Publications: 2025
2025
Slezak, Tomasz; O'Leary, Kelly M; Li, Jinyang; Rohaim, Ahmed; Davydova, Elena K; Kossiakoff, Anthony A
Engineered protein G variants for multifunctional antibody-based assemblies Journal Article
In: Protein Sci, vol. 34, no. 2, pp. e70019, 2025, ISSN: 1469-896X.
@article{pmid39865354,
title = {Engineered protein G variants for multifunctional antibody-based assemblies},
author = {Tomasz Slezak and Kelly M O'Leary and Jinyang Li and Ahmed Rohaim and Elena K Davydova and Anthony A Kossiakoff},
doi = {10.1002/pro.70019},
issn = {1469-896X},
year = {2025},
date = {2025-02-01},
urldate = {2025-02-01},
journal = {Protein Sci},
volume = {34},
number = {2},
pages = {e70019},
abstract = {We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the "knobs into holes" strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent bispecific T-cell engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the "knobs into holes" strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent bispecific T-cell engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research.
Gorelik, Maryna; Miersch, Shane; Sidhu, Sachdev S
Structural Survey of Antigen Recognition by Synthetic Human Antibodies Journal Article
In: Cold Spring Harb Protoc, vol. 2025, no. 2, pp. pdb.over107759, 2025, ISSN: 1559-6095.
@article{pmid38594044,
title = {Structural Survey of Antigen Recognition by Synthetic Human Antibodies},
author = {Maryna Gorelik and Shane Miersch and Sachdev S Sidhu},
doi = {10.1101/pdb.over107759},
issn = {1559-6095},
year = {2025},
date = {2025-02-01},
urldate = {2025-02-01},
journal = {Cold Spring Harb Protoc},
volume = {2025},
number = {2},
pages = {pdb.over107759},
abstract = {Synthetic antibody libraries have been used extensively to isolate and optimize antibodies. To generate these libraries, the immunological diversity and the antibody framework(s) that supports it outside of the binding regions are carefully designed/chosen to ensure favorable functional and biophysical properties. In particular, minimalist, single-framework synthetic libraries pioneered by our group have yielded a vast trove of antibodies to a broad array of antigens. Here, we review their systematic and iterative development to provide insights into the design principles that make them a powerful tool for drug discovery. In addition, the ongoing accumulation of crystal structures of antigen-binding fragment (Fab)-antigen complexes generated with synthetic antibodies enables a deepening understanding of the structural determinants of antigen recognition and usage of immunoglobulin sequence diversity, which can assist in developing new strategies for antibody and library optimization. Toward this, we also survey here the structural landscape of a comprehensive and unbiased set of 50 distinct complexes derived from these libraries and compare it to a similar set of natural antibodies with the goal of better understanding how each achieves molecular recognition and whether opportunities exist for iterative improvement of synthetic libraries. From this survey, we conclude that despite the minimalist strategies used for design of these synthetic antibody libraries, the overall structural interaction landscapes are highly similar to natural repertoires. We also found, however, some key differences that can help guide the iterative design of new synthetic libraries via the introduction of positionally tailored diversity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Synthetic antibody libraries have been used extensively to isolate and optimize antibodies. To generate these libraries, the immunological diversity and the antibody framework(s) that supports it outside of the binding regions are carefully designed/chosen to ensure favorable functional and biophysical properties. In particular, minimalist, single-framework synthetic libraries pioneered by our group have yielded a vast trove of antibodies to a broad array of antigens. Here, we review their systematic and iterative development to provide insights into the design principles that make them a powerful tool for drug discovery. In addition, the ongoing accumulation of crystal structures of antigen-binding fragment (Fab)-antigen complexes generated with synthetic antibodies enables a deepening understanding of the structural determinants of antigen recognition and usage of immunoglobulin sequence diversity, which can assist in developing new strategies for antibody and library optimization. Toward this, we also survey here the structural landscape of a comprehensive and unbiased set of 50 distinct complexes derived from these libraries and compare it to a similar set of natural antibodies with the goal of better understanding how each achieves molecular recognition and whether opportunities exist for iterative improvement of synthetic libraries. From this survey, we conclude that despite the minimalist strategies used for design of these synthetic antibody libraries, the overall structural interaction landscapes are highly similar to natural repertoires. We also found, however, some key differences that can help guide the iterative design of new synthetic libraries via the introduction of positionally tailored diversity.
