Characterization of a highly specific monoclonal antibody against human aldo-keto reductase AKR1C3.

Human aldo-keto reductase AKR1C3 (type 2 3α-hydroxysteroid dehydrogenase/type 5 17ß-hydroxysteroid dehydrogenase) is involved in testosterone and estrogen metabolism. AKR1C3 expression is relatively low in most tissues and high in prostate and mammary glands in regulating androgen and estrogen levels. However, in many cancers, overexpression of AKR1C3 was observed, thus prompting the development of therapeutics targeting AKR1C3. To facilitate the development of AKR1C3 targeting therapeutics, evaluating the expression of AKR1C3 is vital. As AKR1C3 is highly homologous with its family proteins, AKR1C1, AKR1C2, AKR1C4 and other AKR1 proteins, reagents that can unambiguously discriminate these enzymes are needed. In this report, a highly specific monoclonal antibody for AKR1C3, 10B10, was developed and characterized. Compared to other AKR1C3 antibodies, 10B10 is highly specific and sensitive to AKR1C3 in multiple assay formats. Thus, 10B10 will be a valuable tool for the clinical development of AKR1C3 targeting therapeutics and the study of AKR1C3 biology.

[Paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples].

OBJECTIVE: To investigate paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples. METHODS: p16 immunohistochemical staining was performed in 150 cases of chronic cervicitis, 126 cases of low-grade squamous intraepithelial lesions(LSIL), 96 cases of high-grade squamous intraepithelial lesions (HSIL) and 78 cases of cervical cancer from January 2014 to March 2018 in Zhongshan Boai Hospital. The results of p16 protein expression in paraffin sections with thickness of 2, 3, 4, 5 and 6 µm were compared using Logistic regression analysis. RESULTS: With the increase of slice thickness, the staining of p16 protein in nucleus gradually increased. The thickness of cervical slices in chronic cervicitis and cervical cancer samples had no significant effect on the positive rate of p16 protein(χ2=7.817 and 1.332, both P>0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (χ2=17.688 and 10.182, P<0.05 or P<0.01). The stable and reliable results were obtained when the slices were between 3 and 5 µm thick. CONCLUSIONS: Paraffin sections with thickness of 3.0-5.0 µm are recommended for immnohistochemical staining of p16 protein in cervical tissue samples.

Site-specific PEGylation of an anti-CEA/CD3 bispecific antibody improves its antitumor efficacy.

INTRODUCTION: Bispecific antibodies that engage immune cells to kill cancer cells are actively pursued in cancer immunotherapy. Different types of bispecific antibodies, including single-chain fragments, Fab fragments, nanobodies, and immunoglobulin Gs (IgGs), have been studied. However, the low molecular weight of bispecific antibodies with single-chain or Fab fragments generally leads to their rapid clearance in vivo, which limits the therapeutic potential of these bispecific antibodies. MATERIALS AND METHODS: In this study, we used a site-specific PEGylation strategy to modify the bispecific single-domain antibody-linked Fab (S-Fab), which was designed by linking an anticarcinoembryonic antigen (anti-CEA) nanobody with an anti-CD3 Fab. RESULTS: The half-life (t1/2) of PEGylated S-Fab (polyethylene glycol-S-Fab) was increased 12-fold in vivo with a slightly decreased tumor cell cytotoxicity in vitro as well as more potent tumor growth inhibition in vivo compared to S-Fab. CONCLUSION: This study demonstrated that PEGylation is an effective approach to enhance the antitumor efficacy of bispecific antibodies.

A Bispecific Antibody Based on Pertuzumab Fab Has Potent Antitumor Activity.

Human epidermal growth factor receptor 2 (HER2) is frequently overexpressed and activated in metastatic breast cancers. Monoclonal antibodies targeting Her2, such as trastuzumab and pertuzumab, have become important targeted therapies for patients with HER2-positive breast cancer. Both trastuzumab and pertuzumab can reduce Her2 positive tumor burden by inhibiting Her2 signaling and inducing ADCC activities (antibody dependent cell-mediated cytotoxicity). In this study, we have generated a bispecific antibody, Her2(Per)-S-Fab, by linking the pertuzumab Fab to an anti-CD16 single domain antibody. The Her2(Per)-S-Fab can be expressed and purified efficiently from Escherichia coli. In vitro and in vivo experiments showed Her2(Per)-S-Fab had potent cytotoxicity against Her2-positive tumor cells. Thus, Her2(Per)-S-Fab may provide an alternative to treat Her2-positive cancer patients.