Patent Application
20210046008
Geometry and spatial organization are critical components in many biological systems. The importance of geometry and spatial organization can be seen within the immune system in a variety of ways including during the interaction of a T cell with an antigen presenting cell (APC), which is a critical determinant of T cell fate and effector function. With activation, APC, such as dendritic cells (DC), have major changes in their cell morphology, which results in a significant increase in their overall cell surface area. Such changes in cell morphology facilitate interaction with naïve T cells and ultimately affect T cell fate and outcome. T cell activation is further modulated by the formation of a large surface area of close membrane apposition between the DC and T cell membrane termed the “immune synapse.” Grakoui, A., et al., The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221-227 (1999). Monks, C. R., et al., Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82-86 (1998). Lee, K. H., et al., T cell receptor signaling precedes immunological synapse formation. Science 295, 1539-1542 (2002). Thus, taking into account the geometry and spatial organization is important in studying biological responses.
Reductionist systems also have facilitated the study of effective immune responses. One such system has been the development of acellular artificial antigen presenting cells (aAPCs). These systems have been made by coupling proteins required for T cell activation to particles. Minimally, T cell activation requires two sets of receptor-receptor interactions between cells. The first interaction, Signal 1, is the binding of major histocompatibility complexes (MHC) or a surrogate, such as anti-CD3, to the T cell receptor (TCR). The second interaction, Signal 2, is the binding of costimulatory receptors on the APC, such as B7.1, to ligands on the T cell, such as CD28. Accordingly, aAPC have been generated by coupling proteins that deliver Signal 1 and Signal 2 to the surface of microbeads (
Such systems have been broadly applied to tumor immunotherapy, vaccination, and immunosuppression, and are amenable to in vivo or ex vivo T cell stimulation and offer possible novel translational approaches to immunotherapy. Ugel, S., et al., In vivo administration of artificial antigen-presenting cells activates low-avidity T cells for treatment of cancer. Cancer Res 69, 9376-9384 (2009). Ndhlovu, Z. M., et al., Dynamic regulation of functionally distinct virus-specific T cells. Proc Natl Acad Sci USA 107, 3669-3674 (2010). Ito, F., et al., Antitumor reactivity of anti-CD3/anti-CD28 bead-activated lymphoid cells: implications for cell therapy in a murine model. J Immunother 26, 222-233 (2003). Lum, L. G., et al., Immune modulation in cancer patients after adoptive transfer of anti-CD3/anti-CD28-costimulated T cells-phase I clinical trial. J Immunother 24, 408-419 (2001). Taylor, P. A., et al., The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood 99, 3493-3499 (2002). Balmert, S. C. and Little, S. R., Biomimetic delivery with micro- and nanoparticles. Adv Mater 24, 3757-3778 (2012).
While useful, the Signal 1 and Signal 2 paradigms alone do not capture aspects of spatial organization or the geometry of interactions. Previous work developing artificial systems for stimulation of effective in vitro and in vivo T cell responses has not attempted to re-capitulate these aspects of APC behavior. As a result, all particle systems tested thus far have used spherical particles for their aAPC platforms, which unlike DC, minimize surface area for a given volume (
Particle shape has only recently become a design parameter of interest in the field of material design for drug delivery. Shape can play a role in tuning the rate and mechanism of cellular uptake, Wang, J., et al., More effective nanomedicines through particle design. Small 7, 1919-1931 (2011), can dramatically reduce internalization by phagocytic cells, such as macrophages, Champion, J. A. and Mitragotri, S., Role of target geometry in phagocytosis. Proc Natl Acad Sci USA 103, 4930-4934 (2006). Sharma, G., et al., Polymer particle shape independently influences binding and internalization by macrophages. J Control Release 147, 408-412 (2010), can change the biodistribution of the drug delivery vehicle, Champion, J. A., et al., Particle shape: a new design parameter for micro- and nanoscale drug delivery carriers. J Control Release 121, 3-9 (2007). Devarajan, P. V., et al., Particle shape: a new design parameter for passive targeting in splenotropic drug delivery. J Pharm Sci 99, 2576-2581 (2010), and can affect the ability of a particle to bind a cell, in part, by increasing the surface area for interaction. Champion, J. A., et al., Particle shape: a new design parameter for micro- and nanoscale drug delivery carriers. J Control Release 121, 3-9 (2007). Harris, B. J. and Dalhaimer, P., Particle shape effects in vitro and in vivo. Front Biosci (Schol Ed) 4, 1344-1353 (2012). Yoo, J. W. and Mitragotri, S., Polymer particles that switch shape in response to a stimulus. Proc Natl Acad Sci USA 107, 11205-11210 (2010)
In some aspects, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) comprising: (a) a three-dimensional microparticle or nanoparticle having an asymmetrical shape that mimics a shape of a cell or a microorganism; and (b) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell. In certain aspects, the cell or a microorganism is selected from the group consisting of a bacterium, an archaeon, a protozoan, a fungus, an algae, and a virus. In some aspects, the cell or microorganism has a shape selected from the group consisting of a spiral, a cube, a rod, a comma, a star, a square, a column, a polyhedran, a helix, an icosahedran, a cylinder, a tetrahedron, and a pyramid.
In other aspects, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) comprising: (a) a three-dimensional microparticle or nanoparticle having an asymmetrical shape, wherein the asymmetrical shape has at least one surface having a radius of curvature along at least one axis which is in one of the following ranges: (a) about 1 nm to about 10 nm; (b) about 11 nm to about 100 nm; (c) about 101 nm to about 400 nm; (d) about 401 nm to about 1 μm; (e) about 10 μm to about 20 μm; (0 about 20 μm to about 100 μm; and (g) about 101 μm to about—1 mm; and (b) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell.
In yet other aspects, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) comprising (a) a three-dimensional microparticle or nanoparticle having an asymmetrical shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c); and (b) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell. In particular aspects, the asymmetrical shape comprises an ellipsoid, which can be described by one of the following equations: a>b=c (prolate ellipsoid); a>b>c (tri-axial ellipsoid); and a=b>c (oblate ellipsoid).
In another aspect, the presently disclosed subject matter provides a method for modulating a T cell, the method comprising (a) providing an artificial antigen presenting cell (aAPC) comprising: (i) a three-dimensional microparticle or nanoparticle having an asymmetrical shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c); and (ii) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell and one or more molecules capable of interacting with a receptor other than a TCR on the T cell to costimulate the T cell; (b) contacting the T cell with the aAPC; and (c) allowing the T cell to be modulated.
In still another aspect, the presently disclosed subject matter provides a method for making an artificial antigen presenting cell (aAPC) comprising a three-dimensional microparticle or nanoparticle having an asymmetricall shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c); and one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell, the method comprising (a) providing or preparing a plurality of microparticles or nanoparticles; (b) preparing a film comprising the plurality of microparticles or nanoparticles; (c) stretching the film comprising the plurality of microparticles or nanoparticles to form a plurality of three-dimensional microparticles or nanoparticles having an asymmetrical shape; (d) harvesting the plurality of three-dimensional microparticles or nanoparticles having an asymmetrical shape; and (e) coupling to the plurality of three-dimensional microparticles or nanoparticles having an asymmetrical shape one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell.
