OBJECTIVE: The standard treatment for localized prostate cancer involves surgical removal of the prostate with curative intent. However, when tumor cells persist in the operation site, there is high risk of local recurrence and tumor spread, leading to stressful follow-up treatments, impaired quality of life, and reduced overall survival. This study examined photoimmunotherapy (PIT) as a new treatment option for prostate cancer cells.
METHODS: We generated conjugates consisting of either a humanized antibody or Fab fragments thereof targeting the prostate specific membrane antigen (PSMA), along with our silicon phthalocyanine photosensitizer dye WB692-CB1. PSMA-expressing prostate cancer cells were incubated with the antibody dye or Fab dye conjugates and cell binding was measured using flow cytometry. Cells were irradiated with varying doses of red light for dye activation, and cytotoxicity was determined by erythrosin B staining and subsequent analysis using a Neubauer counting chamber.
RESULTS: Specific cytotoxicity was induced with the antibody dye conjugate in the prostate cancer cells in a light dose-dependent manner. Treatment of the cells with the Fab dye conjugate resulted in lower cytotoxicity, which could be attributed to a reduced binding affinity and a reduced dye uptake of the Fab fragment.
CONCLUSIONS: Our new antibody dye and Fab dye conjugates offer potential for future intraoperative PIT in patients with localized prostate cancer, with the aim to ensure complete removal of tumor cells from the surgical area, to avoid local recurrence, and to improve clinical outcome.

Charge heterogeneity among therapeutic monoclonal antibodies (mAbs) is considered an important critical quality attribute and requires careful characterization to ensure safe and efficacious drug products. The charge heterogeneity among mAbs is the result of chemical and enzymatic post-translational modifications and leads to the formation of acidic and basic variants that can be characterized using cation exchange chromatography (CEX). Recently, the use of mass spectrometry-compatible salt-mediated pH gradients has gained increased attention to elute the proteins from the charged stationary phase material. However, with the increasing antibody product complexity, more and more selectivity is required. Therefore, in this study, we set out to improve the selectivity by using a solvent-enriched mobile phase composition for the analysis of a variety of mAbs and bispecific antibody products. It was found that the addition of the solvents to the mobile phase appeared to modify the hydrate shell surrounding the protein and alter the retention behavior of the studied proteins. Therefore, this work demonstrates that the use of solvent-enriched mobile phase composition could be an attractive additional method parameter during method development in CEX.

Yolk immunoglobulin (IgY) is perfect supplement to mammalian immunoglobulin G in passive immune protection but with poor delivery stability. This work succeeded in pH-responsive oral delivery of IgY-Fab fragments with cellulose based multi-network composite hydrogels. Data displayed that the hydrogel 2 showed superior mechanical properties and load performance (encapsulation efficiency of 99.25% and loading capacity of 45.11 mg/100 mg). The stability of the released Fab was confirmed by HPLC with Fab purity up to 79.65% at the end of digestion. The FTIR spectra revealed the potential interactions between Fab and the hydrogel matrix of the formation of hydrogen bonds or electrostatic interactions between the groups of -OH, -CH2, and -COO-. The excellent rehydration of the hydrogels wouldn\'t be impacted by low-temperature freeze drying. In sum, this work is of great significance to the development of Fab-themed health-care food, intensive processing of poultry eggs and the economic construction of related industries.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains\' interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

OBJECTIVE: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL.
METHODS: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts.
RESULTS: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts.
CONCLUSIONS: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

High-purity Fab fragment and immunoglobulin Y (IgY) were prepared to evaluate their anti-inflammatory activity in the lipopolysaccharide (LPS)-induced Raw 264.7 macrophage system. Compared with IgY, the Fab fragment possessed a greater potency in inhibiting the inflammation by nitric oxide (NO)/inducible nitric oxide synthase (iNOS) and prostaglandin-E2 (PGE2)/cyclooxygenase-2 (COX-2) pathways. The Fab fragment attenuated the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) to 38.07 ± 1.86-48.39 ± 11.33 pg/mL (63.1-71.0% inhibition), 31.59 ± 3.91-38.08 ± 4.44 pg/mL (72.4-77.1% inhibition), and 20.62 ± 0.46-21.91 ± 0.65 pg/mL (50-53% inhibition), respectively. Additionally, the Fab fragment significantly inhibited the translocation of nuclear transcription factor-κB (NF-κB) p65 and the phosphorylation of mitogen-activated protein kinase (MAPK) proteins, including ERK1/2 (41.5/33.2%), JNK1/2 (44.2/39.6%), and p38 (42.2%). The Fab fragment could be internalized into cells, and the pretreatment of RAW 264.7 macrophages with the Fab fragment reduced the mRNA expression of the Toll-like receptor (TLR4, 32.7-44.4% inhibition) and αVβ3 integrin (76.1% inhibition). In conclusion, Fab fragments regulated the TLR4 and αVβ3 integrin-mediated inflammatory processes by blocking the NF-κB and MAPKs pathways in the LPS-induced RAW 264.7 macrophage system.

