Optimizing Lysosomal Activation of ADCs Using Novel Cleavable Dipeptide Linkers

Advantages of Dipeptide Linkers in ADCs

The selection of a linker for an ADC significantly impacts its stability in circulation, the rate of lysosomal processing, further catabolic transformation, and the potential bystander cytotoxic activity of the catabolite released from the target cell toward neighboring cancer cells. A peptide linker-conjugated ADC has been reported to show greater cytotoxic activity in vitro, and improved efficacy in several in vivo tumor xenograft models compared to those with noncleavable linkers. A typical example is the Val-Cit-PABC dipeptide linker with the self-immolative p-aminobenzyl carbamate (PABC) spacer, which is cleaved by cathepsin B and rat liver lysosomes and is employed in several ADCs in clinical use. Despite the use of peptide linkers in multiple clinical-stage ADCs, there have been limited reports on optimizing peptide linkers for efficient lysosomal proteolysis and stability in circulation. Therefore, to maximize the rate of lysosomal activation of ADCs, minimize peptide length, and ensure plasma stability, it is essential to assess the impact of varying dipeptide linker sequences in ADCs on lysosomal processing rates and the in vitro and in vivo activities of the ADCs.

With years of experience in recombinant antibody production, antibody engineering, bio-conjugation, and the utilization of the advanced "DrugLnk" organic synthesis platform, Creative Biolabs offers high-quality customized services for the design and synthesis of peptide linkers and drug-peptide linker complexes.

In this study, multiple dipeptide linkers were screened for protease cleavage and compared to the linkers currently under evaluation in clinical trials, namely Val-Cit, Val-Ala, Ala-Ala, and a noncleavable linker. Additioally, l-amino acid dipeptide linkers that can be rapidly and specifically cleaved by lysosomal proteases and cathepsins were incorporated into indolinobenzodiazepine dimer (IGN) payload-bearing ADCs (Fig. 1). The impact of varying l-amino acid dipeptide linker sequences in ADCs on lysosomal processing rates and the in vitro and in vivo activities of the ADCs, including comparison with those bearing a noncleavable linker, was assessed.

Fig. 1 Screening strategy for evaluating a library of dipeptide candidates and antibody-drug conjugates structures explored in this study.¹

Screening of Cbz-P2-P1-AMC Dipeptide Linkers

In the initial screening, peptides were designed and synthesized as N-terminal benzyloxycarbonyl (Cbz) protected dipeptides with a C-terminal fluorophore, 7-amino-4-methylcoumarin (AMC), in Cbz-P2-P1-AMC configuration (Fig.1A). Upon proteolytic cleavage, AMC is released and becomes fluorescent, allowing for a direct time-course measurement of the cleavage of a peptide sequence. It's worth noting that these peptide substrates were not used as ADC linkers in this study. The researchers also screened the dipeptide candidates for cleavage by both individual lysosomal cathepsins and cell lysates. The proteolytic activities of cathepsins and HSC-2 and H292 cell lysates were determined using a widely used substrate, Cbz-l-Phe-l-Arg-AMC. The results showed that among the tested dipeptide-AMCs, these new l-amino acid dipeptides were cleaved at a higher rate by cell lysates and cathepsins compared to those bearing a Val-Ala or Ala-Ala linker.

Preparation and Characterization of Conjugates

First, conjugates containing humanized antibodies and IGN payload with peptide and noncleavable linkers were prepared. Mal-P2-P1-AMC payloads were conjugated by generating thiol groups in an antibody through initial modification using N-succinimidyl-4-(2-pyridyldithio)-butanoate (SPDB), followed by rapid, selective disulfide reduction using DTT. Subsequently, the possible susceptibility of these peptide linkers to cleavage by plasma proteases and the stability of these dipeptides in plasma after conjugation with antibodies were investigated.

The results indicated that: 1) A greater amount of peptide cleavage was observed in mouse plasma, and except for Tyr-Arg and Phe-Arg, the peptides were more stable in cynomolgus monkey plasma but were most stable in human plasma. 2) Antibody conjugates with peptide linkers were considerably more stable in plasma compared to the model AMC-dipeptides.

