Generation of a Human Anti-Tumor Necrosis Factor-α Monoclonal Antibody by in Vitro Immunization with a Multiple Antigen Peptide

We developed the in vitro immunization method to induce antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). However, when we used a peptide as sensitizing antigen, the antigen-specific immune response was found to be weak, and hence, we could not effectively obtain the antigen-specific antibody gene. In the present study, we attempted to improve the in vitro immunization method by augmenting the immune response to the peptide antigen. We used a multiple antigen peptide for sensitization. In vitro immunization of the multivalent antigen elicited a strong antigen-specific immune response in the PBMCs, and we succeeded in obtaining antigen-specific antibody genes by the phage-display method. Further, by combining the variable-region genes and constant-region genes of human IgG, we obtained four independent human monoclonal antibodies specific for tumor necrosis factor-α. This might be a good strategy for generating antigen-specific human monoclonal antibodies using a peptide antigen.     

Direct cDNA cloning of the rearranged immunoglobulin variable region

A major problem in the study of multigene families is the effort required to clone and sequence these genes. We describe a method to rapidly clone and sequence immunoglobulin variable region gene sequences without constructing cDNA libraries. Because immunoglobulin variable-region genes are flanked by conserved sequences, we have been able to apply the polymerase chain reaction (PCR) to clone and sequence both the light- and heavy-chain rearranged immunoglobulin genes from small numbers of hybridoma cells. This method will greatly facilitate the construction of chimeric mouse/human monoclonal antibodies for immunoglobulin structural studies as well as for therapeutic use.     

On the skew distribution of immunoglobulins and the inverted protein-folding problem

The question of antibody specificity is discussed in the framework of the inverted protein-folding problem (i.e. the characterization of protein sequences with a common fold). A stochastic model of the immune response, patterned after a model for the distribution of words in natural languages is proposed. It is shown that the steady-state probability distribution of immunoglobulin variable-region frequencies is the Yule distribution.     

Isoelectric focus analysis of rat anti-phosphocholine antibodies.

Anti-phosphocholine (PC) antibodies in sera from four strains of rats were examined before and afterimmunization with either Streptococcus pneumoniae R36A, which contains PC as a cell wall component, or with PC-coupled keyhole limpet hemocyanin (PC-KLH). PC-specific protein was purified from pooled immune sera and shown by a combination of isoelectric focus (IEF) in acrylamide and crossed immunoelectrophoresis, as well as by molecular weight determination in NaDodSO4-acrylamide, to be immunoglobulin. An additional, small molecular weight, nonimmunoglobulin protein (pI = 7.1-7.3) was present in sera from normal and germ-free rats which had the ability to bind the C-carbohydrate of S. pneumoniae R36A, but without specificity for PC. The IEF profile of normal and immune sera showed marked sharing of bands of anti-PC antibody between individual rats as well as between strains. In addition, other anti-PC antibodies which focused between pH 8.5 and 9.5 were less regularly shared. The uniformity of IEF profile of the bulk of anti-PC antibodies in rats is most consistent with their being the products of germ line genes.     

Novartis Medal Lecture. Antibodies: a paradigm for the evolution of molecular recognition

Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen -- but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.     

Model immune complexes formed between monoclonal antibodies and a novel bivalent affinity label

Murine monoclonal anti-dinitrophenyl antibodies were produced by lymphocyte hybridomas and incubated with a new bivalent affinity label, bis-(dinitrofluorobenzene)-pimelic acid amide. Stable dimers were isolated from the resulting mixture of immune complexes in high purity. Experiments were performed to show that the immune complexes are covalently cross-linked through antibody active sites. Model dimers were also formed with varying ratios of monoclonal anti-hapten antibodies based on immunoglobulin isotype. The potential uses of these complexes for analyses of the biological activites of immune aggregates are discussed.     

Identification of aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection, virus neutralization, and immune complex formation

Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.     

Prevalence and Gene Characteristics of Antibodies with Cofactor-induced HIV-1 Specificity

The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses.     

Abnormalities of B cell phenotype, immunoglobulin gene expression and the emergence of autoimmunity in Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathologic features and the production of typical autoantibodies. In addition, characteristic changes in the distribution of peripheral B cell subsets and differences in use of immunoglobulin variable-region genes are also features of pSS. Comparison of B cells from the blood and parotid gland of patients with pSS with those of normal donors suggests that there is a depletion of memory B cells from the peripheral blood and an accumulation or retention of these antigen-experienced B cells in the parotids. Because disordered selection leads to considerable differences in the B cell repertoire in these patients, the delineation of its nature should provide important further clues to the pathogenesis of this autoimmune inflammatory disorder.     

Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA.     

