Preparation and characterization of murine monoclonal antibodies that express both cold agglutinin and cryoglobulin activities

To obtain murine cold agglutinin (CA) and cryoglobulin antibodies, BALB/c mice were hyperimmunized with heat-killed type XIV Streptococcus pneumoniae. The spleen cells of these mice were fused with either the P3 NS1/Ag4. 1 or P3 X63/Ag.653 cell line. Several stable hybridomas were obtained that produced monoclonal antibodies (Mab) that reacted with rabbit and human erythrocytes only at temperatures below 37 degrees C. Three of these Mab were also cryoglobulins, as evidenced by their insolubility at reduced temperature. All of the antibodies studied were IgM(k) and reacted with purified type XIV S. pneumoniae polysaccharide at room temperature. With one exception, all antibodies were specific for N-acetyl-lactosamine, the immunodominant sugar residue expressed on type XIV polysaccharide. Inhibition experiments demonstrated that both CA activity and cryoprecipitation were inhibited by the same sugar compounds in the same order of efficiency. The data presented strongly suggest these CA antibodies are cross-reactive members of a S. pneumoniae-specific population. Cryoprecipitation persisted in antibodies purified under conditions that would exclude the presence of trapped serum antigens. It is therefore proposed that the cryoprecipitation observed is a result of the interactions of the antibody combining sites with carbohydrate residues of adjacent antibody molecules.     

Human large granular lymphocytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass

We have evaluated the ability of murine monoclonal antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by human lymphoid cells. Purified large granular lymphocytes (LGL) and interleukin 2-dependent cloned LGL lines having a CD2+/CD16+/CD3- phenotype were tested as effector cells in an ADCC assay by using a family of IgG isotype switch variant anti-Thy-1.1 antibodies against 51Cr-labeled Thy-1.1+ murine SL2 thymoma target cells, a system in which human cells have no spontaneous cytotoxicity. Cytotoxicity was greatest when using the IgG3, followed in rank order by the IgG2a and IgG2b. No cytotoxicity was observed with the IgG1 antibody. Because the antigen-binding regions of the antibodies are identical, the differences in cytotoxicity directly reflect the relative affinity of LGL Fc receptors for each antibody isotype.     

A new murine IgG1 anti-Tn monoclonal antibody with in vivo anti-tumor activity

The Tn antigen (GalNAc α-O-Ser/Thr) is heterogeneously synthesized by a variety of tumors and contains an epitope defined by lectins and antibodies as a cluster of αGalNAc carbohydrates synthesized within a peptide sequence, which is rich in serine and/or threonine. The Tn antigen has been utilized as a target in vaccine experiments and also used as a biomarker for prognosis of different cancer forms. In this paper, we present a new monoclonal antibody, GOD3-2C4, with the clear hallmarks of an anti-Tn antibody. It was generated through somatic cell hybridization after immunization of a mouse with a tumor cell line and a Tn carrying mucin. The antibody recognizes synthetic Tn antigen and binds breast, colon, lung, ovarian and pancreas cancer. The GOD3-2C4 antibody has antibody-dependent cellular cytotoxicity activity against Jurkat cells in vitro, and for the first time, it can be shown that an anti-Tn antibody has a significant in vivo effect on a human cancer cell line grown as a xenograft in severe combined immunodeficiency mice.     

Production and biological activity of murine monoclonal antibodies against GnRH

Immunization against GnRH represents a nonsurgical means of castrating domestic species. However, clear target antibody titres for bioactivity have not been established. The aims of this study were to produce characterized anti-GnRH monoclonal antibodies and to determine a threshold titre. Three murine monoclonals were developed which produced IgG2a class immunoglobulins and bound 50% I(125)-GnRH at a 10(6) to 10(7) dilution. The antibodies were specific to GnRH, showed a strong affinity (Ka values from 1.99 to 2.60 x 10(10) litres/mole), and were directed towards the amino terminus. In female mice all 3 antibody clones interrupted ovarian cyclicity, causing an extension in diestrus followed by prolonged estrus/metestrus (12 to 30 d). Throughout this period circulating titres were greater than 15% I(125)-GnRH binding at a 5 x 10(4) dilution. In male mice, immunization with 0.2 ml of ascites significantly reduced testes (P < 0.05), epididymides (P < 0.001) and seminal vesicle (P < 0.01) weights. A 0.1 ml dose (61.4 +/- 18.6% binding at a 10(6) dilution) was ineffective. A serial dilution study indicated that a titre of 50% binding at 2 x 10(6) dilution (antigen binding capacity of 268 +/- 35 ng/ml) was required to completely block GnRH activity. This is a higher tire than threshold levels determined previously. Identification of factors determining the titre required for bioactivity is needed.     

Production and characterization of murine monoclonal antibodies specific for baboon IgG heavy and light chain epitopes

We have generated a panel of murine monoclonal antibodies (MAbs) that recognize baboon IgG epitopes. The reactivity of the MAbs with IgG from other primate species was also examined. Specificity for IgG heavy (H) or light (L) chain epitopes was determined by Western blot analysis. The H chain-specific MAbs were analyzed for IgG subclass specificity and the L chain-specific MAbs for reactivity with baboon IgM and polymeric sIgA. Finally, an ELISA was developed to demonstrate the utility of the MAbs in analysis of humoral immune responses in baboons.     

