In vivo Assessment of the Impact of Molecular Weight on Constructs of 68Ga-DOTA-Manocept in a Syngeneic Mouse Tumor Model.

PURPOSE: Manocept™ constructs are mannosylated amine dextrans (MADs) that bind with high affinity to the mannose receptor, CD206. Tumor-associated macrophages (TAMs) are the most numerous immune cells in the tumor microenvironment and a recognized target for tumor imaging and cancer immunotherapies. Most TAMs express CD206, suggesting utility of MADs to deliver imaging moieties or therapeutics to TAMs. The liver Kupffer cells also express CD206, making them an off-target localization site when targeting CD206 on TAMs. We evaluated TAM targeting strategies using two novel MADs differing in molecular weight in a syngeneic mouse tumor model to determine how varying MAD molecular weights would impact tumor localization. Increased mass dose of the non-labeled construct or a higher molecular weight (HMW) construct were also used to block liver localization and enhance tumor to liver ratios. PROCEDURES: Two MADs, 8.7 kDa and 22.6 kDa modified with DOTA chelators, were synthesized and radiolabeled with 68Ga. A HMW MAD (300 kDa) was also synthesized as a competitive blocking agent for Kupffer cell localization. Balb/c mice, with and without CT26 tumors, underwent dynamic PET imaging for 90 min followed by biodistribution analyses in selected tissues. RESULTS: The new constructs were readily synthesized and labeled with 68Ga with ≥ 95% radiochemical purity in 15 min at 65 °C. When injected at doses of 0.57 nmol, the 8.7 kDa MAD provided 7-fold higher 68Ga tumor uptake compared to the 22.6 kDa MAD (2.87 ± 0.73%ID/g vs. 0.41 ± 0.02%ID/g). Studies with increased mass of unlabeled competitors showed reduced liver localization of the [68Ga]MAD-8.7 to varying degrees without significant reductions in tumor localization, resulting in enhanced tumor to liver signal ratios. CONCLUSION: Novel [68Ga]Manocept constructs were synthesized and studied in in vivo applications, showing that the smaller MAD localized to CT26 tumors more effectively than the larger MAD and that the unlabeled HMW construct could selectively block liver binding of [68Ga]MAD-8.7 without diminishing the localization to tumors. Promising results using the [68Ga]MAD-8.7 show a potential path to clinical applications.

Cy3-tilmanocept labeling of macrophages in joints of mice with antibody-induced arthritis and synovium of human patients with rheumatoid arthritis.

γ-Tilmanocept (99m Tc-tilmanocept) is a receptor-directed, radiolabeled tracer that is FDA-approved for guiding sentinel lymph node biopsy. Tilmanocept binds the C-type lectin mannose receptor (MR, CD206) on macrophages. In this study, nonradioactive, fluorescently-labeled Cy3-tilmanocept was used to detect CD206+ mononuclear cells in the cartilage of mice with antibody-induced arthritis and in the synovial fluid and tissue of human subjects with rheumatoid arthritis (RA) for comparison with osteoarthritis (OA), and healthy volunteer (HV) controls. Murine arthritis was induced by injection of monoclonal anti-cartilage antibody followed by injection of Escherichia coli lipopolysaccharide. Post-arthritis development (7-11 days), the mice were injected intravenously with Cy3-tilmanocept followed by in vivo and ex vivo epifluorescence imaging. Two-photon imaging, immunofluorescence, and immunohistochemistry were used to identify articular and synovial macrophages (CD206, F4/80, and Cy3-tilmanocept binding) in murine tissues. Cy3-tilmanocept epifluorescence was present in arthritic knees and elbows of murine tissues; no radiographic changes were noted in the skeletons. However, inflammatory arthritic changes were apparent by histopathology and immunohistochemistry (F4/80), immunofluorescence (CD206) and Cy3-tilmanocept binding. In human RA synovial fluid, Cy3-tilmanocept staining correlated with CD206+ /CD16+ cells; negligible labeling was observed in OA samples. Cy3-tilmanocept colocalized with CD206 and staining was significantly higher in RA synovial tissue compared to OA or HV. Our results demonstrate that imaging with Cy3-tilmanocept can detect in vivo inflammatory, CD206+ macrophages in an early arthritis animal model and in human RA patients. These data establish a novel tool for preclinical research of early arthritis and have implications for early RA detection and monitoring of therapeutic efficacy in humans.

Mannose receptor high, M2 dermal macrophages mediate nonhealing Leishmania major infection in a Th1 immune environment.

The origin and functional specialization of dermal macrophages in cutaneous infections have been little studied. In this paper, we show that a strain of Leishmania major (L. major Seidman [LmSd]) that produces nonhealing cutaneous lesions in conventionally resistant C57BL/6 mice was more efficiently taken up by M2-polarized bone marrow (BM)-derived macrophages (BMDMs) in vitro and by mannose receptor (MR)hi dermal macrophages in vivo compared with a healing strain (L. major Friedlin V1). Both in steady and in T helper type 1 (Th1) cell-driven inflammatory states, the MRhi dermal macrophages showed M2 characteristics. The dermal macrophages were radio resistant and not replaced by monocytes or adult BM-derived cells during infection, but were locally maintained by IL-4 and IL-10. Notably, the favored infection of M2 BMDMs by LmSd in vitro was MR dependent, and genetic deletion of MR or selective depletion of MRhi dermal macrophages by anti-CSF-1 receptor antibody reversed the nonhealing phenotype. We conclude that embryonic-derived, MRhi dermal macrophages are permissive for parasite growth even in a strong Th1-immune environment, and the preferential infection of these cells plays a crucial role in the severity of cutaneous disease.

