Antibody Molecules Research Articles

Mass spectrometry characterization of antibodies at the intact and subunit levels: From targeted to large-scale analysis

Antibodies are one of the most formidable molecular weapons available to our immune system. Their high specificity against a target (antigen) and capability of triggering different immune responses (e.g., complement system activation and antibody-dependent cell-mediated cytotoxicity) make them ideal drugs to fight many different human diseases. Currently, both monoclonal antibodies and more complex molecules based on the antibody scaffold are used as biologics. Naturally, such highly heterogeneous molecules require dedicated analytical methodologies for their accurate characterization. Mass spectrometry (MS) can define the presence and relative abundance of multiple features of antibodies, including critical quality attributes. The combination of small and large variations within a single molecule can only be determined by analyzing intact antibodies or their large (25–100 kDa) subunits. Hence, top-down (TD) and middle-down (MD) MS approaches have gained popularity over the last decade. In this Young Scientist Feature we discuss the evolution of TD and MD MS analysis of antibodies, including the new frontiers that go beyond biopharma applications. We will show how this field is now moving from the “quality control” analysis of a known, single antibody to the high-throughput investigation of complex antibody repertoires isolated from clinical samples, where the ultimate goal is represented by the complete gas-phase sequencing of antibody molecules without the need of any a priori knowledge.

Preservation of whole antibodies within ancient teeth

Archaeological remains can preserve some proteins into deep time, offering remarkable opportunities for probing past events in human history. Recovering functional proteins from skeletal tissues could uncover a molecular memory related to the life-history of the associated remains. We demonstrate affinity purification of whole antibody molecules from medieval human teeth, dating to the 13th-15th centuries, from skeletons with different putative pathologies. Purified antibodies are intact retaining disulphide-linkages, are amenable to primary sequences analysis, and demonstrate apparent immunoreactivity against contemporary EBV antigen on western blot. Our observations highlight the potential of ancient antibodies to provide insights into the long-term association between host immune factors and ancient microbes, and more broadly retain a molecular memory related to the natural history of human health and immunity.

DISCOVERY OF NOVEL ANTI-SERUM ALBUMIN VHH AS A BUILDING BLOCK FOR PK PROLONGATION

Abstract Background The serum half-life of endogenous albumin is approximately 19 days in humans and 1.5-2.5 days in rodents. This extended half-life in vivo is primarily due to effective recycling upon internalization mediated by the neonatal Fc receptor (FcRn). Many protein therapeutics smaller than 60 kD have short serum half-life, which can be extended by fusing the proteins of interest with an anti-albumin antibody. The fusion proteins then take advantage of FcRn-mediated recycling. Single domain antibody (VHH) molecules (around 15 kD), derived from camelid heavy-chain-only IgG, are attracting increased attention globally in the field of antibody-based therapies due to several features including small size, high stability, low immunogenicity, good tissue penetration and cost effectiveness in manufacturing. Methods an anti-human/cynomolgus monkey/murine/canine/feline serum albumin VHH lead was discovered from a proprietary, large native phage-displayed VHH library, which was then humanized, and affinity matured. The optimized VHH was fused to bispecific and trispecific antibodies for in vivo PK studies. Results An anti-human serum albumin VHH Ab was discovered, and it also bound to cyno and mouse serum albumin with high affinity without affecting the interaction of HSA with FcRn. The VHH showed good developability and, once fused to protein therapeutics, long half-life and anti-tumor activity in rodents. Conclusions a novel VHH against serum albumin of different species was discovered from native VHH libraries, and it can be easily assembled into bispecific Abs and multispecific Abs to prolong the molecules’ PK profile.