Positive darwinian selection observed at the variable-region genes of immunoglobulins

To examine Gojobori and Nei's hypothesis that the immunoglobulin heavy- chain variable-region (VH) genes in mammals are subject to diversity- enhancing selection, we studied the rates of synonymous and nonsynonymous nucleotide substitution in the complementarity- determining regions (CDRs) and in the framework regions (FRs) of mouse and human VH genes. The results obtained indicate that the non- synonymous rate is higher than the synonymous rate in CDRs, whereas the reverse is true in FRs. This observation supports Gojobori and Nei's hypothesis and suggests that diversity-enhancing selection (similar to overdominant selection) operates mainly in CDRs and that this is one of the evolutionary factors that increase antibody diversity.      

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V_H repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V_H3 (41%), V_H4 (39%), and V_H1 (14%) subgroups used more frequently than the V_H5 (3.9%) or V_H7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human V_H genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.     

Abnormalities of B cell phenotype,immunoglobulin gene expression and the emergence of autoimmunity in Sjögren's syndrome

Primary Sj?gren's syndrome (pSS) is an autoimmune disorder characterized by specific pathologic features and the production of typical autoantibodies. In addition, characteristic changes in the distribution of peripheral B cell subsets and differences in use of immunoglobulin variable-region genes are also features of pSS. Comparison of B cells from the blood and parotid gland of patients with pSS with those of normal donors suggests that there is a depletion of memory B cells from the peripheral blood and an accumulation or retention of these antigen-experienced B cells in the parotids. Because disordered selection leads to considerable differences in the B cell repertoire in these patients, the delineation of its nature should provide important further clues to the pathogenesis of this autoimmune inflammatory disorder.     

Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. Implications for the molecular basis of human autoimmune responses

In order to investigate the genetic basis for natural anti-DNA immune responses, we isolated and sequenced the variable gene elements (VH and VL) encoding an anti-DNA antibody expressed by a human hybridoma of normal origin (Kim4.6) and compared these sequences with those reported for four other human anti-DNA antibodies. The Kim4.6 antibody leader and VH segments were identical in nucleotide sequence with the VH1.9III germ-line VH3 gene, and the Kim4.6VL segment showed 98% nucleotide sequence identity with a V lambda I subgroup gene expressed in a Burkitt's lymphoma. Comparative analysis of Kim4.6 and other human hybridoma anti-DNA antibodies indicated that anti-DNA immune responses are diverse in terms of VH and VL gene utilization but may exhibit a bias toward rearrangement of VH genes that are over-represented in the fetal pre-B cell repertoire. Moreover, Kim4.6 and three of four other sequenced human anti-DNA antibodies appear to use a germ-line diversity gene, DXP'1, which may represent a counterpart of the DFL16.1 segment utilized in murine responses to the hapten nitrophenyl. Taken together, our findings indicate that anti-DNA immune responses can be encoded by nonmutated VH genes and that the elements and molecular mechanisms which engender this response are essentially the same among natural and lupus-associated anti-DNA antibodies. Our data also suggest that natural autoimmune responses originate early in B cell ontogeny as is consistent with the hypothesis that autoreactivity plays a major role in shaping the normal immune repertoire.     

Construction of chimeric antibodies: cloning of immunoglobulin genes including their promoter regions by PCR.

In the production of recombinant antibodies, it is necessary to have an immunoglobulin gene promoter for driving the expression of the antibody genes. Here we describe a simple PCR method that allows cloning of the immunoglobulin genes together with their own promoters despite the fact that the sequence of the upstream part of the gene is unknown.     

Antibody engineering for therapeutics

With the acceptance of antibodies as therapeutics, a diversity of engineered antibody forms have been created to improve their efficacy, including enhancing the effector functions of full-length antibodies, delivering toxins to kill cells or cytokines in order to stimulate the immune system, and bispecific antibodies to target multiple receptors. After years of in vitro investigation, many of these are now moving into clinical trials and are showing promise. A potential new type of effector function for antibodies, that is, the generation of reactive oxygen species that may effect inflammation or bacterial killing, has been elucidated. In addition, the field has expanded beyond a concentration on immunoglobulin G to include immunoglobulin A antibodies as potential therapeutics.     

鲨源单域抗体的研究进展

单克隆抗体是重要的生物大分子,在免疫检测、体外诊断以及药物开发等领域获得广泛的应用。但是单克隆抗体的分子量大、结构复杂等固有属性正日益成为制约其进一步发展的关键因素。因此,当前迫切需要开发单克隆抗体的替代品。鲨源单域抗体即鲨源新抗原受体可变区(Variabledomainofimmunoglobulinnew antigenreceptor,VNAR),是基于鲨总目鱼类天然存在的新抗原受体(Immunoglobulinnewantigenreceptor,IgNAR)并通过基因工程技术获得的抗原结合域,其分子量仅为12kDa,是目前已知脊椎动物中尺寸最小的抗原结合域。鲨源单域抗体具有分子量小、亲和力高、稳定性强、溶解度好、组织穿透性强以及可识别隐藏抗原表位等优点,在免疫试剂以及药物开发等领域受到广泛的关注。文中综述了鲨源单域抗体的结构及功能特性、制备及人源化改造技术、亲和力成熟策略以及应用领域,并系统性分析了鲨源单域抗体的优缺点,最后对其发展前景进行了展望。