Page 98 - 2020_11-Haematologica-web

E. Gaudio et al.
AB
C
R= -0.61 P < 0.001
log MEN 1309 IC50. pM
D
Figure 1. The antitumor of MEN1309/OBT076 is correlated to the expression of its target. (A) Correlation in all B-cell lymphoma. y-axis: Log10 MFI values of CD205 as measured by flow cytometry. x-axis: Log10 IC50 values of MEN1309/OBT076 (pM). (B) FARAGE cell lines were treated with MEN1309/OBT076 (0-1.2pM and two different concentrations of MBH1309: 16 and 80nM). (C) Representative histogram of cell cycle distribution in FARAGE cells treated with 100pM of IgG-DM4, MEN1309/OBT076 alone and in combination with MBH1309 (72 hours). (D) Cell cycle distribution after exposure with IgG-DM4, MEN1309/OBT076 or combination with MBH1309. Graph plots show mean±standard deviation for values from two biological replicates. MFI: mean fluorescence intensity.
confirmed the cytotoxic activity of MEN1309/OBT076. Four cell lines (Farage, OCI-LY-1, SU-DHL-4 and SU-DHL- 10) were treated with MEN1309/OBT076 (low concentra- tion and high concentration, reflecting IC50 values for each cell line), and with IgG-DM4 and the unconjugated anti- CD205 antibody for 72 hours. In all the cases, MEN1309/OBT076 induced a strong sub-G0 arrest in the cell lines, while the effect of the two control antibodies (IgG-DM4 and naked antibody MBH-1309) was minimal (Online Supplementary Figure S2). No differences in anti- tumor activity were seen according to DLBCL cell of ori- gin, lymphoma histotype, the presence of MYC transloca- tion or BCL2 translocation or TP53 inactivation (Online Supplementary Table S2). The ADC MEN1309/OBT076 did not exert any apoptotic effects against PBMC from two healthy donors who had CD205 expression on their CD19+ B cells similar to one the most sensitive DLBCL cell lines (171 and 111 vs. 106 of the OCI-LY-10) (Online Supplementary Figure S3).
MEN1309/OBT076 anti-tumor activity was highly correlated with CD205 expression in diffuse large B-cell lymphoma cell lines
We then evaluated whether the activity of
MEN1309/OBT076 was affected by the expression levels of its target. Sensitivity to the ADC was correlated with the cell surface CD205 expression measured by flow cytometry in 41 lymphoma cell lines (Online Supplementary Table S3), as shown by the negative correlation between IC50 values and the mean fluorescence intensity values (P<0.0001) (Figure 1A).
Focusing on 23 DLBCL cells, we also assessed the corre- lation between sensitivity and RNA expression of the LY75 gene, coding for CD205, as measured by gene expression profiling or real-time polymerase chain reac- tion (RT-PCR) (Online Supplementary Table S3). The expres- sion levels obtained with the two modalities were posi- tively correlated between them (P<0.001) and with cell membrane protein expression (P<0.001) (Online Supplementary Figure S4). Similar to protein expression, also RNA levels were negatively correlated with IC50 val- ues (P<0.001) (Online Supplementary Figure S5).
The LY75 gene, coding for CD205, is known to give rise to intergenically spliced transcripts with its centromeric neighboring gene CD302.5 MEN1309/OBT076 activity significantly correlated with the expression of two CD205-CD302 intergenically spliced transcripts, meas- ured by RT-PCR, indicating that this phenomenon does
2586
haematologica | 2020; 105(11)