Evaluation of nimotuzumab Fab2 as an optical imaging agent in EGFR positive cancers.

Molecular-targeted imaging probes can be used with a variety of imaging modalities to detect diseased tissues and guide their removal. EGFR is a useful biomarker for a variety of cancers, because it is expressed at high levels relative to normal tissues. Previously, we showed the anti-EGFR antibody nimotuzumab can be used as a positron emission tomography and fluorescent imaging probe for EGFR positive cancers in mice. These imaging probes are currently in clinical trials for PET imaging and image-guided surgery, respectively. One issue with using antibody probes for imaging is their long circulation time and slow tissue penetration, which requires patients to wait a few days after injection before imaging or surgery, multiple visits and longer radiation exposure. Here, we generated a Fab2 fragment of nimotuzumab, by pepsin digestion and labeled it with IRDye800CW to evaluate its optical imaging properties. The Fab2 had faster tumor accumulation and clearance in mice relative to the nimotuzumab IgG. The fluorescent signal peaked at 2 h post injection and remained high until 6 h post injection. The properties of the Fab2 allow a higher signal to background to be obtained in a shorter time frame, reducing the wait time for imaging after probe infusion.

Rapid Copper-free Click Conjugation to Lipid-Shelled Microbubbles for Ultrasound Molecular Imaging of Murine Bowel Inflammation.

Microbubbles are ultrasound contrast agents that can adhere to disease-related vascular biomarkers when functionalized with binding ligands such as antibodies or peptides. The biotin-streptavidin approach has predominantly been used as the microbubble labeling approach in preclinical imaging. However, due to the immunogenicity of avidin in humans, it is not suitable for clinical translation. What would aid clinical translation is a simple and effective microbubble functionalization approach that could be directly translated from animals to humans. We developed a targeted microbubble to P-selectin, a vascular inflammatory marker, labeled using a strain-promoted [3 + 2] azide-alkyne (azide-DBCO) reaction, comparing its ability to detect bowel inflammation to that of P-selectin targeted microbubbles labeled with a traditional biotin-streptavidin approach. Bowel inflammation was chemically induced using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in Balb/C mice. Each mouse received both non-targeted and P-selectin targeted microbubbles (either biotin-streptavidin or azide-DBCO). Using the biotin-streptavidin reaction, there was a significant increase in the ultrasound molecular imaging signal in inflamed mice using P-selectin targeted (2.30 ± 0.91 a.u.) compared to isotype control microbubbles (1.14 ± 0.7 a.u.) (p = 0.009). Using the azide-DBCO reaction, there was a similar increase in the ultrasound molecular imaging signal in inflamed mice (2.54 ± 0.56 a.u) compared to the isotype control (0.44 ± 0.25 a.u) (p = 0.009). There were no significant differences between the two labeling approaches between non-targeted and P-selectin targeted microbubbles. Mouse inflammatory phenotypes and expression of P-selectin were validated using histology and immunostaining. We constructed P-selectin targeted microbubbles using an azide-DBCO click reaction, which could detect bowel inflammation in vivo. This reaction generated a similar ultrasound molecular imaging signal to biotin-strepavidin-labeled microbubbles. These data show the potential of click chemistry conjugation (azide-DBCO) as a quick, cost-efficient, and clinically translatable approach for developing targeted microbubbles.

Imaging Immune Cells Using Fc Domain Probes in Mouse Cancer Xenograft Models.

Tracking immune responses is complex due to the mixture of cell types, variability in cell populations, and the dynamic environment. Tissue biopsies and blood analysis can identify infiltrating and circulating immune cells; however, due to the dynamic nature of the immune response, these are prone to sampling errors. Non-invasive targeted molecular imaging provides a method to monitor immune response, which has advantages of providing whole-body images, being non-invasive, and allowing longitudinal monitoring. Three non-specific Fc-containing proteins were labeled with near-infrared dye IRDye800CW and used as imaging probes to assess tumor-infiltrating immune cells in FaDu and A-431 xenograft models. We showed that Fc domains localize to tumors and are visible by fluorescent imaging. This tumor localization appears to be based on binding tumor-associated immune cells and some xenografts showed higher fluorescent signals than others. The Fc domain alone bound to different human immune cell types. The Fc domain can be a valuable research tool to study innate immune response.

