A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development.
A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies.


CONCLUSIONS
The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML.
ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
Verena
V
Strassberger
ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
Katrin L
KL
Gutbrodt
ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
Nikolaus
N
Krall
ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
Christoph
C
Roesli
Division of Hematology, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland.
Hitoshi
H
Takizawa
Division of Hematology, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland.
Markus G
MG
Manz
Philochem AG, Libernstrasse 3, 8112 Otelfingen, Switzerland. Electronic address: tim.fugmann@philochem.ch.
Tim
T
Fugmann
ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland. Electronic address: neri@pharma.ethz.ch.
Dario
D
Neri
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000627:therapeutic use; D000912:Antibodies, Neoplasm / Q000276:immunology* / Q000627:therapeutic use; D015703:Antigens, CD / Q000276:immunology* / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal / Q000276:immunology* / Q000378:metabolism; D005326:Fetal Proteins / Q000276:immunology* / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000276:immunology* / Q000378:metabolism; D020543:Proteome / Q000276:immunology* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020410:Activated-Leukocyte Cell Adhesion Molecule
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D015703:Antigens, CD; D015816:Cell Adhesion Molecules, Neuronal; D005326:Fetal Proteins; D009363:Neoplasm Proteins; D020543:Proteome; C105613:ALCAM protein, human; D020410:Activated-Leukocyte Cell Adhesion Molecule
Activated-leukocyte cell adhesion molecule; Acute myeloid leukemia; Antibody–drug conjugates; Biotinylation; Cell surface antigens; Proteomics