Cancer Imaging and Therapy with Bispecific Antibody Pretargeting

Abstract

This article reviews recent preclinical and clinical advances in the use of pretargeting methods for the radioimmunodetection and radioimmunotherapy of cancer. Whereas directly-labeled antibodies, fragments, and subfragments (minibodies and other constructs) have shown promise in both imaging and therapy applications over the past 25 years, their clinical adoption has not fulfilled the original expectations due to either poor image resolution and contrast in scanning or insufficient radiation doses delivered selectively to tumors for therapy. Pretargeting involves the separation of the localization of tumor with an anticancer antibody from the subsequent delivery of the imaging or therapeutic radionuclide. This has shown improvements in both imaging and therapy by overcoming the limitations of conventional, or 1-step, radioimmunodetection or radioimmunotherapy. We focus herein on the use of bispecific antibodies followed by radiolabeled peptide haptens as a new modality of selective delivery of radionuclides for the imaging and therapy of cancer. Our particular emphasis in pretargeting is the use of bispecific trimeric (3 Fab's) recombinant constructs made by a modular method of antibody and protein engineering of fusion molecules called Dock and Lock (DNL).

Figures

Figure 1

Schematic representation of several molecular constructs of antibodies. By splicing the Vh and VL of an IgG’s heavy and light chains (Fv-portion) and linking these polypeptide chains with a linker that is generally 15–18 amino acids in length, the 2 chains will self-associate to form the monovalent scFv (single chain Fv). Progressively shortening the length of the amino acid linker will result in various forms of constructs, such as diabodies to tetrabodies, with multivalent binding ability. Another construct simply removes the CH2 domain that otherwise maintains the parent IgG’s divalent binding structure, but has markedly faster clearance from the blood. The (scFv)2-Fc is another larger construct with fast blood clearance properties due to modifications made in amino acids that are involved in FcRN-binding. The minibody is another example of a fast clearing, smaller engineered construct with divalent antigen binding

Figure 2

Pretargeting methods used clinically. (A) For the bispecific antibody (bsMAb) method, after allowing time for the bsMAb to localize the tumor and clear from the blood, a radiolabeled divalent hapten-peptide is given. Within minutes the hapten-peptide is available for tumor binding, while the vast majority clears from the blood. (B) Conventionally known as the “2-step” streptavidin-pretargeting approach, this method utilizes a streptavidin-scFv fusion protein. It is given 1–2 days to target the tumor, and then the excess in the blood is removed by the administration of a clearing/blocking agent. The complexes are cleared into the liver, and then 1-day later, radiolabeled biotin is given, which very rapidly localizes in the tumor while also quickly clearing from the body. (C) The “3 step” pretargeting method starts with the injection of an IgG-biotin conjugate. After localizing in the tumor, the excess conjugate is removed from the blood using avidin, which clears the conjugate to the liver. A few hours later, streptavidin is given. Streptavidin will bind to the biotin conjugate in the tumor and by the next day, it will have cleared from the blood. Because streptavidin is multivalent, the radiolabeled biotin given the next day can be localized in the streptavidin bound to the IgG-biotin conjugate in the tumor.

Figure 3

Biodistribution of 111In-di-DTPA-FKYK hapten-peptide at 1, 4, 24. 48 and 72 h after injection with radiotracer in mice bearing subcutaneous NU-12 tumors that were pretargeted 3 days earlier with 15 μg G250 x DTIn-1 bsMAb.

Figure 4

Scintigraphic images of three nude mice bearing NU-12 tumors in the upper left flank that were given 15 μg of G250 x DTIn-1 bsMAb and then 3 days later received 50 μCi di-DTPA-FKYK hapten-peptide. Imaging was performed at 5 min (A), 4 hours (B) and 3 days (C) after the radiolabeled diDTPA-peptide was given.

Figure 5

Uptake and retention of the 125I- L-amino acid d-DTPA-peptide (○) and 125I-D-amino acid di-DTPA-peptide (●) in SK-RC-52 tumors. Tumor bearing mice received the radiolabeled peptides 72 h after pretargeting tumors with 15 μg bsMAb G250 x DTIn-1 bsMAb (mean ± SD).

Figure 6

Example of disease stabilization in a patient with MTC treated with AES pretargeted radioimmunotherapy. The patient had undergone 4 different surgeries because of the persistence of calcitonin in blood. After the final surgery, calcitonin continued to increase steadily with a doubling time of 4 years (open boxes). The patient was then enrolled in the first AES radioimmunotherapy clinical trial and treated in May 1996. After radioimmunotherapy, calcitonin serum levels stabilized (“x”-boxes), and the patient’s condition has remained stable ever since. CEA serum levels showed a similar parallel behavior (data not shown).

Figure 7

The “Dock and Lock” method for preparing bsMAb starts with the splicing of a cysteine-modified, 44-amino acid sequence to the CH1domain of an anti-tumor antibody using a flexible amino acid linker. This recombinant protein naturally forms well-defined dimers, and is designed the “DDD2 form. A separate recombinant protein is prepared by splicing a synthetic 17-amino acid peptide to the CH1 of an anti-hapten Fab (designated the AD2 form). This peptide is also modified with cysteine in 2 locations. The dimerized DDD2 forms a pocket in which the AD2 will naturally bind (i.e., Dock), but the strategic placement of cysteine allows these structures to form a more stable bond (i.e., Lock).

Figure 8

Targeting of a human colonic cancer injected intravenously that grows exclusively in the lungs of nude mice. A (transverse sections through the chest) and C (coronal sections) show the 18F-FDG uptake profile 1.5 h after the injection of 2 nude mice that had ~80 to 120 tumor nodules in the lung, all <0.3 mm in diameter. Uptake is seen in the bone marrow (BM) heart wall (H), and urinary bladder (UB), but no evidence of uptake in the lungs could be detected. B (transverse section through the chest) and D (coronal slice) show the targeting of an 124I-diHSG-peptide 1.5 h after its injection in 2 nude having the same tumor-bearing conditions as the mice in A & C. These mice received an anti-CEA x anti-HSG bsMAb prepared by the Dock and Lock method 1 day earlier. Clear evidence of uptake in the lungs (L) is seen in both sets of images. In the coronal view (D) stomach (S) uptake is observed as a result of dehalogenation of the radioiodine. Kidney uptake was also apparent (not shown). E shows a hematoxylin-eosin-stained section of the lungs taken from a different animal, where the tumor was allowed to grow for 10 additional days. Several purple-stained tumor (T) colonies are seen. This animal was given the anti-CEA bsMAb and one day later received an 111In-labeled di-HSG peptide After 3 hours, the animal was necropsied so that the localization of the radioactivity in the lungs could be revealed by autoradiography. F is an overlay of an autoradiographic film over the stained section illustrating the selective uptake in the tumor colonies.