scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.     

The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution.

Stability of therapeutic IgG preparations is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concentration (5-10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amount of additives without endangering quality and stability. Since both biological function and aggregation depend on the conformational properties of individual IgG molecules, two sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of pH and addition of standard stabilizers. It was observed that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0-6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiologically acceptable concentration have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concentration as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of aromatic side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temperature are decisive factors for the long-term stability of IgG solutions. An increase in the dimer content was observed upon storage at 5 degrees C which was partly reverted upon incubation at 37 degrees C. Storage at temperatures higher than 5 degrees C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concentration, it is advisable to omit sugars at intravenous immunoglobulin (IVIG) formulation. Hydrophobic amino acids give promising alternatives.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

冻干低pH静注丙球质量稳定性观察

对１４批静注丙球保留样品进行３年效期内稳定性观察结果表明：出厂后３年内制品的物理检查和热稳定性试验均合格,ｐＨ值没有变化,ＩｇＧ分子仍以单体形式存在,抗补体活性（ＡＣＡ）和ＰＫＡ含量未见升高,白喉抗毒素效价未见下降。表明我所大规模生产的静注丙球质量是稳定可靠的     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.     

Engineering a human IgG2 antibody stable at low pH

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid‐induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti‐2,4‐dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti‐DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain—namely, F300Y, V309L, and T339A (IgG2_YLA)—reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen‐binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.     

静注人免疫球蛋白中白喉抗体效价对其Fc段生物学活性检测结果影响的分析

目的分析静注人免疫球蛋白(IVIG)中白喉抗体效价对其Fc段生物学活性检测的影响。方法检测20批IVIG的白喉抗体效价水平和Fc段生物学活性结果,进一步探讨白喉抗体效价与Fc段生物学活性的关系。结果20批IVIG中白喉抗体效价水平均符合中国药典的要求,在3~60 HAU/g之间,其中有两批IVIG的白喉抗体效价水平相对较高,其他18批IVIG的白喉抗体效价水平变化趋势不明显。20批IVIG的Fc段生物学活性均较高,在60%~140%之间。白喉抗体效价水平高者,其Fc段生物学活性并非高,反之亦然。结论 IVIG的Fc段生物学活性与其白喉抗体效价水平无明显的相关性。     

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.     

冻干静脉注射人血丙种球蛋白的研制

作者参照瑞典ＫａｂｉＶｉｔｒｕｂ药厂的“Ｇａｍｍｏｎａｔｉｖ”的制备方法,以低温乙醇法的ＦⅡ沉淀为原料,经ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０吸附,加入人血白蛋白作为稳定剂制备静脉注射人血丙种球蛋白（ＩＶＩＧ）。该制剂的γ－球蛋白纯度＞９４％,ＩｇＧ单体二聚体含量＞９７％,ＩｇＧ各亚类均存在且构成比与正常人血浆相似。抗补体活性（ＡＣＡ）和前激肽释放酶激活剂活性（ＰＫＡ）符合规程要求,安全性和稳定性好。该制剂为冻干剂型,便于运输和保存。     

Immunoelectron microscopic localization of idiotypes and allotypes on immunoglobulin molecules

We have developed a novel approach to the analysis of antigenic (allotypic and idiotypic) determinants on intact immunoglobulin molecules. Immune complexes composed of IgG in combination with anti-idiotype or anti-allotype antibody were "visualized" by transmission electron microscopy. Individual Fab fragments of anti-idiotype or anti-allotype antibody, when bound to the IgG, altered the "Y" configuration in a reproducible and interpretable manner. Anti-idiotype antibody (either as Fab or IgG) bound to the terminus of the presumed V region of the IgG molecule, thus extending the apparent length of the Fab arms. Analysis of a rabbit VH framework allotype (a1) revealed that the determinant(s) is (are) located on the lateral portion of the V region of IgG. Binding of the anti-a1 Fab fragments was always at approximately right angles to the axis of the Fab arms of IgG. Fab antibody to the rabbit kappa light chain (b4) allotype bound to the lateral portion of the terminal half of the IgG Fab arms. This technique should be of value in localizing less well defined immunoglobulin determinants.     

The measurement of progesterone in serum by a non-competitive idiometric assay

A novel non-competitive idiometric time-resolved fluoroimmunoassay for the determination of serum progesterone was developed, based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary antiprogesterone antibody. The first anti-idiotype, the betatype, competes with progesterone for an epitope of the primary antiprogesterone antibody at the binding site. The second anti-idiotype, the alphatype, binds to the antiprogesterone antibody in the presence of progesterone, but does not bind to the betatype antiprogesterone complex due to epitope proximity. In the present configuration, the biotinylated alphatype was captured onto anti-biotin IgG which was immobilized on microtiter wells. Reaction mixtures containing europium-labeled antiprogesterone antibody complexed sequentially with progesterone in standards or serum samples and with the betatype anti-idiotypic antibody were then reacted with the immobilized alphatype anti-idiotypic antibody. After 30 min of incubation, the fluorescence of europium is measured by time-resolved fluorescence and is proportional to the concentration of progesterone over the range 0–320 nmol/mL. The method demonstrates good sensitivity, precision, and comparability with a direct competitive radioimmunoassay. The idiometric assay for progesterone is suitable for dipstick technology and biosensors.     

