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The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8 N/C EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8 N/C EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8 N/C EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. Cancer therapy using systemically administrated 4-1BB-targeting antibodies is often associated with severe toxicity due to the nonspecific activation of autoreactive T cells. Here, the authors have developed a trimeric antibody targeting both 4-1BB and EGFR, which activates T cells effectively and shows negligible cytotoxicity.

4-1BB (CD137) is an inducible costimulatory receptor that promotes expansion and survival of activated T cells; and IgG-based 4-1BB-agonistic monoclonal antibodies exhibited potent antitumor activity in clinical trials. However, the clinical development of those antibodies is restricted by major off-tumor toxicities associated with FcγR interactions. We have recently generated an EGFR-targeted 4-1BB-agonistic trimerbody that demonstrated strong antitumor activity and did not induce systemic inflammatory cytokine secretion and hepatotoxicity associated with first-generation 4-1BB agonists. Here, we generate a bispecific 4-1BB-agonistic trimerbody targeting the carcinoembryonic antigen (CEA) that is highly expressed in cancers of diverse origins. The CEA-targeted anti-4-1BB-agonistic trimerbody consists of three 4-1BB-specific single-chain fragment variable antibodies and three anti-CEA single-domain antibodies positioned around a murine collagen XVIII-derived homotrimerization domain. The trimerbody was produced as a homogenous, non-aggregating, soluble protein purifiable by standard affinity chromatographic methods. The purified trimerbody was found to be trimeric in solution, very efficient at recognizing 4-1BB and CEA, and potently costimulating T cells in the presence of CEA. Therefore, trimerbody-based tumor-targeted 4-1BB costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions.

A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory mechanism of human A2ML1 and determine the structures of its native and protease-cleaved conformations. The functional inhibitory unit of A2ML1 is a monomer that depends on covalent binding of the protease (mediated by A2ML1’s thioester) to achieve inhibition. In contrast to the A2M tetramer which traps proteases in two internal chambers formed by four subunits, in protease-cleaved monomeric A2ML1 disordered regions surround the trapped protease and may prevent substrate access. In native A2ML1, the bait region is threaded through a hydrophobic channel, suggesting that disruption of this arrangement by bait region cleavage triggers the extensive conformational changes that result in protease inhibition. Structural comparisons with complement C3/C4 suggest that the A2M superfamily of proteins share this mechanism for the triggering of conformational change occurring upon proteolytic activation. A2ML1 is a human protease inhibitor belonging to the A2M protein family. In this study, the authors determine structures of A2ML1 before and after protease inhibition and investigate its mechanism of action.

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (V HHs ) to the N-terminus of a human collagen XVIII trimerization domain (TIE XVIII ) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified V HH -TIE XVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three V HH -TIE XVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem V HH -based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem V HH -based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific V HH -based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.

The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of the C3(H2O) analog, C3MA, in which the C3 thioester is broken by aminolysis is indistinguishable from C3b except for the 77-residue anaphylatoxin (ANA) domain. In contrast, the reaction intermediate C3* formed during C3 adopts a dynamic conformation dramatically different from both C3 and C3MA/C3b. In C3*, unlocking of the macroglobulin (MG) 3 domain creates a large opening in the MG-ring through which the ANA domain translocates. In support of this mechanism, C3MA formation is inhibited by an MG3/MG4-interface-specific nanobody and prevented by linking the ANA domain to the C3 β-chain. Our study reveals an unexpected dynamic behavior of C3 where an exceptional conformational change allows the translocation of an entire domain through a large dynamic opening. These results form the basis for elucidation of the in vivo contribution of C3 hydrolysis to complement activation and offer a rational approach for modulation of C3(H2O) with the potential for preventing complement activation caused by intravascular hemolysis and surface contacts.