Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.

Fusion phage libraries expressing single-chain Fv antibodies were constructed from the peripheral blood lymphocytes of two melanoma patients who had been immunized with autologous melanoma cells transduced the gamma-interferon gene to enhance immunogenicity, in a trial conducted at another institution. Anti-melanoma antibodies were selected from each library by panning the phage against live cultures of the autologous tumor. After two or three rounds of panning, clones of the phage were tested by ELISA for binding to the autologous tumor cells; > 90% of the clones tested showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting antimelanoma antibodies. The panned phage population was extensively absorbed against normal melanocytes to enrich for antibodies that react with melanoma cells but not with melanocytes. The unabsorbed phage were cloned, and the specificities of the expressed antibodies were individually tested by ELISA with a panel of cultured human cells. The first tests were done with normal endothelial and fibroblast cells to identify antibodies that do not react, or react weakly, with two normal cell types, indicating some degree of specificity for melanoma cells. The proportion of phage clones expressing such antibodies was approximately 1%. Those phage were further tested by ELISA with melanocytes, several melanoma lines, and eight other tumor lines, including a glioma line derived from glial cells that share a common lineage with melanocytes. The ELISA tests identified three classes of anti-melanoma antibodies, as follows: (i) a melanoma-specific class that reacts almost exclusively with the melanoma lines; (ii) a tumor-specific class that reacts with melanoma and other tumor lines but does not react with the normal melanocyte, endothelial and fibroblast cells; and (iii) a lineage-specific class that reacts with the melanoma lines, melanocytes, and the glioma line but does not react with the other lines. These are rare classes from the immunized patients' repertoires of anti-melanoma antibodies, most of which are relatively nonspecific anti-self antibodies. The melanoma-specific class was isolated from one patient, and the lineage-specific class was isolated from the other patient, indicating that different patients can have markedly different responses to the same immunization protocol. The procedures described here can be used to screen the antibody repertoire of any person with cancer, providing access to an enormous untapped pool of human monoclonal anti-tumor antibodies with clinical and research potential.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Presence of antibodies to different subunits of replication protein A in autoimmune sera.

A human cDNA expression library was used to investigate the nature of molecules recognized by serum from a patient with Sjögren syndrome that exhibits a mixed immunofluorescence pattern and reacts with multiple components on an immunoblot. The data demonstrated that this serum contains IgG antibodies specific for the 70- and 32-kDa subunits of replication protein A (RPA; RPA-70 and RPA-32, respectively), a highly conserved multisubunit DNA binding protein. Affinity purification of serum autoantibodies demonstrated a complete lack of cross-reactivity between RPA-70 and RPA-32, suggesting a direct participation of the native protein complex in the autoimmune response in this patient. Purified anti-RPA-70 and anti-RPA-32 antibodies labeled nuclear and cytoplasmic components in an immunofluorescence assay, suggesting that RPA is present in both cellular compartments. Additional sera from 55 patients with different autoimmune conditions were screened against purified RPA-70 and RPA-32 recombinant proteins. One of these 55 sera was positive and reacted with only RPA-32. Twenty sera from healthy control individuals did not react with RPA. These results show that RPA is a target for autoantibodies in human autoimmune diseases, although its precise frequency, occurrence in other autoimmune diseases, and pathological significance remain to be fully elucidated.

Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library.

Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries.

Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.

Suppression and promotion of tumor growth by monoclonal antibodies to ErbB-2 differentially correlate with cellular uptake.

Amplification and overexpression of the erbB-2/neu protooncogene are frequently associated with aggressive clinical course of certain human adenocarcinomas, and therefore the encoded surface glycoprotein is considered a candidate target for immunotherapy. We previously generated a series of anti-ErbB-2 monoclonal antibodies (mAbs) that either accelerate or inhibit the tumorigenic growth of erbB-2-transformed murine fibroblasts. The present study extended this observation to a human tumor cell line grown as xenografts in athymic mice and addressed the biochemical differences between the two classes of mAbs. We show that the inhibitory effect is dominant in an antibody mixture, and it depends on antibody bivalency. By using radiolabeled mAbs we found that all of three tumor-inhibitory mAbs became rapidly inaccessible to acid treatment when incubated with tumor cells. However, a tumor-stimulatory mAb remained accessible to extracellular treatments, indicating that it did not undergo endocytosis. In addition, intracellular fragments of the inhibitory mAbs, but not of the stimulatory mAb, were observed. Electron microscopy of colloidal gold-antibody conjugates confirmed the absence of endocytosis of the stimulatory mAb but detected endocytic vesicles containing an inhibitory mAb. We conclude that acceleration of cell growth by ErbB-2 correlates with cell surface localization, whereas inhibition of tumor growth is associated with an intrinsic ability of anti-ErbB-2 mAbs to induce endocytosis. These conclusions are relevant to the selection of optimal mAbs for immunotherapy and may have implications for the mechanism of cellular transformation by an overexpressed erbB-2 gene.

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.