Arginine-Selective Bioconjugation Reagent for Effective 18F-labeling of Native Proteins.

Protein-based 18F-PET tracers offer new possibilities in early disease detection and personalized medicine. Their development relies heavily on the availability and effectiveness of 18F-prosthetic groups. We prepared and evaluated a novel arginine-selective prosthetic group, 4-[18F]fluorophenylglyoxal ([18F]FPG). [18F]FPG was radiosynthesized by a one-pot, two-step procedure with a non-decay-corrected (n.d.c.) isolated radiochemical yield (RCY) of 41 ± 8% (n = 10). [18F]FPG constitutes a generic tool for 18F-labeling of various proteins, including human serum albumin (HSA), ubiquitin, interleukin-2, and interleukin-4 in ∼30-60% n.d.c. isolated RCYs. [18F]FPG conjugation with arginine residues is highly selective, even in the presence of a large excess of lysine, cysteine, and histidine. [18F]FPG protein conjugates are able to preserve the binding affinity of the native proteins while also demonstrating excellent in vivo stability. The [18F]FPG-HSA conjugate has prolonged blood retention, which can be applied as a potential blood pool PET imaging agent. Thus, [18F]FPG is an arginine-selective bioconjugation reagent that can be effectively used for the development of 18F-labeled protein radiopharmaceuticals.

Crosstalk between CD64+MHCII+ macrophages and CD4+ T cells drives joint pathology during chikungunya.

Communications between immune cells are essential to ensure appropriate coordination of their activities. Here, we observed the infiltration of activated macrophages into the joint-footpads of chikungunya virus (CHIKV)-infected animals. Large numbers of CD64+MHCII+ and CD64+MHCII- macrophages were present in the joint-footpad, preceded by the recruitment of their CD11b+Ly6C+ inflammatory monocyte precursors. Recruitment and differentiation of these myeloid subsets were dependent on CD4+ T cells and GM-CSF. Transcriptomic and gene ontology analyses of CD64+MHCII+ and CD64+MHCII- macrophages revealed 89 differentially expressed genes, including genes involved in T cell proliferation and differentiation pathways. Depletion of phagocytes, including CD64+MHCII+ macrophages, from CHIKV-infected mice reduced disease pathology, demonstrating that these cells play a pro-inflammatory role in CHIKV infection. Together, these results highlight the synergistic dynamics of immune cell crosstalk in driving CHIKV immunopathogenesis. This study provides new insights in the disease mechanism and offers opportunities for development of novel anti-CHIKV therapeutics.

Imaging Memory T-Cells Stratifies Response to Adjuvant Metformin Combined with αPD-1 Therapy.

The low response rates associated with immune checkpoint inhibitor (ICI) use has led to a surge in research investigating adjuvant combination strategies in an attempt to enhance efficacy. Repurposing existing drugs as adjuvants accelerates the pace of cancer immune therapy research; however, many combinations exacerbate the immunogenic response elicited by ICIs and can lead to adverse immune-related events. Metformin, a widely used type 2 diabetes drug is an ideal candidate to repurpose as it has a good safety profile and studies suggest that metformin can modulate the tumour microenvironment, promoting a favourable environment for T cell activation but has no direct action on T cell activation on its own. In the current study we used PET imaging with [18F]AlF-NOTA-KCNA3P, a radiopharmaceutical specifically targeting KV1.3 the potassium channel over-expressed on active effector memory T-cells, to determine whether combining PD1 with metformin leads to an enhanced immunological memory response in a preclinical colorectal cancer model. Flow cytometry was used to assess which immune cell populations infiltrate the tumours in response to the treatment combination. Imaging with [18F]AlF-NOTA-KCNA3P demonstrated that adjuvant metformin significantly improved anti-PD1 efficacy and led to a robust anti-tumour immunological memory response in a syngeneic colon cancer model through changes in tumour infiltrating effector memory T-cells.

Imaging Effector Memory T-Cells Predicts Response to PD1-Chemotherapy Combinations in Colon Cancer.

Often, patients fail to respond to immune checkpoint inhibitor (ICI) treatment despite favourable biomarker status. Numerous chemotherapeutic agents have been shown to promote tumour immunogenicity when used in conjunction with ICIs; however, little is known about whether such combination therapies lead to a lasting immune response. Given the potential toxicity of ICI-chemotherapy combinations, identification of biomarkers that accurately predict how individuals respond to specific treatment combinations and whether these responses will be long lasting is of paramount importance. In this study, we explored [18F]AlF-NOTA-KCNA3P, a peptide radiopharmaceutical that targets the Kv1.3 potassium channel overexpressed on T-effector memory (TEM) cells as a PET imaging biomarker for lasting immunological memory response. The first-line colon cancer chemotherapies oxaliplatin and 5-fluorouracil were assessed in a syngeneic colon cancer model, either as monotherapies or in combination with PD1, comparing radiopharmaceutical uptake to memory-associated immune cells in the tumour. [18F]AlF-NOTA-KCNA3P reliably separated tumours with immunological memory responses from non-responding tumours and could be used to measure Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo.

