(C) Flow cytometry analysis of binding of increasing amounts of F7AK3 to primary TNBC cells. primary tumor cells mediated by F7AK3 were determined in vitro. In vivo antitumor activity of F7AK3 was investigated in a xenograft TNBC tumor model, using immunodeficient mice that were reconstituted with human peripheral blood mononuclear cells. Results We demonstrated that F7AK3 binds specifically to human TROP2 and CD3 antigens, as well as TNBC cell lines and primary tumor cells. Human T cells can only be activated by F7AK3 in the presence of target tumor cells. F7AK3 recruits T cells to TROP2+ tumor cells in vitro and into tumor tissues in vivo. Antitumor growth activity of F7AK3 is observed in a xenograft TNBC tumor model. Conclusion This study showed the antitumor potential of an anti-TROP2xCD3 bispecific antibody F7AK3 to TNBC tumor cells both in vitro and in vivo. These data demonstrate that F7AK3 has the potential to treat TNBC patients, which warrants further preclinical and clinical evaluation of the F7AK3 in advanced or metastatic TNBC patients. Keywords: immunotherapy, tumor microenvironment, breast neoplasms, lymphocytes, tumor-infiltrating, antibodies, neoplasm Background Triple negative breast cancer (TNBC) represents a diverse subgroup of breast cancers that are characterized with the absence of expression of the estrogen receptor (ER), progesterone receptor (PR), and ERBB2 (also known as HER2).1 2 TNBC is associated with (+)-Alliin a poor prognosis.1 2 Chemotherapy has been the mainstay for the treatments of TNBC patients, however, most patients experience relapse following an initial response.2C4 Over the last few years, the therapeutic regimens for TNBCs have been expanded.5 6 Olaparib and talazoparib, two poly ADP-ribose polymerase (PARP) inhibitors were approved by U S Food and Drug Administration (FDA) and European Medicines Agency for locally advanced/metastatic TNBC with germline BRCA mutations.7C9 Atezolizumab in combination with nab-paclitaxel was approved in 2019 by FDA as first-line treatment for unresectable locally advanced or metastatic TNBC whose tumors express programmed cell death 1 ligand 1 (PD-L1) and pembrolizumab plus chemotherapy was approved for inoperable locally advanced/metastatic TNBC.10C12 Sacituzumab govitecan, a first-in-class antibody-drug conjugates (ADCs) targeting trophoblast cell surface antigen 2 (TROP2) has been approved in 2020 by FDA for metastatic TNBC (+)-Alliin who have received at least two prior therapies.13C15 TROP2, encoded by gene. Primer sequences are as follows. (figure (+)-Alliin 1D). Of which, MDA-MB-468 had the highest expression (figure 1D). We further evaluated the amounts of TROP2 protein with immunoblot and flow cytometry assays. Comparing to MCF7 cells, all other three cell lines had significant enhanced expression of TROP2 (figure 1E, F, online supplemental figure S1C). Taken together, these data demonstrated that TROP2 is highly expressed in TNBC tumor cells, tumor tissues but not adjacent normal tissues. Open in a separate window Figure 1 TROP2 is highly expressed in TNBC tumor tissues and cells. (A) H&E and IHC staining of TROP2, CD3 and KI67 in TNBC tumor and paratumor tissues and representative images are shown (n=9). Scale bar, 100?m. (B) TROP2 expression scores in TNBC tumor and paratumor tissues measured by IHC (n=9). (C) The percentages of T Rabbit Polyclonal to LRP11 cells in TNBC tumor and paratumor tissues measured by IHC staining with anti-CD3 antibody (n=9). (D) Quantitative PCR analysis of expression in four breast cancer cell lines. (E) TROP2 expression in four breast cancer cell lines was determined by flow cytometry using anti-TROP2 (Biolgend, 363804) and histograms of the MFI (median fluorescence intensity) of TROP2 from three experiments were shown (F). Experiments.