Enzymic degradation of the Fc fragment of rabbit immunoglobulin IgG

The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.     

Higher protective activity of Fab' fragment compared to F(ab')2 and IgG on experimental tetanus

Summary Horse sera containing anti-tetanus whole IgG molecules, bivalent F(ab')₂ fragments and monovalent Fab' fragments were injected in 24 groups of 10–20 mice to compare their protective activity. When tetanus was induced in the mice, either with toxin or with spore suspension ofClostridium tetani 24 or 32 h prior to the injection of the antitoxins, monovalent Fab' was significantly more efficient in conferring protection against tetanus than F(ab')₂ or IgG.

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Resumen Afín de comparar la actividad protectora frente al tétanos de moléculas de IgG enteras, fragmentos F(ab')₂ bivalentes y fragmentos Fab' monovalentes, se inyectaron, con suero de caballo que contenía las distintas moleculas, 24 grupos de 10 a 20 ratones. Se indujo el tétanos en los ratones 24 o 32 horas antes de la inoculación con antitoxinas, mediante la toxina o bien con una suspensión de esporas deClostridium tetani. El fragmento monovalente Fab' confirió una protección frente al tétanos significativamente mayor que F(ab')₂ y IgG.

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Résumé Des sera de cheval contenant soit des molécules d'IgG entier, des fragments bivalents F(ab')₂ ou des fragments monovalents Fab' ont été injectés dans 24 groupes de 10 à 20 souris afin de comparer leur activité protectrice. Lorsque le tétanos a été induit chez les souris, que ce fût avec la toxine ou avec une suspension de spores deClostridium tetani, 24 ou 32 heures avant l'injection des antitoxines, le fragment monovalent Fab' s'est révelé plus efficace de manière significative que le fragment F(ab')₂ ou l'IgG dans l'octroi de l'activité protectrice contre le tétanos.

     

Adsorption of 125I-labeled immunoglobulin G, its F(ab')2 and Fc fragments onto plasma-polymerized films

Plasma-polymerized films were formed on flat glass plates using allylamine, acrylic acid, acrolein, and allylcyanide as monomers. Adsorption of (125)I-labeled-proteins such as immunoglobulin G (IgG), its F(ab')(2) and Fc fragments, and human serum albumin (HSA) was measured on these plasma-polymerized (PP) films covering the glass plates and on commercially available polymer plates. The adsorption isotherm followed the Langmuir equation, from which the binding constant and amount of saturation binding were estimated. We found that, in general, a cationic surface had higher affinity for protein adsorption than an anionic surface. Among the surfaces examined, the PP-allylamine surface showed the highest binding capacity (264.2 nmol/m(2)) for F(ab')(2) fragment: it was remarkably high. Of the surfaces examined, the PP-acrylic acid surface showed the lowest binding capacity (12.8 nmol/m(2)) for F(ab')(2) fragment. The PP-acrylic acid surface also indicated the lowest protein binding capacity for IgG (16.5 nmol/m(2)), Fc-IgG (32.4 nmol/m(2)) and HSA (16.7 nmol/m(2)), respectively. These imply that the PP-acrylic acid film is useful to fabricate as a low protein adsorption material which expected to decrease cell adhesion. Results of our investigation indicate that the plasma-polymerization technique is promising for fabrication of a smart NanoBio-interface which can control the protein adsorption on a solid-phase substrate using a suitable monomer such as allylamine for the large adsorption and acrylic acid for the small adsorption.     

Anti-rabies immunoglobulin preparation based on F(ab')2 fragments

Technology of manufacturing of new anti-rabies immunoglobulin preparation based on F(ab')2 fragments has been developed. This preparation is characterized by low reactogenicity, increased virus-neutralizing activity and stability.     

Distribution, kinetics and immunoscintigraphy of 131-I labelled intact antifibrinogen-fibrin antibody (AbFbg) and its F(ab')2 fragment

131-I-labelled anti fibrin-fibrinogen antibody (AbFbg) was compared with its F(ab')2 fragment in distribution studies and by immunoscintigraphy with a view to tumour visualization in tumour bearing rats. The distribution studies indicated that the intact antibody is more concentrated in tumour tissue than the F(ab')2 fragment. By 168h after injection, when tumour-to-tissue ratios were highest in the majority of tissues, the tumour concentration of intact antibody was 3 to 4 times that of the F(ab')2 fragment. The intact antibody is more suitable than the F(ab')2 fragment for tumour imaging especially in the abdominal region where the highest tumour-to tissue ratios were obtained with intact antibody in liver, spleen, intestines and kidneys.     

Rabies post-exposure prophylaxis in the Philippines: health status of patients having received purified equine F(ab')(2) fragment rabies immunoglobulin (Favirab)

Background

Recommended treatment for severe rabies exposure in unvaccinated individuals includes wound cleaning, administration of rabies immunoglobulins (RIG), and rabies vaccination. We conducted a survey of rabies treatment outcomes in the Philippines.

Methods

This was a case series involving 7,660 patients (4 months to 98 years of age) given purified equine RIG (pERIG) at the Research Institute for Tropical Medicine (Muntinlupa, Philippines) from July 2003 to August 2004 following Category II or III exposures. Data on local and systemic adverse reactions (AR) within 28 days and biting animal status were recorded; outcome data were obtained by telephone or home visit 6–29 months post-exposure.

Results

Follow-up data were collected for 6,464 patients. Of 151 patients with laboratory-confirmed rabies exposure, 143 were in good health 6–48 months later, seven could not be contacted, and one 4-year-old girl died. Of 16 deaths in total, 14 were unrelated to rabies exposure or treatment. Two deaths were considered PEP failures: the 4-year old girl, who had multiple deep lacerated wounds from a rabid dog of the nape, neck, and shoulders requiring suturing on the day of exposure, and an 8-year-old boy who only received rabies PEP on the day of exposure.

