Tumor targeting and SPECT imaging properties of an 111In-labeled galectin-3 binding peptide in prostate carcinoma

IntroductionGalectin-3 (gal-3) is a carbohydrate binding protein that has been implicated in cell adhesion, tumor invasion and metastasis. The objective of this study was to evaluate the tumor targeting and imaging properties of a gal-3 binding peptide selected by phage display in a mouse model of metastatic human prostate carcinoma expressing gal-3.MethodsA gal-3 binding peptide, ANTPCGPYTHDCPVKR, was synthesized with a Gly–Ser–Gly (GSG) spacer and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and then radiolabeled with ¹¹¹In. The in vitro cell binding properties of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR were determined in metastatic human PC3-M prostate carcinoma cells. The pharmacokinetics and single-photon emission computed tomographic (SPECT/CT) imaging with the radiolabeled peptide were evaluated in SCID mice bearing human PC3-M prostate carcinoma tumor xenografts.ResultsThe radiolabeled peptide bound with a 50% inhibitory concentration of 191±10.2 nM to cultured PC3-M prostate carcinoma cells. In vivo tumor uptake and retention coupled with fast whole-body clearance of the peptide were demonstrated in PC3-M tumor-bearing SCID mice. The tumor uptake rates of the radiolabeled peptide were 1.27±0.10%ID/g at 30 min, 0.82±0.15%ID/g at 1 h and 0.57±0.09%ID/g at 2 h. MicroSPECT/CT studies revealed good tumor uptake of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR 2 h postinjection, while uptake in normal organs was low, with the exception of the kidneys.ConclusionsIn vitro cell binding along with tumor uptake of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR in PC3-M human prostate carcinoma tumor-bearing SCID mice suggests the potential of this peptide as a radiopharmaceutical for imaging of gal-3-expressing prostate tumors.     

Optimization of radioimmunotherapy of renal cell carcinoma: labeling of monoclonal antibody cG250 with 131I, 90Y, 177Lu, or 186Re.

Radioimmunotherapy (RIT) can be performed with various radionuclides. We tested the stability, biodistribution, and therapeutic efficacy of various radioimmunoconjugates ((131)I, (88/90)Y, (177)Lu, and (186)Re) of chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) in nude mice with subcutaneous renal cell cancer (RCC) tumors. METHODS: The (88/90)Y and (177)Lu labeling procedures of cG250 conjugated with cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA), isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), or 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) were characterized. Stability of the labeled conjugates in plasma at 37 degrees C was assessed. Biodistribution and therapeutic efficacy of labeled cG250 were compared in nude mice with SK-RC-52 human RCC xenografts. RESULTS: Both SCN-Bz-DTPA and DOTA were stable in vitro (<5% release of the radiolabel during 14 and 21 d of incubation) and in vivo (uptake in bone 相似文献   

ImmunoPET imaging of phosphatidylserine in pro-apoptotic therapy treated tumor models

An immunoPET imaging probe for the detection of phosphatidylserine was developed and tested in animal models of human cancer treated with pro-apoptotic therapy. We hypothesized that the relatively long plasma half-life of a probe based on a full-length antibody coupled with a residualizing radionuclide would be able to catch the wave of drug-induced apoptosis and lead to a specific accumulation in apoptotic tumor tissue.MethodsThe imaging probe is based on a ⁸⁹Zr-labeled monoclonal antibody PGN635 targeting phosphatidylserine. The probe was evaluated pre-clinically in four tumor xenograft models: one studied treatment with paclitaxel to trigger the intrinsic apoptotic pathway, and three others interrogated treatment with an agonistic death-receptor monoclonal antibody to engage the extrinsic apoptotic pathway.ResultsHigh accumulation of ⁸⁹Zr-PGN635 was observed in treated tumors undergoing apoptosis reaching 30 %ID/g and tumor-to-blood ratios up to 13. The tumor uptake in control groups treated with vehicle or imaged with a non-binding antibody probe was significantly lower.ConclusionsThe results demonstrate the ability of ⁸⁹Zr-PGN635 to image drug-induced apoptosis in animal models and corroborate our hypothesis that radiolabeled antibodies binding to intracellular targets transiently exposed on the cell surface during apoptosis can be employed for detection of tumor response to therapy.     