In yet further aspects, the presently disclosed subject matter provides a device for stretching a film.
Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
Control of biological cells is crucial for the study and treatment of many diseases. The presently disclosed subject matter achieves cellular control by developing an innovative class of synthetic micro-and nano-sized particles that mimic the function of target cells. These artificial/synthetic cells are engineered in new ways so that the following parameters are able to be tuned independently: size of the artificial cells, shape of the artificial cells, composition and location of proteins immobilized to the cell surface, composition of soluble factors released from the artificial cells, the time course of this drug release, and the time scale of the biodegradation of the artificial cells themselves (
Specific applications of the artificial cells include their use as artificial antigen presenting cells. The artificial antigen presenting cells could potentially enable new treatments for infectious diseases and cancer. The creation of a new biomaterial class of synthetic micro- and nano-particles that mimic the function(s) of target cells could potentially have both scientific and translational impact.
Previous work developing particle-based acellular, artificial antigen presenting cells (aAPCs) has focused exclusively on spherical platforms. The geometry of the interaction between an activated antigen presenting cell and a T cell, however, is quite different from that of two interacting spheres. To explore the role of shape in this process, the presently disclosed subject matter adopts the approach of altering the shape of microparticles or nanoparticles, e.g., in some embodiments, PLGA microparticles, to generate ellipsoidal microparticles having varying long axis lengths and aspect ratios (ARs). The presently disclosed “stretched” biomimetic non-spherical aAPCs with high AR showed significantly enhanced activity above that seen with spherical aAPCs with particle volume and antigen content held constant.
Without wishing to be bound to any one particular theory, confocal imaging indicates that this effect may be due to improved interaction along the long axis of the stretched aAPCs and that T cells will preferentially migrate to and are activated by interaction with the long axis of the particle. Enhanced activity of non-spherical, high-aspect ratio aAPCs also was observed in vivo with high-aspect ratio aAPCs which, in a representative example, improved melanoma survival compared to non-cognate aAPCs (p=0.004), as well as cognate spherical aAPCs (p=0.05). The presently disclosed subject matter indicates that particle geometry is a critical design criterion in the generation of aAPCs and provides insights into the essential role of geometry involved in the interaction between T cells and biological APCs.
Accordingly, in some embodiments, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) comprising: (a) a three-dimensional microparticle or nanoparticle having an asymmetrical shape that mimics a shape of a cell or a microorganism; and (b) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell. In certain embodiments, the cell or a microorganism is selected from the group consisting of a cell, a bacterium, and a virus.
As used herein, the term “microorganisms” refers to unicellular organisms, such as the prokaryotes, including bacteria and archaea, single-celled protozoa, single-celled fungi, single-celled algae, and viruses.
Examples of shapes of cells or microorganisms include, but are not limited to, spiral, cube-like, rod, comma-shaped, star shaped, square, columnar, polyhedral, helical, icosahedral, cylindrical, tetrahedron, and pyramid.
In other embodiments, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) comprising: (a) a three-dimensional microparticle or nanoparticle having an asymmetrical shape, wherein the asymmetrical shape has at least one surface having a radius of curvature along at least one axis which is in one of the following ranges: (a) 1 nm-10 nm; (b) 11 nm-100 nm; (b) 101 nm-400 nm; (c) 401 nm-1 μm; (d) 10 μm-20 μm; (e) 20 μm-100 μm (f) 101 μm-1 mm.; and (b) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell.
In yet other embodiments, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) comprising (a) a three-dimensional microparticle or nanoparticle having an asymmetrical shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c); and (b) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell.
As the particle becomes flatter, the radius of curvature becomes larger. Conversely, as a surface on the particle becomes more curved, the radius of curvature becomes smaller. In some embodiments, the particle has at least one surface that has a radius of curvature that does not include the range from about 1 micron to about 10 microns.
In some embodiments, the non-spherical shape comprises a prolate ellipsoid, which is defined by the equation a>b=c. In other embodiments, the non-spherical shape comprises a tri-axial ellipsoid, which can be described by the equation a>b>c. In yet other embodiments, the non-spherical shape comprises an oblate ellipsoid, which can be described by the equation a=b>c. In other embodiments, the non-spherical shape has a dimension (a) along the x axis is equal to the dimension (b) along they axis, both of which are much less than dimension (c) along the z-axis, such that a=b<<c and the three-dimensional microparticle or nanoparticle comprises a rod.
As used herein, the term “nanoparticle,” refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and all integers and fractional integers in between). In some embodiments, the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm. In some embodiments, the nanoparticle has a diameter of about 200 nm. In other embodiments, the nanoparticle has a diameter of about 500 nm. In yet other embodiments, the nanoparticle has a diameter of about 1000 nm (1 μm). In such embodiments, the particle also can be referred to as a “microparticle”. Thus, the term “microparticle” includes particles having at least one dimension in the range of about one micrometer (μm), i.e., 1×10−6 meters, to about 1000 μm. The term “particle” as used herein is meant to include nanoparticles and microparticles.
As used herein, an “artificial antigen presenting cell” (aAPC) is an artificial biomimetic particle-based platform that has been made in vitro and has not been made naturally by a body.
As used herein, an “antigen presenting cell” is a cell that comprises a molecule that is capable of binding to the T cell receptor (TCR) on a T cell and has other factors which direct the T cell response. As used herein, the term “capable of” refers to having the capacity or ability, for example, a molecule that is “capable of” binding to a receptor is a molecule that has a three-dimensional structure having the capacity or ability to interact and/or bind with a receptor.
As used herein, the term “molecule” generally refers to two or more atoms held together by covalent bonds. Therefore, a molecule can be relatively small, such as the size of a peptide, or it can be relatively big, such as the size of a protein comprising several polypeptides. As used herein, a molecule is not restricted by size.
As used herein, an “antigen” is a molecule or part of a molecule that can be bound by a MHC and presented to a T cell receptor. Examples of antigens include, but are not limited to, microbial structures, such as bacterial and fungal cell walls, protozoan cell membranes, bacterial and fungal capsules, viral capsids, viral glycoproteins, microbial toxins, allergens, such as dust mites, pollen, hair, dander, bee venom, drugs, and other agents causing allergic reactions, foreign tissue or cells, and the body's own cells that the body fails to recognize as normal, such as cancer cells, infected cells, and cells involved in autoimmune diseases.
In some embodiments, the molecule is an antigen presented by the major histocompatibility complex (MHC). In some embodiments, the major histocompatibility complex is the human leukocyte antigen (HLA). In general, MHC Class II molecules mediate specific immunity to an antigen and Class I molecules mediate destruction of host cells displaying that antigen. In other embodiments, an antigen presenting cell also comprises a molecule that binds to or interacts with another receptor on a T cell.
