WO2011103182A2

WO2011103182A2 - Imaging methods for assessment and quantification of vaccination and in vivo antigen capture

Info

Publication number: WO2011103182A2
Application number: PCT/US2011/025072
Authority: WO
Inventors: Jeff Bulte; Christopher Long; Hyam Levitsky
Original assignee: The Johns Hopkins University
Priority date: 2010-02-16
Filing date: 2011-02-16
Publication date: 2011-08-25
Also published as: WO2011103182A3

Abstract

The invention provides for labeling, imaging, and quantifying an antigen presenting cell in vivo.

Description

IMAGING METHODS FOR ASSESSMENT AND QUANTIFICATION

OF VACCINATION AND IN VIVO ANTIGEN CAPTURE

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/305,056, filed February 16, 2010, the contents of which is incorporated herein by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This work was supported by grant no. P50CA96888 from the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Immunization has eradicated many harmful infectious diseases in the Western world. This is commonly achieved by administering vaccines to individuals containing attenuated pathogens, such as viruses and bacteria, or antigenic peptides/proteins. In addition, cellular vaccines can be used for the therapeutic or prophylactic treatment of chronic pathological conditions, such as cancer and HIV.

Effective vaccine compositions directed against infectious diseases (carrier vehicle and antigen dose) are well defined. The specific antigen makeup is the major variable adjusted each year to cover antigenic drift (e.g., with attenuated influenza virus). In contrast, many variables remain to be optimized for so-called "therapeutic" vaccines, such as those being developed for the treatment of established cancer. Even with the current enthusiasm in the field of cancer immunotherapy for the use of vaccines, the objective response using conventional oncologic criteria for clinical tumor response is only around 2.6%. Clearly, there is a need for understanding the immunological basis of individual successes or failures. Tools to quantify key intermediate biological endpoints that can be targeted for optimization will greatly assist in the development of more effective cancer immunotherapy strategies. A key event corresponding to the magnitude of systemic B and T cell immunity generated by a vaccine is the efficiency of antigen capture and delivery to lymph node, where lymphocytes are primed. Dendritic cells (DCs) are potent antigen-presenting cells that have the capacity to stimulate lymph-node -based naive T helper (Th) cells and initiate primary T cell responses. It is now generally accepted that immature DCs, residing in peripheral tissues, require activation/maturation signals in order to undergo phenotypic and functional changes to acquire a fully competent antigen presenting capacity. Activation/maturation of DCs involves several steps such as a transient increased capacity to take up antigen, migration towards nearby lymph nodes and simultaneous up regulation of molecules including chemokine receptors and co- stimulatory molecules. For successful immunization, it is important for DCs to capture and take up antigen, and traffic to lymph nodes in order to present these antigens to T cells. As such, effective migration of DCs to the secondary lymphoid organs remains an important step for vaccine efficacy and efficient monitoring of DC migration by a non-invasive method will play a role in the development of successful cellular therapeutics.

Dendritic cells have previously been labeled with radionuclides for scintigraphic imaging, which is the only clinical cellular imaging modality approved by the U.S. Food and Drug Administration (FDA). However, scintigraphy has the major drawback of only allowing gross anatomical determination. Additionally, there is currently no non-invasive means to visualize and quantify the process of antigen uptake by DCs, and migration of DCs to the lymph nodes. Thus, little is known about the time course of events, or parameters that improve the efficiency of this process. Histological examination of lymph nodes and enumeration of antigen-presenting DCs is a slow and cumbersome process, and raises ethical issues when using non-human primates. For humans, it is very difficult to obtain lymph node tissue for evaluation. Accordingly, in the field of cancer vaccines, progress is stagnated by the lack of suitable means to evaluate and measure the lymph node homing of antigen- presenting DCs, as well as the quantification of these cell numbers. This has also hampered the development of suitable immune adjuvants that can further boost the immune system through the activation and induced migration of DCs from the site of antigen capture (typically skin or muscle) to the site of lymphocyte priming (draining lymph node). The present invention solves this problem and is expected to accelerate development of vaccines used in conjunction with immunoadjuvants SUMMARY OF THE INVENTION

As described below, this invention provides novel methods for labeling antigens, including vaccines containing an antigen(s), and non-invasively imaging the subsequent capture of the labeled antigens by endogenous antigen presenting cells and their migration to lymph nodes. In addition, the invention also provides a novel image-based method to quantify antigen-presenting cells that deliver antigen within lymph nodes. The invention will be useful to evaluate and quantify the efficacy of vaccination, to aid the development of new vaccines, and to help guide the development of immune adjuvants.

In one aspect, the invention provides methods for labeling a dendritic cell in vivo. In embodiments, the methods involve contacting the dendritic cell with an antigen comprising an imaging agent, thereby labeling the dendritic cell. In embodiments, the antigen is an inactivated tumor cell. In related embodiments, the tumor cell contains SPIO. In further related embodiments, the SPIO is present in an intracellular compartment of the tumor cell, and the SPIO is transferred to the dendritic cell during antigen uptake.

In one aspect, the invention provides methods for visualizing dendritic cell activation and migration in a subject. In embodiments, the methods involve contacting one or more dendritic cells in vivo with an antigen comprising an imaging agent, thereby labeling the dendritic cells at the site of antigen capture. In embodiments, the methods involve detecting the presence of labeled dendritic cells in the subject at the site of antigen capture, during dendritic cell migration, or in a lymph node of the subject. In embodiments, the detecting step is repeated over the course of hours or days following administration of the antigen.

In one aspect, the invention provides methods for labeling an antigen presenting cell in vivo. In embodiments, the methods involve labeling an antigen with an imaging agent in vitro. In embodiments, the methods involve contacting an antigen presenting cell with the labeled antigen in vivo, thereby labeling an antigen presenting cell at the site of antigen capture.

In one aspect, the invention provides methods for visualizing antigen presenting cell activation and migration in a subject. In embodiments, the methods involve labeling an antigen with an imaging agent in vitro. In embodiments, the methods involve contacting one or more antigen presenting cells with the labeled antigen in vivo, thereby labeling an antigen presenting cell at the site of antigen capture. In embodiments, the methods involve detecting the presence of labeled antigen presenting cells in the subject at the site of antigen capture, during antigen presenting cell migration, or in a lymph node of the subject. In embodiments, the detecting step is repeated over the course of hours or days after contacting the antigen presenting cells with the labeled antigen.

In one aspect, the invention provides methods for inducing a detectable immune response in a subject. In embodiments, the methods involve administering to the subject an antigen comprising an imaging agent, thereby inducing a detectable immune response in the subject.

In one aspect, the invention provides methods for monitoring a subject for an immune response. In embodiments, the methods involve administering to the subject an antigen containing an imaging agent, thereby inducing a detectable immune response in the subject. In embodiments, the methods involve detecting the imaging agent in an antigen presenting cell of the subject. In embodiments, the methods involve localizing the imaging agent to a site of antigen uptake, antigen presenting cell migration, or a lymph node. In related embodiments, localization is indicative of the progress of the immune response. In embodiments, the methods involve quantifying the number of antigen presenting cells containing the imaging agent. In related embodiments, an increased number of labeled antigen presenting cells is indicative of a robust immune response. In embodiments, the methods involve quantifying the number of antigen presenting cells containing the imaging agent that are present in the lymph node. In related embodiments, an increased number of labeled antigen presenting cells in the lymph node is indicative of a robust immune response.

In one aspect, the invention provides methods for evaluating the effectiveness of an antigen. In embodiments, the methods involve administering to a subject an antigen containing an imaging agent, thereby inducing a detectable immune response in the subject. In embodiments, the methods involve detecting the imaging agent in an antigen presenting cell of the subject. In embodiments, the methods involve quantifying the number of antigen presenting cells containing the imaging agent. In related embodiments, an increased number of labeled antigen presenting cells is indicative of the effectiveness of the antigen. In one aspect, the invention provides methods for evaluating the effectiveness of an adjuvant. In embodiments, the methods involve administering to a subject an adjuvant and an antigen containing an imaging agent, thereby inducing a detectable immune response in the subject. In embodiments, the methods involve detecting the imaging agent in an antigen presenting cell of the subject. In embodiments, the methods involve quantifying the number of antigen presenting cells containing the imaging agent. In related embodiments, an increased number of labeled antigen presenting cells as compared to a control is indicative of the effectiveness of the adjuvant.

In one aspect, the invention provides methods for selecting a treatment strategy for a subject. In embodiments, the methods involve administering to a subject an immunogenic composition containing a labeled antigen, thereby inducing a detectable immune response in the subject. In embodiments, the methods involve detecting the presence or level of labeled antigen presenting cells in a lymph node of the subject. In related embodiments, detection of the labeled antigen presenting cells in the lymph node of the subject indicates that the subject is mounting an immune response against the antigen. In embodiments, the methods involve treating the subject with the immunogenic composition if labeled antigen presenting cells are detected in the lymph node of the subject.

In one aspect, the invention provides methods for quantifying antigen presenting cells in vivo. In embodiments, the methods involve labeling an antigen with an imaging agent in vitro. In embodiments, the methods involve administering the labeled antigen to a subject, thereby labeling an antigen presenting cell at the site of antigen capture. In embodiments, the methods involve obtaining a spin-echo (SE) image and a multi-gradient echo (MGE) image of the subject after administration of the labeled antigen. In embodiments, the methods involve measuring a parameter in a region of interest on the MGE image. In embodiments, the methods involve determining the number of labeled antigen presenting cells present in the region of interest. In related embodiments, the region of interest is identified from the SE image. In related embodiments, the parameter is selected from the group consisting of the number of black pixels, Tl, Rl, T2, R2, T2*, R2* phase shift, and magnetic susceptibility values. In related embodiments, the number of labeled antigen presenting cells is determined by comparing the the measured parameter to a previously established calibration curve. In one aspect, the invention provides for a kit containing an imaging agent for use in labeling an antigen presenting cell in vivo. In embodiments, the kit contains instructions for labeling an antigen of interest with the imaging agent. In embodiments, the kit contains instructions for labeling and imaging an antigen presenting cell in vivo. In embodiments, the kit contains an antigen. In related embodiments, the antigen is labeled with an imaging agent.

In one aspect, the invention provides for immunogenic compositions containing an antigen. In embodiments, the antigen contains an imaging agent.

In any of the above aspects, the antigen presenting cell is one or more macrophages, B cells, hematopoietic progenitor cells, or dendritic cells. In embodiments, the antigen presenting cell is a dendritic cell.

In any of the above aspects, the antigen is one or more polynucleotides, polypeptides, fragments thereof, infectious agents, or cells. In embodiments, the polynucleotide, polypeptide, or fragment thereof is derived from an infectious agent or a tumor. In embodiments, the infectious agent is a bacteria, fungus, virus, prion, or parasite. In embodiments, the cell is an inactivated cell. In related embodiments, the cell is an immune cell or a cancer cell.

In any of the above aspects, the antigen contains one or more imaging agents. In embodiments, the antigen contains at least two imaging agents that are detected by different imaging modalities (e.g., positron emission tomography ("PET"), single photon emission computed tomography ("SPECT"), computed tomogrpahy ("CT"), X-ray, ultrasound, fluorescence molecular tomography ("FMT"), magnetic resonance imaging ("MRI"), and the like).

In any of the above aspects, the imaging agent is an imaging agent described herein. In embodiments, the imaging agent is suitable for use in MRI, PET, SPECT, CT, X-ray, ultrasound, or FMT. In embodiments, the imaging agent is selected from the group consisting of a metal particle, a perfluorocarbon, an iodinated particle, a brominated particle, a gold-based agent, a silver-based agent, an iron-based agent, a gadolinium-based agent, a fluorescent agent, and a gas bubble. In embodiments, the imaging agent is SPIO. In embodiments, the imaging agent is derivatized with a functional group for the conjugation of a bioactive molecule. In embodiments, the imaging agent is acoustically active. In embodiments, the imaging agent is acoustically reflective.

In any of the above aspects, the labeled dendritic cells or the labeled antigen presenting cells are detected by any of the imaging methods described herein. In embodiments, the labeled dendritic cells or the labeled antigen presenting cells are detected by MRI, CT, FMT, X-ray, ultrasound, PET, or SPECT. In related embodiments, the labeled dendritic cells or the labeled antigen presenting cells are repeatedly detected over the course of hours or days following administration of the antigen and/or labeling of the dendritic cells or the antigen presenting cells.

In any of the above aspects, the labeled dendritic cells or the labeled antigen presenting cells are quantified using any method well-known in the art and any method described herein.

In any of the above aspects, the antigen is labeled by contacting the antigen with the imaging agent. In embodiments, the antigen is a cell and labeling involves contacting the cell with the imaging agent in the presence of a transfection agent. In embodiments, the antigen is a cell and labeling involves electroporating the cell in the presence of the imaging agent

In any of the above aspects, the antigen(s), including labeled antigen(s), are administered to a subject using any method or route known in the art, including the methods and routes described herein. In embodiments, the antigen(s) are administered to the subject systemically. In related embodiments, the antigen(s) are administered to the subject by injection.