166Ho and 90Y labeled 6D2 monoclonal antibody for targeted radiotherapy of melanoma: Comparison with 188Re radiolabel

IntroductionAn approach to radioimmunotherapy (RIT) of metastatic melanoma is the targeting of melanin pigment with monoclonal antibodies (mAbs) to melanin radiolabeled with therapeutic radionuclides. The proof of principle experiments were performed using a melanin-binding antibody 6D2 of IgM isotype radiolabeled with a β emitter ¹⁸⁸Re and demonstrated the inhibition of tumor growth. In this study we investigated the efficacy of 6D2 antibody radiolabeled with two other longer lived β emitters ⁹⁰Y and ¹⁶⁶Ho in treatment of experimental melanoma, with the objective to find a possible correlation between the efficacy and half-life of the radioisotopes which possess high energy β (E_max > 1.5 MeV) emission properties.Methods6D2 was radiolabeled with longer lived β emitters ⁹⁰Y and ¹⁶⁶Ho in treatment of experimental melanoma in A2058 melanoma tumor-bearing nude mice. The immunoreactivity of the radiolabeled 6D2 mAb, its in vitro binding to the MNT1 human melanoma cells, the biodistribution and therapy in A2058 human melanoma bearing nude mice as well as dosimetry calculations were performed.ResultsWhen labeled with the longer lived ⁹⁰Y radionuclide, the 6D2 mAb did not produce any therapeutic effect in tumor bearing mice while the reduction of the tumor growth by ¹⁶⁶Ho-6D2 was very similar to the previously reported therapy results for ¹⁸⁸Re-6D2. In addition, ¹⁶⁶Ho-labeled mAb produced the therapeutic effect on the tumor without any toxic effects while the administration of the ⁹⁰Y-labeled radioconjugate was toxic to mice with no appreciable anti-tumor effect.Conclusions¹⁶⁶Ho-labeled mAb to melanin produced some therapeutic effect on the tumor without any toxic effects while the administration of the ⁹⁰Y-labeled radioconjugate was toxic to mice with no appreciable anti-tumor effect. We concluded that the serum half-life of the 6D2 carrier antibody matched well the physical half-life of ¹⁶⁶Ho to deliver the tumoricidal absorbed dose to the tumor. Further investigation of this radionuclide for RIT of melanoma is warranted.     

ImmunoPET for assessing the differential uptake of a CD146-specific monoclonal antibody in lung cancer

Purpose

Overexpression of CD146 in solid tumors has been linked to disease progression, invasion, and metastasis. We describe the generation of a ⁶⁴Cu-labeled CD146-specific antibody and its use for quantitative immunoPET imaging of CD146 expression in six lung cancer models.

Methods

The anti-CD146 antibody (YY146) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with ⁶⁴Cu. CD146 expression was evaluated in six human lung cancer cell lines (A549, NCI-H358, NCI-H522, HCC4006, H23, and NCI-H460) by flow cytometry and quantitative western blot studies. The biodistribution and tumor uptake of ⁶⁴Cu-NOTA-YY146 was assessed by sequential PET imaging in athymic nude mice bearing subcutaneous lung cancer xenografts. The correlation between CD146 expression and tumor uptake of ⁶⁴Cu-NOTA-YY146 was evaluated by graphical software while ex vivo biodistribution and immunohistochemistry studies were performed to validate the accuracy of PET data and spatial expression of CD146.

Results

Flow cytometry and western blot studies showed similar findings with H460 and H23 cells showing high levels of expression of CD146. Small differences in CD146 expression levels were found among A549, H4006, H522, and H358 cells. Tumor uptake of ⁶⁴Cu-NOTA-YY146 was highest in CD146-expressing H460 and H23 tumors, peaking at 20.1?±?2.86 and 11.6?±?2.34 %ID/g at 48 h after injection (n?=?4). Tumor uptake was lowest in the H522 model (4.1?±?0.98 %ID/g at 48 h after injection; n?=?4), while H4006, A549 and H358 exhibited similar uptake of ⁶⁴Cu-NOTA-YY146. A positive correlation was found between tumor uptake of ⁶⁴Cu-NOTA-YY146 (%ID/g) and relative CD146 expression (r ²?=?0.98, p?<?0.01). Ex vivo biodistribution confirmed the accuracy of the PET data.

