WO2004087876A2

WO2004087876A2 - Use of red blood cells to facilitate cell activation

Info

Publication number: WO2004087876A2
Application number: PCT/US2004/009254
Authority: WO
Inventors: Surendra J. Chavan; Vivek Chitnis; Jayant Thatte; Mohammad A. El-Kalay
Original assignee: Valeocyte Therapies Llc
Priority date: 2003-03-25
Filing date: 2004-03-25
Publication date: 2004-10-14
Also published as: WO2004087876A3

Abstract

The present invention provides methods and compositions which use complexes comprising red blood cells for stimulation of a biological effect in target cells.

Description

USE OF RED BLOOD CELLS TO FACILITATE CELL ACTIVATION

TECHNICAL FIELD

[0001] The present invention relates to the use of complexes comprising red blood cells for stimulation of a biological effect in target cells. It also relates to the administration of the complexes to stimulate a biological effect in a target cell.

BACKGROUND ART

[0002] Induction of a biological effect in a cell can be stimulated or activated in a variety of ways. For example, interactions of cell surface receptors and ligands can generate intracellular signals that result in cell activation or stimulation. The particular biological effect that can result from such activation or stimulation depends on, among other things, the cell receptor(s), the ligand(s), their interaction(s) and the type of cell involved.

[0003] Generally, cell surface receptors transmit signals received on the outside of a cell to the inside through ligand-induced allosteric conforaiational change and/or through ligand- induced association. Receptors can also be stimulated to induce cell activation through interaction with non-ligand molecules, such as antibodies or ligand mimics.

[0004] Studies on the allosterically activated receptor class have yielded many pharmacologically agents that act as antagonists or agonists. Antagonists block the binding of the natural ligand without inducing the conformational change in the receptor thereby blocking a signal transduction pathway. Agonists bind to the receptor in a manner which mimics the natural ligand closely enough to induce the same conformational change as natural ligand thereby initiating a signal transduction pathway.

[0005] In some cases, activation of cell signaling occurs when multiple receptors on the cell surface are brought into close proximity in a process referred to as receptor clustering. Receptor clustering as a means for receptor activation has been well documented, especially for receptor kinases (Ullrich et al. (1990) Cell 61 :203-212; Kolanus et al. (1993) Cell 74: 171- 183).

[0006] Receptors activated by a ligand-induced association or clustering, such as multimerization, including dimerization, include, for example, those for cell growth and differentiation factors. Factors which serve as ligands for these receptors are typically large polypeptide hormone and cytokines such as erythropoietin, granulocyte colony stimulating factor (G-CSF), or granulocyte macrophage colony stimulating factor (GM-CSF), and human growth hormone (hGH). Many of the multimerization-activated receptors have cytoplasmic tails that contain protein kinase domains or docking sites. Ligand-induced multimerization of the extracellular domains of these receptors results in the juxtaposition of their cytoplasmic tails. In some cases, they then presumably phosphorylate each other in trans and thereby initiate the cytosolic signaling pathway. In some cases the cytoplasmic domains of multimerization-activated receptors do not have kinase domains themselves, but function the same as if they did because they associate with protein kinases via docking sites.

[0007] Receptors activated by multimerization or aggregation are frequently found in the immune system. They include, for example, the T cell surface receptors such as CD4, CD8, CD28, CD26, CD45, CD 10, and CD3/TCR (T cell antigen receptor). The ligands for these T cell receptors are most often cell surface proteins themselves, and can be found on antigen presenting cells. Aggregation-activated receptors frequently have short cytoplasmic domains which act to bind and thereby recruit other cell surface and/or cytosolic factors following the aggregation of their extracellular domains. Generally, receptor aggregation or clustering is important in stimulating the signaling pathway in the cell.

[0008] Important in this aggregation process, whether it be dimerization or multimerization, is for the receptors to have some mobility on the cell surface so that they can move or be moved into appropriate proximity for aggregation to occur.

[0009] Non-ligand molecules which interact with receptors, such as antibodies or ligand mimics, can also induce cell stimulation or activation. Such molecules can activate receptors to initiate cytosolic signaling through inducing receptor aggregation and/or through inducing allosteric conformational change. Because antibodies are naturally multivalent, they are often able to mimic the natural ligand in inducing association or clustering of the receptors to induce a biological effect such as, for example, an agonistic effect.

[0010] Interactions of receptors or ligands on cell surfaces with their respective ligand or receptor (i.e., cognate molecule) or with non-cognate molecules can lead to generation of intracellular signaling that ultimately results in the activation or stimulation of the cell.

[0011] Thus, molecular interactions at the cell surface can stimulate biological effects in cells which can be of use in treating diseases and/or disorders and/or in generating useful biological reagents. For example, such interactions can lead to stimulation of proliferation in a particular cell population or to induction of programmed cell death in a cancer cell. There remains a need for improved ways to stimulate or activate cells to induce a desired biological effect. [0012] All publications and patent applications cited herein are hereby incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

[0013] The invention, in one aspect is directed to methods and compositions for stimulating a biological effect in a target cell, comprising contacting a target cell with a first complex comprising a first moiety coupled to the surface of a red blood cell (RBC), wherein the first moiety interacts with a receptor on the surface of the target cell and wherein the interaction of the first moiety with the receptor stimulates a biological effect in the target cell. Optionally the method further includes contacting the target cell with a second complex comprising a second moiety coupled to the surface of an RBC, wherein the second moiety interacts with a second receptor on the surface of the target cell. In one embodiment, the method stimulates T cell proliferation and/or T cell differentiation. In one embodiment, the method stimulates apoptosis. Optionally, the method and compositions further include the RBC loaded with an agent which stimulates a biological effect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1 is a graph depicting T cell growth as fold change from day 0 over time. The triangles represent cells in the culture stimulated with RBCs + antibody complex A, the circles represent cells in the culture stimulated with RBCs + antibody complex B, and the diamonds represent cells in the culture stimulated with paramagnetic beads coated with antibodies (SOP)

[0015] Fig. 2 is a graph depicting T cell growth as cell number in culture over time. The numbers at the left of the graph indicate the fold change at day 9 in cell growth from day 0. The solid circles represent the cells in the culture stimulated with RBCs-conj (W), the solid triangles represent cells in the culture stimulated with RBCs-conj, the solid squares represent cells in the culture stimulated with paramagnetic beads coated with antibodies (SOP), the solid diamonds represent cells in the culture stimulated with antibody complexes only, the open squares represent cells in the culture stimulated with RBCs only, and the open diamonds represent unstimulated cells in the culture.

[0016] Fig. 3 contains histograms from the FACS analysis of RBCs coupled to Streptavidin-SMCC (SA-SMCC) and stained with FITC labeled anti-streptavidin antibody.

[0017] Fig. 4 is a graph depicting CD4+ T cell growth as cell number in culture over time. After 10 days of expansion, the cell number increased an average of 32-118 fold. [0018] Fig. 5 contains histograms from the FACS analysis of cell surface staining of the population of T cells after a 10 day expansion. The solid lines indicate expression of the markers detected by antigen-specific antibodies and the dotted lines indicate background staining on these cells using isotype control antibodies. Fig. 5 A depicts expression of the cell phenotype markers indicated: CD4, CD3, CD45RO and CD45RA. Fig. 5B depicts expression of the cell activation markers indicated: CD25 and CD44.

[0019] Fig. 6 contains histograms from the FACS analysis of cell surface staining of the population of T cells after a 10 day expansion. Depicted is the expression of the adhesion molecules indicated: CD62L, LFA-1, CD 162, CD49D, CLA and integrin α4β7. The solid lines indicate expression of the markers detected by antigen-specific antibodies and the dotted lines indicate background staining on these cells using isotype control antibodies.

[0020] Fig. 7 contains histograms from the FACS analysis of cell surface staining of the population of T cells after a 10 day expansion. Depicted is the expression of the chemokine receptors indicated: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR3, CXCR5 and CXCR6. The solid lines indicate expression of the markers detected by antigen- specific antibodies and the dotted lines indicate background staining on these cells using isotype control antibodies.

[0021] Fig. 8 is a graph depicting the TCR Vβ repertoire of the CD4+ T cells before and after the population expansion. The lighter bars indicate the percentage of the T cells with the indicated Vβ allele in the T cell population before expansion (day 0). The darker bars indicate the percentage of the T cells with the indicated Vβ allele in the T cell population after expansion (day 13).

