Ultrapotent Broadly Neutralizing Human-llama Bispecific Antibodies against HIV-1.

Broadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV-1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion-stabilized HIV-1 envelope (Env) trimer, BG505 DS-SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4-binding site (CD4bs) of vulnerability. Two of the vaccine-elicited CD4bs-targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a-hinge region and human IgG1-constant region (G36×3-IgG2a and R27×3-IgG2a), neutralized 96% of a multiclade 208-strain panel at geometric mean IC80s of 0.314 and 0.033 µg mL-1, respectively. Cryo-EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV-1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2-apex-targeting broadly neutralizing antibody, CAP256V2LS. The resultant human-llama bispecific antibody CAP256L-R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV-1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn-Fc mice similar to the parent CAP256V2LS. Vaccine-elicited llama nanobodies, when combined with V2-apex broadly neutralizing antibodies, may therefore be able to fulfill anti-HIV-1 therapeutic and prophylactic clinical goals.

Extrapolating differential scanning calorimetry data for monoclonal antibodies to low temperatures.

For irreversible denaturation transitions such as those exhibited by monoclonal antibodies, differential scanning calorimetry provides the denaturation temperature, Tm, the rate of denaturation at Tm, and the activation energy at Tm. These three quantities are essential but not sufficient for an accurate extrapolation of the rate of denaturation to temperatures of 25 °C and below. We have observed that the activation energy is not constant but temperature dependent due to the existence of an activation heat capacity, Cp,a. It is shown in this paper that a model that incorporates Cp,a is able to account for previous observations like, for example, that increasing the Tm does not always improve the stability at low temperatures; that some antibodies exhibit lower stabilities at 5 °C than at 25 °C; or that low temperature stabilities do not follow the rank order derived from Tm values. Most importantly, the activation heat capacity model is able to reproduce time dependent stabilities measured by size exclusion chromatography at low temperatures.

Directing-Group-Free Arene C(sp2)-H Amination Using Bulky Aminium Radicals and DFT Analysis of Regioselectivity.

A hydroxylamine-derived electrophilic aminating reagent produces a transient and bulky aminium radical intermediate upon in situ activation by either TMSOTf or TFA and a subsequent electron transfer from an iron(II) catalyst. Density functional theory calculations were used to examine the regioselectivity of arene C-H amination reactions on diversely substituted arenes. The calculations suggest a simple charge-controlled regioselectivity model that enables prediction of the major C(sp2)-H amination product.

Potent and pan-neutralization of SARS-CoV-2 variants of concern by DARPins.

We report the engineering and selection of two synthetic proteins - FSR16m and FSR22 - for possible treatment of SARS-CoV-2 infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon and exhibit broad spectrum neutralization of SARS-Cov-2 strains. The IC 50 values of FSR16m against authentic B.1.351, B.1.617.2 and BA.1.1 variants are 3.4 ng/mL, 2.2 ng/mL and 7.4 ng/mL, respectively, comparable to currently used therapeutic antibodies. Despite the use of the spike protein from a now historical wild-type virus for design, FSR16m and FSR22 both exhibit increased neutralization against newly-emerged variants of concern (39- to 296-fold) in pseudovirus assays. Cryo-EM structures revealed that these DARPins recognize a region of the receptor binding domain (RBD, residues 455-456, 486-489) overlapping a critical portion of the ACE2-binding surface. K18-hACE2 transgenic mice inoculated with a B.1.617.2 variant and receiving intranasally-administered FSR16m were protected as judged by less weight loss and 10-100-fold reductions in viral burden in the upper and lower respiratory tracts. The strong and broad neutralization potency make FSR16m and FSR22 promising candidates for prevention and treatment of infection by current and potential future strains of SARS-CoV-2.

Evaluation of the Inhibitory Effects of Pyridylpyrazole Derivatives on LPS-Induced PGE2 Productions and Nitric Oxide in Murine RAW 264.7 Macrophages.

A series of thirteen triarylpyrazole analogs were investigated as inhibitors of lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 macrophages. The target compounds 1a-m have first been assessed for cytotoxicity against RAW 264.7 macrophages to determine their non-cytotoxic concentration(s) for anti-inflammatory testing to make sure that the inhibition of PGE2 and NO production would not be caused by cytotoxicity. It was found that compounds 1f and 1m were the most potent PGE2 inhibitors with IC50 values of 7.1 and 1.1 µM, respectively. In addition, these compounds also showed inhibitory effects of 11.6% and 37.19% on LPS-induced NO production, respectively. The western blots analysis of COX-2 and iNOS showed that the PGE2 and NO inhibitory effect of compound 1m are attributed to inhibition of COX-2 and iNOS protein expression through inactivation of p38.