Separation of bispecific antibody related impurities with mixed-mode chromatography

The complex structure of bispecific antibody molecules and the rapid increase in upstream expression pose a significant challenge for downstream purification. Compared to mAbs, bsAbs typically contain a higher proportion of aggregates and fragments that are difficult to remove. Extensive study and optimization are often required because the properties of impurities are close to the target molecules. In this investigation, ion exchange resins and mixed-mode resins were used to evaluate the effectiveness in removing byproducts. Mixed-mode anion chromatography was found to be effective in polishing step of bsAbs with strong hydrophobicity. Hydroxyapatite chromatography using arginine hydrochloride as an eluent provided excellent separation performance compared to that of cation exchange resins and conventional mixed-mode resins. The separation process was optimized and verified in a 200 L pilot batch. The final products were obtained with excellent purity (99.0 %) and yield (80.70 %). This work provides a new idea for high-yield polishing in downstream processing, which improves the economics of large-scale production.

Protein structural insights into a rare PCSK9 gain-of-function variant (R496W) causing familial hypercholesterolemia in a Saudi family: whole exome sequencing and computational analysis.

Familial hypercholesterolemia (FH) is a globally underdiagnosed genetic condition associated with premature cardiovascular death. The genetic etiology data on Arab FH patients is scarce. Therefore, this study aimed to identify the genetic basis of FH in a Saudi family using whole exome sequencing (WES) and multidimensional bioinformatic analysis. Our WES findings revealed a rare heterozygous gain-of-function variant (R496W) in the exon 9 of the PCSK9 gene as a causal factor for FH in this family. This variant was absent in healthy relatives of the proband and 200 healthy normolipidemic controls from Saudi Arabia. Furthermore, this variant has not been previously reported in various regional and global population genomic variant databases. Interestingly, this variant is classified as "likely pathogenic" (PP5) based on the variant interpretation guidelines of the American College of Medical Genetics (ACMG). Computational functional characterization suggested that this variant could destabilize the native PCSK9 protein and alter its secondary and tertiary structural features. In addition, this variant was predicted to negatively influence its ligand-binding ability with LDLR and Alirocumab antibody molecules. This rare PCSK9 (R496W) variant is likely to expand our understanding of the genetic basis of FH in Saudi Arabia. This study also provides computational structural insights into the genotype-protein phenotype relationship of PCSK9 pathogenic variants and contributes to the development of personalized medicine for FH patients in the future.

Tumor Site-Specific Cleavage Improves the Antitumor Efficacy of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) play important roles in tumor therapy. However, traditional ADCs are limited by the extremely large molecular weight of the antibody molecules, which results in low permeability into solid tumors. The use of small ADCs may be expected to alleviate this problem, but this switch brings the new limitation of a greatly shortened blood circulation half-life. Here, we propose a new cleavable ADC design with excellent tumor tissue permeability and a long circulation half-life by fusing the small ADC ZHER2-MMAE with the Fc domain of the antibody for circulation half-life extension, and inserting a digestion sequence between them to release the small ADC inside tumors for better tumor penetration. The experimental results showed that the designed molecule Fc-U-ZHER2-MMAE has a significantly increased blood circulation half-life (7.1 h, 59-fold longer) compared to the small ADC ZHER2-MMAE, and significantly improved drug accumulation ability at tumor sites compared to the conventional full-length antibody-coupled ADC Herceptin-MMAE. These combined effects led to Fc-U-ZHER2-MMAE having significantly enhanced tumor treatment ability, as shown in mouse models of NCI-N87 gastric cancer and SK-OV-3 ovarian cancer, where Fc-U-ZHER2-MMAE treatment achieved complete regression of tumors in all or a portion of animals with no obvious side effects and an MTD exceeding 90 mg/kg. These data demonstrate the therapeutic advantages of this cleavable ADC strategy, which could provide a new approach for ADC design.

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles’ inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.

