ABSTRACT
PURPOSE: Noninvasive quantifying activated hepatic stellate cells (aHSCs) by molecular imaging is helpful for assessing disease progression and therapeutic responses of liver fibrosis. Our purpose is to develop platelet-derived growth factor receptor ß (PDGFRß)-targeted radioactive tracer for assessing liver fibrosis by positron emission tomography (PET) imaging of aHSCs. METHODS: Comparative transcriptomics, immunofluorescence staining and flow cytometry were used to evaluate PDGFRß as biomarker for human aHSCs and determine the correlation of PDGFRß with the severity of liver fibrosis. The high affinity affibody for PDGFRß (ZPDGFRß) was labeled with gallium-68 (68Ga) for PET imaging of mice with carbon tetrachloride (CCl4)-induced liver fibrosis. Binding of the [68Ga]Ga-labeled ZPDGFRß ([68Ga]Ga-DOTA-ZPDGFRß) for aHSCs in human liver tissues was measured by autoradiography. RESULTS: PDGFRß overexpressed in aHSCs was highly correlated with the severity of liver fibrosis in patients and CCl4-treated mice. The 68Ga-labeled ZPDGFRß affibody ([68Ga]Ga-DOTA-ZPDGFRß) showed PDGFRß-dependent binding to aHSCs. According to the PET imaging, hepatic uptake of [68Ga]Ga-DOTA-ZPDGFRß increased with the accumulation of aHSCs and collagens in the fibrotic livers of mice. In contrast, hepatic uptake of [68Ga]Ga-DOTA-ZPDGFRß decreased with spontaneous recovery or treatment of liver fibrosis, indicating that the progression and therapeutic responses of liver fibrosis in mice could be visualized by PDGFRß-targeted PET imaging. [68Ga]Ga-DOTA-ZPDGFRß also bound human aHSCs and visualized fibrosis in patient-derived liver tissues. CONCLUSIONS: PDGFRß is a reliable biomarker for both human and mouse aHSCs. PDGFRß-targeted PET imaging could be used for noninvasive monitoring of liver fibrosis in mice and has great potential for clinical translation.
Subject(s)
Gallium Radioisotopes , Liver Cirrhosis , Positron-Emission Tomography , Receptor, Platelet-Derived Growth Factor beta , Liver Cirrhosis/diagnostic imaging , Liver Cirrhosis/metabolism , Animals , Positron-Emission Tomography/methods , Humans , Receptor, Platelet-Derived Growth Factor beta/metabolism , Mice , Male , Hepatic Stellate Cells/metabolism , Heterocyclic Compounds, 1-Ring/chemistryABSTRACT
PURPOSE: Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel-targeted molecular imaging might be effective for early diagnosis of HCC. Herein, we developed a novel trimeric affibody (ZTRI) with highly specific binding to the platelet-derived growth factor receptor beta (PDGFRß). The aim of this study is to evaluate the feasibility of 68Ga-radiolabeled ZTRI ([68Ga]Ga-DOTA-ZTRI) as PET tracer for diagnosis of HCC. METHODS: The bioinformatics analysis of clinical database and immunoblotting of clinical specimens were performed to validate the potential of PDGFRß as HCC biomarker. The trimeric affibody ZTRI was conjugated with DOTA-NHS-ester and radiolabeled with 68Ga to produce [68Ga]Ga-DOTA-ZTRI conjugate. Immunoreactivity and specific uptake of [68Ga]Ga-DOTA-ZTRI were assessed by dose-dependent cell binding, autoradiography, and biodistribution analysis. [68Ga]Ga-DOTA-ZTRI PET/CT scanning of diethylnitrosamine (DEN)-induced primary HCC rats and a rare case of idiopathical HCC rhesus monkey was performed to evaluate the imaging capability and radiation dosimetry of [68Ga]Ga-DOTA-ZTRI in vivo. RESULTS: Excessive PDGFRß was validated as a representative biomarker of HCC neovascularization. The radiolabeling of [68Ga]Ga-DOTA-ZTRI was achieved at more than 95% radiochemical yield. In vitro assays showed specific uptake of [68Ga]Ga-DOTA-ZTRI in HCC tumor vessels by autoradiography. Animal PET/CT imaging with [68Ga]Ga-DOTA-ZTRI successfully visualized the tumor lesions in primary HCC rats and rhesus monkey, and indicated radiation absorbed dose of 2.03E-02 mSv/MBq for each scanning. CONCLUSIONS: Our results demonstrated that [68Ga]Ga-DOTA-ZTRI conjugate could be applied as a promising PET tracer for early diagnosis of hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Rats , Animals , Positron Emission Tomography Computed Tomography/methods , Carcinoma, Hepatocellular/diagnostic imaging , Gallium Radioisotopes/chemistry , Tissue Distribution , Macaca mulatta , Cell Line, Tumor , Liver Neoplasms/diagnostic imaging , Positron-Emission Tomography/methods , BiomarkersABSTRACT
Antibody-based near-infrared photoimmunotherapy (NIR-PIT) is an attractive strategy for cancer treatment. Tumor cells can be selectively and efficiently killed by the targeted delivery of an antibody-photoabsorber complex followed by exposure to NIR light. Glycoprotein A33 antigen (GPA33) is highly expressed in most human colorectal cancers (CRCs) and is an ideal diagnostic and therapeutic target. We previously produced a single-chain fragment of a variable antibody against GPA33 (A33scFv antibody). Here, we investigate the efficacy of NIR-PIT by combining A33scFv with the NIR photoabsorber IR700 (A33scFv-IR700). In vitro, recombinant A33scFv displayed specific binding and delivery of an NIR dye to GPA33-positive tumor cells. Furthermore, A33scFv-IR700-mediated NIR-PIT was successful in rapidly and specifically killing GPA33-positive colorectal tumor cells. NIR-PIT treatment induced the release of lactate dehydrogenase from tumor cells, followed by cell necrosis, rather than apoptosis, through the promotion of reactive oxygen species accumulation in tumor cells. In mice bearing LS174T tumor grafts, A33scFv selectively accumulated in GPA33-positive tumors. Following only a single injection of the conjugate and subsequent illumination, A33scFv-IR700-mediated NIR-PIT induced a significant increase in therapeutic response in LS174T-tumor mice compared with that in the non-NIR-PIT groups (p < 0.001). Because the GPA33 antigen is specifically expressed in CRC tumors, A33scFv-IR700 might be a promising antibody fragment-photoabsorber conjugate for NIR-PIT of CRC.
