Monoclonal Antibody B72 Research Articles

Inorganic colour pigments

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311–322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of -19, -29, -39 and -59 amino acids were designed to disrupt the α-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76 kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion.

Primate antibody response to immunotoxin: serological and computer-aided analysis of epitopes on a truncated form of Pseudomonas exotoxin.

NLysPE38 is a 38-kDa derivative of Pseudomonas exotoxin (PE) in which domain Ia (amino acids 1 to 252) and part of domain Ib (365 to 380) are deleted and an 11-amino-acid N-terminal peptide is added. LMB-1 is an immunotoxin in which the monoclonal antibody B3 is coupled to NLysPE38 near its N terminus. LMB-7 is a single-chain immunotoxin in which the Fv fragment of B3 is fused to PE38. To identify the antigenic regions of PE38, 12 polyclonal serum samples from monkeys immunized with the immunotoxins LMB-1 (six monkeys) and LMB-7 (six monkeys) were tested for their reactivity to a panel of 120 synthetic, overlapping peptides representing the amino acid sequence of NLysPE38. The antibody responses to peptides were similar among the 12 serum specimens, identifying several major immunodominant B-cell epitopes. Predominant reactivity was seen in six locations: amino acids 272 to 287, 341 to 359, 504 to 516, 540 to 564, and 573 to 591 and the C-terminal amino acids 591 to 613. The sera did not react with approximately 75% of the peptides. Furthermore, a computer-aided analysis was done to predict the immunologically relevant areas and revealed the same antigenic regions defined by serum reactivity to peptides. Competition enzyme-linked immunosorbent assays and neutralization assays were performed with domain II, III, or III plus Ib of PE38 and confirmed the immunodominance of domain III. To analyze the role of specific amino acids in antibody binding, individual amino acids of PE38 with large accessible surface areas were altered by site-directed mutagenesis. These results also show that the predicted areas of immunogenicity agree with the reactivity of the anti-PE38 antibodies to peptides and to the mutants of PE.

The role of the I domain in ligand binding of the human integrin alpha 1 beta 1.

We report here the analysis of potential ligand binding domains within the human integrin alpha 1 subunit, a known collagen/laminin receptor. This integrin is effectively blocked by the mouse monoclonal antibody 1B3.1. A truncated version of the alpha 1 subunit lacking the NH2-terminal half of the extracellular domain is not recognized by monoclonal antibody 1B3.1. Furthermore, we have isolated a cDNA containing the I domain from chicken alpha 1 bearing significant homology to the human and rat alpha 1 sequences. Replacing the human I domain with its chicken counterpart led to the surface expression of a functional heterodimer with endogenous mouse beta 1 on NIH 3T3 cells. However, 1B3.1 does not bind to the chicken/human chimera, demonstrating that the human alpha 1 I domain is required for epitope recognition. Mutation of Asp253 within the I domain to alanine resulted in surface expression of an alpha beta heterodimer recognized by 1B3.1 but with markedly reduced binding to collagen IV or laminin. Since a previously reported mutation of a homologous Asp in the Mac-1 I domain has similar consequences, these results suggest a central role for the I domain in ligand recognition for all integrin alpha subunits containing this domain.

Evaluation of immunoreactivity of normal tissues from dogs, using monoclonal antibody B72.3

Summary Monoclonal antibody (MAB) B72.3, which recognizes human tumor-associated glycoprotein-72, has immunoreactivity for malignant epithelial neoplasms in human beings and dogs. To further characterize the range of immunoreactivity of MAB B72.3 in canine tissues, MAB B72.3 and 2 other tumor-associated glycoprotein-72 antibodies (MAB CC49 and CC83) were tested against a wide spectrum of normal tissues from dogs. Immunoreactivity was detected, using an avidin-biotin-complex immunoperoxidase method. Monoclonal antibody B72.3 did not stain most types of normal canine tissues, but various types of epithelial cells within the gastrointestinal and respiratory tract mucosae, salivary gland, esophagus, epididymis, uterus, thymus, hair follicle, and apocrine glands of the anal sac had variable staining with MAB B72.3. A similar range of immunoreactivity in comparable types of normal tissues was seen for MAB CC49 and CC83; however, MAB CC49, but not MAB B72.3 and CC83, stained the endothelium of capillaries and small vessels in most normal tissues. Staining of frozen and paraffin-embedded tissues was similar. In conclusion, we found that MAB B72.3, CC49, and CC83 had selected immunoreactivity for specific types of normal canine epithelial cells, especially those involved with mucin production.