Preclinical evaluation of novel HER3-targeting radioconjugates for the imaging and treatment of HER3-expressing cancers.

e15114 Background: HER3 overexpression in tumors is associated with poor survival and is implicated in drug resistance mechanisms of HER1 (EGFR) and HER2 targeted therapies. Although antibodies against HER3 have been developed and evaluated in clinical trials, no HER3 targeted therapy has been approved, underscoring the unmet need for novel HER3 therapeutic approaches. We hypothesized that HER3 radioligand targeted therapies could provide enhanced therapeutic efficacy against HER3-positive cancers through the energy deposited by alpha or beta emitting radionuclides in tumor cells. Here we describe the development and preclinical evaluation of novel anti-HER3 antibody radioconjugates (ARCs), 225Ac-HER3 and 177Lu-HER3, using the alpha-emitting Actinium-225 (225Ac) and beta-emitting Lutetium-177 (177Lu) radioisotopes, respectively. Methods: ARCs were prepared by radiolabeling HER3 antibody with 225Ac or 177Lu using S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) to yield 225Ac or 177Lu-HER3-ARCs. DOTA modified antibodies were characterized by HPLC and MALDI-TOF and HER3-ARCs were fully characterized by instant thin-layer chromatography (iTLC) and HPLC. The in vitro binding properties of the HER3-ARCs were evaluated using HER3 recombinant proteins and cells expressing HER3 in ELISA and flow cytometry. The in vivo pharmacological properties of the HER3-ARCs were evaluated by molecular imaging using Zirconium-89 (89Zr) Positron Emission Tomography (PET), biodistribution and antitumor efficacy studies. Results: HER3 antibody was successfully modified with p-SCN-Bn-DOTA to yield a conjugate with approximately 8 DOTAs per antibody molecule. Results from the in vitro binding assays of ARCs to either HER3 human recombinant protein or cells expressing HER3 demonstrated similar binding properties of 225Ac-HER3 and 177Lu-HER3 to unmodified HER3 antibody. The biodistribution study in NCI-H1975 tumor bearing mice showed that both 225Ac-HER3 and 177Lu-HER3-ARCs specifically accumulate in HER3-expressing tumors relative to other tissues. In agreement with the biodistribution studies, PET images revealed specific uptake of 89Zr-HER3 in NCI-H1975 tumors. Furthermore, 225Ac and 177Lu-HER3-ARCs showed significantly more potent NCI-H1975 tumor growth inhibition compared to controls. Conclusions: Our findings suggest that a HER3 targeted radiotherapy approach can enhance therapeutic response and overcome the limitations of previously explored HER3 targeted drug modalities. Thus, further preclinical evaluation of 225Ac-HER3 and 177Lu-HER3 is warranted, including in settings of tumor drug resistance, which can provide the basis for investigating HER3-ARCs as an effective treatment approach in future clinical trials.

Noncovalent antibody catenation on a target surface greatly increases the antigen-binding avidity.

Immunoglobulin G (IgG) antibodies are widely used for diagnosis and therapy. Given the unique dimeric structure of IgG, we hypothesized that, by genetically fusing a homodimeric protein (catenator) to the C-terminus of IgG, reversible catenation of antibody molecules could be induced on a surface where target antigen molecules are abundant, and that it could be an effective way to greatly enhance the antigen-binding avidity. A thermodynamic simulation showed that quite low homodimerization affinity of a catenator, e.g. dissociation constant of 100 μM, can enhance nanomolar antigen-binding avidity to a picomolar level, and that the fold enhancement sharply depends on the density of the antigen. In a proof-of-concept experiment where antigen molecules are immobilized on a biosensor tip, the C-terminal fusion of a pair of weakly homodimerizing proteins to three different antibodies enhanced the antigen-binding avidity by at least 110 or 304 folds from the intrinsic binding avidity. Compared with the mother antibody, Obinutuzumab(Y101L) which targets CD20, the same antibody with fused catenators exhibited significantly enhanced binding to SU-DHL5 cells. Together, the homodimerization-induced antibody catenation would be a new powerful approach to improve antibody applications, including the detection of scarce biomarkers and targeted anticancer therapies.