Mann, Samuel I; Lin, Zhi; Tan, Sophia K; Zhu, Jiaqi; Widel, Zachary X W; Bakanas, Ian; Mansergh, Jarrett P; Liu, Rui; Kelly, Mark J S; Wu, Yibing; Wells, James A; Therien, Michael J; DeGrado, William F
De Novo Design of Proteins That Bind Naphthalenediimides, Powerful Photooxidants with Tunable Photophysical Properties Journal Article
In: J Am Chem Soc, 2025, ISSN: 1520-5126.
@article{pmid39982408,
title = {De Novo Design of Proteins That Bind Naphthalenediimides, Powerful Photooxidants with Tunable Photophysical Properties},
author = {Samuel I Mann and Zhi Lin and Sophia K Tan and Jiaqi Zhu and Zachary X W Widel and Ian Bakanas and Jarrett P Mansergh and Rui Liu and Mark J S Kelly and Yibing Wu and James A Wells and Michael J Therien and William F DeGrado},
doi = {10.1021/jacs.4c18151},
issn = {1520-5126},
year = {2025},
date = {2025-02-01},
urldate = {2025-02-01},
journal = {J Am Chem Soc},
abstract = { protein design provides a framework to test our understanding of protein function and build proteins with cofactors and functions not found in nature. Here, we report the design of proteins designed to bind powerful photooxidants and the evaluation of the use of these proteins to generate diffusible small-molecule reactive species. Because excited-state dynamics are influenced by the dynamics and hydration of a photooxidant's environment, it was important to not only design a binding site but also to evaluate its dynamic properties. Thus, we used computational design in conjunction with molecular dynamics (MD) simulations to design a protein, designated NBP (DI inding rotein), that held a naphthalenediimide (NDI), a powerful photooxidant, in a programmable molecular environment. Solution NMR confirmed the structure of the complex. We evaluated two NDI cofactors in this protein using ultrafast pump-probe spectroscopy to evaluate light-triggered intra- and intermolecular electron transfer function. Moreover, we demonstrated the utility of this platform to activate multiple molecular probes for protein labeling.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
protein design provides a framework to test our understanding of protein function and build proteins with cofactors and functions not found in nature. Here, we report the design of proteins designed to bind powerful photooxidants and the evaluation of the use of these proteins to generate diffusible small-molecule reactive species. Because excited-state dynamics are influenced by the dynamics and hydration of a photooxidant's environment, it was important to not only design a binding site but also to evaluate its dynamic properties. Thus, we used computational design in conjunction with molecular dynamics (MD) simulations to design a protein, designated NBP (DI inding rotein), that held a naphthalenediimide (NDI), a powerful photooxidant, in a programmable molecular environment. Solution NMR confirmed the structure of the complex. We evaluated two NDI cofactors in this protein using ultrafast pump-probe spectroscopy to evaluate light-triggered intra- and intermolecular electron transfer function. Moreover, we demonstrated the utility of this platform to activate multiple molecular probes for protein labeling.
Holmes, Brandon B; Weigel, Thaddeus K; Chung, Jesseca M; Kaufman, Sarah K; Apresa, Brandon I; Byrnes, James R; Kumru, Kaan S; Vaquer-Alicea, Jaime; Gupta, Ankit; Rose, Indigo V L; Zhang, Yun; Nana, Alissa L; Alter, Dina; Grinberg, Lea T; Spina, Salvatore; Leung, Kevin K; Condello, Carlo; Kampmann, Martin; Seeley, William W; Coutinho-Budd, Jaeda C; Wells, James A
β-Amyloid Induces Microglial Expression of GPC4 and APOE Leading to Increased Neuronal Tau Pathology and Toxicity Journal Article
In: bioRxiv, 2025, ISSN: 2692-8205.
