Efficacy of a therapeutic cocaine vaccine in rodent models.

Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.

Functional and biochemical characterization of a secreted I-J(+) suppressor factor that binds to immunoglobulin.

A secreted product of a T cell leukemic cell line, LH-8, was examined for its biochemical and biological properties. The factor that we have termed Immunoglobulin-Binding T cell Suppressor Factor (IgB-TsF) was shown to be suppressive for the in vitro and in vivo humoral response to a variety (but not all) antigens tested. The cell surface phenotype of the LH-8.1 subclone was M.Ig(-), Thy-1(+), L3T4(-), Lyt-2(+), FcR(-), MAC-1(-), and H-2b(+). In addition, both the cell surface and secreted factor, IgB-TsF, of LH-8.1 expressed determinants that were recognized by anti-I-Jb mAbs but not by an anti-I-Jd monoclonal. The same factor also retained an affinity for the Fc portion of approximately 30% of randomly selected, purified mAbs. This binding could be abolished if the Fab or F(ab')2 fragments of these mAb were used, but was found to be unrelated to isotype of the respective mAbs. Using subclones that expressed quantitative differences in their ability to exert suppression as sources of biosynthetically labeled IgB-TsF, we have shown the suppressor activity correlated with a single, 28 kD protein. Furthermore, comparisons of these same subclones that differ in their suppressor activity, do not show any direct correlation of this biological activity with the expression of the previously described T cell receptor genes. It also suggests that at least some suppressor cell subsets may use the same or related family of T cell receptor genes for their recognitive stage of activation as helper and cytotoxic T cell subsets, but not for their effector stage of immunologic suppression.

Somatically mutated forms of a major anti-p-azophenylarsonate antibody variable region with drastically reduced affinity for p-azophenylarsonate. By-products of an antigen-driven immune response?

The pivotal role played by antigen in the clonal selection of B cells for initial participation in an immune response is well established. Antigen selective mechanisms ensure that antigen-binding antibodies are produced during all stages of the immune response. However, antibodies that lack specificity for the immunogen might also be produced during the course of an antigen-driven immune response . It has been suggested that, through idiotype-antiidiotype network interactions within the immune system, production of antibodies that lack specificity for the immunogen but that share idiotopes with antigen-binding antibodies could result (1). In addition, data obtained by a number of investigators suggest that somatic mutation of antibody V region genes occurs at a rate of 10(-3)/basepair/cell division in B cells participating in an immune response (2, 3). One outcome of such V region structural alteration could be antibodies that lack, or have drastically reduced affinity for the immunogen . We sought to identify and characterize some of the antibody by-products of the antigen-driven immune response that are expected to be created by the mechanisms described above.

Control of immunoglobulin secretion in the murine plasmacytoma line MOPC 315.

Cells of the 315LV-1 (derived from NP1) variant line of MOPC 315 contain approximately 1% the normal intracellular level of the heavy (alpha) chain of IgA and no detectable light (lambda2) chain. The synthesis rate of alpha-chain in the variant, however, is similar to that in cells of the parent line. Moreover the relative amount of translatable alpha-chain mRNA that can be extracted from 315LV-1 cells is about the same as for parental cells. No light-chain synthesis can be detected either in vivo or in vitro in a wheat germ cell-free system. The 315LV-1 heavy chain synthesized in vivo or in vitro has slightly greater electrophoretic mobility than normal H chain and turns over rapidly intracellularly. The variant fails to secrete any of its heavy chain, despite the fact that its H chain mRNA is bound to membranes, as one would expect for a secretory protein message. Fusion of 315LV-1 cells with cells of a kappa-producing MPC 11 variant line leads to stabilization of the intracellular H chain and also to full recovery of secretion of the H chain as an H2L2 molecule.

Parallel evolution of antibody variable regions by somatic processes: consecutive shared somatic alterations in VH genes expressed by independently generated hybridomas apparently acquired by point mutation and selection rather than by gene conversion.

We identified, in independently generated hybridoma antibodies, blocks of shared somatic alterations comprising four consecutive amino acid replacements in the CDR2s of their heavy chain variable regions. We found that the nucleotide sequences encoding the shared replacements differed slightly. In addition, we performed genomic cloning and sequencing analyses that indicate that no genomic sequence could encode the block of shared replacements in any one of the antibodies and thus directly serve as a donor by a recombinational process. Finally, in a survey of other somatically mutated versions of the same heavy chain variable gene, we found several examples containing one, two, or three of the shared CDR2 mutations in various combinations. We conclude that the shared somatic alterations were acquired by several independent events. This result, and the fact that the antibodies containing the four shared mutations were elicited in response to the same antigen and are encoded by the same VH and VK gene segments, suggests that an intense selection pressure has fixed the shared replacements by favoring the clonal expansion of B cells producing antibodies that contain them. The basis of this selection pressure is addressed elsewhere (Parhami-Seren, B., L. J. Wysocki, M. N. Margolies, and J. Sharon, manuscript submitted for publication).

Identical peptides recognized by MHC class I- and II-restricted T cells.

