Antibody-drug conjugates (ADCs) combine the cytotoxic capacity of small-molecule medicines and the specificity of monoclonal antibodies (mAbs). This method has been effective in the treatment of hematological malignancies such as B-cell non-Hodgkin lymphomas (NHLs), including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL).
The B-cell receptor (BCR) complex component CD79b is expressed on B-cells but not on plasma cells in B-cell malignancies, making it an ideal target. The microtubule inhibitor MMAE is delivered by Polivy (an ADC targeting CD79b) through a heterogeneous conjugation approach, which frequently results in inconsistent drug-to-antibody ratios (DARs) and reduced safety or efficacy. This study shows an effective semi-synthetic approach to create a second-generation class of ADCs based on tiancimycins (TNMs).
With the use of dual-variable domain (DVD) IgG1 antibodies and a site-specific conjugation technology, these next-generation ADCs offer improved cytotoxicity, homogeneity, and target precision against B-cell malignancies. Natural products (NPs) show potential in the form of ADC payloads due to their anticancer properties, though their diversity is limited. The 1,5-diyne-3-ene core of enediynes consists of nine or ten-membered rings, possesses potent DNA-damaging properties, supported by various peripheral functional groups.
This approach combines AFE-based payloads with the DVD IgG1 antibody format, utilizing site-specific conjugation to create next-generation ADCs. A bacterial strain called Streptomyces sp. CB03234-S was developed by the researchers to produce TNM A and its precursor TNM D (compound 8) at high yields (~19 mg/L). Comprehensive nuclear magnetic resonance (NMR) analysis confirmed the structural identity of TNM A, which and keto-TNM A were obtained through large-scale fermentation, oxidation, and purification.
The linkers were installed using effective copper-catalysed azide-alkyne cycloaddition (CuAAC) reactions, including a β-lactam moiety required for site-specific conjugation to DVD IgG1 antibodies. The five keto-TNM A payloads were successfully conjugated to the anti-CD79b DVD IgG1 and a control anti-HER2 DVD IgG1 by using β-lactam chemistry. Mass spectrometry and size-exclusion chromatography confirmed the production of homogenous ADCs with high conjugation efficiencies of 92%.
A CD79b-negative T-cell line and B-cell lymphoma cell lines were used to evaluate the cytotoxic capacity of the keto-TNM A payloads and ADCs. Keto-TNM A demonstrated sub-nanomolar IC50 values across B-cell lines and exhibited greater potency than both TNM A and MMAE derivatives. Flow cytometry revealed that CD79b expression in CLL samples was consistently lower than in MCL and DLBCL cell lines. This suggests that B-cell malignancies with higher CD79b expression correlate with increased therapeutic potential for CLL treatment.
Single-cell analysis measuring mean fluorescence intensity (MFI) and surface expression of CD79b in patient-derived primary CLL B-cells showed variability in target expression and distribution. The efficiency, site-specificity of payload conjugation, and stability of the resulting ADCs are crucial in creating ADCs with optimal payload potency. These factors assure their homogeneity, consistency of action, and favourable pharmacokinetic profiles of the product. Due to the biocatalysts, this platform is capable of manufacturing and assessing homogeneous AFE- and DVD IgG1-based ADCs, representing the next generation of immunotherapies designed to target a broad range of cancer-specific antigens.
Reference: Kiefer AF, Thirugnanasambantham P, Jin Y, et al. Second Generation Tiancimycin-Based Antibody–Drug Conjugates Enabled by Highly Efficient Semi-synthetic Approach Specifically Targeting B-Cell Malignancies. JACS Au. 2025;5(7):1330-1348. doi:10.1021/jacsau.5c00353