Haploinsufficiency of CYP8B1 associates with increased insulin sensitivity in humans.

BACKGROUNDCytochrome P450 family 8 subfamily B member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) that are associated with insulin resistance in humans.METHODSTo determine whether reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays.RESULTSMutation carriers had lower plasma 12α-hydroxylated/non-12α-hydroxylated BA and cholic acid (CA)/chenodeoxycholic acid (CDCA) ratios compared with age-, sex-, and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to mutation-carrier CA/CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We found that reduced CYP8B1 activity associates with increased insulin sensitivity in humans.CONCLUSIONOur findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization.FUNDINGBiomedical Research Council/National Medical Research Council of Singapore.

Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response.

Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer; however, only a relatively small proportion of recipients show a durable response to treatment. Strategies that reliably differentiate durably-responding tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a new Kv1.3 targeting radiopharmaceutical, [18F]AlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic colon cancer model, we compared tumour retention of [18F]AlF-NOTA-KCNA3P with changes in the tumour immune microenvironment determined by flow cytometry. Imaging with [18F]AlF-NOTA-KCNA3P reliably differentiated tumours responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially Kv1.3-expressing CD8+ effector memory T cells.

Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model.

Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [18F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.

PET Imaging of Translocator Protein as a Marker of Malaria-Associated Lung Inflammation.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a severe complication of malaria that occurs despite effective antimalarial treatment. Currently, noninvasive imaging procedures such as chest X-rays are used to assess edema in established MA-ARDS, but earlier detection methods are needed to reduce morbidity and mortality. The early stages of MA-ARDS are characterized by the infiltration of leukocytes, in particular monocytes/macrophages; thus, monitoring of immune infiltrates may provide a useful indicator of early pathology. In this study, Plasmodium berghei ANKA-infected C57BL/6 mice, a rodent model of MA-ARDS, were longitudinally imaged using the 18-kDa translocator protein (TSPO) imaging agent [18F]FEPPA as a marker of macrophage accumulation during the development of pathology and in response to combined artesunate and chloroquine diphosphate (ART+CQ) therapy. [18F]FEPPA uptake was compared to blood parasitemia levels and to levels of pulmonary immune cell infiltrates by using flow cytometry. Infected animals showed rapid increases in lung retention of [18F]FEPPA, correlating well with increases in blood parasitemia and pulmonary accumulation of interstitial inflammatory macrophages and major histocompatibility complex class II (MHC-II)-positive alveolar macrophages. Treatment with ART+CQ abrogated this increase in parasitemia and significantly reduced both lung uptake of [18F]FEPPA and levels of macrophage infiltrates. We conclude that retention of [18F]FEPPA in the lungs is well correlated with changes in blood parasitemia and levels of lung-associated macrophages during disease progression and in response to ART+CQ therapy. With further development, TSPO biomarkers may have the potential to accurately assess the early onset of MA-ARDS.

An Improved Synthesis of N-(4-[18F]Fluorobenzoyl)-Interleukin-2 for the Preclinical PET Imaging of Tumour-Infiltrating T-cells in CT26 and MC38 Colon Cancer Models.

Positron emission tomography (PET) imaging of activated T-cells with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL-2) may be a promising tool for patient management to aid in the assessment of clinical responses to immune therapeutics. Unfortunately, existing radiosynthetic methods are very low yielding due to complex and time-consuming chemical processes. Herein, we report an improved method for the synthesis of [18F]FB-IL-2, which reduces synthesis time and improves radiochemical yield. With this optimized approach, [18F]FB-IL-2 was prepared with a non-decay-corrected radiochemical yield of 3.8 ± 0.7% from [18F]fluoride, 3.8 times higher than previously reported methods. In vitro experiments showed that the radiotracer was stable with good radiochemical purity (>95%), confirmed its identity and showed preferential binding to activated mouse peripheral blood mononuclear cells. Dynamic PET imaging and ex vivo biodistribution studies in naïve Balb/c mice showed organ distribution and kinetics comparable to earlier published data on [18F]FB-IL-2. Significant improvements in the radiochemical manufacture of [18F]FB-IL-2 facilitates access to this promising PET imaging radiopharmaceutical, which may, in turn, provide useful insights into different tumour phenotypes and a greater understanding of the cellular nature and differential immune microenvironments that are critical to understand and develop new treatments for cancers.

Granzyme B PET Imaging of Combined Chemotherapy and Immune Checkpoint Inhibitor Therapy in Colon Cancer.