Conclusion

The strong correlation between tumor uptake of ⁶⁴Cu-NOTA-YY146 and CD146 expression demonstrates the potential use of this radiotracer for imaging tumors that elicit varying levels of CD146. In the future, this tool may promote enhanced monitoring of therapeutic response and improved patient stratification.

     

Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy

IntroductionIn spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium(¹⁸⁸Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy.MethodsMice bearing A2058 melanoma xenografts were treated with either 1.5 mCi ¹⁸⁸Re-6D2 antibody, saline, unlabeled 6D2 antibody or ¹⁸⁸Re-labeled non-specific IgM.ResultsOn Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P < 0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers — chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells.ConclusionsThese results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5 + and JARID1B + cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers.     

Quantitative studies of monoclonal antibody targeting to disialoganglioside GD2 in human brain tumors

Iodine-131 3F8, a murine IgG₃ monoclonal antibody that targets to G_D2-bearing tumors, was administered intravenously to 12 patients with brain tumors. Six patients received 2 mCi (0.74 Bq) of¹³¹I-3F8, five patients 10 mCi (3.7 Bq)/1.73 m² of¹³¹I-3F8, and one patient 2.6 mCi (0.96 Bq) of¹²⁴I-3F8, with no side-effects. Nine of 11 malignant gliomas and the single metastatic melanoma showed antibody localization, with the best tumor delineation on single-photon emission tomography (SPET) following 10 mCi (3.7 Bq)/1.73 m² dose. No nonspecific uptake in the normal craniospinal axis was detected. There was no difference in the pharmacokinetics of low-dose versus the higher-dose antibody groups; plasma and total-body half-lives were 18 h and 49 h, respectively. Surgical sampling and time-activity curves based on quantitative imaging showed peak uptake in high-grade glioma at 39 h, with a half-life of 62 h. Tumor uptake at time of surgery averaged 3.5×10^–3 %ID/g and peak activity by the conjugate view method averaged 9.2×10^–3 %ID/g (3.5–17.8). Mean radiation absorption dose was 3.9 rad per mCi injected (range 0.7–9.6) or 10.5 cGy/Bq (range 1.9–26). There was agreement on positive sites when immunoscintigraphy was compared with technetium-99m glucoheptonate/diethylene triamine penta-acetic acid planar imaging, thallium-201 SPET, and fluorine-18 fluorodeoxyglucose positron emission tomography. Taken together, these data suggest that quantitative estimates of antibody targeting to intracranial tumors can be made using the modified conjugate view method.     

Radioimmunotherapy of head and neck cancer xenografts using 131I-labeled antibody L19-SIP for selective targeting of tumor vasculature.

The extra domain B of fibronectin (ED-B) is a marker of tumor angiogenesis. The human monoclonal antibody (mAb) L19-SIP (approximately 80 kDa; SIP is "small immunoprotein") has been selected for targeting of ED-B. The aim of this study was to evaluate the potential of radioimmunotherapy (RIT) with L19-SIP, either alone or in combination with cetuximab, for treatment of head and neck squamous cell carcinoma (HNSCC). Combination with cetuximab was considered because this anti-EGFR (epidermal growth factor receptor) mAb has proven value for the treatment of HNSCC. METHODS: HNSCC xenograft lines FaDu and HNX-OE were evaluated for ED-B and EGFR expression. L19-SIP was radiolabeled with 2 candidate radionuclides for RIT, 177Lu and 131I (or 125I as substitute). The biodistribution of coinjected 177Lu-L19-SIP and 125I-L19-SIP was assessed in FaDu-bearing nude mice, whereas 131I-L19-SIP was evaluated in both xenograft lines. After labeling with high-dose 131I (623-789 MBq/mg), the maximum tolerated dose (MTD) was assessed. The efficacy of RIT with injected 131I-L19-SIP, either alone or in combination with unlabeled cetuximab (1 mg 2 times a week intraperitoneally for 4 wk), was evaluated in both xenograft lines. RESULTS: Xenograft lines expressed both antigens, with similar EGFR expression and the highest ED-B expression in FaDu. Radioiodinated L19-SIP performed better than 177Lu-L19-SIP and was further exploited. The biodistribution of 131I-L19-SIP was most favorable in FaDu-bearing mice, with tumor uptake values at 24, 48, and 72 h after injection of 8.6 +/- 1.6, 5.8 +/- 0.4, and 3.4 +/- 0.2 %ID/g (%ID/g is percentage injected dose per gram of tissue), respectively, and ratios of tumor to normal tissues that gradually increased in time, such as for blood from 4.4 +/- 1.8 at 24 h to 21.4 +/- 1.7 at 72 h, after injection. RIT at the MTD level of 74 MBq caused significant tumor growth delay and improved survival in both lines. Although FaDu was most sensitive for RIT, with size reduction of all tumors, HNX-OE was most sensitive for treatment with cetuximab. The best survival and cure rates were obtained, however, when RIT and cetuximab were combined. CONCLUSION: RIT with 131I-L19-SIP appeared efficacious in HNSCC xenografts. The efficacy of RIT was enhanced by combination with cetuximab, without increase of toxicity.     

