GUCY2C-targeted cancer immunotherapy - Springer Link

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Oct 30, 2011 - use of chimeric antigen receptor (CAR)-expressing T cells, for treatment of patients with advanced colorectal cancer for whom Ad5-GUCY2C ...
Immunol Res (2011) 51:161–169 DOI 10.1007/s12026-011-8253-7

CURRENT IMMUNOLOGY RESEARCH AT JEFFERSON

GUCY2C-targeted cancer immunotherapy: past, present and future Adam E. Snook • Michael S. Magee • Scott A. Waldman

Published online: 30 October 2011 Ó Springer Science+Business Media, LLC 2011

Adam E. Snook

Abstract For the last decade, we have focused on guanylyl cyclase C (GUCY2C) as a potentially ideal target antigen for colorectal cancer immunotherapy. GUCY2C is expressed only in intestinal epithelial cells and by nearly 100% of colorectal cancers. We have developed and tested a recombinant adenoviral vector possessing GUCY2C (Ad5-GUCY2C) as a candidate vaccine for colorectal cancer patients. Murine studies have revealed that this vaccine is safe and effective against GUCY2C-expressing targets, and Ad5-GUCY2C is poised for phase I clinical testing in colorectal cancer patients with minimal residual disease. Moreover, we are developing second-generation GUCY2C-targeted therapeutics, including the use of chimeric antigen receptor (CAR)-expressing T cells, for treatment of patients with advanced colorectal cancer for whom Ad5-GUCY2C immunization is not appropriate. Thus, a family of GUCY2C-targeted immunotherapeutics may bridge the gap in effective treatments for the 500,000 patients worldwide who die annually from colorectal cancer. Keywords

Guanylyl cyclase C  GUCY2C  Immunotherapy  Adoptive cell therapy  Cancer

Adenocarcinoma of the colon and rectum (colorectal cancer) is the third most frequent cause of cancer and second leading cause of cancer mortality in the United States [1]. Furthermore, colorectal cancer is the third most frequent cancer and fourth most common cause of cancer mortality in the world, resulting in *1 million cases and *500,000 deaths annually [2, 3]. Colorectal cancer prognosis reflects progression of the disease upon diagnosis. Patients with localized disease (stages I and II) have a favorable prognosis, and surgery is presumptively curative—indeed, these patients do not receive adjunct chemotherapy. Patients with metastases in regional

Disclosures: SAW is the Chair of the Data Safety Monitoring Board for the C-Cure TrialTM sponsored by Cardio Biosciences and the Chair (uncompensated) of the Scientific Advisory Board to Targeted Diagnostics and Therapeutics, Inc., which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work. A. E. Snook (&)  M. S. Magee  S. A. Waldman Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, 1020 Locust Street, JAH 348A, Philadelphia, PA 19107, USA e-mail: [email protected]

lymph nodes (stage III) have a less-favorable prognosis, and approximately 50% survive 5 years following standard chemotherapy regimens. Patients with distant metastases have a poor prognosis, and most succumb to the disease. Importantly, among the stage I and stage II patients, who are ostensibly free of metastases in regional lymph nodes (pN0), approximately 33% develop metastases during the course of their disease [4]. This occurs because surgery removes detectable tumor, but undetectable micrometastases may result in relapse. Overall, *50% of surgically treated patients suffer recurrent disease, due to either undetectable micrometastases (stages I and II) or therapeutic failure in the context of detectable metastases (stage III). Thus, there is a critical need for therapeutics to effectively treat those patients with unappreciated systemic metastases who will develop recurrent disease. Due to the low risk of toxicity and potentially high efficacy of immunotherapy, it remains an attractive solution for treating these patients.

Colorectal cancer immunotherapy To date, colorectal cancer immunotherapy has been largely ineffective. While virtually all varieties of immunotherapy

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have been tested—including vaccines targeting mutant K-ras or p53 [5–7], oncofetal antigens [8], survival and growth factors [9, 10]—the greatest vaccine success occurred using viral vector vaccines against carcinoembryonic antigen (CEA) [11]. While CEA is highly expressed by normal and neoplastic mucosa, it is also found in various normal tissues including prostate, uterus, bladder, spleen, lymph nodes and blood [12, 13]. Thus, a high level of CEA-specific tolerance may exist, dampening CEA-specific responses [14]. Conversely, if CEA-specific tolerance was broken, the resulting CEA-specific immune responses could produce widespread pathology targeting CEA in normal tissues [15].