Label-Free Electrochemical Biosensing Architecture Based on a Protein-Decorated Pt@BSA Nanocomposite for Sensitive Immunoassay of Pathogenic Escherichia coli O157:H7

Electrochemical immunosensors equipped with high specificity, superior analysis sensitivity, low cost, and convenient operations have been widely applied in the food industry, clinical medicine, biomonitor technique, and environmental protection. Because of the pressing requirements for detecting the pathogen Escherichia coli (E. coli) O157:H7 in various practical systems, an efficient and specific immunosensor was delicately designed based on the Pt@BSA nanocomposite with the surface conjugation of affinity-purified antibody molecules to achieve immune recognition. Amplified signal response toward probe molecules and favorable biocompatibility for bacteria attachment could be obtained at the assembled Pt@BSA biosensing interface. With the formation of immune complexes, specific binding of E. coli O157:H7 cells under a certain concentration range onto the antibody-immobilized interface resulted in electrochemical signal responses at different levels, which could be directly measured by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Herein, E. coli O157:H7 cells were quantitatively monitored by the DPV technique in a linear concentration range from 2.5 × 101 to 5.0 × 108 cfu mL–1 with a low detection limit of 9 cfu mL–1. This fabricated bacteria immunosensor indicated high specificity for E. coli determination, favorable selectivity among the interfering bacteria, excellent storage stability, satisfactory preparation reproducibility with a relative standard deviation (RSD) of 3.5%, and acceptable detection accuracy in practical food samples, providing a promising approach for ultrasensitive monitoring of hazardous substances and disease markers at an extremely low level in clinical diagnosis.

Pre-clinical pharmacologic and tolerability characterization of a novel KIRxCD8 targeting bispecific CD8 Treg modulator

Abstract We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. Here we describe the binding and specificity profiles of a novel bispecific CD8 Treg modulator in vitro, ex vivo, and in vivo, and initial assessment of tolerability and pharmacology in a humanized mouse model. We evaluated the binding, pharmacokinetics (PK), and early tolerability of the bispecific CD8 Treg modulator targeting CD8 and KIR2DL1/2/3. Despite single arm binding to NK cells via the KIR targeting arm and CD8 T cells by the CD8 targeting arm of the bispecific, we did not detect activation of immune cells or increased pro-inflammatory cytokines in vitro, nor did we observe any contribution of NK cells to the elimination of pathogenic CD4 T cells. In healthy mice, the CD8 Treg modulator had a PK profile consistent with antibody molecules; treatment delayed disease onset in an acute GVHD setting. In humanized CD34-engrafted NSG-IL-15 transgenic mice, supporting engraftment of NK cells, no activation of immune cells in peripheral blood or terminal tissues or induction of proinflammatory cytokines in the serum was observed following a single dose of the CD8 Treg modulator. The recently described CD8 Treg network appears dysfunctional in autoimmune diseases. The KIRxCD8 targeting bispecific modulator targets this network, engages and activates CD8 Treg and reduces inflammation without increasing unwanted immune cell activation or pro-inflammatory cytokines, both in vitro and in vivo. Our early data suggest that KIR-mediated enhancement of CD8 Treg function using a bispecific modulator is a broadly applicable and a promising CD8 Treg specific therapeutic modality for the treatment of autoimmune disease.

Silver nanoparticle decorated graphene-based SERS electrode towards procalcitonin detection

Monitoring procalcitonin levels (PCT) is critical for early diagnosis of sepsis, chronic disease, COVID-19 and other time-dependent pathologic cases. In this work, silver nanoparticles (AgNps) doped graphene-based Surface-enhanced Raman scattering (SERS) electrode was demonstrated for the first time to rapid detection of PCT biomarkers. An excellent combination of AgNps and single-layer graphene (SLG) was achieved, resulting in effective SERS enhancement. Due to the smooth and large surface area of SLG, many antibody molecules bind to the surface of SERS electrode and interact with PCT protein. The SERS enhancement factors (EFs) for R6G and PCT protein molecules were calculated to be 1012 and 1.6 × 109, respectively. The limits of detection (LODs) for R6G and PCT protein molecules were reported to be 10−13 M and 4 ngmL−1, respectively. These high EFs and lower LODs indicate that the fabricated AgNp/Gr@ ITO SERS electrodes have perfect SERS performance. SERS studies showed that there is a perfect interaction between PCT protein and AgNps and SLG, which leads to the enhancement of electromagnetic mechanism (EM) and chemical mechanism (CM) in the fabricated SERS materials. The developed eco-friendly SERS platform can provide reliable and sensitive test results, faster analysis compared to conventional biosensors, mobile applications and cost-effective clinical management.

