Pretargeted molecular imaging and radioimmunotherapy

Abstract

Pretargeting is a multi-step process that first has an unlabeled bispecific antibody (bsMAb) localize within a tumor by virtue of its anti-tumor binding site(s) before administering a small, fast-clearing radiolabeled compound that then attaches to the other portion of the bsMAb. The compound's rapid clearance significantly reduces radiation exposure outside of the tumor and its small size permits speedy delivery to the tumor, creating excellent tumor/nontumor ratios in less than 1 hour. Haptens that bind to an anti-hapten antibody, biotin that binds to streptavidin, or an oligonucleotide binding to a complementary oligonucleotide sequence have all been radiolabeled for use by pretargeting. This review will focus on a highly flexible anti-hapten bsMAb platform that has been used to target a variety of radionuclides to image (SPECT and PET) as well as treat tumors.

Keywords: bispecific antibody; cancer detection; pretargeting; radioimmunodetection; radioimmunotherapy..

Figure 1

The HSG-hapten binding system for binding a variety of radionuclides. (A) The initial bsMAb pretargeting systems utilized antibodies that bound to a specific chelate-metal complex, e.g., indium-loaded DTPA. As such, the anti-(In)DTPA antibody had a much lower binding affinity for DTPA loaded with another metal, such as ⁹⁰Y. Thus, to bind another radiometal, like ⁹⁰Y, a new anti-chelate antibody would need to be prepared. An antibody against a hapten that does not bind the radionuclide, such as the anti-HSG (histamine-succinyl-glycine) antibody, would open the possibility for using different radionuclides or other compounds with one system. (B) In the HSG-binding system, a peptide core of D-amino acids (aa) has 2 HSG haptens to enhance tumor-binding stability. The remaining structure can be modified to accommodate any radionuclide-binding agent. A tyrosine in the peptide core could be radioiodinated. We have also modified the carboxyl end of the peptide to allow for binding of fluorescent dyes. (C) For pretargeting, the bsMAb is first localized in the tumor, and after clearing from the blood and tissues, the radiolabeled divalent hapten-peptide is given. The divalent hapten structure enhances its binding avidity to the bsMAb, and it can potentially enhance the binding avidity of the bsMAb to the tumor as well. Trivalent bsMAbs with divalent binding to the tumor are preferred.

Figure 2

Bispecific antibody conjugates/constructs. Chemically conjugated bsMAb were prepared from Fab' fragments of the anti-tumor (α-T) and the anti-hapten (α-H) antibodies. The anti-tumor Fab' was coupled first to the bifunctional N,N”-o-phenylene-dimaleimide (PDM), and then the anti-hapten Fab' was added to form a stable Fab' x Fab' bsMAb. Molecularly engineered bsMAb used by our group include the hBS14 (anti-CEACAM5 x anti-679 HSG) and several tri-Fab (TF) constructs, each binding to a different tumor antigen, but all using the anti-HSG hapten-binding antibody. Whereas the hBS14 construct is prepared in a single antibody-producing clone, the tri-Fab Dock-and-Lock constructs are formed from 2 separate clones. One produces the anti-tumor fusion protein, using the docking and dimerization domain (DDD) peptide sequence that has a strategically placed cysteine (designated DDD2). The Fab-DDD2 forms a dimer, which has a docking domain that will bind to the anchoring domain sequence, which is also modified to include 2 cysteines (AD2). Thus, all these constructs have bivalent binding to the tumor and monovalent binding to the hapten.