Guanylyl cyclase C Thus, we sought to identify more efficacious targets for colorectal cancer immunotherapy. The ideal target would be universally expressed by cancer, induce robust immune responses against tumors, but have a low risk of autoimmunity. Previous observations of immune system compartmentalization suggested that this could be achieved by targeting mucosa-specific differentiation antigens [16–27]. Indeed, the adaptive immune system is comprised of specialized compartments that serve distinct, but overlapping, functions, that often contain unique cell populations and that frequently do not communicate with one another. Thus, immune responses can be compartment-specific. This has been revealed largely through studies to produce HIV vaccines for which systemic administration (typically intramuscular or subcutaneous) generates systemic responses measurable in blood or spleen, but not in mucosal tissues, such as intestine [25–27]. Therefore, we hypothesized that if immune responses targeting intestinal self-antigens could be induced in systemic tissues, these should not produce immunity (to tumor or self) in intestine. However, because colorectal cancer metastasizes from the mucosa into the systemic compartment, these metastases may be targeted by systemic immunity. Moreover, sequestration of mucosal self-antigens may limit their exposure within the systemic compartment, reducing the quantity or altering the quality of tolerance to these antigens, possibly allowing for more robust responses and effective immunity. The antigen we selected to test this hypothesis is guanylyl cyclase C (GUCY2C; GCC). GUCY2C is a type I transmembrane protein expressed by intestinal epithelial cells from the duodenum to rectum [28]. Importantly, GUCY2C expression is maintained through all stages of neoplastic transformation from pre-cancerous polyps to distant colorectal cancer metastases. A number of physiologic processes including enterocyte proliferation [29],

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differentiation [29] and metabolism, [30] are regulated by GUCY2C signaling. GUCY2C signaling is induced by the endogenous hormones guanylin and uroguanylin (GUCA2A and GUCA2B, respectively) and by the exogenous bacterial heat-stable enterotoxin, ST, to produce the second messenger, cGMP, activating the cGMP-dependent protein kinase (PKG). The initial discovery of GUCY2C revealed its presence by RNA northern blot analysis in intestine, but not in nonintestinal tissues [31]. This restricted expression has since been confirmed by in situ ligand binding, immunoblotting, immunohistochemistry and RT-PCR in animals [32–38] and in humans [32, 38–40]. Within the GI tract, GUCY2C expression is restricted to intestinal epithelial cells and occurs only from the duodenum through the rectum but does not occur in normal gastric and esophageal tissues [28, 35, 41–43]. Importantly, RT-PCR studies have demonstrated that GUCY2C expression persists in both primary and metastatic colorectal tumors in humans [39, 40, 44, 45]. Because GUCY2C is normally confined to intestine, its persistent expression in disseminated metastatic cancers originating in the intestine makes GUCY2C a sensitive and specific molecular marker for identifying and staging colorectal cancer in patients [32, 38, 40]. The prognostic utility of GUCY2C qRT-PCR (quantitative reverse transcriptase polymerase chain reaction) to detect occult metastases in lymph nodes was recently explored in a prospective multicenter blinded clinical trial [46]. Thirteen percent of patients who were considered cured by conventional pathologic assessment had lymph nodes negative for GUCY2C [pN0(mol-)], and nearly all remained free of disease during follow-up (recurrence rate 6.3%). Conversely, 87.5% of patients had lymph nodes positive for GUCY2C [pN0(mol?)], and 20.9% developed recurrent disease. Multivariate analyses demonstrated that GUCY2C in lymph nodes was the most powerful independent marker of prognosis, and patients whose lymph nodes were positive for GUCY2C [pN0(mol?)] exhibited earlier time to recurrence and reduced disease-free survival. Thus, GUCY2C is a powerful marker to identify occult metastases independently associated with prognosis in pN0 colorectal cancer [46] due to its highly restricted expression pattern. As a means to generate GUCY2C-specific immune responses, we selected recombinant adenovirus as a vector. Recombinant adenovirus, primarily human serotype 5 (rAd5), has served as a vector for gene therapy and vaccine delivery in clinical trials for more than 15 years [47] involving thousands of patients with only rare adverse events [48]. Our GUCY2C vaccine vector employs recombinant Ad5, a stable nonenveloped icosahedral virus with a linear double-stranded DNA genome of *38 kb, encoding more than 30 proteins [49]. The rAd5 possesses