Modulation and intracellular transport of CD20 and CD21 antigens induced by B1 and B2 monoclonal antibodies in RAJI and JOK-1 cells—An immunofluorescence and immunoelectron microscopy study

By fluorescence microscopy (FM), flow cytometry (FCM) and immunoelectron microscopy (IEM) we have shown that B1 and B2 monoclonal antibodies (MoAbs) were able to induce modulation of CD20 and CD21 in RAJI and JOK-1 cell lines. Redistribution and internalization of both antigens (Ags) after binding with MoAbs was readily demonstrated by FM, and by IEM CD20 and CD21 were found to be processed by the pathway of receptor-mediated endocytosis. The rate of intracellular transport varied: CD21 > CD20 and RAJI > JOK-1. Approximately 65 and 55% of CD20 and 60 and 45% of CD21 were cleared from the surface of RAJI and JOK-1 cells, respectively (FCM and IEM). These values, however, clearly exceeded those corresponding to internalization (11, 9, 24 and 16%) indicating shedding of Ag-MoAb complexes. No evidence of recycling was found. The present data support the hypothesis that the kinetics of modulation vary from one Ag to another and probably also reflect the stage of differentiation of the malignant B-cells. The results are discussed in the context of the possible usefulness of B1 and B2 MoAbs in the therapy of B-cell malignancies.

Characterization of Mouse Monoclonal Antibody B1.4 Reactive with Human Invasive Bladder Cancer and Some Other Malignant Tumors but Not with Normal Urinary Epithelium

Immunohistochemical analysis by indirect immunoperoxidase staining demonstrated that monoclonal antibody (mAb) B1.4 derived from a mouse immunized with a bladder cancer cell line EJ‐1 was reactive with a high proportion of high‐grade and invasive bladder tumors, but not with the majority of low‐grade and superficial bladder tumors, or normal urinary epithelium. Among 71 primary bladder tumors classified by pathological grading, positive stainings were observed in 1 of 34 tumors (3%) of grade 1, 8 of 20 tumors (40%) of grade 2 and 14 of 17 tumors (82%) of grade 3. When the tumors were classified by pathological staging, positive stainings were observed in only 8 of 54 (15%) superficial tumors of stages Ta and T1, but in 15 of 17 (88%) invasive tumors of stages T2 and T3. mAb B1.4 showed restricted positive stainings with normal tissues including renal glomerulus, vascular endothelium, squamous epithelium of esophagus, glandular epithelium of prostate, and epithelium of pancreatic acinar gland and minute duct, while positive stainings were observed in a range of tumor tissues other than bladder tumor. Mixed hemadsorption assays with a panel of cell cultures showed also that the antigen recognized by mAb B1.4 was expressed on a range of tumor cell lines. These findings suggest that the antigen recognized by mAb B1.4 may appear after malignant transformation, and be an indicator of malignant potential of bladder cancer.

Mutations of two lysine residues in the CDR loops of a recombinant immunotoxin that reduce its sensitivity to chemical derivatization.

B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of monoclonal antibody B3 is connected to a truncated form of Pseudomonas exotoxin. It would be desirable to use the lysine residues of the molecule for chemical modification so that it can be derivatized with poly(ethylene glycol) to achieve reduced immunogenicity or with the Bolton-Hunter reagent for biodistribution studies. We found that derivatizing lysine residues of B3(Fv)-PE38 causes a marked loss of specific target cell cytotoxicity and/or immunoreactivity. Here we show that two lysine residues in the antibody-combining region of B3(Fv)-PE38 can be replaced with arginines, with only a small loss of cytotoxicity and no change in specificity. This mutant molecule is 3-fold more resistant to inactivation by derivatization with succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) or Bolton-Hunter reagent.