Abstract LB209: An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells

Abstract Introduction: Multiple myeloma (MM) is a neoplastic malignancy characterized by the abnormal proliferation of plasma cells with excessive antibody production in the bone marrow. It accounts for approximately 1.8% of all new cancer cases and 2.1% of cancer deaths in the United States and occurs most frequently among older adults (age > 75). While there are a number of treatments with high response rates, MM relapses frequently within a few years and is considered incurable. Herein, experimental studies and results of a new therapeutic agent under development, TE-1146, are reported. TE-1146 is an antibody drug conjugate (ADC) composed of two therapeutic agents already in use in MM, namely, an anti-CD38 antibody, daratumumab (Dara), and lenalidomide (Lena). Method: TE-1146 is designed by employing “HIDAR” technology platform, which enables the preparation of homogeneous ADCs with high DAR (drug to antibody ratio). In the TE-1146 molecules, the Fab is replaced by scFv and a cysteine-containing Zn2+-binding motif is engineered at the C-termini of the H chains. Lena molecules are assembled into drug bundles containing 3 Lena molecules and a maleimide group. Two drug bundles are conjugated site-specifically to the Zn2+-activated SH groups of the reconfigured antibody molecule, creating an ADC with DAR of 6. TE-1146 was investigated for cytolytic effects against human MM cell lines H929 in cell cultures in vitro and transplanted H929 tumor in NOD-SCID mice in vivo, in comparison with Dara, Lena, and their combination. Results: In human plasma, TE-1146 has stability comparable with Dara, and the Lena molecules in the drug bundles remain conjugated. In cultures of H929 cells, TE-1146 caused the lysis of H929 cells at least 100 times more effectively than Dara, Lena, and Dara/Lena combination, based on IC50 comparison. It was shown that H929 cells internalized and degraded TE-1146 and freed Lena. In NOD-SCID mice, the subcutaneously transplanted H929 cells were allowed to grow into solid tumors in 14 days, and TE-1146 and other agents for comparison were administered intraperitoneally. It was found that one single dose of TE-1146 at 20 nmol/kg (conjugated with Lena at 120 nmol/kg) could retard the growth of the transplanted tumor and ultimately eliminate the tumor over 28-42 days, while the combination of one dose Dara at 20-80 nmol/kg and Lena of 46 μmol/kg given daily could not eliminate the tumor. The amount of Lena used in the combination treatment over a 28-day course is 10,700 times that of Lena in TE-1146. Conclusion: Lenalidomide is extremely powerful in killing multiple myeloma cells if brought into the MM cells. It is estimated that a very minute amount, probably less than 0.01%, of intraperitoneally injected lenalidomide gets into the transplanted MM tumor in the mouse model. TE-1146 may be a more effective and less toxic drug than Daratumumab/Lenalidomide combination in treating patients with hard-to-treat MM. Citation Format: Yueh-Hsiang Yu, Ming-Yu Hsieh, Pei-Wen Wu, Hui-Ju Lee, Pei-Hsuan Lin, Carmay Lim, Hsing-Mao Chu, Tse Wen Chang. An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB209.

