Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

Reviewer #1 (Public review):

Summary:

This manuscript describes the use of computational tools to design a mimetic of the interleukin-7 (IL-7) cytokine with superior stability and receptor binding activity compared to the naturally occurring molecule. The authors focused their engineering efforts on the loop regions to preserve receptor interfaces while remediating structural irregularities that destabilize the protein. They demonstrated the enhanced thermostability, production yield, and bioactivity of the resulting molecule through biophysical and functional studies. Overall, the manuscript is well written, novel, and of high interest to the fields of molecular engineering, immunology, biophysics, and protein therapeutic design. The experimental methodologies used are convincing; however, the article would benefit from more quantitative comparisons of bioactivity through titrations.

Reviewer #2 (Public review):

Summary:

This manuscript presents the computational design and experimental validation of Neo-7, an engineered variant of interleukin-7 (IL-7) with improved folding efficiency, expression yield, and therapeutic activity. The authors employed a rational protein design approach using Rosetta loop remodeling to reconnect IL-7's functional helices through shorter, more efficient loops, resulting in a protein with superior stability and binding affinity compared to wild-type IL-7. The work demonstrates promising translational potential for cancer immunotherapy applications.

Strengths:

(1) The integration of Rosetta loop remodeling with AlphaFold validation represents an established computational pipeline for rational protein design. The iterative refinement process, using both single-sequence and multimer AlphaFold predictions, is methodologically sound.

(2) The authors provide thorough characterization across multiple platforms (yeast display, bacterial expression, mammalian cell expression) and assays (binding kinetics, thermostability, bioactivity), strengthening the robustness of their findings.

(3) The identification of the critical helix 1 kink stabilized by disulfide bonding and its recreation through G4C/L96C mutations demonstrates deep structural understanding and successful problem-solving.

(4) The MC38 tumor model results show clear therapeutic advantages of Neo-7 variants, with compelling immune profiling data supporting CD8+ T cell-mediated anti-tumor mechanisms.

(5) The transcriptomic profiling provides valuable mechanistic insights into T cell activation states and suggests reduced exhaustion markers, which are clinically relevant.

Weaknesses:

(1) While computational predictions are extensive, the manuscript lacks experimental structural validation of the designed Neo-7 variants. The term "Structural Validation" should not be used in the header.

(2) The authors observe slower on/off-rates for Neo-7 variants compared to wild-type IL-7. Could the authors speculate about the potential biological impacts of the slow off-rate, especially focusing on downstream signaling pathways that might be differentially affected by the altered binding kinetics of Neo-7 variants?

(3) While computational immunogenicity prediction is provided, these methods are very limited.