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. 2021 Apr 1;13(7):1632.
doi: 10.3390/cancers13071632.

An Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity against B Cell Acute Lymphoblastic Leukemia

Fabian Riegg  1   2 , Martina S Lutz  1   2 , Bastian J Schmied  1   2 , Jonas S Heitmann  1   2 , Manon Queudeville  3 , Peter Lang  3 , Gundram Jung  4 , Helmut R Salih  1   2 , Melanie Märklin  1   2
Affiliations

An Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity against B Cell Acute Lymphoblastic Leukemia

Fabian Riegg et al. Cancers (Basel). .

Abstract

In recent decades, antibody-dependent cellular cytotoxicity (ADCC)-inducing monoclonal antibodies (mAbs) have revolutionized cancer immunotherapy, and Fc engineering strategies have been utilized to further improve efficacy. A promising option is to enhance the affinity of an antibody's Fc-part to the Fc-receptor CD16 by altering the amino acid sequence. Herein, we characterized an S239D/I332E-modified CD133 mAb termed 293C3-SDIE for treatment of B cell acute lymphoblastic leukemia (B-ALL). Flow cytometric analysis revealed CD133 expression on B-ALL cell lines and leukemic cells of 50% (14 of 28) B-ALL patients. 293C3-SDIE potently induced NK cell reactivity against the B-ALL cell lines SEM and RS4;11, as well as leukemic cells of B-ALL patients in a target antigen-dependent manner, as revealed by analysis of NK cell activation, degranulation, and cytotoxicity. Of note, CD133 expression did not correlate with BCR-ABL, CD19, CD20, or CD22, which are presently used as therapeutic targets in B-ALL, which revealed CD133 as an independent target for B-ALL treatment. Increased CD133 expression was also observed in MLL-AF4-rearranged B-ALL, indicating that 293C3-SDIE may constitute a particularly suitable treatment option in this hard-to-treat subpopulation. Taken together, our results identify 293C3-SDIE as a promising therapeutic agent for the treatment of B-ALL.

Keywords: ADCC; B-ALL; CD133; Fc engineering; NK cells; acute lymphoblastic leukemia; immunotherapy; prominin-1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of 293C3-SDIE in B cell acute lymphoblastic leukemia (B-ALL). (A–C) B-ALL cell lines and primary leukemic cells of B-ALL patients were incubated with anti-human CD133 antibody clone 293C3 or mIgG2b as isotype control (both 10 µg/mL) and analyzed by flow cytometry. (A,B) Exemplary data of CD133 expression on the B-ALL cell lines REH, SEM, and RS4;11 and three B-ALL patients (B-ALL1–3) (shaded peaks, anti-CD133; open peaks, control). Specific fluorescence intensity (SFIs) levels are depicted in the upper right corner. (C) Pooled data of CD133 expression on leukemic cells of primary B-ALL patients (n = 28) depicted as % CD133+ B-ALL blasts (left, dotted line: 20% surface expression) and SFI levels (right, dotted line: SFI = 1.5). (D) The B-ALL cell lines SEM and RS4;11, as well as the leukemic cells of an exemplary B-ALL patient (B-ALL3), were incubated with mouse anti-human CD133 antibody clones 293C3, AC133, W6B3C1, or mIgG1 and mIgG2b as isotype controls (all 1 µg/mL) and analyzed by flow cytometry. (E) Schematic illustration of 293C3-SDIE. (F) The B-ALL cell line SEM was incubated with increasing concentrations of the mouse anti-human CD133 antibody 293C3 or 293C3-SDIE and mIgG2b or iso-SDIE as isotype controls (10 µg/mL) and analyzed by flow cytometry. (G) The B-ALL cell line RS4;11 and the leukemic cells of two exemplary B-ALL patients (B-ALL3 and B-ALL4) were incubated with increasing concentrations of 293C3-SDIE or iso-SDIE (10 µg/mL) and analyzed by flow cytometry.
Figure 2
Figure 2
Induction of natural killer (NK) cell reactivity against CD133+ B-ALL cell lines. (A–D) The B-ALL cell lines SEM and RS4;11 were co-cultured with purified NK cells or PBMCs of healthy donors (effector to target (E:T) ratio of 2.5:1 or indicated E:T ratio) in the presence or absence of 293C3-SDIE and iso-SDIE (both 1 µg/mL) for 24 h (activation), 4 h (degranulation), or 2 h (Europium assay). To determine the NK cell activation and degranulation, the NK cells were identified as CD56+CD3− lymphocytes and stained with CD69 or CD107a with subsequent flow cytometric analysis. Target cell lysis of different B-ALL cell lines was analyzed by Europium assays. (A) Exemplary data of purified NK cells tested against the B-ALL cell line SEM. (B,C) Exemplary data from one PBMC donor (left panel) and pooled results of three PBMC donors tested with SEM (red) and RS4;11 (blue) (right panel) are shown (mean ± SEM). (D) Exemplary data from one PBMC donor (left panel) and pooled results of three PBMC donors tested with SEM (red) and RS4;11 (blue) at an E:T ratio of 80:1 (right panel) are shown (mean ± SEM). ns, not significant; * significant (p < 0.05).
Figure 3
Figure 3
Induction of NK cell reactivity against CD133+ primary B-ALL cells. (A–D) Cells of CD133+ B-ALL patients were co-cultured with purified NK cells or PBMCs of healthy donors (E:T ratio of 2.5:1 or indicated E:T ratio) in the presence or absence of 293C3-SDIE and iso-SDIE (both 1 µg/mL) for 24 h (activation), 4 h (degranulation), or 2 h (Europium assay). To determine the NK cell activation and degranulation, the NK cells were identified as CD56+CD3− lymphocytes and stained with CD69 or CD107a with subsequent flow cytometric analysis. Target cell lysis of different B-ALL cell lines was analyzed by Europium assays. (A) Exemplary data of purified NK cells tested against primary B-ALL cells (B-ALL3). (B,C) Exemplary data from one PBMC donor (upper panel) and pooled results of three PBMC donors tested with two B-ALL patients (B-ALL3: red; B-ALL4: blue) (lower panel) are shown (mean ± SEM). (D) Exemplary data from one PBMC donor (left panel) and pooled results of three PBMC donors tested with two B-ALL patients (B-ALL3: red; B-ALL4: blue) at an E:T ratio of 80:1 (right panel) are shown (mean ± SEM). ns, not significant; * significant (p < 0.05).
Figure 4
Figure 4
Association of CD133 expression with B-ALL-related antigens and clinical data. (A–C) Primary leukemic cells of B-ALL patients were incubated with anti-human CD133 antibody clone 293C3 or mIgG2b as isotype control (both 10 µg/mL) and analyzed by flow cytometry. (A) Association of CD133 expression with the B-ALL antigens CD10, CD19, CD20, CD22, CD34, and clinical data. (B) BCR-ABL positivity and MLL-AF4 positivity; (C) risk stratification according to GMALL, complete response, and one-year-survival on primary B-ALL cells. P, p-value; r, Pearson’s correlation coefficient; SR, standard risk; HR, high risk; VHR, very high risk; CR, complete remission; ns, not significant; * significant (p < 0.05).
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