A Bispecific, Tetravalent Antibody Targeting Inflammatory and Pruritogenic Pathways in Atopic Dermatitis.

Inhibition of IL-4/IL-13 signaling has dramatically improved the treatment of atopic dermatitis (AD). However, in many patients, clinical responses are slow to develop and remain modest. Indeed, some symptoms of AD are dependent on IL-31, which is only partially reduced by IL-4/IL-13 inhibition. Thus, there is an unmet need for AD treatments that concomitantly block IL-4/IL-13 and IL-31 pathways. We engineered NM26-2198, a bispecific tetravalent antibody designed to accomplish this task. In reporter cell lines, NM26-2198 concomitantly inhibited IL-4/IL-13 and IL-31 signaling with a potency comparable with that of the combination of an anti-IL-4Rα antibody (dupilumab) and an anti-IL-31 antibody (BMS-981164). In human PBMCs, NM26-2198 inhibited IL-4-induced upregulation of CD23, demonstrating functional binding to FcγRII (CD32). NM26-2198 also inhibited the secretion of the AD biomarker thymus and activation-regulated chemokine (TARC) in blood samples from healthy human donors. In male cynomolgus monkeys, NM26-2198 exhibited favorable pharmacokinetics and significantly inhibited IL-31-induced scratching at a dose of 30 mg/kg. In a repeat-dose, good laboratory practice toxicology study in cynomolgus monkeys, no adverse effects of NM26-2198 were observed at a weekly dose of 125 mg/kg. Together, these results justify the clinical investigation of NM26-2198 as a treatment for moderate-to-severe AD.

An engineered T-cell engager with selectivity for high mesothelin-expressing cells and activity in the presence of soluble mesothelin.

Mesothelin (MSLN) is an attractive immuno-oncology target, but the development of MSLN-targeting therapies has been impeded by tumor shedding of soluble MSLN (sMSLN), on-target off-tumor activity, and an immunosuppressive tumor microenvironment. We sought to engineer an antibody-based, MSLN-targeted T-cell engager (αMSLN/αCD3) with enhanced ability to discriminate high MSLN-expressing tumors from normal tissue, and activity in the presence of sMSLN. We also studied the in vivo antitumor efficacy of this molecule (NM28-2746) alone and in combination with the multifunctional checkpoint inhibitor/T-cell co-activator NM21-1480 (αPD-L1/α4-1BB). Cytotoxicity and T-cell activation induced by NM28-2746 were studied in co-cultures of peripheral blood mononuclear cells and cell lines exhibiting different levels of MSLN expression, including in the presence of soluble MSLN. Xenotransplant models of human pancreatic cancer were used to study the inhibition of tumor growth and stimulation of T-cell infiltration into tumors induced by NM28-2746 alone and in combination with NM21-1480. The bivalent αMSLN T-cell engager NM28-2746 potently induced T-cell activation and T-cell mediated cytotoxicity of high MSLN-expressing cells but had much lower potency against low MSLN-expressing cells. A monovalent counterpart of NM28-2746 had much lower ability to discriminate high MSLN-expressing from low MSLN-expressing cells. The bivalent molecule retained this discriminant ability in the presence of high concentrations of sMSLN. In xenograft models, NM28-2746 exhibited significant tumor suppressing activity, which was significantly enhanced by combination therapy with NM21-1480. NM28-2746, alone or in combination with NM21-1480, may overcome shortcomings of previous MSLN-targeted immuno-oncology drugs, exhibiting enhanced discrimination of high MSLN-expressing cell activity in the presence of sMSLN.

Engineering of a trispecific tumor-targeted immunotherapy incorporating 4-1BB co-stimulation and PD-L1 blockade.

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 EC50 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.

Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies.

Despite their favorable pharmacokinetic properties, single-chain 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 x 10(-9) to 1.5 x 10(-11) m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 x 10(-10) to <1.8 x 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 degrees 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.

Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase.

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.

Yeast as a screening tool.

The versatile genetic malleability of yeast, and the high degree of conservation between its cellular processes and those of human cells, have made it the model of choice for pioneering research in molecular and cell biology over the past four decades. These characteristics of yeast, taken together with technical advantages such as simple growth conditions, rapid cell division and the development of a wealth of genetic tools for analysis of biological functions, have expanded the application of yeast as screening tool to the field of drug discovery.:

Quenching accumulation of toxic galactose-1-phosphate as a system to select disruption of protein-protein interactions in vivo.

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.

Enhanced apoptotic activity of a structurally optimized form of galectin-1.

Galectin-1 is a homodimeric protein with potent anti-inflammatory properties due to its ability to induce apoptosis in thymocytes and T cells. The galectin-1 subunits are not covalently linked but the monomers are in a dynamic equilibrium with the dimeric form. Since the affinity of the monomers for each other is rather low (in the range of 10(-5)M), the in vivo efficacy of galectin-1 is limited because the equilibrium is shifted towards the inactive monomeric form at lower concentrations. In order to overcome this problem, we designed a covalently linked form of the dimer based on the galectin-1 crystal structure. Here we show that this irreversibly dimeric form of galectin-1 is a potent inducer of apoptosis in murine thymocytes as well as murine mature T cells at concentrations 10-fold lower than wild-type galectin-1. This structurally optimized form of galectin-1 may therefore be a potentially powerful tool to treat chronic inflammatory diseases.