FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models.

Purpose: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic.

Experimental Design: A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb.

FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1, Enhances T-Cell Activation Resulting in Potent Antitumor Activity.

Purpose: Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody.

Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases.

Biologic treatment options such as tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of inflammatory diseases, including rheumatoid arthritis. Recent data suggest, however, that full and long-lasting responses to TNF inhibitors are limited because of the activation of the pro-inflammatory TH17/interleukin (IL)-17 pathway in patients. Therefore, dual TNF/IL-17A inhibition is an attractive avenue to achieve superior efficacy levels in such diseases.

A HER2-specific Modified Fc Fragment (Fcab) Induces Antitumor Effects Through Degradation of HER2 and Apoptosis.

FS102 is a HER2-specific Fcab (Fc fragment with antigen binding), which binds HER2 with high affinity and recognizes an epitope that does not overlap with those of trastuzumab or pertuzumab. In tumor cells that express high levels of HER2, FS102 caused profound HER2 internalization and degradation leading to tumor cell apoptosis. The antitumor effect of FS102 in patient-derived xenografts (PDXs) correlated strongly with the HER2 amplification status of the tumors.

Linker length matters, fynomer-Fc fusion with an optimized linker displaying picomolar IL-17A inhibition potency.

Fynomers are small binding proteins derived from the human Fyn SH3 domain. Using phage display technology, Fynomers were generated inhibiting the activity of the proinflammatory cytokine interleukin-17A (IL-17A). One specific Fynomer called 2C1 inhibited human IL-17A in vitro with an IC50 value of 2.

Generation, characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain.

The serine protease chymase (EC = 3.4.21.

Experimental evidence for a frustrated energy landscape in a three-helix-bundle protein family.

Energy landscape theory is a powerful tool for understanding the structure and dynamics of complex molecular systems, in particular biological macromolecules. The primary sequence of a protein defines its free-energy landscape and thus determines the folding pathway and the rate constants of folding and unfolding, as well as the protein's native structure. Theory has shown that roughness in the energy landscape will lead to slower folding, but derivation of detailed experimental descriptions of this landscape is challenging.

Different members of a simple three-helix bundle protein family have very different folding rate constants and fold by different mechanisms.

The 15th, 16th, and 17th repeats of chicken brain alpha-spectrin (R15, R16, and R17, respectively) are very similar in terms of structure and stability. However, R15 folds and unfolds 3 orders of magnitude faster than R16 and R17. This is unexpected.

Studying the folding of multidomain proteins.

There have been relatively few detailed comprehensive studies of the folding of protein domains (or modules) in the context of their natural covalently linked neighbors. This is despite the fact that a significant proportion of the proteome consists of multidomain proteins. In this review we highlight some key experimental investigations of the folding of multidomain proteins to draw attention to the difficulties that can arise in analyzing such systems.

The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain.

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known.

Distinguishing specific and nonspecific interdomain interactions in multidomain proteins.

Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices.

The folding and evolution of multidomain proteins.

Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent.

Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains.

Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be.

Complex folding kinetics of a multidomain protein.

Spectrin domains are three-helix bundles, commonly found in large tandem arrays. Equilibrium studies have shown that spectrin domains are significantly stabilized by their neighbors. In this work we show that domain:domain interactions can also have profound effects on their kinetic behavior.

Cooperative folding in a multi-domain protein.

Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array.

The folding of spectrin domains I: wild-type domains have the same stability but very different kinetic properties.

The study of proteins with the same architecture, but different sequence has proven to be a valuable tool in the protein folding field. As a prelude to studies on the folding mechanism of spectrin domains we present the kinetic characterisation of the wild-type forms of the 15th, 16th, and 17th domains of chicken brain alpha-spectrin (referred to as R15, R16 and R17, respectively). We show that the proteins all behave in a two-state manner, with different kinetic properties.