Volume 343, Issue 2, Pages (February 2014)

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Volume 343, Issue 2, Pages 275-285 (February 2014) A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Yara C.P. Maia, Angela A.S. Sena, Carlos Ueira-Vieira, Ana Paula Carneiro, Juliana F. Almeida, Paulo R. de Faria, Donizeti W. Santos, Luanda Calábria, Tânia M. Alcântara, Fernando A. Soares, Luiz R. Goulart Cancer Letters Volume 343, Issue 2, Pages 275-285 (February 2014) DOI: 10.1016/j.canlet.2013.09.029 Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Schematic outline of the construction the Fab repertoire using phage display. Total RNA was first isolated (A) and IgG and IgM heavy chain were amplified including five VH families (B) followed by Vκ (C), CH and Cκ (D) amplifications. The purified amplicons were assembled in a second round of PCR (E) which was assembled to generate the human Fab fragment (F). After SfiI digestion and ligation of the Fab to the phagemid, the ligated vector was induced into E. coli. The culture was infected by VCSM13 helper and displayed as fusion to pIII coat protein. Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 VH and VL repertoire of non-amplified original Fab library. The graph shows the frequency of VL (A) and VH (B) gene segment family usage (%). The sequences were aligned to their closest germline, using the Ig-Blast to identify the V family. Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Characterization of selected clones after induction by IPTG. The presence of soluble Fab fragments was evaluated by anti-HA antibody, which recognized the hemaglutin tag fused to VH chain. Slot blot analysis demonstrated Fab expression in 98% of selected clones (A). Clone 84 corresponds to pComb3X vector supernatant without Fab fragment. SDS–PAGE (B) and Western-Blot assays (C) was performed to confirm the presence of human Fab in culture supernatants which corresponds to ∼28kDa. M shows Prestained SDS–PAGE Standards, Broad Range (Bio-Rad) and C1–C4 shows randomly selected clones from expression plates. Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Evaluation of the binding selectivity for the induced clones using a pre-screening ELISA in total protein extracted from normal, benign and tumor tissue samples. Absorbance in 492nm is described in panel A with 8 reactive clones obtained from supernatant of bacterial medium after IPTG induction. ANOVA test demonstrated that all clones discriminated breast cancer from benign samples. FabC4 clone was selected for additional procedures, based on its reactivity ratio between cancer/benign and cancer/control (B), which was higher than the other clones. ELISA assay between the recombinant CK10 and the FabC4 antibody for antigen validation (C). Absorbance was significantly different between the three groups of proteins extracted from BC; BBT and N patients. BC protein did not differed from CK10 absorbance and was positive for HPLC-purified FabC4 detection. The other groups presented significantly lower absorbance compared to CK10 recombinant protein. BC: breast cancer; BBT: benign breast tumor; N: normal tissue. ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001. Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Mass spectrometry sequencing for FabC4 target detection comparing breast cancer and benign tissues. It was detected three potential targets: CK1 (A), CK9 (B) and CK10 (C). According to peptide matches and comparing the two groups CK10 was characterized as FabC4 target. In yellow: peptide matches. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Immunoaffinity of FAbC4 against breast cancer tissue antigens. (A) Invasive adenocarcinoma showing striking labeling in the nucleus and cytoplasm from ductal cells. (B) Moderate cytoplasmic immunoreactivity of ductal epithelial cells in benign breast tissue with fibroadenoma. (C) Mamoplasty sample tissue showing none labeling with FAbC4. (D) Negative control of immunohistochemistry assay. (E–H) None immunoreactivity was observed in other cancer types, such as prostate, stomach, pancreas and lymphoma. Counterstaining: Hematoxylin. Magnification: 200×. Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 7 Disease-free survival and overall survival curves according to FabC4 immunoreactivity. P values were determined by log-rank test. Cancer Letters 2014 343, 275-285DOI: (10.1016/j.canlet.2013.09.029) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions