Site-Specifically Labeled Antibody-Drug Conjugate for Simultaneous Therapy and ImmunoPET

Abstract

The conjugation of antibodies with cytotoxic drugs can alter their in vivo pharmacokinetics. As a result, the careful assessment of the in vivo behavior, and specifically the tumor-targeting properties, of antibody-drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide-alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin's F_c domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal ⁸⁹Zr ( t_1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the ⁸⁹Zr-trastuzumab-MMAE immunoconjugate were validated in vivo using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days.

Keywords: ADC; PET; antibody−drug conjugate; click chemistry; heavy chain glycans; positron emission tomography; site-specific bioconjugation; strain-promoted azide−alkyne click chemistry.

Figure 1.

(A) The chemoenzymatic approach used to create the ^IEADC; (B) Schematic structures of MMAE-DIBO and DFO-DIBO.

Figure 2.

Denaturing polyacrylamide gel electrophoresis of (1) native trastuzumab, (2) DFO-^sstrastuzumab, and (3) DFO:MMAE-^sstrastuzumab, demonstrating the site-specific conjugation on the heavy chain. HC: heavy chains, LC: light chains.

Figure 3.

Planar (left) and maximum intensity projection (right) PET images of athymic nude mice bearing HER2-expressing BT474 breast cancer xenografts (white arrow) following the injection of ⁸⁹Zr:MMAE-^sstrastuzumab and ⁸⁹Zr-^sstrastuzumab (150 μCi, 60–65μg). The coronal slices intersect the center of the tumors.

Figure 4.

Biodistribution data collected 120 h after the administration of ⁸⁹Zr:MMAE-^sstrastuzumab and ⁸⁹Zr-^sstrastuzumab (150 μCi; 60–65 μg) to athymic mice bearing HER2-expressing, subcutaneous BT474 human breast cancer xenografts. Stomach, small intestine, and large intestine values include contents.

Figure 5.

Relative tumor volume of athymic nude mice (n = 10 per group) bearing subcutaneous HER2-expressing BT474 xenografts following the injection (t = 0) of saline (blue), 10 mg/kg native trastuzumab (red), a 0.1 mg/kg MMAE (grey), 2.5 mg/kg DFO:MMAE-^sstrastuzumab (green), and 10 mg/kg DFO:MMAE-^sstrastuzumab (purple). The solid lines are the moving averages.