Radiolocalisation of an anti-CEA monoclonal antibody (FO23C5) and its fragments in a colon carcinoma xenograft model

A new monoclonal antibody designated FO23C5 against a protein component of carcinoembryonic antigen (CEA) has been developed. A xenograft system of human colon cancer was used to compare the intact monoclonal IgG with its fragments (Fab')2 and Fab) and with an established anti-CEA antibody (MAb35) and the antibody AUA1 raised against the colon carcinoma cell line. We demonstrate that FO23C5 compares well with the existing anti-CEA antibody and with AUA1, and that F(ab')2 fragments perform best in achieving optimal tumour to normal tissue ratios compared with intact IgG and Fab fragment.     

Immunological properties of a radioiodinated monoclonal antibody and its F(ab')2mu fragments directed against the polymorphic epithelial mucin of human breast cancer.

The purification of the IgM monoclonal antibody 436 against a breast tumor antigen from mouse ascitic fluid is reported. The purified immunoglobulin was radioiodinated and the resulting product assessed for its binding capacity and binding specificity. Purified IgM-436 served for F(ab')2 mu preparation which was tested for its antigen binding capacity. Radioiodinated IgM-436 and its F(ab')2 mu retained their immunological activity which was never lower than those of the corresponding cold products.     

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.     

抗CD20嵌合抗体片段F(ab′)2突变体在大肠杆菌中的高效分泌表达

构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,研究其在大肠杆菌中的高效表达及其表达产物的生物学活性。采用PCR法构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,并用双脱氧终止法测定DNA序列 ;采用 19L发酵罐高密度发酵抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,采用亲和色谱和分子筛色谱法纯化表达产物 ,并用SDS-PAGE和薄层激光扫描鉴定纯化产物 ;采用活细胞间接免疫荧光法测定纯化产物与靶细胞的结合活性 ;MTT法测定纯化产物对Raji细胞的生长抑制作用 ,并研究其作用机理。DNA序列测定结果表明 ,抗CD20嵌合抗体片段F(ab′)₂ 突变体已成功构建 ,表达可溶性产物的产量达 360mg L ,具有与Raji细胞 (CD20⁺)结合的活性 ,并抑制Raji细胞的生长 ,其作用机理为诱导Raji细胞凋亡。此突变体有望成为治疗非何杰金氏B细胞淋巴瘤的药物。     

Pretargeting for cancer radioimmunotherapy with bispecific antibodies: role of the bispecific antibody's valency for the tumor target antigen

The use of a divalent effector molecule improves bispecific antibody (bsMAb) pretargeting by enabling the cross-linking of monovalently bound bsMAb on the cell surface, thereby increasing the functional affinity of a bsMAb. In this work, it was determined if a bsMAb with divalency for the primary target antigen would improve bsMAb pretargeting of a divalent hapten. The pretargeting of a (99m)Tc-labeled divalent DTPA-peptide, IMP-192, using a bsMAb prepared by chemically coupling two Fab' fragments, one with monovalent specificity to the primary target antigen, carcinoembryonic antigen (CEA), and to indium-loaded DTPA [DTPA(In)], was compared to two other bsMAbs, both with divalency to CEA. One conjugate used the whole anti-CEA IgG, while the other used the anti-CEA F(ab')(2) fragment to make bsMAbs that had divalency to CEA, but with different molecular weights to affect their pharmacokinetic behavior. The rate of bsMAb blood clearance was a function of molecular weight (IgG x Fab' < F(ab')(2) x Fab' < Fab' x Fab' conjugate). The IgG x Fab' bsMAb conjugate had the highest uptake and longest retention in the tumor. However, when used for pretargeting, the F(ab')(2) x Fab' conjugate allowed for superior tumor accretion of the (99m)Tc-IMP-192 peptide, because its more rapid clearance from the blood enabled early intervention with the radiolabeled peptide when tumor uptake of the bsMAb was at its peak. Excellent peptide targeting was also seen with the Fab' x Fab' conjugate, albeit tumor uptake was lower than with the F(ab')(2) x Fab' conjugate. Because the IgG x Fab' bsMAb cleared from the blood so slowly, when the peptide was given at the time of its maximum tumor accretion, the peptide was captured predominantly by the bsMAb in the blood. Several strategies were explored to reduce the IgG x Fab' bsMAb remaining in the blood to take advantage of its 3-4-fold higher tumor accretion than the other bsMAb conjugates. A number of agents were tested, including those that could clear the bsMAb from the blood (e.g., galactosylated or nongalactosylated anti-id antibody) and those that could block the anti-DTPA(In) binding arm [e.g., DTPA(In), divalent-DTPA(In) peptide, and DTPA coupled to bovine serum albumin (BSA) or IgG]. When clearing agents were given 65 h after the IgG x Fab' conjugate (time of maximum tumor accretion for this bsMAb), (99m)Tc-IMP-192 levels in the blood were significantly reduced, but a majority of the peptide localized in the liver. Increasing the interval between the clearing agent and the time the peptide was given to allow for further processing of the bsMAb-clearing agent complex did not improve targeting. At the dose and level of substitution tested, galacosylated BSA-DTPA(In) was cleared too quickly to be an effective blocking agent, but BSA- and IgG-DTPA(In) conjugates were able to reduce the uptake of the (99m)Tc-IMP-192 in the blood and liver. Tumor/nontumor ratios compared favorably for the radiolabeled peptide using the IgG x Fab'/blocking agent combination and the F(ab')(2) x Fab' (no clearing/blocking agent), and peptide uptake 3 h after the blocking agent even exceeded that of the F(ab')(2) x Fab'. However, this higher level of peptide in the tumor was not sustained over 24 h, and actually decreased to levels lower than that seen with the F(ab')(2) x Fab' by this time. These results demonstrate that divalency of a bsMAb to its primary target antigen can lead to higher tumor accretion by a pretargeted divalent peptide, but that the pharmacokinetic behavior of the bsMAb also needs to be optimized to allow for its clearance from the blood. Otherwise, blocking agents will need to be developed to reduce unwanted peptide uptake in normal tissues.     

