Selective Noble Gas Inclusion in Pentagon-Dodecahedral X20-Cages.

Using DFT-based computational chemistry calculations (ωB97XD/def2-tzvp//ωB97XD/def2-svp/svpfit + ZPE(ωB97XD/def2-svp/svpfit)), binding energies of noble gases encapsulated in a series of dodecahedrane molecules (general formula: X20H20 where X = C, Si, Ge, Sn and Pb, and X20 where X = N, P, As, Sb and Bi) were calculated to learn about the noble gas selectivity. Based on calculated binding energies, the Sn20H20 cage can best accommodate noble gases with a medium size radius (Ar and Kr), while the Pb20H20 dodecahedrane cage is best suited for noble gases with the larger radii (Xe and Rn). On the other hand, from the elements of the V main group of the periodic table, the Bi20 cage has shown the best results to selectively encapsulate Ar and Kr, with the amounts of energy being released being -5.24 kcal/mol and -6.13 kcal/mol, respectively. By monitoring the geometric changes of all here-reported host cages upon encapsulating the noble gas guest, the host has shown minor to no flexibility, testifying to the high rigidity of the dodecahedrane structure which was further reflected in very high encapsulating energies.

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.

Design of antibody variable fragments with reduced reactivity to preexisting anti-drug antibodies.

Upon reformatting of an antibody to single-chain variable fragment format, a region in the former variable/constant domain interface of the heavy chain becomes accessible for preexisting (PE) anti-drug antibody (ADA) binding. The region exposed because of this reformatting contains a previously hidden hydrophobic patch. In this study, mutations are introduced in this region to reduce PE ADA reactivity and concomitantly reduce the hydrophobic patch. To enhance our understanding of the importance of individual residues in this region with respect to PE ADA reactivity, a total of 50 molecules for each of two antibodies against different tumor-associated antigens were designed, produced, and characterized by an arsenal of biophysical methods. The aim was to identify suitable mutations that reduce, or completely eliminate, PE ADA reactivity to variable fragments, without compromising biophysical and pharmacodynamic properties. Computational methods were used to pinpoint key residues to mutate and to evaluate designed molecules in silico, in order to reduce the number of molecules to produce and characterize experimentally. Mutation of two threonine residues, Thr101 and Thr146 in the variable heavy domain, proved to be critical to eliminate PE ADA reactivity. This may have important implications in optimizing early drug development for antibody fragment-based therapeutics.

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.

Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise.

Caspases play important roles during apoptosis, inflammation and proliferation. The high homology among family members makes selective targeting of individual caspases difficult, which is necessary to precisely define the role of these enzymes. We have selected caspase-7-specific binders from a library of DARPins (designed ankyrin repeat proteins). The DARPins D7.18 and D7.43 bind specifically to procaspase 7 and active caspase 7, but not to other members of the family. Binding of the DARPins does not affect the active enzyme, but interferes with its activation by other caspases. The crystal structure of the caspase 7-D7.18 complex elucidates the high selectivity and the mode of inhibition. Combining these caspase-7-specific DARPins with the previously reported caspase-3-inhibitory DARPin D3.4S76R reduces the activity of caspase 3 and 7 in double-transfected HeLa cells during apoptosis. In addition, these cells showed less susceptibility to TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in living cell experiments. D7.18 and D7.43 are therefore novel tools for in vitro studies on procaspase 7 activation as well as for clarifying the role of its activation in different cellular processes. If applied in combination with D3.4S76R, they represent an excellent instrument to increase our understanding of these enzymes during various cellular processes.