Page 97 - 2020_11-Haematologica-web

CD205 targeting in lymphomas
body directed against CD205, with no direct anti-tumor effect, conjugated through a cleavable N-succinimidyl-4-(2- pyridyldithio) butanoate linker to the potent maytansinoid microtubule disruptor DM4.7 The ADC has shown potent in vivo anti-tumor activity with durable responses and com- plete tumor regressions in many models derived from triple negative breast cancer, pancreatic and bladder cancer cell lines and primary cells.7 MEN1309/OBT076 is now under early clinical investigation for patients with solid tumors and lymphoma (CD205-Shuttle study; clinicaltrials.gov identifier: NCT03403725).9 Here, we present the first preclinical data sustaining CD205 as a novel ther- apeutic target for lymphomas.
Methods
Cell lines
A total of 42 lymphoma cell lines derived from germinal center B-cell type (GCB, n=17) or activated B-cell-like (n=7) diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL, n=10), marginal zone lymphoma (MZL, n=6), and chronic lymphocytic leukemia (CLL, n=2) were used and cultured as previously described.10 Cell line identity was validated by STR DNA finger- printing using the Promega GenePrint 10 System kit (B9510) (Online Supplementary Table S1). BCL2, MYC and TP53 status were defined as previously described.11
Compounds
MEN1309/OBT076, MBH1309 and IgG-DM4 for proliferation assay and MEN1309-PE for FACS analysis were provided by Menarini. Idelalisib, bortezomib, lenalidomide, venetoclax were purchased from Selleckchem (Houston, TX, USA) and rituximab from Roche (Basel, Switzerland).
Proliferation and apoptosis assays
Anti-proliferative activity of MEN1309/OBT076 or IgG-DM4 was assessed as before12 and are described in the Online Supplementary Appendix. Methods for apoptosis detection are defined in the Online Supplementary Appendix. Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by using the Ficoll-Paque PLUS (Ge Heathcare Lifesciences) reagent according to its guidelines. CD19+ B-cell lymphocytes were isolat- ed from PBMC using the CD19 MicroBeads (MACS Miltenyi Biotec). In vitro combinations were assessed as previously described.10,12 Based on the Chou-Talalay Combination Index,13 the effect of the combinations was defined as beneficial if syner- gistic (<0.9) or additive (0-9-1.1).
Western blotting analysis
Protein extraction, separation and immunoblotting were per- formed as previously described.10 The following antibodies were used: anti-β-Tubulin (cst-2146, Cell Signaling Technology), anti- PARP1 (sc-8007, Santa Cruz Biotechnology), anti-BCL2 (sc-492), anti-MCL1 (cst-5453), anti-β BCLXL (sc-8392), and anti-CD20 (ab9475, Abcam).
Immunohistochemistry
Immunohistochemical staining was performed as described in the Online Supplementary Appendix.
CD205 surface expression by cytofluorimetry
CD205 expression was determined by flow cytofluorimetry (FACS) on fresh cells, as described in the Online Supplementary Appendix.
Real-time-polymerase chain reaction
Total RNA was extracted from cells by using TRIZOL. cDNA was prepared by using the Super script double strand cDNA syn- thesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The expression levels of both CD205 and the reported intergenically spliced forms were analyzed as described in the Online Supplementary Appendix. RNA expression levels obtained with the HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, CA, USA) were retrieved from our previous publication (GSE94669).10
Data mining
Associations in two-way tables were tested for statistical signif- icance using either the c2 test or Fisher exact test (two-tailed), as appropriate. Binomial exact 95% confidence intervals (95%CI) were calculated for median percentages. Differences in IC50 values among subtypes were calculated using the Wilcoxon rank-sum test. Baseline gene expression levels of CD205 and of CD302 were extracted from the GSE9466910 dataset obtained using the HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, CA, USA). The degree of correlation among genes was calculated by standard Pearson correlation coefficients. P<0.05 was considered statistically significant. Statistical analyses were conducted using Stata/SE 12.1 for Mac (Stata Corporation, College Station, TX, USA).
Animal studies
Mice maintenance and animal experiments were performed under the institutional guidelines established for the Animal Facility and with study protocols approved by the local Cantonal Veterinary Authority (license TI-22-2015). Methods are described in the Online Supplementary Appendix.
Results
Cluster of differentiation 205 (CD205) is expressed in most diffuse large B-cell lymphoma
Expression of CD205 was assessed using immunohisto- chemistry (IHC) on formalin-fixed paraffin embedded sec- tions of clinical specimens of hematologic cancers derived from lymphomas (n=370), acute myeloid leukemia (n=26), and multiple myeloma (n=14) (Table 1). The antigen CD205 was expressed in the vast majority of the cases, and in entities comprising larger number of samples (DLBCL, MALT lymphomas) expression was detected in 73% and 88% of the cases with moderate-intense expres- sion in 20-50% of the samples. Similar distribution was seen among T-cell lymphomas. CD205 exhibited both membranous and cytoplasmic localization in the lym- phoma cells; expression varied within individual cases (Online Supplementary Figure S1).
MEN1309/OBT076 has in vitro anti-tumor activity in diffuse large B-cell lymphoma
Based on the expression data, we exposed 42 B-cell lym- phoma cell lines derived from DLBCL, MCL, MZL, and CLL, to the anti-CD205 ADC MEN1309/OBT076 (Online Supplementary Table S2). The compound demonstrated a strong anti-tumor activity with a median IC50 of 200 pM (95%CI: 15 pM-20 nM). The control ADC, human IgG conjugated to the DM4 toxin, was 100 times less active (20 nM; 95%CI: 13-70 nM) (Table 2). The anti-tumor activity of MEN1309/OBT076 was mostly cytotoxic: apoptosis induction was observed in 25 of 42 (59.5%) cell lines at a concentration of 1 nM. Cell cycle analysis further
haematologica | 2020; 105(11)
2585