Aligning physics and physiology: Engineering antibodies for radionuclide delivery

Abstract

The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy.

Keywords: ImmunoPET; antibody engineering; antibody fragment; diagnostics; protein scaffold; radioimmunotherapy; radiolabeling; therapeutics.

Figure 1. Intact antibody and engineered antibody fragments

Compared to A, intact IgG antibody, engineered antibody fragments range in size, specificity, avidity, and therefore pharmacokinetic properties. Several fragments and scaffolds for radionuclide delivery have been explored in preclinical and clinical studies, including B, monovalent antibody fragments, C, bivalent monospecific fragments, and D, bispecific fragments. E, Schematic of pretargeting strategy using bispecific tri-F(ab′): (1) Tri-F(ab′) binds to target, and subsequently (2) radiolabeled hapten is administered, which rapidly localizes to the Tri-F(ab′) and binds the anti-hapten Fab. Abbreviations: CH= constant heavy, CL= constant light, Db = diabody, Fv= fragment variable, Fab= fragment-antigen binding, Fc= fragment crystallizable, IgG = immunoglobulin G, Mb = minibody, scFv = single chain fragment variable, sdAb= single domain antibody, SIP = small immunoprotein, Tri-F(ab′) = trivalent Fab, VH= variable heavy, VHH= variable heavy, VL= variable light.

Figure 2. Schematic representation of therapeutic radionuclides

Beta-emitters have a low linear energy transfer (LET) of 0.2 keV/mm and a path length of 1 to 10 mm, and beta emission may reach throughout the tumor and surrounding healthy tissue. Alpha-emitters have a high LET (80–100 keV/μm) and a path length of 50–90 μm, which corresponds to a few cell diameters. Auger-emitters have low energy but high LET (4–26 keV/μm) and a short path length of 2 to 500 nm. Intracellular deposit near the nucleus is necessary for therapeutic effect.

Figure 3. Antibody fragment-based immunoPET imaging of immune cells

A, VHHDC13 (anti-mouse CD11b) is a single domain antibody with an LPETG sortase recognition moiety that can be undergo a transpeptidase reaction to produce VHHDC13-tetrazine. This allows for site specific radiolabeling using ¹⁸F-TCO. ¹⁸F-VHHDC13 immunoPET shows high contrast targeting of CD11b-positive cells in mice bearing B16 melanoma s.c. tumors. In comparison, ¹⁸F-FDG PET shows low contrast between the tumor and background tissues. B, Mice bearing CTD26 xenografts received anti-PD-L1 checkpoint blockade therapy and divided into two groups, responders and nonresponders. ⁸⁹Zr-anti-CD8 169 cys-diabody immunoPET shows uptake in the rim of the tumor of anti-PD-L1 nonresponders, and intratumoral uptake in responders.

Figure 4. Clinical evaluation of antibody fragments for immunoPET

A, First in human Phase I study of ⁶⁸Ga-2Rs15d, an anti-HER2 Nanobody, was conducted in patients with breast cancer. Uptake is detected in HER2 primary lesions as shown by PET/CT (top) and PET (bottom) in two patients. B, First-in-human Phase I study of ⁸⁹Zr-anti-PSMA-IAB2M minibody was conducted in patients with metastatic prostate cancer. ^99mTc-methylene diphosphonate (MDP) bone scan detected lesions in the vertebrae and ribs, ¹⁸F-fluorodeoxyglucose (FDG) imaging detected lesions in femur and vertebrae, and ⁸⁹Zr-IAB2M immunoPET detected soft tissue and bone lesions not identified by the previous two methods. C, Clinical evaluation of bispecific trivalent antibody (BsMAb) TF2 and ⁶⁸Ga-IMP288 in patients with medullary thyroid carcinoma (MTC). TF2 is composed of two anti-carcinoembryonic antigen (CEA) Fab fragments and one anti-histamine-succinyl-glycine (HSG) Fab fragment, and was administered as the pretargeting agent. IMP288 is a bivalent HSG hapten, and the preferred ⁶⁸Ga-IMP288 parameter used was a BsMab-to-peptide molar ratio of 20 and 30 h pretargeting delay. ImmunoPET images were acquired 1- and 2- hours post-IMP288-injection. Arrows point to uptake in cervical node, lumbar node and femoral bone foci. D, Clinical evaluation of ⁶⁸Ga-anti-HER2 ABY-025 Affibody in patients with metastatic breast cancer. The patient shown had HER2-negative primary tumor, and ¹⁸F-FDG PET showed uptake in 3 lesions located in the liver, lymph node, and cervix. ⁶⁸Ga-ABY-025 immunoPET detected the liver lesion, which was confirmed to be HER2-positive by immunohistochemistry (IHC), while the other two lesions were confirmed to be true negative. Red arrow indicates location of portacath used for injection.

Figure 5. Clinical and preclinical applications of antibody-based radioimmunotherapy

A, ¹³¹I-L19-SIP was administered to patients for RIT, this example patient with diffuse large B-cell lymphoma showed a complete response. After a single administration of ¹³¹I-L19-SIP, baseline transaxial SPECT and ¹⁸F-FDG PET show high uptake in the left inguinal lymphoma, and 1 month post-RIT no uptake is detected by ¹⁸F-FDG PET. B, In a patient with refractory Hodgkin’s lymphoma, baseline ¹⁸F-FDG PET shows uptake in lesions in the neck and mediastinum corresponding to uptake seen at day 4 post-injection of ¹³¹I-F16-SIP SPECT. At 1- and 2-month post-injection, ¹⁸F-FDG PET images show reduction in uptake. C, Pretargeting strategy with anti-TROP2 × anti-HSG bispecific TF12 and radiolabeled IMP288 HSG hapten. TF12 and ⁶⁸Ga-IMP288 PET/CT of a BALB/c nude mice bearing s.c. PC3 tumors shows uptake in the tumor, kidneys, and bladder at 1-hour post-injection. Mice were treated with TF12 and ¹⁷⁷Lu-IMP288, and survival was extended compared to ¹⁷⁷Lu-control vehicle and ¹⁷⁷Lu-IMP288 without pretargeting.