HER2-directed antibodies, affibodies and nanobodies as drug-delivery vehicles in breast cancer with a specific focus on radioimmunotherapy and radioimmunoimaging.

PURPOSE: The aim of the present paper is to review the role of HER2 antibodies, affibodies and nanobodies as vehicles for imaging and therapy approaches in breast cancer, including a detailed look at recent clinical data from antibody drug conjugates and nanobodies as well as affibodies that are currently under development. RESULTS: Clinical and preclinical studies have shown that the use of monoclonal antibodies in molecular imaging is impaired by slow blood clearance, associated with slow and low tumor uptake and with limited tumor penetration potential. Antibody fragments, such as nanobodies, on the other hand, can be radiolabelled with short-lived radioisotopes and provide high-contrast images within a few hours after injection, allowing early diagnosis and reduced radiation exposure of patients. Even in therapy, the small radioactively labeled nanobodies prove to be superior to radioactively labeled monoclonal antibodies due to their higher specificity and their ability to penetrate the tumor. CONCLUSION: While monoclonal antibodies are well established drug delivery vehicles, the current literature on molecular imaging supports the notion that antibody fragments, such as affibodies or nanobodies, might be superior in this approach.

Efficacy of Alveolar Ridge Preservation: A Randomized Controlled Trial.

Alveolar ridge preservation (ARP) therapy is indicated to attenuate the physiologic resorptive events that occur as a consequence of tooth extraction with the purpose of facilitating tooth replacement therapy. This randomized controlled trial was primarily aimed at testing the efficacy of ARP as compared with unassisted socket healing. A secondary objective was to evaluate the effect that local phenotypic factors play in the volumetric reduction of the alveolar bone. A total of 53 subjects completed the study. Subjects were randomized into either the control group, which involved only tooth extraction (EXT n = 27), or the experimental group, which received ARP using a combination of socket grafting with a particulate bone allograft and socket sealing with a nonabsorbable membrane (dPTFE) following tooth extraction (ARP n = 26). A set of clinical, linear, volumetric, implant-related, and patient-reported outcomes were assessed during a 14-wk healing period. All linear bone assessments (horizontal, midbuccal, and midlingual reduction) revealed that ARP is superior to EXT. Likewise, volumetric bone resorption was significantly higher in the control group (mean ± SD: EXT = -15.83% ± 4.48%, ARP = -8.36% ± 3.81%, P < 0.0001). Linear regression analyses revealed that baseline buccal bone thickness is a strong predictor of alveolar bone resorption in both groups. Interestingly, no significant differences in terms of soft tissue contour change were observed between groups. Additional bone augmentation to facilitate implant placement in a prosthetically acceptable position was deemed necessary in 48.1% of the EXT sites and only 11.5% of the ARP sites (P < 0.004). Assessment of perceived postoperative discomfort at each follow-up visit revealed a progressive decrease over time, which was comparable between groups. Although some extent of alveolar ridge remodeling occurred in both groups, ARP therapy was superior to EXT as it was more efficacious in the maintenance of alveolar bone and reduced the estimated need for additional bone augmentation at the time of implant placement (ClinicalTrials.gov NCT01794806).

Early Phase I Study of a 99mTc-Labeled Anti-Programmed Death Ligand-1 (PD-L1) Single-Domain Antibody in SPECT/CT Assessment of PD-L1 Expression in Non-Small Cell Lung Cancer.