Pre-clinical study of IRDye800CW-nimotuzumab formulation, stability, pharmacokinetics, and safety.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a target for cancer therapy as it is overexpressed in a wide variety of cancers. Therapeutic antibodies that bind EGFR are being evaluated in clinical trials as imaging agents for positron emission tomography and image-guided surgery. However, some of these antibodies have safety concerns such as infusion reactions, limiting their use in imaging applications. Nimotuzumab is a therapeutic monoclonal antibody that is specific for EGFR and has been used as a therapy in a number of countries. METHODS: Formulation of IRDye800CW-nimotuzumab for a clinical trial application was prepared. The physical, chemical, and pharmaceutical properties were tested to develop the specifications to determine stability of the product. The acute and delayed toxicities were tested and IRDye800CW-nimotuzumab was determined to be non-toxic. Non-compartmental pharmacokinetics analysis was used to determine the half-life of IRDye800CW-nimotuzumab. RESULTS: IRDye800CW-nimotuzumab was determined to be non-toxic from the acute and delayed toxicity study. The half-life of IRDye800CW-nimotuzumab was determined to be 38 ± 1.5 h. A bi-exponential analysis was also used which gave a t1/2 alpha of 1.5 h and t1/2 beta of 40.8 h. CONCLUSIONS: Here, we show preclinical studies demonstrating that nimotuzumab conjugated to IRDye800CW is safe and does not exhibit toxicities commonly associated with EGFR targeting antibodies.

89Zr-Labeled Domain II-Specific scFv-Fc ImmunoPET Probe for Imaging Epidermal Growth Factor Receptor In Vivo.

Epidermal growth factor receptor I (EGFR) is overexpressed in many cancers. The extracellular domain of EGFR has four binding epitopes (domains I- IV). All clinically approved anti-EGFR antibodies bind to domain III. Imaging agents that bind to domains other than domain III of EGFR are needed for accurate quantification of EGFR, patient selection for anti-EGFR therapeutics and monitoring of response to therapies. We recently developed a domain II-specific antibody fragment 8709. In this study, we have evaluated the in vitro and in vivo properties of 89Zr-8709-scFv-Fc (105 kDa). We conjugated 8709-scFv-Fc with the deferoxamine (DFO) chelator and radiolabeled the DFO-8970-scFv with 89Zr. We evaluated the binding of 89Zr-DFO-8709-scFv-Fc in EGFR positive and negative cell lines DLD-1, MDA-MB-231 and MDA-MB-435, respectively, and in mouse xenograft models. Simultaneously, we have compared the binding of 89Zr-8709-scFv-Fc with 111In-nimotuzumab, a domain III anti-EGFR antibody. DFO-8709-scFv-Fc displayed similar cell binding specificity as 8709-scFv-Fc. Saturation cell binding assay and immunoreactive fraction showed that radiolabeling did not alter the binding of 8709-scFv-Fc. Biodistribution and microPET showed good uptake of 89Zr-8709-scFv-Fc in xenografts after 120 h post injection (p.i). and was domain-specific to EGFR domain II. 89Zr-8709-scFv-Fc did not compete for binding in vitro and in vivo with a known domain III binder nimotuzumab. The results show that 89Zr-8709-scFv-Fc is specific to domain II of EGFR making it favorable for quantification of EGFR in vivo, hence, patient selection and monitoring of response to treatment with anti-EGFR antibodies.

Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

Development and preclinical evaluation of cixutumumab drug conjugates in a model of insulin growth factor receptor I (IGF-1R) positive cancer.

Overexpression of insulin growth factor receptor type 1 (IGF-1R) is observed in many cancers. Antibody drug conjugates (ADCs) with PEGylated maytansine (PEG6-DM1) show promise in vitro. We developed PEG6-DM1 ADCs with low and high drug to antibody ratios (DAR) using an anti-IGF-1R antibody cixutumumab (IMC-A12). Conjugates with low (cixutumumab-PEG6-DM1-Low) and high (cixutumumab-PEG6-DM1-High) DAR as 3.4 and 7.2, respectively, were generated. QC was performed by UV spectrophotometry, HPLC, bioanalyzer, and biolayer-interferometry. We compared the in vitro binding and internalization rates of the ADCs in IGF-1R-positive MCF-7/Her18 cells. We radiolabeled the ADCs with 111In and used microSPECT/CT imaging and ex vivo biodistribution to understand their in vivo behavior in MCF-7/Her18 xenograft mice. The therapeutic potential of the ADC was studied in vitro and in mouse xenograft. Internalization rates of all ADCs was high and increased over 48 h and EC50 was in the low nanomolar range. MicroSPECT/CT imaging and ex vivo biodistribution showed significantly lower tumor uptake of 111In-cixutumumab-PEG6-DM1-High compared to 111In-cixutumumab-PEG6-DM1-Low and 111In-cixutumumab. Cixutumumab-PEG6-DM1-Low significantly prolonged the survival of mice bearing MCF-7/Her18 xenograft compared with cixutumumab, cixutumumab-PEG6-DM1-High, or the PBS control group. Cixutumumab-PEG6-DM1-Low ADC was more effective. The study highlights the potential utility of cixutumumab-ADCs as theranostics against IGF-1R positive cancers.

111In- and 225Ac-Labeled Cixutumumab for Imaging and α-Particle Radiotherapy of IGF-1R Positive Triple-Negative Breast Cancer.

Insulin growth factor receptor (IGF-1R) is overexpressed in many cancers of epithelial origin, where it confers enhanced proliferation and resistance to therapies targeted at other receptors. Anti-IGF-1R monoclonal antibodies have not demonstrated significant improvements in patient outcomes in clinical trials. Humanized monoclonal antibody cixutumumab (IMC-A12) binds to IGF-1R with low nM affinity. In this study, cixutumumab was conjugated with p-SCN-Bn-DOTA and radiolabeled with 111In or 225Ac for imaging or radiotherapy using a triple-negative breast cancer (TNBC) model SUM149PT. The antibody conjugate showed low nM affinity to IGF-1R, which was not affected by conjugation and radiolabeling procedures. Cixutumumab immunoconjugates were effectively internalized in SUM149PT and were cytotoxic to the cells with an EC50 of 225Ac-cixutumumab (0.02 nM) that was almost 5000-fold less than that of unlabeled cixutumumab (95.2 nM). MicroSPECT imaging of the SUM149PT xenograft showed the highest tumor uptake occurred at 48 h post injection and was 9.9 ± 0.5% injected activity per gram (%IA/cc). In radiotherapy studies, we evaluated the effect of the specific activity of 225Ac-cixutumumab on efficacy following a tail vein injection of two doses (days 0 and 10) of the investigation agent or controls. Cixutumumab (2.5 mg/kg) prolonged the survival of the SUM149PT tumor-bearing mice with a median survival of 87 days compared to the PBS control group (median survival of 62 days). Median survival of high specific activity 225Ac-cixutumumab (8 kBq/µg, 225 nCi, 0.05 mg/kg) was 103.5 days compared to 122 days for low specific activity 225Ac-cixutumumab (0.15 kBq/µg, 225 nCi, 2.5 mg/kg). Additionally, low specific activity radioimmunoconjugate led to complete tumor remission in 2/6 mice. The data suggest that the efficacy of cixutumumab can be enhanced by radiolabeling with 225Ac at a low specific activity.

Near infrared imaging of epidermal growth factor receptor positive xenografts in mice with domain I/II specific antibody fragments.

