Development of specific anti-mouse atypical chemokine receptor 4 monoclonal antibodies.

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (K D) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10-9 M, 1.3 × 10-8 M, and 1.7 × 10-9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.

Anti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor Activity in Mouse Xenograft Models of Oral Squamous Cell Carcinomas.

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3-10-overexpressed CHO-K1 (CHO/CD44v3-10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3-10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3-10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3-10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Antitumor activities of anti­CD44 monoclonal antibodies in mouse xenograft models of esophageal cancer.

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti­CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody­drug conjugates and chimeric antigen receptor­T cell therapy. Anti-pan­CD44 mAbs, C44Mab­5 and C44Mab­46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG2a version of the anti­pan­CD44 mAbs (5­mG2a and C44Mab­46­mG2a) to evaluate the antitumor activities against CD44­positive cells. Both 5­mG2a and C44Mab­46­mG2a recognized CD44s­overexpressed CHO­K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5­mG2a and C44Mab­46­mG2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5­mG2a and C44Mab­46­mG2a significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control mouse IgG2a. These results indicate that 5­mG2a and C44Mab­46­mG2a could exert antitumor activities against CD44­positive cancers and be a promising therapeutic regimen for tumors.

A Machine Learning Method for Allocating Scarce COVID-19 Monoclonal Antibodies.

Importance: During the COVID-19 pandemic, the effective distribution of limited treatments became a crucial policy goal. Yet, limited research exists using electronic health record data and machine learning techniques, such as policy learning trees (PLTs), to optimize the distribution of scarce therapeutics. Objective: To evaluate whether a machine learning PLT-based method of scarce resource allocation optimizes the treatment benefit of COVID-19 neutralizing monoclonal antibodies (mAbs) during periods of resource constraint. Design, Setting, and Participants: This retrospective cohort study used electronic health record data from October 1, 2021, to December 11, 2021, for the training cohort and data from June 1, 2021, to October 1, 2021, for the testing cohort. The cohorts included patients who had positive test results for SARS-CoV-2 and qualified for COVID-19 mAb therapy based on the US Food and Drug Administration's emergency use authorization criteria, ascertained from the patient electronic health record. Only some of the qualifying candidates received treatment with mAbs. Data were analyzed between from January 2023 to May 2024. Main Outcomes and Measures: The primary outcome was overall expected hospitalization, assessed as the potential reduction in overall expected hospitalization if the PLT-based allocation system was used. This was compared to observed allocation using risk differences. Results: Among 9542 eligible patients in the training cohort (5418 female [56.8%]; age distribution: 18-44 years, 4151 [43.5%]; 45-64 years, 3146 [33.0%]; and ≥65 years, 2245 [23.5%]), a total of 3862 (40.5%) received mAbs. Among 6248 eligible patients in the testing cohort (3416 female [54.7%]; age distribution: 18-44 years, 2827 [45.2%]; 45-64 years, 1927 [30.8%]; and ≥65 years, 1494 [23.9%]), a total of 1329 (21.3%) received mAbs. Treatment allocation using the trained PLT model led to an estimated 1.6% reduction (95% CI, -2.0% to -1.2%) in overall expected hospitalization compared to observed treatment allocation in the testing cohort. The visual assessment showed that the PLT-based point system had a larger reduction in 28-day hospitalization compared with the Monoclonal Antibody Screening Score (maximum overall hospitalization difference, -1.0% [95% CI, -1.3% to -0.7%]) in the testing cohort. Conclusions and Relevance: This retrospective cohort study proposes and tests a PLT method, which can be linked to a electronic health record data platform to improve real-time allocation of scarce treatments. Use of this PLT-based allocation method would have likely resulted in fewer hospitalizations across a population than were observed in usual care, with greater expected reductions than a commonly used point system.

Anti-HER2 Cancer-Specific mAb, H2Mab-250-hG1, Possesses Higher Complement-Dependent Cytotoxicity than Trastuzumab.

Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody (mAb), H2Mab-250/H2CasMab-2. In flow cytometry and immunohistochemistry, H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2Mab-250 (H2Mab-250-hG1) possesses compatible in vivo antitumor effects against breast cancer xenografts to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H2Mab-250-hG1 and trastuzumab. Both H2Mab-250-hG1 and trastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed where trastuzumab showed a more significant ADCC effect compared to H2Mab-250-hG1. Importantly, H2Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H2Mab-250-hG1 and trastuzumab, and indicate a future direction for the clinical development of H2Mab-250-hG1 against HER2-positive tumors.

The Effect of Medicare Annual Wellness Visits on Breast Cancer Screening and Diagnosis.

OBJECTIVE: The Medicare Annual Wellness Visit (AWV)-a prevention-focused annual check-up-has been available to beneficiaries with Part B coverage since 2011. The objective of this study was to estimate the effect of Medicare AWVs on breast cancer screening and diagnosis. DATA SOURCES AND STUDY SETTING: The National Cancer Institute's Surveillance, Epidemiology, and End Results cancer registry data linked to Medicare claims (SEER-Medicare), HRSA's Area Health Resources Files, the FDA's Mammography Facilities database, and CMS "Mapping Medicare Disparities" utilization data from 2013 to 2015. STUDY DESIGN: Using an instrumental variables approach, we estimated the effect of AWV utilization on breast cancer screening and diagnosis, using county Welcome to Medicare Visit (WMV) rates as the instrument. DATA COLLECTION/EXTRACTION METHODS: 66,088 person-year observations from 49,769 unique female beneficiaries. PRINCIPAL FINDINGS: For every 1-percentage point increase in county WMV rate, the probability of AWV increased by 1.7 percentage points. Having an AWV was associated with a 22.4-percentage point increase in the probability of receiving a screening mammogram within 6 months ( P <0.001). There was no statistically significant increase in the probability of breast cancer diagnosis (overall or early stage) within 6 months of an AWV. Findings were robust to multiple model specifications. CONCLUSIONS: Performing routine cancer screening is an evidence-based practice for diagnosing earlier-stage, more treatable cancers. The AWV effectively increases breast cancer screening and may lead to more timely screening. Continued investment in Annual Wellness Visits supports breast cancer screening completion by women who are most likely to benefit, thus reducing the risk of overscreening and overdiagnosis.

Development of a Sensitive Anti-Mouse CCR5 Monoclonal Antibody for Flow Cytometry.

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10-8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.

Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C8Mab-2 Using Flow Cytometry.

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2.

Development of Sensitive Anti-Mouse CCR5 Monoclonal Antibodies Using the N-Terminal Peptide Immunization.

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.

Conscience-Based Barriers to Medical Aid in Dying: A Survey of Colorado Physicians.

BACKGROUND: Approximately 20% of the United States' population lives in a state or jurisdiction where medical aid in dying (MAiD) is legal. It is unknown how physicians' own barriers are associated with their provision of the spectrum of MAiD services. OBJECTIVE: To measure physicians' religious and/or ethical barriers to providing MAiD services and how such barriers relate to physicians' intentions and behaviors. DESIGN: Three-wave cross-sectional survey fielded in Colorado in 2020-2021. PARTICIPANTS: Physicians providing care to patients likely clinically eligible for MAiD according to probabilistic sampling. MAIN MEASURES: Physicians self-reported barriers to their own participation in MAiD. We considered large ethical and/or religious barriers to be conscience-based barriers. We measured physicians' self-reported intention to participate and self-reported prior participation in MAiD since it was legalized in Colorado in 2017. We estimated differences in intention and behavior outcomes according to presence of conscience-based barriers, adjusting for physician gender, race/ethnicity, time in practice, and specialty. KEY RESULTS: Among 300 respondents, 26% reported "large" ethical and/or religious barriers to their involvement in MAiD. Physicians with longer time in practice and those identifying as non-White were more likely to report conscience-based barriers to MAiD. Comparing physicians with and without conscience-based barriers to MAiD, we found no difference in ancillary participation (discussing, referring) but significant differences in direct participation (serving as consultant [5% vs. 31%] or attending [0% vs. 22%]). CONCLUSIONS: Approximately one-quarter of physicians likely to care for MAiD-eligible patients in Colorado reported religious and/or ethical barriers to MAiD. Despite religious and/or ethical barriers, the vast majority of physicians were willing to discuss MAiD and/or refer patients seeking MAiD services. These data provide important empirical foundation for policy from hospitals and health systems as well as medical specialty groups with official positions on MAiD.