Subject(s)
Cell Death/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/radiotherapy , Immunoconjugates/therapeutic use , Immunotherapy/methods , Membrane Glycoproteins/immunology , Phototherapy/methods , Single-Chain Antibodies/immunology , Animals , Cell Death/immunology , Colorectal Neoplasms/immunology , HT29 Cells , Humans , L-Lactate Dehydrogenase/metabolism , Mass Spectrometry , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Necrosis/metabolism , Photosensitizing Agents/therapeutic use , Reactive Oxygen Species/metabolism , Single-Chain Antibodies/radiation effects , Single-Chain Antibodies/toxicity , Xenograft Model Antitumor AssaysABSTRACT
Clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly limited by its inefficient apoptosis induction in tumor cells, which might be improved by using molecular superglue-mediated hyperoligomerization to increase its valency. Here, the minimal superglue peptide pairs, including Snoopligase-catalyzed SnoopTagJr/SnoopDogTag and SpyStapler-catalyzed SpyTag/SpyBDTag, were individually fused at the N- or C-terminus of the TRAIL promoter to produce superglue-fusion TRAIL variants. Similar to native trivalent TRAIL, these superglue-fusion TRAIL variants were highly expressed in Escherichia coli (E. coli) and spontaneously trimerized. In the presence of Snoopligase or SpyStapler, the trivalent superglue-fusion TRAIL variants were predominantly crosslinked into hexavalent TRAIL variants. Nevertheless, Snoopligase was more efficient than SpyStapler in the production of hexavalent TRAIL variants. In particular, Snoopligase-catalyzed trivalent TRAIL variants with N-terminal fusion of SnoopTagJr/SnoopDogTag produced hexavalent SnHexaTR with the highest yield (â¼70%). The in vitro cytotoxicity of SnHexaTR was 10-40 times greater than that of TRAIL in several tumor cells. In addition, compared to trivalent TRAIL, hexavalent SnHexaTR showed a longer serum half-life and greater tumor uptake, which resulted in eradication of 50% of tumor xenografts of TRAIL-sensitive COLO 205. In mice bearing TRAIL-resistant HT-29 tumor xenografts, hexavalent SnHexaTR combined with bortezomib encapsulated in liposomes also showed robust tumor growth suppression, indicating that hyperoligomerization mediated by minimal molecular superglue significantly increased the cytotoxicity and antitumor effect of TRAIL. As a novel anticancer agent candidate, the hexavalent SnHexaTR has great potential for clinical application in cancer therapy.
Subject(s)
Antineoplastic Agents , TNF-Related Apoptosis-Inducing Ligand , Animals , Humans , Mice , Apoptosis , Catalysis , Escherichia coli , Ligands , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Necrosis Factor-alpha , Xenograft Model Antitumor Assays , HT29 Cells , Antineoplastic Agents/pharmacologyABSTRACT
Prompting higher-order death receptor (DR) clustering by increasing the valency of DR agonist is efficient to induce apoptosis of tumor cells. As an attractive DR agonist with superior biosafety, the trimeric tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts limited antitumor effect in patients, which is predominantly attributed to its low DR clustering ability and short serum half-life. Previous antibody scaffolds-based engineering strategies to increase the valency and/or prolong the serum half-life of TRAIL improve apoptosis induction, however, often produce large proteins with poor tumor penetration. Covalent protein ligation mediated by small molecular superglues such as SpyTag/SpyCatcher might be a novel strategy to assemble higher-order TRAIL variants. Upon fusion to TRAIL promotor, SpyTag/SpyCatcher molecular superglue preferentially ligated two trimeric TRAIL to produce a hexameric TRAIL variant, HexaTR, exhibiting a significantly increased apoptosis induction. In addition, an albumin-binding HexaTR, ABD-HexaTR, with a prolonged serum half-life by binding to endogenous albumin was also produced using the same strategy. Compared to the trimeric TRAIL, the hexameric HexaTR and ABD-HexaTR showed 20-50 times greater in vivo antitumor effect, resulting in eradication of several types of large (150-300 mm3) tumor xenografts. Combination with bortezomib carried by liposome further improved the antitumor effects of the hexavalent HexaTR and ABD-HexaTR in refractory cancer. Our results indicate that the superglue-mediated higher-order assembly is promising to improve the DR clustering and proapoptotic signaling of TRAIL, showing great advantages in constructing the next generation of DR agonists for cancer therapy.