PURPOSE: Chemotherapeutic adjuvants, such as oxaliplatin (OXA) and 5-fluorouracil (5-FU), that enhance the immune system, are being assessed as strategies to improve durable response rates when used in combination with immune checkpoint inhibitor (ICI) monotherapy in cancer patients. In this study, we explored granzyme B (GZB), released by tumor-associated immune cells, as a PET imaging-based stratification marker for successful combination therapy using a fluorine-18 (18F)-labelled GZB peptide ([18F]AlF-mNOTA-GZP). METHODS: Using the immunocompetent CT26 syngeneic mouse model of colon cancer, we assessed the potential for [18F]AlF-mNOTA-GZP to stratify OXA/5-FU and ICI combination therapy response via GZB PET. In vivo tumor uptake of [18F]AlF-mNOTA-GZP in different treatment arms was quantified by PET, and linked to differences in tumor-associated immune cell populations defined by using multicolour flow cytometry. RESULTS: [18F]AlF-mNOTA-GZP tumor uptake was able to clearly differentiate treatment responders from non-responders when stratified based on changes in tumor volume. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumor-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells and GZB+ NK+ cells. CONCLUSIONS: [18F]AlF-mNOTA-GZP tumor uptake, driven by changes in immune cell populations expressing GZB, is able to stratify tumor response to chemotherapeutics combined with ICIs. Our results show that, while the immunomodulatory mode of action of the chemotherapies may be different, the ultimate mechanism of tumor lysis through release of Granzyme B is an accurate biomarker for treatment response.

Granzyme B PET Imaging Stratifies Immune Checkpoint Inhibitor Response in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a notoriously difficult cancer to treat. The recent development of immune checkpoint inhibitors has revolutionised HCC therapy; however, successful response is only observed in a small percentage of patients. Biomarkers typically used to predict treatment response in other tumour types are ineffective in HCC, which arises in an immune-suppressive environment. However, imaging markers that measure changes in tumour infiltrating immune cells may supply information that can be used to determine which patients are responding to therapy posttreatment. We have evaluated [18F]AlF-mNOTA-GZP, a radiolabeled peptide targeting granzyme B, to stratify response to ICIs in a HEPA 1-tumours, a syngeneic model of HCC. Posttherapy, in vivo tumour retention of [18F]AlF-mNOTA-GZP was correlated to changes in tumour volume and tumour-infiltrating immune cells. [18F]AlF-mNOTA-GZP successfully stratified response to immune checkpoint inhibition in the syngeneic HEPA 1-6 model. FACS indicated significant changes in the immune environment including a decrease in immune suppressive CD4+ T regulatory cells and increases in tumour-associated GZB+ NK+ cells, which correlated well with tumour radiopharmaceutical uptake. While the immune response to ICI therapies differs in HCC compared to many other cancers, [18F]AlF-mNOTA-GZP retention is able to stratify response to ICI therapy associated with tumour infiltrating GZB+ NK+ cells in this complex tumour microenvironment.

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.

Dopamine transporter neuroimaging accurately assesses the maturation of dopamine neurons in a preclinical model of Parkinson's disease.

BACKGROUND: Significant developments in stem cell therapy for Parkinson's disease (PD) have already been achieved; however, methods for reliable assessment of dopamine neuron maturation in vivo are lacking. Establishing the efficacy of new cellular therapies using non-invasive methodologies will be critical for future regulatory approval and application. The current study examines the utility of neuroimaging to characterise the in vivo maturation, innervation and functional dopamine release of transplanted human embryonic stem cell-derived midbrain dopaminergic neurons (hESC-mDAs) in a preclinical model of PD. METHODS: Female NIH RNu rats received a unilateral stereotaxic injection of 6-OHDA into the left medial forebrain bundle to create the PD lesion. hESC-mDA cell and sham transplantations were carried out 1 month post-lesion, with treated animals receiving approximately 4 × 105 cells per transplantation. Behavioural analysis, [18F]FBCTT and [18F]fallypride microPET/CT, was conducted at 1, 3 and 6 months post-transplantation and compared with histological characterisation at 6 months. RESULTS: PET imaging revealed transplant survival and maturation into functional dopaminergic neurons. [18F]FBCTT-PET/CT dopamine transporter (DAT) imaging demonstrated pre-synaptic restoration and [18F]fallypride-PET/CT indicated functional dopamine release, whilst amphetamine-induced rotation showed significant behavioural recovery. Moreover, histology revealed that the grafted cells matured differently in vivo producing high- and low-tyrosine hydroxylase (TH) expressing cohorts, and only [18F]FBCTT uptake was well correlated with differentiation. CONCLUSIONS: This study provides further evidence for the value of in vivo functional imaging for the assessment of cell therapies and highlights the utility of DAT imaging for the determination of early post-transplant cell maturation and differentiation of hESC-mDAs.

