Therapeutic potential of SGN-CD19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies

Abstract

Patients with relapsed/refractory B-cell malignancies such as non-Hodgkin lymphoma (B-NHL) or acute lymphoblastic leukemia have a poor prognosis. Despite measurable clinical activity with new targeted therapies, many patients do not achieve a complete or durable response suggesting an opportunity to improve upon existing therapies. Here we describe SGN-CD19B, a pyrrolobenzodiazepine (PBD)-based anti-CD19 antibody drug conjugate (ADC) being investigated for treatment of B-cell malignancies, which has improved potency compared with other ADCs. CD19-expressing tumor cells rapidly internalize SGN-CD19B, and the released PBD drug induces DNA damage, resulting in G2/M cell cycle arrest and cell death. SGN-CD19B demonstrated activity against a broad panel of malignant B-cell lines and induced durable regressions in mice bearing xenografts derived from these B-cell malignancies. A single dose of SGN-CD19B induced durable regressions at 300 μg/kg (3 μg/kg drug equivalents); combination with rituximab decreased the curative dose to 100 μg/kg (1 μg/kg drug equivalents). These doses are significantly lower than the level of drug required with other ADC payloads. In cynomolgus monkeys, SGN-CD19B effectively depleted CD20+ B lymphocytes in peripheral blood and lymphoid tissues confirming that SGN-CD19B is pharmacodynamically active at well-tolerated doses. In summary, preclinical studies show SGN-CD19B is a highly active ADC, which releases a DNA cross-linking agent rather than a microtubule inhibitor. The distinct mechanism of action, broad potency, and potential to combine with rituximab suggest that SGN-CD19B may offer unique clinical opportunities in B-cell malignancies. A phase 1 clinical trial is in progress to investigate the therapeutic potential of SGN-CD19B in relapsed/refractory B-NHL. This trial was registered at www.clinicaltrials.gov as #NCT02702141.

Conflict of interest statement

Conflict-of-interest disclosure: All authors are current or former employees of Seattle Genetics, Inc. All studies were paid for by Seattle Genetics, Inc.

© 2017 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

In vitro cytotoxicity of SGN-CD19B on malignant B-cell lines. SGN-CD19B activity was evaluated across a broad panel of B-NHL and B-ALL cell lines using a 96-hour cell viability assay. Malignant B-cell lines are plotted based on IC50 values (y-axis). The x-axis shows ranking of SGN-CD19B sensitivity from lowest to highest. Unique symbols denote tumor type or subtype. Double-hit or triple-hit lymphoma cell lines, as reported in the literature, are indicated on the x-axis using an asterisk.

Figure 2.

Mechanism of action of SGN-CD19B. (A) Cell surface and intracellular localization of SGN-CD19B in WSU-DLCL2 lymphoma cells was detected by fluorescence microscopy. SGN-CD19B binds to CD19 on the cell surface following treatment of 1 hour at 4°C and is distinguishable from LAMP-1 (upper panels). Following a 4-hour incubation at 37°C (t = 4 hours), SGN-CD19B shows evidence of internalization (lower left) and colocalization with LAMP-1 (lower right). Arrows indicate areas of colocalized signal (yellow) observed in the merged image (lower right). Original magnification ×600. WSU-DLCL2 lymphoma cells were incubated with SGN-CD19B (ng/mL) or SGD-1882 (nM) for 48 or 72 hours prior to western blot analyses with antibodies specific for γ-H2AX (B) or cleaved PARP (E). Antibodies recognizing total H2AX (B) or β-actin (E) were used to confirm equivalent protein loading. (C) Caspase 3/7 activation was assessed by treating cells with SGN-CD19B (blue closed triangles) or a nonbinding control ADC (red open circles) for 72 hours prior to evaluation of caspase 3/7 activity. (D) Caspase 3/7 activation following treatment of cells with free drug (SGD-1882) at 48 hours (red open circles) and 72 hours (blue closed triangles).

Figure 3.

SGN-CD19B induces cell cycle arrest. (A) Representative flow cytometry plots for untreated cells (top) and SGN-CD19B-treated cells (bottom). Cells were labeled with BrdU (y-axis) and propidium iodide (x-axis). Gates are indicated with boxes. (B) Cell cycle profile at 48-hour time point. Cells were untreated or incubated with control ADC (10 ng/mL), SGN-CD19B (10 ng/mL), or 0.1 nM PBD. Bar graphs represent results from 3 different experiments. Accumulation of cells in G2/M phase following treatment with SGN-CD19B or free PBD drug (SGD-1882) is statistically significant when compared with untreated and control ADC groups.

Figure 4.

In vivo activity of SGN-CD19B. Xenograft models for B-NHL were established by subcutaneous injection of WSU-DLCL2 (A), Ramos (B), and DoHH2 (C) cell lines, which are derived from DLBCL, follicular lymphoma, and Burkitt lymphoma, respectively. SGN-CD19B or control ADC was administered at a single dose as indicated by the arrowhead when tumor volume reached 100 mm3. Tumor delays occurred at 100 μg/kg dose, and complete durable regressions were achieved at the higher dose of 300 μg/kg. (D) DoHH2 (follicular lymphoma) xenografts were treated with 100 μg/kg of SGN-CD19B (single dose) in the presence or absence of 10 mg/kg of rituximab (every fourth day for 4 injections). Durable cures were attained in 100% of mice treated with SGN-CD19B plus rituximab. (E) A disseminated disease model for ALL was established by injecting 5 × 106 NALM-6 cells per mouse IV into female SCID mice (10 mice per group). Treatment of NALM-6 tumor-bearing mice was started 7 days post tumor implant only once at the doses indicated. Mice treated with ≥10 μg/kg of SGN-CD19B showed significantly improved survival out to >115 days, whereas mice in the untreated or control groups survived <40 days.

Figure 5.

CD20+B-cell depletion in NHPs. (A) CD20+ B cells were measured in serum over time (CD20+ B-lymphocytes mean). SGN-CD19B results in dose-dependent depletion of CD20+ B cells compared with control. Treatment groups are indicated by distinct symbols: vehicle control (red closed circles); 10 μg/kg (blue closed squares); 30 μg/kg (light green closed triangles); 100 μg/kg (purple open squares); 300 μg/kg (orange closed diamonds); and 600 μg/kg (green open circles). (B) Immunohistochemical staining of CD20+ B cells in spleen at day 11 and 113. Comparison is shown for untreated (left), 30 μg/kg SGN-CD19B (middle), or 250 μg/kg SGN-CD19B (right). At day 11, monkeys treated with 30 μg/kg or 250 μg/kg of SGN-CD19B showed moderate (upper middle) to marked (upper right) depletion. Recovery was evident by day 113 (lower middle and right panels). Original magnification ×10.