Fast on-rates of chimeric antigen receptors enhance the sensitivity to peptide MHC via antigen rebinding.

Chimeric antigen receptor (CAR) is a synthetic receptor that induces T cell-mediated lysis of abnormal cells. As cancer driver proteins are present at low levels on the cell surface, they can cause weak CAR reactivity, resulting in antigen sensitivity defects and consequently limited therapeutic efficacy. Although affinity maturation enhances the efficacy of CAR-T cell therapy, it causes off-target cross-reactions resulting in adverse effects. Preferentially expressed antigen in melanoma (PRAME) is an intracellular oncoprotein that is overexpressed in various tumors and restricted in normal tissues, except the testis. Therefore, PRAME could be an ideal target for cancer immunotherapy. In this study, we developed an experimental CAR system comprising six single-chain variable fragments that specifically recognizes the PRAMEp301/HLA-A*24:02 complex. Cell-mediated cytotoxicity was demonstrated using a panel of CARs with a wide range of affinities (KD = 10-10-10-7 M) and affinity modulation. CAR-T cells with fast on-rates enhance antigen sensitivity by accelerating the killing rates of these cells. Alanine scanning data demonstrated the potential of genetically engineered CARs to reduce the risk of cross-reactivity, even among CARs with high affinities. Given the correlation between on-rates and dwell time that occurs in rebinding and cell-mediated cytotoxicity, it is proposed that CAR-binding characteristics, including on-rate, play a pivotal role in the lytic capacity of peptide-major histocompatibility complex-targeting CAR-T cells, thus facilitating the development of strategies whereby genetically engineered CARs target intracellular antigens in cancer cells to lyse the cells.

Preclinical evaluation of a novel CAR-T therapy utilizing a scFv antibody highly specific to MAGE-A4p230-239/HLA-A∗02:01 complex.

Despite the revolutionary success of chimeric antigen receptor (CAR)-T therapy for hematological malignancies, successful CAR-T therapies for solid tumors remain limited. One major obstacle is the scarcity of tumor-specific cell-surface molecules. One potential solution to overcome this barrier is to utilize antibodies that recognize peptide/major histocompatibility complex (MHCs) in a T cell receptor (TCR)-like fashion, allowing CAR-T cells to recognize intracellular tumor antigens. This study reports a highly specific single-chain variable fragment (scFv) antibody against the MAGE-A4p230-239/human leukocyte antigen (HLA)-A∗02:01 complex (MAGE-A4 pMHC), screened from a human scFv phage display library. Indeed, retroviral vectors encoding CAR, utilizing this scFv antibody as a recognition component, efficiently recognized and lysed MAGA-A4+ tumor cells in an HLA-A∗02:01-restricted manner. Additionally, the adoptive transfer of T cells modified by the CAR-containing glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related receptor (GITR) intracellular domain (ICD), but not CD28 or 4-1BB ICD, significantly suppressed the growth of MAGE-A4+ HLA-A∗02:01+ tumors in an immunocompromised mouse model. Of note, a comprehensive analysis revealed that a broad range of amino acid sequences of the MAGE-A4p230-239 peptide were critical for the recognition of MAGE-A4 pMHC by these CAR-T cells, and no cross-reactivity to analogous peptides was observed. Thus, MAGE-A4-targeted CAR-T therapy using this scFv antibody may be a promising and safe treatment for solid tumors.

Exploring TCR-like CAR-Engineered Lymphocyte Cytotoxicity against MAGE-A4.

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.

CAR-Modified Vγ9Vδ2 T Cells Propagated Using a Novel Bisphosphonate Prodrug for Allogeneic Adoptive Immunotherapy.

The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αß T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of 'off-the-shelf' CAR-T cell products for successful allogeneic adoptive immunotherapy.

Antitumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination.

The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.

αß-T cell receptor transduction gives superior mitochondrial function to γδ-T cells with promising persistence.