Epidermal growth factor receptor (EGFR) is a transmembrane cell surface receptor that is frequently overexpressed and/or mutated in many cancers. Therapies targeting EGFR have poor outcomes due to the lack of reliable diagnostic tests to monitor EGFR. Current in vitro EGFR diagnostic methods are invasive, requiring biopsies, which limits tumor sampling and availability. EGFR molecular imaging provides non-invasive whole-body images capable of detecting primary tumors and metastases, which can be used to diagnose and monitor response to therapy. Methods: We evaluated properties of two anti-EGFR fragments, 8708 and 8709, as molecular-targeted imaging probes. 8708 and 8709 are anti-EGFR antigen binding fragments (Fabs) that recognize domain I/II of EGFR, which is distinct from epitopes recognized by current anti-EGFR therapeutic antibodies. We used complementarity determining region sequences from 8708 and 8709 Fabs to generate an anti-EGFR IgG and (scFv)2 and scFv-Fc antibody fragments. We expressed, purified, and labeled the IgG and fragments with IRDye800CW and used them to image EGFR-positive and -negative xenografts in CD-1 nude mice. 8709 scFv-Fc was also tested for competitive binding with the therapeutic anti-EGFR antibody nimotuzumab and for quantifying ratios of EGFR and EGFRvIII deletion mutant. Results: IRDye800CW-labeled 8708 (scFv)2 and 8709 scFv-Fc imaging probes showed high levels of accumulation and good retention in EGFR-positive xenografts, with peak accumulation occurring at 24 and 48 hours post injection, respectively. IRDye680RD-labeled 8709 scFv-Fc did not compete with IRDye800CW-labeled nimotuzumab for EGFR binding as assayed by flow cytometry using an EGFR-positive cell line. IRDye680RD-labeled 8709 scFv-Fc and IRDye800CW-labeled nimotuzumab used in combination were able to determine the ratio of cells expressing EGFR and a deletion mutant EGFRvIII. Conclusion: IRDye800CW-labeled 8708 (scFv)2 and 8709 scFv-Fc had desirable binding affinities, clearance times, and tumor accumulation to be used for imaging in combination with current EGFR targeted therapies. This study highlights the potential for using 8708 (scFv)2 and 8709 scFv-Fc as EGFR diagnostic and therapy monitoring tools.

Therapeutic potential of nimotuzumab PEGylated-maytansine antibody drug conjugates against EGFR positive xenograft.

Nimotuzumab is a humanized anti-epidermal growth factor receptor I (EGFR) monoclonal antibody. We have developed antibody drug conjugates (ADCs) with nimotuzumab conjugated to PEGylated-maytansine (PEG6-DM1). We generated conjugates with low (nimotuzumab-PEG6-DM1-Low: DAR = 3.5) and high (nimotuzumab-PEG6-DM1-High: DAR = 7.3) drug to antibody ratios (DAR). Quality control was performed using UV spectrophotometry, size exclusion HPLC, bioanalyzer, biolayer interferometry (BLI), and flow cytometry in EGFR-positive DLD-1, MDA-MB-468 (high density EGFR), and HT-29 (very low EGFR density) cells. Control antibody drug conjugates were developed using a human anti-maltose binding protein (MBP) antibody. BLI showed that the binding of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High was slightly but significantly affected by conjugation of the drug (nimotuzumab KD 0.89 ± 0.02 nM < nimotuzumab-PEG6-DM1-Low KD 1.94 ± 0.02 nM < nimotuzumab-PEG6-DM1-High KD 3.75 ± 0.03 nM). In vitro cytotoxicity was determined following incubation of cells with the immunoconjugates and IC50 values were determined. Nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High were used to treat EGFR positive KRAS mutant DLD-1 colorectal cancer xenograft. DLD-1 cells were transduced with a red fluorescent protein (iRFP702) to allow the use of near infrared imaging (NIR) for tumor response monitoring. In vitro potency correlated with the number of drugs on antibody, with nimotuzumab-PEG6-DM1-High showing higher activity than nimotuzumab-PEG6-DM1-Low. Three doses (15 mg/kg) of the ADCs prolonged the survival of DLD-1-iRFP-702 tumor bearing mice as monitored by NIR. Nimotuzumab-PEG6-DM1-Low resulted in 4/6 complete cure while nimotuzumab-PEG6-DM1-High resulted in 2/5 complete cure. The novel ADCs were very effective in a colorectal cancer model in vivo.

