Advertisement

ADCT-402, a PBD dimer–containing antibody drug conjugate targeting CD19-expressing malignancies

Francesca Zammarchi, Simon Corbett, Lauren Adams, Peter C. Tyrer, Konstantinos Kiakos, Narinder Janghra, Teresa Marafioti, Charles E. Britten, Carin E. G. Havenith, Simon Chivers, Francois D’Hooge, David G. Williams, Arnaud Tiberghien, Philip W. Howard, John A. Hartley and Patrick H. van Berkel

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Characterization of ADCT-402. (A) Structure of ADCT-402. (B) ADCT-402 characterized by size exclusion chromatography. (C) Reduced reverse phase liquid chromatography depicting reduced heavy and light chains of ADCT-402. (D) Hydrophobic interaction chromatography depicting drug–antibody ratio forms of ADCT-402.

  • Figure 2.

    Correlation between in vitro cytotoxicity and cell surface CD19 density. (A) IC50 values (mean of 3 independent determinations) of ADCT-402 against the named cell lines are plotted against the measured cell surface CD19 levels expressed as sites/cell. The line is the regression through all data points. (B) The equivalent analysis performed for the naked PBD dimer warhead SG3199. Linear relationship between indicated variables was calculated by Pearson correlation coefficient r test.

  • Figure 3.

    Mechanism of action of ADCT-402. (A) Merged immunofluorescent images of CD19+, human Burkitt lymphoma-derived Ramos cells treated with 2 μg/mL ADCT-402 for 1 hour; washed and fixed after T = 0, 1, 2, 4, or 24 hours in medium at 37°C; and stained with labeled antihuman IgG (green), anti-LAMP-1 (red), and Hoechst 33342 (blue) nuclear stain. Yellow indicates colocalization, with specific sites of colocalization indicated by stars. Original magnification ×63. (B) Time course of DNA interstrand crosslinking measured as the % reduction on Olive Tail Moment in Ramos cells treated for 2 hours with ADCT-402 (40 pM), SG3199 (10 pM), or a nontargeted ADC (40 pM) followed by postincubation in drug-free medium for the indicated time. Results are the mean ± standard deviation from at least 3 independent experiments. (C) Percentage viability of CD19 Karpas-299 cells after 96 hours of exposure to media transferred from ADCT-402–treated CD19+ Ramos cells or CD19 NCI-N87 cells for 1, 2, or 3 days. Statistical analysis was done with unpaired t test with Welch’s correction (not assuming equal standard deviations). P values are 2-tailed. *P ≤ .05, **P ≤ .01. ns, not significant.

  • Figure 4.

    Comparative antitumor activity of ADCT-402 in the human CD19-expressing Burkitt lymphoma-derived Ramos model. (A) In vivo antitumor activity of ADCT-402 in SC implanted Ramos xenograft model. ADCT-402 was administered IV at a group mean tumor volume of 120 mm3 as a single dose at 0.33, 0.66, or 1 mg/kg. (B) In vivo antitumor activity of ADCT-402 in SC implanted Ramos xenograft model in comparison with ADC RB4v1.2-DM4 and hBU12-mc-MMAF. All 3 ADCs were compared at a single dose of 1 mg/kg; in addition, RB4v1.2-DM4 and hBU12-mc-MMAF were tested at 3.3 mg/kg every 4 days × 2 and 3 mg/kg every 4 days × 4, respectively. (C) In vivo antitumor activity of ADCT-402 in SC implanted Ramos xenograft model where ADCT-402 was administered at a single dose of 1 mg/kg or fractionated dosing of 0.33 mg/kg given either every week × 3 or every 4 days × 3. (A-C) Data are shown as mean ± SEM from animal group sizes of 10 mice. (D) In vivo antitumor activity of ADCT-402 in a disseminated Ramos model. Kaplan-Meier survival plots show percentage animal survival over 91 days in an experiment in which ADCT-402 was administered at a single dose of 0.33 mg/kg or 1 mg/kg in comparison with vehicle or nontargeting ADC administered as a single dose of 1 mg/kg (each group, n = 10). PBS, phosphate-buffered saline; SEM, standard error of the mean.

