Protein Engineering Design & Selection最新文献

Several technologies leverage avidin/biotin interactions for complexation including the MAPS vaccine technology which utilizes rhizavidin, a biotin-binding protein derived from the proteobacterium Rhizobium etli, to complex antigens with biotinylated polysaccharides. Rhizavidin possesses five potential N-linked glycosylation sites which are not glycosylated in the native bacterium or when rhizavidin is recombinantly expressed in Escherichia coli. However, when expressed in eukaryotic cell systems, these sites undergo variable and non-physiologically relevant glycosylation that complicates purification. To overcome these challenges, we engineered rhizavidin with substitutions that abolish unnatural N-linked glycosylation while maintaining rhizavidin's biotin-binding functionality. As a proof of concept, this construct was genetically fused to the receptor binding domain of the spike protein from the SARS-CoV-2 virus, expressed in mammalian cells, and was successfully incorporated into MAPS technology-based vaccines. This newly engineered rhizavidin enhances the versatility of the MAPS technology, enabling the targeting of viruses and tumor-associated antigens that often require mammalian post-translational modifications.

Antibody-dependent cellular cytotoxicity is a key mechanism for antibody-based therapeutics, and current engineering strategies to enhance ADCC primarily rely on two approaches: Fc mutations to improve the antibody's intrinsic CD16a affinity, or the fusion of binding-modules targeting NK cell receptors. The former often compromises antibody stability and induces CD16a downregulation; the latter occupies sites critical for target association and limits the assembly of multi-specific therapeutics. Here, we introduce a novel Fc engineering approach wherein the CH2 domain of the Fc region is replaced with an anti-NKp46 VHH named VIF-Ig. It has been reported that NKp46 expression remains unaltered after NK cell activation across various tumor microenvironments, addressing a key limitation of CD16a-dependent strategies. The novel molecules exhibit potent ADCC, high thermal stability, and retain FcRn binding for favorable pharmacokinetic profiles. Furthermore, we demonstrate that VIF-Ig can accommodate VHHs to target various epitopes. Thus, this versatile modular platform is suitable for developing next-generation NK cell or even other cell engagers with enhanced efficacy and tunable specificity, especially for emerging multi-specific immune cell engagers.

The rapid evolution of SARS-CoV-2 presents substantial challenges to maintaining diagnostic accuracy. Single-domain antibodies (variable domains of camelid heavy-chain antibodies, VHHs), commonly referred to as nanobodies, are attractive tools for diagnostic applications due to their high specificity, stability, affinity and expression yield. In this study, three high-affinity VHHs that recognize the receptor-binding domain (RBD) of the SARS-CoV-2 BA.2 variant were isolated from a large naïve alpaca VHH phage display library. These nanobodies exhibited nanomolar affinities and high thermal stability. Notably, reformatting one VHH into a bivalent VHH-Fc fusion construct, K115.2-Fc, enhanced binding affinity 24-fold, achieving an equilibrium dissociation constant of 68.3 picomolar. Functional characterization demonstrated that K115.2-Fc cross-reacted with the Wuhan SARS-CoV-2 RBD and multiple variants, including Alpha, Beta, Gamma, Delta, Kappa, BA.1, BA.2, and BA.4/5. In indirect enzyme-linked immunosorbent assay, K115.2-Fc detected full-length spike protein with a limit of detection of 8.1 pM. Moreover, it enabled sensitive detection in western blotting and flow cytometry, highlighting its applicability across diverse diagnostic applications. Collectively, these findings identify K115.2-Fc as a promising candidate with potential for SARS-CoV-2 diagnostic utility.

Model organisms significantly advance research because scientific communities recognize their value, develop infrastructure around them, and utilize them to address questions relevant to multiple species. I propose adopting a similar approach at the molecular level by establishing an official model protein system. This system would acknowledge five proteins that are already informally recognized as models in protein science: GFP, lysozyme, hemoglobin/myoglobin, RNase A, and bacteriorhodopsin. Formal recognition of these proteins would create standardized reporting requirements, shared benchmarks, and reference datasets, enhancing reproducibility, comparability, and educational resources. GFP serves as a prime example of this concept: it is a single-gene, genetically portable protein with a conserved structure and a measurable phenotype that effectively connects computation and experimental research. This paper identifies the criteria for model protein designation, presents a minimal reporting checklist, and outlines initial steps for implementation at the system level.

The evolution of antibody engineering has significantly enhanced the development of antibody-based therapeutics, enabling the creation of novel antibody formats tailored for specific applications. Since the introduction of the Kabat numbering scheme in 1977, various schemes have been developed and modified, forming the foundation for multiple antibody engineering projects. The tools associated with these schemes further facilitate the engineering process. However, discrepancies among current numbering schemes can lead to confusion. This study examines various numbering schemes and related tools, providing new insights into antibody variable domains. Improved understanding of antibody numbering and related tools holds significant potential for more precise and efficient antibody design, thereby advancing antibody-based therapeutics and diagnostics.