Abstract 825: Targeted nanospheres for the treatment of cancer

Abstract Conventional cancer therapy exploits sensitivity of dividing cells to DNA damage leading to apoptosis and tumor kill. Off-target normal tissue toxicity limits the maximum tolerated dose (MTD) and potential tumor ablation. We developed a targeted nanoparticle therapeutic platform that maximizes delivery of small molecules to tumor cells, including across the blood-brain barrier that normally prevents effective therapy with such agents. The core technology is a novel nanoparticle formulation that maximizes bioavailability and preferentially targets tumor cells while minimizing delivery to normal cells. Plasma irinotecan levels remain readily detectable for at least 48 hours, while free irinotecan is no longer detectable at 2 hours. Nanoparticles are readily imaged in tumor cells when virtually none are detectable in normal tissues. Targeting is achieved with antibodies that cause the nanoparticles to preferentially bind to tumor cells versus normal by a factor of at least 10. Levels of drug by quantitative mass spectroscopy are at least 15-fold greater in tumor vs normal tissue. As a result, the minimum effective dose (MED) is typically reduced ten-fold. Typical payloads include irinotecan, doxorubicin, and gemcitabine. Targeting antibodies include CD276 (B7H3) and CD99 (MIC2) for solid tumors. Compared to antibody-drug conjugates (ADCs), far greater drug doses per antibody molecule are delivered. In addition, the nanoparticles readily pass through the blood-brain barrier at least 15 times better than free drug alone. Consequently, the platform technology shows promise for treatment of both extra-cranial and intra-cranial tumors. In pre-clinical animal studies, survival of human PDX glioblastoma (GBM) bearing mice was prolonged by at least three-fold compared to free-drug treated animals when treated at a sub-optimal dose of 10mg/kg of irinotecan thrice weekly. Ewing sarcoma bearing mice showed indefinite survival at 2 mg/kg bi-weekly dosing, even for relapsed, treatment-resistant tumors. Treatment of hepatocellular carcinoma bearing mice biweekly at 5mg/kg resulted in no detectable tumor and 100% survival at 80 days, when all free irinotecan treated mice at the same dose were dead, with control animals dead at 50 days. Similarly, prostate cancer bearing mice treated at the same dose and regimen were all alive at 58 days when all untreated tumor bearing mice were dead. A similar result was obtained with ovarian cancer and the same treatment regimen, but tumor growth in treated animals was observed at 58 days when all untreated animals were dead. Perhaps most significantly, treated mice bearing pancreatic cancer showed 100 % survival at 80 days when all untreated animals were dead at day 59. At no time did any animal show evidence of treatment-related toxicity. Taken together, these results support a non-toxic cancer treatment approach with existing small molecule therapy that preferentially delivers drug to tumor with markedly improved efficacy. Citation Format: HyungGyoo Kang, Bryon Upton, Paul Scott, Mark Lewis, Jon Nagy, Timothy Triche. Targeted nanospheres for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 825.

Abstract 3590: Targeted detection of ovarian cancer using functionalized iron oxide nanoparticles

Abstract SuperParaMagnetic Relaxometry (SPMR) is a highly sensitive detection technology that can differentiate the magnetic signature of nanoparticles (NP) bound to tumor cells from unbound nanoparticles. Nanoparticles that reach and bind to the target cells are measurable by superconducting quantum interference device (SQUID) magnetometers (MRX instrument developed in house), while unbound nanoparticles such as those freely circulating in the bloodstream are not detected and bone and normal tissue do not produce any magnetic signal. We have developed a protocol to produce high precision 25nm+1nm (<7% dispersion) Fe3O4 nanoparticle core. These core nanoparticles are coated by a polymer shell functionalized with carboxylate groups. Antibody are then conjugated on the surface providing molecular targeting capabilities and PEG is also attached to the surface to reduce opsonization. In previous studies, we have demonstrated that when conjugated with anti-HER2 antibody such as Herceptin, these nanoparticles exhibited great specificity and selectivity towards HER2+ tumor cells in vitro and in vivo. In current studies, we expanded our nanoparticles applications to other type of cancers, such as ovarian cancer. The CA125 is a tissue-specific antigen expressed in ovarian cancer. It is associated with greater than 80% of epithelial ovarian neoplasms. OC125, a murine monoclonal antibody, reacts with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. OC125 antibody is conjugated to our nanoparticles using the same strategy developed for Herceptin nanoparticles. Each nanoparticle contains one to three OC125 antibody molecules covalently attached to the surface based on ELISA analysis. Our results have shown that OC125-NP can distinguish CA125+ and CA125- cell lines, OVCAR3 and HeyA8 respectively. Positive signal can be competed out by pre-incubation with free OC125 antibody and negative cell line produce undetectable SPMR signal, demonstrating good sensitivity, specificity and selectivity. Antigen glypican-1 (GPC1) is a proteoglycan located on cell surface composed of a membrane-associated protein core anchored to the cytoplasmic membrane. GPC1 may play a functional role in the control of cell division and growth regulation. The expression of GPC1 has been found to be elevated in many cancer cells, including ovarian. Applying the same conjugation strategy developed for Herceptin NP, humanized anti-GPC1 antibody is conjugated to the nanoparticles with minor modification. GPC1-NP generate appreciable signal using SKOV3 cell line (GPC1+ ovarian cell lines) and the signal can be competed out by the presence of excess free GPC1 antibody. Together, these results suggest that in additional to breast cancer application, our antibody functionalized nanoparticle system can be developed for other targeted cancer detection, such as ovarian cancer. Citation Format: Marie Zhang, Eric Smith-Nguyen. Targeted detection of ovarian cancer using functionalized iron oxide nanoparticles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3590.