Adoptive cell therapy using allogeneic γδ-T cells is a promising option for off-the-shelf T cell products with a low risk of graft-versus-host disease (GVHD). Long-term persistence may boost the clinical development of γδ-T cell products. In this study, we found that genetically modified Vγ9+Vδ2+ T cells expressing a tumor antigen-specific αß-TCR and CD8 coreceptor (GMC) showed target-specific killing and excellent persistence. To determine the mechanisms underlying these promising effects, we investigated metabolic characteristics. Cytokine secretion by γδ-TCR-stimulated nongene-modified γδ-T cells (NGMCs) and αß-TCR-stimulated GMCs was equally suppressed by a glycolysis inhibitor, although the cytokine secretion of αß-TCR-stimulated GMCs was more strongly inhibited by ATP synthase inhibitors than that of γδ-TCR-stimulated NGMCs. Metabolomic and transcriptomic analyses, flow cytometry analysis using mitochondria-labeling dyes and extracellular flux analysis consistently suggest that αß-TCR-transduced γδ-T cells acquire superior mitochondrial function. In conclusion, αß-TCR-transduced γδ-T cells acquire superior mitochondrial function with promising persistence.

TCR-Engineered Lymphocytes Targeting NY-ESO-1: In Vitro Assessment of Cytotoxicity against Tumors.

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling.

FMS-related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70-100% of cases of AML and in virtually all cases of B-lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small-molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off-target toxicities and drug resistance. The development of anti-FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)-induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor-dependent cell line independently of FL addition. In addition, A2 showed complement-dependent cytotoxicity activity, but was devoid of Ab-dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell-based immunotherapies.

Neutralizing anti-gH antibody of Varicella-zoster virus modulates distribution of gH and induces gene regulation, mimicking latency.

The anti-glycoprotein H (gH) monoclonal antibody (anti-gH-MAb) that neutralizes varicella-zoster virus (VZV) inhibited cell-to-cell infection, resulting in a single infected cell without apoptosis or necrosis, and the number of infectious cells in cultures treated with anti-gH-MAb declined to undetectable levels in 7 to 10 days. Anti-gH-MAb modulated the wide cytoplasmic distribution of gH colocalized with glycoprotein E (gE) to the cytoplasmic compartment with endoplasmic reticulum (ER) and Golgi markers near the nucleus, while gE retained its cytoplasmic distribution. Thus, the disintegrated distribution of gH and gE caused the loss of cellular infectivity. After 4 weeks of treatment with anti-gH-MAb, no infectious virus was recovered, even after cultivation without anti-gH-MAb for another 8 weeks or various other treatments. Cells were infected with Oka varicella vaccine expressing hepatitis B surface antigen (ROka) and treated with anti-gH-MAb for 4 weeks, and ROka was recovered from the quiescently infected cells by superinfection with the parent Oka vaccine. Among the genes 21, 29, 62, 63, and 66, transcripts of gene 63 were the most frequently detected, and products from the genes 63 and 62, but not gE, were detected mainly in the cytoplasm of quiescently infected cells, in contrast to their nuclear localization in lytically infected cells. The patterns of transcripts and products from the quiescently infected cells were similar to those of latent VZV in human ganglia. Thus, anti-gH-MAb treatment resulted in the antigenic modulation and dormancy of infectivity of VZV. Antigenic modulation by anti-gH-MAb illuminates a new aspect in pathogenesis in VZV infection and the gene regulation of VZV during latency in human ganglia.

Selection and analysis of anti-cancer antibodies for cancer therapy obtained from antibody phage library.

The search for effective antibodies (Ab) for curable cancer immunotherapy has been a quest of many research groups in order to find an effective target that exists on the cancer cell surface. So far there have been no conclusive answers to shed light on the search. This study therefore aimed to bridge the gap of cancer therapy. Screening against 49 kinds of cell lines belonging to 11 kinds of solids cancers was performed. Isolation and characterization for approximately 4200 monoclonal antibodies (mAb) was also performed thereafter. Of those mAb 488 clones that turned out to bind to 29 tumor-associated antigens (TAA) were subjected to immunohistochemical (IHC) analyses. Selection of target antigens (Ag) and a potential antibody for cancer therapy was conducted prior to clinical examinations. In order to find predictably effective targets for therapeutic Ab against solid cancers, expression of the Ag on the surface of cancer and normal cells was extensively examined by IHC analyses using fresh cancer specimens resected from patients. In this study, the tendencies of all staining patterns and distribution of the Ab are reported. While all of the TAA appeared to be involved in tumorigenesis, their expression was not restricted to some specific tumor types but rather randomly distributed among various cancers. Some kinds of Ab including anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) indicated the frequency of expression in normal cells was generally low. We concluded that identification of 488 mAb and the accumulated results of IHC analyses in this study could be the key for further therapeutic Ab against cancers. The targets that showed cancer-specific expression are expected to be better for therapeutic Ab than the other Ab. Moreover, further investigation into the growth of cancer cell lines using full human IgG form of Ab shows available efficacy in specific cases.

Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic.

Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG(1) Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG(1) revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.

Characterization of neutralizing epitopes of varicella-zoster virus glycoprotein H.

Varicella-zoster virus (VZV) glycoprotein H (gH) is the major neutralization target of VZV, and its neutralizing epitope is conformational. Ten neutralizing human monoclonal antibodies to gH were used to map the epitopes by immunohistochemical analysis and were categorized into seven epitope groups. The combinational neutralization efficacy of two epitope groups was not synergistic. Each epitope was partially or completely resistant to concanavalin A blocking of the glycomoiety of gH, and their antibodies inhibited the cell-to-cell spread of infection. The neutralization epitope comprised at least seven independent protein portions of gH that served as the target to inhibit cell-to-cell spread.

Isolation of antigen/antibody complexes through organic solvent (ICOS) method.

We developed a method termed ICOS (isolation of antigen-antibody complexes through organic solvent) for comprehensive isolation of monoclonal antibodies (mAbs) bound to molecules on the cell surface. By mixing a large number of phage particles of an antibody (Ab) library with living cells, antigen (Ag)-Ab complexes were formed on the cell surface. The mixture was overlaid on organic solution in a tube and subjected to centrifugation. Phages bound to cells were recovered from the precipitate. The phage fraction isolated turned out to contain mAbs that bind to very heterogeneous epitopes and show strong binding activity to Ags. The ICOS method was applied to isolation of human mAbs that may be therapeutic against cancers. Sixty percent of clones isolated by the screening of a phage Ab library against cancer cells turned out to bind to various kinds of tumor-associated Ags. The precise protocol of ICOS method and the rationale of efficient screening were described.

Frequent overexpression of CADM1/IGSF4 in lung adenocarcinoma.

We reported comprehensive screening for antigens (Ags) overexpressed on various carcinomas via isolation of human monoclonal antibodies (mAbs) that may be therapeutic in a previous paper (Proc. Natl. Acad. Sci. USA 105, 7287-7292, 2008). Twenty-one distinct Ags highly expressed on several carcinomas were identified and 356 mAbs with unique sequences turned out to bind to one of the 21 Ags. Among them CADM1/IGSF4 which had been originally referred to as tumor suppressor lung cancer 1 (TSLC1) was included. Therefore we examined the expression of CADM1 in lung cancers in this study. Eight different anti CADM1 mAbs were used for immunohistochemical analysis of 29 fresh lung cancer specimens. Staining patterns were categorized to six groups based on the extent of positive staining and the localization of stained portions. While overexpression of CADM1 was observed on the cell surface of adenocarcinomas at a high frequency, around 60%, positive stainings were rarely observed on that of other lung carcinomas including squamous cell carcinomas. Moreover, some clones among the eight mAbs gave different staining patterns from those by the other clones against the same fresh specimen, suggesting presence of variant forms of CADM1 differentiated by mAbs.

Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types.

Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases.

Dendrodendritic electrical synapses between mammalian retinal ganglion cells.