Immunotherapy with checkpoint inhibitor programmed cell death 1 (PD-1)/programmed death ligand-1 (PD-L1) antibodies demonstrates improvements in treatment of advanced non-small cell lung cancer. Treatment stratification depends on immunohistochemical PD-L1 measurement of biopsy material, an invasive method that does not account for spatiotemporal heterogeneity. Using a single-domain antibody, NM-01, against PD-L1, radiolabeled site-specifically with 99mTc for SPECT imaging, we aimed to assess the safety, radiation dosimetry, and imaging characteristics of this radiopharmaceutical and correlate tumor uptake with PD-L1 immunohistochemistry results. Methods: Sixteen patients (mean age, 61.7 y; 11 men) with non-small cell lung cancer were recruited. Primary tumor PD-L1 expression was measured by immunohistochemistry. NM-01 was radiolabeled with [99mTc(OH2)3(CO)3]+ complex binding to its C-terminal hexahistidine tag. Administered activity was 3.8-10.4 MBq/kg, corresponding to 100 µg or 400 µg of NM-01. Whole-body planar and thoracic SPECT/CT scans were obtained at 1 and 2 h after injection in all patients, and 5 patients underwent additional imaging at 10 min, 3 h, and 24 h for radiation dosimetry calculations. All patients were monitored for adverse events. Results: No drug-related adverse events occurred in this study. The mean effective dose was 8.84 × 10-3 ± 9.33 × 10-4 mSv/MBq (3.59 ± 0.74 mSv per patient). Tracer uptake was observed in the kidneys, spleen, liver, and bone marrow. SPECT primary tumor-to-blood-pool ratios (T:BP) varied from 1.24 to 2.3 (mean, 1.79) at 1 h and 1.24 to 3.53 (mean, 2.22) at 2 h (P = 0.005). Two-hour primary T:BP ratios correlated with PD-L1 immunohistochemistry results (r = 0.68, P = 0.014). Two-hour T:BP was lower in tumors with ≤1% PD-L1 expression (1.89 vs. 2.49, P = 0.048). Nodal and bone metastases showed tracer uptake. Heterogeneity (>20%) between primary tumor and nodal T:BP was present in 4 of 13 patients. Conclusion: This first-in-human study demonstrates that 99mTc-labeled anti-PD-L1-single-domain antibody SPECT/CT imaging is safe and associated with acceptable dosimetry. Tumor uptake is readily visible against background tissues, particularly at 2 h when the T:BP ratio correlates with PD-L1 immunohistochemistry results.

Mouse Monoclonal Antibodies Against Progranulin (PGRN/GEP) as Therapeutics in Preclinical Cancer Models.

The use of monoclonal antibody (mAb) has become a unique means of targeted therapy for human cancers. mAb-based therapies have shown survival benefits by applying alone or in combination with chemotherapeutics. Being a humanized biomolecule with exquisite target specificity, mAb demonstrated effects in a relatively lower dose range with limited off-target harm to the patients. Nowadays, novel targets involved in tumorigenic mechanisms and biomarkers expressed exclusively on cancer cell surface are being constantly discovered. The potential effects of their specific mAb could be investigated in the preclinical cancer model. In this chapter, we outlined our experimental procedures in determining the feasibility of novel mAb in the preclinical cancer model, with an example of progranulin (PGRN/GEP) mAb against hepatocellular carcinoma (HCC) tumor in mouse model. This chapter included the establishment of subcutaneous and orthotopic HCC tumor in mouse model, the injection of the mouse monoclonal antibody in combination with cytotoxic chemotherapeutics, the assessment of tumor development, and the analyses of the molecular changes of the tumor cells.

Granulin-epithelin precursor renders hepatocellular carcinoma cells resistant to natural killer cytotoxicity.

Immunoevasion is an emerging hallmark of cancer. Impairment of natural killer (NK) cytotoxicity is a mechanism to evade host immunosurveillance. Granulin-epithelin precursor (GEP) is a hepatic oncofetal protein regulating growth, invasion, and chemoresistance in hepatocellular carcinoma (HCC). We examined the role of GEP in conferring HCC cells the ability to evade NK cytotoxicity. In HCC cell lines, GEP overexpression reduced, whereas GEP suppression enhanced sensitivity to NK cytotoxicity. GEP downregulated surface expression of MHC class I chain-related molecule A (MICA), ligand for NK stimulatory receptor NK group 2 member D (NKG2D), and upregulated human leukocyte antigen-E (HLA-E), ligand for NK inhibitory receptor CD94/NKG2A. Functionally, GEP augmented production of soluble MICA, which suppressed NK activation. Matrix metalloproteinase (MMP)2 and MMP9 activity was involved partly in the GEP-regulated MICA shedding from HCC cells. In primary HCCs (n = 80), elevated GEP (P < 0.001), MICA (P < 0.001), and HLA-E (P = 0.089) expression was observed when compared with those in nontumor (n = 80) and normal livers (n = 10). Serum GEP (P = 0.010) and MICA (P < 0.001) levels were higher in patients with HCC (n = 80) than in healthy individuals (n = 30). High serum GEP and/or MICA levels were associated with poor recurrence-free survival (log-rank test, P = 0.042). Importantly, GEP blockade by mAbs sensitized HCC cells to NK cytotoxicity through MICA. In summary, GEP rendered HCC cells resistant to NK cytotoxicity by modulating MICA expression, which could be reversed by GEP blockade using antibody. Serum GEP and MICA levels are prognostic factors and can be used to stratify patients for targeted therapy.

Antibody against granulin-epithelin precursor sensitizes hepatocellular carcinoma to chemotherapeutic agents.

