Novel conjugates of single-chain Fv antibody fragments specific for stem cell antigen CD123 mediate potent death of acute myeloid leukaemia cells

Br J Haematol. 2010 Mar;148(6):879-89. doi: 10.1111/j.1365-2141.2009.08033.x. Epub 2010 Jan 8.

Abstract

Four new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor alpha) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K(D)) 4.5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity Fcgamma-receptor III (CD16), created a bispecific single chain Fv (bsscFv). This bsscFv [123 x ds16] mediated potent lysis of AML-derived MOLM-13, THP-1 and SKNO-1 cells in antibody-dependent cellular cytotoxicity (ADCC) reactions at picomolar concentrations. The recruitment of CD16-positive effector cells for the lysis of AML cells via CD123 represents a novel combination with attractive prospects for future clinical testing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases / immunology
  • Animals
  • Antibody-Dependent Cell Cytotoxicity
  • Apoptosis / immunology
  • Bacterial Toxins / immunology
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Immunologic
  • Epitopes
  • Exotoxins / immunology
  • Humans
  • Immunoglobulin Fragments / immunology*
  • Immunotoxins / immunology
  • Interleukin-3 Receptor alpha Subunit / immunology*
  • Leukemia, Myeloid, Acute / immunology*
  • Leukemia, Myeloid, Acute / pathology
  • Mice
  • Mice, Inbred BALB C
  • Single-Chain Antibodies / immunology*
  • Tumor Cells, Cultured
  • Virulence Factors / immunology

Substances

  • Bacterial Toxins
  • Epitopes
  • Exotoxins
  • Immunoglobulin Fragments
  • Immunotoxins
  • Interleukin-3 Receptor alpha Subunit
  • Single-Chain Antibodies
  • Virulence Factors
  • immunoglobulin Fv
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa