Rapid production of protein-based tumorspecific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 g͞ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumorspecific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.Most B cell malignancies are incurable and are increasing in frequency in the populations of industrial nations (1, 2). Although B cell tumors are characterized by extreme variability in treatment and prognosis (3), they share a common feature that makes them ideal for the development of patientspecific cancer vaccines. Each clone of malignant B cells expresses a unique cell surface immunoglobulin (Ig)-a tumorspecific marker. The tumor's surface Ig, when made immunogenic by conjugation to keyhole limpet hemocyanin (KLH) and administered with an adjuvant, has been used to vaccinate patients in chemotherapy-induced remission (4, 5). When an immune response is triggered by such vaccination, patients have a superior clinical outcome (6, 7).Unfortunately, Igs are difficult proteins to produce. Currently, Igs for patient therapies are created by fusion of tumor cells to a transformed human͞mouse heteromyeloma cell (8, 9). Hybridomas are screened for secreted patient tumorspecific Ig and expanded for large-scale production of the protein. Besides the labor and expense involved, a drawback of hybridoma production systems is the unpredictable loss of chromosomes and of tumor-specific Ig protein expression over time. This phenomenon currently limits the application of the therapy, in terms of both the quantity of vaccine per patient and the total number of patients that can be treated. To expand the scope of individualized non-Hodgkin's lymphoma (NHL) therapies, a source of abundant, safe, easily purified vaccine needs to be generated in a time frame of weeks rather than months or years.An appealing alternative to multichain whole Ig vaccines is singe-chain variable region (scFv) vaccines. Consisting of just the hypervariable domains from the tumor-specific Ig, these proteins recreate the antigen-binding site of the native Ig (10-12), are a fraction of the size, and can be expressed in several expression systems (13-17), including transgenic plants (...

Needle-free liquid jet injectors were invented >50 years ago for the delivery of proteins and vaccines. Despite their long history, needle-free liquid jet injectors are not commonly used as a result of frequent pain and bruising. We hypothesized that pain and bruising originate from the deep penetration of the jets and can potentially be addressed by minimizing the penetration depth of jets into the skin. However, current jet injectors are not designed to maintain shallow dermal penetration depths. Using a new strategy of jet injection, pulsed microjets, we report on delivery of protein drugs into the skin without deep penetration. The high velocity (v >100 m/s) of microjets allows their entry into the skin, whereas the small jet diameters (50 -100 m) and extremely small volumes (2-15 nanoliters) limit the penetration depth (Ϸ200 m).In vitro experiments confirmed quantitative delivery of molecules into human skin and in vivo experiments with rats confirmed the ability of pulsed microjets to deliver therapeutic doses of insulin across the skin. Pulsed microjet injectors could be used to deliver drugs for local as well as systemic applications without using needles.MEMS ͉ nanotechnology ͉ noninvasive ͉ piezoelectric ͉ transdermal

Soluble extracellular protein antigens are notoriously poor stimulators of CD8+ cytotoxic T-lymphocyte (CTL) responses, largely because these antigens have inefficient access to an endogenous cytosolic pathway of the major histocompatibility complex (MHC) class I-dependent antigen presentation. Here, we present a strategy that facilitates antigen penetration into the cytosol of antigen-presenting cells (APC) by addition to the antigen of charge-modifying peptide sequences. As a result of this intervention, the charge modification enhances antigen uptake into APC by counteracting the repulsive cell surface charge, and then endosomal membranes are disrupted with a subsequent release of antigen into the cytosol. This technology significantly improves MHC class I-dependent antigen presentation to CTL, enabling a more efficient generation of specific CTL immunity in vivo. The strategy described here has potential for use in developing efficient vaccines for antigen-specific immunotherapy of human malignancies.

The canine transmissible venereal tumor is a naturally occurring neoplastic disease that affects the external genitalia of both sexes and is transmitted during coitus. Cytogenetic and immunologic studies demonstrated that tumors from different parts of the world are very similar, suggesting that they are transferred from one animal to another by the transplantation of viable cells. We found that the c-MYC oncogene was rearranged in this tumor by the insertion of a transposable genetic element sequence (known as LINE, long interspersed element) 5' to the first exon. The amplification of a DNA segment located in thejunction of the LINE genome and c-MYC upstream sequences enabled the testing-of the similarit of transmissible venereal tumor samples collected independently in different parts of the world. Oligonucleotide primers flanking the LINE/c-MYC junction were used to amplify a 340-base-pair segment and nested primers amplified a 280-base-pair segment. A fifth oligonucleotide used as a probe contained the actual junction sequence.-All "of the tumors analyzed revealed the existence of the specific bands, which were absent in normal canine DNA samples. The amplified segments obtained from all of the tumors analyzed were identical in size and nucleotide sequence, suggesting tranmilssion of the original rearranged cell itself, as opposed to independent events of LINE insertion in a "hot spot."

A search for a correlation between the clinical stage of chronic lymphocytic leukaemia (CLL) and the pattern of immunoglobulin heavy chain gene rearrangements was undertaken. DNA samples from the leukaemic cells of 38 CLL patients were analysed by Southern blot hybridization. Using probes for the immunoglobulin heavy chain J (JH) and C mu regions a marked heterogeneity of the hybridization patterns was observed in both regions. The number of JH hybridization bands varied from one to four and more than two were found in 58% of the patients. In 42% of the patients no germline JH genes were found. One to three additional C mu bands were observed in 34%, but the germline was preserved in all samples. There was no correlation between the clinical stage and the number of hybridizing JH bands; however, a significant correlation was found between the loss of JH germline band or a C mu multiband pattern and advanced stage of the disease. The genetic events in the immunoglobulin genes observed in advanced CLL patients are assumed to result from clonal evolution and tumour progression.