@article{pmid40060520,
title = {β-Amyloid Induces Microglial Expression of GPC4 and APOE Leading to Increased Neuronal Tau Pathology and Toxicity},
author = {Brandon B Holmes and Thaddeus K Weigel and Jesseca M Chung and Sarah K Kaufman and Brandon I Apresa and James R Byrnes and Kaan S Kumru and Jaime Vaquer-Alicea and Ankit Gupta and Indigo V L Rose and Yun Zhang and Alissa L Nana and Dina Alter and Lea T Grinberg and Salvatore Spina and Kevin K Leung and Carlo Condello and Martin Kampmann and William W Seeley and Jaeda C Coutinho-Budd and James A Wells},
doi = {10.1101/2025.02.20.637701},
issn = {2692-8205},
year = {2025},
date = {2025-02-01},
urldate = {2025-02-01},
journal = {bioRxiv},
abstract = {To elucidate the impact of Aβ pathology on microglia in Alzheimer's disease pathogenesis, we profiled the microglia surfaceome following treatment with Aβ fibrils. Our findings reveal that Aβ-associated human microglia upregulate Glypican 4 (GPC4), a GPI-anchored heparan sulfate proteoglycan (HSPG). In a amyloidosis model, glial GPC4 expression exacerbates motor deficits and reduces lifespan, indicating that glial GPC4 contributes to a toxic cellular program during neurodegeneration. In cell culture, GPC4 enhances microglia phagocytosis of tau aggregates, and shed GPC4 can act to facilitate tau aggregate uptake and seeding in neurons. Additionally, our data demonstrate that GPC4-mediated effects are amplified in the presence of APOE. These studies offer a mechanistic framework linking Aβ and tau pathology through microglial HSPGs and APOE.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To elucidate the impact of Aβ pathology on microglia in Alzheimer's disease pathogenesis, we profiled the microglia surfaceome following treatment with Aβ fibrils. Our findings reveal that Aβ-associated human microglia upregulate Glypican 4 (GPC4), a GPI-anchored heparan sulfate proteoglycan (HSPG). In a amyloidosis model, glial GPC4 expression exacerbates motor deficits and reduces lifespan, indicating that glial GPC4 contributes to a toxic cellular program during neurodegeneration. In cell culture, GPC4 enhances microglia phagocytosis of tau aggregates, and shed GPC4 can act to facilitate tau aggregate uptake and seeding in neurons. Additionally, our data demonstrate that GPC4-mediated effects are amplified in the presence of APOE. These studies offer a mechanistic framework linking Aβ and tau pathology through microglial HSPGs and APOE.
Ngo, Wayne; Peukes, Julia; Baldwin, Alisha; Xue, Zhiwei Wayne; Hwang, Sidney; Stickels, Robert R; Lin, Zhi; Satpathy, Ansuman T; Wells, James A; Schekman, Randy; Nogales, Eva; Doudna, Jennifer A
Mechanism-guided engineering of a minimal biological particle for genome editing Journal Article
In: Proc Natl Acad Sci U S A, vol. 122, no. 1, pp. e2413519121, 2025, ISSN: 1091-6490.
@article{pmid39793042,
title = {Mechanism-guided engineering of a minimal biological particle for genome editing},
author = {Wayne Ngo and Julia Peukes and Alisha Baldwin and Zhiwei Wayne Xue and Sidney Hwang and Robert R Stickels and Zhi Lin and Ansuman T Satpathy and James A Wells and Randy Schekman and Eva Nogales and Jennifer A Doudna},
doi = {10.1073/pnas.2413519121},
issn = {1091-6490},
year = {2025},
date = {2025-01-01},
urldate = {2025-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {122},
number = {1},
pages = {e2413519121},
abstract = {The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.
Rabe, Daniel C; Choudhury, Adarsh; Lee, Dasol; Luciani, Evelyn G; Ho, Uyen K; Clark, Alex E; Glasgow, Jeffrey E; Veiga, Sara; Michaud, William A; Capen, Diane; Flynn, Elizabeth A; Hartmann, Nicola; Garretson, Aaron F; Muzikansky, Alona; Goldberg, Marcia B; Kwon, Douglas S; Yu, Xu; Carlin, Aaron F; Theriault, Yves; Wells, James A; Lennerz, Jochen K; Lai, Peggy S; Rabi, Sayed Ali; Hoang, Anh N; Boland, Genevieve M; Stott, Shannon L
Ultrasensitive detection of intact SARS-CoV-2 particles in complex biofluids using microfluidic affinity capture Journal Article
In: Sci Adv, vol. 11, no. 2, pp. eadh1167, 2025, ISSN: 2375-2548.
