A Novel Synthetic Antibody Library with Complementarity-Determining Region Diversities Designed for an Improved Amplification Profile.

Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries.

A Fully-Human Antibody Specifically Targeting a Membrane-Bound Fragment of CADM1 Potentiates the T Cell-Mediated Death of Human Small-Cell Lung Cancer Cells.

Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. Despite the earlier identification of membrane-proximal cleavage of cell adhesion molecule 1 (CADM1) in cancers, the role of the membrane-bound fragment of CAMD1 (MF-CADM1) is yet to be clearly identified. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1-4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCI-H187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.

Anti-Inflammatory Actions of Soluble Ninjurin-1 Ameliorate Atherosclerosis.

BACKGROUND: Macrophages produce many inflammation-associated molecules, released by matrix metalloproteinases, such as adhesion molecules, and cytokines, as well, which play a crucial role in atherosclerosis. In this context, we investigated the relationship between Ninjurin-1 (Ninj1 [nerve injury-induced protein]), a novel matrix metalloproteinase 9 substrate, expression, and atherosclerosis progression. METHODS: Ninj1 expression and atherosclerosis progression were assessed in atherosclerotic aortic tissue and serum samples from patients with coronary artery disease and healthy controls, and atheroprone apolipoprotein e-deficient (Apoe-/-) and wild-type mice, as well. Apoe-/- mice lacking systemic Ninj1 expression (Ninj1-/-Apoe-/-) were generated to assess the functional effects of Ninj1. Bone marrow transplantation was also used to generate low-density lipoprotein receptor-deficient (Ldlr-/-) mice that lack Ninj1 specifically in bone marrow-derived cells. Mice were fed a Western diet for 5 to 23 weeks, and atherosclerotic lesions were investigated. The anti-inflammatory role of Ninj1 was verified by treating macrophages and mice with the peptides Ninj11-56 (ML56) and Ninj126-37 (PN12), which mimic the soluble form of Ninj1 (sNinj1). RESULTS: Our in vivo results conclusively showed a correlation between Ninj1 expression in aortic macrophages and the extent of human and mouse atherosclerotic lesions. Ninj1-deficient macrophages promoted proinflammatory gene expression by activating mitogen-activated protein kinase and inhibiting the phosphoinositide 3-kinase/Akt signaling pathway. Whole-body and bone marrow-specific Ninj1 deficiencies significantly increased monocyte recruitment and macrophage accumulation in atherosclerotic lesions through elevated macrophage-mediated inflammation. Macrophage Ninj1 was directly cleaved by matrix metalloproteinase 9 to generate a soluble form that exhibited antiatherosclerotic effects, as assessed in vitro and in vivo. Treatment with the sNinj1-mimetic peptides, ML56 and PN12, reduced proinflammatory gene expression in human and mouse classically activated macrophages, thereby attenuating monocyte transendothelial migration. Moreover, continuous administration of mPN12 alleviated atherosclerosis by inhibiting the enhanced monocyte recruitment and inflammation characteristics of this disorder in mice, regardless of the presence of Ninj1. CONCLUSIONS: Ninj1 is a novel matrix metalloproteinase 9 substrate in macrophages, and sNinj1 is a secreted atheroprotective protein that regulates macrophage inflammation and monocyte recruitment in atherosclerosis. Moreover, sNinj1-mediated anti-inflammatory effects are conserved in human macrophages and likely contribute to human atherosclerosis.

Antibody Phage Display.

Antibody phage display has become an indispensable tool for the discovery and optimization of target-specific monoclonal antibodies suitable for demanding applications including therapeutic reagents. The in vitro nature of the technology enables the rapid and efficient identification of specific binders, as well as greater control over selection parameters that facilitates the isolation of antibodies with unique, desirable functional characteristics. In this chapter, the technological background and the state of the art in the field of antibody phage display is discussed.

A Human Antibody That Binds to the Sixth Ig-Like Domain of VCAM-1 Blocks Lung Cancer Cell Migration In Vitro.

Vascular cell adhesion molecule-1 (VCAM-1) is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung tissue, and high VCAM-1 expression correlated with poor survival in lung cancer patients. VCAM-1 knockdown reduced migration of A549 human lung cancer cells into Matrigel, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6) demonstrated that the VCAM-1-D6 domain was critical for VCAM-1 mediated A549 cell migration into Matrigel. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab), which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed the migration of A549 and NCI-H1299 lung cancer cell lines into Matrigel. Taken together, these results suggest that VCAM-1-D6 is a key domain for regulating VCAM-1-mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.

Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells.

Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140-205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs.

A single-domain antibody library based on a stability-engineered human VH3 scaffold.

Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.

5-aminosalicylic acid suppresses osteoarthritis through the OSCAR-PPARγ axis.

Osteoarthritis (OA) is a progressive and irreversible degenerative joint disease that is characterized by cartilage destruction, osteophyte formation, subchondral bone remodeling, and synovitis. Despite affecting millions of patients, effective and safe disease-modifying osteoarthritis drugs are lacking. Here we reveal an unexpected role for the small molecule 5-aminosalicylic acid (5-ASA), which is used as an anti-inflammatory drug in ulcerative colitis. We show that 5-ASA competes with extracellular-matrix collagen-II to bind to osteoclast-associated receptor (OSCAR) on chondrocytes. Intra-articular 5-ASA injections ameliorate OA generated by surgery-induced medial-meniscus destabilization in male mice. Significantly, this effect is also observed when 5-ASA was administered well after OA onset. Moreover, mice with DMM-induced OA that are treated with 5-ASA at weeks 8-11 and sacrificed at week 12 have thicker cartilage than untreated mice that were sacrificed at week 8. Mechanistically, 5-ASA reverses OSCAR-mediated transcriptional repression of PPARγ in articular chondrocytes, thereby suppressing COX-2-related inflammation. It also improves chondrogenesis, strongly downregulates ECM catabolism, and promotes ECM anabolism. Our results suggest that 5-ASA could serve as a DMOAD.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Construction of Rabbit Immune Antibody Libraries.

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Estimation of Cooling Rate of High-Strength Thick Plate Steel during Water Quenching Based on a Dilatometric Experiment.

The microstructure and hardness along the thickness direction of a water-quenched, high-strength thick plate with a thickness of 40 mm were investigated with three specimens from the thick plate: surface, 1/4t, and 1/2t (center) thickness, and the phase transformation behavior of the thick plate according to the cooling rate was analyzed through dilatometric experiments. Finally, the cooling rate for each thickness of the thick plate was estimated by comparing the microstructure and hardness of the thick plate along with the thickness with those of the dilatometric specimens. Martensite microstructure was observed on the surface of the water-quenched thick plate due to the fast cooling rate. On the other hand, an inhomogeneous microstructure was transformed inside the thick plate due to the relatively slow cooling rate and central segregation of Mn. A small fraction of bainite was shown at 1/4t thickness. A banded microstructure with martensite and bainite resulting from Mn segregation was developed at 1/2t; that is, the full martensite microstructure was transformed in the Mn-enriched area even at a slow cooling rate due to high hardenability, but a bainite microstructure was formed in the Mn-depleted area owing to relatively low hardenability. A portion of martensite with fine cementite at the surface and 1/4t was identified as auto-tempered martensite with a Bagaryatskii orientation relationship between the ferrite matrix and cementite. The microstructure and hardness as well as dilatation were investigated at various cooling rates through a dilatometric experiment, and a continuous cooling transformation (CCT) diagram was finally presented for the thick plate. Comparing the microstructure and hardness at the surface, 1/4t, and 1/2t of the thick plate with those of dilatometric specimens cooled at various cooling rates, it was estimated that the surface of the thick plate was cooled at more than 20 °C/s, whereas the 1/4t region was cooled at approximately 5~10 °C/s during water quenching. Despite the difficulty in estimation of the cooling rate of 1/2t due to the banded structure, the cooling rate of 1/2t was estimated between 3 and 5 °C/s based on the results of an Mn-depleted zone.

Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis.

Osteoarthritis (OA) is the most common joint disease that causes local inflammation and pain, significantly reducing the quality of life and normal social activities of patients. Currently, there are no disease-modifying OA drugs (DMOADs) available, and treatment relies on pain relief agents or arthroplasty. To address this significant unmet medical need, we aimed to develop monoclonal antibodies that can block the osteoclast-associated receptor (OSCAR). Our recent study has revealed the importance of OSCAR in OA pathogenesis as a novel catabolic regulator that induces chondrocyte apoptosis and accelerates articular cartilage destruction. It was also shown that blocking OSCAR with a soluble OSCAR decoy receptor ameliorated OA in animal models. In this study, OSCAR-neutralizing monoclonal antibodies were isolated and optimized by phage display. These antibodies bind to and directly neutralize OSCAR, unlike the decoy receptor, which binds to the ubiquitously expressed collagen and may result in reduced efficacy or deleterious off-target effects. The DMOAD potential of the anti-OSCAR antibodies was assessed with in vitro cell-based assays and an in vivo OA model. The results demonstrated that the anti-OSCAR antibodies significantly reduced cartilage destruction and other OA signs, such as subchondral bone plate sclerosis and loss of hyaline cartilage. Hence, blocking OSCAR with a monoclonal antibody could be a promising treatment strategy for OA.

