Development of unique cytotoxic chimeric antigen receptors based on human scFv targeting B7H6

Protein Eng Des Sel. 2017 Oct 1;30(10):713-721. doi: 10.1093/protein/gzx051.

Abstract

As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated. A representative clone re-formatted as an scFv-CH1-Fc molecule demonstrated specific binding to both B7H6-Ig and native membrane-bound B7H6 on tumor cell lines with a binding avidity comparable to the previously characterized B7H6-targeting antibody, TZ47. Furthermore, these clones recognized an epitope distinct from that of TZ47 and the natural NK cell ligand NKp30, and demonstrated specific activity against B7H6-expressing tumor cells when expressed as a chimeric antigen receptor (CAR) in T cells.

Keywords: Antibody; B7H6; cancer; chimeric antigen receptor; immunotherapy; single chain fragment variable.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Antibodies, Neoplasm / biosynthesis
  • Antibodies, Neoplasm / chemistry*
  • Antibodies, Neoplasm / genetics
  • B7 Antigens / chemistry*
  • B7 Antigens / genetics
  • B7 Antigens / immunology
  • Biomarkers, Tumor / chemistry*
  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / immunology
  • Cell Line, Tumor
  • Cell Surface Display Techniques
  • Cytotoxicity, Immunologic
  • Epitopes / chemistry
  • Epitopes / genetics
  • Epitopes / immunology
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Killer Cells, Natural / cytology
  • Killer Cells, Natural / immunology
  • Mice
  • Models, Molecular
  • Mutant Chimeric Proteins / chemistry*
  • Mutant Chimeric Proteins / genetics
  • Mutant Chimeric Proteins / immunology
  • Mutation
  • Natural Cytotoxicity Triggering Receptor 3 / chemistry
  • Natural Cytotoxicity Triggering Receptor 3 / genetics
  • Natural Cytotoxicity Triggering Receptor 3 / immunology
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Receptors, Antigen, T-Cell / chemistry*
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Single-Chain Antibodies / biosynthesis
  • Single-Chain Antibodies / chemistry*
  • Single-Chain Antibodies / genetics

Substances

  • Antibodies, Neoplasm
  • B7 Antigens
  • B7-H6 antigen, human
  • Biomarkers, Tumor
  • Epitopes
  • Mutant Chimeric Proteins
  • NCR3 protein, human
  • Natural Cytotoxicity Triggering Receptor 3
  • Receptors, Antigen, T-Cell
  • Single-Chain Antibodies