Preparation and preclinical evaluation of 177Lu-nimotuzumab targeting epidermal growth factor receptor overexpressing tumors

ObjectivesNimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) which recognizes the external domain of the epidermal growth factor receptor (EGFR) with high specificity. It was demonstrated that h-R3 has a unique clinical profile for immunotherapy of adult gliomas and pediatric pontine gliomas. The aim of this work was to evaluate the conjugate ¹⁷⁷Lu-h-R3 as a potential radioimmunoconjugate for radioimmunotherapy (RIT) of tumors overexpressing EGFR.Methodsh-R3 was modified with the macrocylcic ligand S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and the acyclic ligand S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA); the immunoconjugates were labeled with no-carried added ¹⁷⁷Lu. Specificity and affinity were tested using radioimmunoassays in a cell line overexpressing EGFR. Biodistribution in mice, healthy or bearing A431 epithelial carcinoma xenografts, was performed for 11 days. Tumor uptake, the influence of the nature of the chelate and the way of administration were studied. Absorbed dose in tumor and selected organs was calculated using the OLINDA/EXM software; the data from the animals was extrapolated to humans.Results¹⁷⁷Lu-h-R3 conjugates were obtained with specific activity up to 915 MBq/mg without significant loss of immunoreactivity. The binding of ¹⁷⁷Lu-h-R3 conjugates to A431 cells showed to be EGFR specific, and the affinity was similar to native h-R3. Tumor uptake reached a maximum value of 22.4±3.1 %ID/g at 72 h and remained ～20% ID/g over 1 week. Locoregional application showed better tumor/nontumor ratios than intravenous application.Conclusions¹⁷⁷Lu-h-R3 should be considered for further evaluations as a potential radiopharmaceutical for RIT of tumors overexpressing EGFR.     

Intratumoral distribution of radiolabeled antibody and radioimmunotherapy in experimental liver metastases model of nude mouse.

The biodistribution and intratumoral distribution of radiolabeled anticarcinoembryonic antigen (CEA) monoclonal antibody in experimental liver metastases and the therapeutic effect of 131I-labeled anti-CEA antibody on the metastases were studied. METHODS: Three weeks after an intrasplenic injection of human colon cancer cells, mice received an intravenous injection of 125I- or 111In-labeled anti-CEA antibody F33-104. The biodistribution and tumor penetration of radiolabeled antibody were examined by using quantitative autoradiography. To evaluate the therapeutic effect, 5.55, 9.25 or 11.1 MBq (150, 250 or 300 microCi) 131I-labeled F33-104 were injected into groups of mice that had micrometastases smaller than 1 mm. Control groups were injected with phosphate-buffered saline or 131I-labeled control antibody. Mice were killed 3 wk later to determine the size of liver metastases. RESULTS: 1251-labeled F33-104 showed a high accumulation in the liver metastases (percentage of injected dose per gram of metastases [%ID/g] >24%, metastasis-to-liver ratio >9.8, metastasis-to-blood ratio >2.1); however, its accumulation was heterogeneous or peripheral in the nodules more than 1 mm in diameter. When the antibody dose was increased, antibody penetration was improved, but tumor uptake of radioactivity and specificity ratios decreased. In mice with large metastases, radioactivity in the normal tissue was lower than that in mice with small metastases, resulting in higher metastasis-to-background ratios. 111In-labeled antibody showed even higher tumor uptake than 125I-labeled antibody (>51 %ID/g). Metastases formation was suppressed in a dose-dependent manner by 131I-labeled F33-104 injection (5 of 8 mice had no macroscopic tumor after an injection of 5.55 MBq (150 microCi), and all mice had no visible metastasis after an injection of 9.25 or 11.1 MBq [250 or 300 microCi]), whereas tumor progression was seen in the control groups. CONCLUSION: Liver metastases had easy accessibility to the antibody. Micrometastases of less than 0.5 mm in diameter showed homogeneous intratumoral distribution of injected antibody and were successfully treated with 131I-labeled antibody. Very high uptake and satisfactory metastasis-to-liver ratios with 111In-labeled antibody suggest that the use of a radiometal with high beta-energy, such as 90Y or 188Re, is preferable for the successful radioimmunotherapy of metastases larger than 1 mm.     

