CD28 and CTLA-4 are homologous T cell receptors of the immunoglobulin (Ig) superfamily, which bind B7 molecules (CD80 and CD86) on antigen-presenting cells and transmit important costimulatory signals during T cell activation. Here we have investigated the subunit structure of CTLA-4 and the stoichiometry of its binding to B7 molecules. We demonstrate CTLA-4 is a homodimer interconnected by one disulfide bond in the extracellular domain at cysteine residue 120. Each monomeric polypeptide chain of CTLA-4 contains a high affinity binding site for B7 molecules; soluble CTLA-4 and CD86 form complexes containing equimolar amounts of monomeric CTLA-4 and CD86 (i.e. a 2:2 molecular complex). Thus, CTLA-4 and probably CD28 have a receptor structure consisting of preexisting covalent homodimers with two binding sites. Dimerization of CTLA-4 and CD28 is not required for B7 binding, nor is it sufficient to trigger signaling.

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.

The iron-binding prcpertios of melanotransferrin, the tumour-associated antigen also known as ~97, have been investigated by UWvisible and Ruorescencc spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other vansferrins. mclanotransferrin binds only one Fe'+ ion per molecule. The binding properties of its N-terminal site arc similar to other transrcrrins, but its C-terminal site does not bind iron at all. The differences ca:l be related to spe;i!ic amino acid changes in the C-terminal site.Iron binding; Melanotransfcrrin; Transferrin; Melanoma cell antigen I, INTRODUCTIONMelanotransferrin, (MT& also known as the tumour-associated antigen, p97, is a monomeric glycoprotein, Ii/f, 97,000, which is expressed by human melanoma cells and certain other tissues [l-3], but is present only in trace amounts in normal adult tissues [4]. Its amino acid sequence [S] shows a high level of identity with proteins of the transferrin family (-40% identity with human transferrin or lactoferrin), it binds iron [6] and it has been proposed to play a role in iron translocation [5].MTf does, however, differ significantly from other transferrins. It is membrane-bound, and it differs in certain key amino acid residues which in other transferrins are involved in iron binding. Examination of the MTf sequence in relation td the three-dimenqional structures of human lactoferrin [7,83 and rabbit serum transferrin [9] has Ied to the proposal that it has an intact transferrin-type iron binding site in its N-terminal half but a possibly defective site in its C-terminal half [lo]. We have tested this proposal by investigation of the iron-binding properties of MTf, and discuss the results in the light of its presumed three-dimensional structure. MATERIALS AND METHODS I. isolation of MT/MTf was purified from cell culture supcrnatanls of transfected mouse melanoma clone 2a cells contained in ten IO-shelf cell factories. The cultures were allowed to incubate at 37°C for 10 days at which time the cells had grown to confluence and were showing signs of deterioration. Cell debris was removed by filtration and the filtrate was concentrated IO-to 1%fold with a Pellicon ultrafiltration system (Millipore) using a 5 sq. ft. 3O,ooO NMWL casctte. The retentate (approx. 2 I) was 0.2 pm filtered and applied overnight to a column (1.6 x 16 cm) of immobilize4 96.5 mAb [3] that had been previously washed with 0. I M citricacid. pH 2.2, and equilibrated in PBS, pH 7.2. The column was washed thoroughly with PBS and the bound MTf was cluted with 0.1 M citrate bulTer, pH 4.0, followed by immediate neutralization with Tris. The purified MTf (approx. IO mg) was dinlysed into PBS and sterile filtered. Batch analysis consisted of gel filtration HPLC, SDS-PAGE, isoelectric focusing and double-determinant binding ELISA, Mcamrawtrs of irott bittdingIron was added as ferric nitrilotriacetatc (FcNTA) to solutions of MTf in 0.025 M Tris-HCI, pH 7.8, containing 0.01 M NaHCO, and 0. I M NaCI. The protein concentration was estimated usin...

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-liiked 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.

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