[0022] Fig. 9 contains histograms from the FACS analysis of cells expressing IFN-γ and IL- 4 as detected by intracellular cytokine staining. The numbers in the inset of the histograms indicate the percentage of the cells in the respective quadrants.

MODES FORCARRYINGOUTTHEINVENTION

[0023] We have discovered that presenting a target cell directed moiety on the surface of an erythrocyte or red blood cell (RBC) to the target cell is particularly effective in stimulating a biological effect in the target cell. The target cell directed moiety interacts with a receptor on the target cell and a biological effect is stimulated in the target cell. The moiety is coupled, either directly or indirectly, to the surface of an RBC. The use of an RBC as a component of a target cell stimulating complex offers distinct benefits for and advantages to stimulating a target cell. Presentation of a target cell directed moiety on the surface of an RBC generally provides a local concentration of the moiety to the target cell through the presence of a number of moieties on the surface of an RBC. In some instances, coupling of the target cell directed moiety to the surface of an RBC allows for some mobility of the moiety when interacting with the target cell receptor and, accordingly, for mobility and/or aggregation of the target cell receptor. For some cell receptors, the ability to aggregate and/or move on the cell surface is important for effective cell signaling. In some instances, the RBC complexes can also be used to deliver agents (e.g., drugs, antigens, cytokines, chemokines, hormones) to particular cells and/or tissues. In addition, the use of RBCs in the presentation of target cell directed moieties to target cells provide a source of oxygen to the cells in culture or in the individuals to which the complexes are administered.

General Techniques [0024] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.. 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed._? 1987); Methods in Enzymology (Academic Press, Inc.); Handbook o ^'Experimental Immunology (Weir el al., eds.); Gene Transfer Vectors for Mammalian Cells (Miller et al., eds., 1987); Current Protocols in Molecular Biology (Ausubel et al., eds., 1987); Antibodies: A Laboratory Manual (Harlow et ah, eds., 1988), Cold Spring Harbor Laboratory Press; PCR: The Polymer ase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (Coligan et al., eds., 1991); The Immunoassay Handbook (Wild, ed., Stockton Press NY, 1994); and Methods of Immunological Analysis (Masseyeff et al., eds., Weinheim: VCH Verlags gesellschaft mbH, 1993)

Definitions [0025] As used herein, a "target cell directed moiety" is a moiety that interacts with a receptor on a target cell. The target cell directed moiety can be an entire molecule or a portion of a molecule. The target cell directed moiety can be included within or attached to another molecule as long as the target cell directed moiety is capable of interacting with the receptor on the target cell.

[0026] As used herein, a "target cell receptor" is a molecule on the surface of a target cell that, upon interaction with a target cell directed moiety/RBC complex, participates in and/or contributes to the stimulation a of biological effect in the target cell.

[0027] As used herein, entities that are "coupled" are joined, linked, attached, or connected, either directly or indirectly.

[0028] As used herein, "red blood cells" includes hemoglobin-containing ei throcytes, erythroblasts and reticulocytes, as well as hemoglobin-depleted red blood cell "ghosts."

[0029] As used herein, "T cells" are CD4-positive or CD8-positive lymphocytes that express the CD3 antigen.

[0030] As used herein, "activated T cells" are T cells that have undergone differentiation to a particular subset of T cell. Activated T cells include, but are not limited to, Thl, Th2, ThO, Tel and Tc2 subsets. Activated T cells include any T cell subtype and are not limited to any particular defined cytokine profile. As used herein, activated T cells may refer to either polyclonal or monoclonal populations of T cells.

[0031] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site. An "antibody combining site" or "binding domain" is formed from the folding of variable domains of an antibody molecule(s) to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows an immunological reaction with the antigen. An antibody combining site may be formed from a heavy and/or a light chain domain (VH and VL, respectively), which form hypervariable loops which contribute to antigen binding. The term "antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, univalent antibodies, the Fab proteins, and single domain antibodies.

[0032] As used herein, the term "bead" refers to a solid phase particulate composition which is insoluble in water. Beads generally have a size of less than about 100 μm and are typically spherical, ellipsoid or rod-shaped in shape. Beads may be particles formed from naturally occurring polymers, synthetic polymers, synthetic copolymers, such as agarose or synthetic agarose, as well as other biodegradable materials known in the art. Beads may also be particles formed from polymers or other materials which are non-erodible and/or non- degradable under mammalian physiological conditions, such as polysytrene, polypropylene, silica, ceramic, polyacrylamide, gold, latex, magnetic and paramagnetic material.

[0033] An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, humans, farm animals, sport animals, rodents and pets.

[0034] An "effective amount" or a "sufficient amount" of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. An effective amount can be administered in one or more administrations.

[0035] As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise. For example, "a" target cell includes one or more target cells.

Methods of the Invention

[0036] The invention relates to the use of complexes comprising RBCs and target cell directed moieties in methods of stimulating a biological effect in target cells. As described herein, the complexes comprise target cell directed moieties coupled to the surface of an RBC. In the methods, when the complex contacts the target cell, the moiety interacts with a receptor on the target cell and the interaction stimulates a biological effect in the target cell. The target cell directed moieties are either directly or indirectly coupled to the surface of the RBC.

[0037] The degree and/or type(s) of biological effect stimulated in a target cell depends on a number of factors, including, for example, the target cell type, the target cell receptor, the target cell directed moiety and the presentation of the moiety to the target cell receptor. In some cases, interaction of a target cell directed moiety alone with a target cell receptor may stimulate a biological effect in the target cell. In other cases, presentation of the target cell directed moiety on the surface of an RBC to the target cell is necessary to stimulate a measurable biological effect in the target cell. In either case, presentation of a target cell directed moiety coupled to the surface of an RBC to the target cell receptor is an effective way to stimulate a biological effect in the target cell. As exemplified herein, contacting a target cell with a target cell directed moiety coupled to a linker coupled to an RBC is more effective in stimulating a biological response than contacting the target cell with the target cell directed moiety alone or with the target cell directed moiety coupled to a bead.

[0038] In methods of the invention, stimulation of a biological effect in the target cell results from contacting the target cell with at least one target cell directed moiety attached to the surface of an RBC. In some embodiments, stimulation of a biological effect in the target cell requires interaction of more than one target cell directed moieties coupled to RBCs with one or more target cell receptors. In some embodiments, stimulation of a biological effect in the target cell requires interaction of more than two target cell directed moieties coupled to RBCs with one or more target cells receptors.

[0039] Examples of target cells for the invention include, but are not limited to, cells of the immune system, bone marrow cells, stem cells, infected cells, hyperplastic cells and tumor and/or cancer cells. Exemplary target cells include T cells, natural killer (NK) cells, tumor infiltrating lymphocytes (TIL), lymphokine-activated killer (LAK) cells, B cells, monocytes, granulocytes, macrophages, immature and mature dendritic cells. The target cell may also be any non-cancerous cell that could provide a direct or indirect therapeutic response.

[0040] Examples of biological responses that can be stimulated in cells of the immune system, bone marrow cells, and/or stem cells, include, but are not limited to, activation, proliferation, differentiation, and/or induction of cytokine production. Examples of biological responses that can be stimulated in non-immune system cells include, but are not limited to, production of hormones, neurotransmitters and/or other biological response molecules. Examples of target cell directed moieties that can stimulate such biological effects in such cells are listed herein.

[0041] Examples of biological responses that can be stimulated in infected cells, hyperplastic cells, tumor cells and/or cancer cells include, but are not limited to, anti- proliferative responses, cytotoxic effects, apoptosis, and necrosis. Examples of target cell directed moieties that can stimulate such biological effects in such cells are listed herein.

[0042] Methods of the invention are appropriate for use in vitro and/or in vivo. For example, target cells in culture can be contacted with the complexes according to the methods and, once the biological effect is stimulated, the cells and/or culture media can be harvested for further use. In some embodiments, methods of the invention can be used for ex vivo purposes, for example, where cells are collected from an individual and put in culture conditions as needed, the biological effect is stimulated according to the methods of the invention and the resultant cells and/or cell products, are administered to an individual in need thereof.

