Fc-Small Molecule Antibody Mimetics.

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

Background: The Src pathway is known to regulate tumor matastasis and plays a role in epithelial-mesenchymal transition(EMT). Lapatinib, although, has improved clinical outcome for HER2 positive breast cancer patients, acquired resistance to lapatinib remains an important reason influencing its clinical efficacy. Our previous research shows lapatinib acquired resistance HER2 positive breast cancer cell lines, SKBR3-R and BT474-R have EMT phenomenon and multiple pathway activated. Niclosamide is an FDA-approved antihelminthic agent, which is found has cytotoxicity effect on tumor stem cells recently. We wished to determine the effect of niclosamide on EMT and lapatinib resistance cells and investigate niclosamide as a potential therapeutic agent for HER2 positive breast cancer. Methods: SKBR3 and BT474, two HER2 positive breast cancer cell lines, were continuously exposed to increasing doses of lapatinib to establish two stable cell lines resistant to lapatinib, SKBR3-R and BT474-R. Cell proliferation was determined by CCK8 assay. Protein expression was determined by western-blotting. Invasion ability was analyzed by transwell assay. FITC staining flow cytometry (FCM) was conducted to observe the percentage of apoptosis. Results: Both two HER2 positive lapatinib resistant cell lines, SKBR3-R and BT474-R had EMT phenomenon. Niclosamide had a stronger inhibition effect on lapatinib resistant cell lines than parental cell lines, and induced more apoptosis by FCM. Western-blot showed niclosamide could reverse the EMT phenomenon of SKBR3-R and BT474-R with E-cadherin up-regulated and snail,vimentin down-regulated at the concentration of 0.5-1μM. A significant reduction of Src signaling was also confirmed, as well as the downstream Akt and Erk pathway. After adding niclosimide for 48 hours, SKBR3-R and BT474-R’s capability of invasion were inhibited. Conclusion: Our results suggested that niclosamide had a strong cytotoxic effect on HER2 positive lapatinib resistant breast cancer cell lines. The EMT induced by lapatinib resistance could be reversed by niclosamide, associated with the inhibition of Src pathway activation, as well as the downstream pathways. Niclosamide treatment produced reduced levels of invasion, serving as a novel therapeutic way for lapatinib-resistant breast cancer patients. Citation Format: Junjun Liu, Xiaosong Chen, Yan Mao, Kunwei Shen. Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-06.

Background:Ultraviolet (UV) light exposure has been one of the major inducers of apoptosis. UV exposure has caused pyrimidine dimers and DNA fragmentation which might lead to cell cycle arrest and apoptosis signals activation. UV induced apoptosis has investigated in MDA-MB 468 as an ER negative breast adenocarcinoma and MCF-7 as an ER positive breast cancer cell line. Apoptosis induction rate by UV might be different in these two types of cells due to different biological characteristics of the cell.Objectives:In this paper we have evaluated serial dose of UV-B exposure on ER positive and ER negative breast cancer cell lines and its effect on apoptosis or necrosis induction in these cells.Materials and Methods:MDA-MB468 and MCF-7 cell lines have cultured for 24 hours and UV exposure has carried out at 290 nm at dose of 154 J/m2 to 18 KJ/m2 using UV lamp. UV exposed cells have incubated in cell culture condition for 24 or 48 hours following UV exposure and the cells have stained and analyzed by flow cytometry for apoptosis evaluation by Annexin V/PI method.Results:Apoptosis rate (PI and Annexin V double positive cells) after 24 hours incubation was higher in 24 hours in comparison with 48 hours incubation in both cell lines. The frequency of PI positive MDA-MB 468 cells was higher than PI and Annexin V double positive cells after 48 hours. PI positive MDA-MB 468 cells were significantly higher than MCF-7 cells in 24 hours incubation time.Conclusions:The results have shown that MDA-MB 468 cells were more sensitive to UV exposure and DNA fragmentation and necrosis pathway was dominant in these cells.