用免疫单扩散法测定低pH静注丙球lgG含量及其影响因素初探

本文报道了低 pH静注丙球IgG含量测定免疫单扩散法的建立 ,并对其影响因素进行了初步试验。结果表明 ,该试验方法较为稳定 ,简单易行。在低pH静注丙球IgG含量检测时 ,进口Sigma琼脂糖效果优于国产琼脂糖(P <0 .0 1) ,国产 (东海 )琼脂糖不适于IgG含量的定量检测 ;应先将静注丙球pH调至中性后再进行IgG含量测定(P <0 .0 5 ) ;样品中含有的 10 %麦芽糖对试验结果无影响 (P >0 .0 5 )。     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Design and expression of a dimeric form of human immunodeficiency virus type 1 antibody 2G12 with increased neutralization potency

The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

Dimerization and other changes of aminoethylcysteine ketimine.

High performance liquid chromatography and gas liquid chromatography have been used for the study of the stability of aminoethylcysteine-ketimine (AECK) in different experimental conditions. Concentration and acidic pH lead to the formation of the dimer of AECK which is very sensitive to temperature and is slowly converted to the corresponding decarboxylated dimer even at room temperature. In the presence of air at neutral and alkaline pH AECK is converted to an unknown compound having spectral characteristics similar to AECK and to other compounds not detectable by HPLC. Under nitrogen at neutral pH AECK is more stable and only undergoes the dimerization reaction to some extent.     

A close look at human IgG sialylation and subclass distribution after lectin fractionation

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti‐inflammatory properties depend on sialylation of the N‐glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti‐inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.     

Production of l-serine from methanol and glycine by resting cells of a methylotroph under automatically controlled conditions

Serine production from methanol and glycine was tried using frozen-thawed resting cells of a methylotroph, Protomonas extorquens NR-1 under multi-variable controlled conditions. The conditions for l-serine formation were optimized at 30°C. The production of l-serine in 0.4% CaCl₂ solution (initial pH 8.2) was the same as in 0.1 M Tris-HCl buffer (initial pH 8.3). Increasing the initial glycine concentration promoted l-serine formation. A high aeration rate decreased l-serine production. The optimum concentrations of dissolved oxygen and methanol were 0.5 ppm and 10 g/l, respectively. The highest l-serine, 24.9 g/l, was obtained at 24 h from 30.94 gl (as dry weight) resting cells using 100 g/l initial glycine with controlled pH. The relationship between the initial rate of l-serine formation and cell concentration indicated an unusual curve due to the effects of the added NaOH which was used for controlling the pH. In similar experiments without control of pH, a normal profile was observed with respect to the relationship between the initial rate of l-serine formation and cell concentration. The highest l-serine, 54.5 g/l, was obtained at 48 h by 36.4 g/l (as dry weight) resting cells. The yield (mol of l-serine/mol of added substrate) of l-serine from methanol and glycine were 8.3% and 39.3%, respectively. The selectivity of l-serine (mol of l-serine/mol of glycine consumed) was 67.9%. The stoichiometry of the maximum l-serine formation showed that the resting cells carried the highly active methanol dehydrogenase while serine transhydroxymethylase was rather low. Serine glyoxalate aminotransferase was not completely inhibited by the high concentration of glycine (about 68% of synthesized l-serine was detected in the supernatant.     

Synthesis of site-specific methotrexate-IgG conjugates

Summary With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6–7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° C, pH 4.6, for 24 h led to the loss of 50%–60% of the bound drug from hydrazide conjugates compared to 20%–30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and R _F on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51° C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage. Abbreviations used: aBSA, rabbit anti-(bovine serum albumin) IgG; EDCI, 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbant assay; IC₅₀, concentration giving 50% inhibition; MTX, methotrexate; MTXAE, N-hydroxy-succinimide-based active ester of MTX; MTXAE-IgG, MTX-IgG conjugate prepared by the active-ester method; MTXH, methotrexate hydrazide; MTXH-IgG, MTX-IgG conjugate prepared by the hydrazide method; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride; TLC, thin-layer chromatography     

Immunofluorescent localization, by use of anti-idiotypic antibody, of monoclonal anti-progesterone antibody in the mouse uterus before implantation

Mouse monoclonal anti-progesterone IgG1 antibody designated DB3 has an anti-fertility effect when injected into female mice shortly after mating. In BALB/c mice, pregnancy is blocked, probably as a result of progesterone withdrawal with inhibition of implantation. Rabbit polyclonal anti-idiotype raised against DB3 has been used in an indirect immunofluorescence method on frozen tissue sections to demonstrate the presence of DB3 on the surface of uterine luminal and glandular epithelia before implantation. DB3 was only detectable 30-60 h after a single parenteral injection (9 nmol antibody per mouse i.p. or i.v. at 32 h post coitum). Immunolocalization was both pregnancy-dependent and anti-progesterone antibody-specific, as it was not seen in pseudopregnant mice or mice treated with P3 (mouse myeloma IgG1 protein, using polyclonal anti-P3 anti-idiotype as a probe) or saline. The immunofluorescent reaction was completely blocked by addition of DB3 idiotype in vitro. The results indicate that anti-progesterone antibody binds to an antigen associated with luminal and glandular epithelia which may locally inhibit the uterine uptake of progesterone and disrupt the process of implantation.