Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions

A chimeric human-mouse anti-T lymphocyte mAb (CHT2; SDZ 214-380) has been constructed by cloning the variable region exons of both the L and H chains from the murine hybridoma RFT2 which have CD7 specificity and reactivity with a 40-kDa Ag. The variable L chain exon was joined to the human C kappa, and the variable H chain exon was joined to the human IgG1 region exon encoding the human allotype nGlm(z), nGlm(a). The gene constructs were introduced by electroporation into SP2/0, a non-Ig-producing murine myeloma. The identical tissue reactivity of the newly made CHT2 and the original murine RFT2 mAb (CD7) was confirmed by blocking experiments as well as by immunohistology and flow cytometry. Because this new mAb may have clinical use, the CD7 Ag expression of T lineage cells has also been quantitated in double and triple immunofluorescence assays in combinations with mAb to restricted forms of leukocyte common Ag that designate unprimed (CD45R+) and primed T lymphocyte populations (UCHL1+). CHT2 shows very strong reactivity with large thymic blast cells and cortical thymocytes from which T-ALL originates. Strong staining is seen on CD45R+ unprimed "virgin" T lymphocytes, whereas the expression on UCHL1+ primed "memory" cell types is weaker unless these cells are reactivated by mitogens or Ag. Thus CHT2 may spare a substantial population of resting memory T cells which is relevant to its potential therapeutic use. In addition the chimeric antibody had a greater in vitro antibody dependent cytotoxicity and a prolonged half-life (4.2 to 5.0 days) in Rhesus monkeys.     

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions.

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.     

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Interleukin 21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4⁺ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.Key words: interleukin 21, IL-21, mAb, human Ig transgenic mice, autoimmunity     

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.     

Epitope mapping and characterization of monoclonal antibodies to human protein C

Six monoclonal antibodies specific to human protein C were characterized. Epitopes of these antibodies were determined on isolated proteolytic peptides of protein C by immunological methods. Three antibodies bound light chain of protein C: PC01 bound the γ-carboxyglutamic acid domain calcium-dependently, while PC02 and PC08 bound the first epidermal growth factor-like domain in calcium-dependent and independent manners, respectively. The other three antibodies bound the heavy chain of protein C: PC13 bound activation peptide, PC04 recognized the activation site and PC09 bound the region close to a disulfide bond connecting light and heavy chains. Activation of protein C with thrombin-thrombomodulin complex was inhibited strongly by PC04 and moderately by PC08, PC09 and PC13. PC04 and PC13 may directly block the activation site. On the other hand, epitopes of PC08 and PC09 may be involved in interaction between protein C and thrombin-thrombomodulin complex, or locate close to activation site on the tertiary structure of protein C. Anticlotting activity of protein C was inhibited strongly by PC01 and moderately by PC02, PC08 and PC09, while amidolytic activity was inhibited only by PC09. The epitopes described here may constitute part of protein-C-specific sites, which are important for the function of protein C.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants.

To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Epitope regions in the heavy chain of Clostridium botulinum type E neurotoxin recognized by monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) were previously established against the heavy chain (Hc) of botulinum type E neurotoxin in BALB/c mice immunized with the type E toxoid. Five MAbs (LE15-5, LE34-6, EK19-7, EK21-4, and AE27-9) showed toxin-neutralizing activity in mice. Two of the five MAbs, EK19-7 and EK21-4, recognized the regions located at amino acid positions 731 to 787 and 811 to 897, respectively. One of the remaining three antibodies (LE34-6) reacted with the amino acid sequence VIKAIN, at amino acid positions 663 to 668, closed by the ion channel-forming domain. It is suggested that the ion channel-forming domain may also be associated with the blocking of acetylcholine release. Furthermore, the amino acid sequence YLTHMRD within 30 residues of the C-terminal region of the Hc component seemed to be recognized by LE15-5. It has been reported that the binding domain of the type E toxin is located on the C-terminal half of the Hc component. Therefore, the neutralizing activity of LE15-5 antibody may be attributed to its ability to block the binding of neurotoxin to the receptor of target cells.     

C-terminal-specific monoclonal antibodies against the human red cell glucose transporter. Epitope localization with synthetic peptides

Mice were immunized with human red cell glucose transporter for production of monoclonal antibodies. Four peptides were synthesized that correspond to relatively hydrophilic segments of the human HepG2 glucose transporter (Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W. J., Lienhard, G.E., and Lodish, H.F. (1985) Science 229, 941-945), including a C-terminal segment. After identification of hybridomas that were positive for the red cell glucose transporter, enzyme-linked immunosorbent assays were done with the synthetic peptides in solution to detect peptide-binding monoclonals. The very hydrophilic C-terminal peptide 478-492 (P2), but no other peptide, gave strong and selective inhibition of antibody binding to the glucose transporter. Two C-terminal-specific monoclonal antibodies were selected. The binding of these two antibodies to immobilized inside-out vesicles of human red cell membranes could be inhibited with the peptide P2. The antibodies did not react with right-side-out vesicles. The binding of these C-terminal-specific antibodies to the glucose transporter, to immobilized vesicles, and to the peptide P2 was enhanced by the presence of the peptide 218-232 (P1), although the peptide P1 alone showed no reaction with these antibodies. This suggests that the C terminus and the segment 218-232 of the red cell glucose transporter are exposed at the cytoplasmic face of the membrane and interact in the transporter. The C-terminal-specific monoclonal antibodies reacted strongly in Western blotting with the human red cell glucose transporter.     