Background We investigated the expression of TLR genes and the effects of CpG ODN in Estrogen Receptor positive, Progesterone Receptor positive breast cancer cell line (T47D) and a triple-negative breast cancer cell line (MDA-MB-468) followed by studying the immunostimulatory activity of CpG oligonucleotides in breast cancer cell lines T47D and MDA-MB-468. Materials and Methods We evaluated the expression pattern of TLR genes (TLR1 to TLR9) in T47D and MDA-MB-468 cells using Real-time qPCR analysis. The intracellular TLR9 protein expression was studied by flow cytometry. The effect of CpG ODN on cell viability was tested using MTT assay. The relative expression of pro-inflammatory (IL6 and TNFα) and anti- inflammatory/immunosuppressive cytokines genes (IL10 and TGF beta1) were examined by Real-time qPCR. Results We found that MDA-MB-468 cells expressed TLR2, TLR3, TLR6, TLR8, and TLR9 genes and T47D cells expressed TLR3, TRL5, TLR8, and TLR9 genes. Stimulation of TLR9 in vitro with CpG significantly reduced the cell viability of T47D and MDA-MB-468 cells. IL6 cytokine gene expression was significantly reduced in both CpG treated T47D cells and MDA-MB-468 cells. TNFα gene expression was significantly reduced after treatment with CpG in MDA-MB-468 cells but not in T47D cells. IL10 and TGFβ1 expression were downregulated in CpG treated T47D cells. Whereas, IL10 and TGFβ1 were elevated in CpG treated MDA-MB-468 cells. Conclusion Our in vitro finding gives preliminary evidence that triggering TLR9 using CpG ODN decreases the cell proliferation and alters the pro-inflammatory cytokines in favor of inhibition of hormone receptor positive breast cancer cells T47D and triple negative breast cancer cells MDA-MB-468.

Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.

Ovarian cancer (OvCa) is the second most common type of gynecological cancer. More seriously, the prognosis for survival is relatively poor if an early OvCa diagnosis is not achieved. However, it is extremely challenging to diagnose very early stage OvCa, when treatments are the most effective, because of the lack of specific and sensitive biomarkers. Therefore, in order to achieve early detection of OvCa, screening and identifying biomarkers with high specificity and affinity are greatly needed. In this study, an integrated microfluidic system capable of performing cell-based systematic evolution of ligands by an exponential enrichment (Cell-SELEX) process was developed for automatic, high-throughput screening of multiple cell lines to competitively select aptamer-based biomarkers for OvCa. This on-chip Cell-SELEX process only required five rounds of aptamer selection, which is much faster than using a conventional SELEX process (22 rounds). Using this on-chip process, 13 aptamers specific to OvCa cells were successfully screened and three of them showed high affinity towards target cells with dissociation constants of 1.8 nM, 8.3 nM, and 1.3 nM. Analysis of stained fluorescence images and competitive testing against multiple cancer cell lines (cervical cancer, breast cancer, lung cancer, and liver cancer) were performed to verify the specificity of these selected aptamers. The results demonstrated that this developed system could perform the on-chip Cell-SELEX selection successfully and could be applied for personalized aptamer screening or targeted therapy monitoring in the near future.

Aptamer: A theranostic approach towards breast cancer

KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A044.

Concomitant Association of T-DM1 and Radiation: An In Vitro Study on HER2 Positive Cells of Breast Cancer

With the development of immunotherapy in recent years, therapeutic antibodies have become a major player combating cancer with the limitation of recognizing cell surface or secreted molecules. In contrast, the human T cell receptor (TCR) recognizes its cognate peptide antigen when presented on human leukocyte antigen (HLA) molecules, and thus detects a broader range of targets including intracellular proteins. Here we established an innovative strategy of generating fully human TCR mimic (TCRm) antibodies that can recognize antigen with a higher specificity and affinity. Previously, Biocytogen established RenMab™ and Renlite࣪ mice (RenMice). These humanized immunoglobulin mouse models generate fully human antibodies that carry human Ig heavy chain and human kappa light chain variable domains. Here, HLA genes were introduced into RenMice to build tolerance towards HLA. When immunized with HLA- peptide complexes, these mice generate antibodies that specifically target the HLA-Peptide complex, with no reactivity to HLA alone nor other peptide-HLA complexes. Compared to natural TCRs, these TCRm antibodies show higher affinity and specificity. Using optimized immunization methods and high-throughput screening, our antibody discovery platform generated fully human TCRm antibodies from HLA/RenMab™ and HLA/Renlite™ mice. Producing TCR-like binders with this unique TCRm technology can become a very powerful tool in the immunotherapy field. Citation Format: Jun Du, Nannan Wang, Li Hui, Ruixue Wang, Taolin Liu, Qingcong Lin. Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2885.