An internalizing antibody targeting of cell surface GRP94 effectively suppresses tumor angiogenesis of colorectal cancer.

Colorectal cancer (CRC) is one of the life-threatening malignancies worldwide. Thus, novel potential therapeutic targets and therapeutics for the treatment of CRC need to be identified to improve the clinical outcomes of patients with CRC. In this study, we found that glucose-regulated protein 94 (GRP94) is overexpressed in CRC tissues, and its high expression is correlated with increased microvessel density. Next, through phage display technology and consecutive in vitro functional isolations, we generated a novel human monoclonal antibody that specifically targets cell surface GRP94 and shows superior internalizing activity comparable to trastuzumab. We found that this antibody specifically inhibits endothelial cell tube formation and simultaneously promotes the downregulation of GRP94 expression on the endothelial cell surface. Finally, we demonstrated that this antibody effectively suppresses tumor growth and angiogenesis of HCT116 human CRC cells without causing severe toxicity in vivo. Collectively, these findings suggest that cell surface GRP94 is a novel potential anti-angiogenic target in CRC and that antibody targeting of GRP94 on the endothelial cell surface is an effective strategy to suppress CRC tumor angiogenesis.

Development and Characterization of Phage-Display-Derived Novel Human Monoclonal Antibodies against the Receptor Binding Domain of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in an ongoing global pandemic crisis, caused by the life-threatening illness coronavirus disease 2019 (COVID-19). Thus, the rapid development of monoclonal antibodies (mAbs) to cope with COVID-19 is urgently necessary. In this study, we used phage display to develop four human mAbs specific to the receptor-binding domain (RBD) of SARS-CoV-2. Our intensive in vitro functional analyses demonstrated that K102.1, an anti-SARS-CoV-2 RBD-specific mAb, exerted potent neutralizing activity against pseudoviral and live viral infection and the interaction between SARS-CoV-2 RBD and human angiotensin-converting enzyme 2. Monotherapy with K102.1 also revealed the therapeutic potential against SARS-CoV-2 infection in vivo. Further, this study developed a sandwich enzyme-linked immunosorbent assay with a non-competing mAb pair, K102.1 and K102.2, that accurately detected the RBDs of SARS-CoV-2 wild-type and variants with high sensitivity in the picomolar range. These findings suggest that the phage-display-based mAb selection from an established antibody library may be an effective strategy for the rapid development of mAbs against the constantly evolving SARS-CoV-2.

Pharmacophore-based strategy for the development of general and specific scFv biosensors for abused antibiotics.

We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFv's bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.

Tensile Properties and Damping Capacity of Cold-Rolled Fe-20Mn-12Cr-3Ni-3Si Damping Alloy.

The tensile properties and damping capacity of cold-rolled Fe-20Mn-12Cr-3Ni-3Si alloys were investigated. The martensitic transformation was identified, including surface relief with a specific orientation and partial intersection. Besides, as the cold rolling degree increased, the volume fraction of ε-martensite increased, whereas α'-martensite started to form at the cold rolling degree of 15% and slightly increased to 6% at the maximum cold rolling degree. This difference may be caused by high austenite stability by adding alloying elements (Mn and Ni). As the cold rolling degree increased, the tensile strength linearly increased, and the elongation decreased due to the fractional increment in the volume of martensite. However, the damping capacity increased until a 30% cold rolling degree was approached, and then decreased. The irregular tendency of the damping capacity was confirmed, depicting that it increased to a specific degree and then decreased as the tensile strength and elongation increased. Concerning the relationship between the tensile properties and the damping capacity, the damping capacity increased and culminated, and then decreased as the tensile properties and elongation increased. The damping capacity in the high-strength area tended to decrease because it is difficult to dissipate vibration energy into thermal energy in alloys with high strength. In the low-strength area, on the other hand, the damping capacity increased as the strength increased since the increased volume fraction of ε-martensite is attributed to the increase in the damping source.

