Perioperative treatments for and postoperative activity of daily living of patients with early-stage breast cancer according to age group: A retrospective observational study.

INTRODUCTION: The number of older patients with breast cancer is increasing worldwide. However, no studies have clarified to what extent activities of daily living (ADL) decline in older patients after surgery. This study aimed to identify perioperative treatments and the proportion of patients with a postoperative decline in ADL among those with early-stage breast cancer, according to age groups (<65, 65-74, ≥75 years). MATERIALS AND METHODS: This retrospective study used healthcare utilization data of women aged ≥40 years who were diagnosed with breast cancer in 431 Japanese hospitals. Patients who underwent breast conserving surgery and mastectomy at clinical stages 0-III were included. ADL were assessed using the Barthel index (100 points indicated independent ADL). RESULTS: Overall, 37,161 patients were analyzed, including 17,313 undergoing a breast conserving surgery and 19,848 undergoing a mastectomy. The difference in the proportion of patients with a postoperative decline in ADL between those in the <65-year and ≥75-year group who underwent mastectomy was approximately 1%. In each age group, a higher proportion of patients received adjuvant chemotherapy (9.4-27.5% for breast conserving surgery; 15.6-40.3% for mastectomy) than neoadjuvant chemotherapy (breast conserving surgery, 2.1-12.0%; mastectomy, 3.0-18.1%). A lower proportion of patients in the ≥75-year group underwent radiotherapy than that in the <65-year group. CONCLUSION: Physical burden of surgery was low in both younger and older patients. Low proportions of patients in the ≥75-year group who underwent surgery received neoadjuvant and adjuvant chemotherapy and adjuvant radiotherapy. Healthcare providers should inform this to patients.

Post-traumatic growth and cancer-related communication among adolescents having mothers with breast cancer.

PURPOSE: This study aimed to clarify the characteristics of post-traumatic growth (PTG) among adolescents having mothers diagnosed with breast cancer and the relationship between PTG and cancer-related communication with breast cancer survivors. METHODS: A cross-sectional study was conducted using anonymous self-report questionnaires with breast cancer survivors and adolescent children. PTG in adolescents was measured using the Japanese version of the revised PTG Inventory for Children (PTGI-C-R-J). Furthermore, hierarchical multiple regression analysis was implemented. To evaluate the impact of cancer-related communication on each subscale, the total score of cancer-related communication was switched with other subscales individually within the constructed model. RESULTS: A total of 97 breast cancer survivors and their adolescent children were included. The mean scores of the total PTGI-C-R-J and subscale scores for "personal strength," "new possibilities," "relating to others," "appreciation of life," and "spiritual change" were 9.0, 1.7, 1.8, 2.3, 2.4, and 0.9, respectively. The connection between PTG and cancer-related communication was partially clarified. The PTGI-C-R-J score was higher when adolescents shared more information regarding breast cancer with their mothers and lower when adolescents expressed more negative feelings toward their mothers. Communication regarding relationships with mothers was not correlated with PTG. CONCLUSIONS: Of all PTG domains, "relating to others" and "appreciation of life" were comparatively higher in adolescents. Health professionals should support breast cancer survivors to ensure that they convey appropriate information regarding their treatment plans and side effects to their adolescent children. Health professionals should help adolescent children express their negative feelings calmly and clearly.

Radiation-induced angiosarcoma of the breast: individual participant meta-analysis of Japanese population.

BACKGROUND: Radiation-induced angiosarcoma (RIAS) of the breast is a very rare and poor prognostic disease. According to previous studies, the efficacy of chemotherapy for RIAS is still controversial. However, no study has assessed the prognosis of RIAS and the prognostic impact of preoperative or postoperative chemotherapy in Japanese patients. Our study aimed to assess them in Japanese people using publication data with our three patients. METHODS: Thirty-nine patients diagnosed with RIAS, including 36 patients from 34 published case series, and three patients from our hospital were used for analysis. Disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS) were assessed. RESULTS: Among the 39 patients, 36 patients (92.3%) underwent surgery. The median DFS and OS periods were 14 months (range 1-75 months) and 23 months (range 4-84 months), respectively. Chemotherapy with taxane-based regimen was administered in 13 cases (33.2%) pre- or post-operatively. DFS was significantly improved with chemotherapy in addition to surgery (p = 0.037). However, addition of chemotherapy to surgery did not improve DDFS (p = 0.09) and OS (p = 0.878). In multivariate analysis, age ≥ 70 years was an independent but poor prognostic factor of DFS. Additionally, a lack of chemotherapy showed a trend to be associated with worse DFS. There was no independent variable contributing to DDFS and OS. CONCLUSIONS: Chemotherapy may have reduced the recurrence rate of RIAS in Japanese patients but did not improve OS. Further data are needed to confirm the efficacy and proper regimen of chemotherapy.

