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Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities. Nevertheless, cytokines are crucial mediators of innate and adaptive antitumor immunity and have the potential to enhance immunotherapeutic approaches to treat cancer. Development of immune checkpoint inhibitors and combination immunotherapies has reinvigorated interest in cytokines as therapeutics, and a variety of engineering approaches are emerging to improve the safety and effectiveness of cytokine immunotherapy. In this review we highlight recent advances in cytokine biology and engineering for cancer immunotherapy.

Sequence changes that arise over evolution can alter various features of a protein's energy landscape, including its global stability and folding pathway. A comprehensive understanding of how the amino acid sequence dictates the energy landscape is critical to our understanding of biophysical properties of proteins, particularly for protein engineering applications. Here, we use ancestral sequence reconstruction (ASR) to examine the evolutionary history of homologous proteins by inferring and generating ancestral states and characterizing their energy landscapes. A systematic analysis of a protein family's evolutionary history should reveal how thermodynamic and kinetic features of the landscape are derived and maintained. This allows us to identify the sequence determinants of stability and folding pathways and reveal how different selective pressures alter the energy landscape and determine trajectories in sequence space.To access a protein family's evolutionary history, we utilized ASR to generate ancestral states along the lineages of mesophilic and thermophilic ribonucleases H. We observe pronounced trends that reveal the divergence in thermostability along the mesophilic and thermophilic lineages. Despite a clear selection for stability, the underlying mechanism was found to be variable, which we attribute to thermodyanmic system drift. Characterization of the ancestral folding pathway by kinetics, fragment models, and hydrogen-exchange mass spectrometry (HX-MS) reveals a subtly but distinctly changing landscape for the ribonuclease H family, revealing the role of critical mutations that alter the structure of partially folded intermediates and modulate barrier heights along the folding pathway.

The process by which proteins adopt their native structure has been an active field of study for decades. However, many basic questions about this protein folding process remain unanswered. For instance, the role of sequence in determining the pathway through which a protein folds has been difficult to characterize since most techniques to characterize pathways are slow, require large amounts of protein, or only probe folding in a small region of the protein. Hydrogen exchange measured by mass spectrometry (HX/MS) has proven to be a useful tool that can quickly determine folding pathway of a protein by measuring the buildup of hydrogen bonding along the protein backbone. In addition to its relative speed, HX/MS is able to measure to probe folding across the entire protein sequence allowing a pathway to be determined from a single experiment and uses nanomolar quantities of protein. Using HX/MS to determine the folding pathway for the RNases H from E. coli and T. thermophilus shows that despite folding to the same final structure these proteins fold through distinct pathways. Applying this technique to proteins from an ancestral sequence reconstruction of the RNase H family further refined the sequence changes responsible for this shift in folding pathways. This has led to a model that intrinsic helicity of stretches of the RNase H sequence may determine the earliest steps in the folding pathway.