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.

Next-generation sequencing-guided identification and reconstruction of antibody CDR combinations from phage selection outputs.

Next-generation sequencing (NGS) technologies have been employed in several phage display platforms for analyzing natural and synthetic antibody sequences and for identifying and reconstructing single-chain variable fragments (scFv) and antigen-binding fragments (Fab) not found by conventional ELISA screens. In this work, we developed an NGS-assisted antibody discovery platform by integrating phage-displayed, single-framework, synthetic Fab libraries. Due to limitations in attainable read and amplicon lengths, NGS analysis of Fab libraries and selection outputs is usually restricted to either VH or VL. Since this information alone is not sufficient for high-throughput reconstruction of Fabs, we developed a rapid and simple method for linking and sequencing all diversified CDRs in phage Fab pools. Our method resulted in a reliable and straightforward platform for converting NGS information into Fab clones. We used our NGS-assisted Fab reconstruction method to recover low-frequency rare clones from phage selection outputs. While previous studies chose rare clones for rescue based on their relative frequencies in sequencing outputs, we chose rare clones for reconstruction from less-frequent CDRH3 lengths. In some cases, reconstructed rare clones (frequency ∼0.1%) showed higher affinity and better specificity than high-frequency top clones identified by Sanger sequencing, highlighting the significance of NGS-based approaches in synthetic antibody discovery.

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.

A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system.

BACKGROUND: Advances in antibody engineering provide strategies to construct recombinant antibody-like molecules with modified pharmacokinetic properties. Multermerization is one strategy that has been used to produce antibody-like molecules with two or more antigen binding sites. Multimerization enhances the functional affinity (avidity) and can be used to optimize size and pharmacokinetic properties. Most multimerization strategies involve genetically fusing or non-covalently linking antibody fragments using oligomerization domains. Recent studies have defined guidelines for producing antibody-like molecules with optimal tumor targeting properties, which require intermediates size (70-120 kDa) and bi- or tri-valency. RESULTS: We described a highly modular antibody-engineering platform for rapidly constructing synthetic, trivalent single chain variable fragments (Tri-scFv) using the SpyCatcher/SpyTag protein ligase system. We used this platform to construct an anti-human epidermal growth factor receptor 3 (HER3) Tri-scFv. We generated the anti-HER3 Tri-scFv by genetically fusing a SpyCatcher to the C-terminus of an anti-HER3 scFv and ligating it to a synthetic Tri-SpyTag peptide. The anti-HER3 Tri-scFv bound recombinant HER3 with an apparent KD of 2.67 nM, which is approximately 12 times lower than the KD of monomeric anti-HER3 scFv (31.2 nM). Anti-HER3 Tri-scFv also bound endogenous cell surface expressed HER3 stronger than the monomer anti-HER3 scFv. CONCLUSION: We used the SpyTag/SpyCatcher protein ligase system to ligate anti-HER3 scFv fused to a SpyCatcher at its C-termini to a Tri-SpyTag to construct Tr-scFv. This system allowed the construction of a Tri-scFv with all the scFv antigen-binding sites pointed outwards. The anti-HER3 Tri-scFv bound recombinant and endogenously expressed HER3 with higher functional affinity (avidity) than the monomeric anti-HER3 scFv. The Tri-scFv had the size, valency, and functional affinity that are desired for therapeutic and imaging applications. Use of the SpyTag/SpyCatcher protein ligase system allows Tri-scFvs to be rapidly constructed in a simple, modular manner, which can be easily applied to scFvs or other antibody fragments targeting other antigens.

Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein-Ligase System.

Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio-distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody-like "devices" by using a bottom-up approach to build them from well-behaved discrete recombinant antibody domains or "parts". Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. We used the SpyTag/SpyCatcher protein ligase to join these parts together, thereby creating devices with desired properties based on summed properties of parts and in configurations that cannot be obtained by using genetic engineering. This strategy will create optimized recombinant antibody devices at reduced costs and with shortened development times.

A Single-Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable Complementarity-Determining Regions.

Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage-displayed antigen-binding fragment (Fab) library built on a modified trastuzumab framework with four fixed and two diversified complementarity-determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most commonly observed "canonical" CDR conformation preferred by the modified trastuzumab Fab framework. The library diversity was engineered within CDRs L3 and H3 by use of custom-designed trinucleotide phosphoramidite mixes and biased towards human antibody CDR sequences. The library contained ≈7.6 billion unique Fabs, and >95 % of the library correctly encoded both diversified CDR sequences. We used this library to conduct selections against the human epidermal growth factor receptor-3 extracellular domain (HER3-ECD) and compared the CDR diversity of the naïve library and the anti-HER3 selection pool by use of next-generation sequencing. The most commonly observed CDR combination isolated, named Her3-3, was overexpressed and purified in Fab and immunoglobulin G (IgG) formats. Fab HER3-3 bound to HER3-ECD with a KD value of 2.14 nm and recognized cell-surface HER3. Although HER3-3 IgG bound to cell-surface HER3, it did not inhibit the proliferation of HER3-positive cells. Near-infrared imaging showed that Fab HER3-3 selectively accumulated in a murine HER3-postive xenograft, thus providing a lead for the development of HER3 imaging probes.

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.

Allosteric lariat peptide inhibitors of Abl kinase.

Going against tradition: although most kinase inhibitors are ATP competitive, lariat peptides inhibit Abl kinase activity in an ATP-uncompetitive manner. Further, lariat peptides discriminated Src family kinases, and recognize the allosteric region that lies adjacent to the ATP binding pocket in the Abl kinase catalytic cleft.

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.

Mitochondrial dysfunction reactivates α-fetoprotein expression that drives copper-dependent immunosuppression in mitochondrial disease models.

Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identified a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that resulted in atrophy of the spleen and thymus and caused a peripheral white blood cell deficiency. We demonstrated that the leukopenia was caused by α-fetoprotein, which required copper and the cell surface receptor CCR5 to promote white blood cell death. We further showed that α-fetoprotein expression was upregulated in several cell types upon inhibition of oxidative phosphorylation. Collectively, our data argue that α-fetoprotein may be secreted by bioenergetically stressed tissue to suppress the immune system, an effect that may explain the recurrent or chronic infections that are observed in a subset of mitochondrial diseases or in other disorders with secondary mitochondrial dysfunction.

Short Read-Length Next Generation DNA Sequencing of Antibody CDR Combinations from Phage Selection Outputs.

Phage display is commonly used to select target-binding antibody fragments from large libraries containing billions of unique antibody clones. In practice, selection outputs are often highly heterogenous, making it desirable to recover sequence information from the selected pool. Next Generation DNA Sequencing (NGS) enables the acquisition of sufficient sequencing reads to cover the pool diversity, however read-lengths are typically too short to capture paired antibody complementarity-determining regions (CDRs), which is needed to reconstruct target-binding antibody fragments. Here, we describe a simple in vitro protocol to bring the DNA encoding the antibody CDRs closer together. The final PCR product referred to as a "CDR strip" is suitable for short read-length NGS. In this method, phagemid ssDNA is recovered from antibody phage display biopanning and used as a template to create a heteroduplex with deletions between CDRs of interest. The shorter strand in the heteroduplex is preferentially PCR amplified to generate a CDR strip that is sequenced using NGS. We have also included a bioinformatics approach to analyze the CDR strip populations so that single antibody clones can be created from paired CDR sequences.

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.

Generation of synthetic antibody fragments with optimal complementarity determining region lengths for Notch-1 recognition.

Synthetic antibodies have been engineered against a wide variety of antigens with desirable biophysical, biochemical, and pharmacological properties. Here, we describe the generation and characterization of synthetic antigen-binding fragments (Fabs) against Notch-1. Three single-framework synthetic Fab libraries, named S, F, and modified-F, were screened against the recombinant human Notch-1 extracellular domain using phage display. These libraries were built on a modified trastuzumab framework, containing two or four diversified complementarity-determining regions (CDRs) and different CDR diversity designs. In total, 12 Notch-1 Fabs were generated with 10 different CDRH3 lengths. These Fabs possessed a high affinity for Notch-1 (sub-nM to mid-nM KDapp values) and exhibited different binding profiles (mono-, bi-or tri-specific) toward Notch/Jagged receptors. Importantly, we showed that screening focused diversity libraries, implementing next-generation sequencing approaches, and fine-tuning the CDR length diversity provided improved binding solutions for Notch-1 recognition. These findings have implications for antibody library design and antibody phage display.

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.

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.

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.

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.