Subject(s)
Apoptosis , TNF-Related Apoptosis-Inducing Ligand , Humans , Cell Line, Tumor , Ligands , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Albumins/pharmacologyABSTRACT
Multidrug resistance (MDR), which is common in colorectal cancer (CRC), induces high mortality in patients. Due to its robust and selective apoptosis induction in some CRC cells with MDR, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is attractive as a novel tool for CRC therapy. However, TRAIL is limited by its poor tumor-homing ability and inefficient apoptosis induction in CRC cells expressing low levels of death receptor (DR). Here, the tumor-homing RGR peptide (CRGRRST) was fused to TRAIL to produce RGR-TRAIL. Compared with TRAIL, RGR-TRAIL showed greater cell binding and cytotoxicity in CRC cells. In addition, RGR-TRAIL exerted significantly enhanced tumor uptake and growth suppression in mice bearing CRC tumor xenografts. Notably, RGR-TRAIL eradicated all tumor xenografts of DR-overexpressing COLO205 cells. However, TRAIL only showed mild tumor growth suppression under the same conditions, indicating that RGR fusion significantly increased the antitumor effect of TRAIL in DR-overexpressing CRC cells by improving tumor homing. Nevertheless, RGR fusion did not significantly enhance the antitumor effect of TRAIL in HT29 cells expressing low levels of DR. We found that DR expression in HT29 cells was enhanced by epidermal growth factor receptor (EGFR)-targeted photodynamic therapy (PDT). Moreover, both the in vitro and in vivo antitumor effects of RGR-TRAIL were significantly improved by combination with PDT. HT29 tumor xenografts (â¼20%) were even eradicated by combination therapy. These results indicate that it is valuable to further evaluate the combination therapy of RGR-TRAIL and tumor-targeted PDT for clinical therapy of CRC with MDR.
Subject(s)
Colorectal Neoplasms , Photochemotherapy , Animals , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Heterografts , Humans , Mice , Receptors, TNF-Related Apoptosis-Inducing Ligand , TNF-Related Apoptosis-Inducing Ligand/pharmacologyABSTRACT
Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRß affibody, which targets platelet-derived growth factor receptor ß (PDGFRß), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRß fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRß fused to the IgBD. ZPDGFRß-modified αPD-L1 bound to both PDGFRß and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRß+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRß fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.
Subject(s)
Drug Delivery Systems , Immune Checkpoint Inhibitors , Immunoglobulin G/immunology , Neoplasms , Animals , Immunotherapy , Mice , Neoplasms/drug therapy , Receptor, Platelet-Derived Growth Factor betaABSTRACT
Chemotherapeutic multidrug resistance (MDR) is the major hindrance for clinical therapy of colorectal cancer (CRC). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with selective cytotoxicity might overcome MDR of CRC cells. Unfortunately, cross-resistance to TRAIL has been detected in many CRC cells, suggesting the need to combine TRAIL with sensitizers to combat refractory CRC. Our purpose is to explore the potential of combination therapy of TRAIL and tumor-cell targeted photodynamic therapy (PDT) in combating CRC with both chemotherapeutic MDR and TRAIL resistance. Methods: Tumor cell-targeted PDT was performed using a Ze-IR700 photosensitizer with high affinity for epidermal growth factor receptor (EGFR). The impact of PDT on the gene expression of CRC cells was revealed by RNA sequencing. The synergistic antitumor effect of long-acting TRAIL and PDT was evaluated in mice bearing tumor grafts of CRC cells with both chemotherapeutic MDR and TRAIL resistance. Results: Chemotherapeutic MDR and TRAIL resistance are common in CRC cells. Pretreatment of CRC cells with tumor cell-targeted PDT significantly (10-60 times) increased the sensitivity of these CRC cells to TRAIL by upregulating death receptors. Combination therapy, but not monotherapy, of long-acting TRAIL and PDT greatly induced apoptosis of CRC cells, thus efficiently eradicated large (~150 mm3) CRC tumor xenografts in mice. Conclusions: Tumor cell-targeted PDT extensively sensitizes CRC cells to TRAIL. Combination therapy of long-acting TRAIL and PDT is promising to combat CRC with both chemotherapeutic MDR and TRAIL resistance, which might be developed as a novel strategy for precision therapy of refractory CRC.