Passive immunization against murine malaria with an IgG3 monoclonal antibody

Spleen cells of BALB/c mice that were immune to the 17X strain of P. yoelii were fused with P3X63Ag8 myeloma cells. Two hundred fifty-three of 1053 hybrid cells produced antibodies reactive with disrupted 17X parasites in a solid phase radioimmunoassay. One of these antibodies, McAb 302, reacted with the merozoites of the 17X (nonlethal) and 17XL (lethal) variants of P. yoelii. Of greater significance, McAb 302 passively protected mice against challenge infection with the lethal variant. Mice treated with this antibody before infection developed low-grade parasitemia (less than 0.3%) of short duration when challenged with P. yoelii 17XL . In contrast, control mice that had been untreated or injected with ascites fluid lacking McAb 302 uniformly died with fulminating malaria upon challenge with the same parasite. In other experiments, McAb 302 was shown capable of controlling blood parasite levels when administered to mice with patent P. yoelii 17XL infections. Although all control mice died, mice protected with a single dose of McAb 302 ultimately cleared their infections. Regardless of how passive immunization was performed, mice given McAb 302 were resistant to subsequent challenge with P. yoelii 17XL , indicating they had developed significant immunity during their initial controlled infections. McAb 302 also showed pronounced passive protective activity against the nonlethal 17X strain of P. yoelii, which is a parasite of reticulocytes. The protection afforded by McAb 302 was specific, because mice passively immunized with this antibody died when challenged with the unrelated P. vinckei. McAb 302 was shown to possess the IgG3 isotype and precipitated a 230-kd protein plus several smaller polypeptides from metabolically labeled parasite antigen preparation derived from both variants of P. yoelii. It did not react with similar preparations of other murine plasmodial species.     

Production and characterization of hybrid monoclonal antibodies with IgG1/IgG3 double isotype

In order to obtain bispecific monoclonal antibodies, two hybridoma cell lines were fused: E13 producing an IgG1 anti-Cytomegalovirus, and 70B12 producing an IgG3 anti-Chlamydia. This experimental model was chosen to test the possibility of association between gamma 1 gamma 3 heavy chains. This association was shown by using an ELISA.     

Cell-mediated lysis of tumor targets directed by murine monoclonal antibodies of IgM and all IgG isotypes

Seven murine monoclonal antibodies to antigens expressed on T lymphoma targets were tested for directing antibody-dependent cellular cytotoxicity (ADCC). Peptone-induced peritoneal exudate macrophages, the LPS-stimulated RAW264.10 cell line, and human blood nonadherent mononuclear leukocytes were used as effector cells. All six IgG monoclonals tested, representing the four murine IgG isotypes and directed against four antigens (Thy-1.2, H-2k, Ly-2.1, Ly-9.2), were all active in ADCC. In contrast, an IgM anti-Thy-1.2 showed no activity despite very high C-cytotoxic titers. Thus, there does not seem to be any restriction among IgG classes for directing ADCC to tumor targets mediated by murine macrophages or human K cells.     

Biological activity in the human system of isotype variants of oligosaccharide-Y-specific murine monoclonal antibodies

Summary The capacity of isotype variants of BR55-2, an anti-tumor monoclonal antibody directed against Y oligosaccharide, to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in the human system was evaluated using freshly isolated peripheral blood mononuclear cells, lymphocytes, monocytes and complement. The ADCC activities of the BR55-2 IgG3 isotype and its switch variants (IgG1, IgG2b, and IgG2a) with human monocytes were high for all isotypes, whereas the activity of all isotypes was lower with freshly isolated lymphocytes, IgG1 being the least effective. The CDC on the other hand was strong with IgG3, IgG2b and IgG2a and negative with the IgG1 variant. The IgG3 and IgG2a isotypes were selected for further development. Their strong ADCC and CDC activity against mammary carcinoma, colon carcinoma and small-cell lung tumor cell lines was confirmed quantitatively using proteins highly purified from tissue-culture supernatants. Significant variations in ADCC and CDC competence was observed among human donors.This work was supported by grants CA 10 815, CA 25 874 and CA 21124 from the National Institutes of Health     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (V_H) of a human beta₂ glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (V_L) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4V_H and paired V_L in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of V_H and V_L sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived V_L sequences were expressed with IS4V_H and the V_H of an anti-dsDNA antibody, B3. Six variants of IS4V_H, containing different patterns of arginine residues in CDR3, were paired with B3V_L and IS4V_L. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4V_H CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, V_L containing B3V_L CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in V_L CDR2 or V_L CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Detection of immunoglobulin heavy chain IgG3 polymorphism in wild mice with xenogeneic monoclonal antibodies

