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J R Couto

Publications and source records attributed to J R Couto.

At least 19 recordsLinked to original sources
PubMed1997

Lactadherin (formerly BA46), a membrane-associated glycoprotein expressed in human milk and breast carcinomas, promotes Arg-Gly-Asp (RGD)-dependent cell adhesion.

Lactadherin, a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and expressed in human breast carcinomas. Previously, we have shown that the mature protein, formerly known as BA46, has three domains: an epidermal growth factor (EGF)-like domain containing an Arg-Gly-Asp (RGD) cell adhesion sequence and C1 and C2 domains similar to those found in coagulation factors V and VIII. An alignment of lactadherin with its bovine (MGP57/53) and murine (MFG-E8) homologs shows that the RGD sequence has been conserved during evolution, suggesting that the RGD sequence is not fortuitous. We demonstrate that lactadherin purified using Triton X-114 phase partitioning promotes RGD-dependent cell attachment of green monkey kidney cells (MA104), mouse fibroblast cells (3T3-L1), and breast carcinoma cells (ELL-G). A lactadherin-specific monoclonal antibody, Mc3, inhibits attachment to purified lactadherin, suggesting that contaminants in the purification are not responsible for binding. In addition, the anti-integrin alpha(v)beta3 monoclonal antibody LM609 inhibits cell attachment of MA104 cells to lactadherin. These results demonstrate that lactadherin promotes RGD-dependent cell adhesion via integrins. Denaturation of lactadherin with heat and reducing conditions diminished cell attachment, suggesting that optimal cell attachment to RGD is dependent on the structural presentation of the sequence.

3T3 Cells↗
PubMed1997

Biodistribution of anti-breast mucin HuBrE3-derived inverted Fabs (IFabs) in nude mice carrying MX-1 xenografts.

In radioimmunotherapy, the long circulation times of antibody radioconjugates correlate with high relative radiation doses to nontumor tissues. Tumor/normal tissue ratios can be significantly improved by using targeting molecules with shorter circulation times. IFabs are multimers of VH-CH1-linker-VK-CK monomers. The lack of the Fc region in IFabs should lead to circulation times that are shorter than those of IgG molecules. The monomers assemble into disulfide-bond-stabilized multimers, 90% of which are 100 kDa dimers (IFab2). IFab2s should not be rapidly eliminated through kidney filtration because their molecular weight is above the threshold for renal passage. We report the first experimental in vivo tests for 125I-IFab radioconjugates derived from a humanized version of the anti-breast mucin monoclonal antibody BrE-3. Biodistributions are reported for athymic nude mice carrying human mammary tumor MX-1 xenografts. The T1/2 beta's for the different tissues ranged from 13.3 h for blood to 19.9 h for tumor. Therefore, IFab radioconjugates cleared the body with a rate comparable to that of F(ab')2 fragments. Except for stomach, tumor/nontumor dose ratios were significantly better for IFabs than for the parent antibody (BrE-3)4 days after injection.

Animals↗
PubMed1996

Cloning and sequence analysis of human butyrophilin reveals a potential receptor function.

Human butyrophilin was cloned and sequenced from a human breast cDNA library. The derived amino acid sequence shows 84% sequence identity and identical domain arrangements with the previously reported bovine sequence. Sequence analysis reveals an immunoglobulin constant (IgC) domain that was not previously identified in the bovine sequence. The extracellular domain composition of butyrophilin suggests a cell surface receptor function.

Amino Acid Sequence↗
PubMed1996

Inverted Fab2s (IFab2s): engineering and expression of novel, dimeric molecules, with a molecular weight of 100 000.

The IFab2 molecule described is expressed by a single gene construct that encodes the VH-CH1 and the Vk-Ck modules of a humanized version of the anti-breast cancer antibody BrE-3, joined via a short peptide linker. It can be shown that this basic genetic construct can lead to the expression of monomers, dimers (IFab2s) and other multimer species, depending on the length of the linker. A 21-residue linker promotes the principal formation of the desired dimer IFab2 molecules. It was also shown that in binding competition assays, purified IFab2 completely retained the affinity and specificity of the original monoclonal antibody.

Amino Acid Sequence↗
PubMed1996

Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain.

The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation.

Amino Acid Sequence↗
PubMed1996

Cloning and expression of cDNAs encoding the variable domains of the antibreast carcinoma antibody Mc5.

