A bovine antibody possessing an ultralong complementarity-determining region CDRH3 targets a highly conserved epitope in sarbecovirus spike proteins

Burke, Matthew J.; Scott, James N.; Minshull, Thomas C.; Gao, Zeqian; Manfield, Iain W.; Savic, Sinisa; Stockley, Peter G.; Calabrese, Antonio N.; Boyes, Joan

doi:10.1016/j.jbc.2022.102624

Cited by 5 publications

(8 citation statements)

References 46 publications

(79 reference statements)

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“…To further differentiate on this observation, each third of the knob in H12 was mutated to an irrelevant sequence resulting in three different mutants, which showed that the C-terminal third of this knob is more important for binding than the other two thirds 24 . A similar observation was made by applying an analogical mutational approach on a SARS-CoV-specific bovine ulCDR-H3 29 . Furthermore, alanine scanning in the knob of NC-Cow1 identified the importance of individual residues.…”

Section: Knobsupporting

confidence: 64%

“…Mammalian cell surface display has also been used to discover antigen-specific ulCDR-H3 (Fig. 2c) 29 . Burke et al developed an scFv system for mammalian surface display of ulCDR-H3s employing a pBovShow vector where the heavy chains are paired with an invariant lambda light chain.…”

Section: Macpherson Et Al Immunized Two Adult Holstein Cattle With Th...mentioning

confidence: 99%

“…x 10 4 ). Strikingly, despite the small library size, the approach yielded a bovine ulCDR-H3 with a moderate binding affinity to the spike proteins of SARS-CoV and SARS-CoV-2 29 .…”

Section: Macpherson Et Al Immunized Two Adult Holstein Cattle With Th...mentioning

confidence: 99%

“…Recent studies have demonstrated that the antibodies with ulCDRs hold enormous potential for therapeutic applications and rational engineering of new target-binding proteins, for example, as antiviral 28,29 , anticancer 30,31 or immunomodulatory 32,33 agents. Here we review the exceptional features of antibodies with ulCDRs.…”

Section: Introductionmentioning

confidence: 99%

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Principles of antibodies with ultralong complementarity-determining regions and picobodies

Passon

Smedt

Svilenov

2023

Biotechnology Advances

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Section: Knobsupporting

confidence: 64%

Section: Macpherson Et Al Immunized Two Adult Holstein Cattle With Th...mentioning

confidence: 99%

“…x 10 4 ). Strikingly, despite the small library size, the approach yielded a bovine ulCDR-H3 with a moderate binding affinity to the spike proteins of SARS-CoV and SARS-CoV-2 29 .…”

Section: Macpherson Et Al Immunized Two Adult Holstein Cattle With Th...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Principles of antibodies with ultralong complementarity-determining regions and picobodies

Passon

Smedt

Svilenov

2023

Biotechnology Advances

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“…The molecular mass of the smallest fusion that we obtained is 14.5 kDa, which is about the mass of a single VHH fragment and four times as small as the most compact bispecific platform based on conventional antibody fragments – a diabody ( 23 ). Due to their size, the knob domain fusions may offer superior tissue penetration properties and improved access to cryptic epitopes ( 75 ). Intrinsic biophysical properties make knob domains especially well-suited to local delivery in potent combinations, where their short circulating half-lives may be expected to preclude systemic toxicity.…”

Section: Discussionmentioning

confidence: 99%

Modular design of bi- and multi-specific knob domain fusions

Kuravsky,

Gibbons,

Joyce

et al. 2024

Front. Immunol.

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IntroductionThe therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively.MethodsAutonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights.ResultsKnob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference.DiscussionCompared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

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Evolution of immunogenetic components encoding ultralong CDR H3

et al. 2023

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The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5–10 million years ago. Supplementary Information The online version contains supplementary material available at 10.1007/s00251-023-01305-9.

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A bovine antibody possessing an ultralong complementarity-determining region CDRH3 targets a highly conserved epitope in sarbecovirus spike proteins

Cited by 5 publications

References 46 publications

Principles of antibodies with ultralong complementarity-determining regions and picobodies

Principles of antibodies with ultralong complementarity-determining regions and picobodies

Modular design of bi- and multi-specific knob domain fusions

Evolution of immunogenetic components encoding ultralong CDR H3

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