Synthesis and evaluation of multivalent nitroimidazole-based near-infrared fluorescent agents for neuroblastoma and colon cancer imaging.

Hypoxia is one of key characteristics of microenvironments of solid tumors, and evaluation of hypoxia status in solid tumors is important to determine cancer stage and appropriate treatment. In the present study, novel, multivalent, near-infrared (NIR) fluorescent imaging agents were developed to measure tumor hypoxia. These agents were synthesized using an amino acid as a backbone to connect mono-, bis-, or tris-2-nitroimidazole as a hypoxia-sensitive moiety to enhance uptake by the tumor and to attach sulfo-Cyanine 5.5 as an NIR fluorophore to visualize tumor accumulation. Studies of physical characteristics demonstrated that the novel NIR imaging agents showed suitable optical properties for in vitro and in vivo imaging and were stable in serum. In vitro cellular uptake studies in SK-N-BE(2) and SW620 cell lines demonstrated that NIR imaging agents bearing 2-nitroimidazole structures showed significantly higher tumor uptake in hypoxic cells than in normoxic cells. Moreover, in vivo optical imaging studies using SK-N-BE(2) and SW620 xenografted mice demonstrated that novel, multivalent, 2-nitroimadazole NIR imaging agents with two or three 2-nitroimidazole moieties showed higher uptake in tumor than the control agents with only one 2-nitroimidazole. These observations suggest that novel, multivalent, NIR agents could serve as potential optical imaging agents for evaluating tumor hypoxia.

Rational Engraftment of Quaternary-Interactive Acidic Loops for Anti-HIV-1 Antibody Improvement.

Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection.IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS.

Late-Stage 18F/19F Isotopic Exchange for the Synthesis of 18F-Labeled Sulfamoyl Fluorides.

Synthesis of sulfamoyl [18F]fluorides has been a challenging topic owing to the inefficient nucleophilic radiofluorination of sulfamoyl derivatives. Herein, we report an 18F/19F isotopic exchange approach to synthesize various sulfamoyl [18F]fluorides, otherwise inaccessible via direct synthesis from amines, with high radiochemical yields up to 97% (30 examples). This late-stage labeling protocol offers an efficient route to yield functionalized molecules by diversifying the chemical library possessing sulfamoyl functionalities through nucleophilic 18F incorporation within nitrogen-containing sulfur(VI) frameworks.

18F-labelled BODIPY dye as a dual imaging agent: Radiofluorination and applications in PET and optical imaging.

Dual Positron emission tomography (PET)/optical imaging techniques have captured scientific interest for clinical applications due to their potential as an effective tool for visualizing in vivo information such as disease processes. 4,4'-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye has been considered an ideal platform strategy to achieve dual PET/optical imaging due to its photochemical nature and chemical structure. Various radiofluorination methods to prepare [18F]BODIPY dye have been developed and established, ranging from nucleophilic substitution reactions to isotope exchange reactions. In addition, 18F-labelled BODIPY dyes for biologically important targets have been used for in vivo and ex vivo studies. These studies proved the practicality of [18F]BODIPY dyes as a hybrid PET/optical imaging probe. In this review, recent advances in the synthesis and biological evaluation of 18F-labelled BODIPY dyes are described.

The covalent SNAP tag for protein display quantification and low-pH protein engineering.

Yeast display has become an important tool for modern biotechnology with many advantages for eukaryotic protein engineering. Antibody-based peptide interactions are often used to quantify yeast surface expression (e.g., by fusing a target protein to a FLAG, Myc, polyhistidine, or other peptide tag). However, antibody-antigen interactions require high stability for accurate quantification, and conventional tag systems based on such interactions may not be compatible with a low pH environment. In this study, a SNAP tag was introduced to a yeast display platform to circumvent disadvantages of conventional antibody display tags at low pH. SNAP forms a covalent bond with its small-molecule substrate, enabling precise and pH-independent protein display tagging. We compared the SNAP tag to conventional antibody-based peptide fusion and to direct fluorescent domain fusion using antibody fragment crystallizable (Fc) gene libraries as a case study in low pH protein engineering. Our results demonstrated that covalent SNAP tags can effectively quantify protein-surface expression at low pH, enabling the enrichment of Fc variants with increased affinity at pH 6.0 to the neonatal Fc receptor (FcRn). Incorporation of a covalent SNAP tag thus overcomes disadvantages of conventional antibody-based expression tags and enables protein-engineering applications outside of physiological pH.

Synthesis of 18F-Labeled Aryl Fluorosulfates via Nucleophilic Radiofluorination.

