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Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher.

ABSTRACT: Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially added to the antigen, their proximity on the antigen results in covalent bond formation and generation of a biparatopic antibody. We validated the system with purified recombinant antigen. Results in antigen-overexpressing cells were promising although further optimization will be required. Because this strategy results in high-affinity targeting with a bipartite molecule that has considerably lower molecular weight than an antibody, this technology is potentially useful for diverse applications.

SUBMITTER: Akiba H

PROVIDER: S-EPMC6976922 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher.

Akiba Hiroki H Takayanagi Kensuke K Kusano-Arai Osamu O Iwanari Hiroko H Hamakubo Takao T Tsumoto Kouhei K

Biotechnology reports (Amsterdam, Netherlands) 20200111

Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially a ...[more]

PMID: 31993343

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Project description:We report a simple strategy for the creation of lentiviral vectors specific to any desired target cells. SpyTag is inserted into an engineered Sindbis virus envelope protein and displayed on the lentivirus surface to create Sindbis virus-SpyTag pseudoparticles (Sind-SpyTag-pp). The SpyTag serves as the covalent anchoring site for a target-cell-specific cell-binding protein (CBP) that is fused to a truncated SpyCatcher (SpyCatcher?). Target-cell-specific lentiviruses are created by mixing the Sind-SpyTag-pp and CBP-SpyCatcher? in vitro We first used a HER2-binding designed ankyrin repeat protein (DARPin.9.26) as the model CBP. The DARPin-conjugated lentivirus transduced HER2+ SKOV3 cells with an infectious titer of 5.2 × 106 IU/ml, >500-fold higher than the unfunctionalized "naked" virions (<104 IU/ml). The ability of the DARPin-conjugated lentivirus to transduce HER2+ cells correlated with the surface expression level of HER2. Furthermore, these lentiviruses preferentially transduced HER2+ cells in cocultures containing HER2+ and HER2- cells. To enable the use of commercially available monoclonal antibodies (MAbs) as the CBP, we developed a convenient click chemistry-based approach to conjugate MAb-derived Fab fragments to a variant SpyCatcher? protein containing a nonnatural amino acid, 4-azido-l-phenylalanine (AzF). Using the HER2-binding trastuzumab as a model cell-specific MAb, we created Fab-conjugated lentiviral vectors that transduced HER2+ SKOV3 cells with an infectious titer of 2.8 × 106 IU/ml, on par with the result achieved using the DARPin-SpyCatcher? fusion protein. The ability to create cell-specific lentiviral vectors through chemical conjugation of a CBP should make this approach generalizable to any antibody, giving it broad utility for a wide range of research and clinical applications.IMPORTANCE Lentiviral vectors hold great potential in gene therapy. However, it remains a major hurdle to robustly engineer cell-specific lentiviral vectors. This article reports a simple and effective strategy to functionalize lentiviral vectors with cell-binding proteins, thus retargeting these viruses to cells expressing the binding partner of the CBP. The CBP is genetically or chemically linked to the SpyCatcher. The SpyTag is displayed on the virion surface as a fusion to an engineered Sindbis virus envelope protein and is used as the anchorage site for SpyCatcher-linked CBP. Using this strategy, we created lentiviral vectors highly infectious toward HER2+ cancer cells. The ability to rapidly create cell-specific lentiviral vectors targeting a wide range of cell types should accelerate the development of custom lentiviral vectors for many research and clinical applications.

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Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher.

Publications

Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher.

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