Tumor‐Associated glycoprotein expression in salivary gland mucoepidermoid carcinomas: An immunohistochemical study using the monoclonal antibody B72.3

The monoclonal antibody B72.3 recognizes a mucin-like glycoprotein which is expressed in a variety of human malignancies including adenocarcinomas of the breast, colon, lung, endometrium, pancreas, and ovary. The antibody, an immunoglobulin G (IgG1) subclass monoclonal antibody, was generated from membrane-enriched fractions of metastatic mammary carcinoma. Because the antigen detected by B72.3 is expressed in a broad spectrum of human neoplasms, B72.3 is a potentially useful marker for glandular neoplasms. Despite its potential usefulness, there is only one previous comprehensive study which has examined the distribution of the tumor-associated glycoprotein (TAG-72), as detected by B72.3, in salivary neoplasms. We examined 21 mucoepidermoid carcinomas with the antibody. Twenty (95%) of the 21 carcinomas stained positively. The only carcinoma that did not stain was initially thought to be a high-grade mucoepidermoid carcinoma based on immunohistochemical staining. Further review, however, indicated that this lesion was most likely a pure squamous cell carcinoma, and the absence of TAG-72 antigen expression was helpful in this regard. Among those neoplasms which stained with B72.3, the strongest staining was seen in the low-grade carcinomas. These results suggest that B72.3 may be a useful marker for glandular differentiation in mucoepidermoid carcinomas.

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.

Retention of B-Cell-Specific Monoclonal Antibodies by Human Lymphoma Cells

AbstractThe rates of endocytosis, intracellular degradation, and cell-surface shedding of 125I-labeled monoclonal antibodies (MoAbs) HD-37 (anti-CD19), B1 (anti-CD20), MB-1 (anti-CD37), BC8 (anti-CD45), and DA4-4 (anti-μ) by B-lym-phoma cells were compared by cellular radioimmunoassay, ultrastructural autoradiography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin layer chromatography using biopsy specimens from 12 patients with non-Hodgkin’s lymphomas. 125I-BC8 was stably retained on the surface of lymphoma cells without appreciable internalization or shedding, whereas 125I-DA4-4 underwent rapid endocytosis and degradation. 12BI-B1 was not internalized or degraded by tumor cells, but rapidly dissociated from the cell surface in intact form. Moderate rates of endocytosis, intracellular metabolism, and cell-surface shedding were shown by 125I-HD37 and 125I-MB-1. The 3 patients with diffuse, small cleaved-cell lymphomas internalized and degraded antibodies more slowly than did patients with other histologic subtypes. These kinetic differences may be important in the selection of MoAbs for immunotoxin and radioimmunoconjugate therapy of B-cell malignancies.

Retention of B-cell-specific monoclonal antibodies by human lymphoma cells

The rates of endocytosis, intracellular degradation, and cell-surface shedding of 125I-labeled monoclonal antibodies (MoAbs) HD-37 (anti- CD19), B1 (anti-CD20), MB-1 (anti-CD37), BC8 (anti-CD45), and DA4–4 (anti-mu) by B-lymphoma cells were compared by cellular radioimmunoassay, ultrastructural autoradiography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin layer chromatography using biopsy specimens from 12 patients with non- Hodgkin's lymphomas. 125I-BC8 was stably retained on the surface of lymphoma cells without appreciable internalization or shedding, whereas 125I-DA4–4 underwent rapid endocytosis and degradation. 125I-B1 was not internalized or degraded by tumor cells, but rapidly dissociated from the cell surface in intact form. Moderate rates of endocytosis, intracellular metabolism, and cell-surface shedding were shown by 125I- HD37 and 125I-MB-1. The 3 patients with diffuse, small cleaved-cell lymphomas internalized and degraded antibodies more slowly than did patients with other histologic subtypes. These kinetic differences may be important in the selection of MoAbs for immunotoxin and radioimmunoconjugate therapy of B-cell malignancies.

