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Optimizing the anti-tumor efficacy of protein-drug conjugates by engineering the molecular size and half-life
Fabian Brandl1, Sarah Busslinger2, Uwe Zangemeister-Wittke1
1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; Institute of Pharmacology, University of Bern, Inselspital INO-F, CH-3010 Bern, Switzerland.
Abstract:
Despite some approvals of antibody-drug conjugates for cancer therapy, their clinical success rate is unsatisfactory because of very small therapeutic windows, influenced by on-target and off-target toxicities of conjugate and liberated toxin. Additional formats with systematically investigated molecular parameters must therefore be explored to increase their therapeutic window. Here we focused on the effective molecular weight. To generate conjugates with exactly defined drug loads and tunable pharmacokinetics, we used Designed Ankyrin Repeat Proteins (DARPins), fused to unstructured polypeptides of different lengths, to produce proteins with any desired half-life, to identify those with the best efficacy. We generated an EpCAM-targeting DARPin-MMAF conjugate, fused to PAS or XTEN of different lengths, and a matched series of controls of a non-binding DARPin to account for the enhanced permeability and retention (EPR) effect, covering half-lives of minutes to 20.6 h in mice. All conjugates were produced at high purity, and demonstrated high specificity and cytotoxicity in human tumor cell cultures, with IC50 values in the low nM range, independent of the polypeptide type and length. Due to their more facile purification, the PASylated conjugates were tested in nude mice bearing HT29 tumor xenografts. Independent of their size, all PASylated conjugates were very well tolerated after repeated systemic administration of 300 nmol/kg. We found that the conjugates with intermediate size and half-life showed the strongest anti-tumor effects, and deduced that this effect is a compromise of serum half-life and diffusion within the tumor, as on-rates and affinities are essentially identical, with extravasation playing only a very minor role.
Insights
New antibody-drug conjugates (ADCs) using Designed Ankyrin Repeat Proteins (DARPins) show promise for cancer therapy. Optimizing molecular weight and half-life improved anti-tumor efficacy and tolerability in preclinical models.
Area of Science:
- Biotechnology
- Pharmacology
- Oncology
Background:
- Antibody-drug conjugates (ADCs) offer targeted cancer therapy but face limitations due to narrow therapeutic windows and toxicity.
- Exploring novel formats and systematically investigating molecular parameters is crucial for enhancing ADC efficacy and safety.
- Effective molecular weight is a key parameter influencing the therapeutic window of antibody-drug conjugates.
Purpose of the Study:
- To develop and evaluate novel antibody-drug conjugates (ADCs) with tunable pharmacokinetics and defined drug loads.
- To investigate the impact of molecular weight and half-life on the efficacy and tolerability of EpCAM-targeting ADCs.
- To identify optimal molecular parameters for improved anti-cancer therapeutic strategies.
Main Methods:
- Generation of EpCAM-targeting Designed Ankyrin Repeat Protein (DARPin)-MMAF conjugates fused to PAS or XTEN polypeptides of varying lengths.
- In vitro assessment of specificity and cytotoxicity in human tumor cell lines.
- In vivo evaluation of pharmacokinetics, tolerability, and anti-tumor efficacy in HT29 tumor xenograft models in nude mice.
Main Results:
- All generated conjugates exhibited high purity, specificity, and potent cytotoxicity (low nM IC50) in vitro, irrespective of polypeptide.
- PASylated conjugates were well-tolerated in vivo after repeated systemic administration.
- Conjugates with intermediate molecular size and half-life demonstrated the strongest anti-tumor effects in preclinical models.
Conclusions:
- Designed Ankyrin Repeat Protein (DARPin)-based conjugates offer a promising platform for developing targeted cancer therapies with improved therapeutic windows.
- Optimal anti-tumor efficacy is achieved through a balance between serum half-life and tumor diffusion, rather than solely relying on rapid tumor penetration.
- This study highlights the importance of systematically optimizing molecular parameters like effective molecular weight for next-generation antibody-drug conjugates.
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