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Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial m...

Glycoprotein CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of hyaluronic acid to CD44 activates cell signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded as an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific antibody fragment and evaluated it for imaging CD44-positive cancers using PET. Methods: A human single-chain fragment variable (scFv) was generated by phage display, using the extracellular domain of recombinant human CD44. The specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell–expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray. The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG(1)–fragment crystallizable (Fc). The scFv-Fc-CD44 was radiolabeled with (64)Cu and (89)Zr. The purified reagents were injected into athymic nude mice bearing CD44-positive human tumors (MDA-MB-231, breast cancer, triple-negative). Biodistribution studies were performed at different times after injection of [(64)Cu]Cu-NOTA-scFv-Fc-CD44 or [(89)Zr]Zr-DFO-scFv-Fc-CD44. PET/CT imaging was conducted with [(89)Zr]Zr-DFO-scFv-Fc-CD44 on days 1 and 7 after injection and compared with a scFv-Fc control antibody construct targeting glycophorin A. Results: Epitope mapping of the scFv binding site revealed a linear epitope within the extracellular domain of human CD44, capable of blocking binding to native hyaluronic acid. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumor-to-blood ratios for [(89)Zr]Zr-DFO-scFv-Fc-CD44 between days 5 and 7. PET imaging confirmed excellent tumor specificity for [(89)Zr]Zr-DFO-scFv-Fc-CD44 when compared with the control scFv-Fc. Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with (64)Cu or (89)Zr, it demonstrated specific uptake in CD44-expressing MDA-MB-231 tumors. The high tumor uptake (∼56% injected dose/g) warrants clinical investigation of [(89)Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with CD44-positive tumors.

Therapies targeting reductive/oxidative (redox) metabolism hold potential in cancers resistant to chemotherapy and radiation. A redox imaging marker would help identify cancers susceptible to redox-directed therapies. Copper(II)-diacetyl-bis(4-methylthiosemicarbazonato) (Cu-ATSM) is a PET tracer developed for hypoxia imaging that could potentially be used for this purpose. We aimed to demonstrate that Cu-ATSM signal is dependent on cellular redox state, irrespective of hypoxia. Methods: We investigated the relationship between (64)Cu-ATSM signal and redox state in human cervical and colon cancer cells. We altered redox state using drug strategies and single-gene mutations in isocitrate dehydrogenases (IDH1/2). Concentrations of reducing molecules were determined by spectrophotometry and liquid chromatography–mass spectrometry and compared with (64)Cu-ATSM signal in vitro. Mouse models of cervical cancer were used to evaluate the relationship between (64)Cu-ATSM signal and levels of reducing molecules in vivo, as well as to evaluate the change in (64)Cu-ATSM signal after redox-active drug treatment. Results: A correlation exists between baseline (64)Cu-ATSM signal and cellular concentration of glutathione, nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NADH). Altering NADH and NADPH metabolism using drug strategies and IDH1 mutations resulted in significant changes in (64)Cu-ATSM signal under normoxic conditions. Hypoxia likewise changed (64)Cu-ATSM signal, but treatment of hypoxic cells with redox-active drugs resulted in a more dramatic change than hypoxia alone. A significant difference in NADPH was seen between cervical tumor orthotopic implants in vivo, without a corresponding difference in (64)Cu-ATSM signal. After treatment with β-lapachone, there was a change in (64)Cu-ATSM signal in xenograft tumors smaller than 50 mg but not in larger tumors. Conclusion: (64)Cu-ATSM signal reflects redox state, and altering redox state impacts (64)Cu-ATSM metabolism. Our animal data suggest there are other modulating factors in vivo. These findings have implications for the use of (64)Cu-ATSM as a predictive marker for redox therapies, though further in vivo work is needed.

Objective: Waldenstrom Macroglobulinemia (WM) is a rare B-cell malignancy characterized by secretion of immunoglobulin M and cancer infiltration in the bone marrow. Chemokine receptor such as CXCR...