Preclinical Evaluation of 111In-Labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-Positive Cancer Models.

Epidermal growth factor receptor I (EGFR) is overexpressed in most cancers of epithelial origin. Antibody drug conjugates (ADCs) with PEGylated-maytansine (PEG-DM1) show promise in vitro and in vivo. However, in vivo biodistribution data for ADCs with PEG-DM1 have not been reported. Development of methods to understand the real-time in vivo behavior of these ADCs is needed to move these compounds to the clinic. Methods: Here we have used noninvasive small-animal SPECT/CT imaging and ex vivo biodistribution to understand the in vivo behavior of PEG6-DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG6-DM1. We generated immunoconjugates with low (nimotuzumab-PEG6-DM1-Low) and high (nimotuzumab-PEG6-DM1-High) drug-to-antibody ratios. The drug-to-antibody of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High was 3.5 and 7.3, respectively. Quality control was performed using ultraviolet spectrophotometry, size-exclusion high-performance liquid chromatography, bioanalyzer, biolayer interferometry, and flow cytometry in EGFR-positive DLD-1 cells. These immunoconjugates were conjugated with DOTA and radiolabeled with 111In. The in vitro binding and internalization rates of 111In-nimotuzumab, 111In-nimotuzumab-PEG6-DM1-Low, and 111In-nimotuzumab-PEG6-DM1-High were characterized. Furthermore, the pharmacokinetics, biodistribution, and imaging characteristics were evaluated in normal and DLD-1 tumor-bearing mice. Results: Flow cytometry and biolayer interferometry showed a trend toward decreasing EGFR affinity with increasing number of PEG6-DM1 on the antibody. Despite the lower overall cellular binding of the PEG6-DM1 radioimmunoconjugates, internalization was higher for PEG6-DM1 ADCs than for the non-PEGylated ADC in the following order: 111In-nimotuzumab-PEG6-DM1-High > 111In-nimotuzumab-PEG6-DM1-Low > 111In-nimotuzumab. Nuclear uptake of 111In-nimotuzumab-PEG6-DM1-High was 4.4-fold higher than 111In-nimotuzumab. Pharmacokinetics and biodistribution showed that 111In-nimotuzumab-PEG6-DM1-High had the slowest blood and whole-body clearance rate. Uptake in DLD-1 tumors of 111In-nimotuzumab was similar to 111In-nimotuzumab-PEG6-DM1-Low but was significantly higher than for 111In-nimotuzumab-PEG6-DM1-High. Tumor-to-background ratios for 111In-nimotuzumab and 111In-nimotuzumab-PEG6-DM1-Low were higher than for 111In-nimotuzumab-PEG6-DM1-High. Conclusion: The results show that conjugation of multiple PEG6-DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG6-DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of 111In-nimotuzumab-PEG6-DM1-High, with a slow overall whole-body clearance rate. These data provide insights for evaluating the pharmacokinetics and normal -tissue toxicity and in determining dosing rate of PEGylated ADCs.

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging.

PURPOSE: Construction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system. PROCEDURES: We used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. RESULTS: Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts. CONCLUSIONS: These results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities.

Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts.