In any of the above aspects, the antigen(s) are administered in combination with one or more pharmaceutically acceptable carriers, excipients, or diluents.

In any of the above aspects, the antigen(s) are administered in combination one or more adjuvants.

In another aspect, the invention provides in vivo labeled dendritic cells or antigen presenting cells for in vivo imaging produced by any of the methods described herein. In a related aspect, the invention provides methods for isolating such cells by i) obtaining a biological sample from a subject, and ii) isolating the labeled dendritic cell or antigen presenting cell from the biological sample.

In one aspect, the invention provides methods for labeling a dendritic cell in vivo. In embodiments, the method involves contacting the dendritic cell with an inactivated tumor cell comprising SPIO in an intracellular compartment of the tumor cell. In embodiments, the SPIO is transferred to the dendritic cell during antigen uptake, thereby labeling the dendritic cell.

In one aspect, the invention provides methods for visualizing dendritic cell activation and migration in a subject. In embodiments, the method involves contacting one or more dendritic cells in vivo with an inactivated tumor cell comprising SPIO in an intracellular compartment of the tumor cell. In embodiments, the SPIO is transferred to the dendritic cell during antigen uptake, thereby labeling the dendritic cells. In embodiments, the methods involve detecting the presence or level of labeled dendritic cells in the subject at the site of antigen capture, during dendritic cell migration, or in a lymph node of the subject.

In one aspect, the invention provides methods for quantifying dendritic cells in vivo. In embodiments, the methods involve contacting one or more dendritic cells in vivo with an inactivated tumor cell comprising SPIO in an intracellular compartment of the tumor cell. In embodiments, the SPIO is transferred to the dendritic cell during antigen uptake, thereby labeling the dendritic cells. In embodiments, the methods involve obtaining a spin-echo (SE) image and a multi-gradient echo (MGE) image of the subject after labeling the dendritic cells. In embodiments, the methods involve identifying a region of interest (ROI) from the SE image. In embodiments, the methods involve measuring the number of black pixels in the corresponding ROI on the MGE image. In embodiments, the methods involve determining the number of labeled dendritic cells present in the ROI by comparing the measured number of black pixels to a previously established calibration curve.

Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations disclosed herein, including those pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention.

Definitions

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

As used herein, the singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a ribonucleoside chain terminator" includes reference to more than one ribonucleoside chain terminator.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

As used herein, the terms "comprises," "comprising," "containing," "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Antigen presenting cells" (APCs) are cells that are capable of activating T cells, and include, but are not limited to, certain macrophages, B cells, hematopoietic progenitor cells, and dendritic cells.

The term "dendritic cell" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of MHC-class II expression (Steinman et al, Ann. Rev. Immunol. 9:271-296 (1991), which is hereby incorporated by reference). Dendritic cells can be isolated from a number of tissue sources, including but not limited to, peripheral blood.

As used herein, the term "imaging" refers to the use of technology to visualize a detectable agent, e.g., an imaging agent, after administration to a cell, tissue, or organ. In one embodiment, imaging is carried out by measuring the signal from the compound after localization of the compound following administration. Imaging technologies, such as PET, SPECT, CT, X-ray, ultrasound, FMT, MRI, and the like are applied.

An "imaging agent," as used herein, refers to a detectable moiety. In one embodiment, the imaging agent is employed to improve the visibility of a cell in an image, e.g., a PET, SPECT, CT, X-ray, ultrasound, FMT, MR image, and the like. In one embodiment, an imaging agent is a contrast agent. Contrast agents can be internalized by a cell or attached to a cell by any method that is well-known in the art.

By "labeling" is meant fixing a detectable moiety to a substance. In one embodiment, the detectable moiety is covalently or non-covalently attached to an antigen. In another embodiment, the detectable moiety is internalized in an APC, e.g., a dendritic cell.

As used herein, the term "particles" include, for example, liposomes, micelles, bubbles containing gas and/or gas precursors, lipoproteins, halocarbon, nanoparticle and/or hydrocarbon nanoparticles, halocarbon and/or hydrocarbon emulsion droplets, hollow and/or porous particles and/or solid nanoparticles. The particles themselves may be of various physical states, including solid particles, solid particles coated with liquid, liquid particles coated with liquid, and gas particles coated with solid or liquid. Various particles useful in the invention have been described in the art as well as means for coupling targeting components to those particles in the active composition. Such particles are described, for example, in U.S. Pat. Nos. 6,548,046; 6,821,506; 5,149,319; 5,542,935; 5,585,112; 5,149,319; 5,922,304; and European publication 727,225, all incorporated herein by reference with respect to the structure of the particles. These documents are merely exemplary and not all-inclusive of the various kinds of particulate vehicles that are useful in the invention. While nanoparticles are generally described herein, it is understood that the embodiments of the invention are not limited to nanoparticles, and that the compositions and methods described herein are similarly useful for other types of particles. The term "adjuvant" has its usual meaning in the art of vaccine technology, i.e., a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combination of vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.

The term "cell" is understood to mean embryonic, fetal, pediatric, or adult cells or tissues, including but not limited to, stem cells, precursors cells, and progenitor cells. Examples of cells include but are not limited to immune cell, stem cell, progenitor cell, islet cell, bone marrow cells, hematopoietic cells, tumor cells, lymphocytes, leukocytes, granulocytes, hepatocytes, monocytes, macrophages, fibroblasts, neural cells, mesenchymal stem cells, neural stem cells, or other cells with regenerative properties and combinations thereof.

The term "antigen" as used herein refers to any substance capable of eliciting an immune response when introduced into a subject. An immune response, includes for example, the formation of antibodies and/or cell-mediated immunity. Exemplary antigens include, but are not limited to, infectious agents (e.g., bacteria, viruses, fungi, prion, parasite, and the like), polypeptides (e.g., proteins), polynucleotides (e.g., DNA, RNA), cancer cells, including molecules expressed by cancer cells, and vaccines containing antigens.

The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non- human primate, murine, bovine, equine, canine, ovine, or feline.

"Administering" is defined herein as a means of providing a labeled antigen to a subject in a manner that results in the labeled antigen being inside the subject's body. Such an administration can be by any route including, without limitation, oral, transdermal, mucosal (e.g., vagina, rectum, oral, or nasal mucosa), by injection (e.g., subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), or by inhalation (e.g., oral or nasal). Pharmaceutical preparations are, of course, given by forms suitable for each administration route.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like, refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition, e.g., infection or cancer.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disease or condition, e.g., infection or cancer, and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated.

"Pharmaceutically acceptable" refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

"Pharmaceutically acceptable excipient, carrier or adjuvant" refers to an excipient, carrier or adjuvant that can be administered to a subject, together with a labeled antigen, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the labeled antigen.

The term "therapeutically effective amount" refers to an amount of a labeled antigen effective to "treat" a disease or infection in a subject or mammal. In the case of cancer, the therapeutically effective amount of the labeled antigen can reduce the number of cancer cells; reduce the tumor size; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor metastasis; inhibit and stop tumor growth; relieve to some extent one or more of the symptoms associated with the cancer, reduce morbidity and mortality; extend the time before cancer recurrence; prolong survival; improve quality of life; or a combination of such effects. In the case of infection by an infectious agent, therapeutically effective amount of the labeled antigen refers to one or more of the following: 1) reduction in the number of infected cells; 2) reduction in the concentration of the infectious agent present in serum; 3) inhibiting (e.g., slowing to some extent, preferably stopping) the rate of infectious agent replication; 4) relieving or reducing to some extent one or more of the symptoms associated with infection; and 6) improving quality of life (e.g., relieving or reducing the side effects associated with the administration of other drugs).

The term "immune response" refers to a cytotoxic T lymphocyte and/or a helper T lymphocyte response to an antigen. The immune response may also include a B lymphocyte antibody response which has been facilitated by the stimulation of helper T cells.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compounds, compositions, or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

DESCRIPTION OF THE DRAWINGS

Figures 1A-1D show superparamagnetic iron oxide (SPIO) labeling of B16 melanoma cells following overnight incubation. Figure 1A includes images of cell staining. The amount of SPIO present in B16 cells was visualized with Prussian Blue and FITC-anti- dextran staining. Figure IB includes a graph showing the results from a Ferrozin-based spectrophotometric iron assay. The assay was used to calculate the mean concentration of Fe per cell, and the graph represents the mean of three independent experiments done in triplicate. Error bars represent standard deviation from the mean. Figures 1C and ID include FACS results. In Figure 1C, FACS analysis of side scatter profile and FITC-anti-dextran intra-cellular staining of B16 melanoma cells indicate that nearly all cells are labeled with SPIO. In Figure ID, FITC-anti-dextran staining of SPIO-labeled cells with or without permeabilization shows that SPIO labeling is predominantly localized to intracellular compartments.

Figure 2 is a schematic outline for labeling and detecting activated APCs in vivo. (Clockwise from top left) B16 melanoma cells are incubated with SPIO overnight, washed, irradiated and combined with irradiated B78H1-GM-CSF cells. Vaccine cells are then injected into the footpads of C57BL/6 mice. Dendritic cells survey the site of the vaccination, are labeled in vivo by the capture of SPIO associated with apoptotic material released from labeled tumor cells, and traffic to popliteal LNs where axial images (black rectangle) are later obtained by MRI (top right). After in vivo imaging, the MR signal is quantified, the LNs are resected, and single-cell suspensions are magnetically separated. FACS analysis and T-cell proliferation assays are then conducted with magnetically separated cells.

Figures 3A-3D are MR images showing in vivo MR monitoring of trafficking of cells that have taken up SPIO in vivo after intra-dermal injection of GM-CSF tumor cell vaccine into footpads of mice. Included to the right of each MR image is a magnification of the inset in each figure. Open arrows indicate draining popliteal LNs from footpads receiving unlabeled GM-CSF vaccines. Closed arrows indicate draining popliteal LNs from footpads receiving SPIO-labeled GM-CSF vaccines. On multi-gradient echo images, SPIO-containing LNs have decreased signal intensity. On Day 1 (Figure 3A), neither popliteal LNs showed any evidence of hypo-intensities. On Day 3 (Figure 3B), there was a decreased signal intensity apparent in LN corresponding to popliteal LN of SPIO-labeled vaccine. At day 4 (Figure 3C) and day 8 (Figure 3D), the signal decrease persisted and there was an actual increases in popliteal LN. Images are representative of three independent experiments with 5 mice each.

Figures 4A-4F include Prussian blue iron staining of excised lymph nodes following footpad vaccination or injection of free Feridex. Figure 4A shows cells from excised lymph node 24 hours post free Feridex injection. A distinct subcapsular distribution was visualized, which became even more apparent when magnified further (Figure 4B). Figure 4C shows the cells 7 days after injection of free Feridex. A subcapsular ring of iron is still present with no iron present within the T cell zones of the lymph node. Figure 4D shows cells from excised lymph node of a mouse that received a Feridex labeled GM-CSF vaccine. No iron is present in subcapsular area. Upon consecutive magnifications (Figures 4E and 4F), iron positive cells are easily visualized within T cell zones of the lymph nodes.

Figures 5A-5C show the phenotypic and functional characterization of popliteal LN single cell suspensions. GM groups were given SPIO-labeled vaccines and control groups were from unvaccinated lymph nodes. Figure 5 A includes FACS results. Following LN resection, single cell suspensions were created from 10 pooled LNs per group and run over MACS columns. Both magnet-positive and magnetic -negative fractions were analyzed by FACS for expression of dendritic cell markers CDl lb and CDl lc (GM group is shown). Cells containing SPIO (magnet positive) displayed high levels of dendritic cell markers as well as an increased forward vs. side scatter profile. Figures 5B and 5C include graphs. As shown in Figure 5B, FACS analysis of dendritic cell markers CDl lb and CDl lc displayed a strong magnetic enrichment of dendritic cells following foot pad injection of SPIO-labeled vaccines. Error bars indicate the standard deviation of 5 experiments. Figure 5C shows the results from proliferation assays using tyrp-1 transgenic CD4⁺ T cells co-cultured with magnet-positive cells versus CDl lc-sorted, non- vaccinated dendritic cells with or without 100 μg/ml tyrp- l io6-i3o peptide. The graph shows the counts per minute after [ H]-thymidine incorporation and is representative of three experiments. Error bars show the standard deviation of triplicates.

Figures 6A-6C include FACS plots showing the effects of double magnet enrichment on dendritic cell isolation. In Figure 6A, the dendritic cell population in vaccinated lymph nodes was enriched over seven fold after one run on the magnetic column but still contained a significant portion of non-dendritic cells. As shown in Figure 6B, after running the positive cells from the first pass over the magnetic column, a greater enrichment was achieved with the remaining cells mainly being B cells (bottom graph shows cells found in red inset). As shown in Figure 6C, cells that were removed in the second magnetic enrichment seemed to be heavily B cell rich but also included a significant number of T cells not found in the double magnet-positive fraction.

