Mavanur R. Suresh

Bispecific and bifunctional single chain recombinant antibodies

Bispecific and bifunctional monoclonal antibodies as second generation monoclonals, produced by conventional chemical or somatic methods, have proved useful in the immunodiagnosis and immunotherapy of cancer and other diseases. Recombinant antibodies produced by genetic engineering techniques have also become available for use in preclinical and clinical studies. Furthermore, through genetic engineering, it is possible to remove or add on key protein domains in order to create designer antibody molecules with two or more desired functions. This review summarizes the strategies for development of single chain variable fragment (scFv) bifunctional and bispecific antibodies. The advantages and disadvantages as well as the problems of generating the various bispecific and bifunctional antibody constructs are reported and discussed. Since conventionally prepared bispecific and bifunctional monoclonal antibodies have already shown promise in clinical trials and results from preclinical studies of recombinant bispecific antibodies are encouraging, clinical trials in humans of recombinant bispecific and bifunctional antibodies, as a new generation of biologicals, are likely to be the thrust in the next decade and beyond.

Specificity and Affinity of 26 Monoclonal Antibodies against the CA 125 Antigen: First Report from the ISOBM TD-1 Workshop

The specificity of 26 monoclonal antibodies against the CA 125 antigen was investigated in two phases of the ISOBM TD-1 workshop. The binding specificity was studied using CA 125 immunoextracted by specific antibodies immobilized on various solid phases, or on the surface of human cell lines. Immunometric assays using all possible antibody combinations were used to study the topography of antibody binding sites on the antigen. We conclude that the CA 125 antigen carries only two major antigenic domains, which classifies the antibodies as OC125-like (group A) or M11-like (group B). One antibody, OV 197, showed binding specificity related to some of the OC125-like antibodies, but was classified into a separate group C. The OC125-like group of antibodies has four subgroups with different binding specificities. These are A1 = OC 125 and K 95, A2 = K 93, A3 = B43.13, and A4 = ZS 33, B27.1 and CCD 247. Binding of nonlabelled OC 125 or K 95 to CA 125 caused a marked increase in binding of labelled OV 197 to the complex. This conformational change was not observed with any other antibody combinations. Antibody B43.13 could form immunometric assay combinations particularly with antibodies of subgroup A4, indicating that the B43.13 epitope is in the periphery of the binding area of OC125-like antibodies. The M11-like group of antibodies is more homogenous with strong cross-inhibition between most antibodies. Only one antibody, ZR 38, would form an immunoassay combination with other M11-like antibodies and thus represents a distinct subgroup. The main group of M11-like antibodies are M 11, ZR 45, MA602-6, K 91, OV 185, K 101, K 90, K 96, K 97, K 102, CCD 242, 145-9, and 130-22. Antibody OV 197 binds to a domain designated C and is unique, as stated above. Antibody pairs from any two of the three groups may be used in immunometric assays. Three antibodies were not studied by complete cross-inhibition due to low affinity (OV 198 and K 100) or lack of material (MA602-1). OV 198 and K 100 are most likely OC125-like and MA602-1 is M11-like. Antibody affinity was estimated with labelled antigen in solution or with antigen absorbed on microtiter wells. Western blot analysis showed staining both in the stacking gel and corresponding to a molecule of 200 kDa. There was a marked difference between the antibodies in their ability to bind to CA 125 immobilized on a membrane. Strongest binding was observed with the M11-like antibodies, particularly M 11, K 96, K 97, MA602-6, 145-9. Antibodies belonging to the subgroup A4 were the only OC 125-like antibodies which reacted well with CA 125 in Western analysis. Digestion of CA 125 with proteolytic enzymes showed it to be particularly sensitive to trypsin cleavage. However, no low molecular weight fragments with preserved immunoreactivity were found.

Synthesis of Celecoxib Analogues Possessing a N-Difluoromethyl-1,2-dihydropyrid-2-one 5-Lipoxygenase Pharmacophore: Biological Evaluation as Dual Inhibitors of Cyclooxygenases and 5-Lipoxygenase with Anti-Inflammatory Activity

A novel class of 1-(4-methanesulfonylphenyl and 4-aminosulfonylphenyl)-5-[4-(1-difluoromethyl-1,2-dihydropyrid-2-one)]-3-trifluoromethyl-1H-pyrazole hybrid cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory anti-inflammatory agents was designed. Replacement of the tolyl ring present in celecoxib by the N-difluoromethyl-1,2-dihydropyrid-2-one moiety provided compounds showing dual selective COX-2/5-LOX inhibitory activities. 1-(4-Aminosulfonylphenyl)-5-[4-(1-difluoromethyl-1,2-dihydropyrid-2-one)]-3-trifluoromethyl-1H-pyrazole exhibited good anti-inflammatory (AI) activity (ED(50) = 27.7 mg/kg po) that compares favorably with the reference drugs celecoxib (ED(50) = 10.8 mg/kg po) and ibuprofen (ED(50) = 67.4 mg/kg po). The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel 5-LOX pharmacophore for the design of cyclic hydroxamic mimetics for exploitation in the development of COX-2/5-LOX inhibitory AI drugs.

