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S M Glaser

Publications and source records attributed to S M Glaser.

15 recordsLinked to original sources
PubMed1997

Determination of the relative affinities of antibody fragments expressed in Escherichia coli by enzyme-linked immunosorbent assay.

We have previously utilized an M13 phage expression system and codon-based mutagenesis to increase the avidity of the tumor-specific antibody, BR96, 65-fold (Yelton et al., 1995, J. Immunol. 155, 1994-2004). Mutants with improved affinity were identified by screening phage-expressed antibodies on a carcinoma cell line. In this study we describe a more broadly applicable assay which permits rapid and quantitative comparison of affinities of related antibodies produced in an M13 phage expression system. BR96 variants displaying a range of affinities were expressed as soluble antibody fragments (Fabs) in the periplasmic space of bacteria and isolated from small-scale cultures grown in a 96-well format, yielding between 142 ng and 1.06 microg of Fab. Although the small-scale cultures expressed variable levels of Fab, the lower quantities were sufficient to saturate microtiter plates coated with a limiting amount of anti-human Fab antibody, resulting in the capture of uniform quantities of the Fab variants. The relative affinities of the variants were then compared by assessing binding to biotinylated antigen followed by detection with streptavidin-alkaline phosphatase conjugates. This approach permitted the direct comparison of the relative affinities of large numbers of antibody variants in a single step without multiple antibody dilutions. The assay is readily adaptable for screening phage-expressed antibody libraries against any biotinylated target and does not require purified antigen. For instance, the biotinylated BR96 antigen utilized in these studies was from a total cell extract prepared by labeling the surface of live tumor cells followed by detergent extraction. Thus, this approach may be applicable to the screening of antibody libraries against other cell surface antigens, such as transmembrane receptors, which are difficult to purify.

Alkaline Phosphatase↗
PubMed1996

A combinatorial library strategy for the rapid humanization of anticarcinoma BR96 Fab.

We have used a combinatorial mutagenesis strategy to humanize BR96, a monoclonal antibody that binds to the Lewis Y class of tumor antigens. This approach allows simultaneous assessment of hundreds of humanized variable regions to identify the molecules that best preserve affinity, thus overcoming the major drawback of current humanization procedures, the requirement to construct and analyze each humanized antibody separately. Murine residues of BR96 were mutated to human if they were solvent-exposed residues that did not participate in the formation of the antigen binding site and were not at the interface of the light and heavy chain. At positions that might be involved in binding to antigen, the choice between the murine and human residue was more difficult. Murine and human alternatives were incorporated into a combinatorial library at positions representing buried residues that might affect the structural integrity of the antigen binding site. By encoding this library of humanized BR96 Fabs in an M13 phage vector, we rapidly identified several candidates with nearly identical antigen binding, within 2-fold, of the chimeric Fab. Additional mutagenesis directed at sites suggested in the literature as potentially important for antigen binding in a similar anti-Lewis Y antibody yielded no further improvements.

Amino Acid Sequence↗
PubMed1995

Affinity maturation of the BR96 anti-carcinoma antibody by codon-based mutagenesis.

We have increased up to 65-fold the avidity of BR96, a mAb recognizing Lewis Y (Le(y))-related Ags expressed on the surface of many human carcinomas. Libraries of mutations in the complementarity-determining regions (CDRs) of BR96 were constructed in an M13 phage Fab expression vector by codon-based mutagenesis, a method that efficiently introduces large numbers and potentially all combinations of amino acid substitutions. Two mutants that improved the affinity of BR96 to tumor Ag were identified by screening the libraries on carcinoma cell lines. One mutant, M1, at position 97 (Asp to Ala) in CDR3 of the heavy chain, resulted in an 8- to 10-fold improvement in Ag binding, as assessed by ELISA. A second mutant, M2, at position 53 (Gly to Asp) in CDR2 of VH increased binding three- to fivefold. When these mutations were combined, the resulting Fab M3 was improved approximately 30-fold. An additional library was constructed in CDR1 of M1. M4, a mutation with three amino acid substitutions in CDR1, was isolated by screening the library with an enzyme conjugate of synthetic Le(y) tetrasaccharide (sLe(y)). This mutant improved BR96 Fab affinity to sLe(y) an estimated 15- to 20-fold by ELISA, and 14-fold as measured by surface plasmon resonance. The M4 IgG had 65-fold improved avidity to sLe(y) relative to the BR96 IgG. The mutants will be useful for comparison of the efficacy of Abs with different affinities for delivery of cytotoxic agents to tumor cells.

