Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.

Ultrasensitive Electrochemiluminescence Biosensor Based on Efficient Signal Amplification of Copper Nanoclusters Induced by CaMnO3 for CD44 Trace Detection.

Metal nanoclusters (Me NCs) have become a research hotspot in the field of electrochemiluminescence (ECL) sensing analysis. This is primarily attributed to their excellent luminescent properties and biocompatibility along with their easy synthesis and labeling characteristics. At present, the application of Me NCs in ECL mainly focuses on precious metals, whose high cost, to some extent, limits their widespread application. In this work, Cu NCs with cathode ECL emissions in persulfate (S2O82-) were prepared as signal probes using glutathione as ligands, which exhibited stable luminescence signals and high ECL efficiency. At the same time, CaMnO3 was introduced as a co-reaction promoter to increase the ECL responses of Cu NCs, thereby further expanding their application potential in biochemical analysis. Specifically, the reversible conversion of Mn3+/Mn4+ greatly promoted the generation of sulfate radicals (SO4•-), providing a guarantee for improving the luminescence signals of Cu NCs. Furthermore, a short peptide (NARKFYKGC) was introduced to enable the fixation of antibodies to specific targets, preventing the occupancy of antigen-binding sites (Fab fragments). Therefore, the sensitivity of the biosensor could be significantly enhanced by releasing additional Fab fragments. Considering the approaches discussed above, the constructed biosensor could achieve sensitive detection of CD44 over a broad range (10 fg/mL-100 ng/mL), with an ultralow detection limit of 3.55 fg/mL (S/N = 3), which had valuable implications for the application of nonprecious Me NCs in biosensing analysis.

Successful management of massive digoxin overdose using DIGIFab and therapeutic plasma exchange: a case report.

BACKGROUND: Despite the efficacy and safety of DIGIFab, it is relatively expensive and has limited availability. In addition, alternative interventions, such as therapeutic plasma exchange, may need to be considered in massive digoxin overdoses. Although few case reports describe its efficacy. CASE PRESENTATION: We report a case of a 17-year-old white male patient brought by family members to our emergency department in Riyadh, Saudi Arabia. After intentionally ingesting 48 mg of digoxin tablets to commit suicide, the patient's initial digoxin serum level was 8.04 ng/mL. The patient was resuscitated in the emergency department. After admission to the intensive care unit, the patient underwent therapeutic plasma exchange, because of insufficient DIGIFab doses. Afterward, the serum digoxin levels drastically decreased, and his symptoms reverted. The patient was successfully managed and discharged 7 days after admission. CONCLUSION: Despite insufficient evidence and a limited number of case reports describing the use of extracorporeal treatment in digoxin overdose, we noted the significant impact of therapeutic plasma exchange on our patient. However, therapeutic plasma exchange's use in routine treatment requires stronger evidence to confirm its benefits.

Eliminating drug target interference with specific antibody or its F(ab')2 fragment in the bridging immunogenicity assay.

Background: DB-1003 is a humanized anti-IgE monoclonal antibody with higher affinity than omalizumab. In the affinity capture elution (ACE)-based bridging electrochemiluminescent immunoassay (ECLIA) for antibodies to DB-1003, monkey serum IgE caused false-positive results. Materials & methods: The target-specific antibody or its F(ab')2 fragment was used to mitigate drug target interference in an ACE-based bridging ECLIA for the detection of anti-DB-1003 antibodies. Results: The sensitivity of the developed assay was at least 100 ng/ml. When the anti-drug antibody concentration was 250 ng/ml, the assay tolerated at least 20.0 µg/ml of the monkey IgE. Conclusion: Incorporating the target-specific antibody or its F(ab')2 fragment can overcome the interference from monkey serum IgE in ACE-based bridging ECLIA for anti-DB-1003 antibody detection.

Utilizing fab fragment-conjugated surface plasmon resonance-based biosensor for detection of Salmonella Enteritidis.