Figures 7A-7D show dendritic cell trafficking after addition of the adjuvant imiquimod. lxlO⁶ SPIO-labeled vaccine cells were injected into the hind footpads of mice with one footpad receiving 10 μΐ of imiquimod cream (closed arrows) and the other receiving 10 μΐ control (open arrows). Figure 7 A includes a RARE spin echo axial image of popliteal LNs 7 days following vaccination. RARE spin echo technique is much less sensitive to SPIO and allows the borders of each LN to be clearly visualized. Figure 7B includes an MGE axial image of popliteal LNs after vaccination in same location as under Figure 7A, which shows decreased signal intensity in each LN, with a greater decrease observed on the side of the imiquimod treated footpad. Figure 7C includes the MGE image shown in Figure 7B with composite signal intensity LNs displaying the extent of SPIO coverage in each LN. Figure 7D includes a graph showing the total SPIO-positive and SPIO-negative DC counts per LN from popliteal LNs following injection of PBS, GM-CSF vaccine, or GM-CSF vaccine plus addition of 10 μΐ imiquimod cream. DC numbers were calculated by multiplying CDl lc⁺ fraction by total cell population retrieved from each group. Twenty LNs were pooled per group and the average taken. The graph is representative of three experiments.

Figures 8A-8D show the correlation between black pixels and dendritic cell number. Identical GM-CSF tumor vaccines were injected into both right and left footpads of 15 C57BL/6 mice. After 4 days, each mouse was imaged and a spin-echo (SE) and multi- gradient echo (MGE) image was taken for each mouse. A region of interest (ROI) was drawn around each LN from the SE and this ROI was copied onto the MGE image. Histograms were produced from each ROI and a low threshold pixel value was calculated from the surrounding muscle tissue. All pixels falling below this threshold value were counted as black pixels and these black pixel counts were correlated to the number of magnet-positive DCs found in each LN. Figure 8A includes an SE image of mouse with both LNs highlighted (left) and an MGE image (right) with SE ROI overlain on each LN. Figure 8B includes histograms showing left and right LN pixel distributions and those pixels falling below the threshold (shaded red). Note that very few pixels in the right LN fell below the threshold. Figure 8C includes a graph showing the correlation (R = 0.998) between the number of black pixels and the number of DCs per LN showing a strong positive correlation between the two values. The graph represents the mean of two independent experiments done with 15 mice each. Error bars represent standard deviation. Figure 8D includes a graph showing the black pixel values for mice followed serially after vaccination. Mice evaluated on day thirteen post-vaccination were placed in same response grouping as initially determined on day four. A similar response trend was found even at the later time point. The graph represents the mean of two independent experiments done with ten mice each. Error bars represent standard deviation. DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, on the discovery that antigen presenting cells (APCs) can be labeled in vivo. Accordingly, the invention provides for APCs that are labeled in vivo. The invention also provides for the cellular imaging of endogenous APC trafficking in vivo as well as the isolation of the APC populations that have processed the relevant antigens and initiated an immune response. The invention further provides a method for quantitatively assessing labeled APC populations non-invasively in vivo.

Generally, the method of this invention relies on administering an antigen that has been labeled with an imaging agent in vitro. By prelabeling the antigens, the imaging agent is transferred to APCs, e.g., dendritic cells, simultaneously with the capture of the antigen, at the site of administration. Applicants are the first to describe in vivo cell labeling through APC antigen uptake, rather than pre-labeling APCs in vitro before administration (which has now reached the clinical stage, see de Vries et ah, Nat. Biotechnol. 23:1407-13 (2005)). By labeling the antigenic component, rather than introducing ex vivo labeled APCs, the immune system is left to respond to the immunization naturally with APCs recruited to the site of administration, therefore providing a more accurate picture of the immune response.

The vaccine can consist of polynucleotides, polypeptides, fragments thereof, infectious agents, immune cells, and cancer cells. Vaccine labeling can occur by any method known to those skilled in the art including, but not limited to, cross-linking, conjugation, pinocytosis, phagocytosis, electroporation, and transfection. The imaging agent and imaging modality used can be by any method known to those skilled in the art including, but not limited to, Feridex for MR imaging; (brominated) perfluorocarbons for ¹⁹F MRI, X-ray, gas bubbles for ultrasound, radioactively or positron-labeled colloids or nanoparticles for SPECT imaging; iodinated particles for CT imaging; and gold nanoparticles for X-ray and ultrasound imaging. In the specific examples below, cancer cells were labeled in vitro with magnetic Feridex particles for detection by MRI.

The invention also provides a method of cell quantification using MRI. In a conventional T2-weighted spin echo image, a region of interest is drawn covering the lymph node of interest. On a multi-gradient echo image, that is sensitized to the relaxation effects of the magnetic nanoparticles, a signal threshold is determined based on measurements of adjacent normal tissue (e.g., muscle tissue). The number of pixels below the threshold is counted from a pixelgram. Using a previously established calibrated curve, the number of cells can be determined.

Digital medical device

The present invention is applicable to digital medical images. One example of such an image is an MR image. MRI generally relies on the relaxation properties of excited nuclei in water, for instance hydrogen, fluorine, or sodium. In conventional ¹H MRI, when the tissues or organs to be imaged are placed in a powerful, uniform magnetic field, the spins of the hydrogen protons within the tissues or organs align along the axis of the magnetic field. The tissue is then briefly exposed to pulses of electromagnetic energy (RF pulse) in a plane perpendicular to the magnetic field, causing some of the magnetically aligned hydrogen nuclei to assume a temporary non-aligned high-energy state. As the high-energy nuclei relax and realign, they emit energy which is recorded to provide information about their environment. MRI takes advantage of the fact that water relaxation characteristics vary from tissue to tissue, and this tissue-dependent relaxation effect provides image contrast, which in turn allows the identification of various distinct tissue types. In order to create an image, spatial information must be recorded along with the received tissue relaxation information. A computer applies an inverse Fourier transform to this information so that it is converted into real space to obtain the desired image. This produces detailed anatomical information of the tissues or organs under inspection.

Another such example is a CT scan image. CT scanners operate by projecting fan- shaped or cone-shaped X-ray beams from an X-ray source. The X-ray source emits X-rays at numerous view angle positions about an object being imaged, such as a patient, which attenuates the X-ray beams as they pass through. The attenuated beams are detected by a set of detector elements, which produce signals representing the intensity of the incident X-ray beams. The signals are processed to produce data representing the line integrals of the attenuation coefficients of the object along the X-ray paths. These signals are typically called "projection data" or just "projections". By using reconstruction techniques, such as filtered backprojection, useful images may be formulated from the projections. The images may in turn be associated to form a volume rendering of a region of interest.

Another such example is an ultrasound image. Ultrasound imaging is done using low- intensity ultrasound waves. Conventional ultrasound devices typically have an array of transmitter-receiver pairs. In operation, each pair only "sees" along a line, commonly referred to as a scanline, that extends from the pair into the medium. With such an assumption, a feature along that scanline can be brought into "focus" by determining the propagation times of the transmitted and reflected signals to and from the feature. A propagation time t can be calculated as t=d/v where v is the velocity of the signal in the medium, and d is the distance of interest (e.g., from the feature to the receiver). The distance d can be determined by dividing the scanline into discrete elements in a predetermined manner, such that the location of each element is known. The velocity v can either be assumed as a constant in the medium, or can be calculated in a manner generally known in the art.

Another such example is a PET image. A PET apparatus images the internal distribution of a radioactive isotope given into an object to be examined. In a diagnostic method in which the PET apparatus is used, first a positron emitting radioactive compound is introduced into the body of the object to be examined. The radioactive compound introduced in the body of the object to be examined is metabolized and accumulated in a specific portion of the body. At this time, the positron is emitted from the radioactive isotope. The emitted positron and a vicinage electron are annihilated and two gamma ray photons are emitted in opposite directions. An image is reconstructed by computer-processing from information about the detected photons.

Another such example is a SPECT image. Single Photon Emission Computed Tomography ("SPECT") is a technique where the radioactive substances ("mTc, ²⁰¹T1, ^U1ln, and ⁶⁷Ga) have longer decay times than those used in PET (ⁿC, ¹³N, ¹⁵0, ¹⁸F, ⁸²Rb, ⁶²Cu, and ⁶⁸Ga), and emit single instead of double gamma rays.

The invention is not limited to MRI, CT, ultrasound, PET, and SPECT images, and may be applied to other digital medical images that are well-known in the art, including, but not limited to, optical images, FMT images, and projection X-ray images. Conventional X- ray images may be developed on an X-ray film prior to being digitized. Imaging agents for use in MRI, CT, ultrasound, PET, SPECT, optical, FMT, and X- ray imaging as well as their mechanisms of action are well-known in the art. Such agents are described in Krause, 2002, Contrast Agents I: Magnetic Resonance Imaging (Topics in Current Chemistry), First Edition, Springer, Germany; Krause, 2002, Contrast Agents II: Optical, Ultrasound, X-ray and Radiopharmaceutical Imaging (Topics in Current Chemistry), First Edition, Springer, Germany; Krause, 2005, Contrast Agents III: Radiopharmaceuticals - From Diagnostics to Therapeutics (Topics in Current Chemistry), First Edition, Springer, Germany; Goldberg et ah, 2001, Ultrasound Contrast Agents: Basic Principles and Clinical Applications, Second Edition, Informa Healthcare, U.K.; and Merbach and Toth, 2001, The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, First Edition, Wiley, Chichester, which are hereby incorporated by reference.

The invention provides methods for imaging labeled antigen presenting cells using one or more imaging modalities. In embodiments, the antigen presenting cells are labeled with a single imaging agent. In embodiments, the antigen presenting cells are labeled with multiple imaging agents. In related embodiments, the single labeling agent is a multimode- detectable agent. Examples of multimode-detectable agents are described in International Application No. PCT/US2008/006380, which is hereby incorporated by reference.

The imaging modalities are performed at any time during or after administration of the labeled antigen. For example, the imaging studies may be performed during administration of the labeled antigen to aid in guiding the delivery of the labeled antigen to a specific location. The imaging studies may be performed after administration to monitor antigen uptake and trafficking of labeled APCs in vivo.

Additional imaging modalities may be performed concurrently with the first imaging modality, or at any time following the first imaging modality. For example, additional imaging modalities may be performed about 1 sec, about 1 hour, about 1 day, or any longer period of time following completion of the first imaging modality, or at any time in between any of these stated times. In embodiments, multiple imaging modalities are performed concurrently such that they begin at the same time following administration of the labeled antigen. One of ordinary skill in the art would be familiar with performance of the various imaging modalities contemplated by the present invention. In embodiments of the invention, the same imaging device is used to perform a first imaging modality and a second imaging modality. In embodiments, different imaging devices are used to perform the different imaging modalities. One of ordinary skill in the art would be familiar with the imaging devices that are available for performance of the imaging modalities described herein.

Imaging agents

In general, the invention includes methods for labeling antigen presenting cells in vivo with an imaging agent. The imaging agent is any compound that provides for the detection of a cell. In one embodiment, the imaging agent is amenable to detection in an image, e.g., a PET, SPECT, CT, X-ray, ultrasound, MR, FMT, optical image, and the like.

Suitable imaging agents for use in the invention are well-known in the art and are described in Krause, Contrast Agents I, supra; Krause, Contrast Agents II, supra; Krause, Contrast Agents III, supra; Goldberg, supra; and Merbach and Toth, supra. For example, imaging agents can be magnetic particles. The magnetic particles include a metal oxide particle and a coating material that is in contact with the surface of the metal oxide particle. The metal of the metal particle may include transition or lanthanide metals. Illustrative transition or lanthanide metals include iron, cobalt, gadolinium, europium and manganese. The magnetic -responsive metal oxide particles may be paramagnetic, ferrimagnetic, superparamagnetic, or anti-ferromagnetic. The coating material may be in contact with the metal oxide particle surface via any type of chemical bonding and/or physical attractive force such as, for example, covalent bonding, ionic bonding, hydrogen bonding, colloidal mixtures, or complexing. Illustrative coating materials include polysaccharides (e.g., starch, cellulose, glycogen, dextran, aminodextran and derivatives thereof), polyvinyl alcohols, polyacrylates, polystyrenes, and mixtures and copolymers thereof.

