Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Summary

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Abstract

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent — dubbed ‘PODS’ — as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

Introduction

Radiopharmaceutical chemists have long exploited the selectivity and specificity of antibodies for biomarkers of disease for both nuclear imaging and targeted radiotherapy¹. Far and away the most common approach to the radiolabeling of antibodies is predicated on the indiscriminate attachment of radiolabeled prosthetic groups or radiometal chelators to amino acids — most often lysines — within the structure of the immunoglobulin (Figure 1A)². While this strategy is certainly effective, its random, non-site-specific nature can create problems. Specifically, traditional bioconjugat....

Protocol

1. The synthesis of 4-[5-(methylthio)-1,3,4-oxadiazol-2-yl]-aniline (1)

NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.

1. In a 10 mL round bottom flask, dissolve 100 mg (0.517 mmol, 1 equivalent) of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol in 3 mL of methanol.
2. To this solution, add 360 μL of diisopropylethylamine (DIPEA; 2.07 mmol; 4 equivalents; anhydrous) and a small magnetic stir bar. Cover the flask with a rubber stopper and stir the solution for 10 minutes at room temperature.
3. Using a 1 mL glass syringe, poke a hole through the rubber stopper and quick....

Results

The first four steps of this protocol — the synthesis of PODS — have been designed to be robust and reliable. The deprotonation and substitution of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol to form the desired thioether product affords the thioether in >99% yield after just 45 minutes. Next, the ligation between 1 and N-Boc-N'-succinyl-4,7,10-trioxa-1,13-tridecanediamine was achieved via a standard peptide coupling procedure, resulting .......

Discussion

In this report, we have chosen not to include any protocols for radiolabeling or in vivo experimentation. Our reasons are straightforward. With respect to the former, the radiolabeling of a PODS-based immunoconjugate does not differ at all from that of an immunoconjugate synthesized using other bioconjugation strategies, and these procedures have been comprehensively reviewed elsewhere². With regard to the latter, the specifics of preclinical in vivo experiments (i.e., mouse models, doses, etc.) c.......

Materials

Name	Company	Catalog Number	Comments
5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol	Sigma-Aldrich	675024
1.5 mL LoBind Microcentrifugal Tube	Eppendorf	925000090
1.5 mL Microcentrifugal Tube	Fisherbrand	05-408-129
Acetonitrile	Fisher Scientific	A998-4
Amicon Ultra-2 Centrifugal Filter Unit	EMD Millipore	EN300000141G
Cyclohexane	Fisher Scientific	C556-4
Dichloromethane	Fisher Scientific	AC383780010
Diisopropylethylamine	MP Biomedicals, LLC	150915
Dimethylsulfoxide	Fisher Scientific	31-727-5100ML
Ethyl Acetate	Fisher Scientific	E145 4
Hydrochloric Acid	Fisher Scientific	A144-500
Iodomethane	Sigma-Aldrich	289566-100G
Magnesium Sulfate	Acros Organics	413485000
m-chloroperbenzoic acid	Sigma-Aldrich	273031
Methanol	Fisher Scientific	A412 1
NBoc-N′-succinyl-4,7,10-trioxa-1,13-tridecanediamine	Sigma-Aldrich	671401	Store at -80 °C
N-ethyl-N′- [3- (dimethylamino)propyl] carbodiimide hydrochloride	Sigma-Aldrich	3450
Phosphate Buffered Saline	Sigma-Aldrich	P5493	10× Concentration
p-SCN-Bn-DOTA	Macrocyclics	B-205	Store at -80 °C
Sephadex G-25 in PD-10 Desalting Columns	GE Healthcare	17085101
Sodium Carbonate	Sigma-Aldrich	S7795
Sodium Hydroxide	Fisher Scientific	S318-1
TCEP	ThermoFischer Scientific	20490
Triethylamine	Fisher Scientific	AC157911000
Trifluoroacetic Acid	Fisher Scientific	A116-50