Antibody–drug conjugates as novel anti-cancer chemotherapeutics

Over the past couple of decades, antibody–drug conjugates (ADCs) have revolutionized the field of cancer chemotherapy. Unlike conventional treatments that damage healthy tissues upon dose escalation, ADCs utilize monoclonal antibodies (mAbs) to specifically bind tumour-associated target antigens and deliver a highly potent cytotoxic agent. The synergistic combination of mAbs conjugated to small-molecule chemotherapeutics, via a stable linker, has given rise to an extremely efficacious class of anti-cancer drugs with an already large and rapidly growing clinical pipeline. The primary objective of this paper is to review current knowledge and latest developments in the field of ADCs. Upon intravenous administration, ADCs bind to their target antigens and are internalized through receptor-mediated endocytosis. This facilitates the subsequent release of the cytotoxin, which eventually leads to apoptotic cell death of the cancer cell. The three components of ADCs (mAb, linker and cytotoxin) affect the efficacy and toxicity of the conjugate. Optimizing each one, while enhancing the functionality of the ADC as a whole, has been one of the major considerations of ADC design and development. In addition to these, the choice of clinically relevant targets and the position and number of linkages have also been the key determinants of ADC efficacy. The only marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), have demonstrated their use against both haematological and solid malignancies respectively. The success of future ADCs relies on improving target selection, increasing cytotoxin potency, developing innovative linkers and overcoming drug resistance. As more research is conducted to tackle these issues, ADCs are likely to become part of the future of targeted cancer therapeutics.     

Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody–drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.     

Antibody–drug conjugates (ADCs) for cancer therapy: Strategies,challenges, and successes

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody–drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.     

A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study

There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.     

Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker

The minor groove binder beta-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a substrate for Escherichia coli beta-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug 1. The ADCs were largely monomeric at intermediate loading levels (4-5drug/mAb), in contrast to highly aggregated p-aminobenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble beta-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.     

Novel peptide linkers for highly potent antibody-auristatin conjugate

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.     

Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.     

Trastuzumab-monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2-positive human breast cancer

Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody–drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV–vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.     

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin β₁₃. It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V_H CDR3 peptide from mAb A4 and V_L CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.     

Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions

 Gangliosides GD3, GD2 and GM2, which are the major gangliosides expressed on most human cancers of neuroectodermal and epithelial origin, have been focused on as effective targets for passive immunotherapy with monoclonal antibodies. We previously developed a chimeric anti-GD3 mAb, KM871, and a humanized anti-GM2 mAb, KM8969, which specifically bound to the respective antigen with high affinity and showed potent immune effector functions. Humanization of anti-ganglioside antibody is expected to enhance its use for human cancer therapy. In the present study, we generated a chimeric anti-GD2 mAb, KM1138, and further developed the humanized form of anti-GD2 and anti-GD3 mAbs by the complementarity-determining regions grafting method. The resultant humanized anti-GD2 mAb, KM8138, and anti-GD3 mAb, KM8871, showed binding affinity and specificity similar to those of their chimeric counterparts. In addition, both humanized mAbs had functional potency comparable to the chimeric mAbs in mediating the immune effector functions, consisting of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The production of these humanized anti-ganglioside mAbs, with potent effector functions and low immunogenicity, precedes the evaluation of the therapeutic value of anti-ganglioside mAbs in passive immunotherapy and the target validation for ganglioside-based vaccine therapy. Received: 30 November 2000 / Accepted: 30 January 2001     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography–mass spectrometry

The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography–mass spectrometry (LC–MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.     

Activity-Modulating Monoclonal Antibodies to the Human Serine Protease HtrA3 Provide Novel Insights into Regulating HtrA Proteolytic Activities

Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential in vivo utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H_N). Epitope mapping data were used to design LC-H_N domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H_N domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.     

Blocking monoclonal antibodies of TIM proteins as orchestrators of anti-tumor immune response

Monoclonal antibody (mAb)-based treatment of cancer has a significant effect on current practice in medical oncology, and is considered now as one of the most successful therapeutic strategies for cancer treatment. MAbs are designed to initiate or enhance anti-tumor immune responses, which can be achieved by either blocking inhibitory immune checkpoint molecules or triggering activating receptors. TIM gene family members are type-I surface molecules expressed in immune cells, and play important roles in the regulation of both innate and adaptive arms of the immune system. Therapeutic strategies based on anti-TIMs mAbs have shown promising results in experimental tumor models, and synergistic combinations of anti-TIMs mAbs with cancer vaccines, adoptive T-cell therapy, radiotherapy and chemotherapy will have great impact on cancer treatment in future clinical development.     

Generation of monoclonal antibodies against non-structural protein 3AB of foot-and-mouth disease virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV), BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained, named C6 and E7 respectively. The microneutralization titer was 1:1024 for mAb C6, and 1:512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens. The titers in abdomen liquor were 1:5×10⁶ for C6 and 1:2×10⁶ for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.