Cryptococcosis remains one of the most severe invasive fungal diseases worldwide, causing high morbidity and mortality, particularly among immunocompromised individuals. Despite the availability of antifungal drugs, current therapeutic regimens are limited by drug toxicity, prolonged treatment, antifungal resistance, biofilm-associated tolerance, and frequent disease relapse, highlighting the need for novel therapeutic approaches. Monoclonal antibodies (mAbs) have emerged as promising immunotherapeutic agents because of their ability to specifically target fungal components while modulating host immune responses. This review provides a comprehensive overview of recent advances in monoclonal antibody-based strategies against Cryptococcus spp. Additionally, recent innovations in antibody engineering, including radioimmunotherapy, Fc engineering, nanobody technology, immune checkpoint modulation, and chimeric antigen receptor-(CAR)-based cellular therapies, are highlighted as emerging approaches with the potential to enhance antifungal efficacy and overcome current therapeutic limitations. Finally, we discuss the major challenges associated with the clinical translation of antifungal mAbs, including pharmacokinetic barriers, production costs, limited clinical evidence, and regulatory constraints. Collectively, the available evidence supports mAbs as promising adjunctive immunotherapeutic agents and underscores the importance of continued advances in antibody engineering and clinical research to facilitate their incorporation into future strategies for the treatment of cryptococcosis.
Cadherin-5 (CDH5), also known as vascular endothelial cadherin (VE-cadherin), plays crucial roles in endothelial cell adhesion, vascular barrier function, and signaling. CDH5 manages endothelial cell–cell junctions during vascular remodeling, which is vital for both vascular homeostasis and adaptive responses to pathological stimuli. Although anti-CDH5 monoclonal antibodies (mAbs) are used for specific applications such as flow cytometry, Western blotting, and immunohistochemistry (IHC), highly sensitive and versatile anti-CDH5 mAbs suitable for all these applications remain limited. Here, new anti-human CDH5 mAbs, called Ca 5 Mabs, were developed through a flow cytometry-based high-throughput screening. Among them, clone Ca 5 Mab-8 (mouse IgG 2a , kappa) recognized CDH5-overexpressed Chinese hamster ovary-K1 (CHO/CDH5) cells in flow cytometry. Moreover, Ca 5 Mab-8 also recognized endogenous CDH5-expressing human endothelial cell lines (HUVEC/TERT2 and HDMVEC/TERT164-B) and a cervical cancer cell line (HeLa). These reactivities exceeded those of a commercial anti-CDH5 mAb (clone BV9). The apparent dissociation constant of Ca 5 Mab-8 for CHO/CDH5 was measured as 6.1 × 10 −9 M. Ca 5 Mab-8 can detect endogenous CDH5 in Western blotting. In addition, Ca 5 Mab-8, but not BV9, can be used for IHC to detect endothelial cells in formalin-fixed paraffin-embedded tissues. These findings suggest that Ca 5 Mab-8 is a versatile basic research tool in vascular biology and has potential for clinical applications including tumor diagnosis and therapy.
The CXC chemokine receptor 2 (CXCR2) is a member of the G-protein-coupled receptor superfamily and regulates a diverse range of immune responses and tumor progression. CXCR2 is expressed on immune cells, especially neutrophils, and is involved in various immune responses by interacting with its chemokine ligands. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR2 has been desired for treatment and diagnosis. This study established a novel sensitive anti-mouse CXCR2 (mCXCR2) mAb; Cx 2 Mab-5 (rat IgG 2a , κ), using the mCXCR2 synthetic N-terminus peptide immunization method. In flow cytometry, Cx 2 Mab-5 recognized mCXCR2-overexpressed Chinese hamster ovary-K1 cells (CHO/mCXCR2) and WEHI-3B (murine myelomonocytic leukemia cell) cells, which express endogenous mCXCR2. Cx 2 Mab-5 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. Cx 2 Mab-5 showed a moderate binding affinity for both CHO/mCXCR2 and WEHI-3B. Furthermore, Cx 2 Mab-5 detected mCXCR2 in Western blot and immunohistochemistry in CHO/mCXCR2 cells, but a commercially available anti-mCXCR2 mAb (clone SA045E1) did not. Hence, Cx 2 Mab-5 can be a valuable tool for analyzing mCXCR2-positive cells in mouse tissues.