[0043] For example, target cells can be contacted in culture with target cell directed moiety/RBC complex(es) to stimulate an increased level of production and/or secretion of a variety of cytokines. The cytokine(s) in the cell culture supernatant can be separated from the target cells and RBC complexes and used for a variety of purposes including administration to a subject in need thereof. RBC complexes of the invention can be used to stimulate cytokine production from a homogeneous cell population (e.g., a population enriched for a particular subset of cells, e.g., CD4+ T cells) or from a heterogeneous cell population. The particular cytokine(s) stimulated by the target cell directed moiety/ RBC complexes depends on the target cell population and on the target cell directed moiety used for the stimulation. For example, cells from the immune system can be stimulated to produce cytokines including, but not limited to, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18, IL-27, TRAIL, FasL, IFN-γ, TNF-α and TNF- β. Target cell directed moieties for stimulation of cytokine production include those described herein, such as a lectin (e.g., PHA) or an anti-target cell receptor antibody (e.g., anti-CD3 and anti-CD28 antibodies).

[0044] In stimulating cytokine production or secretion from the cells in culture, the target cell directed moiety/RBC complexes may be added to the cells once or repeatedly. Separation of the cytokine-containing culture supernatant from the cells and RBC complexes can be done using separation technologies including filtration, precipitation, fractionation and sedimentation: Preferably, the culture supernatant containing the desired cytokine(s) is removed from the cells and RBC complexes prior to substantial cell lysis and without causing substantial cell lysis. If the surface of the RBCs in the complexes is streptavidinated (i.e., coupled with streptavidin), biotinylated magnetic particles and an application of a magnetic field can be used to remove the RBCs, and any cells attached to the RBCs, from the culture supernatant. This can be accomplished in a batch mode (e.g., using a permanent magnet) or in a continuous mode by flowing the mixture of RBC complexes, target cells and cell culture supernatant over a permanent magnet. Where contact with the target cell directed moiety/RBC complex causes the target cells to proliferate, the target cell culture can be saturated by the addition of excess RBC complex prior to removal of the cells and complexes from the supernatant. Cytokines of the cell culture supernatant can be further purified using techniques known in the art, including, for example, using cytokine-specific affinity columns.

[0045] In another example of ex vivo stimulation, T cells can be isolated from peripheral blood mononuclear cells (PBMCs) and stimulated to proliferate and/or differentiate into, for example, Thl, Th2, ThO, Tel, Tc2 or any activated T cell subtype not limited to a particular defined cytokine producing profile. As exemplified herein, T cells can be isolated from PBMCs and stimulated to proliferate and differentiate into activated T cells according to a method of the invention. The activated T cells so generated can then be administered to individuals, for example, for adoptive immunotherapy. [0046] For example, target T cells can include those of any antigen specificity, including non-antigen specific, and include T cell populations that are monoclonal or polyclonal. T cells that result from the methods of the invention include those of any antigen specificity, including non-antigen specific, and monoclonal or polyclonal T cell populations. Also, methods of the invention can be used to generate T cells of any effector profile including any surface marker profile or any cytokine profile.

[0047] In the methods described herein, the target cells can be stimulated with the target cell directed moiety/RBC complex once or repeatedly until the desired effect is obtained. In some embodiments, following stimulation of the target cells with the target cell directed moiety/RBC complex, the cells can be stimulated with other agents that serve to further result in the desired effect. As demonstrated herein, stimulation of CD4+ T cells first with anti- CD3/anti-CD28/RBC complexes and subsequently with anti-CD3/anti-CD28/streptavidin complexes (SA-CD3/CD28) resulted in effective expansion and differentiation of the population of T cells. Thus, in some embodiments, the methods further comprise contacting the target cells with target cell directed moiety/SA complexes, once or repeatedly.

[0048] In some embodiments, methods of the invention are performed in vivo. In such methods, the target cell is contacted after the target cell directed moiety/RBC complex(es) is administered to an individual. The administered complexes contact the target cell and stimulate a biological effect in the individual. Many of the complexes described herein are appropriate for use in vivo, including, but not limited to, those that are particularly selective for the target cell and that stimulate target cell growth arrest or apoptosis, that stimulate target cell proliferation and that stimulate target cell differentiation.

[0049] In one aspect of the invention, methods are provided for modulating immune system function. The complexes and/or compositions of the invention are administered to subjects in need of immune system modulation in amounts effective to modulate immune system function. Modulation of immune system function includes, but is not limited to, increasing immune function such as by specifically stimulating T cells (including cytotoxic T lymphocytes (CTL)), B cells, NK cells, bone marrow cells, monocytes, macrophage, immature dendritic cells, mature dendritic cells, stem cells and/or early lineage progenitor cells to produce a prophylactic or therapeutic result relating to infectious disease, cancer, and the like. Specifically included is the use of particular complexes of the invention for the treatment of disorders characterized by reduced T cell levels in vivo, e.g., HIV and other disorders associated with a compromised immune system. Modulation of immune system function also includes, but is not limited to, decreasing immune function such as by suppressing specifically the immune system to treat autoimmune disease, allergy and the like. In one embodiment, the complexes of the invention are used to shift a Th2-type immune response toward a Thl-type immune response through the stimulation of Thl cell production, as described herein. In another embodiment, the compositions of the invention are used to stimulate blood cell proliferation and/or differentiation.

[0050] The invention provides methods and compositions for increasing the size of a subpopulation of T cells. In such methods, "increasing size of a subpopulation of T cells" refers to stimulating the expansion of a T cell subpopulation by contacting the T cells with at least one complex comprising a T cell directed moiety coupled to an RBC where the interaction of the T cell directed moiety with a T cell receptor stimulates proliferation or expansion of the T cell subpopulation of cells. Preferably, the number of T cells belonging to the subpopulation that are present after this contacting is at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 70, 90, 150, 500, 5000, 50,000 or 100,000 fold greater than the number of these cells present without administration of the complexes or after the corresponding control incubation in the absence of the complexes. More preferably, the number of T cells belonging to the subpopulation that are present after this contacting is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 1000, 10,000 or 100,000 fold greater than the number of these cells present after the corresponding in vivo contact or in vitro incubation in the presence of the same T cell directed moiety attached to the surface of a bead. It is also contemplated that the percentage may remain the same but the actual numbers of the relevant subset may increase if the total number of T cells increases. In some instances, the change in the percentage of cells that belong to the subpopulation of T cells is at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10,000 or 100,000 fold greater than corresponding change in the percentage of cells that belong to the subpopulation of T cells in absence of administration of the complexes, in a control sample that has not be incubated with the complexes or after the corresponding incubation in the presence of the same T cell directed moiety attached to the surface of a bead.

[0051] For example, methods are provided for stimulating production of Thl or Th2 cells, subsets of T helper cells. The Thl subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs). Thus, the Thl subset may be particularly suited to respond to viral infections, intracellular pathogens, and tumor cells because it secretes IFN-γ and other cytokines, which activate other components of the immune system, such as CTLs. The Th2 subset suppresses the cellular immune response and functions more effectively as a helper for B-cell activation and eosinophilic inflammation. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively.

[0052] Differences in the cytokines secreted by Thl and Th2 cells are believed to reflect different biological functions of these two subsets. See, for example, Romagnani (2000) Ann. Allergy Asthma Immunol. 85:9-18. In general, since distinct patterns of cytokines are secreted by Thl and Th2 cells, one type of response can moderate the activity of the other type of response. A shift in the Thl/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response. Methods of the invention can alsσ be used to redirect a Th2 immune response.

[0053] In one embodiment, for example, the invention provides methods for producing a population of Thl cells from a blood sample in the absence of exogenous growth or differentiation factors, such as IL-2 or IFN-gamma. Mononuclear cells collected from a blood sample, for example, by leukapheresis, serve as a source material for production of Thl cells in culture. In an exemplary embodiment, CD4+ T cells are first purified from the source material. Such a purification can be accomplished by, for example, positive selection. The starting population of T cells are then contacted with the complexes of the invention to stimulate the desired biological effect in the T cells. In one embodiment, the cells are activated by simultaneous contact with a first moiety/RBC complex that interacts with the CD3 receptor complex on the T cells and a second moiety/RBC complex which interacts with the CD28 receptor on the T cells. In an exemplary embodiment, the activation is accomplished by co- incubating the starting population of T cells with anti-CD3 antibodies coupled to the surface of RBCs and anti-CD28 antibodies coupled to the surface of RBCs. In these embodiments, the anti-CD3 and/or anti-CD28 antibodies may be directly or indirectly coupled to the surface of the RBC.

