Noninvasive evaluation of PD-L1 expression in non-small cell lung cancer by immunoPET imaging using an acylating agent-modified antibody fragment.

PURPOSE: The aim of this study was to explore an effective 124I labeling strategy and improve the signal-to-noise ratio when evaluating the expression of PD-L1 using an 124I-iodinated durvalumab (durva) F(ab')2 fragment. METHODS: The prepared durva F(ab')2 fragments were incubated with N-succinimidyl-3-(4-hydroxyphenyl) propionate (SHPP); after purification, the HPP-durva F(ab')2 was iodinated using Iodo-Gen method. After the radiochemical purity, stability, and specific activities were determined, the binding affinities of probes prepared using different labeling strategies were compared in vitro. The clinical application value of [124I]I-HPP-durva-F(ab')2 was confirmed by PET imaging. To more objectively evaluate the in vivo distribution and clearance of tracers, the pharmacokinetics and biodistribution assays were also performed. RESULTS: After being modified with SHPP, the average conjugation number of SHPP per durva-F(ab')2 identified by LC-MS was about 8.92 ± 2.84. The prepared [124I]I-HPP-durva F(ab')2 was obtained with a satisfactory radiochemical purity of more than 98% and stability of more than 93% when incubated for 72 h. Compared with unmodified [124I]I-durva F(ab')2, the specific activity of [124I]I-HPP-durva-F(ab')2 was improved (52.91 ± 5.55 MBq/mg and 15.91 ± 0.74 MBq/mg), while the affinity did not significantly change. The biodistribution experiments and PET imaging showed that the prepared [124I]I-HPP-durva-F(ab')2 exhibited an accelerated clearance and improved tumor-to-background ratio compared with [124I]I-durva-F(ab')2. The specificity of [124I]I-HPP-durva-F(ab')2 to PD-L1 was well demonstrated both in vitro and in vivo. CONCLUSIONS: A PD-L1 PET imaging probe [124I]I-HPP-durva F(ab')2 was successfully synthesized through the SHPP modification strategy. The prepared probe was able to accurately evaluate the PD-L1 expression level through high-contrast noninvasive imaging.

124I-Labeled Monoclonal Antibody and Fragment for the Noninvasive Evaluation of Tumor PD-L1 Expression In Vivo.

Lung cancer is a highly heterogeneous cancer and is divided broadly into small and nonsmall cell lung cancer (SCLC or NSCLC). In all NSCLC patients, it is estimated that 50%-60% are programmed cell death ligand 1 (PD-L1) positive, and anti-PD-1/PD-L1 therapies have shown their clinical application prospects in advanced NSCLC. To avoid unnecessary adverse effects and provide anti-PD-1/PD-L1 therapy to the most appropriate patient population, the PD-L1 expression in patients preparing for treatment must be evaluated accurately and in real time. In this study, we noninvasively evaluate the PD-L1 expression in an NSCLC xenograft using 124I-labeled F(ab')2 fragments of durvalumab (Durva) and compared it with the 124I-labeled intact antibody in terms of the biodistribution and dosimetry. The aim is to develop a nuclide labeled molecular probe with better performance for PD-L1 immunoPET imaging. After cleaving using IdeS protease, the F(ab')2 fragments of Durva were labeled with 124I. The radioligand showed a high radiochemical purity (>96%) and outstanding stability. Western blot, quantitative real-time polymerase chain reaction, and flow cytometry were performed on the two selected NSCLC cell lines to measure the in vitro PD-L1 expression. The H460 cells showed a much higher PD-L1 expression than the A549 cells, both at the protein level and the mRNA level. In the following cell binding experiment and binding specificity assay, the labeled radioligand showed good affinity to high PD-L1 expression cells and could be blocked with excess unlabeled intact Durva. The results of the biodistribution and the positron emission tomography (PET) image showed that the peak tumor uptake of 124I-Durva-F(ab')2 was close to 124I-Durva, but much earlier (5.29 ± 0.42% ID/g for 124I-Durva-F(ab')2 at 12 h vs 5.18 ± 0.73% ID/g for 124I-Durva at 48 h). Compared with 124I-Durva, an accelerated blood clearance was observed for 124I-Durva-F(ab')2. The faster blood clearance allowed for a higher tumor-to-background ratio, which was reflected on the image in contrast. The H460 tumors showed excellent contrast as early as 4 h after injection with 124I-Durva-F(ab')2, and for 124I-Durva, the xenograft could not be distinguished clearly until 24 h after injection. Interestingly, 124I-Durva-F(ab')2 showed lower accumulations compared to other metal isotopes labeled PD-L1 antibodies in bone, liver, spleen etc., which will be beneficial for metastasis detection. Another benefit of accelerated blood clearance was a reduction in the radiation dose. According to the results of the OLINDA/EXM, the effective dose for the total body of 124I-Durva was 4.25-times greater than that of 124I-Durva-F(ab')2 (186 µSv/MBq vs 43.8 µSv/MBq). All of these data indicated that 124I-Durva-F(ab')2 is a promising immunoPET tracer for evaluating the in vivo PD-L1 levels in an NSCLC model and is expected to be successful in future clinical application.

