Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 109 members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

Interactions of protein antigens with antibodies.

There are now several crystal structures of antibody Fab fragments complexed to their protein antigens. These include Fab complexes with lysozyme, two Fab complexes with influenza virus neuraminidase, and three Fab complexes with their anti-idiotype Fabs. The pattern of binding that emerges is similar to that found with other protein-protein interactions, with good shape complementarity between the interacting surfaces and reasonable juxtapositions of polar residues so as to permit hydrogen-bond formation. Water molecules have been observed in cavities within the interface and on the periphery, where they often form bridging hydrogen bonds between antibody and antigen. For the most part the antigen is bound in the middle of the antibody combining site with most of the six complementarity-determining residues involved in binding. For the most studied antigen, lysozyme, the epitopes for four antibodies occupy approximately 45% of the accessible surface area. Some conformational changes have been observed to accompany binding in both the antibody and the antigen, although most of the information on conformational change in the latter comes from studies of complexes with small antigens.

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library

Cancer/testis (CT) antigens—immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types—are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5′ end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

Humoral response to herpes simplex virus is complement-dependent

The complement system represents a cascade of serum proteins, which provide a major effector function in innate immunity. Recent studies have revealed that complement links innate and adaptive immunity via complement receptors CD21/CD35 in that it enhances the B cell memory response to noninfectious protein antigens introduced i.v. To examine the importance of complement for immune responses to virus infection in a peripheral tissue, we compared the B cell memory response of mice deficient in complement C3, C4, or CD21/CD35 with wild-type controls. We found that the deficient mice failed to generate a normal memory response, which is characterized by a reduction in IgG antibody and germinal centers. Thus, complement is important not only in the effector function of innate immunity but also in the stimulation of memory B cell responses to viral-infected cell antigens in both blood and peripheral tissues.

Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an “internal image” monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 × 109 M−1 and 2.4 × 107 M−1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 × 106 M−1 and 3.8 × 106 M−1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.

Mycobacterial infection of macrophages results in membrane-permeable phagosomes

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid α-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, α-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

B cell receptor-associated protein α4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A

Recently, TAP42 was isolated as a high copy suppressor of sit4−, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine α4 protein, which was discovered independently by its association with Ig-α in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)–α4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a “pull-down” assay. In an overlay assay, the GST–α4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of α4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The α4–C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant α4 cleaved from GST was phosphorylated by p56lck tyrosine kinase and protein kinase C. A FLAG-tagged α4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-α4. The results reveal a novel heterodimer α4–C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein

A challenge for subunit vaccines whose goal is to elicit CD8+ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.

Key residues revealed in a major conformational epitope of the U1-70K protein

Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, we have used diversely mutated recombinant Drosophila melanogaster 70K proteins as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Our results reveal that amino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119–126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains, and tertiary structure modeling indicates that the residues 119–126 are situated at easily accessible positions in the end of an α-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.