Although antibodies, a key element of biorecognition, are frequently used as biosensor probes, the use of these large molecules can lead to adverse effects. Fab fragments can be reduced to allow proper antigen-binding orientation via thiol groups containing Fab sites that can directly penetrate Au sites chemically. In this study, the ability of the surface plasmon resonance (SPR) sensor to detect Salmonella was studied. Tris(2-carboxyethyl)phosphine was used as a reducing agent to obtain half antibody fragments. Sensor surface was immobilized with antibody, and bacteria suspensions were injected from low to high concentrations. Response units were changed by binding first reduced antibody fragments, then bacteria. The biosensor was able to determine the bacterial concentrations between 103 and 108 CFU/mL. Based on these results, the half antibody fragmentation method can be generalized for faster, label-free, sensitive, and selective detection of other bacteria species.

SEC-MS in denaturing conditions (dSEC-MS) for in-depth analysis of rebridged monoclonal antibody-based formats.

Disulfide rebridging methods are emerging recently as new ways to specifically modify antibody-based entities and produce future conjugates. Briefly, the solvent-accessible disulfide bonds of antibodies or antigen-binding fragments (Fab) thereof are reduced under controlled conditions and further covalently attached with a rebridging agent allowing the incorporation of one payload per disulfide bond. There are many examples of successful rebridging cases providing homogeneous conjugates due to the use of symmetrical reagents, such as dibromomaleimides. However, partial rebridging due to the use of unsymmetrical ones, containing functional groups with different reactivity, usually leads to the development of heterogeneous species that cannot be identified by a simple sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) due to its lack of sensitivity, resolution and low mass accuracy. Mass spectrometry coupled to liquid chromatography (LC-MS) approaches have already been demonstrated as highly promising alternatives for the characterization of newly developed antibody-drug-conjugate (ADC) and monoclonal antibody (mAb)-based formats. We report here the in-depth characterization of covalently rebridged antibodies and Fab fragments in-development, using size-exclusion chromatography hyphenated to mass spectrometry in denaturing conditions (denaturing SEC-MS, dSEC-MS). DSEC-MS was used to monitor closely the rebridging reaction of a conjugated trastuzumab, in addition to conjugated Fab fragments, which allowed an unambiguous identification of the covalently rebridged products along with the unbound species. This all-in-one approach allowed a straightforward analysis of the studied samples with precise mass measurement; critical quality attributes (CQAs) assessment along with rebridging efficiency determination.

Stabilization of adalimumab Fab through the introduction of disulfide bonds between the variable and constant domains.

Fab is a promising format for antibody drug. Therefore, efforts have been made to improve its thermal stability for therapeutic and commercial use. So far, we have attempted to introduce a disulfide bond into the Fab fragment to improve its thermal stability and demonstrated that it is possible to do this without sacrificing its biochemical function. In this study, to develop a novel stabilization strategy for Fab, we attempted to introduce a disulfide bond between the variable and constant domains and prepared three variants of Fab; H:G10C + H:P210C, L:P40C + L:E165C, and H:G10C + H:P210C + L:P40C + L:E165C. Differential scanning calorimetry measurements showed that each of these variants had improved thermal stability. In addition, the variants with two disulfide bonds demonstrated a 6.5 °C increase in their denaturation temperatures compared to wild-type Fab. The introduction of disulfide bonds was confirmed by X-ray crystallography, and the variants retained their antigen-binding activity. The variants were also found to be less aggregative than the wild type. Our results demonstrate that the introduction of a disulfide bond between the variable and constant domains significantly improves the thermal stability of Fab.

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.

Cytoplasmic production of Fabs in chemically defined media in fed-batch fermentation.

Fragment of antigen-binding region (Fab) of antibodies are important biomolecules, with a broad spectrum of functionality in the biomedical field. While full length antibodies are usually produced in mammalian cells, the smaller size, lack of N-glycosylation and less complex structure of Fabs make production in microbial cell factories feasible. Since Fabs contain disulfide bonds, such production is often done in the periplasm, but there the formation of the inter-molecular disulfide bond between light and heavy chains can be problematic. Here we studied the use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) to express two Fabs (Herceptin and Maa48) in the cytoplasm of E. coli in fed-batch fermentation using a generic chemically defined media. We were able to solubly express both Fabs with purified yields of 565 mg/L (Maa48) and 660 mg/L (Herceptin) from low density fermentation. Both proteins exhibited CD spectra consistent with natively folded protein and both were biologically active. To our knowledge this is the first demonstration of high-level production of biological active Fabs in the cytoplasm of E. coli in industrially relevant fermentation conditions.

Preclinical Evaluation of 99mTc-MAG3-5-Fab Targeting TREM2 in Lung Cancer Mouse Models: A Comparison with 99mTc-MAG3-5-F(ab')2.

Triggering receptor expressed on myeloid cells-2 (TREM2), which is expressed on the surface of tumor-associated macrophages (TAMs), has been found to play a major role in the diagnosis and treatment of tumors. TREM2 expression is significantly upregulated in tumor tissues, and therefore, targeting TREM2 for tumor imaging may be of value. Previously, we performed TREM2 targeting imaging by using 68Ga-NOTA-COG1410 or a 124I-labeled monoclonal antibody (mAb) and F(ab')2 in mouse models of colon and gastric tumors. However, some of the shortcomings of these probes (i.e., the high uptake of 68Ga-NOTA-COG1410 in the liver, the difficulty of obtaining iodine-124, and the long half-life of iodine-124) have hindered their clinical use. Herein, we sought to synthesize novel molecular probes targeting TREM2 that are more conducive to clinical translation, eliminating the interference of isotope availability and in vivo probe biodistribution issues. Therefore, we established A549 cell lines with negative human TREM2 (hTREM2) expression (GFP tag; hTREM2- A549) or upregulated hTREM2 expression (GFP tag; hTREM2+ A549) using lentiviral transfection and confirmed these with Western blotting and immunocytochemistry. We then prepared a mouse anti-human TREM2 (5-mAb) by immunizing with the hTREM2 antigen. The antibody fragments 5-F(ab')2 and 5-Fab were prepared from 5-mAb, and 99mTc-MAG3-5-F(ab')2 and 99mTc-MAG3-5-Fab were then synthesized with excellent stability and specificity. 99mTc-MAG3-5-F(ab')2 had a slightly higher in vitro affinity than 99mTc-MAG3-5-Fab (Kd = 3.32 ± 0.05 nmol versus 4.62 ± 0.85 nmol). 99mTc-MAG3-5-F(ab')2 and 99mTc-MAG3-5-Fab both showed excellent specificity: after adding a 100-fold precursor, the two probes binding to the cells were almost blocked. In vivo pharmacokinetics showed that the distribution and elimination half-lives of 99mTc-MAG3-5-Fab (T1/2α = 1.25 ± 0.30 min and T1/2ß = 21.98 ± 2.80 min, respectively) were significantly reduced compared to those of 99mTc-MAG3-5-F(ab')2 (T1/2α = 2.64 ± 0.37 min and T1/2ß = 86.55 ± 26.86 min, respectively). In micro single-photon emission computed tomography/computed tomography (micro-SPECT/CT) imaging, the tumor was clearly displayed at 1 h after 99mTc-MAG3-5-Fab injection, while the blood background was extremely low at 3 h, and the probe was mainly excreted through the kidneys and biliary tract. 99mTc-MAG3-5-F(ab')2 uptake was also detected at the tumor site, although the blood background was consistently high. The biodistribution results were consistent with the micro-SPECT/CT imaging results. 99mTc-MAG3-5-Fab could clearly display hTREM2+ A549 tumors in a short time (1 h) with low uptake in nontumor organs and tissues and thus has clinical application prospects.

Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1.

