Tribody [(HER2)2xCD16] Is More Effective Than Trastuzumab in Enhancing γδ T Cell and Natural Killer Cell Cytotoxicity Against HER2-Expressing Cancer Cells

Abstract

An enhanced expression of human epidermal growth factor receptor 2 (HER2, ErbB2) often occurs in an advanced stage of breast, ovarian, gastric or esophageal cancer, and pancreatic ductal adenocarcinoma (PDAC). Commonly, HER2 expression is associated with poor clinical outcome or chemoresistance in ovarian and breast cancer patients. Treatment with humanized anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, has improved the outcome of patients with HER2-positive metastatic gastric or breast cancer, but not all patients benefit. In this study, the bispecific antibody [(HER2)2xCD16] in the tribody format was employed to re-direct CD16-expressing γδ T lymphocytes as well as natural killer (NK) cells to the tumor-associated cell surface antigen HER2 to enhance their cytotoxic anti-tumor activity. Tribody [(HER2)2xCD16] comprises two HER2-specific single chain fragment variable fused to a fragment antigen binding directed to the CD16 (FcγRIII) antigen expressed on γδ T cells and NK cells. Our results revealed the superiority of tribody [(HER2)2xCD16] compared to trastuzumab in triggering γδ T cell and NK cell-mediated lysis of HER2-expressing tumor cells, such as PDAC, breast cancer, and autologous primary ovarian tumors. The increased efficacy of [(HER2)2xCD16] can be explained by an enhanced degranulation of immune cells. Although CD16 expression was decreased on γδ T cells in several PDAC patients and the number of tumor-infiltrating NK cells and γδ T cells was impaired in ovarian cancer patients, [(HER2)2xCD16] selectively enhanced cytotoxicity of cells from these patients. Here, unique anti-tumor properties of tribody [(HER2)2xCD16] are identified which beyond addressing HER2 overexpressing solid tumors may allow to treat with similar immunoconstructs combined with the adoptive transfer of γδ T cells and NK cells refractory hematological malignancies. A major advantage of γδ T cells and NK cells in the transplant situation of refractory hematological malignancies is given by their HLA-independent killing and a reduced graft-versus-host disease.

Bispecific antibodies enhance tumor-infiltrating T cell cytotoxicity against autologous HER-2-expressing high-grade ovarian tumors

“…Recently, we reported that the application of bsscFv [HER2xCD3] and tribody [(HER2) 2 xVγ9] enhanced the granzyme B release of PBL‐derived T cells of healthy donors and pancreatic cancer patients, whereas the intracellular expression of granzyme B was nearly comparable in unstimulated versus bsAb‐activated cells . By comparing the intracellular granzyme A and B expression of ex vivo isolated PBL versus TIL of ovarian cancer patients without stimulation, we observed an enhanced production of both cytotoxic mediators in CD8‐TIL compared to CD8‐PBL, whereas the production in Vδ1 and Vδ2 T cells was similar in PBL and TIL suggesting that γδ T cells in the analyzed ovarian cancer patients are in an pre‐activated stage.…”

“…We applied tumor cells initially generated out of the tumor from patient OC11, which revealed an intermediate cisplatin response (Fig. 3) and a high expression of HER‐2 . We cocultured these tumor cells with autologous PBL or TIL ex vivo isolated from patient OC11 after adjuvant therapy followed by three further cycles with carboplatin/paclitaxel and a relapse of the disease.…”

“…[HER2xCD3] and [CD19xCD3] molecules were purified by affinity chromatography using nickel‐nitrilotriacetic acid (Ni‐NTA) agarose beads (Qiagen, Hilden, Germany). Heterodimeric molecules [(HER2) 2 xVγ9] and [(HER2) 2 xCD89] composed of a light chain and a heavy chain derivative and tagged with a C‐terminal hexa‐histidine motif were purified from supernatant by two successive steps of affinity chromatography using CaptureSelect Fab kappa affinity matrix (BAC B.V., Naarden, Netherlands) and nickel‐nitrilotriacetic acid (Ni‐NTA) agarose beads (Qiagen) as described earlier . Purity and integrity of bsAbs were verified by capillary electrophoresis using an Experion automated electrophoresis system (Bio‐Rad, Kabelsketal, Germany) and size exclusion chromatography.…”

Unleashing Natural Killer Cells in the Tumor Microenvironment–The Next Generation of Immunotherapy?

“…Another effective strategy to improve NK cell ADCC is incorporation of Trastuzumab in the bispecific antibody format (discussed below). Bispecific Trastuzumab antibody [(HER2)2xCD16] significantly enhanced ADCC compared to Trastuzumab treatment (214).…”

Removal of the translocation domain and the furin cleavage site decreases the relative hepatotoxicity of the targeted antitumor toxins

“…Для сравнения цитотоксичность направленных токсинов также измеряли с использованием линий клеток с различным содержанием рецептора ERBB2. Использовали линии клеток BT474 и SCOV3 с высоким количеством рецептора ERBB2, линии клеток HEK293 и AsPc1 с низким количеством рецептора ERBB2 и клетки RAJI, на поверхности которых рецептор практически отсутствует [9]. Тестирование направленных токсинов проводили путём добавления токсинов в лунки с клетками с последующей инкубацией в течение 5 дней.…”