Ryan Stafford

While the kinetics and mechanisms of the reaction of O3 with alkenes in the gas and condensed phases are reasonably well understood, those with unsaturated organics in the intermediate regime at the air−water interface are not. Studies of the reaction of ozone at room temperature with the unsaturated phospholipid, 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) at the air−water interface, and, for comparison, the fully saturated dipalmitoyl-l-α-phosphatidylcholine (DPPC) were carried out. The phospholipids were exposed at varying surface areas per phosphocholine molecule on a water subphase to a flow of O3 in air (0.25−1 ppm), and atmospheric pressure ionization mass spectrometry (API-MS) was used to monitor the formation of gaseous products in real time. Nonanal was detected as a major gas-phase product of the reaction of ozone with OPPC; no volatile products were observed in the case of DPPC. The yield of nonanal, defined as the nonanal produced per phosphocholine molecule reacted at the air−water interface, was 51 ± 13% (2σ) over this range of ozone concentrations, after correcting for the solubility of nonanal in the subphase. The nonanal yield was also independent of the available area per molecule over the range from 40 to 158 Å2 molecule-1, 56 ± 11% (2σ), at a constant O3 concentration of 1 ppm. Cyclohexane was used as a scavenger for any OH generated in the reaction, but no evidence for gas-phase OH radical production in the OPPC−O3 reaction was found using this technique. The time-dependence of the generation of nonanal shows that these reactions are enhanced kinetically at the air−water interface compared to that expected for analogous gas-phase reactions. Molecular dynamics simulations of OPPC at the air−water interface show that the insensitivity of the time dependence and yield of nonanal production to the extent of film compression, as well as the kinetic enhancement, can be understood in terms of the structure of the fatty acid chains at the interface. These studies illustrate the utility of real-time monitoring of the gas-phase products of reactions at the air−water interface and the insights into kinetics and mechanisms which can be obtained by combining these experimental data with molecular dynamics simulations.

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Although direct interactions are important, the contribution of indirect interactions toward cooperative assembly of Hox and Exd remains unresolved. Here we use minor groove binding polyamides as structural wedges to induce perturbations at specific base steps within the Exd binding site. We find that allosteric modulation of DNA structure contributes nearly 1.5 kcal/mol to the binding of Exd to DNA, even in the absence of direct Hox contacts. In contrast to previous studies, the sequence-targeted chemical wedges reveal the role of DNA geometry in cooperative assembly of Hox-Exd complexes. Programmable polyamides may well serve as general probes to investigate the role of DNA modulation in the cooperative and highly specific assembly of other protein-DNA complexes.