Pretargeting CWR22 prostate tumor in mice with MORF-B72.3 antibody and radiolabeled cMORF

Purpose We have now applied our MORF/cMORF pretargeting technology to the targeting of CWR22 prostate tumor in nude mice. Methods The antiTAG-72 antibody B72.3 was conjugated with an 18 mer MORF while the cMORF was radiolabeled with ^99mTc. The specific binding of the antibody to the CWR22 cells was first confirmed in an assay placing the radiolabeled B72.3 antibody in competition with increasing concentrations of native B72.3. Thereafter, a group of four CWR22 tumored mice intravenously received the MORF-B72.3 and, 3 days later, the ^99mTc-cMORF, and were killed at 3 h postradioactivity injection. The dosage of the labeled cMORF was selected on the basis of previous experience in LS174T tumored mice. As controls, four animals received only the radiolabeled cMORF and another four received only the ¹¹¹In-B72.3. The maximum percent tumor accumulation (MPTA) of the labeled cMORF was subsequently determined by a dosage study of labeled cMORF. Both a multipinhole SPECT image and a planar gamma camera image were obtained of a representative mouse. Results The CWR22 tumor was confirmed to be TAG-72-positive. The MPTA of the labeled cMORF in the CWR22 tumor was 2.22%ID/g compared to only 0.12%ID/g in control mice without pretargeting. Both the planar and tomographic images confirmed the success of the CWR22 pretargeting. Conclusions The MORF/cMORF pretargeting approach has been successfully applied to tumor targeting of the prostate xenograft CWR22. However, the MPTA in this tumor model is lower than that in the LS174T tumor model investigated earlier, possibly due to a lower tumor blood supply. Financial support: CA107360 and CA94994.     

18F]-fluoroestradiol quantitative PET imaging to differentiate ER+ and ERα-knockdown breast tumors in mice

IntroductionThe purpose of this study was to develop a noninvasive model in tumor-bearing mice to investigate the use of 16α-[¹⁸F]fluoro-17β-estradiol (FES) positron emission tomography (PET) imaging as a tool to discriminate between tumors having different estrogen receptor (ER) α status.MethodsMC7-L1 and MC4-L2 murine mammary adenocarcinoma cell lines (ER+) received a small hairpin RNA targeting the ERα gene by lentiviral infection. In vitro assessment of ERα levels of the new cell lines (MC7-L1 and MC4-L2 ERα-knockdown; ERαKD), compared to the parental cell lines, was performed by immunoblotting (?75% ERα protein) and binding assays (?50% estrogen binding). These cell lines were implanted subcutaneously in Balb/c mice and allowed to grow up to a volume of at least 20 mm³. FES and [¹⁸F]fluorodeoxyglucose (FDG) PET images were acquired to measure FES and FDG uptake in the various tumors.ResultsFES uptake as assessed by PET imaging was 1.06±0.21 percent injected dose per gram of tissue (%ID/g) for MC7-L1 tumors and 0.47±0.08 %ID/g for MC7-L1 ERαKD tumors. MC4-L2 tumors had a FES uptake of 1.03±0.30 %ID/g, whereas its ERαKD equivalent was 0.51±0.19 %ID/g. Each ERαKD tumor had a significantly lower %ID/g value, by ～50%, than its ER+ counterpart. Biodistribution studies confirmed these findings and gave %ID/g values that were not significantly different from PET imaging data. FDG PET showed no significant uptake difference between the ER+ and ERαKD tumors, indicating that the metabolic phenotype of the ERαKD cell lines was not altered.ConclusionFES PET imaging was able to reliably differentiate between tumors having differences in their ERα expression in vivo, in a mouse model. Quantitative data obtained by FES PET were in concordance with biodistribution studies and in vitro assays. It is concluded that FES PET imaging can likely be used to monitor subtle ER status changes during the course of hormone therapy.     

