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Letters to the Editor
Bi38-3 is a novel CD38/CD3 bispecific T-cell engager with low toxicity for the treatment of multiple myeloma
Monoclonal antibodies targeting CD38, such as daratu- mumab, have shown good therapeutic efficacy in multi- ple myeloma (MM), both alone1 and in combination with normal standard-of-care regimens.2,3 However, many patients eventually relapse because of resistance mecha- nisms, including FcγR-dependent downregulation of CD38 on tumor cells as well as inhibition of comple- ment-dependent cytotoxicity, antibody-dependent cell- mediated cytotoxicity and antibody-dependent cellular phagocytosis.4
Bispecific T-cell engaging (BiTE) antibodies belong to a new class of immunotherapeutic agents that can recog- nize, on the one hand, a specific antigen on the surface of the target cells (i.e., tumor antigen) and, on the other hand, the CD3e chain on T lymphocytes.5 By activating T cells via the CD3 complex and recruiting them in proximity of target cells, BiTE antibodies efficiently induce T-cell-medi- ated cytotoxicity.6 In MM, bispecific antibodies recogniz- ing B-cell maturation antigen or FcRH5 (CD307) have been shown to eliminate tumor plasma cells in preclinical mod- els.7-9 However, FcRH5 expression is not limited to tumor plasma cells and B-cell maturation antigen is abundantly secreted in MM patients.10,11 These two features may limit the specificity or efficiency of these cognate bispecific anti- bodies in vivo. Recently, an anti-CD38 bispecific antibody, AMG424, was shown to eliminate MM cells in preclinical models, but also to trigger “off tumor” T-cell cytotoxicity on B, T and NK cells in vitro,12 Thus, development of an efficient and safer bispecific antibody could contribute to improve the treatment of MM.
We have developed a new BiTE (Bi38-3) that consists of two single-chain variable fragments derived from mouse hybridomas, producing anti-human CD38 and CD3e (Online Supplementary Figure S1A and B). Purified Bi38-3 efficiently and specifically recognizes CD38 on MM cells and binds CD3e−expressing Jurkat T cells (Online Supplementary Figure S1C and D). To assess the effect of Bi38-3 on the cytotoxic activity of T cells, we performed co-culture assays with effector T cells, isolated from peripheral blood mononuclear cells of healthy donors, on firefly luciferase-expressing target KMS11 and MM.1S MM cell lines. We observed that T cells readily killed KMS11 cells (as measured by luciferase activity) in a Bi38-3 dose-dependent manner, with a half maximal effective concentration (EC50) around 5 ng/mL, the equiv- alent of 0.09 nM for this 55.6 Kda protein (Figure 1A). Bi38-3-mediated T-cell cytotoxic activity was also observed in co-culture with MM.1S cells (Figure 1B). However, in this cell line, which expresses heteroge- neous levels of CD38, the EC50 was 10-fold lower (0.5 ng/mL), indicating that Bi38-3 also triggered strong T-cell cytotoxicity in MM cells with weaker CD38 expression. In line with this, stimulation of donor T cells with Bi38- 3 in the presence of MM.1S cells led to robust prolifera- tion, expression of activation markers CD25 and CD69, as well as production of interferon-γ, tumor necrosis fac- tor-α and interleukin-2 in a Bi38-3 dose-dependent man- ner (Online Supplementary Figure S1E-H). The viability of MM.1S or KMS11 MM cells was not affected by co-cul- ture with T cells or Bi38-3 alone (Figure 1A and B, right). In addition, Bi38-3 induced poor T-cell-mediated killing of CD38-deficient MM.1S cells (MM1.S-KO), with around half of CD38-deficient MM.1S cells surviving the co-culture even at the highest dose of Bi38-3 (1 mg/mL)
(Figure 1C). These results indicate that, at lower doses, similar to those that are expected in patients, Bi38-3 directs efficient T-cell cytotoxic activity specifically towards CD38-expressing MM cells.
We next analyzed the potential of Bi38-3 to induce
lysis of target tumor plasma cells, isolated from four
patients at diagnosis, by autologous effector T cells.
Fluorescence-activated cell sorting (FACS) analysis of
overnight effector cell:target cell (E:T) co-cultures
+
revealed that the numbers of viable CD138 MM cells were reduced in a Bi38-3 dose-dependent manner, with the EC50 ranging from 0.028 to 1.29 ng/mL, depending on the patient (Figure 1D and Online Supplementary Figure S2). Importantly, in the absence of T cells, Bi38-3 exhib- ited no toxicity against fresh tumor cells. Bi38-3-induced cytotoxicity of autologous T cells was further investigat- ed on tumor plasma cells from three MM patients at relapse and demonstrated similar efficacy, with EC50 val- ues ranging from 0.2 to 0.86 ng/mL (Figure 1E). These results indicate that Bi38-3 triggered autologous T-cell- mediated killing of tumor plasma cells from patients both at diagnosis and at relapse.
To investigate potential toxic effects of Bi38-3 on blood cells, peripheral blood mononuclear cells from donors were treated with various concentrations of Bi38-3 for 24 h and the mononuclear cell populations were individual- ly analyzed by FACS (Online Supplementary Figure S3). We observed that the percentages of CD14-expressing monocytes included in the live gate were markedly reduced in a Bi38-3 dose-dependent manner (Figure 2A). In contrast, the percentages of CD4 and CD8 T lympho- cytes, which together represented around 60% of total peripheral blood mononuclear cells, slightly increased in response to Bi38-3, probably due to the decrease in live CD14+ cells. Similarly, the B (CD19+) and NK (CD56+) cell populations remained at similar levels (around 10% and 5%, respectively), even at high concentrations of Bi38-3 (100 ng/mL) (Figure 2A).
Next, we investigated whether expression of CD38 at the surface of blood cells was downregulated by Bi38-3. FACS analysis indicated that CD38 mean fluorescence intensity on T, B and NK cells remained similar in cul- tures containing increasing doses of Bi38-3 (Figure 2B). In line with this, CD38 expression was not dramatically reduced on CD14+ monocytes. Of note, this analysis could not be performed at high doses because these cells, which express higher levels of CD38, were sensitive to elevated concentrations of Bi38-3 (above 1 ng/mL). To compare the activity of Bi38-3 on CD38high MM versus CD38int cells, we performed co-culture assays with MM.1S, expressing high levels of CD38, freshly isolated B cells, expressing intermediate levels of CD38 (Figure 2C) and autologous T cells. Following overnight culture, the percentages of viable CD20+ B cells and CD138+ MM.1S cells were analyzed by flow cytometry (Online Supplementary Figure S4A). We observed that the percent- ages of MM.1S cells dropped at Bi38-3 concentrations of 0.1 ng/mL and this reduction was more dramatic at high- er doses (Figure 2C). In contrast, compared to untreated conditions, the percentages of viable CD20+ B cells remained unchanged even at high concentrations of Bi38-3 (Figure 2C).
We developed a similar autologous tri-culture assay to investigate potential toxic effects of Bi38-3 on CD34+ bone marrow hematopoietic progenitors and on regula- tory T cells, which both express low levels of CD38. While Bi38-3 readily induced MM cell killing at low con- centrations (10-2 ng/mL and above), we found that it did not trigger significant T-cell-mediated cytotoxicity on
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