We have generated four xenogeneic rat antimouse IgG₃ monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG₃ molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG₃ isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG₃ molecules). An IgG₃ variant was also isolated from hybridomas derived from Mus spretus.Abbreviations Igh-C immunoglobulin heavy chain constant region - PVC polyvinyl chloride - RIA radioimmune assay - ELISA enzyme linked immunosorbent assay     

应用双抗体夹心ELISA法定量检测人源破伤风毒素单克隆抗体中的IgG含量

采用山羊抗人IgG作为包被抗体,辣根过氧化物酶标记的山羊抗人IgG作为标记抗体,建立一种双抗体夹心法用于定量检测人源破伤风毒素单克隆抗体的IgG含量。以纯化的IgG作标准,用平行线法测得亲和层析纯化的人源破伤风毒素单克隆抗体G2的含量为0．512μg／ml,与分光光度法测得的结果基本一致。因而样品检测采用纯化G2作参考标准,制作标准曲线,测定了已知样品和未知样品的抗体含量。结果表明本法重复性好,特异性强,可定量测定培养及纯化样品中人源单克隆抗体的含量。     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

Spectroscopic and kinetic analysis of a monoclonal IgG cryoglobulin. Effect of mild reduction on cryoprecipitation.

The precipitation of a monoclonal IgG2 crystalline cryoglobulin (WEB) is shown to be highly dependent on temperature and concentration. Below a critical concentration of 0.6 mg/mL there is no cryoprecipitation. The kinetics of the aggregation exhibits a concentration-dependent lag time. This evidence suggests that a nucleation event is important in the precipitation. Circular dichroism (CD) was used to investigate the conformational properties of the protein. At a low concentration (0.12 or 0.15 mg/mL), no detectable spectral changes in the far- and near-UV range were noted between 40 and 3 degrees C. However, at higher concentrations (1.21 mg/mL), a small and rapid CD change was observed in the 250-280-nm region at 3 degrees C. This indicates an intermolecular interaction that precedes the precipitation. Cryoprecipitation of WEB was also shown to be dependent on maintenance of intact interchain disulfide bonds. Only one or two interchain disulfides need be cleaved to abolish cryocrystallization and to significantly diminish the CD change at 3 degrees C. The evidence is consistent with the formation of an initial intermediate that involves interactions near the disulfide bonds in the hinge region of the cryoimmunoglobulin. In this model, cleavage of these disulfides prevents this interaction and abolishes cryoprecipitation.     

Cytolytic activity of murine anti-dog lymphoma monoclonal antibodies with canine effector cells and complement

Peripheral blood leukocytes (PBL), nonadherent lymphocytes, and adherent monocytes separated from freshly isolated blood of 15 dogs were analyzed for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in combination with murine anti-tumor monoclonal antibodies (MAbs). Canine monocytes isolated from most donors by adherence to gelatin-fibronectin-coated plastic surface presented high ADCC activity against the canine lymphoma 17-71 tumor cell line in combination with antilymphoma MAbs 231 (IgG2a) and 234-2a (IgG2a). Canine lymphocytes generally showed lower ADCC activity than total PBL or monocytes. Canine PBL effector cells showed high ADCC activity against the human colorectal carcinoma SW948 cell line using the Y-6-specific MAb isotype switch variants 55-2 IgG3, 55-2 IgG1, 55-2 IgG2b, and 55-2 IgG2a. Analysis of the role of murine MAb isotypes on ADCC activity against tumors by canine cells using anti-human tumor class-switch variant MAbs and a panel of anti-canine lymphoma MAbs of different IgG subclass revealed the highest ADCC activity with MAbs of the IgG2a and IgG3 subclasses. IgG2a antilymphoma MAbs were also able to lyse tumor cells in complement-dependent cytotoxicity (CDC) assay. These results suggest the potential value of MAbs of IgG3 and IgG2a subclasses in immunotherapy against canine lymphoma.     

Human immune response to multiple injections of murine monoclonal IgG

Murine monoclonal antibody infusions in humans should induce a human anti-mouse immunoglobulin (mIgG) immune response, especially if multiple infusions over an extended period of time are necessary for therapeutic efficacy. We have administered multiple infusions of the murine monoclonal antibody T101 to patients with cutaneous T cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL). Five of 10 CTCL patients, compared with zero of six CLL patients, developed antibodies to mIgG. In those CTCL patients who did not demonstrate anti-mIgG antibodies, we were unable to correlate the lack of response to any of a large number of clinical parameters. Anti-mIgG antibodies were of both the mu and gamma isotypes and were detectable 14 days after the first infusion. Multiple infusions were associated with elevated titers. The anti-idiotypic portion of the anti-mIgG titer steadily increased with each infusion until eventually, in one patient receiving eight weekly infusions, well over one-half the serum anti-mIgG recognized only T101 and not four other murine IgG2AK antibodies tested. To increase our confidence in these findings, four separate assay systems were used to make these determinations. The identification of anti-idiotype antibodies as the dominant species of the immune response to multiple infusions of murine monoclonal antibody has major implications for future work with monoclonal antibodies. Although it has been suggested that human monoclonal antibodies would obviate an immune response, our work implies that such antibodies might still induce anti-idiotype antibodies if multiple infusions are administered.     

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.