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Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody–drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.

The development of genetically encoded, wavelength-tunable fluorescent proteins has provided a powerful imaging tool to the study of protein dynamics and functions in cellular and organismal biology. However, many biological functions are not directly encoded in the protein primary sequence, e.g., dynamic regulation afforded by protein posttranslational modifications such as phosphorylation. To meet this challenge, an emerging field of bioorthogonal chemistry has promised to offer a versatile strategy to selectively label a biomolecule of interest and track their dynamic regulations in its native habitat. This strategy has been successfully applied to the studies of all classes of biomolecules in living systems, including proteins, nucleic acids, carbohydrates, and lipids. Whereas the incorporation of a bioorthogonal reporter site-selectively into a biomolecule through either genetic or metabolic approaches has been well established, the development of bioorthogonal reactions that allow fast ligation of exogenous chemical probes with the bioorthogonal reporter in living systems remains in its early stage. Here, we review the recent development of bioorthogonal reactions and their applications in various biological systems, with a detailed discussion about our own work—the development of the tetrazole based, photoinducible 1,3-dipolar cycloaddition reaction.

Labeling endogenous proteins in their natural environment with synthetic probes represents a major challenge in chemical biology. In a recent study, an elegant traceless labeling technique has been reported that allows attachment of biophysical probes to the targeted proteins in vivo .

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 770 million cases and >6.9 million deaths worldwide. We identified a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor-binding domain (RBD) (J. A. Duty, T. Kraus, H. Zhou, Y. Zhang, et al., Med 3:705–721, 2022, https://doi.org/10.1016/j.medj.2022.08.002 ). Representative antibodies from genetically distinct families were evaluated for the inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in the place of VSV-G. One mAb clone denoted FG-10A3 and its therapeutically modified version STI-9167 effectively inhibited infection and in vivo proliferation of early variants of SARS-CoV-2 including Omicron BA.1 and BA.2 and corresponding pseudoviruses and rcVSV-S variants (Duty et al.). To define the epitope of the broadly reactive FG-10A3 mAb, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-electron microscopy (EM). FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif. Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470–490 loop at the Spike RBD tip. Furthermore, neutralization studies using rcVSV-S F486 point mutants and currently-circulating variants Omicron BA.5, XBB.1.5, and BQ.1.1 that contain a V or P at position 486 further supported the model in which residue 486 is an important residue for FG-10A3 inhibition. This work provides an experimental strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Coronavirus disease 2019 (COVID-19) continues to significantly impact the global population, thus countermeasure platforms that enable rapid development of therapeutics against variants of SARS-CoV-2 are essential. We report use of a phage display human antibody library approach to rapidly identify neutralizing antibodies (nAbs) against SARS-CoV-2. We demonstrate the binding and neutralization capability of two nAbs, STI-2020 and STI-5041, against the SARS-CoV-2 WA-1 strain as well as the Alpha and Beta variants. STI-2020 and STI-5041 were protective when administered intravenously or intranasally in the golden (Syrian) hamster model of COVID-19 challenged with the WA-1 strain or Beta variant. The ability to administer nAbs intravenously and intranasally may have important therapeutic implications and Phase 1 healthy subjects clinical trials are ongoing.

Although current treatment strategies for ulcerative colitis have been fairly effective, many ulcerative colitis patients do not respond to such treatments and require a colectomy. Development of new therapeutics is indeed necessary to fulfill unmet medical needs. PDE4 (phosphodiesterase 4) inhibitors, including apremilast and roflumilast, have been demonstrated to be useful in treatment of ulcerative colitis in the pre-clinical animal models, and apremilast is under evaluation in clinical trials for treating ulcerative colitis. However, dose-limiting side effects on the central nervous system (CNS) and gastrointestinal tract (GI), including nausea, emesis, headache, and diarrhea, might impede the establishment of an appropriate therapeutic index for the treatment of ulcerative colitis in a safe and efficacious manner. Given the anti-inflammatory effect of PDE4 inhibitors relies on the suppression of inflammation in various immune cells, all of which express CD11a, we have now developed a targeted therapeutic antibody drug conjugate (ADC), consisting of αCD11a antibody and an analog of a highly potent PDE4 inhibitor GSK256066, to specifically deliver this small molecule into immune cells sparing the neuronal cells of the CNS and GI. This targeted delivery method could be a potential therapeutic strategy for treating ulcerative colitis.MethodsWe generated both human and mouse αCD11a-PDE4 inhibitor ADC, consisting of human αCD11a antibody (Efalizumab) or mouse αCD11a antibody (M17), and an analog of a highly potent PDE4 inhibitor GSK256066. The effect of the ADC in inhibiting inflammation was assessed both in primary human and mouse immune cells in vitro, and in a carrageenan-induced air pouch inflammation mouse model in vivo.ResultsWe show herein human and mouse αCD11a-PDE4 inhibitor ADC suppressed LPS-induced TNFα secretion in human primary monocytes and mouse primary peritoneal cells, respectively. This inhibition of TNFα secretion by mouse αCD11a-PDE4 inhibitor ADC in mouse peritoneal cells was mediated through CD11a, as treatment with excess αCD11a antibody abrogates the ADC's effect and this ADC did not reduce TNFα secretion in MEF cells that were negative for CD11a. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. In addition, mouse αCD11a-PDE4 inhibitor ADC bound to the lamina propria mononuclear cells and reduced TNFα and IFNγ production in these cells isolated from DSS-induced colitis mouse colons.ConclusionsThese results provide evidence for the feasibility of specifically-delivering immune suppressants to immune compartment and the development of PDE4 inhibitor ADC as a promising therapeutic for treating ulcerative colitis.

Bioorthogonal chemistry offers an exciting new strategy for the study of biomolecular dynamics and function in living systems. Compared to ligand-based approach where binding is noncovalent in nature, bioorthogonal chemistry relies upon specific, covalent attachment of probe molecules to the biomolecule of interest. A number of bioorthogonal chemistries were featured in Chapter 1 highlighting the key attributes of each reaction, with a particular emphasis on the reaction mechanism and kinetic studies. Chapter 2 proceeds to outline the development of a new photoinducible bioorthogonal reaction, termed 'azirine ligation' – a 1,3-dipolar azirine-alkene cycloaddition reaction between p-nitrophenylazirine and dimethyl fumarate that provides a rapid (∼2 mins) and highly selective route to protein conjugation at neutral pH and room temperature in biological medium. Chapters 3 to 5 describe the development of sequence-specific bioorthogonal reactions using phage display. In Chapter 3, the feasibility of displaying homoallylglycine (HAG) and homopropargylglycine (HPG) on phage surface was demonstrated. Efforts toward the development of sequence-specific photoclick chemistry and a new efficient strategy to incorporate the unnatural amino acids, HAG and HPG, on the surface of a phage via methionine biosynthesis inhibition (a metabolic approach) were summarized in Chapter 4. Chapter 5 describes the development of sequence-specific copper-free Sonogashira cross-coupling reaction and its utility in functionalizing a metabolically encoded alkyne-containing proteins in aqueous medium, in bacterial cells, and in live mammalian cells. Specifically in this chapter, a short peptide was identified through Cu-free Sonogashira cross-coupling reaction-based selection that led to rate enhancement and lower palladium complex loading.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, 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. Following a campaign of antibody discovery based on the vaccination of Harbour 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. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants 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. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

ABSTRACT We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes. Competing Interest Statement Sorrento authors own options and/or stock of the company. This work has been described in one or more provisional patent applications. HJ is an officer at Sorrento Therapeutics, Inc..