In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling. The purified and bioconjugated molecules have been extensively characterized and tested on Cripto-1-positive cancer cells through in vitro binding assays. These recombinant Fab fragments recognize the target antigen in its native form on intact cells suggesting that they can be further developed as reagents for detecting Cripto-1 in theranostic settings.

Direct Comparison of the Hinge-Cleaving Proteases IgdE and BdpK for LC-MS-Based IgG1 Clonal Profiling.

Human antibodies are heterogeneous molecules primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, such as ELISA, lack the power to resolve abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the constant Fc region of the antibody. Here, we explore an alternative IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest all IgG1s from the same set of plasma samples. Both proteases cleave IgG1 specifically in the hinge, albeit via different mechanisms and at two distinct cleavage sites. Notwithstanding these differences, the Fab fragments generated by IgdE or BdpK produced highly similar clonal repertoires. However, IgdE required ∼16 h of incubation to digest plasma IgG1s, while BdpK required ∼2 h. We authenticated the similarity of the clones by top-down proteomics using electron transfer dissociation. We conclude that BdpK performs very well in digesting polyclonal plasma IgG1s and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge-digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in plasma.

Critical Analysis of Thrombocytopenia Associated With Glycoprotein IIb/IIIa Inhibitors and Potential Role of Zalunfiban, a Novel Small Molecule Glycoprotein Inhibitor, in Understanding the Mechanism(s).

Thrombocytopenia is a rare but serious complication of the intravenous glycoprotein IIb/IIIa (GPIIb/IIIa; integrin αIIbß3) receptor inhibitors (GPIs), abciximab, eptifibatide, and tirofiban. The thrombocytopenia ranges from mild (50 000-100 000 platelets/µL), to severe (20 000 to <50 000/µL), to profound (<20 000/µL). Profound thrombocytopenia appears to occur in <1% of patients receiving their first course of therapy. Thrombocytopenia can be either acute (<24 hours) or delayed (up to ~14 days). Both hemorrhagic and thrombotic complications have been reported in association with thrombocytopenia. Diagnosis requires exclusion of pseudothrombocytopenia and heparin-induced thrombocytopenia. Therapy based on the severity of thrombocytopenia and symptoms may include drug withdrawals and treatment with steroids, intravenous IgG, and platelet transfusions. Abciximab-associated thrombocytopenia is most common and due to either preformed antibodies or antibodies induced in response to abciximab (delayed). Readministration of abciximab is associated with increased risk of thrombocytopenia. Evidence also supports an immune basis for thrombocytopenia associated with the 2 small molecule GPIs. The latter bind αIIbß3 like the natural ligands and thus induce the receptor to undergo major conformational changes that potentially create neoepitopes. Thrombocytopenia associated with these drugs is also immune-mediated, with antibodies recognizing the αIIbß3 receptor only in the presence of the drug. It is unclear whether the antibody binding depends on the conformational change and whether the drug contributes directly to the epitope. Zalunfiban, a second-generation subcutaneous small molecule GPI, does not induce the conformational changes; therefore, data from studies of zalunfiban will provide information on the contribution of the conformational changes to the development of GPI-associated thrombocytopenia.

Anti-VEGFR2 F(ab')2 drug conjugate promotes renal accumulation and glomerular repair in diabetic nephropathy.

Poor renal distribution of antibody-based drugs is the key factor contributing to low treatment efficiency for renal diseases and side effects. Here, we prepare F(ab')2 fragmented vascular endothelial growth factor receptor 2 antibody (anti-VEGFR2 (F(ab')2) to block VEGFR2 overactivation in diabetic nephropathy (DN). We find that the anti-VEGFR2 F(ab')2 has a higher accumulation in DN male mice kidneys than the intact VEGFR2 antibody, and simultaneously preserves the binding ability to VEGFR2. Furthermore, we develop an antibody fragment drug conjugate, anti-VEGFR2 F(ab')2-SS31, comprising the anti-VEGFR2 F(ab')2 fragment linked to the mitochondria-targeted antioxidant peptide SS31. We find that introduction of SS31 potentiates the efficacy of anti-VEGFR2 F(ab')2. These findings provide proof of concept for the premise that antibody fragment drug conjugate improves renal distribution and merits drug validation in renal disease therapy.