In vivo imaging is influenced by the half-life, tissue penetration, biodistribution, and affinity of the imaging probe. Immunoglobulin G (IgG) is composed of discrete domains with known functions, providing a template for engineering antibody fragments with desired imaging properties. Here, we engineered antibody-based imaging probes, consisting of different combinations of antibody domains, labeled them with the near-infrared fluorescent dye IRDye800CW, and evaluated their in vivo imaging properties. Antibody-based imaging probes were based on an anti-HER3 antigen binding fragment (Fab) isolated using phage display. Methods: We constructed six anti-HER3 antibody-based imaging probes: a single chain variable fragment (scFv), Fab, diabody, scFv-CH3, scFv-Fc, and IgG. IRDye800CW-labeled, antibody-based probes were injected into nude mice bearing FaDu xenografts and their distribution to the xenograft, liver, and kidneys was evaluated. Results: These imaging probes bound to recombinant HER3 and to the HER3-positive cell line, FaDu. Small antibody fragments with molecular weight <60 kDa (scFv, diabody, and Fab) accumulated rapidly in the xenograft (maximum accumulation between 2-4 h post injection (hpi)) and cleared primarily through the kidneys. scFv-CH3 (80 kDa) had fast clearance and peaked in the xenograft between 2-3 hpi and cleared from xenograft in a rate comparable to Fab and diabody. IgG and scFv-Fc persisted in the xenografts for up to 72 hpi and distributed mainly to the xenograft and liver. The highest xenograft fluorescence signals were observed with IgG and scFv-Fc imaging probes and persisted for 2-3 days. Conclusion: These results highlight the utility of using antibody fragments to optimize clearance, tumor labeling, and biodistribution properties for developing anti-HER3 probes for image-guided surgery or PET imaging.

99mTc(CO)3+ labeled domain I/II-specific anti-EGFR (scFv)2 antibody fragment for imaging EGFR expression.

Bifunctional chelators (BFCs) are covalently linked to biologically active targeting molecules and radiolabeled with radiometals. Technetium-99 m (99mTc) is the most widely used isotope in nuclear medicine because of its excellent physical properties. The objective of this study was to synthesize and characterize a novel BFC that allows for the labeling of antibodies and antibody fragments using the 99mTc(CO)3+ core which forms a very stable complex with 99mTc in the +1 oxidation sate. This study reports the synthesis of a BFC 1-pyrrolidinyl-2,5-dione-11-(bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino)undecanoic acid (SAAC-CIM NHS ester), and the in vitro and in vivo evaluation of 99mTc(CO)3-SAAC-CIM-DLO6-(scFv)2 (99mTc(CO)3-DLO6-(scFv)2), a domain I/II-specific anti-epidermal growth factor receptor I (anti-EGFR) antibody fragment. The chelator allowed radiolabeling the (scFv)2 antibody fragment in very mild conditions with no significant decrease in binding to EGFR. Radiochemical yields of >50% (radiochemical purity > 95%) of the resulting anti-EGFR (scFv)2 immunoconjugate 99mTc(CO)3-DLO6-(scFv)2 was obtained. The radioimmunoconjugate was stable in histidine challenge experiments with less than 20% transchelation at 24 h after challenge in the presence of a 1500-fold excess of histidine. In vivo biodistribution of 99mTc(CO)3-DLO6-(scFv)2 indicates that the tracer was mainly cleared via renal excretion and to a lesser extent via the hepatobiliary pathway. The microSPECT imaging studies performed in mice confirmed the in vitro affinity results. The 99mTc(CO)3-DLO6-(scFv)2 shows some promising properties and warrants further investigation for imaging EGFR.

89Zr-nimotuzumab for immunoPET imaging of epidermal growth factor receptor I.

RATIONALE: Epidermal growth factor receptor (EGFR) upregulation is associated with enhanced proliferation and drug resistance in a number of cancers. Nimotuzumab is a humanized monoclonal antibody with high affinity for EGFR. The objective of this study was to determine if 89Zr-DFO-nimotuzumab could be suitable for human use as a PET probe for quantifying EGFR in vivo. METHODS: To evaluate the pharmacokinetics, biodistribution, microPET imaging, radiation dosimetry, and normal tissue toxicity in tumor and non-tumor bearing mice of 89Zr-desferoxamine-nimotuzumab (89Zr-DFO-nimotuzumab) of a product prepared under GMP conditions. Nimotuzumab was conjugated to DFO and radiolabeled with 89Zr. 89Zr-DFO-nimotuzumab was characterized by in vitro gel-electrophoresis, biolayer interferometry (BLI) and flow cytometry. 89Zr-DFO-nimotuzumab was evaluated in vivo by microPET and ex vivo by biodistribution in healthy and EGFR-positive tumor bearing mice. RESULTS: Flow cytometry with A431 cells showed no significant difference in the dissociation constant of nimotuzumab (13 ± 2 nM) compared with DFO-nimotuzumab (17 ± 4 nM). PET imaging in mice xenografts showed persistently high tumor uptake with the highest uptake obtained in DLD-1 xenograft (18.3 %IA/cc) at 168 hp.i. The projected human effective dose was low and was 0.184 mSv/MBq (0.679 rem/mCi) in females and 0.205 mSv/MBq (0.757 rem/mCi) in males. There was no apparent normal tissue toxicity as shown by cell blood counts and blood biochemistry analyses at 168-fold and 25-fold excess of the projected human radioactive and mass dose of the agent. CONCLUSION: 89Zr-DFO-nimotuzumab had low organ absorbed dose and effective dose that makes it suitable for potential human use.