[0054] The T cells are stimulated with the T cell directed-RBC complexes one or more times, typically two or more, three or more, four or more, five or more times. In an exemplary embodiment, the T cells are stimulated three times with the anti-CD3/anti-CD28/RBC complexes over the course of 9 days in culture. The repeated stimulation of the cells resulted in an expansion of T cell number in excess of 52 and 76 fold, depending on the particular preparation of complexes used. Stimulation with the antibodies alone resulted in T cell expansion of about 13 fold. In another embodiment demonstrated herein, T cells are stimulated first with anti-CD3/anti-CD28/RBC complexes and subsequently with anti- CD3/anti-CD28/streptavidin complexes (SA-CD3/CD28) over the course of 10 days in culture. The repeated stimulation with these two type of complexes resulted in an expansion of T cell number of 32-118 fold.

[0055] Cells resulting from the exemplified expansions have a Thl phenotype as demonstrated by their production of IFN-gamma, their lack of production of IL-4 and their cell surface markers. Thus, the invention provides methods for increasing the size of a subpopulation of activated T cells. Methods are also provided for producing large numbers of activated T cells.

[0056] Activated T cells, such as Thl cells, would be of use in treating symptoms of individuals with cancers, infectious diseases, allergic diseases and diseases or disorders that are associated with overactive humoral immunity. Individuals with cancer and tumor-bearing animals have been shown to exhibit suppressed cellular immune responses as evidenced by decreased DTH, CTL function and NK activity (Broder et al. (1978) N. Engl. J. Med. 299:1335-1341) apparently due to a lack of Thl cells. Excess production of Th2 cytokines and/or depressed production of Thl cytokines resulting in a Thl/Th2 cytokine imbalance has also been reported in virtually all types of cancer tested. As with asthma and allergies, enhanced Th2 responses are found in a variety of infectious diseases, such as chronic hepatitis C virus infection (Fan et al. (1998) Mediators Inflamm. 7:295), leprosy (Yamamura (1992) Science 255:12), toxoplasmosis (Sher et al. (1992) Immunol. Rev. 127:183) and AIDS (Clerici et al. (1993) Immunol. Today 14:107-111), and autoimmune conditions, such as lupus (Funauchi et al. (1998) Scand. J. Rheumatol. 27:219).

[0057] Therapies that increase Thl cells and/or shift the balance from Th2 to Thl have been shown to have therapeutic utility in treating cancer and infection conditions. For example, down-regulation of the Th2 response in tumor-bearing mice by treatment with anti-IL-4 mAb significantly suppresses growth of murine renal cell carcinoma tumors (Takeuchi et al. (1997) Cancer Immunol. Immunother. 43:375-381), while IL-2 gene transfected murine renal cell carcinoma cells mediate tumor rejection (Hara et al. (1996) Jpn. J. Cancer Res. 87:724-729). IL-2 is a Thl associated cytokine. Adoptive immunotherapy involving transfer of influenza- specific Thl cells was protective against influenza infection, while Th2 infusion failed to induce protection (Graham et al. (1994) J. Exp. Med. 180:1273).

[0058] Accordingly, methods of the present invention are for use in the production of Thl cells that can be used in adoptive immunotherapy for a variety of conditions in which an increase in the population of Thl cells would be of beneficial, such as in treatment of a variety of diseases, including cancer, infectious disease, allergy and diseases characterized by overactive humoral immunity, such as systemic lupus erythematosus. Methods of the invention in which complexes that stimulate differentiation of T cells to Thl cells are administered can be used to shift a Th2 immune response toward a Thl immune response in an individual in need thereof. Methods of the invention can also be used to stimulate production of Thl cells in an individual in need thereof.

[0059] In another embodiment, methods of the present invention involve the use of RBC compositions as artificial antigen presenting cells (APCs) to stimulate T cells to respond a particular antigen, such as a tumor antigen or an antigen associated with infectious disease. RBC compositions that can be used as artificial APCs include those which have a specific antigen, or fragment thereof, coupled to the RBC cell surface. Artificial APCs may also include compositions comprising RBCs having cell surface coupled major histocompatability complex (MHC) molecules (such as, class I or class II molecules) loaded with antigen peptide. Such compositions may be used, for example, in methods to prime T cells in vitro.

[0060] In some embodiments, methods involve the use of RBCs to deliver antigens to cells or to a subject in need thereof, in particular, to deliver antigen to particular cells and/or organs of the immune system, such as lymph nodes and spleen. In these methods, the antigen of interest can be concentrated within the RBC and/or on the surface of the RBC and the antigen- RBC complex administered parenteral^, such as by intravenous delivery. RBCs are collected by the spleen and the antigen can thus be delivered to the T and B cells of the spleen. Also, the RBC can be directed to a particular site through coupling a ligand to the RBC surface that will preferentially direct the RBC to the desired cells and/or organ. For example, coupling LFA-1 and CD62L to the surface of the RBC of the antigen-RBC complex prior to administration would result in the delivery of the antigen-RBC complex to the lymph nodes. Antigen-RBC complexes directed to the lymph nodes may further include an antigen linked to a Tat polypeptide of HIV or any other appropriate signaling peptide which facilitates processing of the antigen. Given the directed aspect of this form of antigen administration, the spread of antigen in the individual would be restricted to the particular desired sites and lower doses of antigen can be delivered since it is preferentially directed to sites where it would be most useful.

[0061] In another aspect of the invention, methods are provided for suppressing proliferation of target cells and/or for inducing cell death in target cells. The complexes and/or compositions of the invention are administered to subjects in need of suppression of cell proliferation and/or induction of cell death in amounts effective to suppress target cell proliferation and/or to induce cell death in the target cell. Such individuals include those with cancer, tumor cells, infected cells and/or diseases or disorders characterized by cell proliferation. Suppressing proliferation (including, for example, through slowing or arresting cell division) and/or inducing cell death (including, for example, through stimulating apoptosis) in target cancer cells, tumor cells, and/or infected cells produces a prophylactic or therapeutic result relating to cancer, infectious disease, and the like.

[0062] In some embodiments, methods for suppressing cell proliferation involve the use of RBC complexes with a coupled target cell directed moiety that interacts with a receptor on the target cell that stimulates suppression of proliferation and/or induction of cell death in the target cell. Such a receptor on the target cell is herein referred to as a "negative signaling" receptor. As used herein, "negative signaling" refers to the inhibition of cell growth, for example, by cell cycle arrest or the induction of apoptosis (programmed cell death).

[0063] Negative signaling receptors and their ligands are known in the art and include, for example, the tumor necrosis factor (TNF) receptor family, such as TNF receptor (TNF-R), TNF-like receptors, lymphotoxin-β receptor (LT-β-R), Fas receptor, and ligands, such as TNF, lymphotoxin-α (LT-α, formerly called TNF-β), lymphotoxin-β (LT-β), TNF-related apoptosis inducing ligand (TRAIL or Aρo-2L) and Fas ligand (FasL). TNF-R signaling is cytotoxic to cells with transfoπned phenotypes or to tumor cells and can lead to selective lysis of tumor cells and virus-infected cells. Like TNF-R, signaling by LT-β-R can activate pathways that lead to cytotoxicity and cell death in tumor cells. Fas receptor (Fas-R) can stimulate cytotoxicity by programmed cell death in a variety of both tumor and non-tumor cells.

[0064] The ligands TNF and LT-α bind to and activate TNF receptors p60 and p80, herein referred to as TNF-R. LT-αl/β2 heterodimeric complex binds the LT-β-R and induces cytotoxic effects on cells bearing the LT-β-R in the presence of an LT-β-R activating agent, such as IFN-gamma. See, for example, U.S. Pat. 6,312,691. Fas ligands are capable of inducing apoptosis in cells that express a Fas receptor. The human and mouse Fas ligand ' genes and cDNAs have been isolated and sequenced (Genbank Accession No. U08137; Takahashi et al. (1994) Intl. Immunol. 6:1567-1574; Takahashi et al. (1994) Cell 76:969-976).

[0065] In addition to stimulation through interaction with specific ligands, antibody binding can also activate negative signaling receptors to signal growth arrest and/or apoptosis. Antibodies that have negative signaling properties include, but are not limited to, anti-Fas, anti- LT-β-R, anti-CD40, anti-Class II MHC, anti-Her-2, anti-CD19, anti-Le^y, anti-idiotype, anti- IgM, anti-CD20, anti-CD21 and anti-CD22 as reported, for example, in Trauth et al. (1989) Science 245:301-305; Funakoshi et al. (1994) Blood 83:2787-2794; Bridges et al. (1987) J Immunol. 139:4242-4249; Scott et al. (1991) J. Biol. Chem. 266:14300-14305); Ghetie et al. (1992) Blood 80:2315-2320; Ghetie et al. (1994) Blood 83:1329-1336; Schreiber et al. (1992) Cancer Res. 52:3262-3266; Levy et al. (1990) J. Natl. Cancer Inst. Monographs 10:61-68; Vitetta et al. (1994) Cancer Res. 54:5301-5309; Page et al. (1988) J Immunol. 140:3717-3726; Beckwith et al. (1991) J Immunol. 147:2411-2418; U.S. Pat. Nos. 6,312,691 and 6,368,596. Furthermore, negative signaling can sometimes be optimized by hypercrosslinking with secondary antibodies or by using "cocktails" of primary antibodies (Marches et al. (1996) Therap. Immunol. 2:125-136).

