Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice

Abstract

Immunotherapy has proven beneficial in many hematologic and nonhematologic malignancies, but immunotherapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is hampered by the lack of tumor-specific targets. We took advantage of the tumor-immunotherapeutic effect of allogeneic hematopoietic stem cell transplantation and searched the B-cell repertoire of a patient with a lasting and potent graft-versus-AML response for the presence of AML-specific antibodies. We identified an antibody, AT1413, that was of donor origin and that specifically recognizes a novel sialylated epitope on CD43 (CD43s). Strikingly, CD43s is expressed on all World Health Organization 2008 types of AML and MDS. AT1413 induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Of note, AT1413 was highly efficacious against AML cells in a humanized mouse model without affecting nonmalignant human myeloid cells, suggesting AT1413 has potential as a therapeutic antibody.

Graphical abstract

Figure 1.

Identification of AML-specific B-cell clones. (A) Subcloning of miniculture 2K23 yielded an AML-specific clone, producing the antibody AT1413 that binds to AML cell lines (French-American-British classification M0-M5). In all experiments, the recombinant antibody was used. (B) AT1413 also bound to a subset of nonmalignant hematopoietic progenitor cells and to peripheral blood-derived nonmalignant monocytes and granulocytes. AT1413 did not bind to blood-, tonsil-, or thymus-derived mature and immature lymphoid cells, nor did it bind to the tissue cell lines HepG2 (liver), Huh7 (liver), H69 (cholangiocytes), Caco2 (colon), or BJ (foreskin fibroblasts), and primary cultured fibroblasts (normal human adult dermal fibroblasts). (C) Immunohistochemistry of AT1413-biotin with streptavidin-HRP as a secondary antibody confirmed binding to mononuclear cells in tonsil and demonstrated binding to endothelial cells in blood vessels and a punctuate staining pattern of macrophage-type cells in the liver. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate diaminobenzidine (DAB). Scale bars, 50 μm. (D) Comparison of AT1413 staining to THP-1 cells (black triangles), granulocytes (white circles), and endothelial cells with FACS analysis. HUVEC, white diamonds; HAEC, white squares; BOEC, blood outgrowth endothelial cells, white triangles. The in-house generated influenza-specific antibody AT1002 was used as a negative control in A-C.

Figure 1.

Identification of AML-specific B-cell clones. (A) Subcloning of miniculture 2K23 yielded an AML-specific clone, producing the antibody AT1413 that binds to AML cell lines (French-American-British classification M0-M5). In all experiments, the recombinant antibody was used. (B) AT1413 also bound to a subset of nonmalignant hematopoietic progenitor cells and to peripheral blood-derived nonmalignant monocytes and granulocytes. AT1413 did not bind to blood-, tonsil-, or thymus-derived mature and immature lymphoid cells, nor did it bind to the tissue cell lines HepG2 (liver), Huh7 (liver), H69 (cholangiocytes), Caco2 (colon), or BJ (foreskin fibroblasts), and primary cultured fibroblasts (normal human adult dermal fibroblasts). (C) Immunohistochemistry of AT1413-biotin with streptavidin-HRP as a secondary antibody confirmed binding to mononuclear cells in tonsil and demonstrated binding to endothelial cells in blood vessels and a punctuate staining pattern of macrophage-type cells in the liver. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate diaminobenzidine (DAB). Scale bars, 50 μm. (D) Comparison of AT1413 staining to THP-1 cells (black triangles), granulocytes (white circles), and endothelial cells with FACS analysis. HUVEC, white diamonds; HAEC, white squares; BOEC, blood outgrowth endothelial cells, white triangles. The in-house generated influenza-specific antibody AT1002 was used as a negative control in A-C.

Figure 2.

The target of AT1413 is a novel sialylated CD43 epitope (CD43s). (A) IP with biotin-labeled, sortase-tagged AT1413 of THP-1 or Jurkat lysate. Imperial Coomassie-stained gel. (B) Western blot analysis of the AT1413 and AT1002 immunoprecipitates of THP-1 and Molm13 lysates with Mem59 mouse-anti-human CD43 antibody. A vertical line has been inserted to indicate the repositioned marker lane. (C) Deglycosylation of THP-1 cells with neuraminidase (sialidase) abrogated binding of antibodies AT1413, Mem59, DF-T1, and 84-3C1 in a dose-dependent manner. Clone L10 does not target a sialylated epitope of CD43. (D) Staining of THP-1 and Jurkat cells with AT1413 and with the commercially available CD43-specific antibodies DF-T1, L10, and Mem59. (E) Competition experiment with AT1413 and commercially available CD43-specific antibodies. THP-1 cells were incubated with indicated antibodies, biotinylated AT1413 and streptavidin PECy7. AT1413 binding to THP-1 target cells was not affected by preincubation of the cells with commercially available CD43 antibodies, but was inhibited in a dose-dependent manner when THP-1 cells were preincubated with unlabeled AT1413. (F) Immunoprecipitation with AT1413 of truncated variants of THP-1 expressed FLAG-tagged CD43. Immunoblotting with FLAG antibody revealed binding of AT1413 to CD43 mutants A-F and no binding to mutants H-K. Truncations were performed as indicated in supplemental Figures 3 and 4. Ctr, control with GFP-transduced THP-1 cells. (G) AT1413 (2.5 µg/mL) binding of the human cell line THP-1 and the murine AML cell line WEHI-3b. (H) Western blot analysis of the AT1413 and AT1002 immunoprecipitates of the mouse AML cell line WEHI-3b lysate with anti-mouse CD43 antibody.

Figure 3.

CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34⁺ and CD38⁺ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS (Table 2). (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138⁺ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45⁺ granulocytes and monocytes and absence of binding to CD45⁺ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.

Figure 3.

CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34⁺ and CD38⁺ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS (Table 2). (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138⁺ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45⁺ granulocytes and monocytes and absence of binding to CD45⁺ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.

Figure 3.

CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34⁺ and CD38⁺ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS (Table 2). (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138⁺ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45⁺ granulocytes and monocytes and absence of binding to CD45⁺ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.

Figure 4.

AT1413 induces ADCC and CDC of malignant myeloid cells in vitro. (A) AT1413 (open squares) induced ADCC and CDC of the AML cell line SH2 with EC50s of 1.1 nM (0.16 μg/mL) and 12.4 nM (1.86 μg/mL), respectively. Control antibody, AT1002 (red dots). (B) AT1413 (blue bars) induced ADCC of AML cells (SH2), but not of HAEC, HUVEC, and granulocytes. Control antibody, AT1002 (red bars). (C) Labeled SH2 cells were incubated with whole blood from a healthy individual and with AT1413 or rituximab. AML cells but not mononuclear cells were killed. As a control experiment, CD20⁺ Ramos cells were incubated with healthy whole blood and AT1413 or rituximab. PMN, polymorphonuclear cells.

Figure 5.

Anti-AML effect of AT1413 in vivo. (A) HIS mice with human AML (luciferase-GFP transduced SH2) received biweekly treatment with AT1413 or control antibody AT1002 (15 mg/kg IV, indicated by asterisk). AML progression was measured by bioluminescence (CPM) after luciferase injection. (B) Bioluminescence of individual organs harvested after mice were sacrificed. (C) Human T cells, B cells, NK cells, and granulocytes, expressed as proportion of human CD45⁺ hematopoietic cells (excluding CD45dim AML cells) in blood and bone marrow of AT1413- and AT1002-treated mice.