Purpose Neuropilin-1 (NRP-1) was initially characterized as a neuronal receptor for specific secreted members of the semaphorin family involved in axon repulsion. However, it was recently reported to play an important role in the immune system. This study aimed to investigate the role of NRP-1 expression on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cell line SKBR3. Methods Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were prepared using Lymphoprep. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD14 and allophycocyanin (APC)-conjugated anti-NRP-1 antibodies, and an NRP-1high and NRP-1low monocyte subset was sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay using SKBR3 as target cells and trastuzumab as an antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. A CD107a degranulation assay was also used to determine the role of NRP-1 expression on monocytes in ADCC. PBMCs were co-cultured with target SKBR3 cells and trastuzumab for 2 h, stained with APC-conjugated anti-NRP-1 and PE-Cy5-conjugated anti-CD107a antibodies, and analyzed by flow cytometry. Results The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted NRP-1 high monocytes (12%) than in NRP-1 low monocytes (4%). Interestingly, microscopic findings showed that most NRP-1 high monocytes covered trastuzumab-treated target cancer cells (showing higher cellular cytotoxicity) while NRP-1 low monocytes did not cover the target cells but changed their morphology from a round to a dendritic shape (showing less cellular cytotoxicity) during ADCC. Moreover, the degranulation assay showed a higher expression of CD107a in NRP-1 high monocytes (17.4%) than in NRP-1 low monocytes (1.16%). Mean fluorescence intensity of CD107a in NRP-1 high and NRP-1 low monocytes was 92.6 and 26.3, respectively. Conclusions These data suggest that NRP-1 high monocytes exhibit higher trastuzumab-mediated ADCC than NRP-1 low monocytes in HER2-positive breast cancer cell lines. It may therefore be possible to differentiate HER2-positive breast cancer patients for whom trastuzumab will be less effective by testing for monocyte NRP-1 expression levels. Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Tomoharu Sugie, Fumiaki Sato, Masakazu Toi. Neurropilin-1 high monocytes play an important role in trastuzumab mediated antibody-dependent cellular cytotoxicity in HER2 positive breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1536. doi:10.1158/1538-7445.AM2013-1536

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl^- and lactate outward currents with an EC₅₀ of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Murine hybridomas were generated by immunizing mice with purified alpha-fetoprotein (AFP) to determine the cross-reactivity of AFP antibodies with human serum albumin (HSA). Eighteen out of 23 hybridoma products were cross-reactive with HSA. All 23 monoclonal antibodies (MAbs) could be subgrouped into 3 groups by their binding affinity to AFP and HSA, and one MAb from each group, IIB3, IIIB1 and IIIC6, were chosen for this study. The affinity to AFP was highest for IIB3 followed by IIIC6 and IIIB1, and that to HSA was in the order IIIB1 greater than IIB3. Immunohistochemical staining revealed no cross-reactions of these antibodies with HSA at the tissue level. IIB3 was the most efficient in the quantitative assays, indicating high affinity and specificity (non-cross-reactivity) of the antibodies, both of which are prerequisites for in vitro assays. Scans with good tumor localization were obtained from mice which received 131I-labeled IIB3 or IIIC6 after xenografting of the human hepatocellular carcinoma. The images obtained with IIB3 were inhibited in the presence of either AFP or HSA in the circulation. These findings indicated that the use of cross-reactive and/or high-affinity antibodies against AFP provides an inhibitory factor for tumor localization. MAb IIIC6, because of its high specificity and moderate affinity to AFP, seems to be the best candidate for immunoscintigraphy of the tumors in future studies.

eP061: Genetic risk for breast and ovarian cancer in a diverse and unselected population

Fluorescence imaging has become an indispensable technique in cancer research because it can reveal informative molecular, cellular, anatomical, and functional insights. Development of advanced fluorescent probes with superior sensitivity and biological selectivity for fluorescence imaging is thus imperative. To move forward in this direction, we developed an easy self-assembly method for fabricating aptamer- anchored rubrene-loaded organic fluorescent nanoprobes. The aptamer-modified organic nanoprobes integrated the best features of the organic light-emitting materials and the aptamers, thus endowing them with excellent cell-targeting capability, high stability, and good biocompatibility. By using this general method, a variety of biocompatible and highly bright organic fluorescent nanoprobes based on novel organic light-emitting materials with specific recognition could be easily constructed for real-time biosensing and long-term biomedical imaging. © 2019 Chinese Chemical Society.