Interrupting Endocrine Therapy to Attempt Pregnancy after Breast Cancer.

BACKGROUND: Prospective data on the risk of recurrence among women with hormone receptor-positive early breast cancer who temporarily discontinue endocrine therapy to attempt pregnancy are lacking. METHODS: We conducted a single-group trial in which we evaluated the temporary interruption of adjuvant endocrine therapy to attempt pregnancy in young women with previous breast cancer. Eligible women were 42 years of age or younger; had had stage I, II, or III disease; had received adjuvant endocrine therapy for 18 to 30 months; and desired pregnancy. The primary end point was the number of breast cancer events (defined as local, regional, or distant recurrence of invasive breast cancer or new contralateral invasive breast cancer) during follow-up. The primary analysis was planned to be performed after 1600 patient-years of follow-up. The prespecified safety threshold was the occurrence of 46 breast cancer events during this period. Breast cancer outcomes in this treatment-interruption group were compared with those in an external control cohort consisting of women who would have met the entry criteria for the current trial. RESULTS: Among 516 women, the median age was 37 years, the median time from breast cancer diagnosis to enrollment was 29 months, and 93.4% had stage I or II disease. Among 497 women who were followed for pregnancy status, 368 (74.0%) had at least one pregnancy and 317 (63.8%) had at least one live birth. In total, 365 babies were born. At 1638 patient-years of follow-up (median follow-up, 41 months), 44 patients had a breast cancer event, a result that did not exceed the safety threshold. The 3-year incidence of breast cancer events was 8.9% (95% confidence interval [CI], 6.3 to 11.6) in the treatment-interruption group and 9.2% (95% CI, 7.6 to 10.8) in the control cohort. CONCLUSIONS: Among select women with previous hormone receptor-positive early breast cancer, temporary interruption of endocrine therapy to attempt pregnancy did not confer a greater short-term risk of breast cancer events, including distant recurrence, than that in the external control cohort. Further follow-up is critical to inform longer-term safety. (Funded by ETOP IBCSG Partners Foundation and others; POSITIVE ClinicalTrials.gov number, NCT02308085.).

Clinicopathological and imaging features of ductal carcinoma in situ in BRCA1/2 mutation carriers.

BACKGROUND: BRCA1/2-associated invasive breast cancer has been extensively studied. However, there are few reports of ductal carcinoma in situ (DCIS). OBJECTIVE: This study aimed to investigate the clinicopathological and imaging findings of DCIS in patients with BRCA1/2 mutations. METHODS: This was a single-institution, retrospective study. We identified patients diagnosed with DCIS with BRCA mutations between September 2003 and December 2020. Clinicopathological data and mammography (MG), magnetic resonance imaging (MRI), and ultrasound (US) findings were reviewed. RESULTS: We identified 30 cancers in 28 patients; 7 (25.0%) patients had BRCA1 mutations, and 21 (75.0%) had BRCA2 mutations. The median patient age was 42 years. Screening was the most common reason for the detection of DCIS (50.0%), followed by occult cancer diagnosed by pathological examination after risk-reducing mastectomy (26.7%). The nuclear grade was most often 1 (46.7%), and 93.3% were estrogen and/or progesterone receptor positive. The detection rates of MG, MRI, and US were 64.3%, 72.0%, and 64.0%, respectively. The most common imaging findings were calcification (100%) on MG, non-mass enhancement (88.9%) on MRI, and hypoechoic area (75.0%) on US. CONCLUSION: BRCA-associated DCIS was more strongly associated with BRCA2, and imaging features were similar to those of sporadic DCIS. Our results are helpful in informing surveillance strategies based on genotypes in women with BRCA mutations.

Prognostic effect of subsequent childbirth after the diagnosis of breast cancer using propensity score matching analysis.

