Low, Philip S.

Abstract Folate receptor (FR) expressing macrophages have been shown to play an integral role in a number of inflammatory diseases. (1) In this report, we characterize these FR+ macrophages and further investigate whether monocytes, the precursors to these macrophages, also express FR. We demonstrate that FR expression is observed on a subset of highly activated peritoneal and synovial macrophages, but not on other infiltrating immune cells or resident macrophages. We further find that a subset of human peripheral blood monocytes also expresses FR, albeit at a lower level than activated macrophages. Surface antigen characterization of the FR+ monocytes suggests markers characteristic of an inflammatory phenotype (CD14hiCD16), while the resident monocytes (CD14loCD16+) express few or no FR. Preliminary clinical data also suggest that the numbers of peripheral FR+ monocytes can be reduced by a folate-targeted immunotherapy. These results suggest that activated macrophages and inflammatory monocytes express a FR that can be targeted with folate-linked drugs without promoting drug uptake by non-activated macrophages and resident monocytes. 1. Xia W, Hilgenbrink AR, et al. Blood. 2008 Oct 24. [Epub ahead of print] This work was supported in part by a grant from Endocyte, Inc.

20043 Background: Studies have demonstrated that the generation of endogenous immunity to specific tumor antigens is associated with improved prognosis for many cancers. Thus, there has been increased efforts to identify tumor-associated antigens to which there is immunity, as these may be tumor rejection antigens. A candidate antigen is the folate receptor alpha (FRα) which is overexpressed in malignancies, especially breast and ovarian cancer. Prior studies involving ovarian and breast cancer patients have demonstrated the presence of FRα-specific cytotoxic T cells in the tumor-associated lymphocyte population. Methods: Using the RANKPEP CD4 T cell epitope prediction algorithm, we predicted promiscuous immunogenic regions of FRα, and tested for immunity in 30 breast or ovarian cancer patients and 18 healthy volunteer donors in order to attain a better understanding of the levels and extent of the endogenous FRα immune response. Immunity was examined using IFN-γ ELIspot analysis and IgG ELISAs. Results: Fourteen peptides were predicted as potential epitopes to which T cells may respond, seven each from the carboxy- and amino-terminus halves of the protein. Greater than 70% of patients demonstrated T cell immunity to at least one of the fourteen peptides. Patients responded to an average of 3 ± 0.7 peptides while healthy donors responded to only 1 ± 0.5 peptides (p = 0.02). Five peptides were recognized by >25% of patients. Responses to three of these peptides were higher (p > 0.05) in the patients than in healthy donors, suggesting that patients generated immunity upon cancer development. Compared to healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor, suggesting natural epitope spreading (p = 0.03). There was no difference between patients and healthy donors in the immune responses to either non-specific stimuli (p = 0.2) or viral peptides (p = 0.5). Patients had detectable FRα-specific IgG antibody consistent with active FRα-specific helper T cell immunity. Conclusions: These findings demonstrate that the FRα is a target of the immune system in patients with breast or ovarian cancer. Understanding the antigens that are naturally targeted by the immune system may be important for diagnosis, prognosis, and immune-based therapies. No significant financial relationships to disclose.