Sulfuryl fluoride gas is a key reagent for SO2F transfer. However, conventional SO2F transfer reactions have limited 18F-radiochemistry translation, due to the inaccessibility of gaseous [18F]SO2F2. Herein, we report the first SO2F2-free synthesis of aryl [18F]fluorosulfates from both phenolic and isolated aryl imidazylate precursors with cyclotron-produced 18F-. The radiochemical yields ranged from moderate to good with excellent functional group tolerance. The reliability of our approach was validated by the automated radiosynthesis of 4-acetamidophenyl [18F]fluorosulfate.

Novel multifunctional 18F-labelled PET tracer with prostate-specific membrane antigen-targeting and hypoxia-sensitive moieties.

Prostate cancer is one of the most frequently found cancers in men worldwide. Prostate-specific membrane antigen (PSMA) is typically highly expressed in prostate cancer, and the Glu-Urea-Lys (GUL) structure has recently received considerable attention as a key unit of PSMA-targeting agents. Additionally, one of the common characteristics of many solid tumors, such as prostate cancer, is hypoxia. In this study, novel multifunctional PSMA inhibitors containing a PSMA-targeting moiety either with or without a hypoxia-sensitive moiety (18F-PEG3-ADIBOT-2NI-GUL and 18F-PEG3-ADIBOT-GUL, respectively; ADIBOT: azadibenzocyclooctatriazole, 2NI: 2-nitroimidazole) were designed and synthesized, and their feasibility as PET tracers for prostate cancer imaging studies was examined. The compounds labelled with 18F via the copper-free click reaction were stable in human serum and showed nanomolar binding affinities in in vitro PSMA binding assays. Micro-PET and biodistribution studies indicate that both 18F-labelled inhibitors successfully accumulated in prostate cancer regions, and 18F-PEG3-ADIBOT-2NI-GUL showed a 2-fold higher tumor-to-total non-target organ ratio than that of 18F-PEG3-ADIBOT-GUL, suggesting that the synergistic effects of the PSMA-targeting GUL moiety and the hypoxia-sensitive 2-nitroimidazole moiety can increase tumor uptake of the novel PET tracers in prostate cancer. These findings suggest that this novel multifunctional PET tracer with an 18F-labelled PSMA inhibitor and a 2-nitroimidazole moiety is a potent candidate to provide better diagnosis of prostate cancer via PET imaging studies.

Synthesis and evaluation of novel potent TSPO PET ligands with 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide.

Translocator protein (TSPO) expression is closely related with neuroinflammation and neuronal damage which might cause several central nervous system diseases. Herein, a series of TSPO ligands (11a-c and 13a-d) with a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide structure were prepared and evaluated via an in vitro binding assay. Most of the novel ligands exhibited a nano-molar affinity for TSPO, which was better than that of DPA-714. Particularly, 11a exhibited a subnano-molar TSPO binding affinity with suitable lipophilicity for in vivo brain studies. After radiolabeling with fluorine-18, [18F]11a was used for a dynamic positron emission tomography (PET) study in a rat LPS-induced neuroinflammation model; the inflammatory lesion was clearly visualized with a superior target-to-background ratio compared to [18F]DPA-714. An immunohistochemical examination of the dissected brains confirmed that the uptake location of [18F]11a in the PET study was consistent with a positively activated microglia region. This study proved that [18F]11a could be employed as a potential PET tracer for detecting neuroinflammation and could give possibility for diagnosis of other diseases, such as cancers related with TSPO expression.

Chemoselective Radiosyntheses of Electron-Rich [18F]Fluoroarenes from Aryl(2,4,6-trimethoxyphenyl)iodonium Tosylates.

Hypervalent diaryliodonium salts have been used to produce various [18F]fluoroarenes. The iodonium salt approach as a labeling precursor has been established to equally afford complex 18F-fluorinated molecules. Because of the inherent two aryl ring system connected to a central iodine atom, safeguarding the chemoselectivity during radiofluorination using diaryliodonium salts is important. Herein, we introduce a superior chemoselective radiosynthesis of [18F]fluoroarenes using an aryl(2,4,6-trimethoxyphenyl)iodonium tosylate as a precursor for 18F-incorporation, even on electron-rich aryl rings.

Synthesis and Evaluation of Multifunctional Fluorescent Inhibitors with Synergistic Interaction of Prostate-Specific Membrane Antigen and Hypoxia for Prostate Cancer.

