Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

Front Immunol. 2024 Aug 29:15:1457887. doi: 10.3389/fimmu.2024.1457887. eCollection 2024.

Abstract

NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted.

Keywords: BiKE; ErbB2; HER2; NK-92; NKG2D; bispecific killer cell engager; chimeric antigen receptor; natural killer cells.

MeSH terms

  • Animals
  • Antibodies, Bispecific* / immunology
  • Antibodies, Bispecific* / pharmacology
  • Cell Line, Tumor
  • Cross Reactions* / immunology
  • Humans
  • Immunotherapy / methods
  • Killer Cells, Natural* / immunology
  • Killer Cells, Natural* / metabolism
  • Mice
  • NK Cell Lectin-Like Receptor Subfamily K* / immunology
  • NK Cell Lectin-Like Receptor Subfamily K* / metabolism
  • Neoplasms / immunology
  • Neoplasms / therapy
  • Receptor, ErbB-2* / immunology
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology

Substances

  • Receptor, ErbB-2
  • NK Cell Lectin-Like Receptor Subfamily K
  • Antibodies, Bispecific
  • ERBB2 protein, human
  • Single-Chain Antibodies
  • KLRK1 protein, human

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported in part by grants from Deutsche Forschungsgemeinschaft (DFG) (WE 2589/6-1), LOEWE Center Frankfurt Cancer Institute (FCI) (HMWK III L 5-519/03/03.001-0015), and institutional funds of Georg-Speyer-Haus. Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health and the Hessian Ministry of Higher Education, Research, Science and the Arts.