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras(G12D) x p53(−/−) sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8(+) T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

The development of somastatin (SS) peptide analogues for the detection and treatment of neuroendocrine tumors has been successful with the recent FDA approval of 68Ga-DOTA-TATE and 177Lu-DOTA-TATE. The structure of these peptide constructs contains the peptide binding motif that binds to the receptor with high affinity, a chelator to complex the radioactive metal, and a linker between the peptide and chelator. However, these constructs suffer from rapid blood clearance, which limits their tumor uptake. In this study, this design has been further improved by incorporating a modification to control the in vivo pharmacokinetics. Adding a truncated Evans Blue (EB) dye molecule into the construct provides a prolonged half-life in blood as a result of its low micromolar affinity to albumin. We compared 177Lu-DOTA-TATE to the modified 177Lu Evans Blue compound (177Lu-DMEB-TATE), in vitro and in vivo in mice bearing A427-7 xenografts. The tumor uptake of 177Lu-DMEB-TATE was significantly greater than the uptake of 177Lu-DOTA-TATE in the biodistribution and SPECT-imaging studies. The therapeutic effect of the 177Lu-DMEB-TATE construct was superior to the that of the 177Lu-DOTA-TATE construct at the doses evaluated.

To evaluate whether tumor uptake of [89Zr]trastuzumab can distinguish HER2-positive from HER2-negative breast cancer. Women with HER2-positive (n = 34) and HER2-negative (n = 16) breast cancer underwent PET/CT 5 ± 2 days following [89Zr]trastuzumab administration. HER2 status was determined based on immunohistochemistry and/or fluorescence in situ hybridization of primary or metastatic/recurrent tumor. Tumor [89Zr]trastuzumab uptake was assessed qualitatively and semiquantitatively as maximum standardized uptake value (SUVmax), and correlated with HER2 status. Additionally, intrapatient heterogeneity of [89Zr]trastuzumab uptake was evaluated. On a per-patient basis, [89Zr]trastuzumab-PET/CT was positive in 30/34 (88.2%) HER2-positive and negative in 15/16 (93.7%) HER2-negative patients. Considering all lesions, the SUVmax was not significantly different in patients with HER2-positive versus HER2-negative disease (p = 0.06). The same was true of when only hepatic lesions were evaluated (p = 0.42). However, after excluding hepatic lesions, tumor SUVmax was significantly higher in HER2-positive compared to HER2-negative patients (p = 0.003). A cutoff SUVmax of 3.2, determined by ROC analysis, demonstrated positive-predictive value of 83.3% (95% CI 65.3%, 94.4%), sensitivity of 75.8% (57.7%, 88.9%), negative-predictive value of 50% (24.7%, 75.3%), and specificity of 61.5% (95% 31.6%, 86.1%) for differentiating HER2-positive from HER2-negative lesions. There was intrapatient heterogeneity of [89Zr]trastuzumab uptake in 20% of patients with multiple lesions. [89Zr]trastuzumab has the potential to characterize the HER2 status of the complete tumor burden in patients with breast cancer, thus obviating repeat or multiple tissue sampling to assess intrapatient heterogeneity of HER2 status.

Summary Here, we utilized spontaneous models of pancreatic and lung cancer to examine how neoantigenicity shapes tumor immunity and progression. As expected, neoantigen expression during lung adenocarcinoma development leads to T cell-mediated immunity and disease restraint. By contrast, neoantigen expression in pancreatic ductal adenocarcinoma (PDAC) results in exacerbation of a fibro-inflammatory microenvironment that drives disease progression and metastasis. Pathogenic TH17 responses are responsible for this neoantigen-induced tumor progression in PDAC. Underlying these divergent T cell responses in pancreas and lung cancer are differences in infiltrating conventional dendritic cells (cDCs). Overcoming cDC deficiency in early-stage PDAC leads to disease restraint, while restoration of cDC function in advanced PDAC restores tumor-restraining immunity and enhances responsiveness to radiation therapy.

