Fab-drug Conjugation by Rebridging Methods

Amongst the plethora of antibody formats, Fab fragments have many advantages over other antibody fragments, including straightforward of preparation via protein expression, standard affinity chromatography purification from culture supernatant using anti-lambda, anti-kappa, or anti-CH1/CL media, high thermostability, and possession of native thiols forming a solvent accessible disulfide bond, which upon reduction can be used as attachment points for conjugation. Creative Biolabs now provides site-specific conjugation for Fab fragment-drug conjugates (Fab-based FDCs) development via disulfide bonds rebridging.

Rebridging Methods

A well-known example for conjugation is the reduction of native disulfide bonds, followed by selective conjugation of the liberated cysteine thiol groups, which is frequently accomplished by the reaction with maleimides. Other sophisticated methods make use of bifunctional reagents, which allow for bridging two thiols generated from the reduction of a disulfide bond. On one hand, bis-reactive electrophilic maleimides were used, to provide a C2-bridge. Alternatively, such rebridging of disulfide bonds could also be accomplished with double-reactive Michael acceptors to give a C1- or C3-bridge. Here, we introduce some typical ways to illustrate how to achieve homogeneous FDC products by rebridging of disulfide bonds.

Case 1: Bis-alkylating Conjugation

Scientists developed a rebridging conjugation approach, using a bis-sulfone linker-drug, in which interchain disulfide bonds in Fab fragments were first partially reduced, followed by bisalkylation to conjugate both thiols of the two native cysteines. The bis-sulfone linker carried a cytotoxic payload monomethyl auristatin E (MMAE) and the rebridging approach resulted in Fab-drug conjugates (Fab-based FDCs) that were more stable in human serum than conjugates prepared using non-rebridging maleimide chemistry.

Fig.1 Bis-alkylating conjugation approach involving a sequence of Michael addition and elimination reactions. (Badescu, 2014)

Case 2 Thiol-Yne Coupling (TYC) Reaction

The Fab is first treated with reducing agents tris (2-carboxyethyl) phosphine (TCEP) to convert the interchain disulfides to free cysteine residues. After reduction of the disulfide bond with TCEP, thiol-yne reaction in a photochemical batch reactor yielded the desired dithioether with quantitative conversion and 49% isolated yield as a mixture of diastereomers in about a 1:1 ratio. The rebridging was proven by mass spectrum (MS) analyses and confirmed by the absence of olefinic protons on ¹H NMR spectra of the resulting products. Finally, this one-pot reduction thiol-yne conjugation was successfully applied to Fab fragment with a promising conversion.

Fig.2 Reduction and TYC for the rebridging of disulfide bonds in peptides and proteins. (Griebenow, 2016)

Case 3: Platinum (II)-based Linker

Study reported a FDC platform technology using a platinum (II)-based linker that can re-bridge the inter-chain cysteines in Fab fragment, post-reduction. The strong platinum-sulfur interaction improves the stability of the FDC when compared with a standard maleimide-linked antibody-drug conjugate (ADC) thereby reducing the linker-drug exchange with albumin significantly. Moreover, due to the precise conserved locations of cysteines, both homogeneity and site-specificity are simultaneously achieved. The Pt-based FDCs can emerge as a simple and exciting proposition to address the limitations of the current ADC linker technologies.

Fig.3 Design of the Pt-based linker-drug and Pt (II)-linked Fab. (Gupta, 2017)

Case 4: Reduction and Functional Rebridging All in One

This case, designing a reagent that could incorporate both reducing and re-bridging functions, presents a significant step towards next-generation disulfide stapling reagents. This strategy has been shown to result in a high local concentration of bridging agent, which has been exploited for the functional rebridging of a multi-disulfide system without disulfide scrambling.

Fig.4 Synthesis of dithioaryl (TCEP) pyridazinedione and the incubation with Fab. (Lee, 2016)

Equipped with extensive experience in the use of maleimides and other technologies for conjugation to cysteines, Creative Biolabs offers comprehensive Fab-based FDC development services using our various rebridging methods. Our services include but not limited to:

• Customized linker design and synthesis
• Homogenous FDCs generation
• In vitro and in vivo evaluation

With advanced disulfide bonds rebridging platform, services from Creative Biolabs will be your best companion in creating customized Fab-based FDCs. Please contact us for more information and a detailed quote.

References

1. Badescu, G.; et al. Bridging disulfides for stable and defined antibody drug conjugates. Bioconjugate chemistry. 2014, 25(6): 1124-1136.
2. Griebenow, N., et al. Site-specific conjugation of peptides and proteins via rebridging of disulfide bonds using the Thiol-Yne coupling reaction. Bioconjugate Chemistry. 2016, 27(4): 911-917.
3. Gupta, N.; et al. Development of a facile antibody-drug conjugate platform for increased stability and homogeneity. Chemical science. 2017, 8(3): 2387-2395.
4. Lee, M. T. W.; et al. Next-generation disulfide stapling: reduction and functional re-bridging all in one. Chemical science. 2016, 7(1): 799-802.

For Research Use Only. NOT FOR CLINICAL USE.