Collins, M., Ibeanu, N, Grabowska, W.R., Awwad, S, Khaw, P.T., Brocchini, S. and Khalili, Hanieh ORCID: https://orcid.org/0000-0002-6612-1628 (2024) Bispecific FpFs: a versatile tool for preclinical antibody development. RSC Chemical Biology.

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Abstract

We previously described FpFs [1 with combining low line] (Fab–PEG–Fab) as binding mimetics of IgGs. FpFs are prepared with di(bis-sulfone) conjugation reagents [3 with combining low line] that undergo disulfide rebridging conjugation with the accessible disulfide of each Fab (Scheme 1). We have now prepared bispecific FpFs [2 with combining low line] (bsFpF and Fab1–PEG–Fab2) as potential bispecific antibody mimetics with the intent that bsFpFs could be used in preclinical antibody development since sourcing bispecific antibodies may be challenging during preclinical research. The di(bis-sulfone) reagent [3 with combining low line] was first used to prepare a bsFpF [2 with combining low line] by the sequential conjugation of a first Fab and then a second Fab to another target (Scheme 2). Seeking to improve bsFpF synthesis, the asymmetric conjugation reagent, bis-sulfone bis-sulfide [1 with combining low line][6 with combining low line], with different thiol conjugation reactivities at each terminus (Scheme 4) was examined and the bsFpFs appeared to be formed at similar conversion to the di(bis-sulfone) reagent [3 with combining low line]. To explore the advantages of using common intermediates in the preparation of bsFpF families, we investigated bsFpF synthesis with a protein conjugation–ligation approach (Scheme 5). Reagents with a bis-sulfone moiety for conjugation on one PEG terminus and a ligation moiety on the other terminus were examined. Bis-sulfone PEG trans-cyclooctene (TCO) [2 with combining low line][8 with combining low line] and bis-sulfone PEG tetrazine (Tz) [3 with combining low line][0 with combining low line] were used to prepare several bsFpFs targeting various therapeutic targets (TNF-α, IL6R, IL17, and VEGF) and tissue affinity targets (hyaluronic acid and collagen II). Surface plasmon resonance (SPR) binding studies indicated that there was little difference between the dissociation rate constant (kd) for the unmodified Fab, mono-conjugated PEG–Fab and the corresponding Fab in a bsFpF. The Fab association rate (ka) in the bsFpF was slower than for PEG–Fab, which may be because of mass differences that influence SPR results. These observations suggest that each Fab will bind to its target independently of the other Fab and that bsFpF binding profiles can be estimated using the corresponding PEG–Fab conjugates.

Item Type:	Article
Identifier:	10.1039/D4CB00130C
Subjects:	Medicine and health
Depositing User:	Marc Forster
Date Deposited:	27 Sep 2024 10:33
Last Modified:	27 Sep 2024 10:45
URI:	https://repository.uwl.ac.uk/id/eprint/12726

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