Live Cells versus Fixated Cells: Kinetic Measurements of Biomolecular Interactions with the LigandTracer Method and Surface Plasmon Resonance Microscopy.

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.

Electrochemical biosensor based on antibody-modified Au nanoparticles for rapid and sensitive analysis of influenza A virus.

The online version contains supplementary material available at 10.1007/s11581-023-04944-w.

Coronary Au@Bi Nanospheres for Highly Impedance‐Sensitive Immunosensing toward CEA

AbstractNovel coronary Au@Bi nanospheres (NSs) are simply synthesized by sparkling gold nanoparticles (Au NPs) on the surface of bismuth (Bi) NSs and successfully used as highly impedance‐sensitive immunosensing platform to develop a new label‐free electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA). Firstly, Bi NSs are prepared by one‐pot hydrothermal method, showing uniform sphere morphology with a dense oxide film formed outside Bi NSs due to the easy oxidation of Bi in the air. The oxide film significantly hinders the transmission of electrons and effectively improves the sensitivity of the proposed electrochemical impedance immunosensor. The surface functionalization of Bi NSs with Au NPs enhances the biocompatibility, stability, as well as active surface area of the produced Au@Bi NSs, preferable for the efficient loading of antibody molecules. The electrochemical impedance spectroscopy (EIS) is selected as the optical technique to measure CEA with the constructed immunosensor based on Au@Bi NSs. Because of the excellent impedance‐responsive performance, this new immunosensor displays highly sensitive detection toward CEA in a linear range of 50 fg mL−1–100 ng mL−1 with an extremely low detection limit of 9.83 fg mL−1 and could be potentially used in early clinical detection of cancer.

An electrochemical immunosensor based on polyaniline microtubules and zinc gallinate for detection of human growth differentiation factor-15.

The incidence rate of cardiovascular diseases (CVDs) remains high, and their mortality rate is significantly higher than that of other diseases. Growth differentiation factor-15 (GDF-15) is a recently developed biomarker for the early diagnosis and prognostic evaluation of CVDs because its concentration in serum increases substantially after a cardiovascular injury or an inflammatory reaction. In this study, a sandwich-type immunosensor was constructed for the sensitive detection of GDF-15. Specifically, peony-like zinc gallinate (ZnGa2O4) prepared using a hydrothermal method, which exhibits excellent electrocatalytic performance, was coupled with Au nanoparticles (NPs) to obtain golden-peony-like ZnGa2O4/Au NPs. Theypreserved the immune activity of GDF-15 antibody molecules and further enhanced the conductivity, thereby realizing additional signal amplification. Hollow polyaniline (PANI) microtubules decorated with Pd NPs were used as the sensing platform (PANI/Pd NPs). The hollow microtubules provided abundant active sites and considerably improved the electron-transfer rate. Under optimal conditions, a linear range and remarkably low detection limit of 100fgmL-1-10ngmL-1 and 42.23fgmL-1, respectively, were achieved. These experimental results indicate that the strategy reported herein can be adopted as a novel approach for the convenient and rapid detection of GDF-15.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.