Characterization and Stabilization of a New 64Cu-Labeled Anti-EGFR Antibody NCAB001 for the Early Detection of Pancreatic Cancer with Positron Emission Tomography

Early diagnosis of pancreatic cancer using current imaging modalities remains challenging. We have developed a new approach to identify tumor lesions ≥ 3 mm in the pancreas by positron emission tomography (PET) with a new intraperitoneally administered 64Cu-labeled anti-epidermal growth factor receptor (EGFR) antibody (encoded as NCAB001), called 64Cu-NCAB001 ipPET. Generally, in clinical research, a radiometal-antibody complex must be prepared immediately before use at the imaging site. To make 64Cu-NCAB001 ipPET available to daily clinical practices in a sustainable way, the NCAB001-chelator conjugate and 64Cu-NCAB001 must be characterized and stabilized. NCAB001 was manufactured under cGMP conditions. NCAB001 was conjugated with a bifunctional chelator (p-SCN-Bn-PCTA), and the antibody-chelator conjugate (PCTA-NCAB001) was characterized by LC/MS and ELISA. Thereafter, to effectively manufacture 64Cu-NCAB001, we developed a new formulation to stabilize PCTA-NCAB001 and 64Cu-NCAB001. An average of three PCTA chelators were conjugated per molecule of NCAB001. The relative binding potency of PCTA-NCAB001 was comparable to cetuximab. The formulation consisting of acetate buffer, glycine, and polysorbate-80 stabilized PCTA-NCAB001 for a year-long storage. Additionally, this formulation enabled the stabilization of 64Cu-NCAB001 for up to 24 h after radiolabeling with a sufficient radioactivity concentration for clinical use. These results may accelerate the future use of 64Cu-NCAB001 ipPET in clinical settings for the early diagnosis and treatment of pancreatic cancer.

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Antibodies as Biosensors’ Key Components: State-of-the-Art in Russia 2020–2021

The recognition of biomolecules is crucial in key areas such as the timely diagnosis of somatic and infectious diseases, food quality control, and environmental monitoring. This determines the need to develop highly sensitive display devices based on the achievements of modern science and technology, characterized by high selectivity, high speed, low cost, availability, and small size. Such requirements are met by biosensor systems—devices for reagent-free analysis of compounds that consist of a biologically sensitive element (receptor), a transducer, and a working solution. The diversity of biological material and methods for its immobilization on the surface or in the volume of the transducer and the use of nanotechnologies have led to the appearance of an avalanche-like number of different biosensors, which, depending on the type of biologically sensitive element, can be divided into three groups: enzyme, affinity, and cellular/tissue. Affinity biosensors are one of the rapidly developing areas in immunoassay, where the key point is to register the formation of an antigen–antibody complex. This review analyzes the latest work by Russian researchers concerning the production of molecules used in various immunoassay formats as well as new fundamental scientific data obtained as a result of their use.

Weak Precipitin Ring from A Fecal Specimen with Markedly Elevated Alpha-1 Antitrypsin Level Measured by Radial Immunodiffusion

Radial immunodiffusion (RID) is a classic methodology for antigen quantification that relies on the development of a distinct precipitin ring from precipitated antigen-antibody complex. As the precipitin ring grows, the precipitate at the inner edge of the ring constantly dissolves due to excess antigen flooding from the point of application, while new precipitate forms at the leading edge of the ring. RID plates with anti-human alpha-1-antitrypsin are prepared in our lab to measure fecal alpha-1 antitrypsin (A1AT) to aid in the diagnosis and monitoring of Protein Losing Enteropathy (PLE). The procedure has routinely produced precipitin rings with small radii and distinct edges after incubating at room temperature for 48 hours. Unexpectedly, a fecal specimen from a patient produced an extremely weak and large precipitin ring that could have been easily overlooked. Dilution studies confirmed a highly elevated A1AT level of 67 mg/g dry stool. The very weak and large precipitin ring was reproduced with a spiked specimen with similar A1AT concentration and kept expanding for several days until a distinct ring was formed eventually. Our data highlights a rare example of high-dose hook effect in RID and calls for meticulous attention and caution when reading and interpreting gel-based immunoassays with unexpected markedly elevated results to avoid additional confirmatory testing. In these cases, we recommend repeat testing with diluted specimens.

The Evolution of the Enzyme Immunoassay/Enzyme-Linked Immunosorbent Assay

50 years ago the Enzyme Immunoassay Enzyme-Linked Immunosorbent Assay, mostly known as ELISA was developed. This is a powerful but simple method that is very widely used in the diagnostic practice, as well as in biomedical research. During this time a number of ELISA modification were developed that significantly increased its properties, especially the senstivity, such as avidin-biotin assay, immuno-PCR, nano-ELISA and finally, the digital ELISA. This short review describes the principles of ELISA and the evolution from a conventional assay to the modern ultra-sensitive method. Most of the immunological methods have two components: antigen and antibody. The high specificity of their interaction gives a possibility to detect one of them if other one is included in the reaction as a specific partner. The simplest method for antigen detection in the presence of the antibody is immune diffusion (radial immune diffusion in that case), which practically the formation of precipitate of the “antigen-antibody” complex, when the target antigen diffuses from well into agarose containing the specific antibody. Unfortunately, this assay, as well as other traditional methods, like hemagglutination or complement fixation, have a low sensitivity and are unwieldy.

