Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC)

Abstract

Trop-2 is a novel target for ADC therapy because of its high expression by many solid cancers. The rational development of IMMU-132 represents a paradigm shift as an ADC that binds a well-known moderately-cytotoxic drug, SN-38, to the anti-Trop-2 antibody. In vitro and in vivo studies show enhanced efficacy, while there is a gradual release of SN-38 that contributes to the overall effect. IMMU-132 is most efficacious at a high drug:antibody ratio (DAR) of 7.6:1, which does not affect binding and pharmacokinetics. It targets up to 136-fold more SN-38 to a human cancer xenograft than irinotecan, SN-38's prodrug. IMMU-132 delivers SN-38 in its most active, non-glucuronidated form, which may explain the lower frequency of severe diarrhea than with irinotecan. Thus, this ADC, carrying a moderately-toxic drug targeting Trop-2 represents a novel cancer therapeutic that is showing promising activity in patients with several metastatic cancer types, including triple-negative breast cancer, non-small-cell and small-cell lung cancers.

Keywords: SN-38; Trop-2; antibody-drug conjugate; solid cancers; triple-negative breast cancer.

Figure 1. Structures of irinotecan, SN-38, and CL2A-SN-38

A. Irinotecan schematic indicates the molecule's 7, 10, & 20 positions. B. IMMU-132 ADC shows SN-38 positioning, site of SN-38 cleavage, PEG moiety to enhance solubility, and site coupled to the antibody. C. In vitro serum stability of IMMU-132 in monkey or human serum.

Figure 2. Hydrophobic interaction chromatography of IMMU-132

Representative HIC-HPLC trace of IMMU-132 resolved with a 15-min gradient of 2.25 M to 1.5 M NaCl. Peaks representing species with DARs of 6, 7 and 8 are indicated.

Figure 3. Therapeutic efficacy of IMMU-132 with different DARs

NCI-N87 human gastric carcinoma xenografts (subcutaneous) were established as described in Materials and Methods. A. Four groups of mice (N = 9) were injected IV with 2 × 0.5 mg (arrows) of IMMU-132 conjugates prepared with a DAR = 6.89, 3.28, or 1.64. Control animals received saline. Therapy began 7 days after tumor cells were administered (size was 0.248 ± 0.047cm³). Survival curves were generated based on the time to progression to ≥ 1.0 cm³, and were analyzed by log-rank test (significance at P ≤ 0.05). B. NCI-N87 tumor-bearing mice (N = 7–9; starting size = 0.271 ± 0.053cm³) were treated with either 0.5 mg IMMU-132 (DAR = 6.89) or 1.0 mg DAR = 3.28 twice weekly for two weeks (arrows). Mice were euthanized and deemed to have succumbed to disease once tumors grew to > 1.0 cm³. Profiles of individual tumor growth were obtained through linear-curve modeling. Statistical analysis of tumor growth was based on area under the curve (AUC) performed up to the time that the first animal within a group was euthanized due to disease progression. An f-test was employed to determine equality of variance between groups prior to statistical analysis of growth curves. A two-tailed t-test was used to assess statistical significance between the various treatment groups and controls, except for the saline control, where a one-tailed t-test was used (significance at P ≤ 0.05).

Figure 4. Immunohistology of TNBC patient specimens

Strong (3+) Trop-2 expression in two TNBC specimens within a tumor microarray.

Figure 5. Therapeutic efficacy of IMMU-132 in TNBC xenograft models

A. Twenty-two days after subcutaneously implantation of MDA-MB-468 tumors (at the onset of treatment, tumors averaged 0.223 ± 0.055 cm³), nude mice (7–8 per group) were injected with IMMU-132 or a control hA20 anti-CD20-SN-38 conjugate twice weekly for two weeks (0.12 or 0.20 mg/kg SN-38 equivalents per dose). Other animals were given irinotecan (10 mg/kg/dose; SN-38 equivalent based on mass = 5.8 mg/kg) every other day for 10 days for a total of five injections. Statistical analysis performed in the same manner as mentioned in Fig 2B. B. Starting on day 56 after treatment initiation, all animals in the control hA20-SN-38 group were given IMMU-132 (4 × 0.2 mg/kg SN-38 equivalents). The size of the tumors in the individual animals of this group from the onset of tumor transplantation is given. Purple arrows indicate when the hA20-SN-38 conjugate was first given, and red arrows indicate when the treatment with IMMU-132 was initiated. C. Mice (N = 12) bearing the MDA-MB-231 TNBC cell line (0.335 ± 0.078 cm³) were treated with IMMU-132 or the control hA20-SN-38 conjugate (0.4 mg/kg SN-38 equivalents), irinotecan (6.5 mg/kg; ∼3.8 mg/kg SN-38 equivalents), or a combination of hRS7 IgG (25 mg/kg) plus irinotecan (6.5 mg/kg).

Figure 6. IMMU-132 mediated pro-apoptosis signaling in human breast cancer lines

MDA-MB-468 or SK-BR-3 cells were exposed to 1 μM SN-38, the SN-38-equivalent of IMMU-132, or protein equivalent of hRS7 for the indicated times. Cells were harvested and Western blots performed as described in Materials and Methods. Untreated control cells (control) were maintained in growth medium alone until harvested after 48 h. Blots shown are representative of one of two separate experiments.

Figure 7. Determination of SN-38 and associated products in serum and Capan-1 tumors taken from animals given irinotecan or IMMU-132

Animals were given irinotecan (773 μg) or IMMU-132 (1.0 mg) and then at 5 intervals, 3 animals from each group were euthanized with serum panel (A) and tumor panel (B) extracted for the products of interest. Data are only shown for sampling intervals where detectable product was measured (e.g., animals given irinotecan did not have any detectable products at the 24-h sampling interval).