In this work, we establish that cocaine binding to the Fab fragment of a recombinant humanized anti-cocaine mAb (h2E2) can be directly and easily quantitated using simple and inexpensive absorption and fluorescence measurements, employing dyes typically used for differential scanning fluorimetry, DASPMI and SYPRO Orange. For concentrated samples of the Fab fragment, absorbance spectroscopy employing these dyes reveals the number of cocaine sites present, using either DASPMI (by measuring the increase in dye absorbance) or SYPRO Orange (by measuring the change in dye maximal absorbance wavelength). Interestingly, we observed that cocaine binding to the Fab fragment had a much different effect on the SYPRO Orange dye absorbance than previously reported for the intact h2E2 mAb, resulting in a large decrease in the total dye absorbance for the Fab fragment, in contrast to previous results with the intact h2E2 mAb. For dilute samples of Fab fragment, a dye fluorescence emission spectroscopy assay was developed to quantitate the number of cocaine (and other high affinity cocaine metabolites) binding sites via the ligand-induced decrease in fluorescence emission of both of these extrinsic dyes. The difference in the cocaine titrations for the high affinity (Kd < 30 nM) ligands, cocaine, cocaethylene and benzoylecgonine and the low affinity (Kd > 30 μM) ligands, norcocaine, ecgonine methyl ester, and ecgonine were obvious using this assay. These simple, direct, and inexpensive techniques should prove useful for evaluation of other small molecule antigen binding Fab fragments, enabling quantitation and rapid biochemical assessments necessary for determining Fab fragment suitability for in vivo uses and other assays and experiments.

Antigen-binding fragments (Fabs) are preferred alternatives to antibodies for medical application, whereas their short half-lives limit therapeutic effectiveness. Albumin binding domain (ABD) with high affinity for albumin possesses a great potential in enhancing in vivo performance of biotherapeutics. In this study, to mitigate the poor pharmacokinetics of adalimumab Fab targeting tumor necrosis factor-α (TNFα), an ABD fusion strategy was applied innovatively using GA3, ABD035, ABD094 and ABDCon with high affinities for albumin. The prokaryotic expression, bioactivities and half-lives of those novel Fab-ABD fusions were investigated in vitro and in vivo. All Fab-ABD fusions were successfully purified, and they retained similar TNFα-binding activities with the unmodified Fab control, also presented high affinities for human/mouse serum albumin (HSA/MSA). Additionally, the simultaneous binding of the difunctional Fab-ABD fusions to TNFα and albumin was verified, and ABD fused to Fab neither interfered with Fab-TNFα binding nor impaired the association between Fc fragment of IgG receptor and transporter (FcRn) and albumin. Based on the highest binding affinity for HSA and maximal yield, Fab-ABDCon was selected for further evaluation. Fab-ABDCon showed similar thermostability with the Fab control and robust stability in human and mouse plasma. Most notably, the pharmacokinetics of Fab-ABDCon in mice was significantly improved with a 22-fold longer plasma half-life (28.2 h) compared with that of Fab control (1.31 h), which have contributed to its satisfactory therapeutic efficacy in murine TNFα-induced hepatonecrosis model. Thus, Fab-ABDCon could be a promising long-acting candidate suitable for drug development targeting TNFα-mediated inflammatory disease.

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise \"uncrystallizable\" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.

The continuous bed bioreactor systems have been used for the production of protein therapeutics, such as IgG, using immobilized enzyme in biopharmaceutical applications. We developed macroporous poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) cryogel-based bioreactor matrix using sodium dodecyl sulfate as surfactans in the presence of ethylene glycol dimethacrylate as cross linking agent by bulk polymerization. The developed polyGMA immobilized bioreactor with papain enzyme was used for specific fragmentation of immunoglobulin G. The catalysis efficiency for immobilized enzyme were investigated in comparison with free enzyme. The immobilized papain displayed broad catalytic activity over a variety of conditions, with maximal activity around pH 7.0 and 70 °C. The Michaelis-Menten kinetic constant (Km), the maximum reaction velocity (Vmax), and the catalytic efficiency (kcat) for free enzyme were 0.1097 mg/mL, 29.9 mg/mL/min, and 92.01 1/min, respectively, whereas for immobilized enzyme, Km, Vmax, and kcat values were 0.1078 mg/mL, 30.53 mg/mL/min, and 94.3 1/min, respectively. In a further step, after digestion, remarkable digestion products of bioreactor, Fab and Fc fragments, produced with immobilized papain bioreactors were analyzed in two ways by SDS-PAGE and reversed-phase HPLC; it was demonstrated that papain immobilized bioreactor successfully used for the digestion of human IgG with high activity. Therefore, the polyGMA cryogel immobilized with papain exhibited a very effective matrix for the bioreactor which can be considered as an alternative bioreactor matrix with great promise in biopharmaceutical applications.