Discovery of novel cMET-targeting antibody Fab drug conjugates as potential treatment for solid tumors with highly expressed cMET.

BACKGROUND: Solid tumors are becoming prevalent affecting both old and young populations. Numerous solid tumors are associated with high cMET expression. The complexity of solid tumors combined with the highly interconnected nature of the cMET/HGF pathway with other cellular pathways make the pursuit of finding an effective treatment extremely challenging. The current standard of care for these malignancies is mostly small molecule-based chemotherapy. Antibody-based therapeutics as well as antibody drug conjugates are promising emerging classes against cMET-overexpressing solid tumors. RESEARCH DESIGN AND METHODS: In this study, we described the design, synthesis, in vitro and in vivo characterization of cMET-targeting Fab drug conjugates (FDCs) as an alternative therapeutic strategy. The format is comprised of a Fab conjugated to a potent cytotoxic drug via a cleavable linker employing lysine-based and cysteine-based conjugation chemistries. RESULTS: We found that the FDCs have potent anti-tumor efficacies in cancer cells with elevated overexpression of cMET. Moreover, they demonstrated a remarkable anti-tumor effect in a human gastric xenograft mouse model. CONCLUSIONS: The FDC format has the potential to overcome some of the challenges presented by the other classes of therapeutics. This study highlights the promise of antibody fragment-based drug conjugate formats for the treatment of solid tumors.

The role of inflammatory biomarkers in the development and progression of pre-eclampsia: a systematic review and meta-analysis.

Background: Pre-eclampsia (PE) is a pregnancy complication associated with maternal and fetal morbidity and mortality. Among the potential pathogenesis discussed, inflammation is considered an essential initiator of PE. Previous studies have compared the levels of various inflammatory biomarkers that indicate the existence of PE; however, the relative levels of pro-inflammatory and anti-inflammatory biomarkers and their dynamic changes during PE progression remain unclear. This knowledge is essential to explain the occurrence and progression of the disease. Objective: We aimed to identify the relationship between inflammatory status and PE using inflammatory biomarkers as indicators. We also discussed the underlying mechanism by which inflammatory imbalance contributes to PE by comparing the relative levels of pro-inflammatory and anti-inflammatory biomarkers. Furthermore, we identified additional risk factors for PE. Methods: We reviewed PubMed, Embase, and the Cochrane Library for articles published until 15th September 2022. Original articles that investigated inflammatory biomarkers in PE and normal pregnancy were included. We selected healthy pregnant women as controls. The inflammatory biomarkers in the case and control groups were expressed as standardized mean differences and 95% confidence intervals using a random-effects model. Study quality was assessed using the Newcastle-Ottawa Scale. Publication bias was assessed using Egger's test. Results: Thirteen articles that investigated 2,549 participants were included in this meta-analysis. Patients with PE had significantly higher levels of C-reactive protein (CRP), interleukin (IL)-4, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) than the controls. CRP and pro-inflammatory cytokine levels were higher than those of anti-inflammatory cytokines. Patients with gestational age > 34 weeks had significantly higher IL-6 and TNF levels. Patients with higher systolic blood pressure had significantly higher IL-8, IL-10, and CRP levels. Conclusion: Inflammatory imbalance is an independent risk factor for PE development. Impairment of the anti-inflammatory system is a crucial initiating factor for PE development. Failed autoregulation, manifested as prolonged exposure to pro-inflammatory cytokines, leads to PE progression. Higher levels of inflammatory biomarkers suggest more severe symptoms, and pregnant women after 34 weeks of gestation are more susceptible to PE.

Discovery and intranasal administration of a SARS-CoV-2 broadly acting neutralizing antibody with activity against multiple Omicron subvariants.

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).

PSMA-targeted bispecific Fab conjugates that engage T cells.

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity.

Chemically generated IgG2 bispecific antibodies through disulfide bridging.

Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture. Full-length antibodies can be conjugated via disulfide bridging with linkers bearing orthogonal groups to produce BsAbs. We report that an αHER2/EGFR BsAb was successfully generated by this approach and retained the ability to bind both antigens with no significant loss of potency.

A 3(10)-helical pentapeptide in water: interplay of alpha,alpha-disubstituted amino acids and the central residue on structure formation.

