Keelara Abiraj

Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.

Bombesin Antagonist–Based Radioligands for Translational Nuclear Imaging of Gastrin-Releasing Peptide Receptor–Positive Tumors

Bombesin receptors are overexpressed on a variety of human tumors. In particular, the gastrin-releasing peptide receptor (GRPr) has been identified on prostate and breast cancers and on gastrointestinal stromal tumors. The current study aims at developing clinically translatable bombesin antagonist–based radioligands for SPECT and PET of GRPr-positive tumors. Methods: A potent bombesin antagonist (PEG4-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 [AR]) was synthesized; conjugated to the chelators DOTA, 6-carboxy-1,4,7,11-tetraazaundecane (N4), 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A); and radiolabeled with 111In, 99mTc, 68Ga, and 64Cu, respectively. The radioconjugates were evaluated in vitro and in vivo in PC-3 tumor–bearing nude mice. Antagonist potency was determined by Ca2+-flux measurements and immunofluorescence. Results: All the conjugates showed high binding affinity to GRPr (inhibitory concentration of 50% [IC50], 2.5–25 nmol/L). The immunofluorescence and Ca2+-flux assays confirmed the antagonist properties of the conjugates. Biodistribution revealed high and specific uptake in PC-3 tumor and in GRPr-positive tissues. Tumor uptake of 64Cu-CB-TE2A-AR (31.02 ± 3.35 percentage injected activity per gram [%IA/g]) was higher than 99mTc-N4-AR (24.98 ± 5.22 %IA/g), 111In-DOTA-AR (10.56 ± 0.70 %IA/g), and 68Ga-NODAGA-AR (7.11 ± 3.26 %IA/g) at 1 h after injection. Biodistribution at later time points showed high tumor-to-background ratios because of the fast washout of the radioligand from normal organs, compared with tumor. High tumor-to-background ratios were further illustrated by PET and SPECT images of PC-3 tumor–bearing nude mice acquired at 12 h after injection showing high tumor uptake, clear background, and negligible or no radioactivity in the abdomen. Conclusion: The chelators do influence the affinity, antagonistic potency, and pharmacokinetics of the conjugates. The promising preclinical results warrant clinical translation of these probes for SPECT and PET.

Evolution of Bombesin Conjugates for Targeted PET Imaging of Tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel 64/67Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC50: 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle14 by Met, and deletion of D-Tyr6, further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6th, 11th, and 14th position of BN induced only slight influences on affinity to mouse GRPr. [CuII]-CPTA-[βAla11] BN(7–14) ([CuII]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [64/67Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [64/67Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [64/67Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (89Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of 89Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of 89Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg 89Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent 89Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of 89Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of 89Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, 89Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.

Tetraamine-derived bifunctional chelators for technetium-99m labelling: synthesis, bioconjugation and evaluation as targeted SPECT imaging probes for GRP-receptor-positive tumours.

Owing to its optimal nuclear properties, ready availability, low cost and favourable dosimetry, (99m)Tc continues to be the ideal radioisotope for medical-imaging applications. Bifunctional chelators based on a tetraamine framework exhibit facile complexation with Tc(V)O(2) to form monocationic species with high in vivo stability and significant hydrophilicity, which leads to favourable pharmacokinetics. The synthesis of a series of 1,4,8,11-tetraazaundecane derivatives (01-06) containing different functional groups at the 6-position for the conjugation of biomolecules and subsequent labelling with (99m)Tc is described herein. The chelator 01 was used as a starting material for the facile synthesis of chelators functionalised with OH (02), N(3) (04) and O-succinyl ester (05) groups. A straightforward and easy synthesis of carboxyl-functionalised tetraamine-based chelator 06 was achieved by using inexpensive and commercially available starting materials. Conjugation of 06 to a potent bombesin-antagonist peptide and subsequent labelling with (99m)Tc afforded the radiotracer (99m)Tc-N4-BB-ANT, with radiolabelling yields of >97% at a specific activity of 37 GBq micromol(-1). An IC(50) value of (3.7+/-1.3) nM was obtained, which confirmed the high affinity of the conjugate to the gastrin-releasing-peptide receptor (GRPr). Immunofluorescence and calcium mobilisation assays confirmed the strong antagonist properties of the conjugate. In vivo pharmacokinetic studies of (99m)Tc-N4-BB-ANT showed high and specific uptake in PC3 xenografts and in other GRPr-positive organs. The tumour uptake was (22.5+/-2.6)% injected activity per gram (% IA g(-1)) at 1 h post injection (p.i.). and increased to (29.9+/-4.0)% IA g(-1) at 4 h p.i. The SPECT/computed tomography (CT) images showed high tumour uptake, clear background and negligible radioactivity in the abdomen. The promising preclinical results of (99m)Tc-N4-BB-ANT warrant its potential candidature for clinical translation.