BACKGROUND: Among younger patients, one of the important concerns is whether they can give birth safely. Although previous studies have investigated this topic, many aspects remain unclear owing to potential biases. We aimed to evaluate the prognostic effect of subsequent childbirth after the diagnosis using propensity score matching. METHODS: A single-center retrospective cohort study was conducted. This study included patients aged ≤ 45 years, diagnosed with breast cancer between 2005 and 2014. Patients with and without subsequent childbirth were assigned to the childbirth and non-childbirth cohorts, respectively. Relapse-free survival (RFS) and overall survival (OS) of the childbirth cohort were compared with those of the non-childbirth cohort. The covariates in the propensity score model included age, tumor size, node status, number of preceding childbirths before the diagnosis, estrogen receptor, and human epidermal growth factor receptor 2 status. RESULTS: 104 patients with childbirth and 2250 without childbirth were assigned to the respective cohorts. At a median follow-up of 82 months, the childbirth cohort showed a significantly longer RFS than the non-childbirth cohort (HR = 0.469 [0.221-0.992]; p = 0.047). There was no significant difference in the OS (HR = 0.208 [0.029-1.494]; p = 0.119). After matching, subsequent childbirth was not significantly associated with RFS (HR = 0.436 [0.163-1.164], p = 0.098) and OS (HR = 0.372 [0.033-4.134], p = 0.402). CONCLUSIONS: Subsequent childbirth was not associated with an increased risk of relapse and mortality. It is important to make younger patients aware of these novel findings and aid them in their decision-making.

Development of novel monoclonal antibodies against nsp12 of SARS-CoV-2.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.

Comparative study of the usefulness of adjunctive tomosynthesis in breast cancer screening by mammography and ultrasound in Japan.

INTRODUCTION: There are few studies have conducted digital breast tomosynthesis (DBT) in addition to digital mammography (2DDM) and ultrasound (US) for screening. The purpose of this study is to determine the possibility of synergistic effects of DBT and US screening for Japanese. METHODS: 5023 examinations of the opportunistic screening using 2DDM and US (2D group: 2581) or 2DDM and US plus DBT (3 group: 2442) were performed at our facility from May 1, 2017 to March 31, 2019. This study was not RCT, and the backgrounds of the two groups were different. RESULTS: The recall rate was 3.1% in the 2D group and 2.6% for the 3D group (p = 0.27). The number of detected cancer cases was 6 (0.23%) in the 2D group and 12 (0.49%) in the 3D group (p = 0.16). The positive predictive value (PPV) was 7.4% for the 2D group and 19.0% for the 3D group (p = 0.045). There was one invasive ductal carcinoma case which had no findings in 2DDM and US, but had a slight distortion in the images of DBT. CONCLUSION: We examined and reported whether DBT was useful for breast cancer screening combined with mammography and US. Compared to the 2D group, the 3D group showed better results of PPV with significant difference. However, due to the non-randomized design and difference between the two groups, the results should be interpreted in caution. Adding DBT in 2DDM and US screening would be acceptable only if the benefits and disadvantages are explained to the women undergoing the screening.

Development of a Novel Anti-Mouse CCR4 Monoclonal Antibody (C4Mab-1) by N-Terminal Peptide Immunization.

The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 that mediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody (mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research, diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C4Mab-1 (rat IgG1, kappa), was established using N-terminal peptide immunization. C4Mab-1 reacted with mCCR4-overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that KDs of C4Mab-1 for CHO/mCCR4, P388, and J774-1 cells were 4.2 × 10-9 M, 5.4 × 10-7 M, and 1.1 × 10-6 M, respectively. C4Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.

Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor by Defucosylated Mouse-Dog Chimeric Anti-Epidermal Growth Factor Receptor Antibody (E134Bf).

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.

Antitumor Activities in Mouse Xenograft Models of Canine Fibroblastic Tumor by Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody.

The epidermal growth factor receptor (EGFR) is involved in tumor malignancy through gene amplification and/or protein overexpression. An anti-human EGFR (hEGFR) monoclonal antibody (clone EMab-134), which explicitly detects hEGFR and dog EGFR (dEGFR), was previously developed. The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, it was shown that 134-mG2a-f reacts with a canine fibroblastic tumor cell line (A-72) using flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f exerted ADCC and CDC on A-72 cell line. The administration of 134-mG2a-f significantly inhibited the A-72 xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects on dEGFR-expressing canine fibroblastic tumors.

