity of the MATCH4 molecule in vitro and in vivo. We demonstrate that the low-affinity bivalent MSLN T cell engager has increased in vitro potency in T cell activation and tumor cell killing, as compared to a high-affinity monovalent counterpart on high MSLN expressing cells. We also demonstrate that the activity on low MSLN expressing cells is reduced for the low-affinity bivalent compared to the high affinity monovalent molecule. Because soluble MSLN is shed from cancer cells into cancer patient serum, we also demonstrate that up to 500 ng/mL of soluble MSLN does not interfere with the cytotoxic activity of the low affinity bivalent T cell engager, compared to the effects of soluble MSLN on a high affinity monovalent T cell engager. Importantly, we demonstrate in vivo that the low-affinity bivalent molecule significantly inhibits tumor growth in a dose-dependent manner.ConclusionsCollectively, these data demonstrate anti-tumor efficacy by this novel multispecific low 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 MSLN expression is low, and yet increase cytotoxicity to MSLN-expressing cancer cells.