Genomic Engineering of Oral Keratinocytes to Establish In Vitro Oral Potentially Malignant Disease Models as a Platform for Treatment Investigation.

Precancerous cells in the oral cavity may appear as oral potentially malignant disorders, but they may also present as dysplasia without visual manifestation in tumor-adjacent tissue. As it is currently not possible to prevent the malignant transformation of these oral precancers, new treatments are urgently awaited. Here, we generated precancer culture models using a previously established method for the generation of oral keratinocyte cultures and incorporated CRISPR/Cas9 editing. The generated cell lines were used to investigate the efficacy of a set of small molecule inhibitors. Tumor-adjacent mucosa and oral leukoplakia biopsies were cultured and genetically characterized. Mutations were introduced in CDKN2A and TP53 using CRISPR/Cas9 and combined with the ectopic activation of telomerase to generate cell lines with prolonged proliferation. The method was tested in normal oral keratinocytes and tumor-adjacent biopsies and subsequently applied to a large set of oral leukoplakia biopsies. Finally, a subset of the immortalized cell lines was used to assess the efficacy of a set of small molecule inhibitors. Culturing and genomic engineering was highly efficient for normal and tumor-adjacent oral keratinocytes, but success rates in oral leukoplakia were remarkably low. Knock-out of CDKN2A in combination with either the activation of telomerase or knock-out of TP53 seemed a prerequisite for immortalization. Prolonged culturing was accompanied by additional genetic aberrations in these cultures. The generated cell lines were more sensitive than normal keratinocytes to small molecule inhibitors of previously identified targets. In conclusion, while very effective for normal keratinocytes and tumor-adjacent biopsies, the success rate of oral leukoplakia cell culturing methods was very low. Genomic engineering enabled the prolonged culturing of OL-derived keratinocytes but was associated with acquired genetic changes. Further studies are required to assess to what extent the immortalized cultures faithfully represent characteristics of the cells in vivo.

Pharmacodynamics and biodistribution of [195mPt]cisplatin(CISSPECT®) in head and neck squamous cell carcinoma.

BACKGROUND: Cisplatin- based chemoradiotherapy is a crucial pillar in the treatment of HNSCC. The use of cisplatin comes with high toxicity rates as 35% of patients cannot sustain the planned dose while response is unpredictable. Unfortunately, there are no clinically applicable biomarkers to predict response. Based on the association of response with the number of DNA adducts and the involved molecular pathway to resolve cisplatin-induced DNA crosslinks in HNSCC, [195mPt]cisplatin (CISSPECT®) might have potential to monitor drug uptake and retention before treatment, and predict cisplatin response. The aim of this study is to investigate this concept by analyzing uptake, retention and biodistribution of [195mPt]cisplatin between known cisplatin-sensitive (VU-SCC-1131) and -resistant (VU-SCC-OE) HNSCC cell lines in vitro and xenografted in mice in vivo. RESULTS: By a variety of experiments in vitro, including cell cycle analyses, and in vivo, the sensitivity of cell line VU-SCC-1131 and resistance of cell line VU-SCC-OE for cisplatin was demonstrated. VU-SCC-OE was able to accumulate more [195mPt]cisplatin in the DNA, and showed an increased capability to repair [195mPt]cisplatin crosslinks compared to VU-SCC-1131. Notably, DNA binding of cisplatin increased even when cisplatin was removed from the medium, likely from intracellular sources. In vivo, [195mPt]cisplatin showed a rapid biodistribution to the large organs such as the liver, with no differences between intravenous and intraperitoneal administration. Most circulating [195mPt]cisplatin was cleared by renal filtration, and accumulation in kidney and liver remained high. Uptake in xenografts was rapid (blood:tumor ratio; 1:1) and highest after 1 h, while decreasing after 6 h in line with the concentration in the blood. Remarkably, there was no significant difference in uptake or retention between xenografts of the cisplatin-sensitive and -resistant cell line. CONCLUSION: VU-SCC-1131 with a known FA deficiency and VU-SCC-OE displayed a significant difference in sensitivity to and recovery from cisplatin treatment, due to S-phase problems in VU-SCC-1131 at low doses, in line with the genetic defect. Using Pt-195m radioactivity analysis, we demonstrated the limited capability of cisplatin crosslink repair in VU-SCC-1131. Unexpectedly, we were not able to translate these findings to a mouse model for sensitivity prediction based on the biodistribution in the tumor, most likely as other factors such as influx counterbalanced repair. These data do not support response prediction by [195mPt]cisplatin, and applications to predict the toxic side-effects of cisplatin and to tailor dosing schemes seem more feasible.

