Disulfide Modified IgG1: An Investigation of Biophysical Profile and Clinically Relevant Fc Interactions

Abstract

Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogenous ADCs, disulfide modification is considered an effective, low-cost and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallisable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibodydependant cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g. CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of inter-chain covalent linkages. It was found that by natively re-bridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g. thermal stability/aggregation), the natively re-bridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to sub-standard non Fc-specific attributes (thermal stability). These results advocate native disulfide re-bridging as an excellent synthetic strategy for forming homogenous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.

Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation

“…Over the past decade, we have reported on the use of bromopyridazinediones (BrPDs) as tools for efficient cysteine bioconjugation, particularly in the synthesis of dually functionalised conjugates, nanoparticle constructs, and therapeutically relevant biomolecules such as ADCs. [20][21][22] Unlike maleimides, conjugates synthesised using BrPDs do not undergo ring hydrolysis, and the unsaturated ring scaffold is otherwise stable under aqueous conditions. 23 We were intrigued, however, as to whether bioconjugates containing a saturated pyridazinedione scaffold (synthesised as previously described) 23 could proceed down a similar reversible retro-Michael deconjugation pathway displayed by classical maleimides and other Michael acceptors, e.g., cyanoacrylates.…”

Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

“…The biggest loss in affinity was observed for a lysine conjugate with an average of 14.2 payloads per mAb, resulting in a 17-fold affinity reduction toward Protein G. It was concluded that a high labeling degree to interchain cysteines results in structural changes, compromising the binding affinity toward the Fc-receptor, and that high labeling degrees using lysine conjugation results in a significant saturation of essential lysines involved in Fc-receptor binding. Also Bahou et al 75 observed a negative impact of highly loaded interchain cysteine conjugates on the CD16a kinetics and antibodydependent cellular cytotoxicity. These data are in agreement with the findings of Anderson and Tomasi 76 and Cunningham-Runles et al 38 who observed a decreasing binding affinity of the mAb to the Fc receptors when PEG was conjugated to lysine residues.…”

Ethynylphosphonamidates for the Rapid and Cysteine‐Selective Generation of Efficacious Antibody–Drug Conjugates

“…We estimated that 16 equiv of 10 per antibody (2 equiv per Cys-residue) are needed to reach aD AR of 4a t1 mg mL À1 antibody concentration. [29] By screening TCEP equivalents needed to modify brentuximab,wefound out that apartial reduction method with 3equiv TCEP,5equiv 10,and 5mgmL À1 brentuximab,a pplied in ao ne pot process, produces an ADC with aD AR of 3.9 without the need for removal of reducing agent prior payload conjugation (Figure S8 in the Supporting Information). [22] To compensate for the slower kinetics,w e increased the antibody concentration in the conjugation reaction to 5mgmL À1 ,aconcentration that was previously also used for maleimide conjugations.…”