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.     

Antigenic determinants of the constant region of human immunoglobin kappa-chain detected by monoclonal antibodies

Ten different monoclonal antibody (monAB) preparations reacting with human IgL chains of the kappa type have been obtained. Nine of the monAB interacted with the kappa-chain C domain, whereas only one monAB reacted with the V domain. It has been determined that monAB against the C domain react with three different epitopes. One epitope is expressed on intact Ig molecules as well as on isolated kappa-chains, whereas the other two epitopes are found only on isolated kappa-chains. The expression of these epitopes in 40 different myeloma kappa-chain preparations belonging to four various subgroups was studied. The level of this C domain epitope expression has been shown to depend on the variable subgroups of kappa-chains indicating a close association between V and C domains. This association leads to the alteration of antigenic activity of some C domain epitopes. The alterations are thought to be local because, as a rule, they involve only one of the three epitopes.     

Epitope analysis of antibodies in Japanese to human cytomegalovirus phosphoprotein 150 with synthetic peptides

Serological detection of antibodies specific to human cytomegalovirus (HCMV) is not reliable because the assay uses the whole HCMV protein fraction. Antigenic materials composed of well-characterized viral proteins are being tried for serodiagnosis in Europe. Epitopes of antibodies to HCMV phosphoprotein 150 (pp150) encoded by UL32 in Japanese individuals were investigated for comparison with the results in Europe. The major epitopes on amino acid residues 496 to 652 of HCMV pp150 were identified and the detection of antibodies with an enzyme-linked immunosorbent assay (ELISA) of synthetic peptides against the main epitopes was established. Fifteen seropositive and five seronegative serum samples for the epitope mapping and 131 seropositive and 50 seronegative samples for ELISA were investigated. Overlapping 15-mer peptides moving by two amino acids through V496-H652 were synthesized. The main epitope regions were V508-D530, L526-Q544, S536-D554, T616-G634, S624-P642, and L632-H652. When each peptide was conjugated with bovine serum albumin for ELISA, 80.9% of the seropositive samples were judged to be positive. The results of this study are the same as those for European sera, so the antigenic materials developed in Europe might be used to replace the whole HCMV protein fraction in Japanese.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.     

Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system

Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14～28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38～52 kDa in BP; 38～60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.     

Antigenically important regions of the relaxin molecule: studies with monoclonal antibodies

A panel of 22 monoclonal antibodies was produced against porcine relaxin. Antibodies were shown to be specific for porcine relaxin and appeared to recognize antigenic determinants associated with at least two different areas of the molecular surface, one of which was the N-terminal region of the A chain.     

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody 92-2

A human-mouse hybridoma clone 92-2 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was established. To detect antibody-binding sequences on Ara h1, we synthesized a series of peptides of the Ara h1 protein on a multi-pin apparatus for the pin-peptide ELISA. The 92-2 human monoclonal antibody was found to recognize a sequence of GREGEQEWGTPGSHVREETS. Further analysis with shorter pin-peptides with eight amino acid-long showed that the sequence of QEWGTPGS was an essential linear sequence of this epitope. When the QEW part of the sequence was replaced by alanine, the 92-2 monoclonal antibody did not bind to the substituted peptide, showing that those amino acids play an important role in the binding of the 92-2 monoclonal antibody.     

Immunochemical and functional analysis of Ia-like antigens on human monocyte-macrophages with monoclonal antibodies

The distribution, structural profile and functional properties of Ia-like antigens synthesized by human monocyte-macrophages have been analyzed using monoclonal antibodies to common determinants of these antigens. Up to 45 and 70%- of monocyte-macrophages isolated from the fluid of blisters induced with cantharidin and from peripheral blood, respectively, react with monoclonal antibodies to human Ia-like antigens. The level of Ia-like antigens on monocytes-macrophages appears to be similar to that on cultured B lymphoid cells. Monoclonal antibodies to common determinants of Ia-like antigens specifically block antigen presentation by monocyte-macrophages to T lymphocytes as well as proliferative response of T lymphocytes to autologous and allogeneic monocytes-macrophages. These results indicate that common determinants of Ia-like antigens play a role in the interaction of monocytes-macrophages with T lymphocytes.     

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.     

Immunohistochemical analysis of human lung cancers with anti-ras p21 monoclonal antibodies

The expression of ras oncogene product p21 in human malignant pleurisy and primary lung cancer was studied immunocyto-histochemically with monoclonal antibodies (MoAbs) rp-28 and rp-35 against ras p21. In pleural effusion cells, cancer cells revealed more intensively positive reaction with MoAb rp-35 than with MoAb rp-28, especially in the plasma membrane, and no positive reaction was obtained in any kind of inflammation cells with the exception of faintly positive reaction in the cytoplasm of macrophages. In primary lung cancers, well or moderately differentiated adenocarcinoma tissues showed higher reactivity with MoAb rp-28 than those of poorly differentiated adenocarcinoma or any other histological subtype of lung cancer. With MoAb rp-35, intensively positive reaction was obtained in most of cases with all different histological subtypes of lung cancer. The staining in cancer cells was usually localized intensively to the plasma membrane and weakly to the cytoplasm with both MoAbs. Normal bronchial epithelial and glandular tissues showed only cytoplasmic staining. These two MoAbs, especially MoAb rp-35, may be useful in clinicopathological applications for the diagnosis of malignant pleurisy and primary lung cancer.