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deletions of the early E1 and E3 regions of the vector, enabling insertion of GUCY2C under the control of the human cytomegalovirus (CMV) immediate-early enhancer/ promoter. Deletion of the E1 gene also renders this virus replication-incompetent [50–52], increasing safety associated with its clinical use [48, 53–58]. GUCY2C is one member of a family of guanylyl cyclase receptors, each of which converts cytosolic GTP to the cGMP. Thus, each possesses a cytosolic enzymatic domain with highly conserved sequence. However, the extracellular ligand binding domain of GUCY2C is antigenically unique, reflecting the unique set of GUCY2C-ligands. Therefore, the vaccine employs only the extracellular domain. In mice, immunization with Ad5-GUCY2C possessing the mouse GUCY2C extracellular domain generates GUCY2C-specific CD8? T-cell responses, but not CD4? T-cell or antibody responses [59, 60]. This pattern is the result of GUCY2C-specific tolerance, because GUCY2Cdeficient (GUCY2C-/-) mice produce responses from all three arms of adaptive immunity. Thus, GUCY2C-specific immunity is characterized by split tolerance that allows development of only CD8? T-cell responses, but not CD4? T-cell or antibody responses. However, split tolerance does not prevent vaccine-induced antitumor immunity [59–61]. Ad5-GUCY2C immunization prevented the development of experimental colorectal cancer metastases in mice following challenge with GUCY2C-expressing CT26 mouse colorectal cancer cells via tail vein to establish lung metastases or via intrasplenic injection to establish liver metastases, modeling the two major sites of metastases in patients. Also, Ad5-GUCY2C administration to mice with established lung metastases was able to reduce metastasis number and extend animal survival when combined with heterologous viral boosts using recombinant vaccinia and rabies viruses [59, 61]. Importantly, while immunization produced CD8? T-cell responses and antitumor immunity, no toxicity was observed [59, 60]. Mice had no signs of gastrointestinal disease following immunization, which could result from GUCY2C-specific T-cell responses in intestine. Similarly, Ad5-GUCY2C immunization did not exacerbate colitis chemically induced with DSS (dextran sulfate sodium). Thus, Ad5-GUCY2C immunization produces CD8? T-cell responses with antitumor activity, but without concomitant autoimmunity, making it an ideal candidate for clinical development. We are in the process of translating Ad5-GUCY2C vaccination to colorectal cancer patients. We have generated cGMP (current good manufacturing practices)-grade vaccine vector possessing the human GUCY2C extracellular domain and anticipate patient enrollment to begin in 2012. However, our clinical approach will not follow the design of previous first-in-man cancer vaccine approaches. Tumor vaccines have been explored primarily in patients

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with advanced disease, with disappointing results. In colorectal cancer, a meta-analysis of 32 trials with 527 patients with advanced (stage IV) colorectal cancer receiving cancer vaccines revealed response rates of less than 1% [62]. Challenges in immunotherapeutic approaches in advanced-stage tumors include the inability of primary T- and B-cell responses to eradicate large volume disease, immune responses impaired by progressive disease and debilitation, and accelerated tolerance opposing immunotherapeutic efficacy [63–65]. In contrast, meta-analyses of clinical trials of active immunotherapy in early-stage (pN0) colorectal cancer patients demonstrated modest effects on clinical outcomes [63–65]. Indeed, there is emerging recognition, echoed in a recent draft guidance from the FDA, that active immunotherapy has the greatest opportunity for efficacy in patients with minimum residual disease [63–65]. Thus, our clinical studies will explore the utility of Ad5-GUCY2C in pN0 colon cancer patients, a cohort with minimum residual disease but a risk of approximately 25% for disease recurrence [46, 66, 67].

