Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcγRI), the high-affinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8+ T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64+ U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC50) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64+ AML cells, whereas CD64− AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924–32]

[1]  H. Kantarjian,et al.  Acute myeloid leukemia , 2018, Methods in Molecular Biology.

[2]  Rainer Fischer,et al.  Eukaryotic expression and secretion of EGFP-labeled annexin A5. , 2008, Protein expression and purification.

[3]  J. Lieberman,et al.  Delivering the kiss of death: progress on understanding how perforin works. , 2007, Current opinion in immunology.

[4]  I. Heisler,et al.  A cleavable molecular adapter reduces side effects and concomitantly enhances efficacy in tumor treatment by targeted toxins in mice. , 2007, Journal of controlled release : official journal of the Controlled Release Society.

[5]  J. Medema,et al.  Granzymes at a glance , 2006, Journal of Cell Science.

[6]  M. Harmsen,et al.  CD64-Directed Immunotoxin Inhibits Arthritis in a Novel CD64 Transgenic Rat Model1 , 2006, The Journal of Immunology.

[7]  W. Wels,et al.  Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition , 2006, Cell Death and Differentiation.

[8]  D. Gilliland,et al.  Drug therapy for acute myeloid leukemia. , 2005, Blood.

[9]  M. Willingham,et al.  Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by Kupffer cells. , 2004, Molecular cancer therapeutics.

[10]  J. Bijlsma,et al.  Depletion of synovial macrophages in rheumatoid arthritis by an anti-FcγRI-calicheamicin immunoconjugate , 2004, Annals of the rheumatic diseases.

[11]  J. Zhao,et al.  Secreted Antibody/Granzyme B Fusion Protein Stimulates Selective Killing of HER2-overexpressing Tumor Cells* , 2004, Journal of Biological Chemistry.

[12]  R. Rudolph,et al.  Killing of target cells by redirected granzyme B in the absence of perforin , 2004, FEBS letters.

[13]  Regina M. Krohn,et al.  Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells. , 2003, Cancer research.

[14]  W. Hittelman,et al.  Targeted delivery of human pro-apoptotic enzymes to tumor cells: In vitro studies describing a novel class of recombinant highly cytotoxic agents. , 2003, Molecular cancer therapeutics.

[15]  M. Rosenblum,et al.  Mechanistic studies of a novel human fusion toxin composed of vascular endothelial growth factor (VEGF)121 and the serine protease granzyme B: directed apoptotic events in vascular endothelial cells. , 2003, Molecular cancer therapeutics.

[16]  J. Bijlsma,et al.  Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fcgamma receptor I-directed immunotoxin. , 2003, Arthritis and rheumatism.

[17]  J. Bijlsma,et al.  Fcγ receptor expression levels on monocytes are elevated in rheumatoid arthritis patients with high erythrocyte sedimentation rate who do not use anti‐rheumatic drugs , 2003 .

[18]  M. Stöcker,et al.  Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. , 2003, Protein expression and purification.

[19]  Yong J. Lee,et al.  Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis , 2003, The Journal of cell biology.

[20]  V. Diehl,et al.  Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma. , 2002, Blood.

[21]  R. Peebles,et al.  Expression of activation markers on alveolar macrophages in allergic asthmatics after endobronchial or whole-lung allergen challenge. , 2002, Clinical immunology.

[22]  L. Case,et al.  Phase I trial of a novel diphtheria toxin/granulocyte macrophage colony-stimulating factor fusion protein (DT388GMCSF) for refractory or relapsed acute myeloid leukemia. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[23]  G. McDonald,et al.  Hepatic sinusoidal obstruction after gemtuzumab ozogamicin (Mylotarg) therapy. , 2002, Blood.

[24]  A. Frankel,et al.  A diphtheria toxin-interleukin 3 fusion protein is cytotoxic to primitive acute myeloid leukemia progenitors but spares normal progenitors. , 2002, Cancer research.

[25]  X. H. Chen,et al.  Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[26]  I. Bernstein,et al.  Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells. , 2001, Blood.

[27]  M. Fanger,et al.  Colocalization of FcγRI-Targeted Antigen With Class I MHC: Implications for Antigen Processing1 , 2001, The Journal of Immunology.

[28]  T. Thepen,et al.  Cytotoxicity of anti-CD64-ricin a chain immunotoxin against human acute myeloid leukemia cells in vitro and in SCID mice. , 2001, Journal of hematotherapy & stem cell research.

[29]  S. Barth,et al.  Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells. , 2001, Journal of immunological methods.

[30]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[31]  E. Knol,et al.  Expression of Fc receptors for IgG during acute and chronic cutaneous inflammation in atopic dermatitis , 2000, The British journal of dermatology.

[32]  T. Logtenberg,et al.  Recombinant lipid-tagged antibody fragments as functional cell-surface receptors , 2000, Nature Medicine.

[33]  J. Trapani,et al.  Anti‐viral strategies of cytotoxic T lymphocytes are manifested through a variety of granule‐bound pathways of apoptosis induction , 1999, Immunology and cell biology.

[34]  A. Krasinskas,et al.  The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation. , 1998, American journal of clinical pathology.

[35]  R. Curnow.  Clinical experience with CD64-directed immunotherapy. An overview , 1997, Cancer Immunology, Immunotherapy.

[36]  J. Perentesis,et al.  Induction of apoptosis in multidrug-resistant and radiation-resistant acute myeloid leukemia cells by a recombinant fusion toxin directed against the human granulocyte macrophage colony-stimulating factor receptor. , 1997, Clinical cancer research : an official journal of the American Association for Cancer Research.

[37]  R. Gottlieb,et al.  New Paradigm for Lymphocyte Granule-mediated Cytotoxicity , 1996, The Journal of Biological Chemistry.

[38]  T. Keler,et al.  Construction and characterization of a humanized anti-gamma-Ig receptor type I (Fc gamma RI) monoclonal antibody. , 1995, Journal of immunology.

[39]  C. Anderson,et al.  Biology of Human Immunoglobulin G Fc Receptors , 1991, Journal of leukocyte biology.

[40]  C. Bloomfield,et al.  Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells. , 1989, Blood.

[41]  R. Graziano,et al.  Cytotoxicity mediated by human Fc receptors for IgG. , 1989, Immunology today.

[42]  A. Lanzavecchia,et al.  Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes. , 1988, Journal of immunology.

[43]  C. Anderson,et al.  Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line. , 1986, The Journal of biological chemistry.

[44]  C. Anderson.  Isolation of the receptor for IgG from a human monocyte cell line (U937) and from human peripheral blood monocytes , 1982, The Journal of experimental medicine.

[45]  V. Diehl,et al.  Ki-4(scFv)-ETA', a new recombinant anti-CD30 immunotoxin with highly specific cytotoxic activity against disseminated Hodgkin tumors in SCID mice. , 2000, Blood.

[46]  T. Thepen,et al.  Resolution of cutaneous inflammation after local elimination of macrophages , 2000, Nature Biotechnology.

[47]  M. Clément,et al.  Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells. , 1990, Nouvelle revue francaise d'hematologie.

[48]  U. G. Dailey.  Cancer,Facts and Figures about. , 2022, Journal of the National Medical Association.