Conclusions

This extensive review of outcomes in persons with Category III exposure shows the recommended treatment schedule at RITM using pERIG is well tolerated, while survival of 143 laboratory-confirmed rabies exposures confirms the intervention efficacy. Two PEP intervention failures demonstrate that sustained education and training is essential in rabies management.     

Anti-DNA activity of IgG F(ab')2 from normal human serum.

The components of normal human serum (NHS) which bound DNA in a standard assay for anti-DNA antibody were characterized. IgG was the major detectable protein isolated from NHS by affinity chromatography on DNA-cellulose. A second adsorption of the whole serum IgG with DNA-cellulose did not remove additional gamma-globulin indicating that only a very small fraction of the IgG was capable of binding DNA. This binding activity was largely restricted to denatured DNA. IgG (Fab')2 bound DNA as well as the intact molecules demonstrating the antibody-like nature of the IgG binding. These results suggest that IgG antibody to denatured DNA is a normal component of human serum.     

抗人肝癌单抗片段抗体HAb18F(ab')_2的冻干工艺研究

选择适合于HAb18F(ab')2片段抗体的冻干保护剂及其冻干工艺,是确保该生物制品的质量关键之一,对不同种类和不同浓度的保护剂进行了研究,结果表明10%蔗糖对HAb18F(ab')2片段抗体冻干样品的剂型、外观、残水量(<3%)、复溶时间(<10s)、纯度(>95%)及稳定性没有影响。研究优化与保护剂相结合的适用于HAb18F(ab')2片段抗体的最佳冻干工艺,为抗体片段扩大生产提供依据。     

Aggregated immunoglobulin and Fc fragment of IgG induce IL-6 release from human monocytes

The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.     

Kinetics of whole serum and prepurified IgG digestion by pepsin for F(ab')2 manufacture

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.     

Immunospecific targeting of immunoliposomes, F(ab')2 and IgG to red blood cells in vivo

In this report a model to study the fate of target cells in the blood circulation after injection of appropriate immunoliposomes is discussed. The effect of intravenous administration of antimouse RBC immunoliposomes, F(ab')2 or IgG on the fate of intravenously injected 51Cr-labelled mouse RBC (Cr-mRBC) in the mouse and, particularly, in the rat was studied. The immunoliposome was of the Fab'-MPBPE-REV type (Fab'-fragments covalently linked to reverse phase evaporation vesicles by maleimido-4-(p-phenylbutyrate)phosphatidylethanolamine). In the rat model a high blood level (80%) of the injected dose of target cells, Cr-mRBC, was maintained for several hours. The elimination by Fab'-liposomes, F(ab')2 or IgG of Cr-mRBC, and subsequent uptake into liver and spleen was dose dependent. Administration of Fab'-liposomes or F(ab')2 resulted in a preferential uptake into the spleen (above a certain dose also, but much lower, uptake into the liver was observed), while after IgG administration 51Cr-label was mainly recovered in the liver. At equal protein doses (+/- 130 micrograms) Fab'-liposomes induced a faster elimination of the Cr-mRBC and a higher uptake into the spleen than F(ab')2. The potential advantage of the use of drug-loaded immunoliposomes to eliminate target cells from the blood stream and to induce a certain pharmacological effect in the target cells, in comparison with the free antibody administration of F(ab')2 or IgG is discussed.     

Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins

The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used. Indeed, at a saturating dose, a cytoreduction of three orders of magnitude was obtained with the fragment immunotoxins vs less than one order of magnitude for the whole immunotoxin, as assessed in a clonogenic assay. This enhancing effect was related to better cell killing kinetics, because with a similar amount of A-chain molecules bound per cell, T101 fragment immunotoxins achieved a twofold faster protein synthesis inactivation rate than the corresponding whole IgG immunotoxin. No significant difference in activity was shown between monovalent (Fab) and divalent (F(ab')2) forms of fragment immunotoxins. The observation that T101 fragment immunotoxins were more potent than intact immunotoxins was extended to another fragment immunotoxin constructed with an antibody (F111.98) directed against a different epitope of the CD5 Ag. In another model (anti-CD22 1G11 antibody on Raji cells), the fragment immunotoxin did not show any superiority over the IgG immunotoxin which was by itself very potent, strongly suggesting an Ag-dependent phenomenon.     

Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc

Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C(H)2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.     

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.     

低pH孵放病毒灭活处理对马破伤风免疫球蛋白F(ab')_2质量的影响

目的考察低pH孵放病毒灭活处理对马破伤风免疫球蛋白F(ab')_2质量的影响。方法取马破伤风免疫球蛋白F(ab')_24份,分别调整pH至3.8、4.1、4.4及6.5,于(25±1)℃条件下放置21 d后取样测定抗体效价、聚合物(二聚体、多聚体)及F(ab')_2含量,评估低pH孵放病毒灭活法对上述质量指标的影响;以1 mL/瓶的规格分装经低pH孵放病毒灭活处理的马破伤风免疫球蛋白F(ab')_2,于(25±1)℃条件下存放6个月,定期取样并进行抗体效价、聚合物(二聚体、多聚体)及F(ab')_2含量检测,评估其稳定性。结果马破伤风免疫球蛋白F(ab')_2经低pH孵放病毒灭活后,其抗体效价、聚合物(二聚体、多聚体)及F(ab')_2含量未发生明显变化;样品于(25±1)℃条件下存放6个月后,抗体效价和F(ab')_2含量均符合《中华人民共和国药典》2015版(三部)的要求。结论低pH孵放病毒灭活法适用于马破伤风免疫球蛋白F(ab')_2病毒的灭活。