Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer.

Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens and the development of targeting agents. Here, we report the development of two novel mAbs against CFPAC-1 human pancreatic cancer cells. Using ELISA, flow cytometry, immunoprecipitation, mass spectrometry, Western blot and immunohistochemistry, we found that the target antigens recognised by the two novel mAbs KU44.22B and KU44.13A, are integrin α3 and CD26 respectively, with high levels of expression in human pancreatic and other cancer cell lines and human pancreatic cancer tissue microarrays. Treatment with naked anti-CD26 mAb KU44.13A did not have any effect on the growth and migration of cancer cells nor did it induce receptor downregulation. In contrast, treatment with anti-integrin α3 mAb KU44.22B inhibited growth in vitro of Capan-2 cells, increased migration of BxPC-3 and CFPAC-1 cells and induced antibody internalisation. Both novel mAbs are capable of detecting their target antigens by immunohistochemistry but not by Western blot. These antibodies are excellent tools for studying the role of integrin α3 and CD26 in the complex biology of pancreatic cancer, their prognostic and predictive values and the therapeutic potential of their humanised and/or conjugated versions in patients whose tumours overexpress integrin α3 or CD26

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers

Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed

Immunohistochemical discrimination of wild-type EGFR from EGFRvIII in fixed tumour specimens using anti-EGFR mAbs ICR9 and ICR10

Background:The human epidermal growth factor receptor (EGFR) is an important therapeutic target in oncology, and three different types of EGFR inhibitors have been approved for the treatment of cancer patients. However, there has been no clear association between the expression levels of EGFR protein in the tumours determined by the FDA-approved EGFR PharmDx kit (Dako) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors.Method:In this study, we investigated the potential of our anti-EGFR monoclonal antibodies (mAbs; ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild-type EGFR and/or the type-III deletion mutant form of EGFR (EGFRvIII) in formalin-fixed, paraffin-embedded human tumour specimens.Results:We found that the anti-EGFR mAb in the EGFR PharmDx kit stained both wild-type and EGFRvIII-expressing cells in formalin-fixed, paraffin-embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR10 and ICR9 were specific for the wild-type EGFR.Conclusion:We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild-type EGFR protein in tumour specimens using immunohistochemistry, and to determine their prognostic significance, as well as predictive value for response to therapy with EGFR antibodies.British Journal of Cancer advance online publication, 7 February 2012; doi:10.1038/bjc.2012.27 www.bjcancer.com

Electroretinogram changes following sequential panretinal photocoagulation for proliferative diabetic retinopathy

Purpose: To evaluate changes in electroretinogram (ERG) response over the course of multiple sessions of panretinal photocoagulation (PRP) in patients with proliferative diabetic retinopathy (PRP). Methods: A prospective cohort study of 11 patients with PDR who required PRP was conducted. PRP was completed over three sessions. Each patient had five ERGs done: baseline, 1 week after each PRP session, and 6 weeks after the last session of PRP. Dark-adapted 0.01 ERG, Dark-adapted 3 ERG, Dark-adapted 10 ERG, Light-adapted 3 ERG, and Light-adapted 30 Hz flicker ERG were done. The mean change in a-and b-wave amplitudes as well as implicit times compared to baseline was analyzed. Results: A significant reduction in peak amplitudes of both a-and b-waves and delay in latencies were observed in all responses (p<0.05). The absolute amplitude reduction and delay in latency were higher for scotopic b-waves (p<0.05). The root mean square (RMS) of Dark-adapted 10.0 ERG (p<0.05) and total mean amplitude changes of a-and b-waves (p<0.001) were reduced after each laser session; however, the magnitude of change was not different between the first, second, or third sessions of PRP, and each session showed a similar deterioration rate of ERG parameters comparing to each other (p=0.4 for RMS and p=0.2 for total mean amplitude changes). In addition, the results indicated recovery of the amplitude and latency of ERG waves after 6 weeks from the final treatment (p<0.001) although not to baseline levels. Conclusion: ERG findings following PRP show reduced retinal function after each session which partially recovers by 6 weeks after the completion of therapy. Clinicians should be mindful of these changes when planning the treatment course for patients with PDR. © 2020 Khojasteh et al

Synergistic effects of various Her inhibitors in combination with IGF-1R, C-MET and Src targeting agents in breast cancer cell lines

Introduction: Overexpression of the receptor tyrosine kinase HER2 has been reported in around 25% of human breast cancers, usually indicating a poor prognosis. As a result, HER2 has become a popular target for therapy. However, despite recent advances in HER2 targeted therapy, many patients still experience primary and secondary resistance to such treatments. It is therefore important to understand the underlying mechanism of resistance and to develop more effective therapeutic interventions for breast cancer. Methods: The sensitivity of a panel of seven breast cancer cell lines to treatment with various types HER-family inhibitors alone, or in combination with a selection of other tyrosine kinase inhibitors (TKIs) or chemotherapeutic agents was determined using the Sulforhodamine B colorimetric assay. Receptor expression, cell-cycle distribution, cell signalling and cell migration were determined using flow cytometry, Western blot and Incucyte Zoom Live-Cell Analysis System respectively. Results: Overall, breast cancer cells were more sensitive to treatment with the irreversible pan-HER family inhibitors, particularly afatinib and neratinib, than treatment with the first-generation reversible inhibitors. Of three HER-2 overexpressing cell lines in this panel, SKBr3 and BT474 were highly sensitive to treatment with HER-family inhibitors (IC50s as low as 3 nM), while MDA-MB-453 was relatively resistant (lowest IC50 = 0.11 μM). When the HER-family inhibitors were combined with other agents such as NVP-AEW541 (an IGF-1R inhibitor), dasatinib (a Src inhibitor) or crizotinib (a c-Met/ALK inhibitor), such combination produced synergistic effects in some of the cell lines examined. Interestingly, co-targeting of Src and HER-family members in MDA-MB-453 cells led to synergistic growth inhibition, suggesting the importance of Src in mediating resistance to HER2-targeting agents. Finally, treatment with the irreversible HER family blockers and dasatinib were also most effective at inhibiting the migration of breast cancer cells. Conclusion: We concluded that the irreversible inhibitors of HER-family members are generally more effective at inhibiting growth, downstream signalling and migration compared with reversible inhibitors, and that combining HER-family inhibitors with other TKIs such as dasatinib may have therapeutic advantages in certain breast cancer subtypes and warrants further investigation