An Antibody-Drug Conjugate Directed to Tissue Factor Shows Preclinical Antitumor Activity in Head and Neck Cancer as a Single Agent and in Combination with Chemoradiotherapy.

Head and neck squamous cell carcinoma (HNSCC) is a solid tumor type that arises in the squamous epithelial cells lining the mucosal surfaces of the upper aerodigestive tract. Long-term survival of patients with advanced disease stage remains disappointing with current treatment options. We show that tissue factor is abundantly expressed on patient-derived HNSCC cell lines, xenograft tumor material, and tumor biopsies from patients with HNSCC. Tisotumab vedotin (TV) is an antibody-drug conjugate (ADC) directed to tissue factor, a protein expressed in many solid tumors. HNSCC cells and xenograft tumors were efficiently eliminated in vitro and in vivo with TV-monotherapy compared with treatment with a control antibody conjugated to monomethyl auristatin E (MMAE). Antitumor activity of TV was also tested in vivo in combination with chemoradiotherapy, standard of care for patients with advanced stage HNSCC tumors outside the oral cavity. Preclinical studies showed that by adding TV to chemoradiotherapy, survival was markedly improved, and TV, not radiotherapy or chemotherapy, was the main driver of antitumor activity. Interestingly, TV-induced cell death in xenograft tumors showed an influx of macrophages indicative of a potential immune-mediated mode-of-action. In conclusion, on the basis of these preclinical data, TV may be a novel treatment modality for patients suffering from head and neck cancer and is hypothesized to improve efficacy of chemoradiotherapy. SIGNIFICANCE: This work shows preclinical in vitro and in vivo antitumor activity of the antibody-drug conjugate Tisotumab vedotin in head and neck cancer models, and enhanced activity in combination with chemoradiotherapy, supporting further clinical development for this cancer type.

NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

Targeting the cell cycle in head and neck cancer by Chk1 inhibition: a novel concept of bimodal cell death.

Head and neck squamous cell carcinomas (HNSCCs) coincide with poor survival rates. The lack of driver oncogenes complicates the development of targeted treatments for HNSCC. Here, we follow-up on two previous genome-wide RNA and microRNA interference screens in HNSCC to cross-examine tumor-specific lethality by targeting ATM, ATR, CHEK1, or CHEK2. Our results uncover CHEK1 as the most promising target for HNSCC. CHEK1 expression is essential across a panel of HNSCC cell lines but redundant for growth and survival of untransformed oral keratinocytes and fibroblasts. LY2603618 (Rabusertib), which specifically targets Chk1 kinase, kills HNSCC cells effectively and specifically. Our findings show that HNSCC cells depend on Chk1-mediated signaling to progress through S-phase successfully. Chk1 inhibition coincides with stalled DNA replication, replication fork collapses, and accumulation of DNA damage. We further show that Chk1 inhibition leads to bimodal HNSCC cell killing. In the most sensitive cell lines, apoptosis is induced in S-phase, whereas more resistant cell lines manage to bypass replication-associated apoptosis, but accumulate chromosomal breaks that become lethal in subsequent mitosis. Interestingly, CDK1 expression correlates with treatment outcome. Moreover, sensitivity to Chk1 inhibition requires functional CDK1 and CDK4/6 to drive cell cycle progression, arguing against combining Chk1 inhibitors with CDK inhibitors. In contrast, Wee1 inhibitor Adavosertib progresses the cell cycle and thereby increases lethality to Chk1 inhibition in HNSCC cell lines. We conclude that Chk1 has become a key molecule in HNSCC cell cycle regulation and a very promising therapeutic target. Chk1 inhibition leads to S-phase apoptosis or death in mitosis. We provide a potential efficacy biomarker and combination therapy to follow-up in clinical setting.

Establishment and Genetic Landscape of Precancer Cell Model Systems from the Head and Neck Mucosal Lining.

Head and neck squamous cell carcinomas (HNSCC) develop in fields of genetically altered cells. These fields are often dysplastic and a subset can be recognized as (erythro)leukoplakia, but most are macroscopically invisible. There is a lack of adequate treatment options to eradicate these fields, whereas they underlie the development of primary tumors as well as part of the local relapses. Unfortunately, there are almost no representative cellular models available to identify suitable treatment options. To this end, clinical biopsy specimens (n = 98) were cultured from normal appearing mucosa of the surgical margins of patients with primary HNSCCs (n = 32) to generate precancer cell culture models. This collection was extended with six previously established precancer cell cultures. Genetic analysis was performed on cultures with an extended life span (≥20 population doublings), the previously established cultures, and some randomly selected cultures. In total, cancer-associated changes were detected in 18 out of 34 (53%) cultures analyzed, which appeared to be independent of life span. A variety of genetic changes were identified, including somatic mutations as well as chromosomal copy-number aberrations (CNA). Loss of CDKN2A/p16Ink4A and mutations in TP53/p53 were most prominent. Remarkably, in some of these precancer cell cultures only chromosomal CNAs were detected, and none of the frequently occurring driver mutations. IMPLICATIONS: The precancer cell cultures, characterized herein, form a representative collection of field models that can be exploited to identify and validate new therapeutic strategies to prevent primary HNSCCs and local relapses.