[0066] In addition to the target cell directed moiety that interacts with the target cell receptor to stimulate a biological effect, the RBC complex can also include a cell targeting molecule that directs the complex to the target cell. Such targeting molecules are components of the complex that enhance the accumulation of the complex at certain tissue or cellular sites in preference to other tissue or cellular sites when administered to an intact individual, organ or cell culture. Such a targeting moiety can be ter alia a peptide, a region of a larger peptide, an antibody specific for a target cell surface molecule or marker, or antigen binding fragment thereof, a nucleic acid, a carbohydrate, a region of a complex carbohydrate, a special lipid, or a small molecule such as a drug, hormone, or hapten, attached to any of the aforementioned molecules. Antibodies with specificity toward cell type-specific cell surface markers are known in the art and are readily prepared by methods known in the art. The complexes can be targeted to any cell type in which a stimulation of the biological effect is desired, e.g., a cell type in which proliferation is to be stimulated or a cell type in which growth arrest is to be induced.

[0067] The methods of the invention may further include delivery of an agent (e.g., a drug) to cells (e.g., in culture or in an individual) or to an individual using the RBCs of the invention as a delivery vehicle for the agent. In this embodiment, the RBCs of the invention are loaded with an agent that will work along with the target cell directed molecule to result in the desired effect. For example, the RBC with a target cell directed moiety designed to stimulate T cell proliferation coupled to its surface can be loaded with a cytokine that further stimulates T cell growth (e.g., IL-2, IL-15, IL-18, IL-27). Thus, the RBC complex provides an additional stimulatory component to support T cell proliferation. In another example, the RBC can be loaded with an anti-apoptosis agent and a target cell directed moiety designed to send an anti- apoptosis signal to the target cell can be coupled to the surface of the RBC.

[0068] In such methods, intact or ghost RBCs can be loaded with one or more agents. Agents and methods for loading agents in RBCs for the invention are described elsewhere herein. RBCs can be loaded with such agents before, during and/or after the target cell directed moiety is coupled to the RBC surface.

Compositions of the invention

[0069] A complex for use in the present invention comprises an RBC coupled, either directly or indirectly, to at least one moiety that interacts with a receptor on a target cell (i.e., "a target cell directed moiety"). For the purposes of the invention, the receptor on the target cell is a molecule that, upon interaction with the target cell directed moiety/RBC complex, stimulates or contributes to stimulation of a biological effect in the target cell.

[0070] Red blood cells for use in the methods and compositions of the invention include red blood cells isolated, for example, from whole blood, bone marrow, fetal liver, cord blood, buffy coat suspensions, pleural and peritoneal effusion, and other tissue or fluid. The RBCs can be autologous or allogeneic relative to the target cell. When administered to an individual, the RBCs can be autologous or allogeneic to the individual.

[0071] For use in the invention, the RBCs can be intact or can be RBCs depleted of hemoglobin, i.e., ghost RBCs. Ghost RBCs can be created by depleting the RBC of hemoglobin using methods known in the art including, for example, through reverse hemolysis using hypotonic/hypertonic solutions.

[0072] The RBCs for use in the invention may have their membranes fixed using a variety of reagents and protocols known in the art including, for example, paraformaldehyde, gluteraldehyde, formamide and the like. Fixing of the RBC membranes can provide some rigidity to the membranes. Ghost or intact RBCs can be fixed and any fixation can occur before, during and/or after the target cell directed moiety is coupled to the RBC surface.

[0073] The choice of the target cell directed moiety and the target cell receptor to which the moiety is directed depends on the target cell and the desired biological effect to be stimulated. Target cell receptors for use in stimulation of a biological effect include, but are not limited to, CD3, CD28, CD2, MHC class I complex (including dimer, tetramer, multimer) loaded with peptide, MHC class II complex (including dimer, tetramer, multimer) loaded with peptide, T cell receptor complexes (including alpha-beta and gamma-delta), CD 16, CD45, CD25, CD27, ICOS, CD40, CD40L, CTLA-4, OX-40, OX40L, CD30, CD30L, CD137, 4-1-BBL, B7.1, B7.2, FasR, FasL, TRAIL, DR4, DR5, DR3, TNFR1, TNFR2, chemokine receptors, receptors of cytokines (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL12P35, IL12P40, IL12P70, IL-13, IL-15, IL-18, IL-23, IL-27, TNF-alpha, TNF-beta, TGF-beta, IFN-gamma, GM-CSF), common gamma chain of IL-2 receptor, and any associated components of cytokine receptors. Additional examples of target cell receptors for use in stimulation of a biological effect include TNF-R, LT-βR, Her-2, CD 19, IgM, CD20, CD21 and CD22. In some instances, target cell receptors for use in stimulation of a biological effect include those that signal the target cell through a tyrosine kinase, such as a src-family tyrosine kinase or a JAK family kinase, through a phosphatidylinositol 3 -OH kinase.

[0074] Accordingly, target cell directed moieties that interact with a receptor on the surface of a target cell include, but are not limited to, natural or non-natural ligands of the receptor and antibodies that bind the receptor. Target cell directed moieties of the present invention include, but are not limited to, those that interact with CD3, CD28, CD2, MHC class I complex (including dimer, tetramer, multimer) loaded with peptide, MHC class II complex (including dimer, tetramer, multimer) loaded with peptide, T cell receptor complexes (including alpha- beta and gamma-delta), CD 16, CD45, CD25, CD27, ICOS, CD40, CD40L, CTLA-4, OX-40, OX40L, CD30, CD30L, CD137, 4-1-BBL, B7.1, B7.2, Fas, FasL, TRAIL, DR4, DR5, DR3, TNFR1, TNFR2, chemokine receptors, receptors of cytokines (e.g., IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-10, IL12P35, IL12P40, IL12P70, IL-13, IL-15, IL-18, IL-23, IL-27, TNF- alpha, TNF-beta, TGF-beta, IFN-gamma, GM-CSF), common gamma chain of IL-2 receptor and any associated components of cytokine receptors. Additional examples of target cell directed moieties for use in stimulation of a biological effect include those that interact with TNF-R, LT-βR, Her-2, CD19, IgM, CD20, CD21 and CD22. In some instances, target cell directed moieties of the invention include those that upon interaction with the target cell receptor result in target cell stimulation through a tyrosine kinase, such as a src-family tyrosine kinase or a JAK family kinase, through a phosphatidylinositol 3-OH kinase. In some instances, target cell directed moieties include a specific antigen, or fragment thereof, including tumor antigen and antigen associated with an infectious disease, such as a viral antigen.

[0075] Target cell directed moieties also include lectins, including lectins which can function as mitogens. In some embodiments, lectins which bind particular cell surface receptors, for example, through interaction with glycosylated moieties on the particular receptor, can be used to induce aggregation of the receptor. Thus, the lectin is a target cell directed moiety that can contribute to stimulation of a biological effect in the target cell. Generally, lectins are glycoproteins that can be extracted from plants, seeds and other sources, and many are commercially available. In some cases, the lectins are biotinylated. Examples of lectins for use in the RBC complexes of the invention include, but are not limited to, Aleuria aurantia lectin, Amaranthus caudatus lectin, Bauhinia purpurea lectin, Concanavalin A (Con A), Succinylated Con A, Datura stramonium lectin, Dolichos biflorus agglutinin, Erythrina cristagalli lectin, Euonymus europaeus lectin, Galanthus nivalis lectin, Griffonia (Bandeiraea) simplicifolia lectin I (GSL I, BSL I), GSL I- isolectin B₄, Griffonia (Bandeiraea) simplicifolia lectin II (GSL II, BSL II), Hippeastrum hybrid lectin, Jacalin, Lens culinaris agglutinin, Lotus tetragonolobus lectin, Lycopersicon esculentum lectin, Maackia amurensis lectin I (MAL I), Maackia amurensis lectin II (MAL II), Madura pomifera lectin, Narcissus pseudonarcissus lectin, Peanut agglutinin, Phaseolus vulgaris agglutinin (PHA-E+L), Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Pisum sativum agglutinin, Psophocarpus tetragonolobus lectin I (PTL I), Psophocarpus tetragonolobus lectin II (PTL II), Ricinus communis agglutinin I (RCA₁₂₀), Ricinus communis agglutinin II (ricin, RCA₆o), Sambucus nigra lectin, Solanum tuberosum lectin, Sophorajaponica agglutinin, Soybean agglutinin, Ulex europaeus agglutinin I (UEA I), Vicia villos lectin, Wheat germ agglutimn (WGA), Succinylated WGA, and Wisteria floribunda lectin.

