Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies

Hybridization with murine myeloma cells P3-X63-Ag8.653 of splenocytes from BALB/c mice immunized with syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 149.53, 225.28, 763.74, and TP41.2 has resulted in the formation of antiidiotypic antibody-secreting hybridomas with a frequency ranging between 1.2% and 5.2%. No marked difference was detected in the frequency of antibody secreting hybridomas in the fusions generated from mice immunized with the four anti-HMW-MAA mAb, suggesting that the idiotopes expressed by each of them display similar immunogenicity in a syngeneic combination. The number of antiidiotypic mAb that did not inhibit the binding of immunizing mAb to melanoma cells was higher than that of those that died, suggesting that idiotopes not associated with the Ag-combining site are more immunogenic than those that are. The idiotopes recognized by mAb were not detected on a large panel of anti-HLA Class I mAb, anti-HLA Class II mAb, and anti-human melanoma-associated Ag mAb. The latter included also mAb that cross-inhibit the immunizing anti-HMW-MAA mAb. The idiotopes recognized by mAb were not detected on the isolated H and L chain of the immunizing anti-HMW-MAA mAb. Cross-blocking experiments with a selected number of antiidiotypic mAb identified three distinct idiotopes on mAb 149.53, 225.28, and TP41.2 and two on mAb 763.74. Three, 5, 2, and 5 antiidiotypic mAb to idiotopes within the Ag-combining site of mAb 149.53, 225.28, 763.74, and TP41.2, respectively, were tested for their ability to induce anti-HMW-MAA antibodies. Serological and immunochemical assays detected anti-HMW-MAA antibodies only in sera from BALB/c mice immunized with mAb MK2-23. Therefore, mAb MK2-23 can be classified as beta, while the remaining 14 can be classified as gamma.     

Detection of private and recurrent idiotopes on natural anti-tubulin antibodies by monoclonal anti-idiotopic antibodies

Two monoclonal anti-idiotopic antibodies (anti-Id) were raised in mice against a human monoclonal IgA,K displaying a monospecific anti-tubulin (anti-alpha- and anti-beta-tubulin) activity. One anti-Id (IgG,K) recognized a private idiotope, TID 3.2, present only on the IgA,K immunogen, close to or within the antigen-combining site. The other anti-Id (IgM,K) recognized a recurrent idiotope, TID 7.1, outside the paratope and present in normal human and BALB/c mouse serum, on 2 of 11 polyspecific human monoclonal immunoglobulins and on 6 of 11 murine natural monoclonal auto-antibodies exhibiting a widespread anticytoskeletal protein-binding activity. Both the idiotopes were absent on two induced anti-tubulin antibodies exhibiting a monospecific anti-alpha- and anti-beta-tubulin specificity. Utilizing competitive and additivity immunoassays, we could show that the polyspecific human and mouse anticytoskeletal antibodies tested, whether bearing the TID 7.1 Id or not, appeared to compete in variable degrees for epitopes on the tubulin molecule recognized by the monoclonal IgA,K but distinct from the epitopes recognized by the induced monospecific anti-tubulin antibodies. The high incidence of the recurrent TID 7.1 idiotope in man and mouse suggests an important physiologic and perhaps regulatory function of this idiotope. Furthermore our data suggest that a restricted family of germ-line genes, highly conserved during phylogeny, may encode for these idiotope-bearing Ig molecules.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with¹²⁵iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b > IgG2a > IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab)₂ preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

In the preceding paper it was suggested that the tumour localisation of 125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced "non-specific" localisation mediated by Fc binding, since it was abolished by the use of F(ab')2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab')2 fragments, or prior "blockade" of Fc receptors by "cold competition" with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab')2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase "absolute" tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8-12-week-old hosts. A further difference was apparent in the subclass "preference" of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal anti-idiotopic antibodies against myasthenia-inducing anti-acetylcholine receptor monoclonal antibodies. Preponderance of nonparatope-directed antibodies affecting antigen binding