Platelet serotonin transporter function after short-term paroxetine treatment in patients with panic disorder.

Dysfunctions in serotonin neurotransmission have been implicated in some psychiatric disorders, and in particular, altered serotonin transporter function has been noted in panic disorder. In this study, the authors compared platelet [(3)H]serotonin uptake parameters, including maximum velocities (V(max)) and affinity constants (K(m)), in patients with panic disorder not undergoing treatment (n=21) and in healthy subjects (n=20). V(max) and K(m) values were re-examined after 12 weeks of paroxetine treatment. Values of V(max) and K(m) were lower in panic disorder patients at baseline than in healthy subjects. After treatment, K(m) normalized in panic patients, whereas V(max) did not change. A significant inverse correlation was found between increased K(m) and changes in anxiety levels. These results support a hypothesis of serotonergic transporter abnormalities in panic disorder, and suggest that increased K(m) values of platelet serotonin transporters parallel clinical improvement after short-term pharmacotherapy in panic disorder.

Bispecific Antibodies and Antibody-Drug Conjugates for Cancer Therapy: Technological Considerations.

The ability of monoclonal antibodies to specifically bind a target antigen and neutralize or stimulate its activity is the basis for the rapid growth and development of the therapeutic antibody field. In recent years, traditional immunoglobulin antibodies have been further engineered for better efficacy and safety, and technological developments in the field enabled the design and production of engineered antibodies capable of mediating therapeutic functions hitherto unattainable by conventional antibody formats. Representative of this newer generation of therapeutic antibody formats are bispecific antibodies and antibody-drug conjugates, each with several approved drugs and dozens more in the clinical development phase. In this review, the technological principles and challenges of bispecific antibodies and antibody-drug conjugates are discussed, with emphasis on clinically validated formats but also including recent developments in the fields, many of which are expected to significantly augment the current therapeutic arsenal against cancer and other diseases with unmet medical needs.

Osteoclast-associated receptor blockade prevents articular cartilage destruction via chondrocyte apoptosis regulation.

Osteoarthritis (OA), primarily characterized by articular cartilage destruction, is the most common form of age-related degenerative whole-joint disease. No disease-modifying treatments for OA are currently available. Although OA is primarily characterized by cartilage destruction, our understanding of the processes controlling OA progression is poor. Here, we report the association of OA with increased levels of osteoclast-associated receptor (OSCAR), an immunoglobulin-like collagen-recognition receptor. In mice, OSCAR deletion abrogates OA manifestations, such as articular cartilage destruction, subchondral bone sclerosis, and hyaline cartilage loss. These effects are a result of decreased chondrocyte apoptosis, which is caused by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in induced OA. Treatments with human OSCAR-Fc fusion protein attenuates OA pathogenesis caused by experimental OA. Thus, this work highlights the function of OSCAR as a catabolic regulator of OA pathogenesis, indicating that OSCAR blockade is a potential therapy for OA.

An Isoform of the Oncogenic Splice Variant AIMP2-DX2 Detected by a Novel Monoclonal Antibody.

AIMP2-DX2, an exon 2-deleted splice variant of AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein 2), is highly expressed in lung cancer and involved in tumor progression in vivo. Oncogenic function of AIMP2-DX2 and its correlation with poor prognosis of cancer patients have been well established; however, the application of this potentially important biomarker to cancer research and diagnosis has been hampered by a lack of antibodies specific for the splice variant, possibly due to the poor immunogenicity and/or stability of AIMP2-DX2. In this study a monoclonal antibody, H5, that specifically recognizes AIMP2-DX2 and its isoforms was generated via rabbit immunization and phage display techniques, using a short peptide corresponding to the exon 1/3 junction sequence as an antigen. Furthermore, based on mutagenesis, limited cleavage, and mass spectrometry studies, it is also suggested that the endogenous isoform of AIMP2-DX2 recognized by H5 is produced by proteolytic cleavage of 33 amino acids from N-terminus and is capable of inducing cell proliferation similarly to the uncleaved protein. H5 monoclonal antibody is applicable to enzyme-linked immunosorbent assay, immunoblot, immunofluorescence, and immunohistochemistry, and expected to be a valuable tool for detecting AIMP2-DX2 with high sensitivity and specificity for research and diagnostic purposes.