In particular embodiments, the three-dimensional microparticle or nanoparticle comprises a prolate ellipsoid, wherein the dimension (a) along the x-axis is greater than the dimension (b) along the y-axis, and wherein the dimension (b) along the y-axis is equal to the dimension (c) along the z-axis, such that the prolate ellipsoid can be described by the equation a>b=c. In other embodiments, the ellipsoid is a tri-axial ellipsoid, wherein the dimension (a) along the x-axis is greater than the dimension (b) along the y-axis, and wherein the dimension (b) along the y-axis is greater than the dimension (c) along the z-axis, such that the tri-axial ellipsoid can be described by the equation a>b>c. In yet other embodiments, the ellipsoid is an oblate ellipsoid, wherein the dimension (a) along the x-axis is equal to the dimension (b) along the y-axis, and wherein the dimension (b) along the y-axis is greater than the dimension (c) along the z-axis, such that the oblate ellipsoid can be described by the equation a=b>c. The presently disclosed asymmetrical particles, however, do not include embodiments in which a=b=c.
In still other embodiments, the microparticle or nanoparticle has an aspect ratio ranging from about 1.1 to about 5. In other embodiments, the aspect ratio has a range from about 5 to about 10. In some embodiments, the aspect ratio has a range from about 1.5 to about 3.5, including 1.5, 2, 2.5, 3, and 3.5.
Generally, the three-dimensional microparticle or nanoparticle comprises a material having one or more of the following characteristics: (i) one or more degradable linkages; (ii) a stretchable Young's modulus ranging from 106-1010 N/m2 and in some embodiments 107-109 N/m2; and (iii) a glass transition temperature such that the material comprising the three-dimensional microparticle or nanoparticle is a solid at room temperature and/or body temperature. The particles can also be composed of copolymers, with one or more constituents being defined as above.
As used herein, “glass transition temperature” refers to the temperature at which amorphous polymers undergo a transition from a rubbery, viscous amorphous liquid, to a brittle, glassy amorphous solid. As used herein, “Young's modulus of elasticity” quantifies the elasticity of the polymer. It is defined, for small strains, as the ratio of rate of change of stress to strain.
As used herein, “biodegradable” compounds are those that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components preferably do not induce inflammation or other adverse effects in vivo. In certain preferred embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.
Generally, to be biodegradable, the presently disclosed materials, e.g., microparticles and/or nanoparticles, contain a degradable linkage. Representative degradable linkages include, but are not limited to:
In some embodiments, the three-dimensional microparticle or nanoparticle comprises a material having one or more of the following characteristics: (i) one or more degradable linkages; (ii) a stretchable modulus; and (iii) a glass transition temperature such that the material comprising the three-dimensional microparticle or nanoparticle is a solid at room temperature and/or body temperature. In other embodiments, the degradable linkage is selected from the group consisting of an ester linkage, a disulfide linkage, an amide linkage, an anhydride linkage, and a linkage susceptible to enzymatic degradation. In particular embodiments, the microparticle or nanoparticle comprises a biodegradable polymer or blends of polymers selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(beta-amino ester) (PBAE), polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), poly(acrylic acid) (PAA), poly-3-hydroxybutyrate (P3HB) and poly(hydroxybutyrate-co-hydroxyvalerate). In other embodiments, nondegradable polymers that are used in the art, such as polystyrene, are blended with a degradable polymer or polymers from above to create a copolymer system. Accordingly, in some embodiments, a nondegradable polymer is blended with the biodegradable polymer.
Other biodegradable polymers suitable for use with the presently disclosed subject matter are provided in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., published Nov. 18, 2010, which is incorporated herein by reference in its entirety.
A T cell or T lymphocyte is a cell that belongs to a group of white blood cells known as lymphocytes and plays a central role in cell-mediated immunity. Different types of T cells include, but are not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells (also known as suppressor cells), and natural killer T cells. A T cell can be distinguished from other lymphocytes by the presence of a T cell receptor (TCR) on its cell surface. A T cell receptor is a protein that is found on the surface of a T cell and it is responsible for recognizing antigens bound to MHC molecules. This recognition ensures that only a T cell with a TCR specific to a particular antigen is activated. In some embodiments, the interaction of the TCR with a MHC:antigen complex is the first signal in the activation or modulation of a T cell. The antigen can be presented to the T cell by a MHC-dimer or -tetramer molecule. The MHC-dimer or -tetramer molecule can be easily loaded with any MHC-restricted peptide of interest. By loaded, it is meant that the peptide is attached in some way to the MHC-dimer or -tetramer, whether by covalent interactions or by noncovalent interactions or both.
In some embodiments, the molecule capable of interacting with the TCR is a peptide. As used herein, a “peptide” is a shorter polymer of amino acid monomers. In some cases, a peptide comprises only a few amino acids and in other embodiments, a peptide comprises hundreds of amino acids. A “polypeptide” refers to a longer polymer of amino acids, generally from about 50 amino acids to a larger protein. In general, there is overlap between the size of a peptide and a polypeptide. In some embodiments, peptides interacting with MHC Class II molecules are typically 13 to 17 amino acids in length, and in other embodiments, shorter or longer peptides are common and allowed. In still other embodiments, peptides interacting with MHC Class I molecules have more stringent requirements of generally less than 15 amino acids length.
In other embodiments, the peptide is loaded onto a MHC-Ig molecule or a HLA:Ig molecule before interacting with the TCR. In some embodiments, the HLA:Ig molecule is a HLA:A2:Ig molecule.
In some embodiments, a second signal is required for T cell activation or modulation. This second signal may be another receptor on the T cell that acts to costimulate the T cell. Examples of receptors include, but are not limited to CD28 (Accession Nos. NP_006130, NP_001230006, NP_001230007), CD2 (Accession No. NP_001758), CD5 (Accession No. NP_055022), CD30 (Accession No. NP_001235), OX40 (NP_003318), 4-1BBL (Accession No. NP_001070977), ICAM-1 (Accession No. NP_000192) and LFA-1 (NP_002200), for example. Accession numbers are given only as examples and similar proteins with other accession numbers also may be used in the presently disclosed subject matter.
A “CD28 receptor” is a protein found on the surface of a T cell. It is activated or induced by stimuli, such as products of pathogens and breakdown products of cells, via the B7 protein. In some embodiments, the CD28 receptor is involved in the second signal required for T cell activation or modulation.
A “B7 protein” is a peripheral membrane protein that when paired with the CD28 receptor on a T cell, enhances or decreases the signal from the interaction of the TCR with the MHC: antigen complex on a T cell. The B7 protein comprises the CD80 (or B7.1) protein and the CD86 protein (or B7.2).
As used herein, the MHC: antigen complex, the Ig molecule, the B7 protein, the anti-CD3 molecule, and any other molecule comprising the aAPC may constitute the whole molecule, protein, or complex or it may constitute the domain of the molecule, protein, or complex that is involved in binding or interacting with the T cell. In other words, in some embodiments, the aAPC of the presently disclosed subject matter can comprise only the part of the molecule, protein, or complex that interacts with the T cell. For example, an aAPC comprising the B7 protein may comprise a full length B7 protein, only one polypeptide of the B7 protein, or only the CD28 receptor binding domain of the B7 protein.
In some embodiments, the T cell is modulated by costimulation of both the TCR and one of the other receptors on a T cell. In some embodiments, costimulation of both the TCR and one of the other receptors results in activation of the T cell, while in other embodiments, costimulation of both the TCR and one of the other receptors results in a decrease in activation of the T cell. Modulation can include activation, inactivation, and other forms of altered cell function.