Animals↗
PubMed1993

The anti-idiotypic response by cynomolgus monkeys to humanized anti-Tac is primarily directed to complementarity-determining regions H1, H2, and L3.

The anti-ld response developed by cynomolgus monkeys to the humanized anti-Tac antibody was analyzed by using 12 humanized anti-Tac variants differing in V region structure. The majority of the monkey response was directed against idiotopes composed wholly or in part of complementarity-determining regions H1, H2, and L3. There was no detectable response directed solely to five single complementarity-determining regions examined or solely to the modified human V region framework.

Animals↗
PubMed1992

Antibody engineering by codon-based mutagenesis in a filamentous phage vector system.

A novel codon-based mutagenesis procedure is described that allows rapid and efficient modification of antibody amino acid sequences expressed as F(ab) fragments in M13. The procedure succeeded in generating a library of mutations in the complementarity-determining regions of chimeric L6, an antibody against a tumor-associated Ag. A set of anti-Id antibodies (anti-Id 1, 3, and 7) that bind near the L6 Ag-binding site served as model Ag. The goal was to select mutant antibody sequences that altered the L6 reactivity with the anti-Id in subtle ways, i.e., to eliminate the binding to one anti-Id while preserving other reactivities or to identify mutants with increased binding. A high frequency of variant M13 phage clones exhibiting altered specificity for the anti-Id were identified. Codon-based mutagenesis in conjunction with the M13 antibody expression and screening system should provide an efficient and general approach for redirecting the specificity and potentially improving the affinity of antibodies in vitro.

Amino Acid Sequence↗
PubMed1992

Application of a filamentous phage pVIII fusion protein system suitable for efficient production, screening, and mutagenesis of F(ab) antibody fragments.

We describe the application of a novel filamentous phage vector system suitable for efficient screening and production of F(ab) antibody fragments. The vector system can concurrently produce free F(ab) fragments and F(ab) displayed on the surface of M13 bacteriophage via a VHCH1-pVIII fusion protein. When expressed in a supO (nonsuppressor) strain of Escherichia coli free F(ab) can be produced. Antibody F(ab) fragments are secreted into culture medium at concentrations up to 0.3 mg/liter and conveniently subjected to detailed analysis with little or no purification. Higher concentrations of F(ab) (approximately 10 mg/liter) were found to accumulate in the periplasmic space. In this report the vector system is shown to produce correctly folded and assembled F(ab) fragments of chimeric L6, a mAb against a tumor-associated Ag expressed by many human carcinomas.

Antibodies, Monoclonal↗
PubMed1992

Dissection of the combining site in a humanized anti-Tac antibody.

The genetically engineered "humanized" anti-Tac antibody (HAT) has been shown to bind the p55 chain of the human IL-2R with an affinity close to that of the murine anti-Tac. Although the HAT molecule contains all six mouse CDR, it was not known which, and to what extent, each of the CDR contributes to Ag binding. These questions were addressed by constructing a series of variant HAT antibodies, each substituting a single HAT CDR with a heterologous CDR. The association constants of the variant HAT antibodies to p55 were determined by competitive binding analysis. We find that CDR 1 and 3 of the H chain and CDR 3 of the L chain are essential for maintaining binding. The remaining three CDR appear to be involved to a lesser degree.

Amino Acid Sequence↗
PubMed1990

A synthetic presequence reversibly inhibits protein import into yeast mitochondria.