Prostate cancer is one of the most common cancers in the world. It is widely known that prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and hypoxia is a common characteristic of many solid tumors, including prostate cancer. In this study, we designed multifunctional fluorescent inhibitors to target PSMA and tumor hypoxia in order to increase the tumor uptake of inhibitors. Novel PSMA inhibitors were prepared using lysine as the backbone to connect three different functional groups: the glutamate-urea-lysine (GUL) structure for inhibiting PSMA, 2-nitroimidazole for the hypoxia-sensitive moiety, and a near-infrared fluorophore (sulfo-Cyanine 5.5). According to the in vitro PSMA binding assay, novel fluorescent inhibitors were demonstrated to have nanomolar binding affinities. Multifunctional inhibitor 2 with one 2-nitroimidazole had a similar inhibitory activity to inhibitor 1 that did not contain the hypoxia targeting moiety, but multifunctional inhibitor 3 with two 2-nitroimidazoles showed lower inhibitory activity than inhibitor 1 due to the bulky structure of the hypoxia-sensitive group. However, in vivo optical imaging and ex vivo biodistribution studies indicated that both multifunctional inhibitors 2 and 3 had higher accumulation in tumors than inhibitor 1 due to a synergistic combination of PSMA and hypoxia targeting moieties. These observations suggest that this novel multifunctional strategy might be a promising approach to improve the diagnosis and therapy of prostate cancer.

Catalyst-Free Aromatic Radiofluorination via Oxidized Iodoarene Precursors.

Oxidized iodoarenes (OIAs), prepared via mCPBA-mediated oxidation, have been demonstrated as versatile precursors for the synthesis of [18F]fluoroarenes in the absence of catalysts. OIAs have been identified as intermediates in single-pot syntheses of iodonium salts and ylides but have never been recognized as radiofluorination precursors. Here, the isolated OIAs were used without any catalysts to produce functionalized [18F]fluoroarenes, regardless of the electronic nature of the arenes. This method was also applied to the production of radiolabeling synthons for use as aromatic 18F-labeled building blocks.

Novel potential pyrazolopyrimidine based translocator protein ligands for the evaluation of neuroinflammation with PET.

Translocator protein (TSPO) is an interesting biological target because TSPO overexpression is associated with microglial activation caused by neuronal damage or neuroinflammation, and these activated microglia are involved in several central nervous system diseases. Herein, novel fluorinated ligands (14a-c and 16a-c) based on a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide scaffold were synthesized, and in vitro characterization of each of the novel ligands was performed to elucidate structure activity relationships. All of the newly synthesized ligands displayed nano-molar affinity for TSPO. Particularly, an in vitro affinity study suggests that 2-(5,7-diethyl-2-(4-(3-fluoro-2-methylpropoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (14a), which exhibited high nano-molar affinity for TSPO and proper lipophilicity, was suitable for in vivo brain studies. Thus, radiosynthesis from tosylate precursor 13a using fluorine-18 was performed, and [18F]14a was obtained in a 31% radiochemical yield (decay-corrected). Dynamic positron emission tomography (PET) imaging studies were performed in a lipopolysaccharide (LPS)-induced neuroinflammation rat model using [18F]14a to identify the location of inflammation in the brain with a high target-to-background signal ratio. In addition, we validated that the locations of inflammatory lesions found by PET imaging were consistent with the locations observed by histological examination of dissected brains using antibodies. These results suggest that [18F]14a is a novel promising PET imaging agent for diagnosing neuroinflammation, and it may also prove to be applicable for diagnosing other diseases, including cancers associated with altered TSPO expression, using PET techniques.

Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.

We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates.

Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation.

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12-14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. Ki values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.

Soluble Prefusion Closed DS-SOSIP.664-Env Trimers of Diverse HIV-1 Strains.

The elicitation of autologous neutralizing responses by immunization with HIV-1 envelope (Env) trimers conformationally stabilized in a prefusion closed state has generated considerable interest in the HIV-1 vaccine field. However, soluble prefusion closed Env trimers have been produced from only a handful of HIV-1 strains, limiting their utility as vaccine antigens and B cell probes. Here, we report the engineering from 81 HIV-1 strains of soluble, fully cleaved, prefusion Env trimers with appropriate antigenicity. We used a 96-well expression-screening format to assess the ability of artificial disulfides and Ile559Pro substitution (DS-SOSIP) to produce soluble cleaved-Env trimers; from 180 Env strains, 20 yielded prefusion closed trimers. We also created chimeras, by utilizing structure-based design to incorporate select regions from the well-behaved BG505 strain; from 180 Env strains, 78 DS-SOSIP-stabilized chimeras, including 61 additional strains, yielded prefusion closed trimers. Structure-based design thus enables the production of prefusion closed HIV-1-Env trimers from dozens of diverse strains.