Surface-Available HER2 Levels Alone Are Not Indicative of Cell Response to HER2-Targeted Antibody-Drug Conjugate Therapies.

HER2-targeting therapies have advanced breast cancer treatment over the past decade. Clinically, eligibility for HER2 therapies is determined by assessing HER2 levels on tumor cell surfaces through immunohistochemistry or by gene regulation through fluorescence in situ hybridization. HER2 therapies are not always effective in patients with elevated levels of HER2, questioning whether the amount of HER2 is sufficiently predictive of patient outcomes. Additionally, the HER2-targeting antibody-drug conjugate (ADC) Enhertu® was recently approved for metastasized HER2-low cancers, confirming the benefits of HER2 treatment for patients with low HER2 levels. To evaluate the correlation between HER2 levels and treatment efficacy, we quantified HER2 on eight cell lines using flow cytometry while simultaneously determining the toxicity of two HER2-targeting ADCs. Both HER2-high cell lines and HER2-low cell lines had significant toxicity responses to ADCs. We quantified HER2 internalization and found no correlation between HER2 levels and the percentage of internalization. We found a useful metric suggesting that a minimum number of HER2 receptors trafficked to lysosomes is sufficient to provide effective treatment. Our results indicate that the current standards of determining eligibility for HER2 therapy could limit patients' access to effective treatment. In conclusion, HER2 levels are not wholly adequate to determine the response to ADC treatment.

Complementation Dependent Enzyme Prodrug Therapy Enables Targeted Activation of Prodrug on HER2-Positive Cancer Cells.

Antibodies have been explored for decades for the delivery of small molecule cytotoxins directly to diseased cells. In antibody-directed enzyme prodrug therapy (ADEPT), antibodies are armed with enzymes that activate nontoxic prodrugs at tumor sites. However, this strategy failed clinically due to off-target toxicity associated with the enzyme prematurely activating prodrug systemically. We describe here the design of an antibody-fragment split enzyme platform that regains activity after binding to HER2, allowing for site-specific activation of a small molecule prodrug. We evaluated a library of fusion constructs for efficient targeting and complementation to identify the most promising split enzyme pair. The optimal pair was screened for substrate specificity among chromogenic, fluorogenic, and prodrug substrates. Evaluation of this system on HER2-positive cells revealed 7-fold higher toxicity of the activated prodrug over prodrug treatment alone. Demonstrating the potential of this strategy against a known clinical target provides the basis for a unique therapeutic platform in oncology.

In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution.

Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody-drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the CH2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.

Palladium-Catalyzed Enantioselective Redox-Relay Heck Alkynylation of Alkenols To Access Propargylic Stereocenters.

An enantioselective redox-relay Heck alkynylation of di- and trisubstituted alkenols to construct propargylic stereocenters is disclosed using a new pyridine oxazoline ligand. This strategy allows direct access to chiral ß-alkynyl carbonyl compounds employing allylic alcohol substrates in contrast to more traditional conjugate addition methods.

Palladium-catalyzed intramolecular C-H arylation of arenes using tosylates and mesylates as electrophiles.

This paper describes a method for the palladium catalyzed intramolecular C-H arylation using tosylates and mesylates as electrophiles. The transformation is efficient for the synthesis of various heterocyclic motifs including furans, carbazoles, indoles, and lactams. Additionally, a protocol for the one-pot sequential tosylation/arylation of phenol derivatives is presented.