Previous data from many groups show that both class I and class II-restricted T cells recognize short synthetic peptides in the context of their respective MHC molecules (9-18), all of the peptides described to date are restricted to only a single class of MHC molecules; however, structural homology between the class I and II MHC molecules and the use of similar TCRs by class I and II-restricted T cells suggest that antigen recognition mechanisms are similar in both systems. To directly compare antigen recognition in the two systems, we analyzed peptides for the ability to function in both a class I and II-restricted system and found that seven of seven individual peptides tested stimulate both class I and II-restricted T cell responses. In addition, two of the peptides can function in different species stimulating both human class I and murine class II T cell responses. Thus, the process of T cell recognition of antigen in the context of MHC molecules was highly conserved in evolution not only between the class I and class II MHC systems, but also between the murine and human species.

Single germline VH and V kappa genes encode predominating antibody variable regions elicited in strain A mice by immunization with p-azophenylarsonate.

We have cloned and sequenced the predominant germline V kappa gene segment expressed by B cells of strain A origin that synthesize antibodies with specificity for Ars. In hybridomas synthesizing anti-Ars antibodies, this V kappa gene segment (V kappa IdCR) has been found exclusively associated with the J kappa 1 gene segment without exhibiting junctional sequence variation. Sequence comparisons of the germline V kappa IdCR gene with expressed derivatives reveals that the latter frequently contain somatically introduced amino acid replacements. Taken together with results of previous structural analyses, these results show that the predominant population of IdCR+ V regions elicited in the secondary immune response is encoded by one or two combinations of V gene segments, has little junctional diversity, and is extensively diversified by somatic mutation in both heavy and light chains.

T lymphocyte-mediated suppression of myeloma function in vitro. III. Regulation of antibody production in hybrid myeloma cells by T lymphocytes.

To investigate the mechanisms by which T lymphocytes regulate myeloma function in vitro, the effects of regulatory T cells on antibody secretion by a hybrid myeloma cell line were examined. Suppressor T cells (Ts) specific for idiotypic determinants on M315 (IgA, lambda 2 anti-2,4-dinitrophenol and anti-2,4,6-trinitrophenol [TNP]) and MPC 11 (IgG2b, kappa) myeloma proteins inhibit antibody secretion by the appropriate parental myeloma cells. When cocultured with a hybrid cell line derived by fusion of MOPC 315 and MPC 11 myelomas, the idiotype-reactive Ts inhibit secretion of only the immunoglobulin (Ig) bearing the relevant idiotype. In contrast, syngeneic TNP-reactive cytolytic T lymphocytes (CTL) inhibit antibody secretion by TNP-binding MOPC 315 cells but not by MPC 11 cells in the presence of soluble TNP-keyhole limpet hemocyanin (KLH), and this inhibition probably represents a prelytic effect of the CTL. Such TNP-reactive CTL, in the presence of TNP-KLH, inhibit both IgA and IgG secretion by the MOPC 315-MPC 11 hybrid, which is consistent with a prelytic effect. Thus, myeloma hybrids are a useful tool for investigating the effector function of regulatory T cells. These results are discussed with reference to the mechanisms of action of regulatory T cells and their relevance to modulation of physiologic humoral immune responses.

Fine specificity of idiotope suppression in the A/J anti-azophenylarsonate response.

Two hapten-inhibitable murine monoclonal antiidiotopic antibodies identified two idiotopes expressed by the heavy chain of hybridoma protein 36-65, whose amino acid sequence is encoded in the germ line of A/J mice. Among cross-reactive idiotype-positive hybridoma proteins and p-azophenylarsonate-immune antibodies, the two idiotopes were not always expressed together; some diversified antibodies expressed one idiotope without the other. Suppression that was induced by the two antiidiotopes was idiotope specific and corresponded to the fine specificities of these two reagents.

T cell receptor gene usage in the response to lambda repressor cI protein. An apparent bias in the usage of a V alpha gene element.

The T cell response to the lambda repressor cI protein is directed to the same region of the protein (residues 12-26) in both BALB/c and A/J mice. A panel of T cell hybridomas specific for P12-26 in the context of either I-Ek or I-Ad have been isolated To further understand the molecular interaction between the TCR and the Ia-P12-26 complex, the primary structures of the TCR of five T cell hybridomas have been determined. Southern and Northern analyses indicate that two members of the V alpha 3 gene family are used by 13 out of 14 I-Ek-restricted T cells. Four different V beta genes are used by these T cell hybridomas, while the majority (8 out of 13) express V beta 1 in combination with the J beta 2.1 element. No clear correlation can be seen in this system between gene usage and MHC restriction. In addition, the fine specificity of I-Ek-restricted T cells to a single amino acid substitution [Phe22/His22]P12-26 is not attributed to the usage of particular V alpha and V beta elements. The V alpha 3 family gene is also used by a few I-Ad-restricted T cells. Interestingly, these I-Ad T cells share a reactivity pattern more similar to that of I-Ek-restricted T cells than other I-Ad-restricted T cells. The nonrandom selection V alpha 3 is also demonstrated by the fact that V alpha 3 is used by P12-26-specific, but not by cytochrome c- or staphylococcal nucleus-specific, I-Ek-restricted T cells. This suggests that although antigen specificity may not be accounted for by either chain of the TCR, the members of V alpha 3 genes may be selected by the antigen (P12-26).