To analyze components of the idiotypic network in experimental autoimmune disease, we produced 17 isogeneic anti-idiotopic monoclonal antibodies (anti-Id) against two experimental autoimmune myasthenia gravis-producing anti-acetylcholine receptor (anti-AChR) monoclonal antibodies. We studied the binding of five of the anti-Id to the anti-AChR monoclonal antibodies bearing the complementary idiotopes (Id-mAb). They bound with Kd values ranging from 0.06 to 0.86 nM, values comparable to those of Id-mAb:AChR complexes (0.26 and 0.34 nM). All of the anti-Id tested moderately inhibited the binding of AChR to Id-mAb, whereas for each anti-Id, AChR either strongly inhibited anti-Id binding or had no effect on anti-Id binding. Hence, the inhibition of Id-mAb:AChR binding by anti-Id was not reciprocal with the inhibition of anti-Id:Id-mAb binding by AChR. For each anti-Id, the relative affinities of anti-Id and AChR for Id-mAb together with the lack of symmetry of inhibition by anti-Id compared to inhibition by AChR indicate that these two "ligands" are not competitive inhibitors. Consequently, anti-Id and AChR do not bind to overlapping sites on the Id-mAb, suggesting that the observed inhibition is mediated allosterically. This may be a common mechanism of anti-Id:Id binding, which would have important implications for the mechanism of anti-Id-induced suppression.     

Characterization of liver-specific expression of rat uricase using monoclonal antibodies and cloned cDNAs

Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.     

Characterization of monoclonal antibodies to Treponema pallidum

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies

The hepatocyte plasma membrane consists of three morphologically and functionally distinct domains, the sinusoidal, the lateral and the canalicular. To study the distribution of antigenic determinants among these domains, we prepared monoclonal antibodies by immunizing mice with a crude, plasma membrane-enriched liver fraction. Four monoclonal antibodies were obtained that recognized various parts of the rat hepatocyte plasma membrane when tested by indirect immunofluorescence and immunoperoxidase assay performed on formaldehyde-fixed liver tissue. Each antibody gave a different staining pattern when analyzed by light and electron microscopy. A59 exclusively labelled the part of the sinusoidal membrane facing the sinusoids. A39 mainly labelled the sinusoidal membrane. B1 mainly labelled the lateral membrane, while the labelling by B10 was almost completely limited to the canalicular membrane. Immunoblotting showed that the antibody B1 recognized an antigen of approximately 100 kilodaltons and that B10 recognized an antigen of approximately 125 to 130 kilodaltons. These antibodies allow us to distinguish the three domains of the hepatocyte plasma membrane.     

Characterization of multi-cellulases of Sporotrichum cellulophilum by using monoclonal antibodies

Cellulases of Sporotrichum cellulophilum, CII1, CII2, CII3, CII5, CIII1, CIII2, and CIV1 were purified to homogeneity by column chromatographies and preparative electrophoresis. The specific activity of CII3 was the highest (100 units/mg), while the activities of CII5, CIII1, and CII2 were low (12–17 units/mg). The molecular weight of CII2 was 19,000, while those of the others ranged from 46,000–63,000. All the cellulases were glycoproteins except for CII2. Three monoclonal antibodies (Mab CII11, Mab CII12, and MabCII13) were prepared against CII1, and Mab CIV11 against CIV1. From the immunological reactivities, it was found that CII1, CIII2, and CIV1 had a common antigenic site recognized by Mab CII11, Mab CII12, and Mab CII13, while CII12 and CVI1 had a different antigenic site that was recognized by Mab CIV11, MabCII11, MabCII12, MabCII13, and MabCIV11 did not react with CII2, CII3, CII5, and CIII1. The classification of these cellulases was discussed.     

Characterization of Leishmania donovani variant clones using anti-lipophosphoglycan monoclonal antibodies

Monoclonal antibodies directed against the lipophosphoglycan of Leishmania donovani were used to characterize two glycosylation variants of the parasite. One of the variants was found to be totally deficient in the synthesis and expression of lipophosphoglycan. The other variant synthesized lower levels (20%) of the glycoconjugate compared to wildtype cells and its lipophosphoglycan was smaller in size. The two lipophosphoglycan-deficient clones will be useful for elucidating the biosynthesis and function of the glycoconjugate.     