Targeting PLK1 as a novel chemopreventive approach to eradicate preneoplastic mucosal changes in the head and neck.

Head and neck squamous cell carcinomas (HNSCC) and local relapses thereof develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, are frequently dysplastic and occasionally macroscopically visible. Currently, no adequate treatment options exist to prevent tumor development. Array-based screening with a panel of tumor-lethal small interfering RNAs (siRNAs) identified Polo-like kinase 1 (PLK1) as essential for survival of preneoplastic cells. Inhibition of PLK1 caused cell death of preneoplastic and HNSCC cells, while primary cells were hardly affected. Both siRNAs and small molecule inhibitors caused a strong G2/M cell cycle arrest accompanied by formation of monopolar spindles. In a xenografted mouse model PLK1 caused a significant tumor growth delay and cures, while chemoradiation had no effect. Thus, PLK1 seems to be a promising target for chemopreventive treatment of preneoplastic cells, and could be applied to prevent HNSCC and local relapses.

A comparative PET imaging study with the reversible and irreversible EGFR tyrosine kinase inhibitors [(11)C]erlotinib and [(18)F]afatinib in lung cancer-bearing mice.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have experienced a tremendous boost in the last decade, where more than 15 small molecule TKIs have been approved by the FDA. Unfortunately, despite their promising clinical successes, a large portion of patients remain unresponsive to these targeted drugs. For non-small cell lung cancer (NSCLC), the effectiveness of TKIs is dependent on the mutational status of epidermal growth factor receptor (EGFR). The exon 19 deletion as well as the L858R point mutation lead to excellent sensitivity to TKIs such as erlotinib and gefitinib; however, despite initial good response, most patients invariably develop resistance against these first-generation reversible TKIs, e.g., via T790M point mutation. Second-generation TKIs that irreversibly bind to EGFR wild-type and mutant isoforms have therefore been developed and one of these candidates, afatinib, has now reached the market. Whether irreversible TKIs differ from reversible TKIs in their in vivo tumor-targeting properties is, however, not known and is the subject of the present study. METHODS: Erlotinib was labeled with carbon-11 and afatinib with fluorine-18 without modifying the structure of these compounds. A preclinical positron emission tomography (PET) study was performed in mice bearing NSCLC xenografts with a representative panel of mutations: an EGFR-WT xenograft cell line (A549), an acquired treatment-resistant L858R/T790M mutant (H1975), and a treatment-sensitive exon 19 deleted mutant (HCC827). PET imaging was performed in these xenografts with both tracers. Additionally, the effect of drug efflux transporter permeability glycoprotein (P-gp) on the tumor uptake of tracers was explored by therapeutic blocking with tariquidar. RESULTS: Both tracers only demonstrated selective tumor uptake in the HCC827 xenograft line (tumor-to-background ratio, [(11)C]erlotinib 1.9 ± 0.5 and [(18)F]afatinib 2.3 ± 0.4), thereby showing the ability to distinguish sensitizing mutations in vivo. No major differences were observed in the kinetics of the reversible and the irreversible tracers in each of the xenograft models. Under P-gp blocking conditions, no significant changes in tumor-to-background ratio were observed; however, [(18)F]afatinib demonstrated better tumor retention in all xenograft models. CONCLUSIONS: TKI-PET provides a method to image sensitizing mutations and can be a valuable tool to compare the distinguished targeting properties of TKIs in vivo.

Genome-wide siRNA Screen Identifies the Radiosensitizing Effect of Downregulation of MASTL and FOXM1 in NSCLC.

Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non-small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle-related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2-M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy.

Development of novel ADCs: conjugation of tubulysin analogues to trastuzumab monitored by dual radiolabeling.

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug conjugates (ADC). For full control over drug-antibody ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting, a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 monoclonal antibody (mAb) trastuzumab, are radiolabeled. (131)I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 > 100 nmol/L) and the potent TUB-OMOM (IC50, ~1 nmol/L), and their direct covalent conjugation to (89)Zr-trastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% ± 5% with radiochemical purity of more than 99.5%. Conjugation of (131)I-tubulysin-NHS esters to (89)Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor-targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM-trastuzumab (DAR 4) showed dose-dependent antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment.

