"Beyond beating heart surgery": heartstring device protects against perioperative neurological events.

Objective: : There is a growing body of evidence favoring off-pump coronary artery bypass surgery (OPCAB) over traditional coronary artery bypass surgery (CABG) with cardiopulmonary bypass as a method for reducing perioperative neurologic events. Aortic manipulation, whether with OPCAB or coronary artery bypass surgery with cardiopulmonary bypass, is strongly linked with adverse neurologic outcomes. Although the aortic "no-touch" technique has merit, most cardiac surgeons are reluctant to base entire myocardial revascularization exclusively on mammary pedicles.

Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization.

We describe here the selection of ultra-potent anti-respiratory syncytial virus (RSV) antibodies for preventing RSV infection. A large number of antibody variants derived from Synagis (palivizumab), an anti-RSV monoclonal antibody that targets RSV F protein, were generated by a directed evolution approach that allowed convenient manipulation of the binding kinetics. Palivizumab variants with about 100-fold slower dissociation rates or with fivefold faster association rates were identified and tested for their ability to neutralize virus in a microneutralization assay.

Reinfusion of aspirated pericardial blood during CPB. Part II. Laparotomy sponges are hazardous parts of the CPB circuit?

Usually, cotton laparotomy sponges are discarded when they become blood soaked. During bypass surgery, however, they are often wrung out into the pericardial sac and the contents of the sac are aspirated into the cardiopulmonary bypass (CPB) circuit. After cardiopulmonary bypass, many patients give evidence of mental confusion, excessive bleeding, and systemic inflammatory response syndrome (SIRS).

Optimization of protein therapeutics by directed evolution.

Directed evolution is a broadly applicable technology platform that is ideally suited to address the need for protein optimization and to fully exploit the therapeutic potential of biologics. The approach takes advantage of the remarkable structural and functional plasticity of proteins and permits the rapid remodeling of biologics into new entities with improved functions. The ability to ameliorate virtually any characteristic of a protein can translate into significant clinical benefits, including decreased immunogenicity, higher potency, greater efficacy and improved safety profile, and can considerably increase the probability of successfully developing and commercializing biotherapeutics.

Cloning, isolation and characterization of human tumor in situ monoclonal antibodies.

A bacterially expressed human antibody (Ab) library (diversity approximately 10(5)) was generated from tumor-infiltrating B lymphocytes present in tissue isolated from a colon tumor. Immunoglobulin (IgG) heavy and light chain variable regions were amplified without isolating or enriching B cells, cloned into a phage-expression vector, and soluble antigen-binding fragment (Fabs) from >10(5) members of the library were screened rapidly by two distinct and complementary methodologies. In the first approach, soluble Fabs were screened by enzyme-linked immunosorbent assay (ELISA) on tumor cell monolayers.

In vitro affinity maturation of human IgM antibodies reactive with tumor-associated antigens.

Human lymphocytes secreting tumor cell-specific IgM antibodies were enriched in vitro following the stimulation of allogeneic human splenocytes from nontumor-bearing donors with cytostatic tumor cells or tumor cell plasma membrane fractions. The antibodies were generally of the IgM class and displayed low intrinsic affinity (K(d) > 100 nM). Nonetheless, the avidity arising from multivalent binding sites permitted the identification of multiple monoclonal antibodies (MAbs) displaying specificity for cultured tumor cells.

A pilot trial of Vitaxin, a humanized anti-vitronectin receptor (anti alpha v beta 3) antibody in patients with metastatic cancer.

The angiogenic response of a progressing malignancy is characterized by a shift in the balance of stimulatory and inhibiting factors of angiogenesis. Recognition of the regulated steps in tumor angiogenesis provides unique targets for developing anti-tumor therapy. Vitaxin is a humanized monoclonal antibody, which has specificity for the integrin alpha v beta 3 (vitronectin receptor).

Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.

Angiogenesis plays a central role in the growth and metastasis of cancers. Strategies aimed at interfering with tumor blood supply offer promise for new cancer therapies. Vitaxin (an anti-alphavbeta3 antibody) interferes with blood vessel formation by inducing apoptosis in newly generated endothelial cells.

Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues.

Optimal protein function often depends on co-operative interactions between amino acid residues distant in the protein primary sequence yet spatially near one another following protein folding. For example, antibody affinity is influenced by interactions of framework residues with complementarity-determining region (CDR) residues. However, despite the abundance of antibody structural information and computational tools the humanization of rodent antibodies for clinical use often results in a significant loss of affinity.

Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody.

A mimotope-guided strategy for engineering antibodies directed against orphan targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb). MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprotein (P-gp). Elevated expression of P-gp on tumor cells is associated with resistance to cytotoxic drugs, a major obstacle in chemotherapy.

Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb.

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alphav beta3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold.

Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries.

An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates.

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.

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.

Improving the copy numbers of antibody fragments expressed on the major coat protein of bacteriophage M13.

Background: Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage).

Objectives: As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis.

Antigen-presenting function of the mouse CD1 molecule.

CD1 molecules are distantly related to major histocompatibility complex (MHC)-encoded class I molecules, and they are coexpressed with beta2 microglobulin (beta2m). In the mouse, CD1 is expressed by intestinal epithelial cells and also by some cells in spleen and lymph node. We have shown that surface expression of mouse CD1 (mCD1) is not dependent upon a functional transporter associated with antigen processing (TAP).

Antigen-presenting function of the TL antigen and mouse CD1 molecules.

The hallmark of all the nonclassical antigen-presenting molecules, including nonclassical class I and nonclassical class II (Karlsson et al. 1992) molecules, is their lack of polymorphism. It is presumed, therefore, that these nonclassical molecules must have a distinct antigen-presenting function in which polymorphism is not advantageous.

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.

We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay.

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.

Peptide binding and presentation by mouse CD1.

CD1 molecules are distantly related to the major histocompatibility complex (MHC) class I proteins. They are of unknown function. Screening random peptide phage display libraries with soluble empty mouse CD1 (mCD1) identified a peptide binding motif.

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 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.

Construction of combinatorial antibody expression libraries in Escherichia coli.

A lambda vector system has been developed for the construction and coexpression of diverse populations of heavy and light chain cDNA sequences. Heavy and light chain sequences within the immunological repertoire are generated by the polymerase chain reaction using primers directed to conserved regions within the variable region framework. Two lambda vectors are employed for the independent construction of heavy and light chain cDNA libraries.