Noninvasive brain cancer imaging with a bispecific antibody fragment,generated via click chemistry

Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)₂] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal “click” ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105^+/+) tumors with ⁶⁴Cu-labeled Bs-F(ab)₂ revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of ⁶⁴Cu-NOTA-Bs-F(ab)₂. ⁶⁴Cu-NOTA-Bs-F(ab)₂ was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1–labeled Bs-F(ab)₂ for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions.Despite advances in diagnostic procedures and clinical patient management, early detection and diagnosis of cancers remains the most important endeavor for reducing cancer morbidity and mortality (1). Although ultrasonography, computed tomography (CT), and magnetic resonance imaging are essential to clinical oncology, tumor detection using these technologies is based primarily on anatomical characteristics, providing limited information about the molecular profile during tumor progression (2). On the other hand, noninvasive molecular imaging techniques, which can be designed to specifically detect alterations in gene amplification or mutations that occur early during cancer progression, have the potential to visualize carcinogenesis at earlier stages (3). Given its excellent sensitivity (picomolar range), adequate spatial resolution, and the ability to accurately quantify the biodistribution of a radiotracer, PET imaging is becoming the modality of choice to noninvasively study the biochemistry of human tumors in situ (4). PET imaging with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), which allows clinicians to scrutinize glucose metabolism in vivo, has largely dominated the clinical diagnostic oncology setting. However, a common disadvantage of the use of ¹⁸F-FDG as an imaging tracer has been its limited sensitivity and specificity, which can lead to confounding diagnosis (5); other pathological processes including inflammation and infection also present high glucose metabolism. Additionally, ¹⁸F-FDG PET often fails at detecting small malignant lesions (<5 mm in diameter) (6). Therefore, there is a pressing need for the implementation of molecular imaging probes that specifically target cancer-associated biological pathways and that can detect earlier such processes at the molecular level (7).Antibodies are of high interest as molecular imaging agents, particularly in oncology, because of their excellent antigen specificity and binding affinity. ImmunoPET probes can be designed to seek and target tumor cell-specific surface epitopes in vivo while maintaining low off-target effects (8). This enables the acquisition of high-quality PET images, which is highly desirable for cancer diagnosis, staging, and therapy response assessment. Compared with ¹⁸F-FDG and several other small-molecule PET tracers, antibodies provide greater specificity and phenotypic information on primary and metastatic diseases that can guide treatment decisions (3). However, the implementation of antibody-based imaging has been limited by practical complications related to long circulation half-lives, slow tumor penetration, immunogenicity, and regulatory hurdles. Fortunately, various protein engineering technologies can alleviate many of these issues. For example, humanized and fully human antibodies are available that minimized the risk of eliciting host immune responses. Furthermore, antibody fragments can exhibit significantly improved pharmacokinetic profiles compared with the intact antibody while retaining excellent antigen-binding affinity. A myriad of such immunoderivatives have been used for immunoPET imaging including monovalent fragments, diabodies, triabodies, minibodies, and single-domain antibodies (9). However, although PET imaging with antibody fragments offers several advantages in terms of radiation exposure, time to image, and multiple/repeated imaging, the fragments typically display significantly reduced tumor uptake and a much higher renal accumulation (10, 11).Given the inherent complexity of cancer, which involves a sophisticated cross-talk and promiscuity between multiple disease-mediating pathways and growth-promoting factors, targeting an isolated process usually fails to provide a satisfactory diagnosis and treatment efficacy (12). On the other hand, bispecific antibody fragments simultaneously targeting two antigens make for a promising alternative to enhance tumor uptake as well as specificity (13, 14). Although the value of bispecific antibodies for combination therapies has been proposed (15, 16), their potential as molecular imaging agents for cancer detection remains largely unexplored.Herein, we developed a bispecific construct, Bs-F(ab)₂—via conjugation of two antibody Fab fragments targeting epidermal growth factor receptor (EGFR) and CD105, respectively—for radiolabeling with ⁶⁴Cu and noninvasive PET imaging. The antibody fragments were obtained by enzymatic digestion of cetuximab (CET), an anti-human EGFR chimeric mAb, and TRC105, a mAb that recognizes both human and murine CD105. To conjugate the two Fab fragments, we exploited the fast reaction kinetics and selectivity of the inverse electron-demand Diels–Alder reaction between electron-deficient tetrazine (Tz) and strained transcyclooctene (TCO) derivatives (17). EGFR has been extensively studied as a target for anticancer therapy, and its activation stimulates tumor proliferation and angiogenesis (18). Similarly, CD105 (also called endoglin) is abundantly expressed on activated endothelial cells, and such overexpression is a negative prognostic factor in many malignant tumor types (19, 20). To date, simultaneous targeting of EGFR and CD105 has not been investigated. We hypothesized that our bispecific Bs-F(ab)₂ will harness the targeting capabilities of CET-Fab and TRC105-Fab and display a synergistic effect via dual targeting of EGFR and CD105. To test our hypothesis, we determined the advantages of dual EGFR/CD105 targeting in terms of tumor-binding affinity and specificity of Bs-F(ab)₂ in a glioblastoma multiforme (GBM) xenograft model, which expresses high levels of both EGFR and CD105 (^+/+). We presented here a generalizable rationale that could be potentially applied to produce bispecific imaging probes from other disease-targeting antibody fragments.     

Targeting of acute myeloid leukaemia by cytokine‐induced killer cells redirected with a novel CD123‐specific chimeric antigen receptor

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T‐cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34⁺ leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine‐induced killer (CIK) cells were transduced with SFG‐retroviral‐vector encoding an anti‐CD123 CAR. Transduced cells were able to strongly kill CD123⁺ cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti‐CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti‐CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123‐low‐expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti‐CD123.CAR. Taken together, our results indicate that CD123‐specific CARs strongly enhance anti‐AML CIK functions, while sparing HSPCs and normal low‐expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.