Accordingly, in some embodiments, the aAPC further comprises a molecule capable of interacting with a receptor other than a TCR on the T cell. In some embodiments, the receptor other than a TCR on the T cell is selected from the group consisting of CD28, CD2, CD5, CD44, OX40, 4-1BBL, ICAM-1, and LFA-1. In particular embodiments, the receptor other than a TCR on the T cell is CD28. In some cases, the CD28 receptor interacts with an anti-CD28 antibody or a B7 protein.
The aAPC can further comprise a drug or therapeutic agent. In some embodiments, the drug or therapeutic agent is a protein. As used herein, a “drug” is a substance that has a physiological effect when introduced into a subject. A drug also can be tested in vitro, for example, in cell culture, to determine its effect on a cell. A “protein drug” is a drug comprising a peptide(s) or polypeptide(s).
In other embodiments, the aAPC further comprises at least one peptide or protein on one or more surfaces of the aAPC and/or within the aAPC.
The presently disclosed subject matter also provides kits comprising the aAPC. In general, the kits comprise aAPCs in an amount sufficient to treat at least one patient at least one time to modulate T cells in the patient. Typically, the aAPCs of the kit will be supplied in one or more container, each container containing a sufficient amount of particles for at least one dosing of the patient.
In other embodiments, the presently disclosed subject matter includes a kit comprising the raw materials for making the presently disclosed aAPCs and the presently disclosed device for stretching the particles (see
The aAPCs of the presently disclosed subject matter can be used to modulate T cells in many different applications. For example, the aAPCs can be used to study the CD8+ T cell response to influenza epitopes in vitro (Lee et al., 2011) and also can be used in methods of preventing and/or treating patients for influenza in vivo. As another example, these aAPCs, such as HLA-A2-Ig-based aAPCs, can be used to stimulate CD8(+) T cells using antigens specific for influenza and measles (Ndhlovu et al., 2010).
As a further example, compositions and methods of the presently disclosed subject matter can use aAPCs to study, prevent, or treat autoimmune diseases, such as Type 1 diabetes mellitus (T1D). In the case of T1D, one potential target is the Fas ligand (FasL) (Xiao et al., 2011).
As still another example, an aAPC based system can be used for ex vivo expansion of human cytomegalovirus specific cytotoxic T lymphocytes for adoptive immunotherapy (Chiu et al., 2011).
As another example, the aAPC based system can be used in a novel cellular microarray assay utilizing soluble peptide-loaded HLA-A2-Ig dimer complexes that optimizes the avidity of peptide-HLA binding by preserving the molecular flexibility of the dimer complex while attaining much higher concentrations of the complex relative to cognate T cell receptors. This high-throughput broad-based assay enables the use of cellular microarrays to determine the stability and flux of antigen-specific T cell responses within and across populations (Li et al., 2011).
Further, the aAPCs can be used to deplete allo- or antigen-specific T cells, such as by taking advantage of the Fas/Fas ligand signaling pathway to efficiently induce antigen-presenting cell-mediated apoptosis in targeted T cells (Scheutz et al., 2010).
Also, aAPCs made by covalently coupling (pep)MHC-Ig dimers and B7.1-Ig molecules to magnetic beads can be used to improve immunotherapy efficacy by rescuing antitumor activity of low-avidity tumor-specific cytotoxic T lymphocytes (Ugel et al., 2009).
In addition, killer artificial APCs (kappaaAPCs) can be generated by coupling an apoptosis-inducing alpha-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These kappaaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)-dependent fashion and therefore have potential for use in treatment of autoimmune diseases and allograft rejection (Schuetz et al., 2008).
As another example, natural killer T (NKT) cells play a pivotal role in maintaining immune homostasis. They recognize lipid antigen in the context of CD1d molecules and subsequently produce cytokines that activate cells of both the innate and adaptive immune responses. Many studies examining patients with autoimmune disease or cancer have shown that there is a reduction in both NKT cell number and function. The aAPC system can facilitate the growth and analysis of NKT cells through the use of CD1d-expressing aAPC. CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical (Valpha14(−)) NKT cells. Importantly, CD1d-Ig aAPC can expand NKT cells from cancer patients and can be used as a novel tool in adoptive immunotherapeutic strategies (Webb et al., 2009)
The aAPCs can also be used in providing a sensitive and specific assay for evaluation of immune responses. Cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. An assay for quantifying virus-specific CD8(+) T cells can combine the use of HLA-A2 immunoglobulin-based aAPCs for stimulation of antigen-specific CD8(+) T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-gamma) mRNA (Ndhlovu et al. 2009). This assay is able to analyze the CD8(+) T cell responses to a wide variety of antigens.
As another example, natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. For example, given that ovarian cancer-associated ascites has been reported to have increased levels of gangliosides, the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells can be examined using aAPCs (Webb et al., 2008).
As a further example, adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells. It has been found that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity, such as in a human/SCID mouse melanoma model (Durai et al., 2008). Therefore, therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL can be used to control tumor growth.
As still another example, lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, aAPCs can be used to study the early stages of lipid overload and antigen presentation by treating aAPCs with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet, for example, and looking at immunity by the effect on MHC I-mediated antigen presentation (Shaikh et al., 2008).
In some embodiments, the presently disclosed subject matter provides a method for modulating a T cell, the method comprising (a) providing an artificial antigen presenting cell (aAPC) comprising: (i) a three-dimensional microparticle or nanoparticle having an asymmetrical shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c); and (ii) one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell and one or more molecules capable of interacting with a receptor other than a TCR on the T cell to costimulate the T cell; (b) contacting the T cell with the aAPC; and (c) allowing the T cell to be modulated.
As used herein, “contacting” or “interacting” means any action that results in at least one molecule affecting another molecule, either by physically contacting, or by getting in close enough proximity that one molecule affects the actions of another molecule. For example, the TCR of a T cell may interact with a MHC: antigen complex in such a way that the antigen of the MHC: antigen complex activates the T cell without physically binding to the TCR. In another example, the antigen physically contacts the TCR.
The method can be practiced in vivo, in which case contacting means exposing at least one T cell in a subject to at least one aAPC. According to the presently disclosed subject matter, contacting includes exposing at least one T cell to at least one aAPC, such as, for example, by administering at least one dose of aAPC to a subject via any suitable route. It also may comprise exposing cells in vitro or ex vivo by introducing, and preferably mixing, at least one aAPC and at least one T cell in a controlled environment, such as in a cell culture dish or a tube. According to the presently disclosed subject matter, contacting includes introducing, exposing, and the like, at least one aAPC at a site distant from the cells to be contacted, and allowing the bodily functions of the subject, or natural (e.g., diffusion) or human-induced (e.g., swirling) movements of fluids to result in contact of the aAPC(s) and the T cell(s). Where practiced ex vivo, the cells also may be re-introduced into a subject, preferably the subject from whom they were obtained.
The aAPCs may be used for multiple manners of T cell modulation including T cell activation and T cell inactivation/inhibition.
T cell activation and/or modulation refers to the engagement of both the T cell receptor and another receptor on the T cell. In the absence of costimulation by another receptor, T cell receptor signaling alone results in anergy, a state in which the T cell cannot activate an effective immune response. Anergy, however, may be taken advantage of for therapeutic uses. For example, the immune response to grafting of transplanted tissue and organs can be minimized without weakening the entire immune system. As another example, anergy may be used to induce activated lymphocytes to become unresponsive with autoimmune diseases, such as diabetes, mellitus, multiple sclerosis, and rheumatoid arthritis.
