Managing the pharmacy benefit in Medicare HMOs: what do we really know?

An estimated five million Medicare beneficiaries received outpatient prescription drug benefits through Medicare + Choice in 1999. However, little is known about how these benefits are managed or about their effects on costs and quality of care. This exploratory study applies a case-study methodology to four large Medicare health maintenance organizations (HMOs) to identify and assess drug-use management strategies. It also poses a number of important issues for consideration by both policymakers and health services researchers, as the debate rages on over the creation and administration of a Medicare outpatient drug benefit, especially in light of the predilection for the use of private pharmacy benefit managers (PBMs) in many of these proposals.

Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation.

The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

Activity of a single-chain immunotoxin that selectively kills lymphoma and other B-lineage cells expressing the CD40 antigen.

We have constructed anti-CD40 immunotoxins consisting of the single chain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a truncated form of Pseudomonas exotoxin, PE40. CD40 is an integral membrane glycoprotein found on the surface of B-lineage cells, including lymphomas and leukemias, as well as certain carcinomas. Two forms of the immunotoxin were constructed, one with the light chain variable (VL) region of the sFv preceding the heavy chain variable region (VH) [G28-5 sFv(VL-VH)-PE40] and the second with the sFv in the opposite orientation [G28-5 sFv(VH-VL)-PE40]. Although both forms of G28-5 sFv-PE40 specifically bound to CD40 in ELISAs, the binding of G28-5 sFv(VL-VH)-PE40 was > 10-fold higher. A number of malignant B- and T-cell lines were screened for CD40 expression and susceptibility to G28-5 sFv(VL-VH)-PE40. All of the B-lineage cells tested were CD40 positive and sensitive to the anti-CD40 immunotoxin, with EC50s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins. Consistent with the antigen-binding results, the VL-VH immunotoxin was > 10-fold more cytotoxic than the VH-VL immunotoxin. The anti-CD40 single-chain immunotoxin fusion protein G28-5 sFv(VL-VH)-PE40 represents a potent and specific cytotoxic agent for the elimination of normal and transformed B-lineage cells expressing CD40.

Human melanotransferrin (p97) has only one functional iron-binding site.

The iron-binding properties of melanotransferrin, the tumour-associated antigen also known as p97, have been investigated by UV/visible and fluorescence spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other transferrins, melanotransferrin binds only one Fe3+ ion per molecule. The binding properties of its N-terminal site are similar to other transferrins, but its C-terminal site does not bind iron at all. The differences can be related to specific amino acid changes in the C-terminal site.

Generation and characterization of murine antiflagellum monoclonal antibodies that are protective against lethal challenge with Pseudomonas aeruginosa.

Two murine monoclonal antibodies, IIG5 (IgG3) and IVE8 (IgG2a), that bind to Pseudomonas aeruginosa type a flagella and type b flagella, respectively, were prepared by conventional hybridoma methodology. Specificity of each monoclonal antibody for type a or type b flagella was demonstrated by enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting. The percentage of P. aeruginosa isolates recognized by each monoclonal antibody was analyzed by enzyme-linked immunosorbent assay. Among a panel of 257 flagellated P. aeruginosa clinical isolates, IIG5 bound to 67.7% of the isolates and IVE8 bound to another 30.7%, for a combined coverage of 98.4%. Inhibition of motility of P. aeruginosa by the monoclonal antibodies was observed in vitro in a soft agar assay and was dose dependent. The protective efficacy of IIG5 and IVE8 was examined in a mouse burn wound sepsis model. The antiflagellum monoclonal antibodies provided specific and significant prophylactic and therapeutic protection against lethal challenge with P. aeruginosa strains.

Isolation of the outer membrane components of Bordetella pertussis which enhance the immunogenicity of Haemophilus influenzae type b capsular polysaccharide polyribosyl ribitol phosphate.

The outer membrane (OM) component(s) from Bordetella pertussis which potentiated the antigenicity of purified Haemophilus influenzae type b capsular polysaccharide, polyribosyl ribitol phosphate (PRP), has been isolated and partially characterized. The OM was isolated from spheroplasting medium by precipitating at pH 5.0; fractionation was carried out by dissolving the crude OM in Triton X-100 followed by selective precipitation of OM in 50% ethanol. Further purification of OM was accomplished by DEAE-Sepharose 6BCL and Sephacryl S-300 chromatography. The biochemical composition and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of various OM preparations have been examined. Combined vaccines consisting of OM components and PRP were given to young Sprague-Dawley rats, and the serum antibody to PRP was measured by an [3H]PRP binding assay. The purified OM containing mainly a 30,000-dalton protein, but not purified lipopolysaccharide or leukocytosis-promoting toxin, exhibited strong enhancement of PRP immunogenicity.