Adoptive Cell Therapy Aside from some recent success, vaccines have largely been ineffective at eliminating bulky, well-established tumors [68]. The inability to generate meaningful antitumor responses in late-stage patients likely results from intricate immunosuppressive networks within the tumor capable of extinguishing productive immune responses. Alternatively, the use of adoptive cell therapy (ACT) allows for the ex vivo activation and expansion of large numbers of tumor-reactive lymphocytes and their reintroduction into lymphodepleted hosts, which effectively removes immunosuppressive cells. In recent clinical trials performed at the National Cancer Institute, ACT using the most aggressive lymphodepleting regimens produced objective response rates, measured by RECIST criteria, in 72% of patients with metastatic melanoma, 56% of which have complete durable responses [69]. These studies utilize isolated and expanded tumor-infiltrating lymphocytes (TIL) from excised patient tumors; however, the ability to effectively expand tumor-reactive lymphocytes from TIL remains limited to melanoma and is further limited by tumor accessibility [70]. Advances in genetic engineering have allowed for the introduction of tumor-specific receptors into T cells through retroviral or lentiviral transduction, eliminating the need for TILs as the starting material for ACT [71]. However, the identification of tumor antigen–specific T-cell receptors (TCR) from human T cells is also limited to patients with melanoma [69]. Alternatively, murine TCRs specific for human proteins such as p53 and CEA have been obtained from HLA-A2.1 transgenic mice

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[72, 73]. The lack of tolerance to human antigens in these mice along with amino acid substitutions in the complementarity-determining regions of these TCRs has led to the production of T cells with high avidities for these tumor antigens. Nevertheless, TCRs generated in this fashion would only be useful in patients with the corresponding HLA-A2.1 haplotype. Furthermore, tumors are known to down-regulate various mechanisms of MHC presentation in order to elude T-cell-mediated destruction [74]. An alternative approach, developed by Eshhar et al. [75], is the use of chimeric antigen receptors (CARs). CARs are single-chain receptors comprised of variable regions from tumor-specific antibodies and various intracellular signaling domains. CAR-expressing T cells, called T bodies, have the advantage of being directed against any extracellular tumor antigen to which monoclonal antibodies can be generated. Moreover, CARs recognize antigen independent of MHC, making their use applicable to all patients, irrespective of MHC type. The majority of CARs are comprised of a single-chain variable fragment (scFv) derived from a monoclonal antibody, an extracellular hinge region and the intracellular portions of one or more signaling receptors [76] (Fig 1). The hinge region is most commonly derived from either CD8a or IgG constant regions (Fc) and acts to promote flexibility of the antigen-recognizing region; however, the appropriate hinge region must be determined empirically based on the location of the target epitope. While hinge regions of extended length have been shown to be beneficial when targeting C-terminal epitopes on large membrane antigens, N-terminal epitopes on large or small antigens are more aptly targeted with short hinge regions or none at all [77]. In addition, heavily glycosylated antigens, such as MUC1, may require more elongated hinges, such as the IgD hinge in conjunction with IgG constant regions, to promote greater flexibility [78]. Initially, CARs contained intracellular activating signals from either the CD3f chains of the T-cell receptor complex or the Fcc receptor. While promoting antigen-specific tumor killing in vitro, these T bodies were largely ineffective at promoting tumor regression in vivo, largely reflecting activation-induced cell death (AICD) [79]. Indeed, early clinical trials testing first-generation CARs were hindered by poor in vivo persistence of T bodies. Second-generation receptors included a secondary costimulatory signal in tandem with the activating signal [76]. The most common addition is the hallmark T-cell costimulatory molecule CD28, although other costimulatory molecules such as ICOS and members of the TNF receptor family including 4-1BB (CD137) and OX40 (CD134) have been used [80]. These molecules classically act to improve T-cell activation and promote pro-survival signaling to counteract AICD that can occur from strong

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Fig. 1 Evolution of chimeric antigen receptors (CARs). First-generation receptors link single-chain variable fragments to the intracellular portion of the CD3f chains. Inserting the CD28 costimulatory molecule in second-generation constructs increases T-cell proliferation, in vivo persistence and cytokine secretion by activating phosphatidylinositol 3-kinases (PI3 K). Addition of a second costimulatory molecule, such as 4-1BB or OX40, in third-generation CARs further increases T-cell survival and in vivo persistence by inducing downstream TRAF signaling (TNF-receptor-associated factor)