Granulin-epithelin precursor (GEP) overexpression has been shown in many cancers with functional role on growth, and recently on regulating chemoresistance and cancer stem cell (CSC) properties. Here, we investigate the combined effect of GEP antibody and chemotherapeutic agent. Combination therapy was compared with monotherapy using hepatocellular carcinoma (HCC) cells in vitro and orthotopic liver tumor models in vivo. CD133 and related hepatic CSC marker expressions were investigated by flow cytometry. Antiproliferative and apoptotic effects and signaling mechanisms were examined by immunohistochemistry, flow cytometry, and Western blot analysis. Secretory GEP levels in the serum and culture supernatant samples were measured by ELISA. We demonstrated that HCC cells that survived under chemotherapeutic agents showed upregulation of hepatic CSC markers CD133/GEP/ABCB5, and enhanced colony and spheroid formation abilities. Importantly, GEP antibody sensitized HCC cells to the apoptosis induced by chemotherapy for both HCC cell lines and the chemoresistant subpopulations, and counteracted the chemotherapy-induced GEP/ABCB5 expressions and Akt/Bcl-2 signaling. In human HCC orthotopic xenograft models, GEP antibody treatment alone was consistently capable of inhibiting the tumor growth. Notably, combination of GEP antibody with high dose of cisplatin resulted in the eradication of all established intrahepatic tumor in three weeks. This preclinical study demonstrated that GEP antibody sensitized HCC cells to apoptosis induced by chemotherapeutic agents. Combination treatment with GEP antibody and chemotherapeutic agent has the potential to be an effective therapeutic regimen for GEP-expressing cancers.

Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.

Granulin-epithelin precursor (GEP) is a pluripotent secretory growth factor which promotes cancer progression in a number of human cancers. However, how cancer cells interact with GEP remains unknown. In this study, we aimed to identify the cell surface-binding partner of GEP on liver cancer cells. Human recombinant GEP (rGEP) was expressed and purified to homogeneity. The rGEP was shown to trigger phosphorylation of AKT and ERK1/2 in liver cancer cells. We demonstrated cell surface attachment of rGEP, which was blocked by prebinding of platelet-derived growth factor-AA, platelet-derived growth factor-BB and fibroblast growth factor-2. Therefore, heparan sulfate (HS) had been reasoned as the binding partner of rGEP. Heparinase digestion validated the role of HS on supporting the attachment. The heparin-binding domain of GEP was mapped to RRH(555-557) in the C-terminal region. Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction. Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT. Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001). This study identified HS, partly through glypican-3, as a novel binding partner of GEP on the surface of liver cancer cells.

Identification and characterization of tropomyosin 3 associated with granulin-epithelin precursor in human hepatocellular carcinoma.

BACKGROUND AND AIM: Granulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry. METHODS AND RESULTS: Specific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: TPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.

Granulin-epithelin precursor and ATP-dependent binding cassette (ABC)B5 regulate liver cancer cell chemoresistance.

BACKGROUND & AIMS: Chemotherapy is used to treat unresectable liver cancer with marginal efficacy; this might result from hepatic cancer cells with stem cell and chemoresistant features. Gene expression profiling studies have shown that hepatic cancer cells express granulin-epithelin precursor (GEP); we investigated its role in hepatic cancer stem cell functions and chemoresistance. METHODS: The effects of GEP and drug transporter signaling on chemoresistance were investigated in hepatic cancer stem cells. We analyzed the expression patterns of 142 clinical samples from liver tumors, adjacent nontumorous liver tissue, and liver tissue from patients who did not have cancer. RESULTS: GEP regulated the expression of the adenosine triphosphate-dependent binding cassette (ABC)B5 drug transporter in liver cancer cells. Chemoresistant cells that expressed GEP had increased levels of ABCB5; suppression of ABCB5 sensitized the cells to doxorubicin uptake and apoptosis. Most cells that expressed GEP and ABCB5 also expressed the hepatic cancer stem cell markers CD133 and EpCAM; blocking ABCB5 reduced their expression. Expression levels of GEP and ABCB5 were correlated in human liver tumor samples. ABCB5 levels were increased in liver cancer cells compared with nontumor liver tissue from patients with cirrhosis or hepatitis, or normal liver tissue. ABCB5 expression was associated with the recurrence of hepatocellular carcinoma after partial hepatectomy. CONCLUSIONS: Expression of GEP and ABCB5 in liver cancer stem cells is associated with chemoresistance and reduced survival times of patients with hepatocellular carcinoma. Reagents designed to target these proteins might be developed as therapeutics and given in combination with chemotherapy to patients with liver cancer.