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Biomedical subjects

M Better

Publications and source records attributed to M Better.

At least 19 recordsLinked to original sources
PubMed2000

Production of antifungal recombinant peptides in Escherichia coli.

Antifungal peptides derived from the human bactericidal/permeability-increasing protein (BPI) were produced in Escherichia coli as fusion proteins with human BoneD. Bacterial cultures transformed with the gene encoding the fusion protein were grown to a high cell density (OD(600)>100), and induced with L-arabinose to initiate product expression. Fusion protein accumulated into cytoplasmic inclusion bodies and recombinant peptide was released from BoneD by acid hydrolysis at an engineered aspartyl-prolyl dipeptide linker. Acid hydrolysis of purified inclusion bodies at pH <2.6 followed Arrhenius kinetics and did not require prior inclusion body solubilization in detergents or denaturants. Surprisingly, at pH <2.6 and 85 degrees C, cell lysis and aspartyl-prolyl hydrolysis with concomitant peptide release occurred simultaneously. Bacterial cultures were, therefore, adjusted to approximately pH 2.6 with HCl directly in the bioreactor and incubated at elevated temperature. Peptide, which is soluble in the aqueous acidic environment, was separated from the insoluble material and purified using column separation techniques. Recombinant peptide was separated from the hydrolyzed bioreactor culture with >76% recovery and a final peptide purity of >97%. Antifungal peptide prepared by recombinant and solid phase synthesis methods demonstrated similar activity against Candida sp. in a broth microdilution assay.

Amino Acid Sequence↗
PubMed1996

T-cell-targeted immunofusion proteins from E. coli.

Antibodies and antibody domains are ideal agents for targeting the surface of cells, and fusion proteins between cell-targeting domains and cytotoxic proteins may be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking the humanized Fab, F(ab')2, or single-chain antibody form of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. To maximize the product yield and simplify the production process, each fusion protein was linked to a bacterial signal sequence for expression in E. coli as a secreted protein. More than 30 fusion genes were assembled with antibody domains and gelonin in various physical orientations. Each immunofusion accumulated in the bacterial culture supernatant in a properly folded, active form. Bacteria transformed with each fusion gene were then grown in a fermentor, and product was recovered from the cell-free fermentation broth by column chromatography. All of the immunofusion proteins were purified by a single process and each was tested for cytotoxicity toward antigen-positive human cells. A compact cGMP fermentation area was built to manufacture these fusion proteins. Our integrated approach to microbial protein production, including molecular genetics, bacterial fermentation, and initial isolation, is described in detail.

Animals↗
PubMed1995

T cell-targeted immunofusion proteins from Escherichia coli.

Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.

Adult↗
PubMed1994

Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties.

We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates.

Amino Acid Sequence↗
PubMed1994

Chimeric immunoglobulin light chains are secreted at different levels: influence of framework-1 amino acids.

Immunoglobulin light chain proteins are generally thought to be readily secreted without their corresponding heavy chains; non-secreted light chains have been viewed as aberrant forms. We have re-examined this assumption by expressing chimeric mouse-human light chains constructed for 12 mouse antibodies (mouse variable regions fused to a human kappa light chain constant region) in Sp2/0 and CHO cells. Five of the 12 light chains were either poorly secreted or not secreted at all. There was approximately a five-fold difference in the levels of secreted light chain between the highest poor secretor and the lowest good secretor. All of these light chains formed functional chimeric IgGs, which were secreted at similar levels, when co-expressed with their respective chimeric mouse-human heavy chains (mouse variable regions fused to a human gamma-1 heavy chain constant region). The influence of variable region amino acids on light chain secretion was examined by replacing the Framework-1 region of three poorly-secreted chimeric light chains with that of a readily-secreted light chain. For two of the light chains, secretion levels increased approximately 30- and 100-fold relative to that of the unmodified light chains. Comparison of the Framework-I amino acid sequence of the poorly- and readily-secreted light chains revealed an asparagine (N) and proline (P) at positions 11 and 12, respectively of these poorly-secreted light chains and a leucine (L) and serine (S) in the same region for some of the readily secreted light chains. Alteration of the NP to LS for one of the poorly-secreted light chains resulted in an approximately seven-fold increase in light chain secretion over that of the native form of the poorly-secreted light chain. We conclude from these studies that poor secretion can be a naturally occurring state for normal light chains and that amino acids within Framework-1 contribute to poor secretion for some of the light chains.

Amino Acid Sequence↗
PubMed1994

Human-engineered monoclonal antibodies retain full specific binding activity by preserving non-CDR complementarity-modulating residues.