The kinetics of antibody binding to membrane antigens in solution and at the cell surface.

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 10(5)--1.1 x 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Receptor aggregation is necessary for activation of the soluble insulin receptor kinase

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. II. Sustained antigen-specific T cell help is required to induce a specific antibody response.

The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.     

Synthesis of bifunctional antibodies for immunoassays

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.     

Anti-idiotypic antibodies in a patient with monoclonal rheumatoid factor after pneumococcal bacteremia

A 51-yr-old Japanese female patient with monoclonal IgM gammopathy with rheumatoid factor activity was admitted because of pneumococcal bacteremia. About 2 wk after admission, her rheumatoid factor activity became undetectable by RAHA test and radioimmunoassay, subsequent to the initial marked elevation. The suppressive capacity of the patient's IgG fraction on the rheumatoid activity of her monoclonal IgM on January 11 was determined. The IgG fraction obtained on February 22 blocked the binding of the rheumatoid factor to rabbit IgG. The suppressive activity in the IgG fraction of February 22 was shown to be localized within the F(ab')2 fragment. Furthermore, the specificity of the suppressive serum factor was shown by the inability to block the binding of SRBC coupled with diazotized phosphorylcholine to anti-pneumococcal antibody. Thus, the marked reduction of rheumatoid factor activity was considered to result from anti-idiotypic antibody transiently appearing in her serum after pneumococcal bacteremia.     

Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. II. Studies of B73.1 antibody-antigen interaction on the lymphocyte membrane

In this paper, we characterize the antigen recognized by the monoclonal antibody B73.1 and the modification occurring at the membrane of the positive cells after interaction with the antibody. The B73.1-defined antigen is a protein of 50,000 to 72,000 daltons that is sensitive to pronase but not to trypsin treatment. B73.1 antibody, and its F(ab')2 fragment, directly block, at high concentrations, the binding of IgG antibody-sensitized erythrocytes to the Fc receptors (FcR) of a subpopulation of lymphocytes and neutrophils. B73.1 antibody dissociates rapidly from the positive cells, but concomitant modulation of both B73.1 antigen and FcR is induced when cells are incubated in the continuous presence of antibody or when B73.1 antibody is cross-linked at the cell membrane with an anti-mouse immunoglobulin antiserum. Reaction of lymphocytes with immune complexes also induces modulation of both FcR and B73.1 antigen, without affecting the expression of other antigens on the positive cells. The possibility that the antigen is internalized and digested by the cell after reaction with the antibody is discussed. B73.1 antibody inhibits antibody-dependent cytotoxicity mediated by lymphocytes (K cells) and neutrophils, whereas it does not affect spontaneous cytotoxicity of NK cells. These results suggest the B73.1-defined antigen might be the FcR or a structure closely related to it on K/NK cells.     