Abstract Background: Folate receptor (FR) is a recently recognized tumor marker that is being investigated for targeted delivery of drugs, imaging of cancer cells, and tumor immunotherapy. The small size of the molecule, simple conjugation chemistry and lack of immunogenicity make folate an ideal candidate for targeted delivery to tumors bearing the FR (Lu Y, Low PS. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv Drug Deliv Rev. 2002 Sep 13;54(5):675–93). FR is overexpressed in 90% of ovarian cancers and in other human tumors including endometrial, colorectal, breast, and neuroendocrine carcinomas, it is negatively or only weakly expressed in most normal tissues including some myeloid cells. In view of the above we wished to learn if overexpression of FR by marrow plasma cells (PC) might differentiate MM from MGUS and SMM. Methods: We obtained marrow biopsy sections from 20 patients each with MM, MGUS, or SMM, and 11 normals and stained them by immunohistochemistry using a FR-beta antibody. Expression was graded 0 (negative), 1 (equivocal), 2 (positive) or 3 (strong positive) based on immunohistochemical staining of marrow PC. We recorded the percent PC, percent positive PC, and the staining of other cell types including myeloid, erythroid, lymphocytes, macrophages, megakaryocytes and control tissues. Results: PC of patients with MM positively expressed FR compared to MGUS, SMM and normal PC (p<0.0001) (Figure 1 and 2). Of interest, 6 of 20 SMM patients showed positive expression. Maturing myeloid cells were also positive, acting as internal positive controls. Macrophages were often positive; some endothelial cells were positive; early erythroid precursors were rarely positive; megakaryocytes, maturing erythroid precursors and lymphocytes did not express FR. The chromosomal location of the FOLR gene cluster is 11q13. Gene expression arrays of purified PC failed to show evidence of overexpression of FOLR1, FOLR2, or FOLR3. We are currently performing Western blots to examine levels of protein product of purified PC. Conclusion: Immunohistochemistal studies show differential expression of FR in MM PC compared to absent or equivocal expression in MGUS and normal PC and variable expression in SMM. The data suggest that FR may be used as a diagnostic tumor marker and has implications for development of folate-based tumor imaging, drug delivery, and immunotherapy. Figure Figure Figure Figure

Abstract Folate receptors (FR) may be of use for targeted delivery of cytotoxic drugs in invasive urothelial carcinoma (iUC), for which improved therapy is needed. FR expression and function in iUC were explored and the antitumor activity and toxicity of a folate-targeted vinblastine conjugate were evaluated in dogs with naturally occurring iUC, an excellent model for human iUC. FR immunohistochemistry was carried out on iUC and normal human and dog bladder tissues together with nuclear scintigraphy in dogs to monitor iUC folate uptake. Dose escalation of a folate-targeted vinblastine compound, EC0905, was conducted in dogs with biopsy-confirmed, FR-positive iUC. FRs were detected by immunohistochemistry (PU17) in most primary iUC and many nodal and lung metastases from dogs, and scintigraphy confirmed folate uptake in both primary and metastatic lesions. The maximum tolerated dose of EC0905 in dogs was 0.25 mg/kg IV weekly, with neutropenia at higher doses. Tumor responses included partial remission (≥50% reduction in tumor volume) in five dogs and stable disease (&lt;50% change in tumor volume) in four dogs. Immunoreactivity to PU17 was similar in humans (78% of primary iUC, 80% of nodal metastases). Less immunoreactivity to mab343 (22% of cases) occurred. FR-β was noted in 21% of human iUC cases. Our findings suggest folate-targeted therapy holds considerable promise for treating iUC, where FR-β may be important in addition to FR-α. Cancer Res; 73(2); 875–84. ©2012 AACR.

Context.— Lung cancer is the number one cause of cancer deaths in the United States and globally. The advent of targeted therapies has offered a new treatment paradigm for lung cancer, but currently validated and emerging drugs are effective in only a small minority of lung cancers, predominantly adenocarcinomas. Folate receptors can serve as targets for drugs attached to folate and are overexpressed in many cancers. Objective.— To determine the frequency of folate receptor overexpression in lung cancers of different cell types as potential targets for folate-linked therapy. Design.— High-density tissue microarrays were constructed from archival formalin-fixed, paraffin-embedded resection specimens from 188 primary stage I or stage II adenocarcinomas or squamous cell carcinomas of the lung with three 0.1-cm cores from each tumor. Tissue microarrays were immunostained for folate receptor α with mAb343 and the results scored (0 to 1+ = weak expression, 2+ to 3+ = strong expression). Results.— Eighty-four of 117 (72%) of the adenocarcinomas were strongly positive for the folate receptor, and 36 of 71 (51%) of the squamous cell carcinomas were strongly positive for the folate receptor. Conclusions.— Our data indicate that a large percentage of lung cancers, including squamous cell carcinomas in addition to adenocarcinomas, strongly express folate receptor. This suggests that folate-linked targeted therapy can potentially be used to treat the majority of lung cancers, both adenocarcinomas and, particularly, squamous cell carcinomas, that do not respond to current targeted therapies.