Glypican-3 (GPC3) belongs to the glypican family of heparan sulfate proteoglycans and is frequently overexpressed in hepatocellular carcinoma (HCC). The overexpression of GPC3 is associated with the poor clinical outcomes, suggesting its potential as a clinically relevant biomarker and therapeutic target. Therefore, anti-GPC3 monoclonal antibodies (mAbs) have been developed in various modalities for tumor diagnosis and therapy. In this study, 88 clones of novel anti-GPC3 mAbs were established using a flow cytometry-based high-throughput screening, the Cell-Based Immunization and Screening (CBIS) method. Among them, a clone G3Mab-25 (IgG1, κ) recognized GPC3-overexpressed Chinese hamster ovary-K1 (CHO/GPC3) but not parental CHO-K1 in flow cytometry. Furthermore, G3Mab-25 recognizes endogenous GPC3 in GPC3-expressing HCC cell lines, including HepG2, HuH-7, and JHH-5. G3Mab-25 specifically recognized only CHO/GPC3, but not other GPC family-overexpressed CHO-K1. The dissociation constant values of G3Mab-25 for CHO/GPC3, HepG2, HuH-7, and JHH-5 were determined to be 1.8 × 10-8 M, 7.3 × 10-9 M, 3.9 × 10-9 M, and 1.4 × 10-9 M, respectively. Moreover, G3Mab-25 detects the N-terminal fragment of GPC3 in western blotting. In immunohistochemistry, G3Mab-25 showed a diverse staining pattern for GPC3 in HCC tissues. G3Mab-25, established by the CBIS method, is a versatile mAb for basic research and is expected to contribute to tumor diagnosis and therapy.
Leukocyte migration is a fundamental process in both innate and adaptive immune responses. This process is tightly regulated by chemokines and their cognate receptors. The bioavailability of chemokines is further modulated by atypical chemokine receptors (ACKRs), a subset of chemokine receptor-like molecules that lack coupling to canonical G protein-mediated signaling pathways. Among these, ACKR4 regulates dendritic cell migration through ligand scavenging and has been implicated in tumor progression in murine models. We previously established anti-mouse ACKR4 (mACKR4) mAbs, A4Mab-1, A4Mab-2, and A4Mab-3, by N-terminal peptide immunization. This study examined the binding epitopes of A4Mabs. Alanine (or glycine) scanning within the N-terminal region (amino acids 2-19) was performed using flow cytometry and Western blotting. Results showed that Tyr12 is required for recognition by A4Mab-1 in flow cytometry, whereas Tyr11, Tyr12, Glu14, Glu15, and Glu17 are required in Western blotting. For A4Mab-2, Tyr12, Glu15, and Asn16 are required in flow cytometry, whereas Tyr11, Tyr12, Tyr13, Glu15, and Asn16 are required in Western blotting. Additionally, Glu14, Asn16, and Glu17 are required for recognition by A4Mab-3 in flow cytometry. These findings contribute to the understanding of mACKR4 recognition by A4Mabs.
The C-C motif chemokine receptor 1 (CCR1) plays key roles in guiding leukocyte movement during immune surveillance and inflammatory responses. Targeting CCR1 is a promising approach for treating autoimmune diseases and cancers. We previously developed monoclonal antibodies against mouse CCR1 (mCCR1) (clones C1Mab-2 and C1Mab-6) for use in flow cytometry and Western blotting. However, the specific binding sites have not yet been identified. This study examined the binding epitope of C1Mab-2 and C1Mab-6 using flow cytometry. Analysis of mCCR1 mutants with altered extracellular domains showed that C1Mab-2 and C1Mab-6 bind to the N-terminal region of mCCR1. Additionally, PA-tag substitution experiments identified the epitope as 1st Met and amino acids 2-13 of mCCR1. Further alanine (or glycine) scanning within the N-terminal region (amino acids 2-13) demonstrated that Glu2, Asp5, and Phe6 are essential for recognition by C1Mab-2, and Glu2, Ile3, and Asp5 are crucial for recognition by C1Mab-6 in flow cytometry and Western blotting. These findings contribute to the understanding of mCCR1 and C1Mab interaction.