In embodiments, the metal particle is a metal oxide particle, e.g., an iron oxide, especially a superparamagnetic iron oxide. Superparamagnetic iron oxides are (on a millimolar metal basis) the most MR-sensitive contrast agents currently available. Superparamagnetic particles possess a large ferrimagnetic moment that, because of the small crystal size, is free to align with an applied magnetic field (i.e., there is no hysteresis). The aligned magnetization then creates microscopic field gradients that dephase nearby protons and shorten the T2 NMR relaxation time, over and beyond the usual dipole-dipole relaxation mechanism that affects both Tl and T2 relaxation times. Examples of superparamagnetic iron oxides include MION-46L (previously available from Harvard Medical School), Feridex (previously commercially available from Berlex Laboratories, Inc. under license from Advanced Magnetic, Inc), Endorem ferumoxides (previously commercially available from Guerbet Group), Clariscan (commercially available from Nycomed Amersham), Resovist (previously commercially available from Schering AG), Combidex (previously commercially available from Advanced Magnetics), Ferumoxytol (commercially available from AMAG pharmaceuticals), and Sinerem (previously commercially available from Guerbet Group under license from Advanced Magnetics).

Suitable imaging agents also include Chemical Exchange Saturation Transfer (CEST) agents and PARACEST agents that contain exchangeable protons (e,g, amides, amines, hydroxyls), including but not limited to polylysines, polyarginines, tryptophan, glycogen, polypeptides, glucose, and polysaccharides.

The coated metal particles can be used as magnetic probes. The magnetic probes can achieve a high degree of intracellular magnetic labeling that is non-specific (i.e., not dependent on targeted membrane receptor binding) and that can be used on virtually any mammalian cell. The magnetic probe could be used to label cells ex vivo.

Chemical modification of the coating on the coated metal oxide particles is not required. Furthermore, the mixing may be accomplished without the presence of an organic solvent. The amount of coated metal oxide particles mixed optionally with a transfection agent should be sufficient to provide uptake of the metal particles by the cell.

Other useful metals also include isotopes of those metals possessing paramagnetism which produce water relaxation properties useful for generating images with MRI devices. Suitable contrast agent metals include, but are not limited to, Mn, Cr, Fe, Gd, Eu, Dy, Ho, Cu, Co, Ni, Sm, Tb, Er, Tm, and Yb. Appropriate chelation ligands to coordinate metals can be readily incorporated into the labeled complexes of this invention. Such chelation ligands can include, but are not limited to, DTPA, EDTA, DOTA, TETA, EHPG, HBED, ENBPI, ENBPA, and other macrocycles that are well-known in the art (see Stark and Bradley, Magnetic Resonance Imaging, C. V. Mosby Co., St Louis, 1988, pp 1516). Suitable imaging agents also include perfluorocarbons (PFCs). Representative perfluorocarbons include bis(F-alkyl) ethanes such as F-44E, i-F-i36E, and F-66E;cyclic f uorocarbons, such as F-decalin, perf uorodecalin or "FDC), F-adamantane ("FA"), F- methyladamantane ("FMA"), F-l,3-dimethyladamantane ("FDMA"), F-di-or F- trimethylbicyclo[3,3,l]nonane ("nonane"); perfluorinated amines, such as F-tripropylamine ("FTPA") and F-tri-butylamine ("FTBA"), F-4-methyloctahydroquinolizine ("FMOQ"), F-n- methyl-decahydroisoquinoline ("FMIQ"), F-n-methyldecahydroquinoline ("FHQ"), F-n- cyclohexylpurrolidine ("FCHP") and F-2-butyltetrahydrofuran ("FC-75" or "RM101"). Brominated perfluorocarbons include 1-bromo-heptadecafluoro-octane (sometimes designated perfluorooctylbromide or "PFOB"), 1-bromopenta-decafluoroheptane, and I- bromotridecafluorohexane(sometimes known as perfluorohexylbromide or "PFHB"). Other brominated fluorocarbons are disclosed in U.S. Pat. No. 3,975,512. Other suitable perfluorocarbons are mentioned in EP 908 178 Al.

In embodiments, the imaging agent is a radiopaque agent. Monobromo and dibromo perfluorocarbons, including both aliphatic and cyclic compounds, exhibit radiopaque properties which make such brominated perfluorocarbons useful.

Suitable imaging agents also include iodinated and brominated particles.

Suitable imaging agents also include gold nanoparticles, cobalt nanoparticles, iron oxide nanopowder, niobium oxide nanopowder, thulium nanoparticles, cobalt oxide nanopowder, lanthanum nanoparticles, palladium nanoparticles, tin nanoparticles, aluminum oxide nanopowder, copper nanoparticles, lanthanum oxide nanopowder, platinum nanoparticles, tin oxide nanopowder, antimony nanoparticles, copper oxide nanopowder, praseodymium nanoparticles, titanium carbide nanoparticles, antimony oxide nanopowder, dysprosium nanoparticles, lithium manganese oxide nanoparticles, praseodymium oxide nanopowder, titanium nanoparticles, antimony tin oxide (ATO) nanoparticles, dysprosium oxide nanopowder, lithium nanoparticles, rhenium nanoparticles, titanium nitride nanoparticles, barium titanate nanoparticles, erbium nanoparticles, lithium titanate nanoparticles, ruthenium nanoparticles, titanium oxide nanopowder, beryllium nanoparticles, erbium oxide nanopowder, lithium vanadate nanoparticles, samarium nanoparticles, tungsten carbide nanoparticles, bismuth oxide nanopowder, europium nanoparticles, lutetium nanoparticles, samarium oxide nanopowder, tungsten nanoparticles, boron carbide nanoparticles, europium oxide nanopowder, magnesium nanoparticles, silicon carbide nanoparticles, tungsten oxide nanopowder, boron nitride nanoparticles, gadolinium nanoparticles, magnesium oxide nanopowder, silicon nanoparticles, vanadium oxide nanopowder, calcium carbonate nanoparticles, gadolinium oxide nanopowder, manganese nanoparticles, silicon nanotubes, ttterbium nanoparticles, calcium chloride nanoparticles, manganese oxide nanopowder, silicon nitride nanoparticles, yttria stabilized zirconia, calcium oxide nanopowder, hafnium oxide nanopowder, molybdenum nanoparticles, silicon oxide nanopowder, yttrium nanoparticles, calcium phosphate nanoparticles, holmium nanoparticles, molybdenum oxide nanopowder, silver nanoparticles, zinc oxide nanopowder, carbon nanohorns, indium nanoparticles, neodymium nanoparticles, strontium carbonate nanoparticles, zirconium nanoparticles, carbon nanoparticles, indium oxide nanopowder, neodymium oxide nanopowder, strontium titanate nanoparticles, zirconium oxide nanopowder, carbon nanotubes, iridium nanoparticles, nickel nanoparticles, tantalum nanoparticles, cerium nanoparticles, iron cobalt nanopowder, nickel oxide nanopowder, tantalum oxide nanopowder, cerium oxide nanopowder, iron nanoparticles, nickel titanium nanopowder, terbium nanoparticles, chromium oxide nanopowder, iron nickel nanopowder, niobium nanoparticles, terbium oxide nanopowder, carbon 60 fullerenes, carbon 70 fullerens and Carbon 85 fullerenes, single wall carbon nanotubes, multi-wall carbon nanotubes, and carbon nano fibers.

Suitable imaging agents can contain a fluorescent or near-infrared label. Fluorescent agents are well-known in the art and are available for use in the invention. Exemplary fluorescent labeling agents include, but are not limited to, Rhodamine 101, Nile Red, Nileblue A, Fluorescein, Sulforhodamine B, Sulforhodamine G, PdTFPP, DiA, 5(6)- Carboxyfluorescein, 2,7-Dichlorofluorescein, l,l\-Diethyl-4,4\-carbocyanine iodide, 3,3- Diethyl-thiadicarbocyanine iodide, Lucifer Yellow CH Dilitium salt 5(6)-Carboxy- tetramethylrhodamine B, N,N-Bis(2,4,6-trimethylphenyl)-3,4:9, 10-perylenebis-

(dicarboximide, Rhodamine B, 2-Di-l-ASP, Dichlorotris(l,10-phenanthroline)ruthenium(II), Tris(4,7-diphenyl-l,10-phenanthroline)-ruthenium(II) TMS, Tris(4,4-diphenyl-2,2-bi- pyridine)ruthenium(II), chloride, Resorufin, Ethyl Eosin, Ethyl Eosin,Coumarin 6, Rhodamine 6G, 8-Benzyloxy-5,7-diphenylquinoline, 8-Benzyloxy-5,7-diphenylquinoline (protonated), DY-500XL, DY-554, DY-633, DY-615, DY-590, DY-650, DY-490XL, DY- 520XL, DY-485XL, DY-480XL, DY-555, DY-590, DY-630, DY-631, DY-635, DY-636, DY-647, DY-651, DY-656, DY-673, DY-675, DY-676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751, DY-776, DY-782, EVOblue-30, Adams Apple Red 680, Adirondack Green 520, Birch Yellow 580, Catskill Green 540 Fort Orange 600, Hemo Red 720, Hops Yellow 560, Lake Placid 490, Maple Red-Orange 620, Snake-Eye Red 900, QD525, QD565, QD585, QD605, QD655, QD705, QD800, ATTO 465, ATTO 425,ATTO 488, ATTO 495, ATTO 520, ATTO 550, ATTO 565, ATTO 590, ATTO 610, ATTO 620, ATTO 635, ATTO 647, ATTO 655, ATTO 680, ATTO 700, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 480, Alexa Fluor 633, 5-FAM, DyLight 549 5-TAMRA, 6-HEX, 6- carboxyrhodamine 6G,6-JOE, 6-TET, BOBO-1, BOBO-3, POPO-1, POPO-3,TOTO-l, TOTO-3, YOYO-1, YOYO-3, aminomethylcoumarin, APC, BCECF, Amplex Gold (product), dichlorofluorescein, TO-PRO-1, TO-PRO-3, SYTO 11, SYTO 13, SYTO 17, SYTO 45, PO-PRO-1, PO-PRO-3, propidium iodide, Pro-Q Diamond,Pro-Q Emerald, quinine, resorufin, rhod-2, rhodamine 110, rhodamine 123, Rhodamine Green,YO-PRO-l, YO-PRO-3, SYTOX Blue, SYTOX Green, SYTOX Orange, Rhodamine Red-X rhodamine, Rhodol Green, R-phycoerythrin, SBFI, Sodium Green sulforhodamine 101, SYBR Green I, SYPRO Ruby, tetramethylrhodamine, Texas Red-X, X-rhod-1, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 610, Alexa Fluor 635 Calcein red-orange, Carboxynaphthofluorescein, DiIC18(3, ELF 97, Ethidium bromide, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610-R-PE, Alexa Fluor 647, Alexa Fluor 647- R-PE Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-APC, Alexa Fluor 680-R-PE, Alexa Fluor 700, Alexa Fluor 750, FITC, Fluo-3, Fluo-4, f uoro-emerald, FM 1-43, FM 4-64, Hoechst 33258, JC-1, JOJO-1, LOLO-1, lucifer yellow CH, LysoSensor Blue DND-192, LysoSensor Green DND-153, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, Yakima Yellow, tdTomato, Tb (Soini), SYTO RNASelect, SYTO RNASelect, Calcofluor white 2MR, DAPI, DDAO, Deep Purple, Diversa Cyan-FP, Diversa Green-FP, Dragon Green Envy Green, Ethidium bromide, Ethyl Nile Blue A, Eu (Soini), Eu203 nanoparticles, EvaGreen, mBanana, mCherry, Methylene Blue, Methylene Blue, Flash Red EX, mHoneyDew, mOrange, mPlum, mRaspberry, mRFP1.2 (Wang), mStrawberry (Shaner), mTangerine (Shaner), Pacific Orange, Plum Purple, Pontamine fast scarlet 4B, Surf Green EX, Suncoast Yellow, Cresyl Violet Perchlorate, DyLight 488, Allophycocyanin, Coumarin 6, C- Phycocyanin, CryptoLight CF1, CryptoLight CF2, CryptoLight CF3, CryptoLight CF4, CryptoLight CF5, CryptoLight CF6, R-phycoerythrin, SensiLight PBXL-1, SensiLight PBXL-3, Spectrum Aqua, Spectrum Blue, Spectrum Fred, Spectrum Gold, Spectrum Green, Spectrum Orange, Spectrum Red, 1,4-Diphenylbutadiene, 1,2-Diphenylacetylene, 1,4- Diphenylbutadiyne, 1,6-Diphenylhexatriene, Ir(Cn)2(acac), 7-Methoxycoumarin-4- Acetic Acid, 9,10-Bis(Phenylethynyl)Anthracene, 9,10-Diphenylanthracene, Acridine Orange, Acridine Yellow, Anthracene, Auramine O, Benzene, Cy3B Biphenyl, C3-Indocyanine, C3- Indocyanine, C3-Oxacyanine, C3-Thiacyanine Dye (EtOH), C3-Thiacyanine Dye (PrOH), C5-Indocyanine, C5-Oxacyanine, C5-Thiacyanine, C7-Indocyanine, C7-Oxacyanine, Coumarin 1, Dye-33, Dye-28, Dye-45, Cy3, DRAQ5, Ethyl -p-Dimethylaminobenzoate, Cy3.5, Cy2, CBQCA, Oregon Green 514, Oregon Green 488, nile red, nile blue, NeuroTrace 500525, NBD-X, monobromobimane, MitoTracker Red CMXRos, MitoTracker Orange CMTMRos, MitoTracker Green FM, Marina Blue, Magnesium Orange, Magnesium Green, LysoTracker Red DND-99, LysoTracker Green DND-26, LysoTracker Blue DND-22, LysoSensor YellowBlue DND-160, LysoSensor Green DND-153, LysoSensor Blue DND- 192, lucifer yellow CH, JC-1, indo-1, fura-2, Fura Red, Coumarin 343, Cy3Cy5 ET, Cy5.5, Cy5, Cy7, CypHer5, Coumarin 30, Coumarin 314, ECF, ECL Plus, PA-GFP (post- activation), PA-GFP (pre-activation), WEGFP (post-activation), CHOxAsH-CCXXCC, FlAsH-CCXXCC, ReAsH-CCXXCC, NIR1, NIR2, NIR3, NIR4, NIR820, SNIR1, SNIR2, SNIR4, AmCyanl, AsRed2, Azami Green monomeric, Azami Green, CFP (Campbell Tsien 2003), Citrine (Campbell Tsien 2003), DsRed, DsRed, DsRed Dimer2 (Campbell Tsien 2003), DsRed-Express Tl, EBFP (Patterson 2001), ECFP (Patterson 2001), EGFP (Campbell Tsien 2003), EGFP (Patterson 2001), Eosin Y, Fluorescein, Fluorescein-Dibase, Hoechst- 33258, Hoechst-33258, Kaede Green, Magnesium Octaethylporphyrin, DyLight 680, AAA, DyLight 649, DyLight 633, Magnesium Phthalocyanine, Magnesium Phthalocyanine, Magnesium Tetraphenylporphyrin Merocyanine 540, Naphthalene, Nile Blue (EtOH), Nile Blue, Nile Red, Octaethylporphyrin, Oxazine 1, Oxazine 170, Perylene, Phenol, Phenylalanine, Phthalocyanine, Pinacyanol-Iodide, Piroxicam, POPOP, Porphin, Lucifer Yellow CH, P-Quaterphenyl, Proflavin, P-Terphenyl, Pyrene, Quinine Sulfate, Rhodamine 123, Ethyl -p-Dimethylaminobenzoate, 1,6-Diphenylhexatriene, 2-Methylbenzoxazole, Rhodamine 6G, Rhodamine B, Riboflavin, Rose Bengal, Squarylium dye III, Stains All, Stilbene, Sulforhodamine 101, Tetrakis(o-Aminophenyl)Porphyrin, Tetramesitylporphyrin, Tetraphenylporphyrin, Tetraphenylporphyrin, Tetra-t-Butylazaporphine, Tetra-t-Butyl- naphthalocyanine, Toluene, Tris(2,2 -Bipyridyl)Ruthenium(II) chloride, and Tryptophan. Suitable imaging agents can contain a radioisotope such as 11 C, 13 N, 15 O, 18 F, 22 Na, ⁸²Rb, ⁶²Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁸¹Kr^m, ^99mTc, ^{U 1}ln, ¹²³I, ¹²⁵I, ¹³¹I, ¹³³Xe, and ²⁰¹T1.