[0076] As described herein, the target cell directed moiety can be included within or attached to another molecule as long as the target cell directed moiety portion of such a hybrid molecule is capable of interacting with the receptor on the target cell. Such molecules include target cell directed moiety - immunoglobulin (Ig) fusion proteins, for example, hybrid molecules containing a target cell directed moiety linked to an Fc fragment of an Ig. Ig fusion proteins are known in the art, including those that contain an Fc fragment that comprises the hinge, CH2 and CH3 regions of human IgG molecules. See, for example, U.S. Pat. No. 5,116,964; Linsley et al. (1991) J Exp. Med. 173:721-730; Linsley et al. (1991) J Ex Med 174:561-569. Fusion proteins within the scope of the invention can be prepared by expression of a nucleic acid encoding the fusion protein in a variety of different systems known in the art and by other means known in the art.

[0077] In the complex, the target cell directed moiety is coupled, either directly or indirectly, to the surface of an RBC. When the target cell directed moiety is coupled directly to the surface of an RBC, the molecule comprising the moiety is either covalently or noncovalently attached to the surface of the RBC. The coupling of the molecule comprising the target cell directed moiety to a moiety on the surface of an RBC can be accomplished using techniques described herein and known in the art, including, but not limited to, direct covalent linkage, covalent conjugation via a crosslinker moiety and noncovalent conjugation (e.g., via a specific binding pair, via electrostatic bonding or via hydrophobic bonding).

[0078] When the target cell directed moiety is indirectly coupled to the surface of an RBC, the target cell directed moiety or the molecule comprising the moiety is attached to a linker and the linker is attached, either directly or indirectly, to a moiety on the surface of the RBC. The target cell directed moiety or the molecule comprising the moiety is either covalently or noncovalently attached to the linker by techniques described herein and known in the art, including, but not limited to, direct covalent linkage, covalent conjugation via a crosslinker moiety (which may include a spacer arm) and noncovalent conjugation (e.g., via a specific binding pair (e.g., biotin and avidin), via electrostatic bonding or via hydrophobic bonding). The linker is either directly or indirectly and either covalently or noncovalently attached to a moiety on the surface of an RBC by techniques described herein and known in the art, including, but not limited to, direct covalent linkage, covalent conjugation via a crosslinker moiety (which may include a spacer arm), noncovalent conjugation via a specific binding pair (e.g., via a specific binding pair (e.g., biotin and avidin), via electrostatic bonding or via hydrophobic bonding).

[0079] Accordingly, in some embodiments, the target cell directed moiety or the molecule comprising the moiety is attached to the surface of an RBC through a linker comprised of a specific binding pair such as biotin or an analogue of biotin, e.g., iminobiotin, and avidin or streptavidin. A biotin group can be attached, for example, to a moiety on the surface of an RBC and avidin or streptavidin incorporated into or attached onto the molecule comprising the target cell directed moiety. Alternatively, a biotin group can be attached to the molecule comprising the target cell directed moiety and avidin or streptavidin attached to the surface of an RBC. In either case, labeling one component with biotin and the other component with avidin or streptavidin allows for the formation of a non-covalently bound complex in which the target cell directed moiety is coupled to a biotin-(strept)avidin linker which is coupled to an RBC. Methods and techniques for attaching biotin, avidin and streptavidin to molecules and cells are well known in the art. See, for example, O'Shannessey et al. (1984) Immunol. Lett. 8:273-277; O'Shannessy et al. (1985) J. Appl. Biochem. 7:347-355; Wade et al. (1985) Biochem. J. 229:785-790; Rosenberg et al. (1986) J. Neurochem. 46:641-648 O'Shannessey et al. (1987) Anal. Biochem. 163:204-209; O'Shannessey et al. (1987) J Immunol. Meth. 99:153- 161; Reisfield et al. (1987) Biochem. Biophys. Res. Com. 142:519-526; Bayer et al. (1988) Anal. Biochem. 170:271-281; Green (1965). Biochem. J. 94:23c-24c; Green (1975) Avidin. Adv. Protein. Chemistry, Academic Press, New York (Anfinsen et al. eds.) 29:85-133.

[0080] In some embodiments, the linker can comprise at least one antibody, or the antigen binding portion thereof. An antibody that serves as a linker can bind both the target cell directed moiety, or the molecule comprising the moiety, and a molecule on the surface of an RBC or a molecule bound to a moiety on the surface of the RBC. A linker could comprise more than one antibody since one antibody could bind both the target cell directed moiety, or the molecule comprising the moiety, and a second antibody and the second antibody could then bind a moiety on the surface of an RBC. In complexes exemplified herein, a first antibody that binds a target cell receptor serves as the target cell receptor moiety is coupled to the surface of an RBC through a second antibody that binds a molecule on the surface of an RBC and a third antibody that binds to both the first and second antibodies.

[0081] Non-covalent associations can also occur through ionic interactions involving a target cell directed moiety and residues within a moiety on the surface of the RBC. Noncovalent associations can also occur through ionic interactions involving a target cell directed moiety and residues within a linker, such as charged amino acids, or through the use of a linker portion comprising charged residues that can interact with both the target cell directed moiety and the RBC surface. For example, non-covalent conjugation can occur between a generally negatively-charged target cell directed moiety or moiety on an RBC surface and positively- charged amino acid residues of a linker, e.g., polylysine, polyarginine and polyhistidine residues.

[0082] Covalent conjugation of the target cell directed moiety to the linker molecule or the linker molecule to the moiety on the RBC or the molecule containing the target cell directed moiety to the moiety on the RBC may be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the target cell directed moiety, linker molecule and/or the moiety on the RBC.

[0083] Target cell directed moieties or molecules containing target cell directed moieties that are polypeptides will contain amino acid side chain moieties containing functional groups such as amino, carboxyl, or sulfhydryl groups that will serve as sites for coupling the target cell directed moiety to the linker. Residues that have such functional groups may be added to the target cell directed moiety if the target cell directed moiety does not already contain these groups. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. In the case of target cell directed moieties or molecules containing target cell directed moieties that are carbohydrate or lipid, functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry. For instance, primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cystamine dihydrochloride followed by reduction with a standard disulfide reducing agent. In a similar fashion, the linker molecule or the moiety on the RBC may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.

[0084] Hydrophilic linkers of variable lengths are useful for connecting peptides or other bioactive molecules to linker molecules. Suitable linkers include linear oligomers or polymers of ethyleneglycol. Such linkers include linkers with the formula

R₁S(CH₂CH₂0)_nCH₂CH₂0(CH₂)_mC0₂R₂ wherein n=0-200, m=l or 2, Ri =H or a protecting group such as trityl, R =H or alkyl or aryl, e.g., 4-nitrophenyl ester. These linkers are useful in connecting a molecule containing a thiol reactive group such as haloaceyl, maleiamide, etc., via a thioether to a second molecule which contains an amino group via an amide bond. These linkers are generally flexible with regard to the order of attachment, i.e., the thioether can be formed first or last.

[0085] Moieties on or at the surface of an RBC to which the target cell directed moiety is coupled, include, but are not limited to molecules that are preferentially expressed on RBCs, such as glycophorin A (GPA), band 3, band 4.1 and spectrin (alpha and/or beta). Accordingly, a target cell directed moiety, or a molecule containing a target cell directed moiety, that interacts specifically with a molecule on the surface of an RBC, such as band 3, may be used in coupling the target cell directed moiety to the RBC. Alternatively, a linker that is specific for binding a molecule on or at the surface of the RBC, such as GPA, such as an anti-GPA antibody, or the GPA binding portion thereof, may be of use in coupling the target cell directed moiety to the RBC.