Mc5, a murine monoclonal antibody that binds to human breast epithelial mucin (BEM), has been shown to be a promising reagent in the diagnosis of breast cancer. We have cloned cDNAs encoding both variable regions of Mc5 (VL and VH) as well as the CL and CH1 constant regions. Mc5 is an IgG1, kappa antibody. We have constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine VH and VL-encoding cDNAs into plasmids encoding human constant domains), and expressed it in SP2/0-Ag14 mouse myeloma cells. The affinity of chimeric Mc5 (chMc5) for BEM is 4.4 x 10(8) M-1. Mc5 binds BEM with an affinity constant of 2.8 x 10(8) M-1. Purified chMc5 and purified Mc5 gave similar competition curves when tested against either 125I-labeled Mc5 or 125I-labeled chMc5 for binding to BEM in a competition radioimmunodetection format. Additionally, chMc5 used in breast carcinoma tissue staining stained as well as the original Mc5.

Amino Acid Sequence↗
PubMed1995

Selection of tumor-specific epitopes on target antigens for radioimmunotherapy of breast cancer.

Evidence is presented for two different breast epithelial antigens that some epitopes have greater tumor specificity and are more effective targets for radioimmunotherapy than others. The two antigens, which are major components of the human milk fat globule membrane, are breast mucin and a M(r) 46,000 glycoprotein (BA46). Of five monoclonal antibodies (Mc5, Mc1, BrE-1, BrE-2, and BrE-3) against breast mucin, all recognize overlapping amino acid epitopes on the tandem repeat domain. However, each have unique and different tissue and tumor specificities and unique epitope structures on the fully glycosylated breast mucin. In preclinical studies, radioimmunoconjugates of all five monoclonal antibodies inhibit growth of transplantable breast tumors in immunodeficient mice. In human clinical trials, radioiodinated Mc5 was very poor in localizing breast tumor metastases. On the other hand, 111In-labeled BrE-3 imaged almost 90% of breast tumors and showed promise in radioimmunotherapy when labeled with 90Y. The failure of Mc5 in clinical trials may be partly attributed to the high levels of its epitope on circulating mucin compared to the epitope of BrE-3. The Mc5 binding affinity increased significantly with glycosylation, while the BrE-3 epitope was masked by glycosylation. The BA46 glycoprotein is a breast tumor-associated membrane antigen containing an NH2-terminal, epidermal growth factor-like domain into which a cell adhesion sequence (RGD) is inserted and a COOH-terminal domain with homology to the phospholipid binding C1/C2 domain of coagulation factors V and VIII. It promotes cell attachment in an RGD-dependent manner. Monoclonal antibody Mc8, which binds to the C2-like domain, is only moderately effective in experimental radioimmunotherapy, while Mc3, which binds an epitope in the EGF-like RGD domain, was highly effective in destroying breast tumors in nude mice. With 90Y-labeled Mc3, 6 of 7 mice are cured of the tumors. These results indicate that by selecting appropriate monoclonal antibodies, a normal antigen can be used as a target for radioimmunotherapy.

3T3 Cells↗
PubMed1995

Biological activity of two humanized antibodies against two different breast cancer antigens and comparison to their original murine forms.

We have humanized two monoclonal antibodies (MoAbs), hu-BrE-3 and hu-Mc3, that are bound to two different antigens of the breast epithelial cell. They bind to the breast epithelial mucin (M(r) 400,000) and the BA46 antigen (M(r) 46,000). They could participate in a joint radioimmunotherapy strategy administering repeated or fractionated dosages, where increased irradiation could be delivered by their simultaneous administration. Both antibodies, hu-BrE-3 and hu-Mc3, had similar reactivity to their antigens and similar binding affinity as those of their original murine forms. However, because humanized MoAbs could have different pharmacokinetic and radioimmunotherapeutic characteristics than their original murine forms, the experimental biodistribution in vivo of both of these two humanized anti-breast tumor MoAbs was compared to their original murine forms. Biodistributions in immunodeficient mice grafted with transplantable human breast tumors, both after radioiodination and 111In labeling via 1-p-isothiocyanatobenzyl-methyl-diethylene-triaminepenta-ace tic acid (MXDTPA), demonstrated comparable tumor:normal tissue ratios for the humanized and murine forms. In radioimmunotherapy, the humanized forms for both MoAbs showed also similar tumoricidal activity as that of the original murine MoAbs. These results show that the new humanized forms are amenable to conjugation and radioisotope labeling without loss of biological activity. Furthermore, they demonstrate that these engineered molecules kept intact, both qualitatively and quantitatively, their binding ability, pharmacokinetics, and radioimmunotherapeutic characteristics after the humanization process.

Animals↗
PubMed1995

Designing human consensus antibodies with minimal positional templates.