Cx1Mab-1: A Novel Anti-mouse CXCR1 Monoclonal Antibody for Flow Cytometry.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

PMab-314: An Anti-Giant Panda Podoplanin Monoclonal Antibody.

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.

Establishment of a Novel Cancer-Specific Anti-HER2 Monoclonal Antibody H2Mab-250/H2CasMab-2 for Breast Cancers.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 monoclonal antibody, named H2Mab-250/H2CasMab-2 (IgG1, kappa). H2Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H2Mab-250 did not react with nontransformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs, such as H2Mab-119 and trastuzumab reacted with both cancer and normal epithelial cells. Immunohistochemical analysis demonstrated that H2Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared with H2Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H2Mab-250. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Patient Perceptions of Chatbot Supervision in Health Care Settings.

This survey study assesses whether patients communicating with a chatbot in a large health care system were able to accurately identify it as an unsupervised computer application.

Epitope Mapping of an Anti-Mouse CD39 Monoclonal Antibody Using PA Scanning and RIEDL Scanning.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275-279, 282-291, and 306-323 aa of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

Establishment of a Novel Anti-Mouse CCR1 Monoclonal Antibody C1Mab-6.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, therapeutic agents for CCR1 have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C1Mab-6 (rat IgG2b, kappa), using the Cell-Based Immunization and Screening method. Flow cytometry and Western blot analyses showed that C1Mab-6 recognizes mCCR1 specifically. The dissociation constant of C1Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9 × 10-9 M, indicating that C1Mab-6 possesses a high affinity to mCCR1. These results suggest that C1Mab-6 could be a useful tool for targeting mCCR1 in preclinical mouse models.

Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies.

A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

Cx3Mab-4: A Novel Anti-Mouse CXCR3 Monoclonal Antibody for Flow Cytometry.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 was determined as 1.3 × 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

Locally misfolded HER2 expressed on cancer cells is a promising target for development of cancer-specific antibodies.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is associated with a poor prognosis, making it an important therapeutic target. Here, we establish a novel cancer-specific anti-HER2 antibody, H2Mab-214. H2Mab-214 reacts with HER2 on cancer cells, but unlike the therapeutic antibody trastuzumab, it does not react with HER2 on normal cells in flow cytometry measurements. A crystal structure suggests that H2Mab-214 recognizes a structurally disrupted region in the HER2 domain IV, which normally forms a ß-sheet. We show that this misfolding is inducible by site-directed mutagenesis mimicking the disulfide bond defects that also may occur in cancer cells, indicating that the local misfolding in the Cys-rich domain IV governs the cancer-specificity of H2Mab-214. Furthermore, we show that H2Mab-214 effectively suppresses tumor growth in xenograft mouse models. Our findings offer a potential strategy for developing cancer-specific therapeutic antibodies that target partially misfolded cell surface receptors.

Epitope Mapping of an Anti-CD44v4 Monoclonal Antibody (C44Mab-108) Using Enzyme-Linked Immunosorbent Assay.

CD44 is a type I transmembrane glycoprotein and possesses various isoforms which are largely classified into CD44 standard (CD44s) and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 monoclonal antibody, C44Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C44Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10) and found that C44Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C44Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C44Mab-108 includes Asp280 and Trp281 of CD44v3-10.