Humanization of murine monoclonal antibodies for human therapy has commonly been achieved by complementarity-determining region (CDR) grafting, in which murine CDR loops are grafted onto human framework regions. Difficulties with that method have revealed the importance of certain framework residues in determining both the 3-D structure of CDR loops and the overall affinity of the molecule for its specific ligand. In the general model of structure-function relationships presented here, each amino acid position in the variable region is classified according to the benefit of achieving a more human-like antibody versus the risk of decreasing or abolishing specific binding affinity. Substitutions of human residues at low-risk positions (exposed to solvent but not contributing to antigen binding or antibody structure) are likely to decrease immunogenicity with little or no effect on binding affinity. Changes at high-risk positions (directly involved in antigen binding, CDR stabilization or internal packing) are avoided to preserve the biological activity of the antibody. Moderate-risk changes are made with caution. This model has been tested experimentally using H65, an anti-CD5 murine monoclonal antibody, whose binding activity had been greatly reduced by two previous attempts at humanization by conventional CDR grafting. The new 'human-engineered' H65 antibody containing 20 low-risk human consensus substitutions (expressed as either IgG or Fab) retains the full binding avidity of parental murine and chimeric H65 antibodies. A human-engineered antibody with an additional 14 moderate-risk substitutions has unexpectedly enhanced avidity (3- to 7-fold). This method is generally applicable to the design of other human-engineered antibodies with therapeutic potential.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a gene encoding gelonin, a ribosome-inactivating protein from Gelonium multiflorum.

A cDNA copy of the gel gene, encoding gelonin (Gel), has been cloned from the seeds of the Asian plant, Gelonium multiflorum. Gel is a type-I ribosome-inactivating protein which has been produced in Escherichia coli as a secreted protein under the transcriptional control of the Salmonella typhimurium araB promoter and linked to the pectate lyase (pelB) leader sequence from Erwinia carotovora. Recombinant, soluble Gel (re-Gel) can be recovered from the E. coli culture supernatant at a yield of greater than 1 mg/ml, and it inhibits protein synthesis in vitro to the same extent as the native protein isolated from plant seeds.

Amino Acid Sequence↗
PubMed1993

Potent anti-CD5 ricin A chain immunoconjugates from bacterially produced Fab' and F(ab')2.

We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30). This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines. An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography. An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab'. Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG. An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30. The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane. These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.

Amino Acid Sequence↗
PubMed1993

Efficacy of anti-CD5 F(ab')2 and Fab' immunoconjugates in human peripheral blood lymphocyte-reconstituted severe combined immunodeficient mice.

A human peripheral blood lymphocyte-reconstituted severe combined immunodeficient (hu-PBL-SCID) mouse model was used to compare in vivo efficacy of immunoconjugates directed against the CD5 antigen present on human T-cells. Four anti-CD5 immunoconjugates were tested, composed of chimeric human-mouse (cH65) F(ab')2 or Fab' fragments chemically linked to recombinant gelonin (rGEL) or the 30,000 M(r) glycoform of ricin A chain (RTA30). Immunoconjugate treatment was initiated approximately 3 weeks after PBL transplantation and consisted of five consecutive daily bolus i.v. injections. Efficacy was subsequently assessed by quantitation of human T-cells in spleens, blood and peritoneal lavage fluid using 3-color flow cytometry. cH65 F(ab')2- and cH65 Fab'-rGEL conjugates were essentially equally effective at depleting human T-cells from SCID mouse tissues, suggesting that bivalent binding is not required for efficacy when rGEL is the cytotoxic moiety. Treatment with unconjugated F(ab')2, unconjugated Fab' or a Fab-rGEL immunoconjugate of irrelevant binding specificity did not result in a significant depletion of T-cells, demonstrating that the cytotoxic moiety and a relevant human T-cell binding moiety are both required for efficacy. In contrast to the results observed with the rGEL conjugates, cH65 Fab'-RTA30 was not as effective as cH65 F(ab')2-RTA30 in depleting human T-cells from SCID mouse tissues. This paralleled in vitro findings in a human PBMC cytotoxicity assay, which demonstrated that cH65 Fab'-RTA30 was 17-fold less potent than cH65 F(ab')2-RTA30 and approximately 50-fold less potent than the rGEL conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Activity of recombinant mitogillin and mitogillin immunoconjugates.

A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.

Adult↗
PubMed1991

Preferential T-cell receptor beta-chain variable gene use in myelin basic protein-reactive T-cell clones from patients with multiple sclerosis.

Multiple sclerosis is an autoimmune disease in which T lymphocytes reactive to myelin basic protein (BP) could play a central role. T cells specific for BP were cloned from the blood of multiple sclerosis patients and normal individuals, and expression of T-cell receptor variable region genes was analyzed. A remarkable bias for use of beta-chain variable region (V beta) 5.2 and, to a lesser extent, V beta 6.1 was seen among BP-specific clones from patients but not from controls. The preferential use of V beta 5.2 for BP recognition did not reflect altered expression of this V beta in the peripheral repertoire. Interestingly, shared V beta 5.2 usage was apparent for clones specific for different BP determinants, even when derived from the same individual. The concurrent demonstration by others (J. R. Oksenberg, M. A. Panzara, A. B. Begovich, H. Erlich, R. Murray, M. Sherritt, S. Stuart, C. C. Bernard, and L. Steinman, personal communication) that T cells within demyelinating areas of multiple sclerosis brains preferentially express V beta 5.2 and V beta 6.1 suggests that the BP-specific clones derived from blood may be relevant to disease pathogenesis. These findings may have important implications for the treatment of multiple sclerosis.