Suitable imaging agents further include contrast agent particles that are filled with gas and coated by a shell, e.g., gas bubbles. Exemplary gases for use in the gas bubbles include but are not limited to air, nitrogen, perfluorocarbon, perfluorobutane, perfluoropropane, and perfluorohexane. Shells for use in the gas bubbles may include but are not limited to liposomes, lipospheres (e.g., acoustically active lipospheres), lipids, phospholipids, albumin, galactose, and polymers. In embodiments, the gas bubble is acoustically active. In embodiments, the gas bubble is acoustically reflective.

In embodiments, the imaging agent is acoustically active. In embodiments, the imaging agent is acoustically reflective.

One of ordinary skill in the art would be familiar with selecting the appropriate imaging agent for use in the various imaging modalities contemplated by the present invention.

Labeled antigens

The invention relates to a method for labeling APCs in vivo. In general, the methods of the invention involve administering to a subject an antigen that has been labeled with an imaging agent in vitro.

The composition contains any antigen of interest. Antigens that can be used in the present invention are compounds which, when introduced into a subject, may result in the formation of antibodies and cell mediated immunity. Suitable antigens for use in the invention include but are not limited to, natural, recombinant or synthetic products derived from viruses, bacteria, fungi, parasites, prions, and other infectious agents in addition to tumor antigens that might be used in prophylactic or therapeutic vaccines. In embodiments, the antigen of interest is a bacterial cell, a fungal cell, a parasitic cell, a virion, a prion, or a particle thereof. In embodiments, the antigen of interest is a polynucleotide, polypeptide, sugar, glycosylated agent, mucopolysaccharide, or a fragment and combinations thereof. In embodiments, the antigen of interest is a cell, including but not limited to an immune cell and a cancer cell. In embodiments, the compositions contains more than one antigen of interest.

In embodiments, the antigen of interest (e.g., polynucleotides, polypeptides, fragments thereof, infectious agents, and cells) is derived or obtained from a clinically relevant virus, including but not limited to Hepadnaviridae, including hepatitis B virus (HBV); Flaviviridae, including human hepatitis C virus (HCV), yellow fever virus, and dengue viruses; Retro viridae, including human immunodeficiency viruses (HIV) and human T lymphotropic viruses (HTLV1 and HTLV2); Herpesviridae, including herpes simplex viruses (HSV-1 and HSV-2), Epstein Barr virus (EBV), cytomegalovirus, varicella- zoster virus (VZV), human herpes virus 6 (HHV-6) human herpes virus 8 (HHV-8), and herpes B virus; Papovaviridae, including human papilloma viruses; Rhabdo viridae, including rabies virus; Paramyxo viridae, including respiratory syncytial virus; Reo viridae, including rotaviruses; Bunyaviridae, including hantaviruses; Filo viridae, including Ebola virus; Adenoviridae; Parvoviridae, including parvovirus B-19; Arenaviridae, including Lassa virus; Orthomyxo viridae, including influenza viruses; Poxviridae, including Orf virus and Monkey pox virus; Togaviridae; Coronaviridae, including corona viruses; and Picornaviridae.

In embodiments, the antigen of interest (e.g., polynucleotides, polypeptides, fragments thereof, infectious agents, and cells) is derived or obtained from a clinically relevant non- viral infectious agent, including but not limited to pathogenic protozoa from the genus Pneumocystis, Trypanosoma, Leishmania, Plasmodia, and Toxoplasma; bacteria from the genus Mycobacteria, Neisseria, Pseudomonas, Klebsiella, and Legioniella; and fungi from the genus Cryptococcus, Histoplasma, and Coccidioides.

In embodiments, the antigen of interest is a polynucleotide, polypeptide, or fragment thereof that is derived from a tumor-associated antigens that is associated with a cancer, including but not limited to melanoma, metastastic cancer, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, and pancreatic cancer. In embodiments, the antigen of interest is a melanoma cancer cell, a metastastic cancer cell, a adenocarcinoma cell, a thymoma cell, a lymphoma cell, a sarcoma cell, a lung cancer cell, a liver cancer cell, a colon cancer cell, a non-Hodgkins lymphoma cell, a Hodgkins lymphoma cell, a leukemia cell, a uterine cancer cell, a breast cancer cell, a prostate cancer cell, an ovarian cancer cell, a cervical cancer cell, a bladder cancer cell, a kidney cancer cell, or a pancreatic cancer cell.

In embodiments of the invention, the antigens of interest are labeled with one or more of the imaging agents described herein. Methods for labeling the antigen of interest are well- known in the art. Non-limiting examples of labeling methods include cross-linking, conjugation, transfection, electroporation (e.g., magnetoelectroporation), magnetofection, sonoporation (e.g., magneto sonoporation), injection (e.g., microinjection), transplantation, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, and lipofection. In some embodiments of the invention, the antigen of interest is a cell, and cellular uptake of the imaging agent occurs by contacting the cell with the imaging agent (e.g., incubation).

In embodiments, the labeled antigen is administered in combination with an adjuvant. An adjuvant is a substance or a composition of matter that when admixed with an antigen and administered into a subject, will induce a more intense immune response to the antigen than when the antigen is administered alone. Effective adjuvants are well-known in the art and include, but are not limited to, aluminum salts such as aluminum hydroxide and aluminum phosphate, muramyl peptides, bacterial cell wall components, saponin adjuvants, and other substances that act as immuno stimulating agents to enhance the effectiveness of the composition (e.g., Toll receptor antagonists). The methods of the present invention can also be used to guide the development of novel immune adjuvants because the methods can be used to evaluate the effectiveness of a particular adjuvant.

In embodiments, the labeled antigen is administered in combination with a pharmaceutically acceptable excipient or carrier. Suitable pharmaceutically acceptable excipients or carriers include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosacchandes, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). Additional pharmaceutically accetable excipients and carriers are described in Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000, which is hereby incorporated by reference.

In embodiments, the labeled antigens disclosed herein can be administered by any route well-known in the art for either local or systemic treatment. Administration can be topical (such as to mucous membranes including vaginal and rectal delivery) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In embodiments, the labeled antigen is administered by injection.

Cancer vaccines

In embodiments, the labeled antigens are labeled cancer vaccines. In related embodiments, the labeled cancer vaccine contains one or more tumor antigens. Multiple antigens can be used in this type of vaccine to vary the immune system response.

Cancer vaccines are useful as therapeutics for the treatment of specific types of cancers. Advantageously, these vaccines may be tailored to treat the cancers of particular individuals, by generating vaccines that target specific tumor antigens expressed on a tumor in a subject. Cancer vaccines typically contain inactivated tumor cells or tumor antigens that stimulate a patient's immune system. The immune system responds to this stimulation by generating immunoresponsive cells that target the cancer. Cancer vaccines can be administered after a subject has been identified as having cancer or as a prophylactic vaccination for individuals at risk of developing cancer. In embodiments, a labeled cancer vaccine is injected near or into the cancerous area of the patient. The cellular imaging methods of the present invention can then be used to monitor the effectiveness of the cancer vaccine, e.g., to evaluate the effectiveness of APCs in capturing the injected antigens and transporting them to the regions of the lymph nodes where T cell priming occurs.

Quantification of APCs using MRI

The invention also provides for a method of quantifying APCs in vivo using MRI. In embodiments, the method involves labeling an antigen with an imaging agent in vitro. In embodiments, the method involves administering the labeled antigen to a subject, thereby labeling the APC in vivo. In embodiments, the method involves obtaining a spin-echo (SE) image and a multi-gradient echo (MGE) image of the subject after administration of the labeled antigen. In embodiments, the method involves measuring the number of black pixels in a region of interest on the MGE image and determining the number of labeled antigen presenting cells present in the region of interest. In related embodiments, the region of interest is identified from the SE image. In related embodiments, the number of labeled antigen presenting cells present in the region of interest (ROI) is determined by comparing the measured number of black pixels to a previously established calibration curve. In embodiments, the calibrated curve is determined from animal studies. Alternatively, other measured parameters within this ROI can be used and compared to previously established calibration curves. These include, but are not limited, to Tl, Rl, T2, R2, T2*, R2* phase shift, and magnetic susceptibility values.

Pharmaceutical compositions

The invention features pharmaceutical compositions that contain a labeled antigen of interest for use to label APCs in vivo. The pharmaceutical compositions useful herein contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to a subject receiving the composition, and which may be administered without undue toxicity. As used herein, the term "pharmaceutically acceptable" means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. These compositions can be useful as a vaccine and/or antigenic compositions for inducing a protective immune response in a subject.

In embodiments, the invention encompasses a pharmaceutical composition containing at least one antigen of interest attached to at least one imaging agent. In related embodiments, the pharmaceutical composition contains two or more antigens of interest. The antigen(s) can be a polynucleotide, a polypeptide, a fragment thereof, an infectious agent, or a cell. In embodiments, the polynucleotide, polypeptide, or fragment thereof is derived from an infectious agent or a tumor antigen. In embodiments, the antigen is a bacterial cell, a fungal cell, a parasitic cell, a virion, a prion, or particles thereof. In embodiments, the cell is an immune cell or a cancer cell. In embodiments, the antigen(s) is attached to one, two, or more imaging agent(s). In related embodiments, the imaging agent(s) is an imaging agent as disclosed herein. In embodiments, the pharmaceutical composition contains an adjuvant. In embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier.

In embodiments, the pharmaceutical composition contains at least on antigen of interest attached to at least one imaging agent, an adjuvant, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. current edition). The formulation should suit the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably is sterile, non-particulate and/or non-pyrogenic.

The composition, if desired, can also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. The composition can be a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In embodiments, the pharmaceutical composition is supplied in liquid form, for example in a sealed container indicating the quantity and concentration of the antigen(s) of interest in the pharmaceutical composition. Preferably, the liquid form of the pharmaceutical composition is supplied in a hermetically sealed container.