Effective T cell activation, which means that both the TCR and another receptor that provides costimulation are stimulated, results in a cascade of events resulting in clonal expansion or proliferation of the T cell to increase the number of T cells capable of binding to the specific antigen. “Clonal expansion”, “T cell proliferation” and “T cell expansion” are used interchangeably herein.
In some embodiments, the molecule capable of interacting with the TCR is a peptide. In other embodiments, the peptide is loaded onto a MHC-Ig molecule or a HLA:Ig molecule before interacting with the TCR.
In some embodiments, the receptor other than a TCR on the T cell is selected from the group consisting of CD28, CD2, CD5, CD44, OX40, 4-1BBL, ICAM-1, and LFA-1. In other embodiments, the receptor other than a TCR on the T cell is CD28. In still other embodiments, the CD28 receptor interacts with an anti-CD28 antibody or the B7 protein.
In some embodiments, the T cell used in the methods is a naïve T cell. A “naïve T cell” is a T cell that can respond to a novel pathogen that the immune system has not yet encountered. Recognition by a naïve T cell of its cognate antigen results in the initiation of an immune response. In other embodiments, modulation of the T cell results in proliferation of the T cell.
The methods for using an aAPC of the presently disclosed subject matter can be in vitro, in vivo, or ex vivo. In some embodiments, contacting occurs in a subject, such as in a human or in a non-human animal. In other embodiments, contacting comprises administering to the subject one or more doses of aAPCs in an amount sufficient to treat a disease, disorder, or dysfunction. A disease, disorder, or dysfunction refers to any condition that affects the health of a subject. As the presently disclosed subject matter affects T cells which are a central part of the immune response, methods and compositions of the presently disclosed subject matter will affect a wide variety of diseases, disorders, or dysfunctions. Examples include cancer and infectious diseases, which may be treated by an increased immune response. Other examples include autoimmune diseases, allergies, and transplanted tissue that may be treated by an attenuated immune response. The presently disclosed subject methods and compositions may be modified to treat a particular disease, disorder, or dysfunction.
Administering may be by oral ingestion, through injection, by infusion, through topical administration, through inhalation, through sublingual absorption, through rectal or vaginal delivery, subcutaneously, and combinations thereof. In some embodiments, the disease, disorder, or dysfunction is cancer or an infectious disease. In other embodiments, the method is an ex vivo method comprising removing at least one T cell from the subject, contacting the at least one T cell with at least one aAPC, and returning the T cell to the subject. In still other embodiments, the in vitro method occurs in cell culture.
In general, the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like. As used herein, a “dose” refers to the amount of aAPCs administered to a subject that is sufficient to treat the subject for a disease, disorder, or dysfunction.
The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein.
The presently disclosed subject matter also provides methods for preparing an aAPC. In some embodiments, the method is a method for making an artificial antigen presenting cell (aAPC) comprising a three-dimensional microparticle or nanoparticle having asymmetrical shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c); and one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell, the method comprising (a) providing or preparing a plurality of microparticles or nanoparticles; (b) preparing a film comprising the plurality of microparticles or nanoparticles; (c) stretching the film comprising the plurality of microparticles or nanoparticles to form a plurality of three-dimensional microparticles or nanoparticles having an asymmetrical shape; (d) harvesting the plurality of three-dimensional microparticles or nanoparticles having an asymmetrical shape; and (e) coupling to the plurality of three-dimensional microparticles or nanoparticles having an asymmetrical shape one or more molecules capable of interacting with one or more T cell receptors (TCRs) of a T cell. In some embodiments, the film is heated before being stretched.
In other embodiments, hyper-dense ligand coated particles that have a surface density greater than what has currently been achieved can be formed. These particles can be formed by stretching microparticles or nanoparticles into an asymmetrical shape, adding the functional ligands to the particles, and then allowing the particles to relax back partially or completely to the original spherical shape. For example, a plurality of three-dimensional microparticles or nanoparticles can be relaxed back partially or completely to a spherical or near spherical shape. In the case of an ellipsoid, the parameters (a), (b), or (c) are approximately equal in a “near spherical shape.”
The reaction scheme for one embodiment of these methods is shown in
Accordingly, in some embodiments, the presently disclosed methods comprise a method wherein a plurality of three-dimensional microparticles or nanoparticles is relaxed back partially or completely to a spherical shape. In some embodiments, relaxing occurs by the addition of heat. In some embodiments, relaxing occurs by the addition of heat. In other embodiments, chemicals can be added to the plurality of particles to allow the particles to resume their partial or complete original shape.
In yet other embodiments, the presently disclosed subject matter provides a device for stretching a film, the device comprising: at least a first and a second block adapted to immobilize at least two edges of a film, the at least first and second blocks comprising one or more surfaces in contact with the film; the at least first and second blocks positioned parallel to one another and perpendicular and in operational communication with at least two parallel rods, wherein at least the second block is adapted to be selectively positioned along the at least two parallel rods relative to the first block; the device further comprising one or more of (i) a cord attached to the second block and (ii) a screw attached to the cord, each of which is adapted to selectively position the second block along the at least two parallel rods relative to the first block.
In some embodiments, the device further comprises one or more cushings in operational communication with the at least two parallel rods. In yet other embodiments of the presently disclosed device, the first and the second block comprise at least one patterned face to facilitate immobilizing the at least two edges of the film. In other embodiments, the at least first and second blocks further comprise one or more rubber pads in operational communication with the one or more surfaces in contact with the film.
Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.
The poly(lactide-glycolide) (PLGA) microparticles were made by dissolving 200 mg of acid-terminated PLGA (50:50 LA/GA, MW 38,000-54,000, Sigma-Aldrich) in 5 ml of dicholoromethane (ACS grade, Sigma-Aldrich). The dissolved PLGA was then added dropwise to 50 mL of an ice-cold 1% poly(vinyl alcohol) (PVA) solution, which was homogenized at 5,000 rpm. After dropwise addition, the solution was allowed to homogenize for an additional minute, and then added to a 100 mL solution of 0.5% PVA, which was stirring at 500 rpm in the cold room (at 4° C.). After stirring for 4 hours to allow for solvent evaporation, the particles were centrifuged (4000 rpm for 5 min) and washed 3×, resuspended in 0.5 mL of deionized water and lyophilized.
Lyophilized microparticles were added to a solution containing 10% PVA and 2% glycerol by weight at 5 mg/mL (particles/mL solution). Then the film was poured on a leveling table and allowed to dry in a chemical hood overnight. After overnight drying at room temp, strips of the film were cut out and placed on a stretching device consisting of two blocks that can be separated by sliding on rods or by pulling on a cord (see
Referring now to
In some embodiments, stretching device 1800 includes cushings (element A in
Stretching device 1800 when fully assembled with the additional elements A-C shown in
Stretching device 1800 as depicted in
In representative embodiments, the film and custom-made stretcher were placed in a 90° C. oven for 10 minutes and then the film was slowly stretched inside of the oven to the desired stretch ratio by separating the two blocks. After stretching, the film was allowed to cool down to room temperature. The stretched section was cut out of the film and dissolved in 10 mL of deionized water, then centrifuged (4000 rpm for 5 min) and washed 3×, and finally resuspended in 200 μL of deionized water and lyophilized. “Non-stretched” particles also are prepared in the same way—they were heated alongside of the stretched particles, but simply not stretched.