TGFβ2 and TGFβ3 isoforms drive fibrotic disease pathogenesis

Transforming growth factor–β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2–anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

Macroprolactin Molecular Heterogeneity and Variable Immunoassay Reactivity

Circulating prolactin (PRL) exhibits molecular heterogeneity the clinical significance of which is not known. Macroprolactin (mPRL) is widely reported, however, its heterogeneity is unknown. In addition to determining its molecular nature, the relative impact of the various forms on routinely available immunoassays needs to be examined. This study, applied various proteomics techniques to define the nature of mPRL and examined the effect of those forms on its measurement. Methods: Samples suspected of false hyperprolactinemia were subjected to; 1) precipitation by polyethylene glycol, 2) gel permeation chromatography, 3) protein-G affinity chromatography to identify presence of PRL-antibody complex and, 4) samples negative for antibody by step (3) were applied onto a lectin concanavalin A column to examine the presence of glycosylated PRL forms. Analysis was according to manufacturer’s instructions. All study samples and their chromatography fractions were measured using highly sensitive ELISA (DuoSet, R&D Systems, MN). Samples identified above as containing different mPRL forms were analyzed using four immunoassay analyzers in routine clinical use, namely; Advia Centaur® and Atellica® (Siemens, PA), Alinity-ci (Abbott Laboratories, IL), and Cobas 6000® (Roche Diagnostics, IN). Results: A total of 13 samples were entered into the study. PRL levels ranged from 21.4 to 1,469 ng/mL (median 48.8). Samples positive for mPRL (n=8) (with <40% recovery by PEG) exhibited predominant (52.3 to 95.0%) PRL activity in the (H) range (≥150kDa), with significant but relatively lower amount (3.6 to 34.1%) in the (M) range (≥30<150kDa) and (1.4 to 34.5%) at the (L) range (<30kDa). Samples negative for mPRL exhibited little PRL activity (1.2 to 5.1%) in (H) range, predominantly (60.0 to 79.4%) in the (M) range and moderate presence (15.4 to 38.9 %) in the (L) range. Two samples indeterminate for mPRL contained prolactin forms at all molecular weight levels, (H) (7.9, 27.1%), (M) (67.0, 40.7%), and (L) (5.9, 51.4%). Samples with mPRL exhibited significant binding to protein G affinity column indicating presence of PRL-antibody complex. Samples with (M) range mPRL forms exhibited significant lectin affinity binding. Samples with (H) mPRL showed markedly high PRL by two of the analyzers (Atellica®, Abbott Laboratories) and Cobas 6000®, (Roche Diagnostics). The deviation was more marked when using the Atellica® compared to Cobas 6000®. Samples with (M) mPRL showed marked deviation using the Roche Cobas 6000® compared to the others. In conclusion: Macroprolactin is heterogenous with antibody-PRL complex and aggregated glycosylated forms. Those forms exhibit variable immunoassay reactivity in different routinely used assays. Knowledge of circulating form as well as of their immunoreactivity is important when suspecting false hyperprolactinemia due to macroprolactin.

IL-2/IL-2R Antibody Complex Enhances Treg-Induced Neuroprotection by Dampening TNF-α Inflammation in an In Vitro Stroke Model

The present in vitro study showed that IL-2/IL-2R antibody complex facilitates Treg-induced neuroprotection in the oxygen glucose deprivation/reoxygenation (OGD/R) model of stroke. First, we examined the role of IL-2/IL-2R-treated Tregs in OGD/R-exposed rat primary cortical cells (PCCs), which represent the cell type of the ischemic gray matter in the stroke brain. Here, OGD/R induced cell death, which was attenuated by Tregs and more robustly by IL-2/IL-2R-treated Tregs, but not by IL-2/IL-2R treatment alone. Second, we next assessed IL-2/IL-2R effects in OGD/R-exposed human oligodendrocyte progenitor cells (OPCs), which correspond to the white matter injury after stroke. Results revealed that a similar pattern neuroprotection as seen in the gray matter, in that OGD/R triggered cell death, which was ameliorated by Tregs and more effectively by IL-2/IL-2R-treated Tregs, but IL-2/IL-2R treatment alone was not therapeutic. Third, as we begin to understand the mechanism underlying IL-2/IL-2R engagement of Tregs, we investigated the inflammatory response in OGD/R-exposed human neural progenitor cells (NPCs), which recapitulate both ischemic gray and white matter damage in stroke. Similar to PCCs and OPCs, OGD/R produced cell death and was blocked by Tregs and more efficiently by IL-2/IL-2R-treated Tregs, whereas IL-2/IL-2R treatment alone did not alter the ischemic insult. Moreover, the inflammatory marker, TNF-α, was upregulated after OGD/R, dampened by both Tregs and more efficiently by IL-2/IL-2R-treated Tregs but more pronounced in the latter, and not affected by IL-2/IL-2R treatment alone, suggesting IL-2/IL-2R-Treg-mediated modulation of inflammatory response in stroke. Altogether, these observations support the use of IL-2/IL-2R treatment in enhancing the anti-inflammatory effects of Tregs in stroke.