A humanized antibody retains from the original murine antibody the variable region amino acid residues that are required for antigen binding. These generally include the grafted complementarity determining regions, as well as a few key framework residues. Although the remainder of the framework sequences are imported from a human antibody, they nevertheless differ at a few positions from the human consensus sequences. These atypical residues, which arose by somatic mutation during the affinity maturation of the chosen human antibody, could elicit an immune response in some of the patients receiving the humanized antibody. Thus, ideally one should, instead, choose human consensus frameworks for humanizing murine antibodies. Because there is a different consensus sequence for each of the subclasses of variable light and heavy chains, a method is needed to choose the most appropriate one. We are developing a minimal positional template for such a purpose. A minimal positional template indicates which positions in the variable region frameworks are absolutely required for maintaining the integrity of the binding domains. Therefore, to choose a human framework for humanization, one screens the available human consensus sequences for those that are most similar to the original murine sequence, specifically at the positions indicated by the template. In the subsequent humanization protocol, one then retains all of the murine residues found in the positions indicated in the template while humanizing the residues found at all other positions. A conservative positional template has been applied to the humanizations of the antibreast epithelial mucin antibodies BrE-3 and KC4-G3 without loss of binding affinity. Now we are using progressive cycles of computer modeling and laboratory testing to develop a minimal template. The first of such cycles produced template B, which has been used successfully in the humanization of the antibreast epithelial antigen BA46 antibody Mc3. This prompted us to design template C, which further approaches the desired minimal template. Future constructs will test the validity of this template as well as the validity of this novel humanization approach.

Amino Acid Sequence↗
PubMed1995

Anti-BA46 monoclonal antibody Mc3: humanization using a novel positional consensus and in vivo and in vitro characterization.

Mc3 is a murine mAb that is highly effective in treating breast tumors in experimental radioimmunotherapy. Mc3 binds to BA46, a 46-kDa glycoprotein of the human milk fat globule membrane that is also expressed in breast carcinoma cells. We cloned and sequenced cDNAs encoding the variable regions of Mc3 and constructed an IgG1, kappa human/mouse chimeric antibody. We then humanized the variable regions of Mc3 using a positional consensus method and retaining residues that might either contact the complementarity-determining regions or the opposite chain. This positional consensus is novel in that it does not include residues with buried side chains. Humanized Mc3 retained full binding affinity, and fully competes with murine Mc3 for antigen binding. Humanized and murine 131I-labeled Mc3 behaved identically in athymic nu/nu mice biodistribution studies. The tumor uptake levels for both antibodies increased over a period of 4 days within a range of 13-20% of the injected dose per g with extremely favorable tumor:normal ratios. Also, a single therapeutic dose of 131I-labeled humanized Mc3 in the same animal model reduced the average tumor size and produced one of five cures while in the uninjected control tumor growth continued unabated. We believe that these results justify the implementation of Phase I human clinical trials for imaging and radioimmunotherapy of breast cancer.

Amino Acid Sequence↗
PubMed1994

Humanization of KC4G3, an anti-human carcinoma antibody.

We have previously constructed a chimeric version of KC4G3, a murine antibody that reacts with several human epithelial cancers and binds to the human breast epithelial mucin. We have now successfully humanized KC4G3 using positional consensus data, previously compiled after examining several other antibody structures, listing residues in the VH and V kappa frameworks that could influence antigen binding. We have previously showed that a fraction of the kappa chains of murine and chimeric KC4G3 migrates abnormally on SDS-PAGE most likely due to N-linked glycosylation in V kappa. The glycosylation signal has now been removed from V kappa, as a consequence of humanization. As expected, the humanized kappa chain migrates normally on SDS-PAGE. We detected no significant differences either in the affinities (1.6 x 10(9) M-1 vs. 1.4 x 10(9) M-1, respectively) or in the ability to compete for antigen binding, between the murine and the humanized antibodies. The humanized version is an IgG1, kappa immunoglobulin produced by mouse myeloma SP2/0-Ag14 cells and is designated HuKC4v2. The HuKC4v2 frameworks conform to the V kappa II and VHIII human consensus in all but six positions in V kappa and three positions in VH.

Amino Acid Sequence↗
PubMed1993

Cloning of cDNAs encoding the variable domains of antibody BrE-3 and construction of a chimeric antibody.

BrE-3 is an IgG1,kappa murine monoclonal antibody that binds to human breast epithelial mucin and that has been shown to be promising for imaging and treatment of breast cancer. We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of BrE-3. VL belongs to group II and resulted from a V kappa-J kappa 1 fusion. VH belongs to group IIIc and arose from a V-D-JH3 non-conservative fusion which left little or nothing of the original D minigene. Thus, the third VH CDR contains only 4 amino-acids. We constructed an IgG1,kappa human/mouse chimeric antibody (by joining the murine variable domains to human constant domains) and expressed it in SP2/0 myeloma cells. This chimeric monoclonal antibody stains breast carcinoma tissue sections by the ABC immunoperoxidase method. Its affinity for the BrE-3 antigen is 2.68 x 10(8) M-1, which, considering the experimental error, is indistinguishable from the affinity of the original murine antibody (3.75 x 10(8) M-1). The VL and VH domains alone are respectively 73%, and 63% identical to the human V kappa II and VHIII consensus sequences. If the CDRs are excluded, these numbers become respectively 82% and 80%. Therefore, we expect the reported chimeric BrE-3 to be considerably less immunogenic to humans than the original murine antibody, while retaining the original binding properties.