At Creative Biolabs, with the advanced "DrugLnk" platform, we are dedicated to providing customized design services using peptide linkers for ADC development.

Quantification of Peptide-Linked ADC Catabolites in Target Cancer Cells

• l-Val-l-Gln and l-Ala-l-Ala were selected for further studies measuring the amount of payload released from the peptide-linked ADC upon processing by target cancer cells HSC-2. The results show that the total catabolite level measured for l-Val-l-Gln linked IGN ADC was much higher than that for l-Ala-l-Ala ADC (Fig. 2). and over time, the catabolite levels for both linkers were similar.
• Cells were incubated with the selective inhibitor bafilomycin A1 (Baf A1) to confirm lysosomal processing. The observed dissociative inhibitory effect of Baf A1 on the peptide line, suggests an important role for lysosomal processing in ADC activity, and a slight advantage for the l-Val-l-Gln peptide linker to be processed more efficiently under limited lysosomal function.
• KB cells, Igrov-1 cells, and JEG-3 cells were exposed to anti-FRα conjugates containing the IGN payload linked via the l-Ala-l-Ala or l-Val-l-Gln linker for a 30 min pulse, followed by incubation for processing studies of anti-FRα conjugates. This research result shows similarity to the observation for ADCs in the HSC-2 cell line.

Fig. 2 Quantitation of IGN Catabolites of l-Ala-l-Ala and l-Val-l-Gln linked anti-EGFR and anti-FRα IGN-conjugates.¹

In Vitro Cytotoxic Activity of Peptide and Noncleavable Linker-Bearing Conjugates

The in vitro cytotoxicity of ADCs with a DNA-alkylating IGN payload, linked to anti-EGFR antibody or anti-FRα antibody via dipeptide linkers (l-Ala-l-Ala and l-Val-l-Gln; P2-P1 linker structure shown in Fig. 1B), was examined in various cell lines expressing EGFR or FRα, respectively. A conjugate with a noncleavable linker (structure shown in Fig.1C) was also evaluated as a control.

In a panel of EGFR-expressing cell lines, two out of the six cell lines tested (H292 and BxPC-3) exhibited similar cytotoxic activity between the peptide linker ADCs and the noncleavable linker ADC. However, in the other four cell lines (HSC-2, PC-9, SAS, and H1975), the peptide linker ADCs demonstrated superior activity (with 10- to 80-fold lower EC₅₀) compared to the noncleavable linker ADC. In six cell lines with varying FRα antigen densities, the two peptide linker ADCs (l-Ala-l-Ala and l-Val-l-Gln) showed comparable in vitro cytotoxicity in KB and NCI-H2110 cell lines.

Fig. 3 In vitro cytotoxicity assay of anti-EGFR ADCs with noncleavable linkers and cleavable dipeptide linkers, and (B) anti-FRα ADCs with cleavable dipeptide linkers.¹

In Vivo Efficacy Study Comparing IGN ADCs Linked With Different Dipeptide Linkers

The antitumor activities of anti-FRα IGN ADCs employing an l-Val-l-Gln, l-Ala-l-Ala, or l-Val-l-Thr dipeptide linker (P2-P1 linker structure shown in Fig.1B) were evaluated in an H2110 xenograft model in immunocompromised SCID mice. At the lower dose of 1.5 μg/kg, all ADCs were inactive. However, at the 3 μg/kg dose, ADCs with the l-Ala-l-Ala and l-Val-l-Gln linker exhibited high activity.

Fig. 4 In vivo activity of anti-FRα-IGN ADCs with dipeptide linkers in NCI-H2110 tumor xenografts in SCID mice.¹

Creative Biolabs provides one-stop ADC development services using peptide linkers tailored to customer needs. We arecommitted to delivering in-depth ADC in vitro and in vivo analysis services.

Reference

1. Salomon PL, Reid EE, Archer KE, et al. Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers. Molecular Pharmaceutics. 2019, 16(12):4817-4825.

For Research Use Only. NOT FOR CLINICAL USE.