Pretargeted immuno-PET and radioimmunotherapy of prostate cancer with an anti-TROP-2 x anti-HSG bispecific antibody

Purpose

TF12 is a trivalent bispecific antibody that consists of two anti-TROP-2 Fab fragments and one anti-histamine-succinyl-glycine (HSG) Fab fragment. The TROP-2 antigen is found in many epithelial cancers, including prostate cancer (PC), and therefore this bispecific antibody could be suitable for pretargeting in this cancer. In this study, the characteristics and the potential for pretargeted radioimmunoimaging and radioimmunotherapy with TF12 and the radiolabeled di-HSG peptide IMP288 in mice with human PC were investigated.

Methods

The optimal TF12 protein dose, IMP288 peptide dose, and dose interval for PC targeting were assessed in nude mice with s.c. PC3 xenografts. Immuno-positron emission tomography (PET)/CT was performed using TF12/⁶⁸Ga-IMP288 at optimized conditions. The potential of pretargeted radioimmunotherapy (PRIT) using the TF12 pretargeted ¹⁷⁷Lu-IMP288 was determined.

Results

TF12 and ¹¹¹In-IMP288 showed high and fast accumulation in the tumor [20.4?±?0.6 %ID/g at 1 h post-injection (p.i.)] at optimized conditions, despite the internalizing properties of TF12. The potential for PRIT was shown by retention of 50 % of the ¹¹¹In-IMP288 in the tumor at 48 h p.i. One cycle of treatment with TF12 and ¹⁷⁷Lu-IMP288 showed significant improvement of survival compared to treatment with ¹⁷⁷Lu-IMP288 alone (90 vs. 67 days, p?<?0.0001) with no renal or hematological toxicity.

Conclusion

TROP-2-expressing PC can be pretargeted efficiently with TF12, with very rapid uptake of the radiolabeled hapten-peptide, IMP288, sensitive immuno-PET, and effective therapy.     

PET imaging of HER1-expressing xenografts in mice with 86Y-CHX-A″-DTPA-cetuximab

Purpose

Cetuximab is a recombinant, human/mouse chimeric IgG₁ monoclonal antibody that binds to the epidermal growth factor receptor (EGFR/HER1). Cetuximab is approved for the treatment of patients with HER1-expressing metastatic colorectal cancer. Limitations in currently reported radiolabeled cetuximab for PET applications prompted the development of ⁸⁶Y-CHX-A″-DTPA-cetuximab as an alternative for imaging HER1-expressing cancer. ⁸⁶Y-CHX-A″-DTPA-cetuximab can also serve as a surrogate marker for ⁹⁰Y therapy.

Methods

Bifunctional chelate, CHX-A″-DTPA was conjugated to cetuximab and radiolabeled with ⁸⁶Y. In vitro immunoreactivity was assessed in HER1-expressing A431 cells. In vivo biodistribution, PET imaging and noncompartmental pharmacokinetics were performed in mice bearing HER1-expressing human colorectal (LS-174T and HT29), prostate (PC-3 and DU145), ovarian (SKOV3) and pancreatic (SHAW) tumor xenografts. Receptor blockage was demonstrated by coinjection of either 0.1 or 0.2 mg cetuximab.