Soluble MHC-Ig dimers were prepared and loaded with peptide as described. Schneck, J. P., J. E. Slansky, S. M. O'Herrin, and T. F. Greten. 2001. Monitoring antigen-specific T cells using MHC-Ig dimers. Current protocols in immunology/edited by John E. Coligan . . . [et al.]. Chapter 17: Unit 17.2.
Briefly, Db-Ig molecules were stripped under mildly acidic conditions (pH 6.5) and refolded in the presence of 40-fold molar excess peptide and 2-fold molar excess of human β2-microglobulin. Peptides GP100 (KVPRNQDWL; the “cognate” peptide) and ASN (ASNENMETH; a “non-cognate” peptide) were purchased from Genscript (Piscataway, N.J.). Protein concentration was determined after labeling by size exclusion High Performance Liquid Chromatography.
Stretched and non-stretched microparticles were resuspended in coupling buffer (0.1M MES pH 6.0), and activated with EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, Sigma-Aldrich) and sulfo-NHS (N-hydroxysulfosuccinimide). As a representative example, 5 mg of microparticles were resuspended in 1 mL of coupling buffer and activated with 10 mg of EDC and 13 mg of sulfo-NHS for 15 min at 1000 rpm on a multitube vortexer (VWR). Activated microparticles were then centrifuged, the supernatant was removed, and the aAPCs were resuspended in 1 mL PBS (pH 7.4) and transferred to a 5 mL glass scintillation vial for coupling. After addition of protein (bivalent cognate (+) or non-cognate (−) peptide-in-MHC and anti-CD28 antibody), the reaction was allowed to proceed in the cold room (4° C.) for 4 hours. After 4 hours, the aAPCs were centrifuged and washed 2× with PBS, then centrifuged again and re-suspended in 200 μL of 0.2 μm-filtered sterilized 90-mg/mL endotoxin-free sucrose solution, frozen, and lyophilized overnight.
Lyophilized particle samples were spread on conductive carbon tape mounted on aluminum SEM mounts (Electron Microscopy Sciences, Hatfield, Pa.). Samples were sputter coated with a chromium sputter coater and imaged on a Leo/Zeiss Field emission SEM in the Johns Hopkins Core Microscopy facility. Particle size and aspect was quantified using ImageJ software. For spherical particles, a single diameter was measured for each particle. For non-spherical particles, two diameters were measured (long-axis and short-axis) and the aspect ratio was calculated by dividing the two.
Surface protein quantification was performed by conjugation of unlabeled anti-CD28 mAb and fluorescently labeled MHC-IgG dimer to the surface of pre-activated 2-fold stretched or spherical PLGA microparticles for 4 hours at 4° C. These aAPC were centrifuged washed 3×, and then their fluorescence was characterized on a Synergy 2 plate reader (Biotek, Winooski, Vt.). Release from surface was characterized by incubating 10 mg of aAPC (stretched, non-stretched) with labeled dimer in 500 μL PBS at 37° C. for 1 week. At 3 days and 7 days, the particles were centrifuged and the supernatant was removed and stored for subsequent analysis. At 7 days, the supernatant was removed, the particles were resuspended, and the fluorescence was characterized.
Six days after primary stimulation with aAPC, T cell functional activity was assessed by re-challenge with peptide-pusled C57B1/6j splenocytes. Splenocytes were pulsed with the indicated concentration of peptide for 2 hours at 37° C. and then washed. 200,000 aAPC-activated T cells were incubated in complete RPMI with 200,000 splenocytes for 4 hours in a round bottom 96-well plate in the presence of 0.2-μL GolgiPlug, 0.2 μL GolgiStop, and anti-CD107a-fitC (BD Biosciences, Mountain View, Calif.). Cells were washed and fixed using a BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions, then stained with anti-IFNγ PE (BioLegend). Cytokine staining was assessed by flow cytometry and frequency of cytokine functional cells was assessed by comparison with an unstimulated control in FlowJo (TreeStar).
aAPC-T Cell Conjugate Formation Evaluation
PLGA microparticles, with encapsulated 5(6)-carboxy-tetramethylrhodamine dye (TAMRA, Nova Biochem, San Diego, Calif.), were synthesized for confocal imaging. The TAMRA was dissolved in dichloromethane at 1 mg/mL. 200 mg of acid-terminated PLGA was dissolved in 4.9 mL of DCM and 100 μl of TAMRA solution was added to the PLGA DCM phase. Particle synthesis otherwise followed the same protocol as the single emulsion particles above. These labeled particles were then cast into a film, and the film stretching and subsequent synthesis of aAPC from stretched and non-stretched TAMRA-loaded particles was accomplished as before. 1×106 CFSE-labeled T cells were incubated with 1 mg stretched or non-stretched aAPC for 60 minutes at 37° C. in a No. 1.5 glass bottom dish (MatTek, Ashland, Mass.). Images were acquired on a Zeiss LSM 510 META (Zeiss, Oberkochen, Germany) laser scanning confocal at 40× magnification at the Johns Hopkins School of Medicine Microscopy Facility.
In Vivo Activity of aAPCs
A subcutaneous B16 melanoma tumor prevention mouse model (
T cells used were obtained from homogenized mouse spleens after depletion of RBC by hypotonic lysis. Cytotoxic lymphocytes were isolated using a CD8 negative isolation kit and magnetic enrichment column from Miltenyi Biotec (Cologne, Germany) and if necessary labeled with Carboxyfluorescein succinimidyl ester (CFSE) for 15 minutes at 37° C., then washed extensively. Cells and particles at the indicated amounts and dosages were mixed and cultured for 4-7 days in complete RPMI media supplemented with T cell factor, a cytokine cocktail harvested from human plasma. Durai, M., et al., In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC). Cancer immunology, immunotherapy: CII. 58: 209-20 (2009). Cell proliferation was quantified by manual cell counting, and final T cell count was divided by the initial T cell count for fold-change data.
Determining the Lengths of a, b, c
For a spheroid that has been elongated in one dimension, since total volume is conserved from a sphere, the length of the short axes is related to the length of the long axis by b=c=1/√{square root over (a)}. . . . The identities of a, b, c, 1, and 2 are illustrated in
Since the geometrical shape corresponds to a prolate spheroid (a>b=c), the surface area of the spheroid can be determined by the following formula, Weisstein, E. W. Prolate Spheroid. in Math World—A Wolfram Web Resource (2012):
This surface area was then normalized by the surface area of a sphere with radius 1.
Equivalent protein density with total protein content held constant is the inverse of the normalized surface area (Density=1/SA).
The radius of curvature is the radius of a circle with the same curvature as the observed curve at that point. Thus, for flatter curves, the radius of curvature increases, as that flatness requires a larger circle to describe it.
The radius extremes of an oblate ellipsoid, also referred to as an oblate spheroid, are the equatorial radius, or semi-major axis, a, and the polar radius, or semi-minor axis, b. An ellipse can be described parametrically by:
For any parameterized equation of the form
the radius of curvature can be calculated from
So for an ellipse:
To calculate the radius of curvature at the tip, since t=0 corresponds to point 1 (
Because the be plane at 2 is already described by a circle, Rc=b=c.