Generally, pharmaceutical compositions of the invention are administered in an effective amount or quantity sufficient to stimulate an immune response against an infectious agent (e.g., virus, bacteria, parasite, prion, and the like) or a cancer. In embodiments, administration of the pharmaceutical composition of the invention elicits an immune response against the infectious agent or cancer. Typically, the dose can be adjusted within this range based on, e.g., the subject's age, the subject's health and physical condition, the capacity of the subject's immune system to produce an immune response, the subject's body weight, the subject's sex, diet, time of administration, the degree of protection desired, and other clinical factors.

Pharmaceutical compositions are administered in a manner compatible with the dose formulation. For example, the pharmaceutical composition can be systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needleless injection device. The pharmaceutical composition can also be systemically administered by intravenous injection using a needle and syringe. Alternatively, the pharmaceutical composition can be administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract or small particle aerosol (less than 10 microns) or spray into the lower respiratory tract. The pharmaceutical composition can also be administered by suppository or orally. Oral compositions may be administered as a solution, suspension, tablet, pill, capsule, or sustained release formulation. A physician or veterinarian having ordinary skill in the art can readily determine the appropriate dose and route necessary to elicit an immune response against an infectious agent or cancer in a subject.

In embodiments, the pharmaceutical composition is a vaccine. Typically vaccines are prepared in an injectable form, either as a liquid solution or as a suspension. Solid forms suitable for injection may also be prepared as emulsions, or with the polypeptides encapsulated in liposomes. Vaccine antigens are usually combined with a pharmaceutically acceptable carrier, which includes any carrier that does not induce the production of antibodies harmful to the subject receiving the carrier. Suitable carriers typically comprise large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactive virus particles. Such carriers are well-known to those skilled in the art. These carriers may also function as adjuvants.

In embodiments, the pharmaceutical composition contains an adjuvant. Adjuvants are immunostimulating agents that enhance vaccine effectiveness. If desired, the pharmaceutical composition contains an adjuvant that enhances the effectiveness of the immune response generated against the antigen(s) of interest. Effective adjuvants are well-known in the art and include, but are not limited to, aluminum salts such as aluminum hydroxide and aluminum phosphate, muramyl peptides, bacterial cell wall components, saponin adjuvants, and other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

Thus, the invention also provides a method for formulating a pharmaceutical composition that induces an immune response to an infectious agent or cancer. In embodiments, the method involves adding an effective dose of at least one antigen of interest to the composition, e.g., a polynucleotide, a polypeptide, a fragment thereof, an infectious agent, or a cell. In embodiments, the polynucleotide, polypeptide, or fragment thereof is derived from an infectious agent or a tumor antigen. In embodiments, the antigen is a bacterial cell, a fungal cell, a parasitic cell, a virion, a prion, or particles thereof. In embodiments, the cell is an immune cell or a cancer cell. In related embodiments, the antigen(s) is attached to at least one imaging agent.

In embodiments, stimulation of an immune response with a single dose is preferred. In embodiments, additional dosages are administered, by the same or different route, to achieve the desired effect. In neonates and infants, for example, multiple administrations may be required to elicit sufficient levels of an immune response. Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against infections. Similarly, adults who are particularly susceptible to repeated or serious infections, such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function or immune systems may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immune protection can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages can be adjusted or repeated as necessary to elicit and maintain desired levels of protection.

Prime Boost

The present invention also include a variety of prime-boost regimens. In these methods, one or more priming immunizations is followed by one or more boosting immunizations. The actual pharmaceutical composition can be the same or different for each immunization, and the route and formulation of the antigens can also be varied. For example, the prime-boost regimen can include administration of a pharmaceutical composition comprising at least one antigen of interest attached to at least one imaging agent. Pharmaceutical compositions of the invention may also be administered on a dosage schedule, for example, an initial administration of the pharmaceutical composition with subsequent booster administrations. In embodiments, a second dose of the composition is administered anywhere from two weeks to one year, preferably from about 1, about 2, about 3, about 4, or about 5 to about 6 months, after the initial administration. Additionally, a third dose may be administered after the second dose and from about three months to about two years, or even longer, preferably about 4, about 5, or about 6 months, or about 7 months to about one year after the initial administration. The third dose may be optionally administered when no or low levels of specific immunoglobulins are detected in the serum, urine, and/or mucosal secretions of the subject after the second dose.

The dosage of the pharmaceutical composition can be determined readily by one of skill in the art, for example, by first identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of antigen specific immunoglobulins or by measuring the inhibitory ratio of antibodies in serum samples, or urine samples, or mucosal secretions. The dosages can be determined from animal studies. A non-limiting list of animals used to study the efficacy of vaccines include the guinea pig, hamster, ferrets, chinchilla, mouse and cotton rat, and non-human primates. Most animals are not natural hosts to infectious agents but can still serve in studies of various aspects of the disease. For example, any of the above animals can be dosed with a vaccine candidate, e.g. labeled antigen of interest, to partially characterize the immune response induced, and/or to determine if any neutralizing antibodies have been produced. In addition, human clinical studies can be performed to determine the preferred effective dose for humans by a skilled artisan. Such clinical studies are routine and well-known in the art. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal test systems.

The labeled antigen(s) of interest of the invention can also be formulated with "immune stimulators." These are the body's own chemical messengers (cytokines) to increase the immune system's response. Immune stimulators include, but are not limited to, various cytokines, lymphokines, and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, and the like. The immunostimulatory molecules can be administered in the same formulation as the labeled antigen(s) of interest, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect. Thus in embodiments, the invention comprises pharmaceutical formulations comprising an adjuvant and/or an immune stimulator.

Pharmaceutical compositions of the invention that are capable of inducing an immune response in a subject are also referred to herein as immunogenic compositions. Immunogenic compositions contain any labeled antigen of interest as described herein. A vaccine can be an immunogenic composition.

Methods of Delivery

The labeled antigen(s) of interest of the invention are useful for preparing compositions that stimulate an immune response. Such compositions are useful for the treatment or prevention of infection by an infectious agent (e.g., bacteria, virus, fungus, parasite, prion, and the like). Such compositions are also useful for the treatment of diseases, including cancer. Both mucosal and cellular immunity may contribute to the immune response against infectious agents and diseases. In embodiments, the invention encompasses a method of inducing an immune response to an infection or a disease by administering to the subject a labeled antigen(s) of interest.

Labeled antigen(s) of interest of the invention can induce a substantial immune response in a subject (e.g., a human) when administered to the subject. The substantial immune response protects, ameliorates symptoms, or reduces the symptoms associated with infection or disease. In some instances, if the subject is infected, the infection will be asymptomatic. The immune response may be not a fully protective response. In this case, if the subject is infected with an infectious agent, the subject will experience reduced symptoms or a shorter duration of symptoms compared to a non-immunized subject. In embodiments, the invention encompasses a method of inducing a substantial immune response to an infection or a disease by administering at least one effective dose of a labeled antigen of the invention.

As mentioned above, labeled antigen(s) of interest of the invention prevent or reduce at least one symptom associated with an infection or a disease in a subject. A reduction in a symptom may be determined subjectively or objectively, e.g., self assessment by a subject, by a clinician's assessment, or by conducting an appropriate assay or measurement (e.g., body temperature), including, e.g., a quality of life assessment, a slowed progression of infection, disease, or additional symptoms, a reduced severity of symptoms; or a suitable assays (e.g., antibody titer and/or T-cell activation assay). The objective assessment comprises both animal and human assessments. In embodiments, the invention encompasses a method of treating an infection or a disease by administering a therapeutically effective amount of a pharmaceutical composition containing at least one labeled antigen of interest. In embodiments, the invention encompasses a method of reducing the symptoms associated with an infection or a disease by administering a therapeutically effective amount of a pharmaceutical composition containing at least one labeled antigen of interest.

Identifying a subject in need of such treatment(s) can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the agents described herein, such as a labeled antigen of interest, to a subject (e.g., animal, human) in need thereof. Such treatment will be suitably administered to subjects, e.g., humans, suffering from, having, susceptible to, or at risk for an infection, a disease, or a symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like).

Kits

The invention provides for a kit for labeling APCs in vivo. In embodiments, the kit contains one or more imaging agents as described herein. In related embodiments, the kit provides instructions for use. The instructions for use can pertain to labeling an antigen of interest with the imaging agent, administration of the labeled antigen, or monitoring the labeling and trafficking of endogenous APC.

In embodiments, the kit contains an antigen of interest labeled with an imaging agent.

In embodiments, the kit contains a cryopreserved cell that is labeled with a detectable label. Cells can be cryopreserved by methods that are known to one of skill in the art. For example, methods for cryopreserving cells are disclosed in U.S. Patent Nos. 6,176,089; 6,361,934; 6,929,948; and 6,951,712, which are hereby incorporated by reference.

EXAMPLES

It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.

Example I: Vaccine labeling properties

In order to evaluate the potential for superparamagnetic iron oxide (SPIO) particles to track antigen transfer by MR imaging, the ability of B16 melanoma tumor cells to efficiently endocytose the dextran-coated particles was first examined. Prussian Blue iron staining as well as fluorescein isothiocyanate (FITC) anti-dextran staining showed that these cells were capable of high levels of SPIO uptake and that a similar labeling efficiency was obtained with or without magnetoelectroporation (Walczak et ah, Magn. Reson. Med. 54:769-74 (2005)) (Figures 1A and IB). To confirm that tumor cells were homogeneously labeled and that SPIO particles predominantly resided within intracellular compartments, staining with anti- dextran antibody was performed with or without cellular permeabilization. A lack of anti- dextran staining in non-permeabilized cells demonstrated a primarily intracellular distribution of the SPIO particles, which were readily detected in permeabilized cells as a clear shift in fluorescence and side scatter, indicating that all cells were labeled (Figures 1C and ID).

Example 2: MRI detection of DCs in draining LNs

Immature DCs naturally sample their surroundings, and are specifically equipped with receptors that mediate the capture of apoptotic material, including antigens naturally present within a cancer cell, as well as substances experimentally placed within a tumor, e.g., a contrast agent such as SPIO. A schematic overview of a general approach for the invention is presented in Figure 2. In vitro labeled, granulocyte-macrophage colony-stimulating factor (GM-CSF) producing irradiated tumor cells are injected into the hind footpad of mice. As these cells begin to die, they release apoptotic bodies associated with SPIO. DCs then take up this material and traffic to the draining popliteal lymph nodes (LNs) (Figure 2, top row). DCs are indirectly labeled in vivo, and DC capture of antigen/SPIO allows the imaging and quantification of endogenous DC trafficking in vivo, as well as the isolation of the DC populations that have processed the relevant immunogens and initiated the immune response.

For example, a SPIO-labeled vaccine mixture was injected into the hind footpads of C57BL/6 mice (Figure 2). Mice were imaged with MRI immediately after and each day following the vaccine injections. The popliteal lymph nodes (LNs) were monitored for signs of antigen presenting cell (APC) migration from the vaccine site using a T2*-weighted multi- gradient echo MR sequence (Figure 2). Additionally, a fat suppression pulse was added in order to eliminate any chemical shift artifacts caused by the position of the popliteal lymph node in a surrounding fat pad. With these imaging parameters in place, a generally bright appearance for the LNs surrounded by a dark region of fat was anticipated. Within the nodes, any decrease in signal intensity (i.e. darkening) would indicate the presence of SPIO-labeled cells.

Approximately 3 days after the mice were injected with SPIO-labeled GM-CSF vaccine cells, localized hypo-intense regions within the lymph nodes could be defined indicating the presence of SPIO-labeled cells (Figure 3B). This negative signal persisted, and in some cases became more abundant, until day 8 (Figures 3C and 3D). As expected, no signal dropout was seen in popliteal nodes corresponding to unlabeled vaccines. The presence of SPIO was never detected before the third day post-vaccination, indicating that this SPIO content was likely transported actively by cells, rather that arriving passively as free SPIO in lymphatic flow. In fact, the injection of free (non-cell associated) SPIO resulted in the rapid appearance of signal that was localized to the subcapsular region of the LNs (Figures 4A-4C), a distribution that is consistent with previous reports (Baumjohann et ah, Eur. J. Immunol. 36:2544-55 (2006)). In contrast, after injection of SPIO-labeled tumor cells, the signal appeared later and was centrally located within the node (Figures 4D-4F), supporting the active cell transfer of SPIO in this model. This does not, however, rule out the possibility that SPIO and antigen could have been delivered partially by diffusion or by transmission from migrating DCs to "resident" DCs (Allan et ah, Immunity 25:153-62 (2006)).