C(alpha,alpha)-disubstituted amino acids (alphaalphaAAs) are widely used to conformationally constrain peptides. A series of pentapeptides containing dipropylglycine (Dpg) at alternating positions and their alpha-amino acid counterpart L-norvaline (Nva) analogues were synthesized to fully investigate the impact of Dpg on peptide backbone structure in aqueous solution. CD, VCD, and NMR spectral analysis suggest that Dpg containing peptides adopt more ordered structures relative to their Nva containing analogues. The central residues (Ala, Thr, Tyr, Val) and the charged side-chains of Glu and Lys play important roles in the degree of peptide folding. Hydrophobic and branched residues (Val, Tyr) at the central position of the peptide produce greater folding as judged by CD and NMR. Variation of the chemical shift with temperature (Deltadelta/DeltaT NH) of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH(2) suggests a series of i --> i + 3 hydrogen bonds between the N-terminal acetyl carbonyl and the Tyr(3) NH, and the Glu(1) carbonyl and the Dpg(4) NH. The solution conformation of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH(2) calculated from NMR-derived constraints shows a 3(10)-helical structure (two repetitive type-III beta-turns) at residues 1-4, which is supported by 2D NMR, CD, and VCD spectra. Analysis of NMR-derived models of these peptides suggest that there is a strong hydrophobic interaction of the pro-S propyl side chain of Dpg(2) and the Tyr(3) side-chain that may be a strong stabilizing force of the peptide folding in water.

Alzheimer's Abeta peptides containing an isostructural backbone mutation afford distinct aggregate morphologies but analogous cytotoxicity. Evidence for a common low-abundance toxic structure(s)?

Amyloid beta (Abeta) peptide amyloidogenesis, involving the formation of numerous distinct quaternary structures, appears to cause Alzheimer's disease. However, the precise identification of the toxic structure(s) and the neurotoxicity mechanism(s) remains elusive. Mutating the Abeta 1-40 Phe19-Phe20 backbone amide bond to an isostructural E-olefin bond enables formation of spherical aggregates to the exclusion of detectable amyloid fibrils. Herein, the fibrillization and toxicity of amide-to-ester mutants of Abeta 1-40 at the 19-20 position and surrounding backbone amide bonds are compared to the fibrillization and toxicity of the 19-20 E-olefin Abeta analogue and wild type Abeta. Whereas isostructural amide-to-E-olefin mutations eliminate both the H-bond donor and acceptor capabilities, isostructural amide-to-ester mutations eliminate the donor while retaining the ester carbonyl as a weakened acceptor. None of the amide-to-ester Abeta 1-40 mutants prevent fibrillization; in fact several exhibit hastened amyloidogenesis. The 18-19 amide-to-ester substitution is the only backbone mutation within the hydrophobic core region of the fibril (residues 17-21) that significantly slows fibrillization. Despite forming different morphologies, the 19-20 E-olefin mutant, the 18-19 amide-to-ester mutant, and WT Abeta 1-40 fibrils all exhibit similar toxicities when applied to PC12 cells at 18 h into the aggregation reactions, as assessed by MTT metabolic activity measurements. This result suggests that a common but low abundance aggregate morphology, that is accessible to these Abeta analogues, mediates toxicity, or that several different aggregate morphologies are similarly toxic.

E-olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: probing the requirements for Alzheimer's amyloidogenesis.

Herein, we report a stereospecific E-olefin dipeptide isostere synthesis that can be used to make gram quantities of the Phe-Phe isostere desired for eliminating a specific backbone H-bond donor and acceptor in the Alzheimer's disease related Abeta peptide. The Phe19-Phe20 E-olefin analogue of Abeta(1-40) was prepared by solid-phase peptide synthesis and was subjected to amyloidogenesis conditions. This analogue can aggregate into spherical morphologies but does not progress on to form protofibrils or fibrils as is the case for the all-amide sequence, providing insight into the structural requirements for amyloidogenesis.

Facile synthesis of alpha,alpha-diisobutylglycine and anchoring its derivatives onto PAL-PEG-PS resin.

alpha,alpha-Diisobutylglycine has been synthesized using a Pd-mediated dialkylation of ethyl nitroacetate as a key first step. The free alphaalphaAA is N(alpha)-protected and has been applied to the assembly of conformationally constrained peptide analogues. Mixed anhydrides from BOP-Cl and Fmoc-alphaalphaAA-OH are used for anchoring alphaalphaAAs onto a trialkoxybenzyl linker on PEG-PS grafted support, upon which a beta-strand mimic with difficult sequence is assembled in a superior quality.