Non-Spherical aAPC Synthesis and Characterization
The single emulsion PLGA (50:50 LA/GA, MW 38,000-54,000) microparticle synthesis resulted in spherical microparticles (
The stability of the non-spherical aAPC particle shape was characterized by analyzing their relaxation rate to the more stable spherical shape. Previous studies have indicated that relaxation rates depend on surface characteristics, molecular weight, polymer composition, and temperature. Yoo, J. W. & Mitragotri, S. Polymer particles that switch shape in response to a stimulus. Proc Natl Acad Sci USA 107, 11205-11210 (2010). Very little shape relaxation was observed for aAPCs fabricated from high aspect ratio particles over one week at 37° C. in PBS, indicating that the shape transition is very slow for non-spherical aAPCs with the chosen lactide to glycolide ratio (
Non-stretched and stretched microparticles were made into aAPCs by EDC-sulfo-NHS mediated covalent coupling of a dimeric MHC-Ig fusion protein, Schneck, J. P., J. E. Slansky, S. M. O'Herrin, and T. F. Greten. 2001. Monitoring antigen-specific T cells using MHC-Ig dimers. Current protocols in immunology/edited by John E. Coligan . . . [et al.]. Chapter 17: Unit 17.2, and an activating against CD28 to free carboxyl groups on the particle surface. For characterization experiments, fluorescently labeled MHC-Ig was used and total protein content for a given quantity of PLGA was assessed by fluorimetry (
While bulk polymer degradation from PLGA microparticles have been well investigated in the drug delivery field, Rothstein, S. N., et al., A unified mathematical model for the prediction of controlled release from surface and bulk eroding polymer matrices. Biomaterials 30, 1657-1664 (2009); Batycky, R. P., et al., A theoretical model of erosion and macromolecular drug release from biodegrading microspheres. J Pharm Sci 86, 1464-1477 (1997); von Burkersroda, F., et al., Why degradable polymers undergo surface erosion or bulk erosion. Biomaterials 23, 4221-4231 (2002), the effect of degradation on release of surface-coupled proteins is less well studied. For aAPCs, presentation of immobilized protein is critical for T cell activation and thus it is important to study release of surface immobilized proteins. Mescher, M. F., Surface contact requirements for activation of cytotoxic T lymphocytes. J Immunol 149, 2402-2405 (1992). To characterize surface degradation, particles bearing fluorescently labeled MHC-Ig were incubated for varying amounts of time at 37° C. Supernatants were recovered through centrifugation of the microparticles and released protein was quantified by protein fluorescence. For both spherical aAPCs and non-spherical aAPCs, 60-70% of the protein that was conjugated to the surface was released, with 30-40% remaining on the surface of the aAPCs (
High Aspect Ratio aAPCs Efficiently Induce T Cell Proliferation
To assess the impact of particle elongation, the ability of aAPC to induce antigen specific T cell expansion of pMEL TCR transgenic T cells was measured. Non-stretched and 2-fold stretched (AR=2.8) aAPCs were synthesized at three different cognate MHC-peptide densities (4 μg, 1 μg, and 0.5 μg of Db-Ig GP100/mg PLGA). Non-stretched aAPCs bearing non-cognate Db-Ig ASN at the highest protein density were used as negative control. The aAPCs were added to pMEL TCR transgenic T cells at three aAPC to cell ratios (1 mg, 0.1 mg and 0.01 mg aAPC/100,000 cells) and proliferation was assessed at days 4, by CFSE dilution and on day 7 by cell counts. Day 4 CFSE dilution for a representative Db-Ig density (4 μg Db-Ig/mg PLGA) is shown at each aAPC:cell ratio (
At a low, subsaturating dose of aAPC, 0.01 mg/100,000 cells, non-spherical, but not spherical, aAPCs induced T cell proliferation as measured by CFSE dilution (
To assess the effects of particle elongation on T cell expansion and viability, the aAPCs were prepared as before, and the aAPCs were added to pMEL TCR transgenic T cells at four aAPC to cell ratios (1.5 mg, 1 mg, 0.5 mg, and 0.1 mg aAPC/100,000 cells). Viability was determined by using a dye exclusion assay (e.g., Trypan blue). Scanning electron micrographs of non-stretched (
Without wishing to be bound to any one particular theory, it is thought that the enhanced proliferation activity due to elongation of aAPC is a result of changes in the density of MHC presented on the particle surface, as well as the increased surface area for cell contact on the particle long axis. The presence of stretched aAPC, however, decreased protein density (Table 1) compared to non-stretched aAPC, whereas enhanced T cell activation is associated with increased density. Bullock, T. N. J., et al., Antigen density presented by dendritic cells in vivo differentially affects the number and avidity of primary, memory, and recall CD8+ T cells. Journal of Immunology. 170:1822-9 (2003). This observation is confirmed by examining proliferation at a 0.01 mg dose of aAPC, where stretched particles induce 11-fold expansion for 1 μg/mg stretched aAPC, a clear proliferation advantage over non-stretched particle whether they present a lower (1.7-fold expansion for 0.5 μg MHC-Ig/mg non-stretched aAPC) or higher (1.3-fold expansion for 4 μg MHC-Ig/mg non-stretched aAPC) density of protein. Again, without wishing to be bound to any one particular theory, enhanced proliferation mediated by stretched aAPC is thought to be due to particle geometry rather than density of surface protein.
To further investigate the importance of stretching on T cell stimulation, PLGA microparticles were synthesized, stretched varying amounts and made into aAPCs. A correlation between increased shape AR and increased T cell proliferation (
T cell quality, as reflected by the amount and diversity of cytokines and cytotoxic markers produced when T cells are re-challenged by antigen, is a critical parameter for assessing responses. Seder, R. A., et al., T cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 8, 247-258 (2008). To determine the functional status of the expanded T cell population, aAPC-activated T cells with peptide-pulsed splenocytes were re-challenged and measured the production of a key cytokine, IFNγ, as well as measured the degranulation marker, CD107a in an intracellular cytokine staining (ICS) assay. Function tracked with proliferation; no significant difference in the quality of T cells generated from spherical or non-spherical aAPCs was observed, as determined by IFNγ or CD107a expression (
High Aspect Ratio Non-Spherical aAPCs Enhance T Cell Conjugate Formation
Antigen recognition on APC is known to trigger coordinated cytoskeletal rearrangements in both T cells and APCs, leading to close apposition of their cellular membranes. The resulting interactions mediate T cell activation and, when visualized by imaging or flow cytometry, are termed cell-cell conjugates. Kroger, C. J. and Alexander-Miller, M. A., Cutting edge: CD8+ T cell clones possess the potential to differentiate into both high- and low-avidity effector cells. J Immunol 179, 748-751 (2007).