Tuning in vivo activity of protein therapeutics can improve their safety. In this vein, it is possible to add a 'mask' moiety to a protein therapeutic such that its ability to bind its target is prevented until the mask has been proteolytically removed, for instance by a tumor-associated protease. As such, new methods to isolate functional masking sequences can aid development of protein therapies. Here, we describe a yeast display-based method to discover peptide sequences that prevent binding of antibody fragments to their antigen target. Our method includes an in situ ability to screen for restoration of binding by scFvs after proteolytic mask removal, and it takes advantage of the antigenic target itself to guide mask discovery. First, we genetically linked a yeast-displayed αPSCA scFv to overlapping 'tiles' of its target. By selecting for reduced antigen binding via flow cytometry, we discovered two peptide masks that we confirmed to be linear epitopes of the PSCA antigen. We then expanded our method towards developing masks for three-dimensional epitopes by using a co-crystal structure of an αHer2 antibody in complex with its antigen to guide combinatorial mask design. In sum, our efforts show the feasibility of employing yeast-displayed, antigen-based libraries to find antibody masks.

Bioconjugates as therapeutic modalities combine the advantages and offset the disadvantages of their constituent parts to achieve a refined spectrum of action. We combine the concept of bioconjugation with the full atomic simulation capability of computational protein design to define a new class of molecular recognition agents: CDR-extended antibodies, abbreviated as CDRxAbs. A CDRxAb incorporates a covalently attached small molecule into an antibody/target binding interface using computational protein design to create an antibody small-molecule conjugate that binds tighter to the target of the small molecule than the small molecule would alone. CDRxAbs are also expected to increase the target binding specificity of their associated small molecules. In a proof-of-concept study using monomeric streptavidin/biotin pairs at either a nanomolar or micromolar-level initial affinity, we designed nanobody-biotin conjugates that exhibited >20-fold affinity improvement against their protein targets with step-wise optimization of binding kinetics and overall protein stability. The workflow explored through this process promises a novel approach to optimize small-molecule based therapeutics and to explore new chemical and target space for molecular-recognition agents in general.

Pompe disease is a tissue glycogen disorder caused by genetic insufficiency of the GAA enzyme. GAA enzyme replacement therapies for Pompe disease have been limited by poor lysosomal trafficking of the recombinant GAA molecule through the native mannose-6-phosphate-mediated pathway. Here, we describe the successful rational engineering of a chimeric GAA enzyme that utilizes the binding affinity of a modified IGF-II moiety to its native receptor to bypass the mannose-6-phosphate-mediated lysosomal trafficking pathway, conferring a significant increase in cellular uptake of the GAA enzyme. We also demonstrate the ablation of binding between our modified IGF-II tag and two off-target receptors: IGF-I receptor and insulin receptor, as well as preserved enzymatic activity of the chimeric GAA molecule.

Clustered regularly interspaced short palindromic repeat interference (CRISPRi), the fusion of nuclease-inactive Cas9 with transcriptional repressor domains, is a powerful platform enabling site-specific gene knockdown across diverse biological contexts. Previously described CRISPRi systems typically utilize two distinct domain classes: (1) Krüppel-associated box domains and (2) truncations of the multifunctional protein, MeCP2. Despite widespread adoption of MeCP2 truncations for developing CRISPRi platforms, individual contributions of subdomains within MeCP2's transcriptional repression domain (TRD) toward enhancing gene knockdown remain unclear. Here, we dissect MeCP2's TRD and observe that two subdomains, the expected NcoR/SMRT interaction domain (NID) and an embedded nuclear localization signal (NLS), can separately enhance gold-standard CRISPRi platform performance beyond levels attained with the canonical MeCP2 protein truncation. Incorporating side-by-side analyses of nuclear localization and gene knockdown for over 30 constructs featuring MeCP2 subdomains or virus-derived NLS sequences, we demonstrate that appending C-terminal NLS motifs to dCas9-based transcriptional regulators, both repressors and activators, can significantly improve their effector function across several cell lines. We also observe that NLS placement greatly impacts CRISPRi repressor performance, and that modifying the subdomain configuration natively found within MeCP2 can also enhance gene suppression capabilities in certain contexts. Overall, this work demonstrates the interplay of two complimentary chimeric protein design considerations, transcriptional domain 'dissection' and NLS motif placement, for optimizing CRISPR-mediated transcriptional regulation in mammalian systems.

ProteinMPNN is widely used in protein design workflows due to its ability to identify amino acid sequences that fold into specific 3D protein structures. In our work, we adjust ProteinMPNN to design proteins for a given 3D protein structure with reduced immune-visibility to cytotoxic T lymphocytes that recognize proteins via the MHC-I pathway. To achieve this, we developed a novel framework that integrates direct preference optimization (DPO)-a tuning method originally designed for large language models-with MHC-I peptide presentation predictions. This approach fosters the generation of designs with fewer MHC-I epitopes while preserving the protein's original structure. Our results demonstrate that DPO effectively reduces MHC-I visibility without compromising the structural integrity of the proteins.