Longitudinal [18F]FB-IL-2 PET Imaging to Assess the Immunopathogenicity of O'nyong-nyong Virus Infection.

O'nyong-nyong virus (ONNV) is an arthritogenic alphavirus that caused two large epidemics in 1959 and 1996, affecting millions of people in Africa. More recently, sero-surveillance of healthy blood donors conducted in 2019 revealed high rates of unreported ONNV infection in Uganda. Due to similar clinical symptoms with other endemic mosquito-borne pathogens in the region, including chikungunya virus, dengue virus and malaria, ONNV infections are often un- or misdiagnosed. Elucidating the immunopathogenic factors of this re-emerging arbovirus is critical with the expanding geographic distribution of competent vectors. This study reports the establishment of an immune competent C57BL6/J mouse model to mechanistically characterize ONNV infection and assess potential treatment efficacy. This mouse model successfully recapitulated arthralgia and viremia profiles seen in ONNV patients. Furthermore, longitudinal in-vivo PET imaging with [18F]FB-IL-2 (CD25+CD4+ binding probe) and histopathological assessment in this model demonstrated the pathogenic role of CD4+ T cells in driving joint pathology. Concordantly, in vivo CD4+ T cell depletion, or suppression with fingolimod, an FDA-approved immunomodulating drug, abrogated CD4+ T cell-mediated disease. This study demonstrates the importance of this immune competent ONNV model for future studies on factors influencing disease pathogenesis, which could shape the discovery of novel therapeutic strategies for arthritogenic alphaviruses.

Examining Immunotherapy Response Using Multiple Radiotracers.

PURPOSE: Cancer immunotherapy has shown huge potential in the fight against cancer, but only a small proportion of patients respond successfully to treatment. Non-invasive methods to stratify responders from non-responders are critically important as immune therapies are often associated with immune-related side effects. Currently, conventional clinical imaging modalities do not provide a useful measure of immune therapy efficacy. Sensitive imaging biomarkers that provide information about the tumoural microenvironment may provide useful insights allowing for improved patient management. PROCEDURES: We have assessed the ability of a number of radiopharmaceuticals to non-invasively measure different aspects of the tumour microenvironment and correlated tumour uptake to immune therapy response in a syngeneic model of colon cancer, CT26-WT. Four radiopharmaceuticals, [18F]FDG (a glucose analogue), [18F]FEPPA (a marker for macrophage activation), [18F]FB-IL2 (a marker for CD25+ cells) and [68Ga] Ga-mNOTA-GZP (a marker for granzyme B, the serine protease downstream effector of cytotoxic T cells), were assessed as potential biomarkers to help stratify response to PD-1 monotherapy or combined anti-PD1 and CLTA4 therapy in vivo correlating tumour uptake with changes in tumour-associated immune cell populations. RESULTS: [18F]FDG, [18F]FEPPA and [18F]FB-IL2 (a marker for CD25+ cells) showed limited ability to determine therapy response and showed little correlation to tumour-associated immune cell changes. However, [68Ga] Ga-mNOTA-GZP showed good predictive ability and correlated well with changes in tumour-associated T cells, especially CD8+ T cells. CONCLUSIONS: [68Ga]Ga-mNOTA-GZP uptake correlates well with changes in CD8+ T cell populations supporting continued development of granzyme B-based imaging agents for stratification of response to immunotherapy. Early assessment of immunotherapy efficacy with [68Ga]Ga-mNOTA-GZP may allow for the reduction of unnecessary side effects while significantly improving patient management.

Author Correction: Lung endothelial cell antigen cross-presentation to CD8+T cells drives malaria-associated lung injury.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lung endothelial cell antigen cross-presentation to CD8+T cells drives malaria-associated lung injury.

Malaria-associated acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are life-threatening manifestations of severe malaria infections. The pathogenic mechanisms that lead to respiratory complications, such as vascular leakage, remain unclear. Here, we confirm that depleting CD8+T cells with anti-CD8ß antibodies in C57BL/6 mice infected with P. berghei ANKA (PbA) prevent pulmonary vascular leakage. When we transfer activated parasite-specific CD8+T cells into PbA-infected TCRß-/- mice (devoid of all T-cell populations), pulmonary vascular leakage recapitulates. Additionally, we demonstrate that PbA-infected erythrocyte accumulation leads to lung endothelial cell cross-presentation of parasite antigen to CD8+T cells in an IFNγ-dependent manner. In conclusion, pulmonary vascular damage in ALI is a consequence of IFNγ-activated lung endothelial cells capturing, processing, and cross-presenting malaria parasite antigen to specific CD8+T cells induced during infection. The mechanistic understanding of the immunopathogenesis in malaria-associated ARDS and ALI provide the basis for development of adjunct treatments.

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.

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.

111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model.

Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.