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.

     

A high-affinity human/mouse cross-reactive monoclonal antibody,specific for VEGFR-2 linear and conformational epitopes

Vascular endothelial growth factors receptor 2 (VEGFR-2) has been implicated in playing an important role in the formation of new blood vessels in tumors and other diseases. A high affinity human/mouse cross-reactive anti-VEGFR-2 monoclonal antibody (mAb) named A8H1 was established by hybridoma technology. Several immunological methods were used to characterize the A8H1, including ELISA, affinity and kinetics assay, MALDI-TOF MS, WB, IP, IF, FASC and IHC. The results suggested that A8H1 could bind with linear and conformational epitopes of the VEGFR-2 antigen. The mAb had good specific reactivity with three forms of VEGFR-2 in HUVEC, and two forms in NIH-3T3 mouse fibroblast cells, which are regarded as non-expressive for VEGFR-2. The A8H1 mAb associated with intracellular and plasma membranes in HUVEC and with the nuclei in NIH-3T3 cells. This mAb also effectively identified VEGFR-2 over-expressing cells in a number of archived human cancer tissues.     

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.     

Origin of the autoreactive anti-type II collagen response. II. Specificities, antibody isotypes and usage of V gene families of anti-type II collagen B cells

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.     

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。     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Differential T cell hyporesponsiveness induced by in vivo administration of intact or F(ab')2 fragments of anti-CD3 monoclonal antibody. F(ab')2 fragments induce a selective T helper dysfunction.

Induction of peripheral T cell anergy associated with stimulation through the TCR complex in vivo has been described in mice using chemically modified APC, staphylococcal enterotoxin B, and intact anti-CD3 mAb. In the latter two models, T cell proliferation, IL-2R expression, and lymphokine production have been demonstrated before subsequent induction of hyporesponsiveness, whereas in the former model, these events have not been observed. To further investigate the relationship between mitogenicity and induction of peripheral hyporesponsiveness, mice were treated with either mitogenic intact anti-CD3 mAb or nonmitogenic F(ab')2 fragments of anti-CD3 mAb. T cells from F(ab')2-treated mice demonstrated a selective decrease in helper functions, with minimal effect on CTL function. Specifically, a marked reduction in ability of Th cells to secrete IL-2 when challenged in vitro with mitogen or alloantigen was observed, which persisted for at least 2 mo after mAb administration and which was independent of T cell depletion. Proliferative function was decreased in CD4+ T cells and could not be fully restored with addition of exogenous IL-2. A helper defect was also evident in vivo, in that F(ab')2-treated mice were deficient in their ability to reject MHC-disparate skin grafts, and in vivo administration of IL-2 reconstituted their ability to reject skin grafts normally. In contrast, T cells from mice treated with intact mAb demonstrated a significant decrease in both CTL and helper functions. A long term reduction in TCR expression on CD4+ cells from F(ab')2-treated mice, and on both CD4+ and CD8+ cells from intact mAb-treated mice was observed. These findings demonstrate that peripheral T cell hyporesponsiveness can be induced in vivo by binding an identical epitope on the TCR complex in the presence or absence of initial proliferation, lymphokine secretion, or IL-2R expression, and that binding to the same epitope can result in varying long term effects on T cell function.     

Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin.

We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography. Chimeric BR96 [IgG and F(ab')2] immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM. Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic. Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability. The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h. Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently. The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins. Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding. This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40. This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.     

A functionally unique anti-CD3 monoclonal antibody cross-reactive with basal keratinocytes

Monoclonal antibodies (mAb) 147, 446, and 454 each recognize different epitopes of CD3. The CD3 epitope recognized by mAb 446 is functionally unique for the T cell. In contrast to mAb 147 and 454, mAb 446 induces modulation of surface CD3 with delayed kinetics and, hence, is impaired in inducing a refractory state in the T cell to subsequent anti-CD3-induced helper function. MAb 446 (but not other anti-CD3 mAb, including mAb 147, 454, OKT3, and anti-Leu4) recognizes a cytoplasmic determinant within basal keratinocytes. Extraction of keratinocytes with nonionic detergent and 2 M NaCl abolished subsequent staining with mAb 446 but enhanced subsequent staining with anti-keratin mAb, suggesting that this cross-reactive determinant is not keratin. Immunoprecipitation of internally labeled keratinocytes with the anti-CD3 mAb 147 and 446 failed to reveal specific bands, whereas these same mAb immunoprecipitated specific bands from internally labeled CD3+ Jurkat cells corresponding to previously identified CD3 subunits, suggesting that the cross-reactive determinant in keratinocytes is also not CD3. The cross-reactivity is not species specific, in that mAb 446 stained a mouse keratinocyte line, nor is it absolutely keratinocyte specific, in that mAb 446 stained one of the two nonkeratinocyte human epithelial cell lines tested. This study raises the possibility that perturbation of unique CD3 epitopes may have unique effects on T cell surface events and subsequent activation and function.     

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.     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.     

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.     

A monoclonal antibody specific for snRNPs U1 and U2

A monoclonal antibody (D-5) is described which selectively precipitates snRNPs U1 and U2. The antibody was derived from a mouse immunized with extracts from chick embryonic nuclei. By immunoblotting on either total proteins from purified snRNPs U1-U6, U2-U6 or U1 only, we could demonstrate that the monoclonal antibody cross-reacts with the U1 RNP specific polypeptide A and the U2 RNP specific polypeptide B", thereby establishing that the two snRNP proteins share at least one epitope. D-5 precipitates snRNPs U1 and U2 from a variety of species, including man, chicken, mouse, rat kangaroo and Xenopus laevis. It will thus be a useful tool for studying structure function relationships of the two snRNP species in different cell systems.     

F(ab')2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

Trypanosoma cruzi calreticulin (TcCRT), described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host/parasite equilibrium. In an in vitro correlate of this situation, we show that the Clq/TcCRT interaction is inhibited by F(ab')2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion Clq, and this Clq is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate Clq could be inhibited by F(ab')2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process.     

A sequence-dependent monoclonal antibody specific for single-chain urokinase

To mimic the sequence spanning the primary site (the Lys158-Ile159 bond) cleaved by plasmin in its conversion of single-chain urokinase plasminogen activator (scuPA) to urokinase, we synthesized the peptide Cys(Acm)-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Phe-Cys [Cys(Acm)scuPA(153-164)Cys]. Immunization of A/J mice with the Cys(Acm)scuPA(153-164)Cys peptide linked to hemocyanin, followed by somatic cell fusion with a myeloma cell line (SP2/0), yielded a monoclonal antibody (SCOOP1) that bound to single-chain urokinase but not to urokinase or plasmin-treated single-chain urokinase. SCOOP1 could discriminate between single-chain urokinase and urokinase by greater than three orders of magnitude. In a radioimmunoassay, Cys(Acm)scuPA(153-164)Cys completely inhibited SCOOP1 binding to single-chain urokinase, whereas an equimolar mixture of two heptapeptides comprising the amino terminal [Cys-scuPA(153-158)] and carboxy terminal [scuPA(159-164)Cys)] halves of the cleavage site peptide did not. Thus the epitope recognized by SCOOP1 includes the Lys158-Ile159 peptide bond.