Electrical synapses between alpha-type ganglion cells were detected using combined techniques of dual patch-clamp recordings, intracellular labeling, electron microscopy, and channel subunit connexin immunocytochemistry in the albino rat retina. After intracellular injection of Neurobiotin into alpha-cells of inner (ON-center) and outer (OFF-center) ramifying types, measurement of tracer coupling resulted in a preferentially homologous occurrence among cells of the same morphological type (n = 19 of 24). In high-voltage as well as conventional electron microscopic analysis, direct dendrodendritic gap junctions (average size, 0.86 mum long) were present in contact sites between tracer-coupled alpha-cells. In simultaneous dual whole-cell recordings from pairs of neighboring alpha-cells, these cells generated TTX-sensitive sustained spiking against extrinsic current injection, and bidirectional electrical synapses (maximum coupling coefficient, 0.32) with symmetrical junction conductance (average, 1.35 nS) were observed in pairs with cells of the same morphological type. Precise temporal synchronization of spike activity (average time delay, 2.7 msec) was detected when depolarizing currents were simultaneously injected into the pairs. To address whether physiologically identified electrical synapses constitute gap junctional connectivity between cell pairs, identified neuronal connexin36 immunoreactivity was undertaken in Lucifer yellow-labeled cell pairs after patch-clamp recordings. All alpha-cells expressed connexin36, and confocal laser-scanning imaging demonstrated that connexin36 is primarily located at dendritic crossings between electrically coupled cells (seven sites in a pair, on average). These results give conclusive evidence for electrical synapses via dendrodendritic gap junctions involving connexin36 in alpha retinal ganglion cells of the same physiological type.

Novel human monoclonal antibody against epidermal growth factor receptor as an imaging probe for hepatocellular carcinoma.

OBJECTIVE: The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, including hepatocellular carcinoma (HCC), and is an attractive target for cancer imaging and therapy. We attempted a novel noninvasive imaging method to evaluate anti-EGFR human monoclonal antibody clones for determining the uptake of therapeutic anti-EGFR antibody in HCC. METHODS: In-vitro cell binding of nine I-labeled antibody clones was compared in the human epidermoid cancer cell line A431, in three HCC cell lines Hep-G2, SK-Hep1, and HuH-7, and in the EGFR-negative control cell line A4. In-labeled or I-labeled 048-006 was subjected to cell binding, competitive inhibition, and internalization assays using A431, SK-Hep1, and HuH-7. Further, In-labeled 048-006 was evaluated in in-vivo biodistribution analysis and single-photon imaging in nude tumor-bearing mice. RESULTS: The 048-006 clone showed the highest binding to EGFR-expressing cells among the nine antibodies. In-labeled or I-labeled 048-006 specifically bound to EGFR-expressing cells with high affinity and was internalized after binding to EGFR. A431 and HuH-7 tumors showed high In-labeled 048-006 uptake, which was visualized by single-photon imaging. CONCLUSION: Radiolabeled human anti-EGFR monoclonal antibody 048-006 has the potential to be a safer imaging probe for predicting tumor uptake of anti-EGFR antibody therapeutic agents in HCC.

Antibody-dependent cell-mediated cytotoxicity is induced by a single-chain Fv-protein III fusion in the presence of a rabbit anti-protein III polyclonal antibody.

The use of phage-displayed antibody libraries has enabled the isolation of several thousand cancer-specific monoclonal antibodies. To further select for clones among these antibodies which have therapeutic potential for cancer, several types of in vitro anti-tumor assay, such as an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, are required. The cytotoxic activities of effector cells are triggered by the binding of the Fc portion of IgG to its receptor, necessitating the conversion of a candidate clone with a single-chain variable fragment (scFv) form into a human IgG form. In the laboratory however, this conversion process is expensive and involves laborious steps such as the cloning of mammalian cells that contain an IgG expression vector, the subsequent production of protein, and affinity purification. In our current study, we show that an original fusion of scFv and protein III, a coat protein of the M13 bacteriophage, can induce ADCC activity towards its target cells in the presence of a rabbit anti-protein III polyclonal antibody. Our modified assay method thus enables the more rapid selection of potentially therapeutic clones from phage-displayed antibody libraries.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.

Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies.

In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. Thereafter, the Ags recognized by the mAbs were identified. For identification of the Ags by MS candidate molecules had to be purified either by immunoprecipitation or by affinity chromatography. We isolated several hundred mAbs that showed cancer-specific staining patterns. In order to identify the Ags that were recognized by the numerous mAbs within a short time we developed two methods. Using the GFC [grouping of clones by flow cytometry (FCM)] method many Abs could be grouped by comparing the staining patterns of FCM. Members in each group turned out to bind to the same molecule in many cases. After a candidate Ag was revealed, the polypeptide corresponding to its extracellular portion was prepared and used for identification of clones that bound to the Ag among all the mAbs by SITE (simultaneous identification of clones through three dimensional ELISA) method. Both methods can be generally applicable to various kinds of membrane proteins and the mAbs against them.