Development of [18F]afatinib as new TKI-PET tracer for EGFR positive tumors.

INTRODUCTION: Afatinib is an irreversible ErbB family blocker that was approved for the treatment of EGFR mutated non-small cell lung cancer in 2013. Positron emission tomography (PET) with fluorine-18 labeled afatinib provides a means to obtain improved understanding of afatinib tumor disposition in vivo. PET imaging with [(18)F]afatinib may also provide a method to select treatment responsive patients. The aim of this study was to label afatinib with fluorine-18 and evaluate its potential as TKI-PET tracer in tumor bearing mice. METHODS: A radiochemically novel coupling, using peptide coupling reagent BOP, was explored and optimized to synthesize [(18)F]afatinib, followed by a metabolite analysis and biodistribution studies in two clinically relevant lung cancer cell lines, xenografted in nude mice. RESULTS: A reliable [(18)F]afatinib radiosynthesis was developed and the tracer could be produced in yields of 17.0 ± 2.5% calculated from [(18)F]F(-) and >98% purity. The identity of the product was confirmed by co-injection on HPLC with non-labeled afatinib. Metabolite analysis revealed a moderate rate of metabolism, with >80% intact tracer in plasma at 45 min p.i. Biodistribution studies revealed rapid tumor accumulation and good retention for a period of at least 2 hours, while background tissues showed rapid clearance of the tracer. CONCLUSION: We have developed a method to synthesize [(18)F]afatinib and related fluorine-18 labeled 4-anilinoquinazolines. [(18)F]Afatinib showed good stability in vivo, justifying further evaluation as a TKI-PET tracer.

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: Prelude to Phase 1 clinical studies.

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with (89)Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake.   Studies with (89)Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44(+) or CD44(-)) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44(+), resp), PL45 (CD44(+), non-resp) and HepG2 (CD44(-), non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose. At 1, 2, 3, and 6 days after injection, (89)Zr-RG7356 uptake in MDA-MB-231 (CD44(+), resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44(-), non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of (89)Zr-RG7356 was similar in MDA-MB-231 (CD44(+), resp) and PL45 (CD44(+), non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. (89)Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356. (89)Zr-RG7356 selectively targets CD44(+) responsive and non-responsive tumors in mice and CD44(+) tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44(+) tumors may not be sufficient in itself to drive an anti-tumor response.

Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo.

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced ß-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Inert coupling of IRDye800CW and zirconium-89 to monoclonal antibodies for single- or dual-mode fluorescence and PET imaging.

IRDye800CW and zirconium-89 ((89)Zr) have very attractive properties for optical imaging and positron emission tomography (PET) imaging, respectively. Here we describe a procedure for dual labeling of mAbs with IRDye800CW and (89)Zr in a current good manufacturing practice (cGMP)-compliant way. IRDye800CW and (89)Zr are coupled inertly, without impairment of immunoreactivity and pharmacokinetics of the mAb. Organ and whole-body distribution of the final product can be assessed by optical and PET imaging, respectively. For this purpose, a minimal amount of the chelate N-succinyldesferrioxamine (N-sucDf) is first conjugated to the mAb. Next, N-sucDf-mAb is conjugated with IRDye800CW, after which the N-sucDf-mAb-IRDye800CW is labeled with (89)Zr. After each of these three steps, the product is purified by gel filtration. The sequence of this process avoids unnecessary radiation exposure to personnel and takes about 5 h. The process can be scaled up by the production of large batches of premodified mAbs that can be dispensed and stored until they are labeled with (89)Zr.

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties.

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

[¹¹C]Sorafenib: radiosynthesis and preclinical evaluation in tumor-bearing mice of a new TKI-PET tracer.