@article{pmid39792670,
title = {Ultrasensitive detection of intact SARS-CoV-2 particles in complex biofluids using microfluidic affinity capture},
author = {Daniel C Rabe and Adarsh Choudhury and Dasol Lee and Evelyn G Luciani and Uyen K Ho and Alex E Clark and Jeffrey E Glasgow and Sara Veiga and William A Michaud and Diane Capen and Elizabeth A Flynn and Nicola Hartmann and Aaron F Garretson and Alona Muzikansky and Marcia B Goldberg and Douglas S Kwon and Xu Yu and Aaron F Carlin and Yves Theriault and James A Wells and Jochen K Lennerz and Peggy S Lai and Sayed Ali Rabi and Anh N Hoang and Genevieve M Boland and Shannon L Stott},
doi = {10.1126/sciadv.adh1167},
issn = {2375-2548},
year = {2025},
date = {2025-01-01},
urldate = {2025-01-01},
journal = {Sci Adv},
volume = {11},
number = {2},
pages = {eadh1167},
abstract = {Measuring virus in biofluids is complicated by confounding biomolecules coisolated with viral nucleic acids. To address this, we developed an affinity-based microfluidic device for specific capture of intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our approach used an engineered angiotensin-converting enzyme 2 to capture intact virus from plasma and other complex biofluids. Our device leverages a staggered herringbone pattern, nanoparticle surface coating, and processing conditions to achieve detection of as few as 3 viral copies per milliliter. We further validated our microfluidic assay on 103 plasma, 36 saliva, and 29 stool samples collected from unique patients with COVID-19, showing SARS-CoV-2 detection in 72% of plasma samples. Longitudinal monitoring in the plasma revealed our device's capacity for ultrasensitive detection of active viral infections over time. Our technology can be adapted to target other viruses using relevant cell entry molecules for affinity capture. This versatility underscores the potential for widespread application in viral load monitoring and disease management.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Measuring virus in biofluids is complicated by confounding biomolecules coisolated with viral nucleic acids. To address this, we developed an affinity-based microfluidic device for specific capture of intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our approach used an engineered angiotensin-converting enzyme 2 to capture intact virus from plasma and other complex biofluids. Our device leverages a staggered herringbone pattern, nanoparticle surface coating, and processing conditions to achieve detection of as few as 3 viral copies per milliliter. We further validated our microfluidic assay on 103 plasma, 36 saliva, and 29 stool samples collected from unique patients with COVID-19, showing SARS-CoV-2 detection in 72% of plasma samples. Longitudinal monitoring in the plasma revealed our device's capacity for ultrasensitive detection of active viral infections over time. Our technology can be adapted to target other viruses using relevant cell entry molecules for affinity capture. This versatility underscores the potential for widespread application in viral load monitoring and disease management.
Górniak, Ireneusz; Stephens, Zachery; Erramilli, Satchal K; Gawda, Tomasz; Kossiakoff, Anthony A; Zimmer, Jochen
Structural insights into translocation and tailored synthesis of hyaluronan Journal Article
In: Nat Struct Mol Biol, vol. 32, no. 1, pp. 161–171, 2025, ISSN: 1545-9985.
@article{pmid39322765,
title = {Structural insights into translocation and tailored synthesis of hyaluronan},
author = {Ireneusz Górniak and Zachery Stephens and Satchal K Erramilli and Tomasz Gawda and Anthony A Kossiakoff and Jochen Zimmer},
doi = {10.1038/s41594-024-01389-1},
issn = {1545-9985},
year = {2025},
date = {2025-01-01},
urldate = {2025-01-01},
journal = {Nat Struct Mol Biol},
volume = {32},
number = {1},
pages = {161--171},
abstract = {Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.