Influence of clonal selection on the expression of immunoglobulin variable region genes.

The humoral immune response of the mouse to certain antigens is characterized by the dominant expression of a single or limited number of related, immunoglobulin variable region (V) structures by antibody-secreting lymphocytes. Such dominance could be due to preferred expression of these V regions in the B cell population prior to the immune response or could result from the action of selective or regulatory mechanisms during the immune response. Expression of a heavy chain variable region (VH) gene segment that partially encodes a V region structure that dominates the immune response to para-azophenylarsonate (Ars) in strain A mice was examined in the B cell population of Ars nonimmune mice. This VH gene segment participates in encoding several hundred thousand different V region structures expressed in this B cell population. The immune system is therefore capable of recurrently selecting a single V region structure from such a repertoire for dominant expression by antibody-secreting lymphocytes during an immune response.

Murine MHC polymorphism and T cell specificities.

The major histocompatibility complex (MHC) genes are polymorphic in mouse and man. The products of these genes are receptors for peptides, which while bound, are displayed to T lymphocytes. When bound peptides from antigens are recognized by T lymphocytes, an immune response is initiated against the antigens. This study assessed the relation of the polymorphic MHC molecules to their peptide specificity. The results indicate that although an individual of the species has a limited ability to recognize antigens, the species as a whole has broad reactivity. This rationalizes the extreme polymorphism observed.

Structure and in vitro transcription of human globin genes.

The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

Immunological self, nonself discrimination.

The ability of immunodominant peptides derived from several antigen systems to compete with each other for T cell activation was studied. Only peptides restricted by a given transplantation antigen are mutually competitive. There is a correlation between haplotype restriction, ability to bind to the appropriate transplantation antigen, and ability to inhibit activation of other T cells restricted by the same transplantation antigen. An exception was noted in that a peptide derived from an antigen, bacteriophage lambda cI repressor, binds to the I-Ed molecule in a specific way, yet is not I-Ed-restricted. Comparison of the sequence of the repressor peptide with that of other peptides able to bind to (and be restricted by) I-Ed and a polymorphic region of the I-Ed molecule itself revealed a significant degree of homology. Thus, peptides restricted by a given class II molecule appear to be homologous to a portion of the class II molecule itself. The repressor-derived peptide is identical at several polymorphic residues at this site, and this may account for the failure of I-Ed to act as a restriction element. Comparison of antigenic peptide sequences with transplantation antigen sequences suggests a model that provides a basis for explaining self, nonself discrimination as well as alloreactivity.

Preliminary crystallographic studies of the Fab fragment of an anti-azophenylarsonate antibody.

Single crystals of the Fab fragment of a murine A/J anti-azophenylarsonate monoclonal antibody have been prepared by the vapor diffusion method. Antibody 3A7 uses the same combination of variable region gene segments (VK, JK, VH, JH) as do anti-azophenylarsonate antibodies bearing a predominant cross-reactive idiotype, but utilizes a different D gene segment. The crystals grow in the presence of beta-octylglucoside as tetragonal bipyramids in the space group of either P4(1)2(1)2 or P4(3)3(1)2 and with unit cell dimensions of a = b = 77.9 A, and c = 146.7 A. They diffract X-rays to better than 2.7 A resolution. Data up to 2.7 A resolution have been collected.

Affinity analysis of idiotype-positive and idiotype-negative Ars-binding hybridoma proteins and Ars-immune sera.

The possibility that idiotype dominance may be associated with increased affinity for hapten was investigated in the murine A/J anti-p-azophenylarsonate (Ars) response. Fluorescence quenching of 14 Ars-binding hybridoma proteins by Ars-tyrosine was measured and Ka calculated using computer-assisted curve fitting. There was a 200-fold range in Ka for idiotype-positive hybridoma proteins, with 2 IgM hybridoma proteins being near the median. No clear difference in Ka was apparent between idiotype-positive (Id+) and idiotype-negative (Id-) hybridoma proteins. Ka was measured by fluorescence quenching on affinity-purified anti-Ars antibodies from 6 conventional antisera; there was no difference between Id+ and Id- (idiotype suppressed) sera. The affinities of the hybridoma proteins were correlated with the ratio of binding to Ars36-BSA and Ars10-BSA by direct radioimmunoassay. With this calibration, functional affinities of Ars-immune sera could be determined from relative binding ratios without the need for prior affinity purification. This was done for 18 Ars-immune sera, and again there was no clear difference between Id+ and Id- sera. Studies from this laboratory have identified the amino acid sequence of a hybridoma protein which corresponds to the germ line DNA sequence for the cross-reactive idiotype family. The present study shows that the protein directly encoded by the germ line gene has low affinity for hapten suggesting that somatic diversification operating on the germ line sequence can produce antibodies with increased affinity for hapten within the cross-reactive idiotype family. The present study also suggests that affinity is not the driving force behind idiotype dominance of the Ars-immune response.