Erythropoietin-producing hepatocellular carcinoma (Eph) receptor A4 (EphA4) binds to a broad range of ephrin ligands and is upregulated in various tumors. EphA4 activation plays crucial roles in the maintenance of cancer invasiveness, immune evasion, and stem cell properties. Furthermore, a novel EphA4 ligand, secretory ribonuclease 1, has been identified, and its interaction stimulates EphA4 signaling, leading to the promotion of cancer stem cell properties. Therefore, EphA4 has been considered an attractive target for cancer therapies. Thus, monoclonal antibodies (mAbs) against EphA4 are essential for basic research and mAb-based treatment in the clinic. In this study, we developed a novel anti-human EphA4 mAb, Ea4Mab-3, using the Cell-Based Immunization and Screening (CBIS) method. Ea4Mab-3 reacted with EphA4-overexpressed Chinese hamster ovary-K1 (CHO/EphA4) and endogenous EphA4-positive lung squamous cell carcinoma LK-2 in flow cytometry. The binding affinities (apparent dissociation constant KD values) were determined as 4.5 × 10-9 M for CHO/EphA4 and 2.5 × 10-9 M for LK-2. Furthermore, Ea4Mab-3 did not exhibit cross-reactivity with other Eph receptor-overexpressed CHO-K1 cells. In addition, Ea4Mab-3 is suitable for immunoblotting and immunohistochemistry. These results indicate that Ea4Mab-3, established using the CBIS method, facilitates basic studies of EphA4 and is expected to be useful for mAb-based tumor diagnosis and therapy.
Cadherin-5 (CDH5), also known as vascular endothelial cadherin (VE-cadherin), plays crucial roles in endothelial cell adhesion, vascular barrier function, and signaling. CDH5 manages endothelial cell-cell junctions during vascular remodeling, which is vital for both vascular homeostasis and adaptive responses to pathological stimuli. Although anti-CDH5 monoclonal antibodies (mAbs) are used for specific applications such as flow cytometry, Western blotting, and immunohistochemistry (IHC), highly sensitive and versatile anti-CDH5 mAbs suitable for all these applications remain limited. Here, new anti-human CDH5 mAbs, called Ca5Mabs, were developed through a flow cytometry-based high-throughput screening. Among them, clone Ca5Mab-8 (mouse IgG2a, kappa) recognized CDH5-overexpressed Chinese hamster ovary-K1 (CHO/CDH5) cells in flow cytometry. Moreover, Ca5Mab-8 also recognized endogenous CDH5-expressing human endothelial cell lines (HUVEC/TERT2 and HDMVEC/TERT164-B) and a cervical cancer cell line (HeLa). These reactivities exceeded those of a commercial anti-CDH5 mAb (clone BV9). The apparent dissociation constant of Ca5Mab-8 for CHO/CDH5 was measured as 6.1 × 10-9 M. Ca5Mab-8 can detect endogenous CDH5 in Western blotting. In addition, Ca5Mab-8, but not BV9, can be used for IHC to detect endothelial cells in formalin-fixed paraffin-embedded tissues. These findings suggest that Ca5Mab-8 is a versatile basic research tool in vascular biology and has potential for clinical applications including tumor diagnosis and therapy.