Idiopathic pulmonary fibrosis (IPF) is a progressive, inflammatory lung disease that is monitored clinically by measures of lung function, without effective molecular markers of disease activity or therapeutic efficacy. Lung immune cells active in the pro-fibrotic process include inflammatory monocyte and interstitial macrophages that express the C-C motif chemokine receptor 2 (CCR2). CCR2+ monocyte lung influx is essential for disease phenotypes in models of fibrosis and identified in lungs from subjects with IPF. Here, we show that our peptide-based radiotracer 64Cu-DOTA-ECL1i identifies CCR2+ inflammatory monocytes and interstitial macrophages in multiple preclinical mouse models of lung fibrosis, using positron emission tomography (PET) imaging. Mice with bleomycin-induced fibrosis treated with blocking antibodies to interleukin-1β, a mediator of fibrosis associated with CCR2+ cell inflammation, or with pirfenidone, an approved anti-fibrotic agent, demonstrated decreased CCR2-dependent interstitial macrophage accumulation and reduced 64Cu-DOTA-ECL1i PET uptake, compared to controls. Lung tissues from patients with fibrotic lung disease demonstrated abundant CCR2+ cells surrounding regions of fibrosis, and an ex vivo tissue-binding assay showed correlation between radiotracer localization and CCR2+ cells. In a phase 0/1 clinical study of 64Cu-DOTA-ECL1i PET, healthy volunteers showed little lung uptake, while subjects with pulmonary fibrosis exhibited increased uptake, notably in zones of subpleural fibrosis, reflecting the distribution of CCR2+ cells in the profibrotic niche. These findings support a pathologic role of inflammatory lung monocytes/macrophages in fibrotic lung disease and the translational use of 64Cu-DOTA-ECL1i PET to track CCR2-specific inflammation for image-guided therapy.One Sentence SummaryPET imaging of CCR2+ cells in lung fibrosis identifies a therapeutic response in mouse models and displays a perifibrotic signal in subjects with IPF.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme in the DNA repair process and the target of several FDA-approved inhibitors. Several of these inhibitors have been radiolabeled for non-invasive imaging of PARP-1 expression or targeted radiotherapy of PARP-1 expressing tumors. In particular, derivatives of olaparib and rucaparib, which have reduced trapping potency by PARP-1 compared to talazoparib, have been radiolabeled for these purposes. Here, we report the first radiosynthesis of [18F]talazoparib and its in vitro and in vivo evaluation. Talazoparib (3a”) and its bromo- or iodo-derivatives were synthesized as racemic mixtures (3a, 3b and 3c), and these compounds exhibit high affinity to PARP-1 (Ki for talazoparib (3a”): 0.65 ± 0.07 nM, 3a: 2.37 ± 0.56 nM, 3b: 1.92 ± 0.41 nM, 3c: 1.73 ± 0.43 nM, known PARP-1 inhibitor Olaparib: 1.87 ± 0.10 nM, non-PARP-1 compound Raclopride: &gt, 20,000 nM) in a competitive binding assay using a tritium-labeled PARP-1 radioligand [3H]WC-DZ for screening. [18F]Talazoparib (3a”) was radiosynthesized via a multiple-step procedure with good radiochemical and chiral purities (98%) and high molar activity (28 GBq/μmol). The preliminary biodistribution studies in the murine PC-3 tumor model showed that [18F]talazoparib had a good level of tumor uptake that persisted for over 8 h (3.78 ± 0.55 %ID/gram at 4 h and 4.52 ± 0.32 %ID/gram at 8 h). These studies show the potential for the bromo- and iodo- derivatives for PARP-1 targeted radiotherapy studies using therapeutic radionuclides.

INTRODUCTION: Poly (ADP-ribose) polymerase-1 (PARP-1) plays many roles in prostate cancer (PC), such as mediating DNA damage repair, transcriptional regulation and nuclear hormone receptor signaling. Because of this, PARP-1 has been targeted for therapy in PC, and non-invasive imaging of PARP-1 could help predict which patients are likely to respond to such therapy. Several PARP-1 positron emission tomography (PET) imaging agents have been developed and show promise for imaging PARP-1 expression in breast, brain, and lung cancer in small animals, but not as yet in prostate cancer. [(18)F]WC-DZ-F is an analogue of [(18)F]Fluor Thanatrace (FTT) and [(125)I]KX1, which are well-established PARP-1 ligands for measuring PARP-1 expression. Herein, we evaluated the potential of [(18)F]WC-DZ-F for the imaging PARP-1 expression in PC. METHODS: [(18)F]WC-DZ-F was synthesized by a two-step sequence. [(18)F]WC-DZ-F was evaluated by in vitro uptake studies in PC-3 cells and by in vivo biodistribution and microPET imaging using PC-3 tumor xenografts. Ex vivo autoradiography of PC-3 tumors after microPET imaging was also performed. RESULTS: [(18)F]WC-DZ-F has high, PARP-1-specific uptake in PC-3 cells. In the microPET imaging study, [(18)F]WC-DZ-F accumulated in PC-3 xenograft tumors over 2 h, and the uptake was significantly reduced by blocking with olaparib. PC-3 tumors were clearly visualized in microPET images, and the imaging results were further confirmed by autoradiography of PC-3 tumors ex vivo. In the biodistribution study [(18)F]WC-DZ-F washed out quickly from most tissues within 2 h, except for the liver in which the uptake was not blockable by olaparib. CONCLUSIONS: We synthesized a novel PARP-1 radioligand, [(18)F]WC-DZ-F. The preliminary evaluation of [(18)F]WC-DZ-F indicates that it is a suitable PET imaging agent for measuring PARP-1 expression in prostate cancer and should be applicable to other types of cancers.