We show that a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence blocked import of pre-subunit IV into yeast mitochondria. The 22-residue peptide pL4-(1-22) did not alter the electrical potential across the mitochondrial inner membrane (the delta psi). Inhibition of import was reversible and could be overcome by the addition of increased amounts of precursor. Two other peptides, pL4-(1-16) and pL4-(1-23), which correspond to, respectively, the N-terminal 16 and 23 residues of the same presequence, also blocked import of pre-subunit IV. However, pL4-(1-16) was a much weaker inhibitor of import, while the inhibitory effect of pL4-(1-23) was due to its ability to completely collapse the delta psi. pL4-(1-22) seems to be a general inhibitor of mitochondrial import, in that it also blocked uptake of several other proteins. These included the precursors of the yeast proteins cytochrome c oxidase subunit Va, the F1-ATPase beta subunit, mitochondrial malate dehydrogenase, and the ATP/ADP carrier. In addition, uptake of two non-yeast precursor proteins (human ornithine transcarbamylase and a cytochrome oxidase subunit IV-dihydrofolate reductase fusion), was also blocked by the peptide. Subsequent studies revealed that pL4-(1-22) did not block the initial recognition or binding of proteins to mitochondria. Rather, our results suggest that the peptide acts at a subsequent translocation step which is common to the import pathways of many different precursor proteins.

Amino Acid Sequence↗
PubMed1990

Localization of a synthetic presequence that blocks protein import into mitochondria.

In the accompanying paper (Glaser, S. M., and Cumsky, M. G. (1990) J. Biol. Chem. 265, 8808-8816) we demonstrated that pL4-(1-22), a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence, blocked protein import into mitochondria. Import inhibition was reversible and occurred at a step subsequent to the initial recognition and binding of precursor proteins to the mitochondrial surface. In the present work we have studied the nature of the association between the peptide and mitochondria, as well as determined its intramitochondrial location. We found that pL4-(1-22) was imported into mitochondria in a manner that was dependent upon the delta psi and that the majority of the mitochondrially associated peptide was in the membrane fraction. Density gradient analysis of total membranes indicated that pL4-(1-22) cofractionated with the inner membrane, although the possibility that it was present in both membranes could not be ruled out. It appeared to be inserted within the bilayer since it could not be extracted with salts, chaotropic agents, or high pH. We observed a steady decrease in the amount of pL4-(1-22) found within peptide-treated mitochondria over time. Coincident with this decrease was an increase in the ability of those mitochondria to import and process precursor proteins, suggesting that the peptide was ultimately turned over. The results presented here correlate well with those of the accompanying paper. Together they suggest that pL4-(1-22) blocks import at the level of the mitochondrial membranes, although the exact nature of the import block is not yet clear.

Antibodies↗
PubMed1990

Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix.

We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient. We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays. We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein. The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se. We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein. Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.

Amino Acid Sequence↗
PubMed1988

Functional analysis of mitochondrial protein import in yeast.

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.

Amino Acid Sequence↗
PubMed1985

SL12 murine T-lymphoma: a new model for tumor cell heterogeneity.

It has been observed that subclones from the spontaneous murine AKR/J T-lymphoma cell line SL12 with similar in vitro growth characteristics exhibit stable differences in tumorigenicity. The cell line is composed of at least three distinct cloned cell types that are highly, moderately, or poorly tumorigenic in syngeneic host animals. When healthy, young, syngeneic host animals were given iv injections with the same number of viable growth phase cells, each cloned cell type had a different tumor incidence, latent period, and pattern of tumor spread. The unusual stability of the cloned cell lines is shown by a similar incidence, latency, and spread of the tumors when studied after more than 1 year of continuous in vitro culture. The SL12 clones also differ in several phenotypic characteristics commonly used to classify thymocyte maturation, e.g., a) the expression of three of seven surface antigens examined, b) the cellular response to glucocorticoid hormone, and c) the expression of terminal deoxynucleotidyl transferase.

Animals↗