Production and Characterization of New Murine Monoclonal Antibodies Reactive toMycobacterium tuberculosis

Murine monoclonal antibodies were raised against Mycobacterium tuberculosis (H37Rv) employing conventional hybridoma procedure. The binding characteristics of the four selected monoclonal antibodies reactive to M. tuberculosis were assessed by enzyme-linked immunosorbent assay (ELISA) using sonic extracts. The immunofluorescence test (IFT) was done using intact M. tuberculosis, 16 other mycobacterial species, and 10 bacteria of other genera. Monoclonal antibody A30 reacted strongly to M. tuberculosis both in ELISA and IFT. Monoclonal antibody A25 showed restricted cross-reactivity with mycobacterial species whereas two other monoclonal antibodies B6 and H2 appeared broadly cross-reactive. In Western blot analysis, A30 reacted with the 30-kD protein antigen, A25 with the 18- and 28-kD protein bands, H2 with the 18-kD antigen alone, and B6 with three bands of 17-19, 22, and 28 kD of M. tuberculosis H37Rv. A30 exhibited high reactivity with virulent M. tuberculosis H37Rv and a clinical South Indian Strain and minimal reactivity with avirulent M. tuberculosis H37Ra strain.

Expression of epitopes of the tumour-associated glycoprotein 72 and clinicopathological correlations in mammary carcinomas

We analysed the immunohistochemical expression pattern of the distinct carbohydrate epitopes of the TAG-72 molecule, defined by the monoclonal antibodies (MAbs) B72.3, CC-49 and CC-83, in 92 breast carcinomas of different histological type, and in other histological components identified in the mammary tissue samples studied. The results were correlated with the clinico-pathological characteristics of the tumours, and with their proliferative activity, assessed by thymidine labelling index (TLI). Expression of the TAG-72 epitopes was detected in all the tumour histotypes analysed, but patterns of immunoreactivity tended to vary in relation to type and level of differentiation. The comparative analysis of the reactivities of the three anti-TAG-72 MAbs revealed differences in their ability to recognise neoplastic lesions. MAb CC-49 reacted with the highest percentage of tumours (82%), and also tended to yield the highest percentages of immunoreactive cancer cells, while B72.3 and CC-83 reacted with lower percentages of tumours (respectively, 55 and 51%), and identified lower percentages of immunoreactive cells. High levels of expression of the three TAG-72 epitopes were detected in areas of in situ ductal carcinoma. Comparatively lower levels of immunohistochemical positivity were found in atypical epithelial hyperplasia, normal mammary epithelium and epithelium with cystic disease. TAG-72 epitope expression was correlated with prognostic parameters. The synchronous expression of the three epitopes significantly correlated with large tumour size (> 2 cm), and with high histological grade. No correlations could be demonstrated between TAG-72 phenotypes and nuclear grade, lymph node status and proliferative activity (high versus low).

An immunotoxin with increased activity and homogeneity produced by reducing the number of lysine residues in recombinant Pseudomonas exotoxin.

Pseudomonas exotoxin A (PE) is a protein composed of 613 amino acids arranged into three major, and one minor, domains. Immunotoxins (ITs) containing PE38, a mutant form of PE which lacks the cell binding domain (Ia, amino acids 1-252) and 16 amino acids from domain Ib (amino acids 365-380), are extremely potent cytotoxic agents which can cause a complete regression of various human carcinomas grown in nude mice. However, these ITs are a mixture of several different chemical forms since the coupling between the antibody and the toxin may occur between either the light or heavy chain of the antibody and one of the four primary amino groups present on the truncated toxin. To modify the toxin with heterobifunctional crosslinking reagents only at specific sites, we replaced lysines 590 and 606 with glutamines and lysine 613 with arginine (PE38QQR). We also added two different peptide sequences, each containing a lysine residue, at the N-terminus of PE38. In one of these the sequence is ANLAEEAFK ("Lys" peptide), and in the other, the sequence is LQGTKLMAEE ("NLys" peptide). The mutant toxins were coupled using a thioether linkage to monoclonal antibody B3 which recognizes an antigen present in large amounts on many human cancers. PE38QQR-containing recombinant toxins can only be linked to an antibody through the N-terminal methionine or the lysine within the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in vimentin distribution in glial cells in culture revealed with an antibody against a phosphorylated epitope