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Replication-Deficient Ebolavirus as a Vaccine Candidate

ABSTRACT Ebolavirus causes severe hemorrhagic fever, with case fatality rates as high as 90%. Currently, no licensed vaccine is available against Ebolavirus. We previously generated a replication-deficient, biologically contained Ebolavirus, EbolaΔVP30, which lacks the essential VP30 gene, grows only in cells stably expressing this gene product, and is genetically stable. Here, we evaluated the vaccine potential of EbolaΔVP30. First, we demonstrated its safety in STAT-1-knockout mice, a susceptible animal model for Ebolavirus infection. We then tested its protective efficacy in two animal models, mice and guinea pigs. Mice immunized twice with EbolaΔVP30 were protected from a lethal infection of mouse-adapted Ebolavirus. Virus titers in the serum of vaccinated mice were significantly lower than those in nonvaccinated mice. Protection of mice immunized with EbolaΔVP30 was associated with a high antibody response to the Ebolavirus glycoprotein and the generation of an Ebolavirus NP-specific CD8+ T-cell response. Guinea pigs immunized twice with EbolaΔVP30 were also protected from a lethal infection of guinea pig-adapted Ebolavirus. Our study demonstrates the potential of the EbolaΔVP30 virus as a new vaccine platform.

T cell recognition of a tumor-associated glycoprotein and its synthetic carbohydrate epitopes: stimulation of anticancer T cell immunity in vivo.

SummaryThe Thomsen, Friedenreich (TF) and Tn carbohydrate antigens are expressed on the vast majority of human adenocarcinomas and are associated with aggressive behavior of certain tumors. TF and Tn antigens are also expressed on certain murine cancer cell lines including TA3-Ha, a highly lethal, transplantable mammary adenocarcinoma. TF and Tn cancer-associated carbohydrate haptens were synthesized, conjugated to protein carriers and used to demonstrate that delayed-type hypersensitivity (DTH) effector T cells can specifically recognize and respond to carbohydrate determinants on the TA3-Ha tumor-associated glycoprotein, epiglycanin. The effector cells were shown to have the helper DTH phenotype (Lytl+, Lyt2−, Thyl+) and it was demonstrated that they respond to specific carbohydrate determinants in an MHC-restricted fashion. These experiments provide the rationale for the use of synthetic tumor-associated glycoconjugates (S-TAGs) to stimulate anticancer T cell immunity. In support of this hypothesis, it was shown that preimmunization with the appropriate S-TAGs could provide a degree of protection against a subsequent tumor transplant and that antitumor effector Lytl+, Lyt2− T cells could be generated in vitro using the appropriate S-TAGs as antigens.

Synthesis and evaluation of 1,5-diaryl-substituted tetrazoles as novel selective cyclooxygenase-2 (COX-2) inhibitors

A series of 1,5-diaryl-substituted tetrazole derivatives was synthesized via conversion of readily available diaryl amides into corresponding imidoylchlorides followed by reaction with sodium azide. All compounds were evaluated by cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Tetrazoles 3a-e showed IC(50) values ranging from 0.42 to 8.1 mM for COX-1 and 2.0 to 200 μM for COX-2. Most potent compound 3c (IC(50) (COX-2)=2.0 μM) was further used in molecular modeling docking studies.

Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety: Synthesis, biological evaluation and nitric oxide release studies

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a-b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a-b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4-5.8% range). In comparison, the percentage NO released was higher (3.1-8.4% range) when these nitrate prodrugs were incubated in the presence of L-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure-activity relationship data acquired showed that compounds having a MeSO2 COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H2NSO2 substituent. Compounds having a MeSO2 COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED50=132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED50=118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED50=128.7 mg/kg po) but lower than ibuprofen (ED50=67.4 mg/kg po).

Role of B-type natriuretic peptide in epoxyeicosatrienoic acid-mediated improved post-ischaemic recovery of heart contractile function

AIMS This study examined the functional role of B-type natriuretic peptide (BNP) in epoxyeicosatrienoic acid (EET)-mediated cardioprotection in mice with targeted disruption of the sEH or Ephx2 gene (sEH null). METHODS AND RESULTS Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C epsilon and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3beta was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts. CONCLUSION Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events.

An Overview Of Tuberculosis Chemotherapy – A Literature Review

Tuberculosis (TB) is a major threat in global public health. The emergence of HIV and also multi drug resistant (MDR) and extremely drug resistant (XDR)-TB poses a vital challenge to the control of the disease. For the last 40 years, no new anti-TB drug has been discovered. This literature review provides a brief discussion of existing drugs and emerging drug targets, and also of the advantages of incorporating modern drug delivery systems and immune modulators in order to improve the existing treatment regimen in terms of better efficacy, reduced drug administration frequency, shortened period of treatment and reduced drug related toxicity. The investigation for new a drug target is essential in carrying on the fight against MDR and XDR-TB. However, owing to the enormous cost and time involved in new drug development, improvement of the existing treatment regimen is seen to be a valid alternative.