Idiotypic determinants on human T cells and modulation of human T cell responses by anti-idiotypic antibodies

The role of idiotypic anti-idiotypic interactions in the regulation of the human T cell response to tetanus toxoid (TT) antigen was examined in three subjects. Rabbit anti-idiotypic (anti-Id) antisera were raised against IgG (Fab')2 anti-TT obtained 7 to 10 days after booster immunization with TT. F(ab')2 fragments of rabbit-anti-Id IgG were used in conjunction with fluorescein-conjugated goat anti-rabbit Ig in an indirect immunofluorescence assay to determine the frequency of Id-positive cells in T cell-enriched preparations. This frequency was 24, 29, and 38 per 10,000, respectively, in the three subjects studied. Significant contribution of contaminating B cell to fluorescence-staining was ruled out by capping experiments using goat anti-human Ig (GAHIG) and by double staining experiments using rhodamine-conjugated GAHIG. Absorption of anti-Id antisera with Epstein Barr virus (EBV)-transformed B cell lines from the IgG (Fab')2 anti-TT donor, but not with EBV-B cell lines from unrelated donors, removed their reactivity with the T cells. Rabbit anti-Id IgG caused minimal proliferation (two-threefold) of T cells and had no effect on T cell proliferation in response to TT antigen when added to the cultures. Preincubation of T cells for 48 hr with rabbit anti-Id IgG (Fab')2, but not with preimmune rabbit IgG (Fab')2, resulted in the generation of antigen-specific suppressor cells that inhibited T cell proliferation in response to TT, but not in response to diphtheria toxoid (DT). These cells also inhibited the synthesis of IgG anti-TT in response to in vitro stimulation with TT antigen, but not the synthesis of IgG anti-DT in response to DT antigen. Adsorption of T cells over plates coated with rabbit anti-Id IgG (Fab')2 enhanced the proliferative response of the T cells to TT, but not to DT antigen, and enhanced the helper activity of the T cells for the in vitro synthesis of IgG anti-TT but not of IgG anti-DT antibodies. These results suggest that idiotypic-anti-idiotypic interactions play a role in the human T cell response to antigen.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

Conjugation and evaluation of 7E3 x P4B6, a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface.

A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab')2 (7E3 X P4B6) combining the GPIIb/IIIa-specific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab' moieties with the homobifunctional cross-linker bis(maleimido methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA's. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay. 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls.     

Purification of beta cells from rat islets by monoclonal antibody-fluorescence flow cytometry

Fluoresceinated monoclonal antibody plus flow cytometry was used to purify beta cells from mixed pancreatic islet endocrine cell populations. A2B5, a monoclonal antibody to a glycolipid on the surface of cells of neuroendocrine origin, was incubated with single cells dissociated from rat pancreatic islets. Antibody-bound cells were labeled with fluoresceinated goat F(ab')2 antimouse immunoglobulin and highly fluorescent cells were separated from less fluorescent cells on a Coulter EPICS IV cell sorter. Sorted cell populations were analyzed by radioimmunoassay for insulin, glucagon, and somatostatin. The highly fluorescent cell population was enriched sixfold for insulin-containing beta cells, indicating that islet beta cells are relatively enriched in A2B5 antigen and can be partially purified by this method.     

The interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. I. Analysis of the mechanism of binding

We studied the mechanism of binding of radiolabeled, monoclonal anti-H-2 antibodies to mouse spleen cells to determine the number of H-2 antigen molecules per cell. Equilibrium and kinetic data were analyzed in detail according to theoretical models developed for different modes of antibody binding. The results of binding experiments from three monoclonal IgG antibodies (36-7-5, anti-Kk; 27-11-13, anti-DbDd; and 11-4-1, anti-Kk) and their F(ab')2 and F(ab') fragments show that for the IgG and F(ab')2 from all three antibodies, the monovalently and bivalently bound states of the antibody co-exist in rapid equilibrium with one another on the cell surface, with the bivalent state predominating. We show that the relative proportions of the monovalently and bivalently bound species can be estimated from dissociation kinetics experiments, and that once the mode of antibody binding has been established, the density of H-2 determinants on the cell surface can be estimated from equilibrium-binding data. We conclude that the average numbers of H-2K and H-2D molecules on B10.A spleen cells are 5 X 10(4) and 1.1 X 10(5) molecules/cell, respectively.     

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 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.     

Antigen-antibody complexes bound to B-lymphocyte Fc gamma receptors regulate B-lymphocyte differentiation

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.