Cadherin 26 (CDH26) is a recently identified member of the cadherin superfamily. Although CDH26 gene expression has been reported in association with allergic inflammatory responses, the protein expression levels and the signaling pathways mediated through its interactions with other proteins remain poorly understood. This is primarily due to the lack of monoclonal antibodies (mAbs) that can recognize the intact, cell surface-expressed form of CDH26. In this study, we developed an antihuman CDH26 mAb, Ca26Mab-6 (IgM, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Ca26Mab-6 demonstrated high sensitivity and specificity for CDH26 in flow cytometry and did not bind to Chinese hamster ovary (CHO)-K1 cells, which overexpress any of the other type I or type II cadherins. Ca26Mab-6 successfully detected endogenous CDH26 protein expression in HepG2, U-87 MG, MCF7, and 293FT cells. The apparent dissociation constant of Ca26Mab-6 was determined to be 9.8 ± 4.8 × 10-9 M for CDH26-overexpressed CHO-K1 (CHO/CDH26) cells and 3.6 ± 1.0 × 10-7 M for HepG2 cells. The detection of CDH26 expression in cancer cells may offer new insights into the potential relationship between inflammatory responses and malignant transformation. Therefore, Ca26Mab-6, developed using the CBIS method, is expected to facilitate functional studies of CDH26 and contribute to the development of CDH26-targeted antibody-based therapies.
The CXC chemokine receptor 2 (CXCR2) is a member of the G-protein-coupled receptor superfamily and regulates a diverse range of immune responses and tumor progression. CXCR2 is expressed on immune cells, especially neutrophils, and is involved in various immune responses by interacting with its chemokine ligands. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR2 has been desired for treatment and diagnosis. This study established a novel sensitive anti-mouse CXCR2 (mCXCR2) mAb; Cx2Mab-5 (rat IgG2a, κ), using the mCXCR2 synthetic N-terminus peptide immunization method. In flow cytometry, Cx2Mab-5 recognized mCXCR2-overexpressed Chinese hamster ovary-K1 cells (CHO/mCXCR2) and WEHI-3B (murine myelomonocytic leukemia cell) cells, which express endogenous mCXCR2. Cx2Mab-5 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. Cx2Mab-5 showed a moderate binding affinity for both CHO/mCXCR2 and WEHI-3B. Furthermore, Cx2Mab-5 detected mCXCR2 in Western blot and immunohistochemistry in CHO/mCXCR2 cells, but a commercially available anti-mCXCR2 mAb (clone SA045E1) did not. Hence, Cx2Mab-5 can be a valuable tool for analyzing mCXCR2-positive cells in mouse tissues.
Ephrin type-B receptor 3 (EphB3) binds to transmembrane ephrin-B ligands to regulate cell migration, adhesion, and proliferation. EphB3 exhibits a gradient expression pattern in the normal intestine, with the highest levels at the crypt base, and plays a crucial role in the maintenance of normal intestinal epithelium. Therefore, anti-EphB3 monoclonal antibodies (mAbs) are required for basic research and diagnosis. In this study, we developed novel antihuman EphB3, Eb3Mab-5 (IgG1, κ) and Eb3Mab-11 (IgG1, κ), using the Cell-Based Immunization and Screening (CBIS) method. Eb3Mab-5 and Eb3Mab-11 reacted with EphB3-overexpressed Chinese hamster ovary-K1 (CHO/EphB3) and endogenous EphB3-positive colorectal cancer LS174T in flow cytometry. The apparent binding affinity of Eb3Mab-5 for CHO/EphB3 and LS174T was 7.6 × 10-9 M and 1.7 × 10-8 M, respectively. Eb3Mab-11 could detect EphB3 in western blot analysis and immunohistochemistry. Eb3Mab-5 and Eb3Mab-11, established by the CBIS method, may contribute to the diagnosis and therapy of EphB3-positive tumors.