Characterization of mouse monoclonal antibodies to human interferon-gamma

Mouse monoclonal antibodies (mAb) to human interferon-gamma (HuIFN-gamma) were characterized. The mAbs studied--E4-18, G4-15, and SAT-1--which are all IgGl-type, reacted to all HuIFN-gamma molecular species, both glycosylated and non-glycosylated. Affinity constants calculated of E4-18 and G4-15 didn't have considerable differences for both kinds of HuIFN-gamma (1-3 x 10(8) liter/mol), but SAT-1 had a difference--a higher value (10(10) liter/mol) for the former than for the latter (8 x 10(8) liter/mol). In epitope specificity, the results suggested that E4-18 and G4-15 recognized an overlapped region remote from the region of SAT-1. Competition experiment using synthetic peptides suggested that epitope of G4-15 is around N9-26 of the HuIFN-gamma sequence. Those mAbs could be used for sandwich radioimmunoassay of HuIFN-gamma using double mAbs in two combinations, one (G4-15/E4-18) based on dimer forms of HuIFN-gamma and the other (SAT-1/E4-18) based on epitope difference. The mAbs are all neutralizing antibodies in which SAT-1 neutralized at a lower concentration than did G4-15, and at a much lower one than did E4-18. The receptor binding of HuIFN-gamma was inhibited by mAbs G4-15 and SAT-1. Efficacy of G4-15 and SAT-1 for the inhibition correspond with that for neutralization.     

Characterization of monoclonal antibodies directed against the rat neurotensin receptor NTS1

G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a variety of ligands and represent major drug targets. Despite their medical importance, detailed structural information is limited because only one GPCR has been crystallized and its structure determined. To develop tools to aid in the formation of well-ordered crystals, we generated monoclonal antibodies with high affinity to the rat neurotensin receptor. All antibodies bound to the C-terminus of the receptor, which may reflect the selection strategy used to identify high-affinity binders. Further characterization revealed that some antibodies bound to the receptor in a sodium chloride sensitive manner, but others did not. Epitope mapping revealed distinct antigenic regions within the receptor C-terminus. Tight binding of Fab fragments to the receptor was verified by size exclusion chromatography.     

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.     

Characterization of the glucagon receptor and its functional domains using monoclonal antibodies

Four monoclonal antibodies, designated 4H11, 6E10, 2C5, and 3E9 were prepared against partially purified rat hepatic glucagon receptor. These antibodies were characterized by their ability to recognize the glucagon receptor in target tissues using immunoblot and immunoprecipitation procedures. The antibodies recognized a 62-kDa receptor band in rat liver, kidney, and adipose tissue but not in lung, adrenals, and erythrocytes, indicating a high degree of specificity. These antibodies recognize different antigenic determinants; the 6E10 and 2C5 bind protein epitopes, while 4H11 and 3E9 bind carbohydrate epitopes. Furthermore, proteolytic mapping of the glucagon receptor established that monoclonal antibodies 6E10 and 2C5 recognize different domains of the receptor molecule. These antibodies were used to study the immunochemical similarities among the receptors from different species and to assess the topological location of the ligand-binding site. By combining the techniques of affinity cross-linking, proteolytic mapping, and antibody binding, we have identified the location of the glucagon-binding site near to the COOH-terminal domain of the receptor.     

Characterization of the nucleolar protein, B-36, using monoclonal antibodies

A panel of nine monoclonal antibodies has been produced against a major nuclear protein, B-36, purified from the slime mold Physarum polycephalum. B-36, a 34 kD protein biochemically similar to the major structural proteins of mammalian hnRNP particles, was previously shown to be largely associated with the nucleolus. Eight of the monoclonal antibodies are specific for B-36 protein in Physarum and at least three different epitopes are represented among these eight. Using the monoclonal antibodies B-36 has been shown to be localized exclusively to the nucleolus in actively-growing Physarum cultures. The nucleolar localization of B-36 is dependent on the presence of intact RNA, but not DNA, supporting the hypothesis that B-36 is associated with nucleolar RNA, possibly in some analogous manner to the interaction of the related proteins within heterogeneous nuclear ribonucleoproteins (hnRNP) particles. B-36 is apparently a highly conserved nucleolar protein in eukaryotes as all eight of the monoclonal antibodies specific for B-36 in Physarum are also specific for a 34.5 kD nucleolar protein in rat liver. This indicates that a minimum of three distinct epitopes are conserved in B-36 protein from slime mold to rat.