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Biomedical subjects

P Wentworth

Publications and source records attributed to P Wentworth.

At least 19 recordsLinked to original sources
PubMed2001

Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Animals↗
PubMed2001

Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptide-binding motif.

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2-restricted CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Catalytic antibodies: structure and function.

More than 10 years have now elapsed since the first reports confirmed that antibodies could be programmed as catalysts for chemical processes. Much of the initial research focussed on exploring the scope and utility of these new biocatalysts. Recently however, increasing information gleaned from X-ray analyses is allowing an exciting insight into the structural basis of antibody catalyzed reactions. This review details the evolving knowledge of the structure-function relationship for catalytic antibodies that accelerate a range of different reaction classes.

Allergy and Immunology↗
PubMed2001

The therapeutic potential for catalytic antibodies: from a concept to a promise.

More than ten years have now elapsed since the first reports confirmed that antibodies not only label antigenic targets but can also perform catalytic functions. Much of the initial research in this area focussed on exploring the scope and utility of these biocatalysts both as enzyme mimics and as programmable protein catalysts. However, their potential in the biomedical field has also been probed. This review details the present perspective of catalytic antibodies as new tools for immunotherapy and specifically focuses on their application to prodrug activation and drug inactivation.

Animals↗
PubMed2000

Soluble polymer-supported chemoenzymatic synthesis of the C(21)-C(27) fragment of the bryostatins.

A chemoenzymatic synthesis of the C(21)-C(27) fragment of the marine macrolide family of bryostatin antibiotics is presented. The approach commences from achiral starting materials and has as its crucial step the enzymatic resolution of a racemic mixture of soluble polymer-supported alcohols (syn-10 and syn-11). The immobilized lipase from Candida antarctica (Novozym 435) catalyzes the enantioselective acetylation of syn-10 (in 40% conversion and >99% ee), allowing isolation of the key intermediate (R)-14 in enantiomerically pure form following its cleavage from the poly(ethylene) glycol (PEG) scaffold. The PEG matrix is both compatible with the multipolymer enzymatic transformation and allows for rapid purification and facile NMR characterization of all intermediates throughout the synthesis.

Bryostatins↗
PubMed2000

Antibodies have the intrinsic capacity to destroy antigens.

Research throughout the last century has led to a consensus as to the strategy of the humoral component of the immune system. The essence is that, for killing, the antibody molecule activates additional systems that respond to antibody-antigen union. We now report that the immune system seems to have a previously unrecognized chemical potential intrinsic to the antibody molecule itself. All antibodies studied, regardless of source or antigenic specificity, can convert molecular oxygen into hydrogen peroxide, thereby potentially aligning recognition and killing within the same molecule. Aside from pointing to a new chemical arm for the immune system, these results may be important to the understanding of how antibodies evolved and what role they may play in human diseases.

Animals↗
PubMed2000

A soluble polymer-supported triflating reagent: a high-throughput synthetic approach to aryl and enol triflates.

[reaction: see text] The high-yielding synthesis and application of the first example of a polymer-supported reagent for the preparation of trifluoromethanesulfonates (triflates) is described. This new reagent efficiently triflates aryl alcohols and lithium enolates in high yield (>90%). A simple precipitation and filtration to remove the excess reagent and byproduct facilitate purification of the triflate products. The PEG-supported approach is highly efficient, as the PEG-supported byproduct can be quantitatively recovered and recycled into reagent 1.

Indicators and Reagents↗
PubMed1999

Recent developments and applications of liquid-phase strategies in organic synthesis.

Chemistry on soluble polymer supports, termed liquid-phase organic synthesis, is developing into an increasingly viable alternative or adjunct to the classical solid-phase approach across the broad spectrum of polymer-supported organic chemistry. Recent advances in the field include the use of soluble polymers in the combinatorial synthesis of peptide and small-molecule libraries, as catalyst and reagent supports, and as functionalized polymer-quench reagents for purifying solution-phase combinatorial libraries.

Chemistry, Organic↗
PubMed1998

Investigating highly crosslinked macroporous resins for solid-phase synthesis.

The washing efficiencies of a chromophore and the reaction rates of a classical esterification reaction are improved with macroporous resins (MRs) relative to a classical Merrifield resin. Furthermore, Wacker-oxidation of a MR bound alkene yielded the expected methylketone product whereas an alkene bound to a low-crosslinked Merrifield resin gave no product, a function of the relative permeability of each of these resins to the aqueous solvent conditions employed.

Cross-Linking Reagents↗
PubMed1998

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1998

A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis.

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2'-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25 degreesC showed that the most active antibody, MATT.F-1, obeyed classical Michaelis-Menten kinetics with a Km = 104 microM, a kcat = 0.44 min-1, and a kcat/kuncat = 1.7 x 10(3). Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody-catalyst (1.6 x 10(7) M-1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.