Age-specific association of steroid hormone pathway gene polymorphisms with breast cancer risk.

BACKGROUND: Breast cancer (BC) is a complex disease, and the incidence rates for BC increase with age. Both environmental factors and genetics have an impact on the risk of BC. Although the effects of environmental factors may vary with age, it has been assumed generally that the penetrance of single nucleotide polymorphisms (SNPs) is constant throughout life. In the current study, the results demonstrated that certain SNPs exhibit BC risk associations that vary considerably with age. METHODS: SNPs in 12 steroid hormone pathway genes were investigated for associations with BC risk in white women who were enrolled in an age-matched, case-control (1:2 for cases and controls, respectively) study that consisted of a discovery set (n = 5000 women) and an independent validation set (n = 1583 women). RESULTS: Significant age-related trends were identified and confirmed for SNPs in 4 genes associated with BC risk. The cytosine/cytosine (C/C) genotype of cytochrome P450 XIB2 (CYP11B2) was associated with decreased risk at younger ages (ages 30-44 years) but an increased risk at older ages (ages 55-69 years). The homozygous cytosine-guanine (CG/CG) genotype of uridine phosphorylase glycosyltransferase 1A7 (UGT1A7) was associated with increased risk at younger ages but decreased risk at older ages. Associations in cytochrome P450 19 (CYP19) and progesterone receptor (PGR) were confined to middle age (ages 45-54 years). CONCLUSIONS: The identification of age-specific genetic associations may have profound implications for future etiologic studies of BC and for the use of SNP genotyping to accurately predict the risk of BC in women.

Oligogenic combinations associated with breast cancer risk in women under 53 years of age.

Common, but weakly penetrant, functional polymorphisms probably account for most of the genetic risk for breast cancer in the general population. Current polygenic risk models assume that component genes act independently. To test for potential gene-gene interactions, single nucleotide polymorphisms in ten genes with known or predicted roles in breast carcinogenesis were examined in a case-control study of 631 Caucasian women diagnosed with breast cancer under the age of 53 years and 1,504 controls under the age of 53 years. Association of breast cancer risk with individual genes and with two- and three-gene combinations was analyzed. Sixty-nine oligogenotypes from 37 distinct two- and three-gene combinations met stringent criteria for significance. Significant odds ratios (ORs) covered a 12-fold range: 0.5-5.9. Of the observed ORs, 17% differed significantly from the ORs predicted by a model of independent gene action, suggesting epistasis, i.e., that these genes interact to affect breast cancer risk in a manner not predictable from single gene effects. Exploration of the biological basis for these oligogenic interactions might reveal etiologic or therapeutic insights into breast cancer and other cancers.

DD23 Biomarker: a prospective clinical assessment in routine urinary cytology specimens from patients being monitored for TCC.

BACKGROUND: A prospective clinical study was conducted to assess the ability of the DD23 murine monoclonal antibody to enhance detection of bladder cancer in routine alcohol fixed urine cytology samples. METHODS: Prospectively, 308 bladder cytology specimens were obtained from patients with a history of bladder cancer with a mean age of 71.4+/-11.9 (27% female, 73% male). Data included 121 biopsy-confirmed results and 187 cystoscopy results to assess presence or absence of cancer. Thirty-five normal cytology specimens were obtained from asymptomatic men and women between 55-85 years of age. Separate slides from the alcohol fixed cytology specimens were stained using the Papanicolaou (Pap) and Feulgen staining procedures. The DD23 assay was performed using an avidin-biotin alkaline phosphatase immunocytochemical procedure, with a single urothelial cell exhibiting intense immunostaining sufficient to make a positive call. RESULTS: Pap-Feulgen cytopathology for the 308 cases yielded an overall sensitivity of 65.5% and a specificity of 85.1%, and the DD23 biomarker alone yielded a sensitivity of 80.5% and a specificity of 59.7%. Analysis of the voided urines only (n=164) yielded sensitivities of 61.0% and 73.2% and specificities of 86.2% and 67.5% for cytopathology and DD23 alone, respectively. Results in 49 bladder wash urine cytology cases produced a sensitivity of 70.2% and 100% and specificities of 92.3% and 61.5% for cytopathology and DD23 alone, respectively. In 133 patients that underwent biopsy or had positive cystoscopy results, cytopathology yielded a sensitivity of 65.5% and a specificity of 69.6% while DD23 yielded a sensitivity of 80.5% and a specificity of 58.7%. In 25 biopsy-confirmed low-grade cancers, DD23 improved cancer detection from 32% to 72% when compared to cytopathology. The DD23 biomarker had a specificity of 85.7% in 35 age-matched normal asymptomatic control specimens. CONCLUSIONS: The DD23 biomarker is an adjuvant test that provides improved detection of bladder cancer in cytology specimens and enhances the sensitivity of the cytopathology diagnosis, especially in low-grade cancers.