Development of Monoclonal Antibody 281-mG2a-f Against Golden Hamster Podoplanin.

Golden (Syrian) hamster (Mesocricetus auratus) is a small animal model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Pathological analyses of the tissues are required to understand the pathogenesis of SARS-CoV-2 and the evaluation of therapeutic modalities, including neutralizing monoclonal antibodies (mAbs). However, mAbs that recognize the golden hamster-derived antigens and distinguish specific cell types, such as the pneumocytes, are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-Chinese hamster (Cricetulus griseus) PDPN mAb PMab-281 (IgG3, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. A defucosylated mouse IgG2a version of PMab-281 (281-mG2a-f) was also developed. The 281-mG2a-f strongly recognized both the Chinese hamster and the golden hamster PDPN using flow cytometry and could detect lung type I alveolar epithelial cells, lymphatic endothelial cells, and Bowman's capsules in the kidney from the golden hamster using immunohistochemistry. These results suggest the usefulness of 281-mG2a-f for analyzing the golden hamster-derived tissues and cells for SARS-CoV-2 research.

Epitope Mapping of an Anti-CD10 Monoclonal Antibody (MME/1870) Using Enzyme-Linked Immunosorbent Assay.

CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. CD10 is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Antibodies against CD10 are used for the diagnosis of follicular lymphoma. Anti-human CD10 monoclonal antibody (clone MME/1870) can be used for Western blotting and immunohistochemical analyses. This study examined the critical epitope of MME/1870 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and MME/1870 reacted to the 501-520 amino acid sequence of CD10. Next, we analyzed the reaction to 20 point mutants, and MME/1870 did not recognize the alanine-substituted peptides of Y507A, I511A, I512A, and L515A. These results indicate that the binding epitope of MME/1870 includes Tyr507, Ile511, Ile512, and Leu515 of CD10.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody (134-mG2a-f) Exerts Antitumor Activities in Mouse Xenograft Models of Canine Osteosarcoma.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is physiologically essential in normal cells, it contributes to tumor malignancy through gene amplification and/or protein overexpression, which augment signaling cascades in tumor cells. We previously developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), EMab-134 (mouse IgG1, kappa), which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. The mouse IgG2a version of EMab-134 (134-mG2a) has antitumor effects toward mouse xenografts of hEGFR-expressing oral squamous cell carcinomas. Furthermore, 134-mG2a-f, the defucosylated version of 134-mG2a, exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. Herein, the reactivity of 134-mG2a-f against canine cancer cells with endogenous dEGFR was first examined by flow cytometry and immunocytochemistry. In vitro analysis demonstrated that 134-mG2a-f highly exerted ADCC and CDC for a canine osteosarcoma cell line, D-17, which expresses endogenous dEGFR. Moreover, in vivo administration of 134-mG2a-f significantly suppressed the development of D-17 compared with the results in response to control mouse IgG. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine cancers, and could be valuable as part of an antibody treatment regimen for them.

Immunohistochemical Analysis Using Monoclonal Antibody PMab-269 Against Steller Sea Lion Podoplanin.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.

Phosphorylation of hTERT at threonine 249 is a novel tumor biomarker of aggressive cancer with poor prognosis in multiple organs.

Recent evidence indicates that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates expression of target genes and is directly involved in tumor formation in a telomere-independent manner. Non-canonical function of hTERT has been considered as a therapeutic target for cancer therapy. We have previously shown that hTERT phosphorylation at threonine 249 (p-hTERT), which promotes RdRP activity, is an indicator of an aggressive phenotype and poor prognosis in liver and pancreatic cancers, using two cohorts with small sample sizes with polyclonal p-hTERT antibody. To clarify the clinical relevance of p-hTERT, we developed a specific monoclonal antibody and determined the diagnostic and prognostic value of p-hTERT in cancer specimens using a large cohort. A monoclonal antibody for phosphorylated hTERT (p-hTERT) at threonine 249 was developed and validated. The antibody was used for the immunohistochemical staining of formalin-fixed, paraffin-embedded specimens from 1523 cases of lung, colon, stomach, pancreatic, liver, breast, and kidney cancers. We detected elevated p-hTERT expression levels in cases with a high mitotic activity, high pathological grade, and high nuclear pleomorphism. Elevated p-hTERT expression was an independent prognostic factor for lung, pancreatic, and liver cancers. Furthermore, p-hTERT expression was associated with immature and aggressive features, such as adenosquamous carcinoma (lung and pancreas), invasive type of cancer (lung), high serum alpha-fetoprotein level (liver), and triple-negative status (breast). In conclusion, RdRP activity indicated by p-hTERT expression predicts aggressive cancer phenotypes in various types of cancer. Thus, p-hTERT is a novel biomarker for the diagnosis of aggressive cancers with a poor prognosis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Anxiety and related factors among parents of patients with breast cancer after surgery in Japan: A multi-informant and multilevel study.