First-in-Human Phase I Study of Lumretuzumab, a Glycoengineered Humanized Anti-HER3 Monoclonal Antibody, in Patients with Metastatic or Advanced HER3-Positive Solid Tumors.

Purpose: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer. Experimental Design: Twenty-five patients with histologically confirmed HER3-expressing tumors received lumretuzumab (100, 200, 400, 800, 1,600, and 2,000 mg) every two weeks (q2w) in 3+3 dose-escalation phase. In addition, 22 patients were enrolled into an extension cohort at 2,000 mg q2w. Results: There were no dose-limiting toxicities. Common adverse events (any grade) included diarrhea (22 patients, 46.8%), fatigue (21 patients, 44.7%), decreased appetite (15 patients, 31.9%), infusion-related reactions (13 patients, 27.7%), and constipation (10 patients, 21.3%). The peak concentration (Cmax) and area under the concentration–time curve up to the last measurable concentration (AUClast) of lumretuzumab increased more than dose proportionally from 100 mg up to 400 mg. Linear PK was observed with doses ≥400 mg q2w indicating target-mediated drug disposition saturation. Downregulation of HER3 membranous protein was observed in on-treatment tumor biopsies from 200 mg, and was maximal at and above 400 mg. An ex vivo assay demonstrated increased activation potential of peripheral NK lymphocytes with lumretuzumab compared with a non-glycoengineered anti-HER3 antibody. Ten patients (21.3%) had stable disease and remained on study at a median of 111 days (range, 80–225 days). Conclusions: Lumretuzumab was well tolerated and showed evidence of clinical activity. Linear serum PK properties and plateauing of PD effects in serial tumor biopsies indicate optimal biologically active doses of lumretuzumab from 400 mg onwards. Clin Cancer Res; 22(4); 877–85. ©2015 AACR.

Imaging Integrin αvβ3 on Blood Vessels with 111In-RGD2 in Head and Neck Tumor Xenografts