Abstract Introduction: Invasive transitional cell carcinoma of the urinary bladder (InvTCC) kills &gt;14,000 people yearly in the US. Folate receptors (FRs, especially FRα) are being targeted for imaging and therapy across multiple cancer types. This work was performed to define FR expression and uptake in InvTCC, with comparison to normal bladder; and to determine the antitumor activity and toxicity of folate-targeted vinblastine in dogs with naturally-occurring InvTCC where the cancer closely mimics human InvTCC. Canine InvTCC metastasizes frequently, and only 35% of dogs have remission with chemotherapy. Methods: Immunohistochemistry (IHC, mab343, PU17, FRβmab) was performed in InvTCC, bladder tissues adjacent to InvTCC in humans, and in bladder from normal dogs. Folate uptake was determined ex vivo by folate binding assay, and in vivo in dogs with InvTCC by nuclear scintigraphy (99mTc based FR targeted imaging agent). A dose escalation study of folate-targeted vinblastine (EC0905, IV weekly) was performed in dogs with biopsy-confirmed, FR-positive, measurable InvTCC. Toxicity (VCOG criteria) was assessed by CBCs, serum chemistry profiles, urinalyses, physical exams, and dog owner observations. The MTD was defined as the highest dose at which 0 of 6 dogs had grade 4 toxicity and ≤1 of 6 dogs had grade 3 toxicity. Tumor response was assessed by ultrasonography (with detailed bladder mapping) and radiography. Results: FR expression (IHC, PU17) was noted in the majority of canine samples including 56 of 74 (76%) primary tumors, 7 of 12 (58%) nodal metastases, and 10 of 21 (48%) lung metastases, as well as in normal urothelium. Typically, &gt;50% of tumor cells were positive. FR expression was cytoplasmic in 18, membrane in 6, and in both sites in 32 InvTCC cases. Scintigraphy (n= 12 dogs) revealed folate uptake in primary and metastatic lesions. The MTD of EC0905 in dogs was 0.25 mg/kg IV weekly. At doses above the MTD, gastrointestinal upset and myelosuppression occurred. No urologic toxicity was noted. Tumor responses included partial remission (≥50% reduction in tumor volume) in 5 dogs and stable disease (&lt;50% change in tumor volume) in 5 dogs. In human InvTCC, IHC results with PU17 were similar to those in dogs with 29 of 36 (78%) primary tumors, 12 of 15 (80%) nodal metastases, and 5 of 5 tissues adjacent to InvTCC being positive. Immunoreactivity to mab343 was less frequent (22% of primary tumors, 33% of nodal metastases). FRβ was expressed in 3 of 14 (21%) InvTCC cases. Ex vivo folate binding to human and canine InvTCC was detected, although binding varied from case to case and did not always correlate with IHC. Conclusions: Folate-targeted therapy holds promise for the treatment of InvTCC. FR expression was detected in human and canine InvTCC, and folate-targeted vinblastine had excellent antitumor activity against canine InvTCC where the cancer closely mimics human InvTCC. In addition to FRα, FRβ may be important in InvTCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-5. doi:1538-7445.AM2012-LB-5

Abstract Previous work has demonstrated that a subset of macrophages expresses a folate receptor (FR) that can mediate internalization of folate-linked molecules, including imaging and therapeutic agents. To characterize this subset, macrophages were collected from peritoneal cavities of mice injected with saline, thioglycolate, zymosan, heat-killed or live bacteria, and cell-surface markers that coexpress with FR were identified. Virtually no F4/80+ peritoneal macrophages from saline-injected mice expressed FR, whereas numerous macrophages from mice injected with each inflammatory stimulus expressed FR. Examination of cell differentiation antigens that are up-regulated in FR+ macrophages revealed markers characteristic of an activated state (CD80, CD86, Ly-6C/G), whereas macrophages lacking these activation markers expressed few or no FR. FR+ macrophages also produced tumor necrosis factor-α (TNF-α) and reactive oxygen species, and production of reactive oxygen species correlated linearly with expression of FR. Synovial macrophages collected from arthritic patients were found to bind and internalize folate-linked dyes. Moreover, a folate-linked radioimaging agent was shown to image inflamed joints of rheumatoid arthritic patients. These results suggest that FR constitutes a marker for macrophage activation and that FR+ macrophages can be targeted with folate-linked drugs without promoting drug uptake by nonactivated macrophages. This trial was registered at www.clinicaltrials.gov as #NCT00588393.