Results

⁸⁶Y-CHX-A″-DTPA-cetuximab was routinely prepared with a specific activity of 1.5–2 GBq/mg and in vitro cell-binding in the range 65–75%. Biodistribution and PET imaging studies demonstrated high HER1-specific tumor uptake of the radiotracer and clearance from nonspecific organs. In LS-174T tumor-bearing mice injected with ⁸⁶Y-CHX-A″-DTPA-cetuximab alone, ⁸⁶Y-CHX-A″-DTPA-cetuximab plus 0.1 mg cetuximab or 0.2 mg cetuximab, the tumor uptake values at 3 days were 29.3?±?4.2, 10.4?±?0.5 and 6.4?±?0.3%ID/g, respectively, demonstrating dose-dependent blockage of the target. Tumors were clearly visualized 1 day after injecting 3.8–4.0 MBq ⁸⁶Y-CHX-A″-DTPA-cetuximab. Quantitative PET revealed the highest tumor uptake in LS-174T (29.55?±?2.67%ID/cm³) and the lowest tumor uptake in PC-3 (15.92?±?1.55%ID/cm³) xenografts at 3 days after injection. Tumor uptake values quantified by PET were closely correlated (r ²?=?0.9, n?=?18) with values determined by biodistribution studies.

Conclusion

This study demonstrated the feasibility of preparation of high specific activity ⁸⁶Y-CHX-A″-DTPA-cetuximab and its application for quantitative noninvasive PET imaging of HER1-expressing tumors. ⁸⁶Y-CHX-A″-DTPA-cetuximab offers an attractive alternative to previously labeled cetuximab for PET and further investigation for clinical translation is warranted.     

Development of high-specific-activity 68Ga-labeled DOTA-rhenium-cyclized α-MSH peptide analog to target MC1 receptors overexpressed by melanoma tumors

IntroductionA previous report on ⁶⁸Ga-1,4,7,10-tetraazacyclodedecane-N,N′,N″,N′″-tetraacetic acid (DOTA)-Re(Arg¹¹)CCMSH was shown to indicate the imaging agent's potency for early detection of metastatic melanoma. However, the main limiting factor to developing high-specific-activity ⁶⁸Ga-DOTA-Re(Arg¹¹)CCMSH is the short half-life of ⁶⁸Ga, which precludes further purification of the agent. To circumvent this problem, we incorporated the microwave technique to rapidly radiolabel the peptide with ⁶⁸Ga, thereby allowing enough time to include high-performance liquid chromatography (HPLC) purification in the overall procedure.MethodsDOTA-Re(Arg¹¹)CCMSH was radiolabeled with ⁶⁸Ga in <1 min using a circular-cavity microwave apparatus. Reverse-phase HPLC purification was accomplished in less than 20 min. ⁶⁸Ga-DOTA-Re(Arg¹¹)CCMSH was then administered on B16/F1 murine melanoma-bearing C57 mice to study its biodistribution and positron emission tomography (PET) imaging capability.ResultsThe production of high-specific-activity ⁶⁸Ga-DOTA-Re(Arg¹¹)CCMSH resulted in an improved tumor uptake [6.93±1.11%ID/g at 30 min postinjection (p.i.) and 6.27±1.60%ID/g at 1 h p.i.] and tumor retention (5.85±1.32%ID/g at 4 h p.i.). Receptor-mediated tumor uptake was verified by blocking studies. Furthermore, high-resolution PET images of the tumor were obtained, owing to high tumor-to-nontarget organ ratios at an early time point (i.e., at 1 h biodistribution: tumor/blood, 14.3; tumor/muscle, 89.6; tumor/skin, 12.3) and fast clearance of the labeled peptide from kidney and other healthy tissues.ConclusionHigh-specific-activity ⁶⁸Ga-DOTA-Re(Arg¹¹)CCMSH may have a potential role in the early diagnosis of metastasized melanoma.     

Quantitative PET of EGFR expression in xenograft-bearing mice using <Superscript>64</Superscript>Cu-labeled cetuximab,a chimeric anti-EGFR monoclonal antibody

Purpose Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using ⁶⁴Cu-labeled cetuximab. Methods We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), labeled with ⁶⁴Cu, and tested the resulting ⁶⁴Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. Results MicroPET imaging showed that ⁶⁴Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R ² = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. Conclusion The success of EGFR-positive tumor imaging using ⁶⁴Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. Weibo Cai and Kai Chen contributed equally to this work.     