Erythropoietin-producing hepatocellular receptor A1 (EphA1) is one of the Eph receptor family members, the largest group of receptor tyrosine kinases. EphA1 is expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphA1 critically correlates with the pathogenesis in several disorders, including Alzheimer’s disease and cancers. Therefore, establishing sensitive monoclonal antibodies (mAbs) for EphA1 has been desired for basic research, diagnosis, and treatment. In this study, a novel specific and sensitive anti-human EphA1 mAb, clone Ea 1 Mab-30 (mouse IgG 1 , kappa), was established by the Cell-Based Immunization and Screening (CBIS) method. Ea 1 Mab-30 demonstrated reactivity with an EphA1-overexpressed Chinese hamster ovary-K1 cell line (CHO/EphA1), an endogenously EphA1-expressing bladder carcinoma cell line (5637), and a colorectal adenocarcinoma cell line (Caco-2) in flow cytometry. Crossreactivities of Ea 1 Mab-30 with other Eph receptors were not observed. Furthermore, the values of apparent binding affinity for CHO/EphA1 and 5637 were determined to be 8.9 × 10 −9 M and 1.7 × 10 −9 M, respectively. Furthermore, Ea 1 Mab-30 detected EphA1 protein in CHO/EphA1 and 5637 lysates using Western blot analysis. Ea 1 Mab-30 also clearly stained EphA1 of formalin-fixed paraffin-embedded CHO/EphA1 using immunohistochemistry. Ea 1 Mab-30, established by CBIS method, could help analyze the EphA1-contributed cellular functions and have potential applications in pathological diagnosis and treatment with specificity and high affinity for EphA1-expressing cells.
Androgen receptor (AR) is activated by binding to androgens, which leads to nuclear translocation, dimerization, and binding to androgen response elements (AREs) to regulate gene transcription. AR is important in masculinization during mammalian development and is a major driver of tumor growth in prostate cancer, for which AR pathway inhibitors are the standard treatment. However, the mechanisms by which AR participates in these processes remain unclear. In this study, we describe rat monoclonal antibodies (mAbs) that were generated against human and mouse AR. These mAbs recognize endogenous AR and were shown to be effective in the immunofluorescence staining of human cell lines and mouse tissue sections and in immunoprecipitation experiments. We expect these mAbs to be useful for functional analyses of AR.
CD155, also known as poliovirus receptor (PVR) or Necl-5, is an immunoreceptor with three immunoglobulin-like domains in the extracellular portion and is ubiquitously expressed on the cell surface of hematopoietic and nonhematopoietic cells. Human CD155 contains membrane-bound CD155 (mCD155) and soluble CD155 (sCD155) lacking the transmembrane region encoded by splicing variants of genes. The serum levels of sCD155 increased in patients with a variety of cancers. Furthermore, sCD155 suppresses tumor immunity through the blockade of DNAM-1 signaling. Therefore, sCD155 is potentially helpful for the diagnosis of cancer development and a novel therapeutic target in cancer treatment. However, monoclonal antibodies (mAbs) specific to sCD155, but not mCD155, have not yet been developed. Here, we generated 14 mAbs (named TX119 to TX122 and TX126 to TX135) that recognize the cytoplasmic region of CD155. These mAbs bind to sCD155, but not mCD155, when applied from the outside of the cell. Moreover, we established the assay system to quantify the sCD155 concentration in human serum. Thus, these mAbs can be utilized for the targeting and quantification of sCD155 in the human peripheral blood.
Biological fluids collected from crime scenes play a crucial role in solving serious crimes such as murder, rape, burglary, and theft. Identifying human blood using various methods is crucial for linking disparate pieces of evidence and solving crimes. In this study, the Anti-Human Hemoglobin antibody (Ah-HB) from Sigma-Aldrich® USA was internally validated for human blood identification using the Ouchterlony Double Immunodiffusion (ODD) technique at the DNA and Serology Department of the Punjab Forensic Science Agency in Lahore, Pakistan. Additionally, a comparative analysis was conducted with Seratec® HemDirect (S_HD) strips to evaluate the economic feasibility of both methods. The internal validation included assessments of sensitivity, specificity, and an analysis of real-case work samples to determine the viability of the antibody as a confirmatory test for human blood. Although Ah-HB had lower sensitivity in detecting human blood at higher dilutions (1:10,000), it offered a more cost-effective solution per sample compared with S_HD, which demonstrated higher sensitivity (1:2,000,000) but at a significantly higher cost per sample. Specificity tests revealed no cross-reactivity with nonhuman blood for both Ah-HB and S_HD. This study emphasizes the importance of selecting suitable antibodies for forensic analysis by evaluating sensitivity, specificity, and cost-effectiveness. Ah-HB emerges as a valuable tool for forensic laboratories, providing reliable results at a lower cost compared with S_HD, thereby enhancing the efficiency and effectiveness of criminal investigations.