We have previously described that spatial and temporal changes in the organization of vimentin that are correlated with protein kinase C (PKC)-induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski-Treska et al. (1991) J. Neurosci. Res.29, 362–378]. The antibodies were generated to native form of vimentin-containing filaments and antibody B3 reveals a restricted immunostaining of vimentin in glial cells from human, rat and mouse origin. In the present study we show the distribution of epitope B3 analyzed by immunofluorescence within defined populations of rat glial cells. Relatively high immunoreactivity was found in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had low immunoreactivity. Although the results suggested that in Type 1 astrocytes the phosphorylated epitope is prominent, its phosphorylation was not found to be cell cycle-dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the morphological conversion of polygonal to stellate astrocytes. As expected, the immunofluorescence was increased by exposition of astrocyte cultures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC-mediated phosphorylation. In addition, the finding that the immunofluorescence of vimentin was more homogeneous in quiescent, serum-deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycloheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescence studies on cultures enriched in Type 2 astrocytes and in oligodendrocytes indicate that the epitope B3 is hypophosphorylated in glial cells of this lineage and becomes dephosphorylated after terminal differentiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation of vimentin could be related to the down regulation in vimentin expression.

Characterization of a monoclonal antibody against human prolactin receptors

Tamoxifen is the first line of therapy for most human breast cancers. It not only works through the estrogen receptor but also can directly affect the binding of prolactin to its receptor. To define this latter mechanism, the nature of the prolactin receptor needs to be clearly defined. Monoclonal antibody (MAb) B6.2, and IgG1 raised against a membrane-enriched fraction from metastatic human breast cancer cells, was as effective as polyclonal anti-prolactin receptor antibody in inhibiting the binding of prolactin to membranes from human tissue and to T47D human breast cancer cells. Control MAbs, MOPC-2I and the anti-NCA B1.1 MAb, had no effect on binding. Epidermal growth-factor receptors on these same cells were unaffected by B6.2. Prolactin-induced growth of the T47D cells was blocked by addition of B6.2 to the media while the control antibodies were without effect. Specific binding of B6.2 to the cells was completely inhibited by prolactin. Binding of both prolactin and B6.2 was inhibited by growing the T47D cells in the presence of tunicamycin A1 under conditions where protein synthesis was not affected but glycosylation of proteins was. An affinity column of B6.2 was used to purify its antigen from T47D cells. The primary purification product, a M(r) 90,000 protein, specifically bound the lactogenic hormones human prolactin, human growth hormone and ovine prolactin but not the somatogenic hormone, bovine growth hormone and was precipitated by the polyclonal anti-prolactin receptor antibody but not by control MAbs. When tryptic and V8 digests of the B6.2 antigen and purified prolactin receptors were compared, identical electrophoretic profiles were obtained. Mouse 3T3 cells, when stably transfected with the gene for the long form of the human prolactin receptor, reacted with B6.2 and polyclonal anti-prolactin receptor antibody. Parental 3T3 cells, devoid of prolactin receptors, were negative for all antibodies tested. Thus, MAb B6.2 provides a useful tool for further studies on purification and characterization of these receptors from human tissues and may provide new insights into treatment for breast cancer.

Evaluation of a new DTPA-derivative chelator: Comparative biodistribution and imaging studies of 111In-labeled B3 monoclonal antibody in athymic mice bearing human epidermoid carcinoma xenografts