Effect of Glutamate Antibody F(ab)2 Fragments on Memory Changes in Aged C57BL/6 Mice.

Intranasal administration of F(ab)2 fragments of anti-glutamate antibodies to 12-month-old C57BL/6 mice improves passive avoidance conditioning and have no effect on horizontal and vertical locomotor activity in the open-field test. In contrast to full-length antibodies to glutamate, their F(ab)2 fragments significantly increase the number of animals developed a conditioned passive avoidance reflex.

The structure of the teleost Immunoglobulin M core provides insights on polymeric antibody evolution, assembly, and function.

Polymeric (p) immunoglobulins (Igs) serve broad functions during vertebrate immune responses. Typically, pIgs contain between two and six Ig monomers, each with two antigen binding fragments and one fragment crystallization (Fc). In addition, many pIgs assemble with a joining-chain (JC); however, the number of monomers and potential to include JC vary with species and heavy chain class. Here, we report the cryo-electron microscopy structure of IgM from a teleost (t) species, which does not encode JC. The structure reveals four tIgM Fcs linked through eight C-terminal tailpieces (Tps), which adopt a single ß-sandwich-like domain (Tp assembly) located between two Fcs. Specifically, two of eight heavy chains fold uniquely, resulting in a structure distinct from mammalian IgM, which typically contains five IgM monomers, one JC and a centrally-located Tp assembly. Together with mutational analysis, structural data indicate that pIgs have evolved a range of assembly mechanisms and structures, each likely to support unique antibody effector functions.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

The origins of nonideality exhibited by monoclonal antibodies and Fab fragments in human serum.

The development of therapeutic antibodies remains challenging, time-consuming, and expensive. A key contributing factor is a lack of understanding of how proteins are affected by complex biological environments such as serum and plasma. Nonideality due to attractive or repulsive interactions with cosolutes can alter the stability, aggregation propensity, and binding interactions of proteins in solution. Fluorescence correlation spectroscopy (FCS) can be used to measure apparent second virial coefficient (B2,app ) values for therapeutic and model monoclonal antibodies (mAbs) that capture the nature and strength of interactions with cosolutes directly in undiluted serum and similar complex biological media. Here, we use FCS-derived B2,app measurements to identify the components of human serum responsible for nonideal interactions with mAbs and Fab fragments. Most mAbs exhibit neutral or slightly attractive interactions with intact serum. Generally, mAbs display repulsive interactions with albumin and mildly attractive interactions with IgGs in the context of whole serum. Crucially, however, these attractive interactions are much stronger with pooled IgGs isolated from other serum components, indicating that the effects of serum nonideality can only be understood by studying the intact medium (rather than isolated components). Moreover, Fab fragments universally exhibited more attractive interactions than their parental mAbs, potentially rendering them more susceptible to nonideality-driven perturbations. FCS-based B2,app measurements have the potential to advance our understanding of how physiological environments impact protein-based therapeutics in general. Furthermore, incorporating such assays into preclinical biologics development may help de-risk molecules and make for a faster and more efficient development process.

Epitope-Based Specific Antibody Modifications.

Modified antibodies have essential roles in analytic, diagnostic, and therapeutic uses, and thus, these antibodies are required to have optimal physical and biological properties. Consequently, the development of methods for site-selective antibody modification is crucial. Herein, we used epitope-based affinity labeling to introduce a Fab region-selective antibody modification method. Although labeling that exploits the high affinity between an antibody and its epitope may appear straightforward, it remains challenging probably because of the loss of target affinity caused by modification around the epitope-binding site. By thoroughly screening the modifying agent structure, reaction conditions, and purification methods, we developed an efficient method for the selective modification of the Fab region of the antibody while maintaining the high affinity for the epitope.