AIM: Anxiety and its correlates in parents of patients with breast cancer have rarely been studied. We explored anxiety among parents of postoperative patients with breast cancer and its relationship with parents' social support and care needs and patients' anxiety. METHODS: A cross-sectional survey using self-report questionnaires and medical records was conducted among patients with breast cancer after surgery and their parents at four designated cancer care hospitals between September 2015 and June 2016. Anxiety was measured using the Hospital Anxiety and Depression Scale (HADS). Parents provided information about social support and care needs. Multilevel analysis was performed on patient-parent paired data controlling for patient-level variation. RESULTS: Participants included 107 patients, 83 mothers, and 51 fathers. The mean HADS anxiety scores reported by mothers and fathers were 7.2 and 6.5, respectively, which were higher than patients' HADS anxiety scores. Fulfillment of important care needs was related to lower anxiety among mothers and fathers (estimate = -1.38, p = .01). Lower family support and higher patient anxiety were associated with higher anxiety in mothers, but not fathers. CONCLUSIONS: Parents of patients with breast cancer had high anxiety. Communication, providing cancer-related information, and fulfilling care needs can alleviate anxiety in parents of patients with breast cancer after surgery. Furthermore, increasing family support and decreasing patients' anxiety are essential to alleviating mothers' anxiety.

Defucosylated Mouse-Dog Chimeric Anti-EGFR Antibody Exerts Antitumor Activities in Mouse Xenograft Models of Canine Tumors.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, namely EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR monoclonal antibody, namely E134Bf. In vitro analysis revealed that E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against a canine osteosarcoma cell line (D-17) and a canine fibroblastic cell line (A-72), both of which express endogenous dEGFR. Moreover, in vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

Epitope Mapping of an Anti-CD20 Monoclonal Antibody (C20Mab-60) Using Enzyme-Linked Immunosorbent Assay.

CD20 is a glycosylated transmembrane protein and is expressed on normal B cells and B cell malignancies. Therapeutic antibodies against CD20 are developed and used in clinic. The understanding of antibody-antigen binding by revealing the epitope is essential for future application to antibody technology. Previously, we developed an anti-human CD20 monoclonal antibody, C20Mab-60 (IgG2a, kappa), using the Cell-Based Immunization and Screening (CBIS). C20Mab-60 can be used for flow cytometry, Western blot, and immunohistochemical analyses. In this study, we examined the critical epitope of C20Mab-60 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. We performed ELISA with deletion mutants, and C20Mab-60 reacted to the 160-179 amino acids sequence of CD20. Next, we analyzed the reaction to 20 point mutants, and C20Mab-60 did not recognize the alanine-substituted peptides of N171A, P172A, S173A, and E174A. The results indicate that the binding epitope of C20Mab-60, developed by CBIS, includes Asn171, Pro172, Ser173, and Glu174 of CD20.

Epitope Mapping of an Anti-HER2 Monoclonal Antibody (H2Mab-181) Using Enzyme-Linked Immunosorbent Assay.

Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein expressed in various types of normal or cancer cells. Overexpression of HER2 is found in many cancers and is related to cell proliferation, differentiation, and migration. We recently developed a novel anti-HER2 monoclonal antibody, H2Mab-181, by immunizing mice with the purified recombinant extracellular domain of HER2. H2Mab-181 can specifically and sensitively detect HER2 not only in flow cytometry and Western blotting for gastric cancer cell lines, but also in immunohistochemical analyses for gastric cancer tissues. In this study, we analyzed the binding epitope of H2Mab-181 to HER2 using enzyme-linked immunosorbent assay (ELISA). Results showed that the H2Mab-181 epitope was determined to be Gly383, Asp384, Ala386, Asn388, and Pro391 by ELISA.