Bispecific Targeting of PD-1 and PD-L1 Enhances T-cell Activation and Antitumor Immunity.

The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell-mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor-ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti-PD-1 and anti-PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation.

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity.

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.

Binding is not enough: flexibility is needed for photocrosslinking of Lck kinase by benzophenone photoligands.

Benzophenone photophores are employed widely for photoaffinity-labeling studies. Photolabeling with benzophenone, however, is hardly a routine experiment. Even when a photoprobe binds to its target, photocrosslinking does not necessarily occur. This is because photolabeling by benzophenone is affected by many factors other than target-binding, such as conformational flexibility of photoligand. Despite the widespread recognition of such complications, there has been no systematic study to assess the relative importance of individual factors that can affect photolabeling efficiency. In order to gain an insight into this problem, we conducted a structure-activity relationship (SAR) study of benzophenone photoligands for Lck kinase, in which photoligands with varying target-binding affinity and conformational flexibility were compared. The study found that binding-affinity, as indicated by kinase inhibitory potency, did not correlate with photolabeling efficiency. Instead, conformational flexibility was found to be the determining factor for efficient photolabeling by our photoligands. Implication of the current findings, in particular, with regard to selection and optimization of benzophenone photoligands, is discussed.

Selective photolabeling of Lck kinase in complex proteome.

A molecular probe that selectively tags Lck, a Src-family kinase, was developed. This probe was one of many compounds originally designed to target the active site of tyrosine kinases in general. To our surprise, however, the probe almost exclusively labeled Lck even in a lysate of Jurkat cells. This finding led us to further characterize this probe-Lck complex by a series of photolabeling and mass spectrometric analyses. The probe-binding site on Lck was located within the well-conserved region of Src-family kinases, as we originally expected. However, the unexpected selectivity of this probe toward Lck suggests that subtle factors, which are difficult to predict based on static crystal structures, play important roles in probe recognition.

Protein fishing with chiral molecular baits.

Chirality plays a central role in various biological recognition processes. Here a methodology was developed to utilize chiral recognition processes for the selective biotinylation of proteins in crude cell lysates. Two pairs of diastereomeric probes containing benzophenone and biotin were prepared through solid-phase synthesis. Protein-binding selectivity of each probe was examined by photo-cross-linking of cell lysates, followed by SDS-PAGE and Western blot. The study revealed that our approach permits selective labeling of benzophenone-binding proteins in complex proteomes. In addition, it was found that the selectivity depends largely on a single chiral center and substitutions in the vicinity of benzophenone. Taken together, the current work demonstrates that chiral recognition process can be employed to selectively label proteins in complex proteomes. Thus the study opens up the possibility to expand the scope of chemical proteomics research for various applications, including biomarker discovery, drug screening and development.