Methods for the Generation of Single-Payload Antibody-Drug Conjugates

Abstract

Antibody-drug conjugates (ADCs) have emerged as a powerful form of targeted therapy that can deliver drugs with a high level of selectivity towards a specific cell type, reducing off-target effects and increasing the therapeutic window compared to small molecule therapeutics. However, creating ADCs that are stable, homogeneous, and with controlled drug-to-antibody ratio (DAR) remains a significant challenge. Whilst a myriad of methods have been reported to generate ADCs with a DAR of 2, 4, and 8, strategies to generate DAR 1 constructs are seldom reported despite the advantages of low drug loading to tune ADC properties or to allow access to antibody-antibody and antibody-protein constructs. This concept article highlights the diversity of methods that have been employed to access single-payload ADCs and explores the outlook for the field.

Keywords: Antibodies; Antibody-drug conjugates; Cytotoxicity; Drug delivery; Drug-to-antibody ratio.

Scheme 1

Conjugation of a single‐payload to an antibody enables access to DAR 1 ADCs and antibody‐protein constructs.

Scheme 2

Genetic engineering ensures only two cysteines are accessible for bioconjugation. Using dual reactive linker 1, a single payload can be added in a ‘loop’. TCEP=Tris(2‐carboxyethyl)phosphine; PBS=Phosphate buffered saline.

Scheme 3

Glycan engineering installed reactive azide handles which can undergo SPAAC with a bis‐reactive linker to form a ‘loop’ with a single payload attached. TBS=Tris buffered saline; PBS=Phosphate buffered saline.

Scheme 4

‘Knob‐into‐Hole’ antibody engineering allows for single site‐specific modification of a heavy chain C‐terminus. a) Example work flow. Sortase A is used to functionalise a peptide tag with a tetrazine handle that can then undergo IEDDA with a TCO‐payload. b) Combining tetrazine‐ and TCO‐functionalised antibodies allows the creation of antibody‐antibody conjugates via an IEDDA reaction.

Scheme 5

Incomplete conjugation leads to a mixture of mono‐ and di‐reacted ADC. DAR 1 species can be obtained by purifying out the mono‐reacted species.

Scheme 6

Repeated low conversion bioconjugation and starting material recycling allows for the controlled accumulation of single‐payload ADCs. a) Reagent addition cycles followed by purification with a streptavidin affinity column allows unreacted antibody to be recycled whilst capturing mono‐reacted ADC. Treatment of the column with strained alkyne 4 swaps the biotin tag for a payload and releases the desired ADC. b) Structures of the biotin tag and BCN‐Payload click handle. c) Full scheme of the ‘click‐to‐release’ cycloaddition. BCN=Bicyclononye.

Scheme 7

The four reactive DVP motifs of TetraDVP enable the re‐bridging of all four disulphide bonds within a single antibody. a) Initial work using CuAAC led to poor ‘click’ efficiency affording DAR 0.5 ADCs. b) Using SPAAC enabled an increase in DAR to 0.9. c) Structures of the TetraDVPs used. Linker core length was also found to impact the conjugation and click rates. TCEP=Tris(2‐carboxyethyl)phosphine; TBS=Tris buffered saline; PBS=Phosphate buffered saline.