T-cell receptor signals alone. Inclusion of these costimulatory molecules, most commonly CD28 or 4-1BB, has resulted in T bodies with prolonged in vivo persistence that are capable of promoting regression of large tumors in both animal models and the clinic [81–84]. Third-generation constructs have included two costimulatory signals, most commonly CD28 with either 4-1BB or OX40, and the CD3f chains [85, 86]. While third-generation CARs mediate in vitro cytotoxicity similar to second-generation CARs, these third-generation CARs may promote increased antitumor activity and in vivo persistence [85]. Numerous reports have demonstrated efficacy of genetically engineered T cells in immunodeficient mouse models. Initially, this success was not reproduced in the clinic due to poor persistence of adoptively transferred cells. However, lymphodepletion prior to T-cell transfer has improved engraftment by creating immunologic space for transferred T cells to expand. Removal of lymphocytes, using cyclophosphamide, fludarabine, or total body irradiation, causes an increase in production of homeostatic cytokines, such as IL-7 and IL-15, that further act to increase T-cell engraftment and expansion [70]. Additionally, use of CARs containing secondary costimulatory signals drastically increases in vivo persistence [87, 88]. Two recent reports have detailed regression of B-cell cancers in four patients using second-generation CARs [82, 89]. However, to date, no published results have demonstrated regression in any patients with epithelial tumors. In the absence of truly tumor-specific targets, the most commonly targeted proteins are typically tumor-associated

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self-antigens. In that context, the potential for antigenspecific toxicities against normal antigen-expressing tissues must be evaluated for each individual target. For instance, ACT targeting melanoma produces uveitis and vitiligo by destroying melanocytes [90]. Both trials reporting B-cell cancer regression after T-body treatment also observed a prolonged reduction in B cells, as seen in preclinical mouse studies [81, 82, 89]. The adverse effects associated with depletion of melanocytes and B cells are relatively small compared with the antitumor benefits, as neither cell type is essential for survival. Conversely, many antigens targeted in carcinomas of the breast, colon, kidney and lung are abundantly found on normal epithelial cells whose destruction could be fatal to the host. For example, T bodies targeting carbonic anhydrase IX (CAIX) in renal cell carcinomas have resulted in liver toxicities attributed to antigen-specific recognition of CAIX on bile duct epithelial cells in three patients [91]. ACT using murine TCRs targeted to CEA in 3 patients with metastatic colorectal cancer resulted in severe inflammatory colitis targeting CEA-expressing epithelial cells in the intestine [15]. Similar results were also seen in a preclinical mouse study that transferred CEA-specific T cells into CEA-transgenic mice expressing the protein in similar tissues compared to humans [92]. The most severe adverse event occurred targeting Her2 in a patient with metastatic colorectal cancer [93]. This patient received a lymphodepleting regimen consisting of cyclophosphamide and fludarabine followed by an intravenous dose of third-generation Her2-specific T bodies. The patient eventually succumbed to cardiac arrest 5 days after T-body administration. Toxicity was likely caused by complications arising from inflammatory conditions resembling a cytokine storm generated by recognition of Her2 on lung epithelial cells [93]. These toxicities demonstrate the importance of antigen selection as well as appropriate dose-escalation strategies when targeting selfantigens. Multiple strategies have been proposed to safely test self-reactive T cells in the clinic to limit such adverse events. These include lowering T-cell dose, not using cytokine supplementation and eliminating lymphodepletion [94]. Once safety has been demonstrated at early stages, more aggressive regimens can be used. Additionally, second- and third-generation CAR constructs appear to be more potent than first-generation constructs. It may be necessary to begin with first-generation CARs and sequentially increase costimulatory molecules once safety has been confirmed [94]. However, these strategies could eliminate any potential benefits to patients receiving lessaggressive regimens [94]. Suicide genes, such as thymidine kinase and inducible caspase 9, could also be inserted into T cells as a means of eliminating reactive cells in vivo, although these genes may only be useful when managing