  • Figure 5.

    Antitumor activity of ADCT-402 in a range of tumor models. (A) In vivo antitumor activity of ADCT-402 in SC implanted CD19-expressing Burkitt lymphoma-derived Daudi xenograft model. ADCT-402 was administered IV at a group mean tumor volume of 130 mm3 as a single dose at 0.1 or 0.3 mg/kg. (B) In vivo antitumor activity of ADCT-402 in SC implanted CD19-expressing diffuse large B-cell lymphoma-derived WSU-DLCL2 xenograft model. ADCT-402 was administered IV at a group mean tumor volume of 120 mm3 as a single dose at 0.3 or 1 mg/kg. (A-B) Mean ± SEM from animal group sizes of 10 mice. (C) In vivo antitumor activity of ADCT-402 in a disseminated CD19-expressing ALL-derived NALM-6 model. Kaplan-Meier survival plots show percentage of animal survival over 90 days in an experiment in which ADCT-402 was administered at a single dose of 0.33 or 1 mg/kg in comparison with vehicle or nontargeting ADC administered as a single dose of 1 mg/kg (each group, n = 10).

  • Figure 6.

    Pharmacodynamic studies of ADCT-402. (A) Representative scans of FFPE Ramos xenograft tumor sections, obtained from vehicle-treated control mice or mice treated with 0.3 and 1 mg/kg ADCT-402 or 1 mg/kg nontargeting ADC and stained with an anti-CD19 antibody (top), anti-PBD linker antibody (middle), or an anti-γ-H2AX antibody (bottom). (B) Histogram depicting % cells with γ-H2AX in tumor cell suspensions taken from Ramos xenograft tumors 24 hours after injection with vehicle or 0.3 or 1 mg/kg ADCT-402 or 1 mg/kg nontargeted ADC. Data represent the mean and SD from 3 individual mice per data point. **P ≤ .01, ***P ≤ .001. (C) Histogram depicting mean OTM in irradiated (I) or unirradiated (UI) tumor cell suspensions taken from Ramos xenograft tumors 24 hours after injection with vehicle or 0.3 mg/kg or 1 mg/kg ADCT-402 or 1 mg/kg nontargeted ADC. Data represent the mean and SD from 3 individual mice per data point. Statistical analysis was done with unpaired t test with Welch correction (not assuming equal SDs). P values are 2-tailed; *P ≤ .05, **P ≤ .01. SD, standard deviation.

  • Figure 7.

    Pharmacokinetics of ADCT-402 in rat and cynomolgus monkey. (A) Quantitation of total antibody and PBD-conjugated ADCT-402 in rat serum from 3 individual Crl:CD (SD) rats treated with a single IV dose of 1.5 mg/kg. (B) Quantitation of total antibody, PBD-conjugated antibody, and free PBD dimer warhead SG3199 in cynomolgus monkey serum after IV administration of 0.6 mg/kg ADCT-402 on days 1 and 22. The results are mean ± SEM (n = 2/5 male and 2/5 female).

Tables

  • Table 1.

    CD19 expression in matched primary and relapsed clinical samples

    Clinical subtypeCase no., P/RBiopsy typeTime between biopsy (mo)CD19 intensity*
    DBLCL1PBM82
    1RBM3
    2PLN203
    2RMesenteric mass3
    3PTestis123
    3RPenile mass3
    4PLN143
    4RLN3
    5PBM82
    5RLN3
    MCL6PSalivary gland203
    6RSubmandibular3
    7PBM113
    7RLN3
    8PLN153
    8RLN3
    9PGastrointestinal83
    9RDuodenal bulb3
    FL10PLN273
    10RLN3
    11PLN263
    11RLN3
    Small/chronic LL12PBM203
    12RBM2
    • BM, bone marrow; DBLCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; LL, lymphocytic leukemia; LN, lymph node; MCL, mantle cell lymphoma; P, primary; R, relapsed.

    • * 0 = negative, 1 = weak, 2 = moderate, 3 = strong staining as determined by pathologist review.

    • In all samples analyzed, >80% tumor cells were CD19+.