Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Conservative and variable regions of homologous snake phospholipases A2 sequences: Prediction of the taxonspecific peptides structure

Homologous amino acid sequences of phospholipases A₂ (PLA₂) of snakes belonging to the families Elapidae, Viperidae, and Colubridae were considered in order to study the conservative and variable regions location. The PLA₂ sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair similarity matrix. Results of the intergroup comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown that the results of the comparison between two phylogenetic groups of snake PLA₂ did not much depend on the number of each group representatives and did not markedly change if one of the groups was represented by the single sequence. The knowledge of the number and location of conservative and variable regions and their dependence on the phylogenetic relations between compared taxa may be used to predict a synthetic peptide structure to obtain specific antibodies against PLA₂ of one of these taxons. Such prediction is possible if there is a specific region conservative for one taxon but variable for two of them.     

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of¹²⁵I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of¹²⁵I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.     

Cytofluorometric analysis of anti-lymphocyte antibodies in AIDS

Abstract Anti-lymphocyte antibodies (ALA) have been detected in the plasma of 53.8% of HIV-positive patients tested (CD4/CD8 ratios: mean 0.265; range 0.01 to 0.5) using analytical continuous-flow cytofluorometry. IgG from the AIDS plasma was seen to bind to normal PBL in 53.8% of cases (14/26). In double labelling experiments CD4 + lymphocytes, CD8 + lymphocytes, and B lymphocytes were all bound by the ALA, but monocytes were not bound. Pre-adsorption of the diluted AIDS plasma onto an excess of mouse spleen cells did not remove lymphocyte binding activity. No evidence was found for preferential binding to phytohaemagglutinin-stimulated lymphocytes.
ALA could not be detected in the plasma of normal subjects, patients with acute renal failure undergoing renal dialysis, or patients with high levels of circulating immune complexes.     

Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

A hemocyte surface membrane marker (BGH₁) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH₁ epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata. Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH₁^?, compared to a prevalence of 10% BGH₁⁺ cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH₁⁺ cells were morphologically distinguishable from BGH₁^? cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH₁^? hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH₁ epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH₁ surface epitope. There was no apparent age-dependent expression of the BGH₁ determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH₁⁺ and BGH₁^? subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH₁^? hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH₁⁺ cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH₁^? hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.     

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies

Summary Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.