Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.

In contrast to T cell receptors, signal transducing cell surface membrane molecules involved in the regulation of responses by cells of the innate immune system employ structures that are encoded in the genome rather than generated by somatic recombination and that recognize either classical MHC-I molecules or their structural relatives (such as MICA, RAE-1, or H-60). Considerable progress has recently been made in our understanding of molecular recognition by such molecules based on the determination of their three-dimensional structure, either in isolation or in complex with their MHC-I ligands. Those best studied are the receptors that are expressed on natural killer (NK) cells, but others are found on populations of T cells and other hematopoietic cells. These molecules fall into two major structural classes, those of the immunoglobulin superfamily (KIRs and LIRs) and of the C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize, in a functional context, the structures of the murine and human molecules that have recently been determined, with emphasis on how they bind different regions of their MHC-I ligands, and how this allows the discrimination of tumor or virus-infected cells from normal cells of the host.

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.

MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.

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