Example 3: SPIO⁺ cells in the LNs are functional APCs

To determine whether SPIO detected in the draining LN was associated with functional antigen presenting cells, 10 mice were immunized with SPIO-labeled GM-CSF secreting tumor cells in both footpads. Three days post- vaccination, popliteal LNs were isolated from all vaccinated mice and single cell suspensions were obtained. A fraction of cells was stained for both CDl lc and CDl lb, cell surface markers expressed on DC subsets. The remaining unstained cells were applied to a magnetic cell sorting column and cells from both magnet-positive and magnet-negative fractions were subsequently stained with anti- CDl lc and CDl lb antibodies. Both pre-magnetic and post-magnetic cell separation fractions were then analyzed by flow cytometry. The magnetic cell separation process enriched the DC population by over 12-fold (Figure 5A and 5B). This enrichment of CDl lc⁺ and CDl lb⁺ cells indicates that DCs constitute the major population of cells that take up SPIO (Figure 6) and that the MR images have a strong correlation with DC trafficking. Additionally, this simple magnetic cell separation illustrates a major utility of this system to isolate only those cells that have captured material initially contained by the vaccine.

In order to verify that the isolated cells were in fact responsible for stimulating the immune response, magnet-enriched (SPIO⁺) cells were tested for their ability to activate T cells specific for an antigen expressed by B16 melanoma cells (tyrp-1). SPIO⁺ cells isolated from mice three days after vaccination with SPIO-labeled GM-CSF producing B16 cells induced these tyrp-1 specific T cells to undergo robust proliferation ex vivo (Figure 5C). T cell proliferation induced by control DCs isolated from unimmunized mice by magnetic isolation of CDl lc⁺ cells pulsed with or without tyrp-1 peptide served as positive and negative controls, respectively. These data show that DCs that have captured SPIO have also captured endogenous tumor antigen and are capable of activating T cells directly ex vivo. Whereas these cells most likely represent the APCs that captured antigen at the vaccine site, the possibility of secondary presentation by resident DCs cannot be ruled out (Allan et ah, Immunity 25:153-62 (2006)). The specificity of T cell responses was verified by the observation that irradiated control DCs pulsed with the relevant peptide tyrp (106-130) stimulated the transgenic T cells. Tumor antigen- specific T cell proliferation induced by SPIO⁺ cells indicates a co-localization between captured tumor antigen and SPIO by APCs in this system, confirming that the MR images are not simply demonstrating DC trafficking from the site of the vaccine but also reflecting the accumulation of cells that present antigen in the draining LN.

Example 4: Imiquimod enhances DC trafficking into draining LNs

It has been previously shown that murine DCs injected into imiquimod-treated skin resulted in maturation and migration of these DCs (Nair et ah, J. immunol. 171:6275-82 (2003)). To evaluate the capacity of MR imaging to detect differences in DC trafficking, the properties of the Toll Like Receptor 7 (TLR7) agonist imiquimod were exploited to induce DC maturation. SPIO-labeled vaccine was injected into both hind footpads of mice and imiquimod was applied to the right footpad and a control lotion was applied to the left footpad of each mouse 30 minutes post vaccine. Mice were either followed serially for 9 days by MRI or sacrificed after 3 days for magnetic cell separation of LN cells and DC quantification.

As depicted in Figure 7, the application of imiquimod resulted in a modest enhancement in trafficking of DCs to the popliteal LN (Figures 7A-7C). In spite of this modest adjuvant effect however, the sensitivity of MR imaging was such that many LNs draining the imiquimod treated sites were nearly completely blacked out by the increased signal. Accordingly, spin echo (SE) pulse sequences were used, which are not susceptible to iron, to elucidate the borders of the LNs from the surrounding fat (Figure 7A). These SE images were then overlaid on the multi-gradient echo (MGE) image and composite images created to show the extent of DC trafficking (Figures 7B and 7C). MR detection of enhanced trafficking of these antigen bearing DCs into the draining popliteal LN was confirmed by magnetic cell separation (Figure 7D). In some cases, the number of magnet^"1" DCs found in the imiquimod treated group was double that of the control group; however, this response was highly variable. These results demonstrate that imiquimod enhances DC migration in the setting of GM-CSF tumor cell vaccination. More importantly, they establish that MRI detection of DC trafficking has sufficient resolution and sensitivity to evaluate a key attribute of an immune adjuvant, i.e., the ability to initiate a program in resident DCs leading to their migration from the site of antigen capture to the T cell zone of draining lymph nodes.

Example 5: Black pixel count provides DC quantification in LNs

In order to assess the ability to monitor natural biological variability in this system, 15 mice were vaccinated in both hind footpads with a SPIO-labeled GM-CSF tumor cell vaccine. Four days post-vaccination, all mice were imaged and those pixels with a signal intensity falling below a defined threshold ("black pixels") were calculated for each popliteal LN. Figure 8A shows how the LN borders were delineated using a combination of SE and MGE images. Figure 8B displays histograms from the corresponding left and right lymph nodes seen in Figure 8A. These nodes serve as an example of high versus low levels of black pixels. Within 12 hours of the last scan, each popliteal LN was removed and pooled with LNs of similar black pixel counts. Pooling, rather than individual LN analysis was performed due to the limitations of magnetic separation using small numbers of cells. Those nodes with counts above 10,000 black pixels were grouped as "High pixel nodes"; 5,000-10,000 black pixels were considered "Mid pixel nodes"; and any node containing less than 5,000 black pixels was placed in the "Low pixel" group. Single cell suspensions were made from these pools and cells were run over MACS columns to isolate the SPIO⁺ cells. Following flow cytometric analysis, CD1 lc⁺CDl lb⁺ DCs were enumerated for each group. As the number of black pixels increased between groups, the corresponding number of SPIO⁺ DCs present in each LN group sharply increased as well (Figure 8C). These results demonstrate the strong correlation between black pixel count and the number of DCs that have presumably captured antigen and trafficked to the LN. Additionally, when these mice were followed past day 4 to investigate the possibility of delayed DC trafficking in our system, there was no significant change in response status as measured by black pixel counts (Figure 8D). The absolute number of black pixels did increase in each group, but the general response trend remained the same.

The range of DCs traveling to the draining LNs ranged from 5,000 DCs to as many as 40,000, representing a range of biological variation that approaches 8-fold. This biological variation in antigen delivery almost certainly manifests itself as variability in individuals' response to the therapy, and it represents a parameter that is ripe for optimization with newer vaccine formulations and adjuvants. Thus, the present invention is an invaluable tool to prospectively monitor this variability in preclinical as well as clinical studies.

A major parameter limiting immune responses to vaccination is the number of activated antigen presenting cells (APCs) that capture antigen and migrate to draining lymph nodes (LNs). Currently, a quantitative non-invasive technique for monitoring in vivo antigen capture and delivery is lacking. The use of cellular imaging is a promising approach for this purpose; however, cellular imaging currently requires ex vivo pre-labeling of cells with contrast agents followed by reintroduction of cells into the subject being monitored. Described herein is an in vivo labeling method from an antigen labeled with an imaging agent to endogenous antigen presenting cells, in situ, in order to quantify APC delivery to LNs. A subject is immunized with an antigen that is labeled with the imaging agent. APCs that have captured the imaging agent (and the antigen) are imaged over time as they accumulate in LNs. As desmonstrated herein, the present invention is capable of monitoring, in vivo, the trafficking of labeled APCs inducing a antigen- specific immune response, and that these cells can be recovered ex vivo. Excellent correlation is observed between in vivo and ex vivo quantification of APCs, such that the present invention can be used to detect increased APC trafficking elicited by an adjuvant. The present invention is well- suited to monitor the kinetics of antigen delivery for cancer vaccination in a clinically applicable manner using FDA-approved materials.

Dendritic cell tracking using MRI has been evaluated by other groups in the past (Baumjohann et al., Eur. J. Immunol. 36:2544-55 (2006); de Vries et al., Nat. Biotechnol. 23:1407-13 (2005); and Ahrens et al, Nat. Biotechnol. 23:983-7 (2005). However, previous MRI-based approaches used an in vitro labeling method which relied upon the reintroduction of DCs to the subject. The efficacy of this approach is highly dependent upon the administered route and is strictly limited to DC immunization (de Vries et ah, Nat. Biotechnol. 23:1407-13 (2005)). In contrast, the present invention allows for the detection of DCs in vivo following in vivo labeling of the endogenous DC pool generated in response to an injected antigen. More specifically, the present invention relies on cell-to-cell transfer of a contrast agent from antigens labeled with an imaging agent to those DCs responsible for initiating the anti-antigen immune response. By using an appropriate imaging agent, the present invention allows for "antigen tracking" using imaging systems such as MRI, X-ray, ultrasound, CT, and the like.

The present invention can be used to accurately monitor the frequency of antigen- bearing DCs that migrate from vaccine sites to the draining LNs. In addition, the present invention can be used to quantify the DC response to specific antigens and to quantify the impact of immune adjuvants. Furthermore, the use of certain imaging agents permits experimental sorting of cells involved in initiating the immune response in vivo. Thus, the appeals of the present invention are manifold. In the context of immunotherapies, the present invention can be used to image a process that occurs early in the development of an immune response. Unlike existing techniques, it does not require tissue destruction or compromise the natural physiology of the immune response. The use of cellular imaging allows precise anatomical localization of the labeled cells after migration. As an experimental tool, the ability to sort the labeled cells not only confirms the imaging studies, but also provide a means to further characterize features of DCs associated with immune priming, as well as how different forms of tumor cell death impact the maturation program of the APC (Sauter et al, J. Exp. Med. 191:423-34 (2000)).

The results reported herein were obtained using the following methods and materials. Mice

Transgenic mice expressing a T cell receptor specific for Tyrosinase Related Protein- 1 (TPvP-1) have recently been described (Muranski et al, Blood 112:362-73 (2008)). All experiments involving the use of mice were performed in accordance with protocols approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine.

Tumor cells

MHC class I-negative, C57BL/6-derived murine melanoma cell lines B78H1-GM- CSF(33) and B16 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin- streptomycin (50 U/ml), L-glutamine (2 mM), HEPES buffer (5 mM), non-essential amino acids, and 2-mercaptoethanol (100 uM) (complete medium) and grown at 37°C in a humidified 5% C0₂ atmosphere. Additionally, B78H1-GM-CSF cells were maintained in high dose hygromycin (1200 ug/ml) to assure high levels of GM-CSF expression, which averaged 1 μg GM-CSF/10⁶ cells/24 hours.

Preparation of SPIO-labeled tumor cells

For magnetic-labeling of tumor cells, B16 melanoma cells were irradiated with 10,000 rads and incubated for 18 hours in a culture medium containing a ferumoxides injectable solution (335 μg Fe per milliliter, Feridex®, Berlex Laboratories, Wayne, NJ). Initial studies comparing magnetoelectroporated cells were labeled as described previously (Walczak et al., Magn. Reson. Med. 54:769-74 (2005)). The tumor cells were washed three times in phosphate-buffered saline (PBS) to remove any excess particles.

Iron labeling efficiency was verified by Prussian blue staining and anti-dextran staining (Bulte et al, Methods Enzymol. 386:275-99 (2004)). Total iron content of SPIO- labeled cells was assessed by a Ferrozin-based spectrophotometric assay following acid- digestion of labeled cell samples (Bulte et al., Nat. Biotechnol. 19:1141-7 (2001)). The iron content was approximately 8 pg of iron per cell. Cell viability was determined by Trypan blue staining, showing comparable viability (more than 90%) for unlabeled tumor cells and SPIO-labeled tumor cells (data not shown).

MRI analysis

For each MR image, a region of interest (ROI) was manually selected for each popliteal LN using the SE image. These ROIs were then copied into the MGE image of the corresponding slice and a pixel intensity histogram was created using ImageJ software. Pixel intensity histograms were also created for adjacent muscle tissue and the darkest pixels were chosen to represent a low signal threshold for "black pixel" determination. This calibration was done slice by slice for each image containing a lymph node. Lymph node pixels falling below this minimum pixel intensity were summated for all slices and we present these values as "black pixels." For all calculations, black pixel status was defined on Day 4 post vaccine.

In addition to black pixel calculations, color composite images were created for imiquimod treated mice in order to visualize the extent of DC trafficking in blacked out nodes. Again, SE images were used to delineate the borders of lymph nodes and these borders were then copied into MGE images. ImageJ software was used to create color composites of the lymph nodes and these composites were overlain on the same MGE image they were created from.

Dendritic cell isolation

Single cell suspensions were made from popliteal LNs of vaccinated mice on either day 3 or day 5 post-vaccination by mechanical disruption and filtration through nylon mesh. Cells were pooled washed in PBS and then run over MACS MS columns (Miltenyi Biotec, Auburn, CA) for SPIO-positive cell selection. For optimal enrichment, this process was repeated in order to minimize the number of SPIO^" cells. Of the SPIO negative fraction, CDl lc⁺ cells were isolated using CDl lc-coated magnetic beads (Miltenyi Biotec) and MACS MS columns. Antibodies and flow cytometry

Antibodies for immunostaining and flow cytometry were anti-dextran anti-CDl lc (FITC), anti-CDl lb (PE), anti-I-A^b (biotin-conjugated), anti-B220 (PE), anti-NKl.l (APC), and streptavidin (APC). Anti-dextran was obtained from Stem Cell Technologies (Vancouver, Canada). All other antibodies were purchased from BD Biosciences (Mountain View, CA). Intracellular staining for dextran was performed using the Cytofix/Cytoperm kit (PharMingen, San Diego, CA). All FACS analysis was performed on a FACSCalibur (Becton Dickinson, San Jose, California) and analyzed using CellQuest software (Becton Dickinson, San Jose, California).