Antibodies, Catalytic↗
PubMed1998

Pools of lipidated HTL-CTL constructs prime for multiple HBV and HCV CTL epitope responses.

Various peptide-based approaches to simultaneous induction of multiple cytotoxic T lymphocyte (CTL) responses were evaluated as part of ongoing efforts to develop immunotherapeutic vaccines for use in humans. To this end, HLA (human histocompatibility leukocyte antigen)-A2-restricted epitopes from several specific viral proteins were tested in an HLA-A2 transgenic mouse model system, which mimics human CTL responses to these viral proteins. Multiple CTL responses were elicited by immunization with either peptides emulsified in incomplete Freund's adjuvant (IFA), or lipidated peptides administered in phosphate buffered saline (PBS). In the case of lipidated peptides, induction of CTL responses was crucially dependent on the presence of helper T lymphocyte (HTL) epitopes, and most efficient in the case of lipidated covalently linked HTL-CTL epitope constructs. CTL could also be induced by immunization with lipidated HTL epitopes simply mixed with CTL epitopes and formulated in PBS. However, this approach was highly dependent on the particular lipidated HTL/CTL combination utilized, and was marginally effective for simultaneous priming of multiple CTL responses. By contrast, all HTL/CTL combinations were potent immunogens when delivered as lipidated, covalently linked molecules. This was the most effective of the approaches analysed in terms of multi-epitope priming, as demonstrated by the induction of simultaneous CTL responses to a pool of five different epitopes.

Adjuvants, Immunologic↗
PubMed1998

Catalytic antibodies.

Following the end of the first decade in catalytic antibody research, recent efforts are reflecting a more introspective view of the field. Notably, X-ray crystal structure analyses of antibody catalysts are permitting an increased understanding of the evolution and modus operandi of these remarkable biocatalysts. Additionally, the breadth and scope of new antibody-catalyzed reactions and novel hapten design strategies have continued to flourish with support from an increasing number of contributors to this ever expanding area.

Antibodies↗
PubMed1997

Derivation of HLA-A11/Kb transgenic mice: functional CTL repertoire and recognition of human A11-restricted CTL epitopes.

Transgenic mice expressing chimeric human (alpha1 and alpha2 HLA-A11 domains) and murine (alpha3, transmembrane, and cytoplasmic H-2Kb domains) class I molecules were derived. These mice were used as a model system to study the immunogenicity of human CTL epitopes and also to examine the aspects of Ag processing differences of mice vs man. Immunization of these mice with seven known HLA-A11-restricted CTL epitopes emulsified in IFA resulted in vigorous specific CTL responses. A larger panel of 45 A11-binding peptides was used to examine the relationship between immunogenicity in the HLA-A11/Kb transgenic mice and HLA-A11 binding capacity. Twenty-one of 28 (75%) peptides with high binding affinities (50% inhibitory concentration (IC50), 2-50 nM) and 7 of 13 (54%) intermediate binding peptides (IC50, 50-500 nM range) were immunogenic. In parallel, 19 of these peptides were used for in vitro primary immunizations of PBMC derived from HLA-A11 healthy human donors. It was found that 8 of 8 peptides that were able to elicit CTL in primary human in vitro cultures were also immunogenic in HLA-A11/Kb mice. Finally, HLA-A11/Kb transgenic mice were found to generate an A11/Kb restricted CTL response following immunization with influenza virus A/PR/8/34, suggesting that, at least to some extent, A11 epitopes are generated by transgenic mice as a result of natural in vivo processing and presentation.

Animals↗
PubMed1996

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used chemotherapeutic agents. Most ADEPT systems incorporate a bacterial enzyme, and thus their potential is reduced because of the immunogenicity of that component of the conjugate. This limitation can be circumvented by the use of a catalytic antibody, which can be "humanized," in place of the bacterial enzyme catalyst. We have explored the scope of such antibody-directed "abzyme" prodrug therapy, ADAPT, to evaluate the potential for a repeatable targeted cancer chemotherapy. We report the production of a catalytic antibody that can hydrolyze the carbamate prodrug 4-[N,N-bis(2-chloroethyl)]aminophenyl-N-[(1S)-(1,3- dicarboxy)propyl]carbamate (1) to generate the corresponding cytotoxic nitrogen mustard (Km = 201 microM, kcat = 1.88 min-1). In vitro studies with this abzyme, EA11-D7, and prodrug 1 lead to a marked reduction in viability of cultured human colonic carcinoma (LoVo) cells relative to appropriate controls. In addition, we have found a good correlation between antibody catalysis as determined by this cytotoxicity assay in vitro and competitive binding studies of candidate abzymes to the truncated transition-state analogue ethyl 4-nitrophenylmethylphosphonate. This cell-kill assay heralds a general approach to direct and rapid screening of antibody libraries for catalysts.

Aniline Mustard↗