Canine immunoglobulin F(ab')2 fragments protect cats against feline parvovirus virus infection.

Feline parvovirus virus (FPV) causes severe diarrhea and leukopenia in felines, and threatening the health of wild and domestic felines. Currently, specific drugs to treat FPV have not yet been developed. In this study, IgG was extracted from inactivated FPV-immunized dog sera. Canine F(ab')2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. The results showed that canine immunoglobulin F(ab')2 fragments showed efficient neutralizing activity in vitro against FPV and had therapeutic and prophylactic effects in FPV infected cats. The anti-FPV-specific F(ab')2 fragment can significantly alleviate the clinical symptoms of FPV infected cats and reduce the viral loads of the intestinal tract. These results indicated that the F(ab')2 fragment prepared from inactivated FPV-immunized felines may be used as a prophylactic and therapeutic agent for diseases caused by FPV.

Equine immunoglobulin F(ab')2 fragments protect cats against feline calicivirus infection.

Feline calicivirus (FCV) causes upper respiratory tract infections in felines and threatens the health of wild and domestic felines. Clinically, specific drugs to treat FCV have not yet been developed. Here, IgG was extracted from inactivated FCV-immunized horse sera. Equine F(ab')2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. In our study, equine immunoglobulin F(ab')2 fragments showed efficient neutralizing activity in vitro against FCV and had therapeutic and prophylactic effects in FCV-infected cats. The anti-FCV-specific F(ab')2 fragment can significantly alleviate the clinical symptoms of FCV-infected cats and reduce the viral loads of the trachea, lung and spleen. These results indicate that the F(ab')2 fragment prepared from inactivated FCV-immunized horses may be used as a prophylactic and therapeutic agent for diseases caused by FCV.

Quality of horse F(ab')2 antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity.

BACKGROUND: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. METHODS: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. RESULTS: Horse F(ab')2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. CONCLUSIONS: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

Production and characterization of anti-human IgG F(ab')2 antibody fragment.

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

Quality of horse F(ab')2 antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity

Background: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results: Horse F(ab')2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')(2) immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G.

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal's immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits.

Passive immunotherapy for Middle East Respiratory Syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model.

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a coronavirus (CoV), MERS-CoV. With the continuing spread of MERS-CoV, prophylactic and therapeutic treatments are urgently needed. In this study, we prepared purified equine F(ab')2 from horses immunized with MERS-CoV virus-like particles (VLPs) expressing MERS-CoV S, M and E proteins. Both IgG and F(ab')2 efficiently neutralized MERS-CoV replication in tissue culture. Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab')2 for potential use in the prophylactic or therapeutic treatment of exposed or infected patients.

Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding.

Extensive crosslinking of CD22 by epratuzumab triggers BCR signaling and caspase-dependent apoptosis in human lymphoma cells.

Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 µg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 µg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.