Figure 3

Pretargeted ^99mTc-hapten-peptide vs. ^99mTc-Fab'. Nude mice bearing large (1.3 to 1.5 g) human colonic tumor xenografts (arrows in photos) were used to assess the dynamics of a ^99mTc-hapten-peptide alone or pretargeted with an anti-CEA bsMAb given 48 h earlier, and compared to an ^99mTc-anti-CEA Fab' fragment. Dynamic imaging was performed over 60 min, taking images at 2-min intervals. Static images of these same animals were then also taken at 1, 6, and 24 h. Panel A shows the dynamic images taken from 10 to 20 minutes that illustrate the pretargeted ^99mTc-hapten-peptide with uptake in the tumor (T) as early as 10 min after its injection, but at this time, tumor uptake was similar to the heart (H), with more intense uptake in the kidneys (K) and liver (Lv). Substantial activity had already been eliminated in the urinary bladder (UB). Over the next 10 minutes, uptake in heart and liver diminished so that they were not visible, and the tumor's intensity was now similar to the kidneys. In Panel B, the 2-min dynamic ending at 40 min showed tumor uptake had exceeded the kidneys. The static images shown in Panel C illustrate the targeting seen with the ^99mTc-anti-CEA Fab' fragment (top), the ^99mTc-hapten peptide alone (no bsMAb was given; middle), and the pretargeted ^99mTc-hapten-peptide (bottom). The ^99mTc-anti-CEA Fab' showed only a minor blush in the tumor at 1 h, with most of the activity residing in the blood, liver and kidneys, with some elimination in the urinary bladder. The follow-up images show tumor localization, but renal uptake is much more prevalent. Even the ^99mTc-hapten-peptide alone showed some uptake in the tumor over the first 6 h, but by 24 h, all activity was eliminated from the body. In contrast, the animal pretargeted with the anti-CEA bsMAb show strong tumor localization that persisted over time as the normal tissues continued to wash out.

Figure 4

PET imaging of nude mice bearing a s.c. human colonic tumor (T). A (top panel) shows an animal given an anti-CEACAM5 bsMAb pretargeted ¹²⁴I-hapten-peptide, B (middle panel) an ¹²⁴I-labeled anti-CEACAM5 Fab', and C (bottom panel) was given ¹⁸F-FDG. The far left side of each panel shows a transverse section taken through a plane that includes the tumor, with white lines in the adjacent coronal sections indicating the approximate location of the plane. For A and B, another transverse section through the abdomen in the region of the kidneys (K) is also shown, whereas C shows a transverse section that thorough a plane that includes the heart and another lower plane below the heart. The coronal slices are on the posterior side of the animal in order to highlight the tumor that is in on left side of the image. All images are adjusted to the same intensity without background subtraction to give a better appreciation of the relative uptake in the tissues. The animals were not pre-medicated to ameliorate radioiodine uptake in the thyroid (Thy) or stomach (St). ¹⁸F-FDG has extensive uptake in the bone marrow (BM) of mice, as well as in the brain (Br) and heart wall (H).

Figure 5

Localization of micrometastatic tumors the lungs of nude mice injected intravenously with a human colon cancer cell line. Nude mice were given a specific anti-CEACAM5 trivalent bsMAb or a control, non-binding trivalent bsMAb (anti-CD22 x anti-HSG). They then later received an ¹²⁴I-hapten peptide. A separate group of animals received ¹⁸F-FDG. Images A and B are sagittal sections of 2 separate mice and corresponding transverse slices through the chest taken ~1 h after the ¹²⁴I-hapten-injection. The lungs (L) of both animals showed increased uptake, and the transverse sections appear to show distinct uptake in small clusters. The sections were cut in a plane to include one of the kidneys (K) to illustrate uptake in this organ, as well as some activity in the stomach (St) immediately above the kidney. The activity is cleared in the urine (urinary bladder, UB). C shows a coronal section of a tumor-bearing mouse given the control bsMAb that did not find tumor in the chest. D shows coronal slices of 2 animals given ¹⁸F-FDG and imaged ~1 h later. E shows sagittal slices of these same 2 animals, and F and G are transverse sections through 2 different regions of the chest (F is higher than G). Even in the transverse sections, there was no indication of tumor involvement in lungs these animals, but there was intense uptake in the brain (Br), bone marrow (BM; e.g., ribs, scapula, spine, pelvis, femurs) and heart wall (H). The histology sections show lungs taken from one of the ¹⁸F-FDG imaged animals (animal to the right in panel D) that had multiple foci of tumor scattered in several lobes. A portion of the section highlighted in the rectangle is magnified, showing even the largest lesion in the field was only ~0.25 mm in diameter. A separate autoradiography study using animals with more advanced disease that were pretargeted with an anti-CEACAM5 bsMAb followed by an ¹¹¹In-hapten-peptide showed uptake was specifically targeted to the tumor nodules in the lungs.