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Functional antibodies produced in tobacco plants were first reported over a decade ago (1989). The basic protocol used to generate these 'plantibodies' involved the independent cloning of H and L chain antibody genes in Agrobacterium tumefaciens vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole plants expressing individual chains, and their sexual cross. In a 'Mendelian' fashion, a fully assembled and functional antibody was recovered from plant tissue in some double-transgenic plants. In mammalian cells, the antibody H and L chains are produced as precursor proteins that are translocated into the endoplasmic reticulum (ER), under the guidance of signal sequences. Within the ER, the signal peptides are proteolytically cleaved, and several stress proteins act as chaperonins to bind the unassembled antibody chains, and direct subsequent folding and tetramer formation. A similar process occurs in plant cells, and expression can be directed via signal sequences (even of foreign origin) into the aqueous environment of the apoplasm, or to be accumulated in other specific plant tissues, including tubers, fruit, or seed. Plants can facilely assemble secretory IgA, which is comprised of four chains, H and L chains, J chain and secretory component. Plant 'bioreactors' are expected to yield over 10 kg of therapeutic antibody/acre in tobacco, maize, soybean, and alfalfa [(Ann. NY Acad. Sci.)721(1994)235; (Biotechnol. Bioeng.)20(1999)135]. Compared with conventional steel tank bioreactors using mammalian cells, or microorganisms, the costs of GMP plantibodies are expected to perhaps one tenth. The differences in glycosylation patterns of plant and mammalian cell produced antibodies apparently have no effect on antigen-binding or specificity, but there is some concern about potential immunogenicity in humans. N-linked glycans of plants differ from human by having fucose-linked alpha 1,3 and the sugar xylose. No adverse effects or human anti-mouse antibodies (HAMA) have been observed in >40 patients receiving topical oral application of a plant produced secretory IgA specific to Streptococcus mutans, for the control of caries [(Nat. Med.)4(1998)601]. The progressive improvement of expression vectors for plantibodies, and purification strategies, as well as the increase in transformable crop species, is expected to lead to almost limitless availability of inexpensive (even edible forms of) recombinant immunoglobulins free of human pathogens for human and animal therapy, and for novel industrial applications (e.g. catalytic antibodies).

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Recognition of self is emerging as a theme for the immune recognition of human cancer. One question is whether the immune system can actively respond to normal tissue autoantigens expressed by cancer cells. A second but related question is whether immune recognition of tissue autoantigens can actually induce tumor rejection. To address these issues, a mouse model was developed to investigate immune responses to a melanocyte differentiation antigen, tyrosinase-related protein 1 (or gp75), which is the product of the brown locus. In mice, immunization with purified syngeneic gp75 or syngeneic cells expressing gp75 failed to elicit antibody or cytotoxic T-cell responses to gp75, even when different immune adjuvants and cytokines were included. However, immunization with altered sources of gp75 antigen, in the form of either syngeneic gp75 expressed in insect cells or human gp75, elicited autoantibodies to gp75. Immunized mice rejected metastatic melanomas and developed patchy depigmentation in their coats. These studies support a model of tolerance maintained to a melanocyte differentiation antigen where tolerance can be broken by presenting sources of altered antigen (e.g., homologous xenogeneic protein or protein expressed in insect cells). Immune responses induced with these sources of altered antigen reacted with various processed forms of native, syngeneic protein and could induce both tumor rejection and autoimmunity.

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Differentiation antigens on cancer cells are recognized by the immune system. A prototype set of these autoantigens in melanoma cells are the melanosomal glycoproteins, expressed in both melanomas and normal melanocytes. These are intracellular proteins that can be recognized by both antibodies and T lymphocytes. While one can understand how T cells can respond to intracellular proteins, based on cellular requirements for antigen processing and presentation, it is more difficult to understand how antibody responses to melanosomal proteins could lead to tumor rejection. We demonstrate that gp75 is expressed on the cell surface as well as intracellularly in human and mouse melanomas. The surface expression of gp75 can be augmented by IFN-gamma and during tumor growth in vivo. Surface expression of gp75 on mouse melanoma cells correlates with the ability of a monoclonal antibody (mAb) against gp75 to reject melanomas in syngeneic mice. Antibody-mediated rejection seems to require the Fc portion of the antibody, suggesting a role for Fc receptor-positive effector cells such as natural killer cells. However, although NK1.1(+) cells have been implicated in antibody-induced rejection in vivo, cell surface expression of gp75(+) on melanoma does not lead to susceptibility to antibody-dependent cellular cytotoxicity in vitro. The mAb to gp75 induced tumor rejection in mice carrying both scid and bg/bg traits, showing that neither thymus-dependent T cells nor natural killer cytotoxic activity was required in vivo. Long-term treatment of mice with mAb led to patchy depigmentation in the coat. In summary, an intracellular organellar protein can be expressed at the cell surface and provide an antigenic target for antibody therapy and autoimmunity.

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Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.

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The underlying premise of tumor immunology is that the immune system is capable of recognizing cancer cells and that immune recognition can lead to rejection of tumors by the host. We focus on recent advances in tumor immunology in experimental systems, including the molecular identification of tumor antigens, studies of recognition of tumor cells by the immune system, and the development of new strategies for treatment of cancer. Identifying potential target antigens on tumors and defining effector arms of the immune system that mediate tumor rejection (including T cells, natural killer cells, antibodies, complement cells, and cytokines) remain crucial problems for the field.

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