Biodistribution and imaging characteristics of monoclonal antibody (MAb) B3 conjugated to either the 2-( p-isothiocyanatobenzyl)-cyclohexyl-DTPA (CHX-B) or 2-( p-isothiocyanatobenzyl)-6-methyl-DTPA (1B4M) and labeled with 111In, were evaluated in nude mice bearing A431 human epidermoid carcinoma xenografts. MAb B3, is a murine IgG1k reacting with a carbohydrate antigen abundantly expressed by most carcinomas. Both 111In-(CHX-B)-B3 and 111In-(lB4M)-B3 showed good tumor targeting with peak values observed at 72 h with 27.6 ± 7.6 and 25.4 ± 1.7% ID/g, respectively ( P > 0.05). High tumor-to-organ ratios were also observed and, confirmed by the imaging results. In particular, tumor-to-liver ratios increased from 5.0 ± 0.9 at 24 h to 9.2 ± 2.0 at 168 h for 111In-(CHX-B)-B3 and from 4.5 ± 0.6 to 8.9 ± 3.5 for 111In-(1B4M)-B3. This was mainly the result of low liver accumulation of both 111In-(CHX-B)-B3 and 111In-(lB4M)-B3, with only 2.48 ± 0.46 and 2.5 ± 0.9% ID/g at 168 h, respectively ( P > 0.05). Our findings indicate that either CHX-B or 1B4M can be successfully used for 111In-labeling of MAbs and that 111In-B3 may represent a promising radioimmunoimaging agent.

Reactivity of b72.3 with adenocarcinomas. An immunohistochemical study of 476 cases

The monoclonal antibody B72.3 has been shown to react with several common types of adenocarcinoma. However, the distribution of B72.3 reactivity in adenocarcinomas has not been well-enough defined to determine whether B72.3 immunostaining would be helpful in the diagnosis of adenocarcinoma of unknown origin. Formaldehyde fixed, paraffin-embedded tissue from 476 adenocarcinomas from a variety of primary sites were studied using B72.3 and an avidin-biotin complex immunohistochemical technique. B72.3 immunoreactivity was seen in 75-100% of the adenocarcinomas from the ovary, endocervix, endometrium, distal esophagus/stomach, colon, lung, breast, prostate, pancreas, and bile ducts. Positive reactions were unusual in thyroid carcinomas (7%) and renal cell carcinomas (11%). B72.3 reactivity was not seen in the 20 hepatocellular carcinomas or 4 adrenal cortical carcinomas studied. B72.3 reacts with a wide variety of adenocarcinomas. Immunostaining with B72.3 may be helpful in excluding hepatocellular carcinoma in certain clinical settings.

The structure of aggrecan fragments in human synovial fluid. evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis

To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA). Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphate arthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin sulfate-substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal analysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3. Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immunoreactive products were also seen, with an apparent size of > 250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the human aggrecan core protein. The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the human aggrecan core protein at the glutamate 373-alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent pattern of fragments seen on SDS-PAGE and the single predominant N-terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.

Lymphoproliferation in primary Sjögren's syndrome. Evidence of selective expansion of a B cell subset characterized by the expression of cross-reactive idiotypes.

To evaluate the possibility that lymphoproliferation in primary Sjögren's syndrome (SS) arises within a subset of B cells. A panel of monoclonal antibodies (MAb) specific for rheumatoid factor (RF)-associated cross-reactive idiotypes (CRI) and anti-V kappa and anti-VH subgroup antibodies were used to define the clonality of B lymphocytes undergoing neoplastic transformation in 5 patients with primary SS. Anti-CRI antibodies were also used to study longitudinal variations in serum paraprotein levels and in vitro regulation of IgM and IgM-RF production by peripheral blood lymphocytes. The levels of CRI, IgM, and IgM-RF were quantitated in serum and culture supernatants by enzyme-linked immunosorbent assay. Heavy and light chain isotypes and VH subgroups of the paraproteins were determined by immunoelectrophoresis, immunofixation, and Western blotting. Paraproteins from all patients expressed an epitope associated with V kappa IIIb sub-subgroup of light chains. Three of the paraproteins were cryoglobulins with RF activity, all of which expressed the V kappa IIIb-associated CRI (detected by MAb 17-109) and the VHI-associated CRI (detected by MAb G6 and G8). None of the paraproteins expressed the VHIII-associated CRI (detected by MAb B6 and D12). The CRI were consistently expressed over a period of 5-6 years. The anti-CRI and anti-subgroup antibodies substantially inhibited spontaneous production of IgM-RF and IgM by peripheral blood B lymphocytes from 3 of the SS patients. These results suggest that lymphoproliferation in primary SS is highly selective, and that the anti-CRI antibodies can be used as an aid to early diagnosis as well as for monitoring and modulating the lymphoproliferative process in primary SS.