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S.W. Hicks et al.
Table 1. Anti-tumor activity of huB4-DGN462 and SAR3419 in B-cell lymphoma cell lines. huB4-DGN462
SAR3419
All
GCB-DLBCL ABC-DLBCL MCL
MZL
CLL
Number of cell lines
46
19 8 10 6 2
Median IC50
100 pM
100 pM 600 pM 45 pM 65 pM 2250 pM
95%CI
38-214 pM
3 pM-1170 pM 20 pM-3000 pM 5-84 pM 6-910 pM 1500-3000 pM
Median IC50
37 nM
2 nM 6.5 nM 3 nM 5 nM 25 nM
95%CI
2.4-5.1 nM
0.7-3.6 nM 3.8-7.3 nM 1.3-9.7 nM 1.5-9.8 nM 10-40 nM
Wilcoxon rank-sum P <0.0001
0.003 0.001 0.017 0.004 0.130
PMBCL 1 200pM n.d. 10nM n.d. n.d.
GCB-DLBCL: germinal center B-cell type diffuse large B-cell lymphoma; ABC-DLBCL: activated B-cell like diffuse large B-cell lymphoma; MCL: mantle cell lymphoma; MZL: mar- ginal zone lymphoma; CLL: chronic lymphocytic leukemia; PMBCL: primary mediastinal large B-cell lymphoma; n.d.: not determined.
the incorporation of the payload did not appreciably alter antibody binding affinity.
huB4-DGN462 has improved in vitro anti-lymphoma activity compared to SAR3419
To characterize the ability of huB4-DGN462 to kill tumor cells, the in vitro cytotoxicity of huB4-DGN462 was compared to SAR3419 in a subset of B-cell lymphoma and B-ALL tumor cell lines. The tumor cell killing was assayed five days after treatment. While SAR3419 had limited potency, huB4-DGN462 was highly active with IC50s ranging from 1-16 pM (Figure 1D). In addition, unconju- gated antibody was able to block the activity of both huB4-DGN462 and SAR3419 confirming that the cytotox- icity of both conjugates is the result of specific CD19 anti- gen binding on tumor cells.
To further characterize the in vitro potency of huB4- DGN462, we performed a 72-h in vitro cytotoxicity screen on 46 B-cell lymphoma cell lines, mostly derived from DLBCL, exposed also to SAR3419 (Table 1 and Online Supplementary Table S1). huB4-DGN462 was potent with a median IC50 of 100 pM (95%CI: 38-214). The free pay- load, DGN462-SMe, had a median IC50 of 26 pM (95%CI: 1-186) (Online Supplementary Table S1). huB4- DGN462 induced caspase 3/7 activation consistent with an apoptotic mechanism of action (Online Supplementary Table S1). In agreement, cell cycle analysis in two DLBCL cell lines showed a G2-M arrest with subG0 accumulation of cells after huB4-DGN462 and DGN462-SMe treatment (Online Supplementary Figure S1), similar to that reported for IMGN779, a DGN462-containing ADC.24
While the rank order of anti-proliferative activity for huB4-DGN462 and SAR3419 was similar across the cell lines tested (R=0.42, P=0.004) (Online Supplementary Figure S2), huB4-DGN462 was over 30-times more potent than SAR3419 (P<0.0001) (Table 1 and Online Supplementary Figure S3). Increased cytotoxic activity of huB4-DGN462 was also confirmed in six CD19+ B-ALL cell lines (Online Supplementary Table S2).
While a moderate correlation was seen for SAR3419, the response of B-cell lymphoma lines to huB4-DGN462 was not correlated with either surface CD19 protein expression (as measured by FACS) or CD19 RNA expres- sion (as measured by microarray or by targeted RNA-Seq) (Online Supplementary Figures S4 and S5). However, the activity of huB4-DGN462 was positively correlated with the potency of its free payload (R = 0.71, P<0.0001).
Among DLBCL cell lines, the sensitivity to the two
ADCs was not affected by TP53 inactivation (present in 16 of 23 cell lines), nor the presence of MYC or BCL2 translocations (detected in 8 of 24 and 13 of 21 cell lines, respectively). The sensitivity to SAR3419, but not the novel ADC, was lower in activated B-cell-like (ABC) than in germinal center B-cell type (GCB) DLBCL cell lines (P=0.02) (Table 1, Online Supplementary Table S1 and Online Supplementary Figure S6A) This was in agreement with the correlation between SAR3419 activity and CD19 expression and an observed higher CD19 expression in GCB than ABC DLBCL cell lines (Online Supplementary Figure S6B).
huB4-DGN462 has superior in vivo anti-lymphoma activity compared to SAR3419
To examine whether the enhanced antitumor activity of huB4-DGN462 observed in vitro would translate to improved efficacy in vivo, the anti-tumor activity of both of CD19-targeting ADC was evaluated in two DLBCL xenograft models. In the subcutaneous DoHH2 model, a single intravenous dose of huB4-DGN462 resulted in a sig- nificant, dose-dependent tumor growth delay and survival benefit at 1.7 mg antibody (Ab)/kg (5 of 6 partial responses, 3 of 6 complete responses, 3 of 6 tumor-free survivors) com- pared to a non-targeted control IgG-DGN462 ADC (1 of 6 partial responses, 1 of 6 complete responses, 1 of 6 tumor- free survivors) (Figure 2A). The anti-tumor activity of huB4- DGN462 was better than that obtained in a similar experi- ment using SAR3419 given at higher doses. Both huB4- DGN462 and SAR3419 were well-tolerated, with no signif- icant loss of body weight observed (Online Supplementary Figure S7). In the disseminated Farage model, the 1.7 mg/kg dose of the non-specific IgG1-DGN462 control conjugate was inactive while there was a significant dose-dependent increase in survival observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462 (Figure 2B). At 1.7 mg Ab/kg, survival of tumor bearing mice was increased over 400%. As above, the efficacy of huB4-DGN462 was better than that achieved with SAR3419. Taken together and con- sistent with the in vitro findings, treatment with huB4- DGN462 resulted in a substantial enhancement of anti- tumor activity compared to SAR3419.
Discussion
Here we have compared the anti-lymphoma activity of a novel CD19-targeting ADC, huB4-DGN462, with
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