[0086] In addition or alternatively, a ligand can be coupled to the surface of the RBC which serves to preferentially direct the RBC to a particular cell, organ, tissue and/or site within an individual. Such a ligand may serve to increase up-take of the RBC by a particular organ or tissue. Directing ligands can be coupled to the RBC surface through any of the means described herein for the target cell directed moiety. RBCs with coupled directing ligands may or may not have a target cell directed moiety also coupled. Example of such directing ligands include, but are not limited to, ligands which direct the RBC to cells and/or tissue of the immune system, such as lymph nodes and spleen. RBCs preferentially directed to cells and/tissue of the immune system include those containing antigen(s) to which an immune response is desired. Examples of ligands which direct the RBCs to the lymph nodes are CD62L and LFA-1. RBC-antigen complexes directed to the lymph nodes may further include an antigen linked to a Tat polypeptide of HIV to facilitate processing of the antigen.

[0087] In some embodiments, the compositions and/or methods of the invention involve RBCs which have loaded with an agent (e.g., a drug or antigen) and can serve as a delivery vehicle for the agent.

[0088] As used herein, the term "loading" refers to introducing into or onto a red blood cell, either an intact RBC or an RBC ghost, at least one agent. In one embodiment, the agent is loaded by becoming internalized into the cell. In another embodiment, the agent is loaded by becoming coupled onto the surface of the cell and/or embedded in the membrane of the cell. Loading of an RBC with more than one agent may be performed such that the agents are loaded individually (in sequence) or together (simultaneously or concurrently). Loading can occur before, during and/or after the target cell directed moiety is coupled to the surface of the RBC. Loading is generally performed in a procedure separate from the procedure coupling a target cell directed moiety to the surface of the RBC, however, in some cases, the procedures can be concurrent. Agents may be first admixed at the time of contact with the cells or prior to that time.

[0089] As used herein and in this context, an "agent" includes but is not limited to an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate.

[0090] As used herein, the term "biological effector molecule" or "biologically active molecule" refers to an agent that has activity in a biological system, including, but not limited to, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin), a growth factor, an anti- apoptosis agent, an antigen, an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanized, a peptide hormone, a receptor, and a signaling molecule. As described herein, included within the term "immunoglobulin" are intact immunoglobulins as well as antibody fragments such as Fv, a single chain Fv (scFv), a Fab or a F(ab')₂.

[0091] In some embodiments, the RBCs are loaded with agents that promote Thl/Th2 cell growth, including, for example, IL-2, IL-7, IL-15, IL-18, IL-23, IL-27 and the like. In some embodiments, the RBCs are loaded with agents that promote Thl/Th2 cell differentiation, including, for example, IL-4, IL-12 and the like. In some embodiments, the RBCs are loaded with anti-apoptosis agents including, for example, cellular FLICE (FADD-like IL-1 beta- converting enzyme) inhibitory protein (cFLIP), cIAP (inhibitor of apoptosis protein) 1 and 2.

[0092] In some embodiments, the compositions and/or methods of the invention involve RBCs which are a)coupled to target cell directed moieties such as MHC I or MHC II tetramers loaded with a specific peptide or antigen specific for B cells and b) loaded with an agent such as an antigen or drug, e.g., FasL, TRAIL, TNF-alpha, IL-2, IL-15, IL-18, IL-23, or IL-27. Such compositions can thus direct the agent to the targeted B cell.

[0093] Loading may be performed by a procedure known in the art, such as a procedure selected from the group consisting of: iontophoresis, electroporation, sonoporation, microinjection, calcium precipitation, membrane intercalation, microparticle bombardment, lipid-mediated transfection, viral infection, osmosis, dialysis, including hypotonic dialysis, osmotic pulsing, osmotic shock, diffusion, endocytosis, phagocytosis, crosslinking to a red blood cell surface component, chemical crosslinking, mechanical perforation/restoration of the plasma membrane by shearing, single-cell injection, or a combination thereof. For example, a method and system for loading a cell with an agent is described in U.S. Pat. No. 6,495,351.

[0094] Sonoporation as a method for loading an agent into a cell is disclosed in, for example, Miller et al (1998) Ultrasonics 36: 947-952. Iontophoresis uses an electrical current to activate and to modulate the diffusion of a charged molecule across a biological membrane, such as the skin, in a manner similar to passive diffusion under a concentration gradient, but at a facilitated rate. In general, iontophoresis technology uses an electrical potential or current across a semipermeable barrier. By way of example, iontophoresis technology and references relating thereto is disclosed in WO 97/49450. [0095] Another method for loading agents in RBCs is electroporation. Electroporation has been used for encapsulation of foreign molecules in different cell types including red blood cells as described in Mouneimne et al. (1990) FEBS 275, No. 1, 2, pp. 117-120 and in U.S. Pat. No. 5,612,207. The process of electroporation involves the formation of pores in the cell membranes by the application of electric field pulses across a liquid cell suspension containing the cells. During the poration process, cells are suspended in a liquid media and then subjected to an electric field pulse. The medium may be electrolyte, non-electrolyte, or a mixture of electrolytes and non-electrolytes. The strength of the electric field applied to the suspension and the length of the pulse (the time that the electric field is applied to a cell suspension) varies according to the cell type, as is known in the art.

[0096] RBC loading may also take place by way of hypotonic dialysis. The dialysis devices used may be conventional dialysis devices as known in the art. Dialysis devices work on the principle of osmotic shock, whereby loading of an agent into red blood cell, is facilitated by the induction of sequential hypotonicity and recovery of isotonicity. The term "osmotic shock" is intended herein to be synonymous with the term "hypotonic dialysis" or "hypoosmotic dialysis." An exemplary osmotic shock/hypotonic dialysis method is described in Eichler et al. (1986) Res. Exp. Med. 186:407-412. For example, washed red blood cells are suspended in 1 ml of PBS (150 mMNaCl, 5 mM K₂HP0₄/KH₂PO₄, pH 7.4) to obtain a hematocrit of approximately 60%. The suspension is placed in dialysis tubing (molecular weight cut-off 12,000-14,000; Spectra-Por) and cells are dialyzed against 100 ml of 5 mM K HP0 /KH₂P0 , pH 7.4 for 90 minutes at 4 °C, thereby swelling the cells and rendering them permeable to agents to be loaded. Resealing is achieved by further dialysis, e.g., for 15 minutes at 37 °C against 100 ml of PBS containing 10 mM glucose. Cells are then washed in ice cold PBS containing 10 mM glucose using centrifugation.

[0097] Alternatively, other osmotic shock procedures can be implemented such as described, for example, in U.S. Pat. No. 4,478,824. For example, a packed RBC fraction is incubated in a solution containing a compound (such as dimethyl sulphoxide (DMSO) or glycerol) which readily diffuses into and out of cells. The compound rapidly creates a transmembrane osmotic gradient by diluting the suspension of RBCs in the solution with a near-isotonic aqueous medium. By including an anionic agent in the medium which may be an allosteric effector of hemoglobin, such as inosine monophosphate or a phosphorylated inositol (e.g., inositol hexaphosphate), water diffuses into the cells, swelling the cells and increasing the permeability of the outer membranes of the cells. Thus, the method may be used to load cells with anionic agents, as the increase in the RBCs' permeability is maintained for a period of time sufficient only to permit transport of the anionic agent into the cells and diffusion of readily-diffusing compounds out of the cells. However, this is generally not the method of choice where the desired agent to be loaded into cells is not anionic, or is anionic or polyanionic, but is not present in the near-isotonic aqueous medium in sufficient concentration to cause the needed increase in cell permeability without cell destruction. Other methods of loading RBCs with selected agents using an osmotic shock technique are described in U.S. Pat. No. 4,931,276.

[0098] Another technique of loading cells known in the art comprises microparticle bombardment of cells. For example, gold particles are coated with an agent to be loaded, dusting the particles onto a 22 caliber bullet. The bullet is fired into a restraining shield made of a bullet-proof material and having a hole smaller than the diameter of the bullet, such that the gold particles continue in motion toward cells in vitro and, upon contacting these cells, perforate them and deliver the payload (i.e., the agent) to the cell cytoplasm.

[0099] It will be appreciated by one skilled in the art that combinations of methods may be used to facilitate the loading of an RBC with agents of interest. Likewise, it will be appreciated that, when more than one agent is to be loaded, such as a first and second agent, the first and second agent may be loaded concurrently or sequentially, in either order, into an RBC cell.