Near infrared fluorescence imaging of EGFR expression in vivo using IRDye800CW-nimotuzumab.

Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody that is approved in many countries for the treatment of EGFR-positive cancers. Near infrared (NIR) fluorescent dye-labeled antibodies represent an attractive class of image-guided surgical probes because of their high specificity, tumor uptake, and low dissociation from tumor cells that express the antigen. In this study, we developed a NIR fluorescent dye-labeled nimotuzumab immunoconjugate, IRDye800CW-nimotuzumab, and evaluated in vitro binding with EGFR-positive cells, in vivo tumor uptake by NIR fluorescent imaging, and ex vivo biodistribution. There was no difference in binding between nimotuzumab and IRDye800CW-nimotuzumab to EGFR-positive cells. In mice bearing EGFR-positive xenografts, IRDye800CW-nimotuzumab uptake peaked at 4 days post injection and slowly decreased thereafter with high levels of accumulation still observed at 28 days post injection. In EGFR-positive xenografts, IRDye800CW-nimotuzumab showed more than 2-fold higher uptake in tumors compared to IRDye800CW-cetuximab. In addition, liver uptake of IRDye800CW-nimotuzumab was two-fold lower than cetuximab. The lower liver uptake of IRDye800CW-nimotuzumab could have implications on the selected dose for clinical trials of the immunoconjugate. In summary, this study shows that nimotuzumab is a good candidate for NIR fluorescent imaging and image-guided surgery.

Compounds producing an effective combinatorial regimen for disruption of HIV-1 latency.

Highly active antiretroviral therapy (HAART) has improved the outlook for the HIV epidemic, but does not provide a cure. The proposed "shock-and-kill" strategy is directed at inducing latent HIV reservoirs, which may then be purged via boosted immune response or targeting infected cells. We describe five novel compounds that are capable of reversing HIV latency without affecting the general T-cell activation state. The new compounds exhibit synergy for reactivation of latent provirus with other latency-reversing agents (LRAs), in particular ingenol-3-angelate/PEP005. One compound, designated PH02, was efficient at reactivating viral transcription in several cell lines bearing reporter HIV-1 at different integration sites. Furthermore, it was capable of reversing latency in resting CD4+ T lymphocytes from latently infected aviremic patient cells on HAART, while producing minimal cellular toxicity. The combination of PH02 and PEP005 produces a strong synergistic effect for reactivation, as demonstrated through a quantitative viral outgrowth assay (qVOA), on CD4+ T lymphocytes from HIV-1-infected individuals. We propose that the PH02/PEP005 combination may represent an effective novel treatment for abrogating persistent HIV-1 infection.

HIV Provirus Stably Reproduces Parental Latent and Induced Transcription Phenotypes Regardless of the Chromosomal Integration Site.

