High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers

Abstract

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…The importance of site-specific TCO conjugation was evident in our data, as seen by differences in both tumor and blood signal between 7C2-TCO 2 and 7C2-TCO 6 , a comparison that was enabled by recent developments in site-specific conjugation technology. 47 In addition to controlling stoichiometry, this technology also provides pharmacokinetic benefits by avoiding the presence of high antibody conjugation ratio species that, in the case of antibody-drug conjugates, has previously led to increased clearance 54 and may be associated with undesirable biodistribution. Relatively similar antibody binding in vitro suggested that the observed increase in tetrazine signal originated from surface-bound tetrazine (Figure 2).…”

“…Anti-HER2 THIOMAB™ antibodies (clone 7C2) were engineered with cysteine residues at one or more positions 47 (LC:K149C, LC:K149C+HC:L177C or LC:K149C+HC:L177C+HC:Y376C) for conjugation to the click-reactive linker [(4E)-cyclooct-4-en-1-yl] N-[3-[2-[2-[3-[3-(2,5-dioxopyrrol-1-yl)propanoylamino] propoxy]ethoxy]ethoxy]propyl]carbamate (i.e., “TCO-PEG 3 -maleimide”) to give final conjugates with approximately 2, 4, or 6 TCO groups per antibody, respectively. Conjugation was accomplished by first deblocking the THIOMAB™ antibodies followed by conjugation of TCO-PEG 3 -maleimide to the free engineered cysteine residues as previously described.…”

“…We conjugated TCO moieties, via short polyethylene glycol (PEG) linkers, site-specifically to anti-HER2 THIOMAB™ antibodies 46 with varying numbers of engineered cysteines 47 to determine the importance of stoichiometry on the in vivo IEDDA reaction in a HER2-positive KPL-4 tumor model. We employed 111 In (t 1/2 2.8 days) to allow direct comparison between our conventional radioimmunoconjugate and our click reactive tetrazine probe in an attempt to demonstrate proof of concept for same-day imaging with shorter lived radionuclides such as 68 Ga ( t 1/2  = 68 min).…”

Conjugation of Indoles to Antibodies through a Novel Self‐Immolating Linker

“…Importantly, all of the conjugates were derived from cysteine residues engineered into the LC‐K149 sites of the employed antibodies, and this site‐specific conjugation approach afforded highly homogeneous conjugates with drug‐to‐antibody ratios between 1.9 and 2.0 (Table ). In addition, our prior research indicated that attachment of similar disulfide‐containing linkers to the LC‐K149 antibody site provided conjugates with improved in vivo stability properties relative to ADCs produced using many other possible attachment locations . As a prelude to evaluating the conjugates in animals, we assessed their stabilities in whole blood obtained from various species.…”

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions

“…More recent work from Genentech used a scanning approach similar to ours, whereby a cysteine was substituted at every residue in an anti-HER2 IgG1. 28 Once conjugated, these constructs generated a large ADC library that was interrogated in order to identify those with improved stability relative to conventional cysteine conjugates. Then, the team used structural information to evaluate whether solvent accessibility around the conjugation sites contributed to stability; no significant correlation was found between the two features.…”