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) like sorafenib are important anticancer therapeutics with thus far limited treatment response rates in cancer patients. Positron emission tomography (PET) could provide the means for selection of patients who might benefit from TKI treatment, if suitable PET tracers would be available. The aim of this study was to radiolabel sorafenib (1) with carbon-11 and to evaluate its potential as TKI-PET tracer in vivo. METHODS: Synthetic methods were developed in which sorafenib was labeled at two different positions, followed by a metabolite analysis in rats and a PET imaging study in tumor-bearing mice. RESULTS: [methyl-(11)C]-1 and [urea-(11)C]-1 were synthesized in yields of 59% and 53%, respectively, with a purity of >99%. The identity of the products was confirmed by coinjection on HPLC with reference sorafenib. In an in vivo metabolite analysis [(11)C]sorafenib proved to be stable. The percentage of intact product in blood-plasma after 45 min was 90% for [methyl-(11)C]-1 and 96% for [urea-(11)C]-1, respectively. Due to the more reliable synthesis, further research regarding PET imaging was performed with [methyl-(11)C]-1 in nude mice bearing FaDu (head and neck cancer), MDA-MB-231 (breast cancer) or RXF393 (renal cancer) xenografts. Highest tracer accumulation at a level of 2.52 ± 0.33%ID/g was observed in RXF393, a xenograft line extensively expressing the sorafenib target antigen Raf-1 as assessed by immunohistochemistry. CONCLUSION: In conclusion, we have synthesized [(11)C]sorafenib as PET tracer, which is stable in vivo and has the capability to be used as PET tracer for imaging in tumor-bearing mice.

Functional genetic screens identify genes essential for tumor cell survival in head and neck and lung cancer.

PURPOSE: Despite continuous improvement of treatment regimes, the mortality rates for non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) remain disappointingly high and novel anticancer agents are urgently awaited. EXPERIMENTAL DESIGN: We combined the data from genome-wide siRNA screens on tumor cell lethality in a lung and a head and neck cancer cell line. RESULTS: We identified 71 target genes that seem essential for the survival of both cancer types. We identified a cluster of 20 genes that play an important role during G2-M phase transition, underlining the importance of this cell-cycle checkpoint for tumor cell survival. Five genes from this cluster (CKAP5, KPNB1, RAN, TPX2, and KIF11) were evaluated in more detail and have been shown to be essential for tumor cell survival in both tumor types, but most particularly in HNSCC. Phenotypes that were observed following siRNA-mediated knockdown of KIF11 (kinesin family member 11) were reproduced by inhibition of KIF11 using the small-molecule inhibitor ispinesib (SB-715992). We showed that ispinesib induces a G2 arrest, causes aberrant chromosome segregation, and induces cell death in HNSCC in vitro, whereas primary keratinocytes are less sensitive. Furthermore, growth of HNSCC cells engrafted in immunodeficient mice was significantly inhibited after ispinesib treatment. CONCLUSION: This study identified a wide array of druggable genes for both lung and head and neck cancer. In particular, multiple genes involved in the G2-M checkpoint were shown to be essential for tumor cell survival, indicating their potential as anticancer targets.

Rapid visualization of human tumor xenografts through optical imaging with a near-infrared fluorescent anti-epidermal growth factor receptor nanobody.

Given that overexpression of the epidermal growth factor receptor (EGFR) is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

Nanobodies targeting the hepatocyte growth factor: potential new drugs for molecular cancer therapy.

Hepatocyte growth factor (HGF) and its receptor c-Met are associated with increased aggressiveness of tumors and poor prognostic outcome of patients with cancer. Here, we report the development and characterization of therapeutic anti-HGF (αHGF)-Nanobodies and their potential for positron emission tomographic (PET) imaging to assess HGF expression in vivo. Two αHGF-Nanobodies designated 1E2 and 6E10 were identified, characterized, and molecularly fused to an albumin-binding Nanobody unit (Alb8) to obtain serum half-life extension. The resulting Nanobody formats were radiolabeled with the positron emitter zirconium-89 ((89)Zr, t(1/2;) = 78 hours), administered to nude mice bearing U87 MG glioblastoma xenografts, and their biodistribution was assessed. In addition, their therapeutic effect was evaluated in the same animal model at doses of 10, 30, or 100 µg per mouse. The (89)Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showed decreased blood levels of 12.6%ID/g ± 0.6%ID/g, 7.2%ID/g ± 1.0%ID/g, 3.4%ID/g ± 0.3%ID/g, and 0.3%ID/g ± 0.1%ID/g at 1, 2, 3, and 7 days after injection, whereas tumor uptake levels remained relatively stable at these time points: 7.8%ID/g ± 1.1%ID/g, 8.9%ID/g ± 1.0%ID/g, 8.7%ID/g ± 1.5%ID/g, and 7.2%ID/g ±1.6%ID/g. Uptake in normal tissues was lower than in tumor, except for kidneys. In a therapy study, all Nanobody-treated mice showed tumor growth delay compared with the control saline group. In the 100-µg group, four of six mice were cured after treatment with 1E2-Alb8 and 73 days follow-up, and three of six mice when treated with 6E10-Alb8. These results provide evidence that Nanobodies 1E2-Alb8 and 6E10-Alb8 have potential for therapy and PET imaging of HGF-expressing tumors.