Development of Antibody-Drug Conjugates Using DDS and Molecular Imaging

Abstract

Antibody-drug conjugate (ADC), as a next generation of antibody therapeutics, is a combination of an antibody and a drug connected via a specialized linker. ADC has four action steps: systemic circulation, the enhanced permeability and retention (EPR) effect, penetration within the tumor tissue, and action on cells, such as through drug delivery system (DDS) drugs. An antibody with a size of about 10 nm has the same capacity for passive targeting as some DDS carriers, depending on the EPR effect. In addition, some antibodies are capable of active targeting. A linker is stable in the bloodstream but should release drugs efficiently in the tumor cells or their microenvironment. Thus, the linker technology is actually a typical controlled release technology in DDS. Here, we focused on molecular imaging. Fluorescent and positron emission tomography (PET) imaging is useful for the visualization and evaluation of antibody delivery in terms of passive and active targeting in the systemic circulation and in tumors. To evaluate the controlled release of the ADC in the targeted area, a mass spectrometry imaging (MSI) with a mass microscope, to visualize the drug released from ADC, was used. As a result, we succeeded in confirming the significant anti-tumor activity of anti-fibrin, or anti-tissue factor-ADC, in preclinical settings by using DDS and molecular imaging.

Keywords: ADC (antibody-drug conjugate); DDS (drug delivery system); MSI (mass spectrometry imaging); PET (positron emission tomography); antibody delivery; controlled release; molecular imaging.

Figure 1

Structure and drug delivery of antibody-drug conjugate (ADC). ADC has three parts: antibody, linker, and drug. ADC has four action steps: systemic circulation, enhanced permeability and retention (EPR) effect, penetration, and action on cells, like drug delivery system (DDS) drugs.

Figure 2

Fluorescent imaging of antibody delivery. (a) An in vivo imaging analysis of a mouse xenograft model was conducted on fluorescent non-specific IgG, specific IgG, or specific Fab on days one, three, five, and seven after the administration. (b) Left panel, hematoxylin-eosin straining of malignant lymphoma (ML) (upper panel) and immunostaining of pancreatic cancer (PC) (lower panel) in which cancer cells (blue) were surrounded by dense stromal collagen 4 (brown). The middle panel shows the in vivo imaging of fluorescent anti-CD 20 and anti-EpCAM antibody that were injected into the ML and PC model, respectively. The right panel shows the distribution of anti-CD 20 and anti-EpCAM antibody (both green) within a ML tumor and PC tumor. The blood vessels, yellow in the upper panel and magenta in the lower panel, were also observed.

Figure 3

Evaluation of antibody delivery with positron emission tomography (PET) imaging. (a)–(b) PET imaging analysis was conducted using an ⁸⁹Zr-labeled anti-fibrin antibody on day zero, one, two, three, five, and seven after the administration and %ID/g showed the relative value of Day 0 (100%). (c) With autoradiogram examination, the ⁸⁹Zr-labeled anti-fibrin antibody was accumulated within the fibrin-positive tumor stroma, as represented by the dashed black line. (d) In PET/CT, the ⁸⁹Zr-labeled anti-fibrin antibody showed clear and specific accumulation in the tumor. Adapted from Hisada et al. [32].

Figure 4

Mass spectrometry imaging (MSI) with a mass microscope. (a) A schematic representation of our drug imaging system using MSI with a mass microscope. (b) A mass microscope demonstrates the tissue distribution of targeted molecules with a high spatial resolution. Adapted from Yasunaga et al. [50].

Figure 5

Visualization of the controlled release of PTX-incorporated micelle. (a) In tumor tissue, the bright field (upper), reference substance (middle, an arbitrary signal of 824.6 m/z), and PTX (lower, specific signal of 892.3 m/z). (b) In normal tissue, bright field (upper), neuronal marker (middle, sphingomyelin-specific signal of 851.6 m/z), and PTX (lower, specific signal of 892.3 m/z). The neuronal area is delineated by a white line. Adapted from Yasunaga et al. [50].

Figure 6

Visualization of released monomethyl auristatin E (MMAE) from ADC. (a) Drug design of the anti-tissue factor (TF) antibody-drug conjugate (anti-TF-ADC). (b) The MMAE-specific fragment with a size of 496.3 m/z was determined in the MS/MS analysis. (c) In MSI analysis, released MMAE (MMAE alone) was clearly distinguished from MMAE conjugated in ADC (ADC with MMAE). Adapted from Fujiwara et al. [51].

Figure 7

Evaluation of the controlled release of MMAE from ADC using MSI. Tumor samples from the mouse xenograft model were prepared on three, 24, and 72 h after the administration of the control ADC and anti-TF-ADC, respectively. In each ADC, H&E staining (far left) and bright field (left-middle) are shown. The rectangles on the bright field show the measurement area. The released MMAE signals obtained from 496.3 m/z using a mass microscope is shown. The signals of antibody/ADC were acquired from immunostaining with horseradish peroxidase (HRP) labelled each antibody. Adapted from Fujiwara et al. [51].