No abstract

Therapeutic vaccination against idiotype is a promising strategy for immunotherapy of B-cell malignancies. Its feasibility, however, is limited by the requirement for a patient-specific product. Here we describe a novel vaccine formulation prepared by simply extracting cell-membrane proteins from lymphoma cells and incorporating them together with IL-2 into proteoliposomes. The vaccine was pro- IntroductionImmunization with tumor-specific idiotype (Id) protein conjugated to keyhole limpet hemocyanin (KLH) and administered with an adjuvant induces anti-tumor immunity in patients with follicular lymphoma, and is associated with prolonged diseasefree survival. [1][2][3][4] Previously we demonstrated that a liposomal vaccine, made by co-entrapping hybridoma-derived lymphoma idiotype protein and interleukin-2 (IL-2) in a multilamellar coalescence vesicle (MLCV), 5 protected mice against a lethal challenge of lymphoma cells via a CD4 ϩ /CD8 ϩ T-celldependent mechanism. 6 IL-2 was chosen as a vaccine component due to its ability to expand activated T cells. Furthermore, we have previously demonstrated that IL-2 has a specific interaction with small unilamellar lipid vesicles, leading to the formation of MLCV used for vaccines. 5 In a Phase I clinical trial, this liposomal vaccine formulation was found to be safe and immunogenic. 7 Manufacture of individualized Id protein vaccines, however, using either KLH conjugation or liposome entrapment, required an expensive and time-consuming amplification of tumor antigen via hybridoma or recombinant DNA technology. 8,9 The goal of the present study was therefore to streamline the production of patient-specific lymphoma vaccines. We developed a novel liposomal vaccine by replacing the hybridoma-secreted Id with natural tumor-derived cell membrane proteins and tested the formulation in a mouse lymphoma model. Our results indicate that the potency of the vaccine can be vastly increased by inserting in the MLCV liposomes either purified tumor-derived natural Id or cell membrane "patches" that may contain multiple tumor-associated antigens besides idiotype. Based on estimates of the amount of Id available from human tumor biopsies, this simple and rapid approach can be readily translated to clinical evaluation. Materials and methods Mice and tumorSix-to 12-week-old C3H/HeN female mice were from the National Cancer Institute Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, MD. The carcinogen-induced C3H 38C13 B-cell lymphoma has been previously described. 10 Tumor-derived IgMSecreted 38C13 immunoglobulin M (IgM) Id and a control IgM 4C5 were isolated for use in immunoassays and vaccines following somatic cell hyrbridization (R. Levy, Stanford University, CA) and purification from ascites. 6 A control vaccine formulation was prepared by glutaraldehyde conjugation at a 1:1 ratio of 38C13 Id and KLH as described. 11 Liposomal vaccine preparationCell membranes were prepared from 38C13 cells grown in tissue culture or as solid tumors in C3H mice. Membrane proteins...

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (

Near-UV circular dichroism (CD) was used to identify differences in the tertiary structure of human erythrocyte band 3, the chloride/bicarbonate exchange protein, consequent to covalent binding of anion transport inhibitors to the intramonomeric stilbenedisulfonate (ISD) site. Isolated intact band 3 and its membrane domain (B3MD) were compared. Spectral differences were observed which involved intradomain effects, in that they were seen both with intact band 3 and with B3MD, or interdomain effects, in that they were observed only for B3MD, but were inhibited when the cytoplasmic domain was attached. The intradomain effect involved a significant loss in optical activity in the Phe/Tyr region of the spectrum below 280 nm. It was seen only when the ISD site had stilbenedisulfonates bound covalently at pH 7.4. Raising the pH to 9.6 after adduct formation "normalized" this spectral change irreversibly. The interdomain effect was identified in the Trp spectral region at 292 nm. There was a significant increase in optical activity at 292 nm when bulky covalent ligands such as DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) were bound to B3MD, but not when the same ligands were bound to intact band 3. These latter results offer evidence that certain aspects of the conformational response of the integral domain are inhibited by the presence of an attached cytoplasmic domain. The potential significance of interdomain interactions to band 3 function is discussed briefly.