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Background: Adoptive cell cancer therapies aim to re-engineer a patient's immune cells to mount an anti-cancer response. Chimeric antigen receptor T and natural killer cells have been engineered and proved successful in treating some cancers; however, the genetic methods for engineering are laborious, expensive, and inefficient and can cause severe toxicities when they over-proliferate., Results: We examined whether the cell-killing capacity of activated T and NK cells could be targeted to cancer cells by anchoring antibodies to their cell surface. Using metabolic glycoengineering to introduce azide moieties to the cellular surface, we covalently attached a dibenzocyclooctyne-modified antibody using the strain-promoted alkyne azide cycloaddition reaction, creating antibody-conjugated T and NK cells. We targeted the immune cells to tumors possessing the xenoantigen, N-glycolyl neuraminic acid GM3 ganglioside, using the 14F7hT antibody. These activated T and NK cells are "armed" with tumour-homing capabilities that specifically lyses antigen-positive cancer cells without off-target toxicities. Moreover, when exposed to target cells, 14F7hT-conjugated T cells that are not preactivated exhibit increased perforin, granzyme, CD69, and CD25 expression and specific cell killing., Conclusions: This research shows the potential for a non-genetic method for redirecting cytotoxic immune cells as a feasible and effective approach for tumor-targeted cell immunotherapy., (© 2024. The Author(s).)

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 Fab 2 fragment of nimotuzumab, by pepsin digestion and labeled it with IRDye800CW to evaluate its optical imaging properties. The Fab 2 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 Fab 2 allow a higher signal to background to be obtained in a shorter time frame, reducing the wait time for imaging after probe infusion., (© 2023. The Author(s).)

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 t 1/2 alpha of 1.5 h and t 1/2 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.

Overexpression of insulin growth factor receptor type 1 (IGF-1R) is observed in many cancers. Antibody drug conjugates (ADCs) with PEGylated maytansine (PEG 6 -DM1) show promise in vitro. We developed PEG 6 -DM1 ADCs with low and high drug to antibody ratios (DAR) using an anti-IGF-1R antibody cixutumumab (IMC-A12). Conjugates with low (cixutumumab-PEG 6 -DM1-Low) and high (cixutumumab-PEG 6 -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 111 In 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 EC 50 was in the low nanomolar range. MicroSPECT/CT imaging and ex vivo biodistribution showed significantly lower tumor uptake of 111 In-cixutumumab-PEG 6 -DM1-High compared to 111 In-cixutumumab-PEG 6 -DM1-Low and 111 In-cixutumumab. Cixutumumab-PEG 6 -DM1-Low significantly prolonged the survival of mice bearing MCF-7/Her18 xenograft compared with cixutumumab, cixutumumab-PEG 6 -DM1-High, or the PBS control group. Cixutumumab-PEG 6 -DM1-Low ADC was more effective. The study highlights the potential utility of cixutumumab-ADCs as theranostics against IGF-1R positive cancers.

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 PEG 6 -DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG 6 -DM1. We generated immunoconjugates with low (nimotuzumab-PEG 6 -DM1-Low) and high (nimotuzumab-PEG 6 -DM1-High) drug-to-antibody ratios. The drug-to-antibody of nimotuzumab-PEG 6 -DM1-Low and nimotuzumab-PEG 6 -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 111 In. The in vitro binding and internalization rates of 111 In-nimotuzumab, 111 In-nimotuzumab-PEG 6 -DM1-Low, and 111 In-nimotuzumab-PEG 6 -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 PEG 6 -DM1 on the antibody. Despite the lower overall cellular binding of the PEG 6 -DM1 radioimmunoconjugates, internalization was higher for PEG 6 -DM1 ADCs than for the non-PEGylated ADC in the following order: 111 In-nimotuzumab-PEG 6 -DM1-High > 111 In-nimotuzumab-PEG 6 -DM1-Low > 111 In-nimotuzumab. Nuclear uptake of 111 In-nimotuzumab-PEG 6 -DM1-High was 4.4-fold higher than 111 In-nimotuzumab. Pharmacokinetics and biodistribution showed that 111 In-nimotuzumab-PEG 6 -DM1-High had the slowest blood and whole-body clearance rate. Uptake in DLD-1 tumors of 111 In-nimotuzumab was similar to 111 In-nimotuzumab-PEG 6 -DM1-Low but was significantly higher than for 111 In-nimotuzumab-PEG 6 -DM1-High. Tumor-to-background ratios for 111 In-nimotuzumab and 111 In-nimotuzumab-PEG 6 -DM1-Low were higher than for 111 In-nimotuzumab-PEG 6 -DM1-High. Conclusion: The results show that conjugation of multiple PEG 6 -DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG 6 -DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of 111 In-nimotuzumab-PEG 6 -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., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

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 EGFR vIII 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 EGFR vIII . 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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.