Amino Acid Sequence↗
PubMed1993

Cloning of cDNAs encoding the variable domains of antibody KC4G3 and construction of a chimeric antibody.

We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of monoclonal antibody KC4G3. VL belongs to group II and resulted from a V kappa-J kappa 2 recombination. VH belongs to group IIId and arose from a V-D9-JH3 recombination. The VL and VH frameworks are respectively 84% and 83% identical to the corresponding VL and VH human consensus frameworks. The deduced amino acid sequence of VL contains an asparagine-linked glycosylation site in framework 3 (N74 I75 S76). We have determined that a large fraction of the light chains are indeed glycolysated. We constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine KC4G3 Fv-encoding cDNAs into plasmids encoding a human IgG1, kappa Fc domain) and expressed it in SP2/0-Ag14 mouse myeloma cells. This chimeric monoclonal antibody is designated ChiKC4. We have determined that the murine monoclonal antibody KC4G3 binds the human breast mucin with an affinity constant of 1.1 x 10(9) M-1. ChiKC4 binds the same antigen with an affinity constant of 1.2 x 10(9) M-1. ChiKC4 binds the carcinoma tissue sections by the ABC immunoperoxidase method in an identical manner as does KC4G3.

Amino Acid Sequence↗
PubMed1990

A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing.

We previously described a dominant suppressor of the splicing defect conferred by an A----C intron branchpoint mutation in S. cerevisiae. Suppression occurs by increasing the frequency with which the mutant branchpoint is utilized. We have now cloned the genomic region encoding the prp16-1 suppressor function and have demonstrated that PRP16 is essential for viability. A 1071 amino acid open reading frame contains sequence motifs characteristic of an NTP binding fold and further similarities to a superfamily of proteins that includes members with demonstrated RNA-dependent ATPase activity. A single nucleotide change necessary to confer the prp16-1 suppressor phenotype results in a Tyr----Asp substitution near the "A site" consensus for NTP binding proteins. We propose that PRP16 is an excellent candidate for mediating one of the many ATP-requiring steps of spliceosome assembly and that accuracy of branchpoint recognition may be coupled to ATP binding and/or hydrolysis.

Adenosine Triphosphatases↗
PubMed1987

A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation.

Splicing of introns from Saccharomyces cerevisiae pre-mRNA requires the conserved sequence TACTAAC; the 3'-most A residue is utilized as the site of branch formation. We showed previously that the transcript from an actin-HIS4 gene fusion containing the mutation TACTAAC to TACTACC (designated C259) is spliced inefficiently, thereby preventing growth on the histidine precursor histidinol. By selecting for growth on histidinol, we have identified a mutant in which the splicing of the C259 transcript is increased fourfold; splicing of other mutated introns is not significantly improved. The mutant locus encodes a trans-acting suppressor. A single mutation, rna16-1, is sufficient for suppression; however, suppression is maximized in heterozygous diploids containing both rna16-1 and the wild-type allele RNA16. In addition, wild-type pre-mRNAs (and lariat intermediates) accumulate in rna16-1 cells. We propose that the RNA16 locus encodes a component of the splicing machinery.

Genes, Fungal↗
PubMed1984

Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway.

The yeast Saccharomyces cerevisiae temperature-sensitive lethal mutant alg1-1, has been previously shown to lack the activity necessary for the addition of the first mannose residue in the synthesis of lipid-linked precursor oligosaccharide. The gene ALG1 has been cloned by complementation of the temperature-sensitive mutation alg1-1 with a total genomic DNA library. The original DNA fragment isolated was 11,300 base pairs and has been subcloned to a 1,500-base pair fragment which is still capable of complementing alg1-1. The gene ALG1 has been mapped on chromosome II at a distance of 2.1 map units from LYS2. The ALG1 gene product has been shown to catalyze the transfer of a mannosyl residue from GDP-mannose to the lipid-linked acceptor GlcNAc2, yielding Man beta 1-4GlcNAc2-lipid, in lysates from Escherichia coli transformants. This result proves that ALG1 is the structural gene for the first mannosyltransferase in lipid-linked oligosaccharide assembly.

Asparagine↗
PubMed1982

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly.

The formation of N-glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

Asparagine↗