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chronic toxicities and, to date, most toxicities appear to be acute in nature [95, 96]. More adequate testing in preclinical mouse models also may reveal potential toxicities against normal tissues. The vast majority of preclinical ACT models use human tumor xenografts in immunodeficient mice. While these models are acceptable to determine whether human T cells can promote regression of human tumors in vivo, they offer no insight into potential toxicities in the clinical setting, either because human TCRs will not engage murine MHC or because the scFv portion of the CAR is not cross-reactive with the murine antigen homolog. More appropriate models would either utilize HLA-A2.1 transgenic mice with the corresponding TCRs that would eventually be used in humans or CARs that contain scFv that are cross-reactive with the murine homolog of the targeted antigen. Alternatively, completely syngeneic mouse models may provide insight into potential toxicities. For example, mouse syngeneic models of ACT targeting CD19, using CARs, or CEA, using TCRs, have both demonstrated similar toxicities to those seen in the clinic [81, 92]. GUCY2C may have potential as an ACT target for metastatic colorectal cancer [97]. Unlike many targeted antigens, its expression is maintained in greater than 95% of human colorectal cancers, and its expression is largely confined to the apical surfaces of intestinal epithelial cells. Vaccine studies targeting GUCY2C show no evidence of autoimmunity; however, this may be due to generation of T cells with weak avidities as well as the abundance of immunosuppressive cells in gut lymphoid tissues [59, 60]. The use of lymphodepleting regimens in ACT may make the intestine more susceptible to T-cell-specific toxicities. TCR-directed therapy targeting GUCY2C may not be ideal due to MHC-dependent escape mechanisms in cancers, but GUCY2C could be a suitable target for MHC-independent CAR-mediated recognition. GUCY2C expression on the apical surfaces of intestinal epithelial cells, facing the lumen of the intestine, is largely sequestered from the systemic compartment and therefore from T cells, via tight junctions. Accordingly, CARs could potentially mediate antitumor activity against systemic metastatic tumors without causing GUCY2C-directed colitis in the intestine. Since peptide loading of MHC would not discriminate against antigen that is sorted to apical surfaces, TCRdirected therapy could direct toxicities to the basolateral side of epithelial cells. GUCY2C expression outside of the intestine has been recently demonstrated in the salivary glands as well as the hypothalamus and may be another potential site for toxicity [98, 99]. However, GUCY2C expression in the salivary glands is also expressed on apical surfaces of epithelial cells and hence not facing the systemic compartment, and levels in the hypothalamus appear to be tenfold less than intestine [98, 99]. Additionally, the

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hypothalamus may be protected from ACT by the blood– brain barrier. Nevertheless, the use of GUCY2C-deficient (GUCY2C-/-) mice will provide an additional tool to determine whether specific toxicities arise form CARdirected T cells [59]. While still in early-stage testing, GUCY2C-targeted T bodies may be combined with Ad5-GUC2YC, as well as other GUCY2C-targeted therapeutics, to create a family of treatment options to provide maximal benefit for all colorectal cancer patients. While recent ACT results have been promising in the clinic, it must be noted that currently only melanoma and B-cell lymphoma have been effectively treated via ACT. While the development of more potent T cells through the use of later-generation CARs or other techniques may promote better tumor regression, these treatments may be associated with severe toxicity targeting antigens found in tumors and in normal tissues [100, 101]. Achieving the appropriate balance of safety and efficacy employing GUCY2C-targeted therapies, alone or in combination, may require more thorough preclinical testing to determine acceptable levels of toxicity and means to manage adverse events. Notwithstanding the risk of toxicity associated with ACT, GUCY2C-targeted immunotherapeutics are poised to bridge the gap in treatments for the one-half million colorectal cancer patients dying annually. Acknowledgments Financial support was provided by The National Institutes of Health (CA75123, CA95026) and Targeted Diagnostic and Therapeutics Inc. (to S.A.W.), and Measey Foundation Fellowship (to A.E.S.); SAW is the Samuel M.V. Hamilton Endowed Professor. This project was funded, in part, by a grant from the Pennsylvania Department of Health, which specifically disclaims responsibility for any analyses, interpretations or conclusions.

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