In vivo evaluation of 18F-labeled TCO for pre-targeted PET imaging in the brain

IntroductionThe tetrazine-trans-cylooctene cycloaddition using radiolabeled tetrazine or radiolabeled trans-cyclooctene (TCO) has been reported to be a very fast, selective and bioorthogonal reaction that could be useful for in vivo radiolabeling of molecules. We wanted to evaluate the in vivo biodistribution profile and brain uptake of ¹⁸F-labeled TCO ([¹⁸F]TCO) to assess its potential for pre-targeted imaging in the brain.MethodsWe evaluated the in vivo behavior of [¹⁸F]TCO via an ex vivo biodistribution study complemented by in vivo μPET imaging at 5, 30, 60, 90, 120 and 240 min post tracer injection. An in vivo metabolite study was performed at 5 min, 30 min and 120 min post [¹⁸F]TCO injection by RP-HPLC analysis of plasma and brain extracts. Incubation with human liver microsomes was performed to further evaluate the metabolite profile of the tracer.ResultsμPET imaging and ex-vivo biodistribution revealed an high initial brain uptake of [¹⁸F]TCO (3.8%ID/g at 5 min pi) followed by a washout to 3.0%ID/g at 30 min pi. Subsequently the brain uptake increased again to 3.7%ID/g at 120 min pi followed by a slow washout until 240 min pi (2.9%ID/g). Autoradiography confirmed homogenous brain uptake. On the μPET images bone uptake became gradually visible after 120 min pi and was clearly visible at 240 min pi. The metabolite study revealed a fast metabolization of [¹⁸F]TCO in plasma and brain into three main polar radiometabolites.ConclusionsAlthough [¹⁸F]TCO has previously been described to be a useful tracer for radiolabeling of tetrazine modified targeting molecules, our study indicates that its utility for in vivo chemistry and pre-targeted imaging will be limited. Although [¹⁸F]TCO clearly enters the brain, it is quickly metabolized with a non-specific accumulation of radioactivity in the brain and bone.     

EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

IntroductionAs direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging.MethodsHuman epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to ^99mTc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice.ResultsWhereas both ^99mTc-Av-EGF and ^99mTc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. ^99mTc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). ^99mTc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h.Conclusion^99mTc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors.     

99mTc-labeled SWL specific peptide for targeting EphA2 receptor

IntroductionEphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, ^99mTc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated.MethodsThe SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by ^99mTc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with ^99mTc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution.ResultsImmunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. ^99mTc-HYNIC-SWL displayed high binding affinity with A549 cells (K_D = 2.6 ± 0.7 nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44 ± 0.12 %ID/g) than in OCM-1 tumors (0.43 ± 0.20 %ID/g) at 1 h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58 ± 0.20 %ID/g) was seen. These data suggest that ^99mTc-HYNIC-SWL specifically targets EphA2 in tumors.ConclusionsThe expression of EphA2 can be noninvasively investigated using ^99mTc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of ^99mTc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging.     

Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas

IntroductionPodoplanin/aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has been reported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible for cancer relapse. The purpose of this study was to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas.MethodsThe binding affinity of an anti-podoplanin antibody, NZ-1 (rat IgG_2a), was determined by surface plasmon resonance and Scatchard analysis. NZ-1 was radioiodinated with ¹²⁵I using Iodogen [¹²⁵I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[¹³¹I]iodobenzoate ([¹³¹I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of ¹²⁵I-NZ-1(Iodogen) and that of [¹³¹I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts.ResultsThe dissociation constant (K_D) of NZ-1 was determined to be 1.2×10^?10 M by surface plasmon resonance and 9.8×10^?10 M for D397MG glioblastoma cells by Scatchard analysis. Paired-label internalization assays in LN319 glioblastoma cells indicated that [¹³¹I]SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3±0.8% of initially bound radioactivity at 8 h) compared to that from the ¹²⁵I-NZ-1(Iodogen) (10.0±0.1% of initially bound radioactivity at 8 h). Likewise, tumor uptake of [¹³¹I]SGMIB-NZ-1 (39.9±8.8 %ID/g at 24 h) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of ¹²⁵I-NZ-1(Iodogen) (29.7±6.1 %ID/g at 24 h).ConclusionsThe overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.