Proliferation Assay

Single cell lymphocyte suspensions were made from spleens of tyrp-l^RAG^"7" mice by mechanical disruption over nylon mesh. Tyrp-1 transgenic CD4⁺ T cells were enriched by removing CD8⁺ T cells and B220⁺/MHCII⁺ cells, as previously described (Lu et ah, J. Exp. Med. 191:541-50 (2000)). In brief, cells were incubated with biotinylated antibodies against CD8⁺, B220⁺, and MHC II⁺ cells. Cells were then removed by incubation with streptavidin- conjugated magnetic beads (Dynal, Oslo, Norway) and magnetic separation. The CD4⁺ T cell enriched (5 x 10⁴ per well) lymphocytes from above were mixed with SPIO positive cells and CDl lc positive cells from both non-vaccinated LNs and SPIO negative cell fractions of vaccinated LNs at the indicated ratios. All antigen presenting cells were irradiated with 2000 rads prior to addition. Tyrp-1106-130 peptide was added to appropriate wells at 100 μg/mL. 72 hours after incubation, cells were pulsed with [ H] thymidine (1 μΟΛνεΙΙ [0.037 MBq]) and cultured for 12 hr before harvesting and measuring scintillation counts. Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Incorporation by Reference

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method for labeling a dendritic cell in vivo, the method comprising contacting the dendritic cell with an antigen comprising an imaging agent, thereby labeling the dendritic cell.

2. The method of claim 1, wherein the antigen is an inactivated tumor cell.

3. The method of claim 2, wherein the tumor cell comprises superparamagnetic iron oxide (SPIO).

4. The method of claim 3, wherein the SPIO is present in an intracellular compartment of the tumor cell, and wherein the SPIO is transferred to the dendritic cell during antigen uptake.

5. A method for visualizing dendritic cell activation and migration in a subject, the method comprising i) contacting one or more dendritic cells in vivo with an antigen comprising an imaging agent, thereby labeling the dendritic cells at the site of antigen capture; and ii) detecting the presence of labeled dendritic cells in the subject at the site of antigen capture, during dendritic cell migration, or in a lymph node of the subject, thereby visualizing dendritic cell activation and migration in the subject.

6. The method of claim 5, wherein the detecting step is repeated over the course of hours or days following administration of the antigen.

7. A method for labeling an antigen presenting cell in vivo, the method comprising i) labeling an antigen with an imaging agent in vitro; and ii) contacting an antigen presenting cell with the labeled antigen in vivo, thereby labeling an antigen presenting cell at the site of antigen capture.

8. A method for visualizing antigen presenting cell activation and migration in a subject, the method comprising i) labeling an antigen with an imaging agent in vitro; ii) contacting one or more antigen presenting cells with the labeled antigen in vivo, thereby labeling an antigen presenting cell at the site of antigen capture; and iii) detecting the presence of labeled antigen presenting cells in the subject at the site of antigen capture, during antigen presenting cell migration, or in a lymph node of the subject, thereby visualizing antigen presenting cell activation and migration in the subject.

9. The method of claim 8, wherein the detecting step is repeated over the course of hours or days after contacting the antigen presenting cells with the labeled antigen in vivo.

10. The method of any one of claims 1-9, wherein the antigen is selected from the group consisting of polynucleotides, polypeptides, and fragments thereof.

11. The method of claim 10, wherein the polynucleotide, polypeptide, or fragment thereof is derived from an infectious agent or a tumor.

12. The method of claim 11, wherein the infectious agent is selected from the group consisting of bacteria, fungi, viruses, prions, and parasites.

13. The method of any one of claims 1-9, wherein the antigen is an inactivated cell.

14. The method of claim 13, wherein the cell is an immune cell or a cancer cell.

15. The method of any one of claims 1-14, wherein the imaging agent is suitable for use in MRI, PET, SPECT, CT, X-ray, ultrasound, or FMT.

16. The method of any one of claims 1-15, wherein the imaging agent is selected from the group consisting of a metal particle, a perfluorocarbon, an iodinated particle, a brominated particle, a gold-based agent, a silver-based agent, an iron-based agent, a gadolinium-based agent, a fluorescent agent, and a gas bubble.

17. The method of any one of claims 1-16, wherein the imaging agent is SPIO.

18. The method of any one of claims 1-17, wherein the imaging agent is derivatized with a functional group for the conjugation of a bioactive molecule.

The method of any one of claims 5, 6, and 8-18, wherein the labeled dendritic cells or the labeled antigen presenting cells are detected by MRI, CT, FMT, X-ray, ultrasound, PET, or SPECT.

20. The method of claim 7 or 8, wherein step i) of the method comprises contacting the antigen with the imaging agent.

21. The method of claim 20, wherein the antigen is a cell, and wherein step i) of the method comprises contacting the cell with the imaging agent in the presence of a transfection agent or electroporating the cell in the presence of the imaging agent.

22. The method of any one of claims 1-21, wherein the antigen comprises a second imaging agent.

23. The method of claim 22, wherein the dendritic cell(s) or the antigen antigen presenting cell(s) are labeled with the second imaging agent.

24. The method of any one of claims 1-21, wherein the method further comprises contacting the dendritic cell(s) or the antigen antigen presenting cell(s) with a second antigen, wherein the second antigen comprises a second imaging agent, thereby labeling the dendritic cell(s) or the antigen presenting cell(s) with the second imaging agent at the site of antigen capture.

25. The method of any one of claims 22-24, wherein the imaging agent and the second imaging agent are different.

26. The method of claim 25, wherein the imaging agent and the second imaging agent are detected by different imaging modalities.

27. A method for quantifying antigen presenting cells in vivo, the method comprising i) labeling an antigen with an imaging agent in vitro; ii) administering the labeled antigen to a subject, thereby labeling an antigen presenting cell at the site of antigen capture; iii) obtaining a spin-echo (SE) image and a multi-gradient echo (MGE) image of the subject after administration of the labeled antigen; iv) measuring a parameter in a region of interest on the MGE image; and v) determining the number of labeled antigen presenting cells present in the region of interest.

28. The method of claim 27, wherein the region of interest is identified from the SE image.

29. The method of claim 27 or 28, wherein the parameter is selected from the group consisting of the number of black pixels, Tl, Rl, T2, R2, T2*, R2* phase shift, and magnetic susceptibility values.

30. The method of any one of claims 27-29, wherein step v) of the method comprises comparing the the measured parameter to a previously established calibration curve.

31. The method of any one of claims 27-30, wherein the antigen is selected from the group consisting of polynucleotides, polypeptides, fragments thereof, infectious agents, and inactivated cells.

32. The method of claim 31, wherein the polynucleotide, polypeptide, fragment thereof is derived from an infectious agent or a tumor.

33. The method of any one of claims 27-32, wherein the imaging agent is SPIO.

34. The method of any one of claims 5, 6, and 8-33, wherein the antigen(s) are administered to the subject systemically.

35. The method of claim 34, wherein the antigen(s) are administered to the subject by injection.

36. The method of any one of claims 7-35, wherein the antigen presenting cell(s) are selected from the group consisting of a macrophage, a B cell, a hematopoietic progenitor cell, and a dendritic cell.

37. The method of any one of claims 1-36, wherein the antigen(s) are administered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

38. The method of any one of claims 1-37, wherein the antigen(s) are administered in combination with an adjuvant.

39. An in vivo labeled dendritic cell or antigen presenting cell for in vivo imaging produced by the method of any one of claims 1-4 and 7.

40. A method for isolating the in vivo labeled dendritic cell or antigen presenting cell of claim 39, the method comprising i) obtaining a biological sample from a subject, and ii) isolating the labeled antigen presenting cell from the biological sample.

41. A method for labeling a dendritic cell in vivo, the method comprising contacting the dendritic cell with an inactivated tumor cell comprising SPIO in an intracellular compartment of the tumor cell, wherein the SPIO is transferred to the dendritic cell during antigen uptake, thereby labeling the dendritic cell.

42. A method for visualizing dendritic cell activation and migration in a subject, the method comprising i) contacting one or more dendritic cells in vivo with an inactivated tumor cell comprising SPIO in an intracellular compartment of the tumor cell, wherein the SPIO is transferred to the dendritic cell during antigen uptake, thereby labeling the dendritic cells; and ii) detecting the presence or level of labeled dendritic cells in the subject at the site of antigen capture, during dendritic cell migration, or in a lymph node of the subject, thereby visualizing dendritic cell activation and migration in the subject.

43. A method for quantifying dendritic cells in vivo, the method comprising i) contacting one or more dendritic cells in vivo with an inactivated tumor cell comprising SPIO in an intracellular compartment of the tumor cell, wherein the SPIO is transferred to the dendritic cell during antigen uptake, thereby labeling the dendritic cells; ii) obtaining a spin-echo (SE) image and a multi-gradient echo (MGE) image of the subject after labeling the dendritic cells; iii) identifying a region of interest (ROI) from the SE image; iv) measuring the number of black pixels in the corresponding ROI on the MGE image; and v) determining the number of labeled dendritic cells present in the ROI by comparing the measured number of black pixels to a previously established calibrated curve.

44. A kit comprising an imaging agent for use in labeling an antigen presenting cell in vivo.

45. The kit of claim 44, wherein the kit further comprises instructions for labeling an antigen of interest with the imaging agent.

46. The kit of claim 44 or 45, wherein the kit further comprises instructions for labeling and imaging an antigen presenting cell in vivo.

47. The kit of any one of claims 44-46, wherein the kit further comprises an antigen.

48. The kit of claim 47, wherein the antigen is selected from the group consisting of polynucleotides, polypeptides, fragments thereof, infectious agents, and inactivated cells.

49. The kit of any one of claims 47 or 48, wherein the antigen is labeled with the imaging agent.

50. An immunogenic composition comprising an antigen, wherein the antigen comprises an imaging agent.

51. An immunogenic composition comprising an inactivated cell or an infectious agent, wherein the inactivated cell or the infectious agent comprises an imaging agent.

52. The immunogenic composition of claim 51, wherein the cell or infectious agent is a bacterial cell, a virion, or a cancer cell.

53. The immunogenic composition of any one of claims 50-52, wherein the imaging agent is SPIO.

54. A method for inducing a detectable immune response in a subject, the method comprising administering to the subject an antigen comprising an imaging agent, thereby inducing a detectable immune response in the subject.

55. A method of monitoring a subject for an immune response, the method comprising i) administering to the subject an antigen comprising an imaging agent, thereby inducing a detectable immune response in the subject; and ii) detecting the imaging agent in an antigen presenting cell of the subject.

56. The method of claim 55, wherein the method further involves localizing the imaging agent to a site of antigen uptake, antigen presenting cell migration, or a lymph node, wherein the localization is indicative of the progress of the immune response.

57. The method of claim 55 or 56, wherein the method further involves quantifying the number of antigen presenting cells comprising the imaging agent, wherein an increased number is indicative of a robust immune response.

58. The method of claim 57, wherein the method further involves quantifying the number of antigen presenting cells comprising the imaging agent that are present in the lymph node, wherein an increased number is indicative of a robust immune response.

59. A method for evaluating the effectiveness of an antigen, the method comprising i) administering to a subject an antigen comprising an imaging agent, thereby inducing a detectable immune response in the subject; ii) detecting the imaging agent in an antigen presenting cell of the subject; and iii) quantifying the number of antigen presenting cells comprising the imaging agent, wherein an increased number is indicative of the effectiveness of the antigen.

60. A method for evaluating the effectiveness of an adjuvant, the method comprising i) administering to a subject an adjuvant and an antigen comprising an imaging agent, thereby inducing a detectable immune response in the subject; ii) detecting the imaging agent in an antigen presenting cell of the subject; and iii) quantifying the number of antigen presenting cells comprising the imaging agent, wherein an increased number as compared to a control is indicative of the effectiveness of the adjuvant. A method for selecting a treatment strategy for a subject, the method comprising i) administering to a subject an immunogenic composition comprising an antigen, wherein the antigen comprises an imaging agent, thereby inducing a detectable immune response in the subject; ii) detecting the presence or level of labeled antigen presenting cells in a lymph node of the subject, wherein detection of the labeled antigen presenting cells in the lymph node of the subject indicates that the subject is mounting an immune response against the antigen; and iii) treating the subject with the immunogenic composition if labeled antigen presenting cells are detected in the lymph node of the subject.