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S.W. Hicks et al.
IgG1 antibody, huB4, via an N-succinimidyl-4-(2- pyridyldithio)butyrate (SPDB) cleavable linker.9 SAR3419 demonstrated clinical activity in patients with relapsed/refractory B-cell lymphoma as both a single agent and in combination with rituximab.10-13 To further, improve the clinical benefit of targeting CD19, second generation ADC can be developed incorporating new innovative link- ers and payload moieties.20 For example, the addition of a sulfonate group in the disulfide linker SPDB ([N-succin- imidyl 2-sulfo-4-(2-pyridyldithio)butanoate], sulfo-SPDB) can increase the potency of ADC bearing similar payloads.21 The indolinobenzodiazepine pseudodimer DGN462 is a potent DNA-alkylating agent with proven anti-tumor activity in preclinical models of solid tumors and acute myeloid leukemia (AML).22 These two innova- tions have been implemented in the recently developed IMGN779, an anti-CD33 ADC with promising preclini- cal23,24 and early clinical activity25 in AML. Here, we present huB4-DGN462, a novel ADC utilizing the huB4, anti- CD19 antibody, linked via sulfo-SPDB to DGN462. The huB4-ADC incorporating sulfo-SPDB and DGN462 demonstrated improved in vitro and in vivo activity in lym- phoma and leukemia models.
Methods
Cell lines
Lymphoma cell lines, all validated for their identity by short tandem repeat DNA fingerprinting (IDEXX BioResearch, Ludwigsburg, Germany), were used and cultured as previously described.26 BCL2, MYC and TP53 status were defined as report- ed in Online Supplementary Table S1. Additional cell lines used included the Burkitt's lymphoma Ramos (ACC-603; DSMZ), and the human acute lymphoblastic leukemia (ALL) cell lines RS4;11 (CRL-1873, ATCC), TOM-1 (ACC-578, DSMZ), and BALL-1 (JCRB0071, JCRB).
Antibody-drug conjugates and free payload
Antibody-drug conjugates (SAR3419, huB4-DGN462, non-tar- geting IgG-DGN462 control) and cell-permeable unconjugated toxin S-Methyl-DGN462 (DGN462-SMe) were generated by ImmunoGen. huB4-DGN462 and IgG-DGN462 were produced as described previously.22 Briefly, a 10-fold molar excess of in situ mixture containing DGN462 and sulfo-SPDB was added to huB4 or IgG antibody in buffer (50 mM EPPS [4-(2-Hydroxyethyl)-1- piperazinepropanesulfonic acid], pH 8.5) containing 15% dimethylacetamide. Upon completion of conjugation, the reac- tion mixtures were purified and buffer exchanged into 20 mM histidine, 50 mM sodium chloride, 8.5% w/v sucrose, 0.01% Tween-20, 50 μM sodium bisulfite pH 6.2 using NAP desalting columns (Illustra Sephadex G-25, GE Healthcare).
In vitro anti-tumor activity
To determine in vitro cytotoxicity potency, B-cell lymphoma
and B-ALL cell lines were treated with a 3-fold dilution series of conjugate for five days without or with a 100-fold concentration of unconjugated huB4 blocking antibody. The relative number of viable cells in each well was then determined using the WST-8 based Cell Counting Kit-8 (Dojindo Molecular Technologies Inc., Rockville, MD, USA). The surviving fraction of cells was plotted against conjugate concentration and the EC50 of activity was calculated using a non-linear regression analysis (GraphPad Prims 4.0). For higher-throughput in vitro cytotoxicity screening,
lymphoma or leukemia cells (104) were seeded in 96-well plates and the indicated compounds were added to create 4-fold dilu- tion series ranging from 200 nM to 0.19 pM and assayed by MTT following 72 hours (h) of treatment, as previously described.24
Apoptosis was assessed using the luminescence-base Caspase-Glo 3/7 assay kit (Promega) according to the manufac- turer's instructions. Briefly, cells were seeded in 384-well plates, treated with ADC or free toxin at the following concentrations (50 and 1,000 pM for huB4-DGN462 and DGN462-SMe; 100 and 5,000 pM for SAR3419). Apoptosis was defined by at least a 1.5-fold increase in signal activation with respect to controls. Differences among groups were calculated using the Wilcoxon rank-sum test (Stata/SE 12.1 for Mac, Stata Corporation, College Station, TX, USA). P<0.05 was considered statistically signifi- cant.
CD19 expression
Fluorescence-activated cell sorting (FACS) for CD19 antigen expression analysis was performed on fresh low passage lym- phoma cell lines. Cells were washed with ice-cold FACS buffer (PBS + 0.5% BSA) and divided 1x106 cells/tube. A pretreatment with human FcR blocking (Miltenyi Biotec Inc., Auburn, CA, USA) was performed according to the manufacturer’s instruc- tions. CD19-PE (5 μL; 1μg) or control isotype was incubated with cells at 4°C for 30 minutes. Cells were washed twice and re-suspended in FACS buffer. Flow-cytometry analysis was car- ried out with a FACS Canto II instrument (BD Biosciences). Median Fluorescence intensity (MFI) of each sample was deter- mined using FacsDiva v.8.0.1 software (BD Biosciences, Allschwil, Switzerland). Unstained cells and cells stained with isotype control antibody were used as controls. RNA expression levels, obtained with the HumanHT-12 v.4 Expression BeadChip (Illumina, San Diego, CA, USA) and with the HTG EdgeSeq Oncology Biomarker panel (HTG Molecular Diagnostics Inc., Tucson, AZ, USA) were published previously.26 For leukemia cell lines, the median number of CD19 antibody binding sites (ABC) was determined as previously described.24 Pairwise correlations were assessed using Stata/SE 12.1 for Mac. P<0.05 was consid- ered statistically significant.
In vivo experiments
Experiments using subcutaneous (DOHH2) and disseminated
(Farage) xenograft models in female CB.17 severe combined immunodeficient (SCID) mice (Charles River Laboratories, Wilmington, MA, USA) were run following previously described procedures.27 All animal procedures were performed in strict accordance with Immunogen’s Animal Care and Use Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For the DOHH2 model, mice were inoculated subcutaneously with 107 cells and were randomized by tumor volume (TV) into treatment groups (6 mice/group), when the average TV reached 100 mm3. Tumor volumes were recorded 2-3 times weekly by caliper measure- ments of the height (H), length (L), and width (W) of the tumor, following the formula:
TV = (H x L x W)/2
For the disseminated model, mice were inoculated intra-
venously (IV) via the lateral tail vein with 107 Farage cells and were randomized into treatment groups (8 mice/group) by body weight. Animal survival was followed; animals were removed from the study when advanced health distress signs were observed or measured (> 20% loss of body weight, hind leg paralysis, tumor growth on body, or moribund state).
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haematologica | 2019; 104(8)