Arginine-glycine-aspartic acid (RGD)–based imaging tracers allow specific imaging of integrin αvβ3, a protein overexpressed during angiogenesis, leading to the possibility that it might serve as a tool to stratify patients for antiangiogenic treatment. However, these tracers have generally been characterized in xenograft models in which integrin αvβ3 was constitutively expressed by the tumor cells themselves. In the studies presented here, the use of 111In-RGD2 as a tracer to image only integrin αvβ3 expression on blood vessels in the tumor was determined using tumor xenografts in which tumor cells were integrin αvβ3-negative. Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with 111In (111In-RGD2), and biodistribution studies were performed in squamous cell carcinoma of the head and neck (HNSCC) xenograft mouse models to determine the optimal peptide dose to image angiogenesis. Next, biodistribution and imaging studies were performed at the optimal peptide dose in 3 HNSCC mouse models, FaDu, SCCNij3, and SCCNij202. Immunohistochemical analysis of tumor vascular and cell surface expression of integrin αvβ3 and correlation analysis of vascular integrin αvβ3 and autoradiography were completed. Results: All 3 HNSCC xenografts expressed integrin αvβ3 on the vessels only. The optimal peptide dose of 111In-RGD2 was 1 μg or less for specific integrin αvβ3–mediated uptake of the tracer. SPECT/CT imaging showed clear uptake of the tracer in the periphery of the tumors, corresponding with well-vascularized areas of the tumor. Within the tumor, 111In-RGD2 autoradiography coincided with vascular integrin αvβ3 expression, as determined immunohistochemically. Integrin αvβ3–mediated uptake was also detected in nontumor tissues, which, through immunohistochemical analysis, proved positive for integrin αvβ3. Conclusion: 111In-RGD2 allows the visualization of integrin αvβ3 in xenograft models in which integrin αvβ3 is expressed only on the neovasculature, such as in the HNSCC tumors. Thus, 111In-RGD2 allows specific visualization of angiogenesis in tumor models lacking constitutive tumoral integrin αvβ3 expression but may be less useful for this purpose in many tumors in which tumor cells express integrin αvβ3.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

Zr-89-Lumretuzumab PET Imaging before and during HER3 Antibody Lumretuzumab Treatment in Patients with Solid Tumors

Purpose: We evaluated biodistribution and tumor targeting of 89Zr-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)–targeting monoclonal antibody. Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received 89Zr-lumretuzumab and underwent positron emission tomography (PET). In part A, 89Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV). Results: Optimal PET conditions were found to be 4 and 7 days after administration of 89Zr-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4 (±1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis (n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (±8.2), 10.0% (±16.5), and 24.6% (±20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab. Conclusions: PET imaging showed biodistribution and tumor-specific 89Zr-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased 89Zr-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. Clin Cancer Res; 23(20); 6128–37. ©2017 AACR.

Can 111In-RGD2 Monitor Response to Therapy in Head and Neck Tumor Xenografts?

RGD (arginylglycylaspartic acid)–based imaging tracers allow specific imaging of integrin αvβ3 expression, proteins overexpressed during angiogenesis; however, few studies have investigated the potential of these tracers to monitor responses of antiangiogenic or radiation therapy. In the studies presented here, 111In-RGD2 was assessed for its potential as an imaging tool to monitor such responses to therapies. Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with 111In (111In-RGD2), and biodistribution studies were performed in mice with subcutaneous FaDu or SK-RC-52 xenografts after treatment with either antiangiogenic therapy (bevacizumab or sorafenib) or tumor irradiation (10 Gy). Micro-SPECT imaging studies and subsequent quantitative analysis were also performed. The effect of bevacizumab, sorafenib, or radiation therapy on tumor growth was determined. Results: The uptake of 111In-RGD2 in tumors, as determined from biodistribution studies, correlated well with that quantified from micro-SPECT images, and both showed that 15 d after irradiation 111In-RGD2 uptake was enhanced. Specific or nonspecific uptake of 111In-RGD2 in FaDu or SK-RC-52 xenografts was not affected after antiangiogenic therapy, except in head and neck squamous cell carcinoma 19 d after the start of sorafenib therapy (P < 0.05). The uptake of 111In-RGD2 followed tumor volume in studies featuring antiangiogenic therapy. However, the uptake of 111In-RGD2 in FaDu xenografts was decreased as early as 4 h after tumor irradiation, despite nonspecific uptake remaining unaltered. Tumor growth was inhibited after antiangiogenic or radiation therapy. Conclusion: Here, it is suggested that 111In-RGD2 could allow in vivo monitoring of angiogenic responses after radiotherapy and may therefore prove a good clinical tool to monitor angiogenic responses early after the start of radiotherapy in patients with head and neck squamous cell carcinoma. Despite clear antitumor efficacy, antiangiogenic therapy did not alter tumor uptake of 111In-RGD2, indicating that integrin expression was not altered.