Base Sequence↗
PubMed1991

Chimeric mouse-human anti-carcinoma antibodies that mediate different anti-tumor cell biological activities.

Two chimeric mouse-human antibodies, ING-1 (IgG1, kappa) and ING-2 (IgG1, lambda), have been constructed starting from anticarcinoma mouse hybridomas. These antibodies bind to different tumor-associated antigens which are present on human breast carcinoma cell lines at 10(5)-10(6) antigens/cell; ING-1 binds to a 40-kD membrane glycoprotein, while ING-2 binds to a glycoprotein of greater than 300 kD. In competitive binding experiments, both chimeric antibodies have identical binding activity to the parental mouse antibodies. The antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytolysis (CDC) activities of these antibodies were studied on carcinoma target cell lines. ING-1 mediates potent ADCC, but ING-2 had undetectable or very weak ADCC activity. ING-2 ADCC activity was significantly reduced by the addition of human serum, but ING-1 ADCC was unaffected. Neither ING-1 nor ING-2 mediated CDC of breast carcinoma cell lines, but ING-1 mediated CDC of a colon carcinoma cell line. ING-1 antibody-antigen complexes are stable on the target cell surface for at least 2 hours, while much of bound ING-2 is lost from the surface of cells due to internalization or shedding. The activities of these antibodies confirm that the target antigen plays an important role in the biological effector functions triggered by cell-surface-bound antibodies. Both of these chimeric antibodies are candidates for further study as immunoconjugates for cancer diagnosis or therapy, and the unconjugated ING-1 antibody has promise for cancer therapy due to its potent activation of ADCC.

Animals↗
PubMed1988

Escherichia coli secretion of an active chimeric antibody fragment.

A chimeric mouse-human Fab protein that binds specifically to the human carcinoma cell line C3347 has been expressed and secreted from Escherichia coli. This molecule, which contains functionally assembled kappa and Fd proteins, binds as effectively to sites on the surface of C3347 cells as Fab fragments prepared proteolytically from whole chimeric or mouse antibody. The production in Escherichia coli of foreign heterodimeric protein reagents, such as Fab, should prove useful in the management of human disease.

Amino Acid Sequence↗
PubMed1988

Secretion of functional antibody and Fab fragment from yeast cells.

We have constructed yeast strains that secrete functional mouse-human chimeric antibody and its Fab fragment into the culture medium. For chimeric whole antibody, cDNA copies of the chimeric light-chain and heavy-chain genes of an anti-tumor antibody were inserted into vectors containing the yeast phosphoglycerate kinase promoter, invertase signal sequence, and phosphoglycerate kinase polyadenylylation signal. Simultaneous expression of these genes in yeast resulted in secretion of properly folded and assembled chimeric antibody that bound to target cancer cells. Yeast chimeric antibody exhibited antibody-dependent cellular cytotoxicity activity but not complement-dependent cytotoxicity activity. For production of Fab fragments, a truncated heavy-chain (Fd) gene was created by introducing a stop codon near the codon for the amino acid at which papain digestion occurs. Simultaneous expression of the resulting chimeric Fd and light-chain genes in yeast resulted in secretion of properly folded and assembled Fab fragment that bound to target cancer cells.

Amino Acid Sequence↗
PubMed1985

Electron-microscopic examination of the binding of a large RNA polymerase III transcription factor to a tRNA gene.

A Saccharomyces cerevisiae RNA polymerase III transcription factor was previously shown to bind stably to tRNA genes. This transcription factor has been further purified on the basis of its large size and its binding to a S. cerevisiae tRNALeu3 gene has been examined by electron microscopy. Site-specific binding of the factor to the tRNALeu3 gene sharply bends the DNA.

Centrifugation, Density Gradient↗
PubMed1985

Deletion analysis of Rhizobium meliloti symbiotic promoters.

Previous examination of DNA sequences located 5' to Rhizobium meliloti nif transcription units has shown that extensive sequence homology exists among them. Here we have examined these reiterated sequences for their role in symbiotic gene regulation. Promoter deletion analysis has shown that although an extensive upstream DNA sequence 9160 bp) is required for full heterologous activation of the R. meliloti nifHDK promoter by the Klebsiella pneumoniae nifA protein in Escherichia coli, this region is not required for expression of R. Meliloti nif promoters in root nodules from plants grown under greenhouse conditions. In addition, a minimum functional symbiotic promoter sequence is defined, and a DNA sequence difference affecting regulation of two symbiotic promoters is discussed.

Bacterial Proteins↗