UNLABELLED: Understanding the mechanisms of HIV proviral latency is essential for development of a means to eradicate infection and achieve a cure. We have previously described an in vitro latency model that reliably identifies HIV expression phenotypes of infected cells using a dual-fluorescence reporter virus. Our results have demonstrated that ∼50% of infected cells establish latency immediately upon integration of provirus, a phenomenon termed early latency, which appears to occur by mechanisms that are distinct from epigenetic silencing observed with HIV provirus that establishes productive infections. In this study, we have used a mini-dual HIV reporter virus (mdHIV) to compare the long-term stability of provirus produced as early latent or productive infections using Jurkat-Tat T cell clones. Cloned lines bearing mdHIV provirus integrated at different chromosomal locations display unique differences in responsiveness to signaling agonists and chromatin-modifying compounds, and they also produce characteristic expression patterns from the 5' long terminal repeat (LTR) dsRed and internal EIF1α-enhanced green fluorescent protein (EIF1α-eGFP) reporters. Furthermore, reporter expression profiles of single cell sorted subcultures faithfully reproduce expression profiles identical to that of their original parental population, following prolonged growth in culture, without shifting toward expression patterns resembling that of cell subclones at the time of sorting. Comparison of population dispersion coefficient (CV) and mean fluorescence intensity (MFI) of the subcloned lines showed that both untreated and phorbol myristate acetate (PMA)-ionomycin-stimulated cultures produce expression patterns identical to those of their parental lines. These results indicate that HIV provirus expression characteristics are strongly influenced by the epigenetic landscape at the site of chromosomal integration. IMPORTANCE: There is currently considerable interest in development of therapies to eliminate latently infected cells from HIV-infected patients on antiretroviral therapy. One proposed strategy, known as "shock and kill," would involve treatment with therapies capable of inducing expression of latent provirus, with the expectation that the latently infected cells could be killed by a host immune response or virus-induced apoptosis. In clinical trials, histone deacetylase (HDAC) inhibitors were shown to cause reactivation of latent provirus but did not produce a significant effect toward eliminating the latently infected population. Results shown here indicate that integration of HIV provirus at different chromosomal locations produces significant effects on the responsiveness of virus expression to T cell signaling agonists and chromatin-modifying compounds. Given the variety of phenotypes produced by integrated provirus, it is unlikely that any single potential shock-and-kill therapy will be effective toward purging the latently infected population.

An upstream YY1 binding site on the HIV-1 LTR contributes to latent infection.

During HIV-1 infection a population of latently infected cells is established. This population is the major obstacle preventing total eradication of the virus from AIDS patients. HIV-1 latency is thought to arise by various mechanisms including repressive chromatin modifications. Transcription factors such as YY1 have been shown to facilitate repressive chromatin modifications by the recruitment of histone deacetylases. In this study, we identified a novel binding site for YY1 on the HIV-1 LTR, 120 nucleotides upstream of the transcription start site. We show that YY1 can bind to this site in vitro and in vivo and that binding to the LTR is dissociated upon T cell activation. Overexpression of YY1 causes an increase in the proportion of cells that produce latent infections. These observations, in combination with previous results, demonstrate that YY1 plays a prominent role in controlling the establishment and maintenance of latent HIV-1 provirus in unstimulated cells.

The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update.

PhosphoGRID is an online database that curates and houses experimentally verified in vivo phosphorylation sites in the Saccharomyces cerevisiae proteome (www.phosphogrid.org). Phosphosites are annotated with specific protein kinases and/or phosphatases, along with the condition(s) under which the phosphorylation occurs and/or the effects on protein function. We report here an updated data set, including nine additional high-throughput (HTP) mass spectrometry studies. The version 2.0 data set contains information on 20 177 unique phosphorylated residues, representing a 4-fold increase from version 1.0, and includes 1614 unique phosphosites derived from focused low-throughput (LTP) studies. The overlap between HTP and LTP studies represents only ∼3% of the total unique sites, but importantly 45% of sites from LTP studies with defined function were discovered in at least two independent HTP studies. The majority of new phosphosites in this update occur on previously documented proteins, suggesting that coverage of phosphoproteins in the yeast proteome is approaching saturation. We will continue to update the PhosphoGRID data set, with the expectation that the integration of information from LTP and HTP studies will enable the development of predictive models of phosphorylation-based signaling networks. Database URL: http://www.phosphogrid.org/