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Abstract Background In colorectal cancer, survival of patients is drastically reduced when complete resection is hampered by involvement of critical structures. Targeted photodynamic therapy (tPDT) is a local and targeted therapy which could play a role in eradicating residual tumor cells after incomplete resection. Since carcinoembryonic antigen (CEA; CEACAM5) is abundantly overexpressed in colorectal cancer, it is a potential target for tPDT of colorectal cancer. Methods To address the potential of CEA-targeted PDT, we compared colorectal cancer cell lines with different CEA-expression levels (SW-48, SW-480, SW-620, SW-1222, WiDr, HT-29, DLD-1, LS174T, and LoVo) under identical experimental conditions. We evaluated the susceptibility to tPDT by varying radiant exposure and concentration of our antibody conjugate (DTPA-hMN-14-IRDye700DX). Finally, we assessed the efficacy of tPDT in vivo in 18 mice (BALB/cAnNRj-Foxn1 nu/nu ) with subcutaneously xenografted LoVo tumors. Results In vitro, the treatment effect of tPDT varied per cell line and was dependent on both radiant exposure and antibody concentration. Under standardized conditions (94.5 J/cm2 and 0.5 μg/μL antibody conjugate concentration), the effect of tPDT was higher in cells with higher CEA availability: SW-1222, LS174T, LoVo, and SW-48 (22.8%, 52.8%, 49.9%, and 51.9% reduction of viable cells, respectively) compared to cells with lower CEA availability. Compared to control groups (light or antibody conjugate only), tumor growth rate was reduced in mice with s.c. LoVo tumors receiving tPDT. Conclusion Our findings suggest cells (and tumors) have different levels of susceptibility for tPDT even though they all express CEA. Furthermore, tPDT can effectively reduce tumor growth in vivo.

Abstract Background Combining modalities using dual-labeled antibodies may allow preoperative and intraoperative tumor localization and could be used in image-guided surgery to improve complete tumor resection. Trastuzumab is a monoclonal antibody against the human epidermal growth factor-2 (HER2) receptor and dual-labeled trastuzumab with both a fluorophore (IRDye800CW) and a radioactive label (111In) can be used for multimodal imaging of HER2-positive breast cancer. The aim of this study was to demonstrate the feasibility of HER2-targeted multimodal imaging using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast cancer model. Methods Trastuzumab was conjugated with p-isothiocyanatobenzyl (ITC)-diethylenetriaminepentaacetic acid (DTPA) and IRDye800CW-NHS ester and subsequently labeled with 111In. In a dose escalation study, the biodistribution of 10, 30, and 100 μg [111In]In-DTPA-trastuzumab-IRDye800CW was determined 48 h after injection in BALB/c nude mice with orthotopic high HER2-expressing tumors. Also, a biodistribution study was performed in a low HER2-expressing breast cancer model. In addition, multimodal image-guided surgery was performed in each group. Autoradiography, fluorescence microscopy, and immunohistochemically stained slices of the tumors were compared for co-localization of tumor tissue, HER2 expression, fluorescence, and radiosignal. Results Based on the biodistribution data, a 30 μg dose of dual-labeled trastuzumab (tumor-to-blood ratio 13 ± 2) was chosen for all subsequent studies. [111In]In-DTPA-trastuzumab-IRDye800CW specifically accumulated in orthotopic HER2-positive BT474 tumors (101 ± 7 %IA/g), whereas uptake in orthotopic low HER2-expressing MCF7 tumor was significantly lower (1.2 ± 0.2 %IA/g, p = 0.007). BT474 tumors could clearly be visualized with both micro-SPECT/CT, fluorescence imaging and subsequently, image-guided resection was performed. Immunohistochemical analyses of BT474 tumors demonstrated correspondence in fluorescence, radiosignal, and high HER2 expression. Conclusions Dual-labeled trastuzumab showed specific accumulation in orthotopic HER2-positive BT474 breast tumors with micro-SPECT/CT and fluorescence imaging and enabled image-guided tumor resection. In the clinical setting, [111In]In-DTPA-trastuzumab-IRDye800CW could be valuable for preoperative detection of (metastatic) tumors by SPECT/CT imaging, and intraoperative localization by using a gamma probe and fluorescence image-guided surgery to improve radical resection of tumor tissue in patients with HER2-positive tumors.

Abstract Background Image-guided surgery may improve surgical outcome for colorectal cancer patients. Here, we evaluated the feasibility of a pretargeting strategy for multimodal imaging in colorectal cancer using an anti-carcinoembryonic antigen (CEA) x anti-histamine-succinyl-glycine (HSG) bispecific antibody (TF2) in conjunction with the dual-labeled diHSG peptide (RDC018), using both a fluorophore for near-infrared fluorescence imaging and a chelator for radiolabeling. Methods Nude mice with subcutaneous (s.c) CEA-expressing LS174T human colonic tumors and CEA-negative control tumors were injected with TF2. After 16 h, different doses of 111In-labeled IMP-288 (non-fluorescent) or its fluorescent derivative RDC018 were administered to compare biodistributions. MicroSPECT/CT and near-infrared fluorescence imaging were performed 2 and 24 h after injection. Next, the biodistribution of the dual-labeled humanized anti-CEA IgG antibody [111In]In-DTPA-hMN-14-IRDye800CW (direct targeting) was compared with the biodistribution of 111In-RDC018 in mice with TF2-pretargeted tumors, using fluorescence imaging and gamma counting. Lastly, mice with intraperitoneal LS174T tumors underwent near-infrared fluorescence image-guided resection combined with pre- and post-resection microSPECT/CT imaging. Results 111In-RDC018 showed specific tumor targeting in pretargeted CEA-positive tumors (21.9 ± 4.5 and 10.0 ± 4.7% injected activity per gram (mean ± SD %IA/g), at 2 and 24 hours post-injection (p.i.), respectively) and a biodistribution similar to 111In-IMP288. Both fluorescence and microSPECT/CT images confirmed preferential tumor accumulation. At post mortem dissection, intraperitoneal tumors were successfully identified and removed using pretargeting with TF2 and 111In-RDC018. Conclusion A pretargeted approach for multimodal image-guided resection of colorectal cancer in a preclinical xenograft model is feasible, enables preoperative SPECT/CT, and might facilitate intraoperative fluorescence imaging.

Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system’s first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DX-loaded liposomes significantly delayed arthritis development. Here we have shown the proof-of-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.

For intraoperative imaging, antibodies labeled with both a radionuclide and a fluorophore may be used to tag the tumor lesion with a radiolabel and a fluorescent signal at high tumor to background ratios. However, labeling antibodies with fluorescent moieties may affect the in vivo behavior of the antibody depending on the dye to antibody substitution ratio. To investigate the optimal substitution ratio for use in dual-modality image-guided surgery, we conjugated three different antibodies, MN-14 (anti-CEACAM5), girentuximab (anti-CAIX), and cetuximab (anti-EGFR), with both diethylene triamine pentaacetic acid (DTPA, for labeling with 111 In) and IRdye 800CW at dye to antibody ratios of 0, 1, 1.5, 2, and 3 and assessed in vivo behavior. Biodistribution studies showed that at high dye to antibody ratios, liver uptake of the dual-labeled antibodies increased, whereas tumor uptake decreased. Conversely, very low ratios may not be optimal either because in that case, only a few antibody molecules will be dual-labeled (i.e., contain both a DTPA and an IRDye 800CW moiety), which may complicate interpretation of dual-modality data. The present study shows that, provided that the chelator to antibody ratio is high enough, a dye to antibody ratio in the range of 1 to 1.5 is optimal for antibody-targeted dual-modality imaging applications. However, the optimal configuration is antibody dependent and should be determined for each dual-labeled antibody individually.

A new approach in the treatment of clear cell renal carcinoma (ccRCC) is radioimmunotherapy (RIT) using anti-carbonic anhydrase IX (CAIX) antibody G250. To investigate the potential of RIT with lutetium 177 ( 177 Lu)-labeled G250, we conducted a protein dose escalation study and subsequently an RIT study in mice with intraperitoneally growing ccRCC lesions. Mice with intraperitoneal xenografts were injected with 1, 3, 10, 30, or 100 μg of G250 labeled with 10 MBq indium 111 ( 111 In) to determine the optimal protein dose. The optimal protein dose determined with imaging and biodistribution studies was used in a subsequent RIT experiment in three groups of 10 mice with intraperitoneal SK-RC-52 tumors. One group received 13 MBq 177 LU-DOTA-G250, a control group received 13 MBq nonspecific 177 LU-MOPC21, and the second control group was not treated and received 20 MBq 111 In-DOTA-G250. The optimal G250 protein dose to target ccRCC in this model was 10 μg G250. Treatment with 13 MBq 177 LU-DOTA-G250 was well tolerated and resulted in significantly prolonged median survival (139 days) compared to controls (49-53 days, p = .015), indicating that RIT has potential in this metastatic ccRCC model.

Abstract Background Rheumatoid arthritis (RA) is one of the most prevalent and debilitating joint diseases worldwide. RA is characterized by synovial inflammation (synovitis), which is linked to the development of joint destruction. Magnetic resonance imaging and ultrasonography are widely being used to detect the presence and extent of synovitis. However, these techniques do not reveal the activation status of inflammatory cells such as macrophages that play a crucial role in synovitis and express CD64 (Fc gamma receptor (FcγR)I) which is considered as macrophage activation marker. Objectives We aimed to investigate CD64 expression and its correlation with pro-inflammatory cytokines and pro-damaging factors in human-derived RA synovium. Furthermore, we aimed to set up a molecular imaging modality using a radiolabeled CD64-specific antibody as a novel imaging tracer that could be used to determine the extent and phenotype of synovitis using optical and nuclear imaging. Methods First, we investigated CD64 expression in synovium of early- and late-stage RA patients and studied its correlation with the expression of pro-inflammatory and tissue-damaging factors. Next, we conjugated an anti-CD64 antibody with IRDye 800CW and diethylenetriamine penta-acetic acid (DTPA; used for 111In labeling) and tested its binding on cultured THP1 cells, ex vivo RA synovium explants and its imaging potential in SCID mice implanted with human RA synovium explants obtained from RA patients who underwent total joint replacement. Results We showed that CD64 is expressed in synovium of early and late-stage RA patients and that FCGR1A/CD64 expression is strongly correlated with factors known to be involved in RA progression. Combined, this makes CD64 a useful marker for imaging the extent and phenotype of synovitis. We reported higher binding of the [111In]In-DTPA-IRDye 800CW anti-CD64 antibody to in vitro cultured THP1 monocytes and ex vivo RA synovium compared to isotype control. In human RA synovial explants implanted in SCID mice, the ratio of uptake of the antibody in synovium over blood was significantly higher when injected with anti-CD64 compared to isotype and injecting an excess of unlabeled antibody significantly reduced the antibody-binding associated signal, both indicating specific receptor binding. Conclusion Taken together, we successfully developed an optical and nuclear imaging modality to detect CD64 in human RA synovium in vivo.