Humanized anti-Trop-2 IgG-SN-38 conjugate for effective treatment of diverse epithelial cancers: preclinical studies in human cancer xenograft models and monkeys

Abstract

Purpose: Evaluate the efficacy of an SN-38-anti-Trop-2 antibody-drug conjugate (ADC) against several human solid tumor types, and to assess its tolerability in mice and monkeys, the latter with tissue cross-reactivity to hRS7 similar to humans.

Experimental design: Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2-humanized antibody, hRS7. The immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor-xenograft models that expressed Trop-2 antigen. Toxicity was assessed in mice and in Cynomolgus monkeys.

Results: The hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution (∼ 6), cell binding (K(d) ∼ 1.2 nmol/L), cytotoxicity (IC(50) ∼ 2.2 nmol/L), and serum stability in vitro (t/(½) ∼ 20 hours). Exposure of cells to the ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 in p53 and p21 upregulation were noted. Significant antitumor effects were produced by hRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P ≤ 0.05), Capan-1 (P < 0.018), BxPC-3 (P < 0.005), and COLO 205 tumors (P < 0.033) when compared to nontargeting control ADCs. Mice tolerated a dose of 2 × 12 mg/kg (SN-38 equivalents) with only short-lived elevations in ALT and AST liver enzyme levels. Cynomolgus monkeys infused with 2 × 0.96 mg/kg exhibited only transient decreases in blood counts, although, importantly, the values did not fall below normal ranges.

Conclusions: The anti-Trop-2 hRS7-CL2A-SN-38 ADC provides significant and specific antitumor effects against a range of human solid tumor types. It is well tolerated in monkeys, with tissue Trop-2 expression similar to humans, at clinically relevant doses, and warrants clinical investigation.

Figure 1.. Structure and characterization of SN-38 conjugates: hRS7-CL2-SN-38 and hRS7-CL2A-SN-38.

(A) The chemical structure of the linkage of SN-38 (shown in red) to the hRS7 antibody (blue) via the CL-linker. A single amino acid deletion shows the difference between the CL2-SN-38 and CL2A-SN-38 forms of the hRS7 anti-Trop-2 ADC. Comparisons in chemistry and potency are shown in the table. Comparability of hRS7-CL2-SN-38 versus hRS7-CL2A-SN-38 in mice bearing COLO 205 (B) and Capan-1 tumors (C). Animals were treated twice weekly for four weeks as indicated by the arrows. COLO 205 mice (N=6) were treated with 0.4 mg/kg ADC and tumors measured twice a week. Capan-1 mice (N=10) were treated with 0.2 mg/kg ADC and tumors measured weekly.

Figure 2.. Western blot analysis for early and late signaling events mediated by hRS7-CL2A-SN-38.

Cells were plated overnight in 6-well plates before the addition of hRS7-CL2A-SN-38 (1 μM SN-38 equivalents), free SN-38 (1 μM), or the protein equivalent of hRS7 IgG (25 μg/mL). At the indicated times, cells were lysed and 20 μg protein from these lysates subjected to SDS-PAGE (4–20%) followed by blotting with antibodies to p21, p53, β-Actin, or PARP. Time 0 lanes are from untreated cells grown in the plates for 48 h in growth media alone and represent basal expression levels. Changes in expression are normalized to β-actin loading controls and are relative to levels in the untreated cells.

Figure 3.. Therapeutic efficacy of hRS7-SN-38 ADC in several solid tumor-xenograft disease models.

Efficacy of hRS7-CL2-SN-38 and hRS7-CL2A-SN-38 ADC treatment was studied in mice bearing human non-small cell lung, colorectal, pancreatic, and squamous cell lung tumor xenografts. All the ADCs and controls were administered in the amounts indicated (expressed as amount of SN-38 per dose; long arrows = conjugate injections, short arrows = irinotecan injections). (A) Mice bearing Calu-3 tumors (N = 5–7) were injected with hRS7-CL2-SN-38 every four days for a total of four injections (q4dx4). (B) COLO 205 tumor-bearing mice (N=5) were injected eight times (q4dx8) with the ADC or every two days for a total of five injections (q2dx5) with the MTD of irinotecan. Capan-1 (C; N=10) or BxPC-3 tumor-bearing mice (D; N = 10) were treated twice weekly for 4 weeks with the agents indicated. (E) In addition to ADC given twice weekly for 4 week, SK-MES-1 tumor-bearing (N=8) mice received the MTD of CPT-11 (q2dx5).

Figure 4.. Biodistribution and pharmacokinetics of hRS7-CL2A-SN-38 in SK-MES-1 tumor-bearing mice.

(A-D) Mice were injected with either ¹¹¹In-DTPA-hRS7-CL2A-SN-38 or ¹¹¹In-DTPA-hRS7 IgG (N = 5/observation). Data are expressed as % ID/g. (A) Blood clearance and pharmacokinetics parameters, (B) tumor, (C) liver, and (D) spleen are shown. (E) Pre-dosing with 250 μg of hRS7-CL2A-SN-38 three days before receiving ¹¹¹In-DTPA-hRS7-CL2A-SN-38 effect on tumor uptake.

Figure 5.. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice and Cynomolgus monkeys.

(A-B) Fifty-six Swiss-Webster mice were administered two i.p. doses of buffer or the hRS7-CL2A-SN-38 three days apart (4, 8, or 12 mg/kg of SN-38 per dose; 250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 days after the last injection, seven mice from each group were euthanized, with blood counts and serum chemistries performed. Graph show the percent of animals in each group that had elevated levels of AST (A) and ALT (B). Six monkeys per group were injected twice 3 days apart with buffer (control) or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents per dose (60 and 120 mg/kg conjugate protein). All animals were bled on day −1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group, 3 in the 1.92 mg/kg group. Changes in neutrophil (C) and platelet counts (D), respectively in Cynomolgus monkeys.