To evaluate the formation of T cell-aAPC conjugates, naïve T cells were incubated at 37° C. with spherical or non-spherical aAPCs. Cell-particle interactions were visualized after a one-hour incubation by confocal imaging. In the presence of stretched aAPCs bearing cognate MHC/peptide, T cell membranes could be observed in close apposition to the particle's long but not short axis, creating a T cell “cap” T cell appearance, characteristic of conjugate formation (
Time-lapsed imaging revealed a striking reorientation and rearrangement of the T cell surface against the aAPC long axis. Initially, the T cell appears to contact the aAPC particle along the short axis, but with time migrates along the long axis rearranging its membrane against the long axis of the non-spherical aAPCs (
High Aspect Ratio, Non-Spherical aAPCs Enhance Tumor Killing In Vivo
A subcutaneous B16 melanoma tumor prevention model was used to test the activity of high aspect ratio aAPCs in vivo, where a subcutaneous injection of particles was administered 3 days before and a second dose 3 days after tumor injection (see
Stretched cognate aAPC, however, led to a significant increase in survival over non-stretched cognate aAPC (
These results demonstrate that increased aspect ratio acellular aAPCs, which only differ in their shape (and not in their volume or protein content) as compared to spherical controls, not only engender enhanced antigen specific activation in vitro but in vivo as well, and this enhanced activation has functional consequences, which lead to reduced tumor burden and enhanced survival.
Protein Drug Release from Polymer Matrix
The presently disclosed artificial cells also can be designed such that they can release protein drug from within the polymer matrix. As proof of principle, bovine serum albumin (BSA)-loaded spherical microparticles were synthesized using a double-emulsion method. These BSA-loaded particles were embedded into a film and stretched. The release of BSA (a model for a generic protein drug) from non-stretched and stretched particles is shown in
In principle, the shape of nano-sized particles can be altered to generate nano-sized artificial cells. One potential benefit of transitioning to particles that are more nano-sized would be improved utility for intravenous administration in vivo as compared to micron-sized artificial cells. As proof of principle, PLGA nanoparticles were synthesized using a single-emulsion method where the initial emulsion was accomplished via sonication of the oil water solution for 1 min. The resulting nanoparticles had a number averaged hydrodynamic diameter of 240 nm (
A key consideration in cancer immunotherapy remains the efficient stimulation of antigen (Ag)-specific cytotoxic T cells (CTLs). In vivo, a key interaction for generation of activated, effector Ag-specific CTLs is the interaction between antigen presenting cells, such as dendritic cells or macrophages, and naïve T cells. In the development of acellular systems for CTL stimulation, previous research has focused predominantly on the key proteins involved in the interaction between APCs and T cells, Oelke, M., et al., Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med 9, 619-625. PMID: 12074385 (2003); Ugel, S., et al., In vivo administration of artificial antigen-presenting cells activates low-avidity T cells for treatment of cancer. Cancer Res 69, 9376-9384 (2009); Steenblock, E. R. and Fahmy, T. M., A comprehensive platform for ex vivo T cell expansion based on biodegradable polymeric artificial antigen-presenting cells. Mol Ther 16, 765-772. PMID: 18334990 (2008); Curtsinger, J., et al., Artificial cell surface constructs for studying receptor-ligand contributions to lymphocyte activation. J Immunol Methods 209, 47-57 (1997); Maus, M. V., et al., Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T cell receptor, CD28 and 4-1BB. Nat Biotechnol 20, 143-148. (2002); Steenblock, E. R., et al., Antigen presentation on artificial acellular substrates: modular systems for flexible, adaptable immunotherapy. Expert Opin Biol Ther 9, 451-464. PMID: 19344282 (2009), and a recent study extended this focus to paracrine release of a cytokine. Steenblock, E. R., et al., An artificial antigen-presenting cell with paracrine delivery of IL-2 impacts the magnitude and direction of the T cell response. J Biol Chem 286, 34883-34892 (2011). To the inventors' knowledge, all previous systems investigated use of spherical aAPCs as their systems.
The biological interaction between an activated dendritic cell and a naïve T cell, however, is distinctly not an interaction that is most appropriately modeled by two spheres interacting. The presently disclosed subject matter evaluated the effect of one-dimensional stretching of aAPCs on the ability of these acellular, biodegradable aAPCs to induce T cell proliferation in vitro and tumor killing in vivo. At sub-saturating aAPC doses, high aspect ratio non-spherical aAPCs show significantly enhanced activity beyond spherical aAPCs and increasing the aspect ratio of non-spherical aAPCs shows enhanced activity up to AR 6.6. This enhanced activity also was reflected in vivo, where stretched particles showed increased survival in mice compared to stretched non-cognate aAPCs (p=0.004), as well as cognate spherical aAPCs (p=0.05).
With particle stretching, since each stretched and non-stretched particles had stretching the particles results in higher surface areas (Table 1b), stretched particles with equivalent total protein content had reduced protein density (Table 1c). Decreasing total protein content and thus protein density for both stretched and non-stretched particles, however, resulted in decreased proliferation at equivalent doses (
Key physical parameters altered by stretching of particles: (a) Aspect ratio (AR); (b) Surface area (SA) of stretched particles, relative to non-stretched spheres; (c) Protein density on surface of aAPC with equivalent total protein content relative to non-stretched spheres; and (d-f) Radius of curvature, R, for the tip of the ellipsoid (Ra), and for the flat face of the ellipsoid in either direction (Rb, Rc). For equations used, and description of derivations, see Example 7, Supplemental Calculations.
The particles of the presently disclosed subject matter can be used to form hyper-dense ligand coated particles, which have a surface density greater than what has currently been achieved.
A scheme showing one embodiment of these methods is shown in
Accordingly, in some embodiments, the presently disclosed methods comprise a method wherein a plurality of three-dimensional microparticles or nanoparticles is relaxed back partially or completely to a spherical shape. In some embodiments, relaxing occurs by the addition of heat.
The presently disclosed subject matter indicates that shape matters and that particle geometry is a critical design criterion to consider in the synthesis of biomimetic acellular aAPC systems. While particle-based T cell stimulation systems have yielded crucial insights regarding early activation events, Curtsinger, J., et al., Artificial cell surface constructs for studying receptor-ligand contributions to lymphocyte activation. J Immunol Methods 209, 47-57 (1997); Levine, B. L., et al., Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells. J Immunol 159, 5921-5930. (1997), aAPC that more closely mimic endogenous cell-cell interactions may provide a more complete understanding of the underlying process, such as the role of close membrane apposition and a large surface area of contact in the APC/T cell interaction. aAPCs thus may not only be an enabling tool for antigen-specific immunotherapy, but for studying basic aspects of T cell biology.
All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
Weisstein, E. W. Prolate Spheroid. in Math World—A Wolfram Web Resource (2012).
Weisstein, E. W. Radius of Curvature. in Math World—A Wolfram Web Resource (2012).
Lee J B, Oelke M, Ramachandra L, Canaday, and Schneck J P. Decline of influenza-specific CD8+ T cell repertoire in healthy geriatric donors. Immun Ageing. 2011 Aug. 16, 8:6.
Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.
This application claims is a continuation of U.S. patent application Ser. No. 14/363,955, filed Jun. 9, 2014, which is a § 371 national entry application of PCT/US2012/068759, filed Dec. 10, 2012, which claims the benefit of U.S. Provisional Application No. 61/568,751, filed Dec. 9, 2011, each of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61568751 | Dec 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14363955 | Jun 2014 | US |
| Child | 17008086 | US |