[00100] RBCs for use in the invention may be concentrated to facilitate RBC loading, coupling of the target cell directed moiety and/or administration of the RBCs. Methods for concentrating RBCs and other cells are known in the art. Similar methods may also be used in the separation and/or purification of RBCs before or after RBC loading and/or coupling of target cell directed moiety to the RBC surface. Such methods include, for example, filtration and/or centrifugation techniques. Concentration and/or purification methods can be used to prepare the RBCs so that they are at a concentration or cell density useful for the desired purpose.

[00101] Since blood components have magnetization properties, magnetism has been used to separate and/or isolate blood components. Thus, other methods for separating and/or concentrating cells, including RBCs, uses magnetic techniques. Magnetic separation of RBCs is described in detail in U.S. Pat. Nos. 4,910,148, 5,514,340, 5,567,326, 5,541,072, 4,988,618, 4,935,147, 6,132,607, 6,129,848 and 6,036,857. In such methods, for example, magnetic beads or microbeads are coated with a molecule(s) suitable for specifically binding to an RBC, for example an antibody or other binding moiety capable of specifically binding to an RBC antigen, such as a molecule present on the surface of an RBC. RBCs are then mixed with such magnetic beads or microbeads and then transferred to a chamber where a magnetic field is applied to separate beads to which red blood cells are bound from other components. Alternatively, the beads may be provided within a collection device, and the collection device may be transformed into a separation device through the application of a magnetic field.

Antibody compositions

[00102] As described herein, complexes of use in the invention may include an antibody that binds a receptor on the target cell, or the receptor binding portion of the antibody, as a target cell directed moiety. In such complexes, the anti-target cell antibody is coupled, either directed or indirectly, to the surface of an RBC as described herein. As an example, the antibody can be labeled with biotin or avidin and coupled to the surface of an RBC through a biotin-avidin coupling. For example, the RBC can be biotinylated and the antibody can be covalently labeled with avidin or streptavidin using techniques known in the art. Alternatively, an RBC covalently labeled with avidin or streptavidin and the antibody can be biotinylated. In either case, for use in the invention, the target cell directed antibody labeled with biotin or avidin/streptavidin maintains the ability to bind to the target cell receptor.

[00103] Within the context of the present invention, antibodies are understood to include various kinds of antibodies, including, but not necessarily limited to, naturally occurring antibodies, monoclonal antibodies, polyclonal antibodies, antibody fragments that retain antigen binding specificity (e.g., Fab, and F(ab')₂) and recombinantly produced binding partners, single domain antibodies, hybrid antibodies, chimeric antibodies, single-chain antibodies, human antibodies, humanized antibodies, and the like. Generally, antibodies are understood to be reactive against a selected antigen on the surface of a cell if they bind with an affinity (association constant) of greater than or equal to 10 M .

[00104] Polyclonal antibodies against selected antigens on the surface of cells may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as- horses, cows, various fowl, rabbits, mice, or rats. In some cases, human polyclonal antibodies against selected antigens may be purified from human sources.

[00105] Preferably, monoclonal antibodies are used in the antibody compositions of the invention. Monoclonal antibodies specific for selected antigens on the surface of cells may be readily generated using conventional techniques (see, for example, Harlow et al., 1988, supra, and U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an antigen, and monoclonal antibodies can be isolated. Other techniques may also be utilized to construct monoclonal antibodies (see, for example, Huse et al. (1989) Science 246:1275-1281; Sastry et al. (1989) Proc. Natl. Acad. Sci. USA 86:5728-5732; Alting-Mees et al. (1990) Strategies in Molecular Biology 3:1-9).

[00106] Similarly, binding partners may be constructed utilizing recombinant DNA techniques. For example, the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. The primers may be utilized to amplify- heavy or light chain variable regions, which may then be inserted into appropriate expression vectors. These vectors may then be introduced into cells, for example E. coli cells, for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the _H and V domains may be produced (see, for example, Bird et al. (1988) Science 242:423-426). In addition, such techniques may be utilized to change a "murine" antibody to a "human" antibody, without altering the binding specificity of the antibody.

[00107] As used herein, a "single domain antibody" (dAb) is an antibody which is comprised of a V_H domain, which reacts immunologically with a designated antigen. A dAb does not contain a domain, but may contain other antigen binding domains known to exist in antibodies, for example, the kappa and lambda domains. Methods for preparing dAbs are known in the art. See, for example, Ward et al. (1989) Nature 341 :544-546. Antibodies may also be comprised of VH and V_L domains, as well as other known antigen binding domains. Examples of these types of antibodies and methods for their preparation are known in the art (see, e.g., U.S. Pat. No. 4,816,467).

[00108] Further exemplary antibodies include "univalent antibodies", which are aggregates comprised of a heavy chain/light chain dimer bound to the Fc (i.e., constant) region of a second heavy chain. This type of antibody generally escapes antigenic modulation. See, e.g., Glennie et al. (1982) Nature 295:712-714.

[00109] Antibodies can be fragmented using conventional techniques and the fragments (including "Fab" fragments) screened for utility in the same manner as described above for whole antibodies. The "Fab" region refers to those portions of the heavy and light chains which are roughly equivalent, or analogous, to the sequences which comprise the branch portion of the heavy and light chains, and which have been shown to exhibit immunological binding to a specified antigen, but which lack the effector Fc portion. "Fab" includes aggregates of one heavy and one light chain (commonly known as Fab'), as well as tetramers containing the 2H and 2L chains (referred to as F(ab)₂), which are capable of selectively reacting with a designated antigen or antigen family. Methods of producing Fab fragments of antibodies are known within the art and include, for example, proteolysis, and synthesis by recombinant techniques. For example, F(ab')₂ fragments can be generated by treating antibody with pepsin. The resulting F(ab')₂ fragment can be treated to reduce disulfide bridges to produce Fab' fragments. "Fab" antibodies may be divided into subsets analogous to those described herein, i.e., "hybrid Fab", "chimeric Fab", and "altered Fab".

[00110] "Hybrid antibodies" are antibodies wherein one pair of heavy and light chains is homologous to those in a first antibody, while the other pair of heavy and light chains is homologous to those in a different second antibody. Typically, each of these two pairs will bind different epitopes, particularly on different antigens. This results in the property of "divalence", i.e., the ability to bind two antigens simultaneously. Such hybrids may also be formed using chimeric chains, as set forth herein.

[00111] The invention also encompasses "altered antibodies", which refers to antibodies in which the naturally occurring amino acid sequence in a vertebrate antibody has been varied. Utilizing recombinant DNA techniques, antibodies can be redesigned to obtain desired characteristics. The possible variations are many, and range from the changing of one or more amino acids to the complete redesign of a region, for example, the constant region. Changes in the constant region, in general, to attain desired cellular process characteristics. Changes in the variable region may be made to alter antigen binding characteristics. The antibody may also be engineered to aid the specific delivery of a molecule or substance to a specific cell or tissue site. The desired alterations may be made by known techniques in molecular biology, e.g., recombinant techniques, site directed mutagenesis, and other techniques.

[00112] By "humanized" is meant alteration of the amino acid sequence of an antibody so that fewer antibodies and/or immune responses are elicited against the humanized antibody when it is administered to a human. For the use of the antibody in a mammal other than a human, an antibody may be converted to that species format.

[00113] "Chimeric antibodies", are antibodies in which the heavy and/or light chains are fusion proteins. Typically the constant domain of the chains is from one particular species and/or class, and the variable domains are from a different species and/or class. The invention includes chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region. Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. A variety of approaches for making chimeric antibodies have been described and can be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes selected antigens on the surface of differentiated cells or tumor cells. See, for example, Morrison et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 81:6851; Takeda et α/. (1985) Nαtωre 314:452; U.S. Pat. Νos. 4,816,567 and 4,816,397; European Patent Publications EP171496 and EP173494; United Kingdom patent GB 2177096B.

[00114] Bispecific antibodies may contain a variable region of an anti-target cell receptor antibody and a variable region specific for at least one antigen on the surface of an RBC. In other cases, bispecific antibodies may contain a variable region of an anti-target cell receptor antibody and a variable region specific for a linker molecule. In other cases, bispecific antibodies may contain a variable region specific for at least one antigen on the surface of an RBC and a variable region specific for a linker molecule. Bispecific antibodies may be obtained forming hybrid hybridomas, for example by somatic hybridization. Hybrid hybridomas may be prepared using the procedures known in the art such as those disclosed in Staerz et al. (1986, Proc. Natl. Acad. Sci. U.S.A. 83:1453) and Staerz et al. (1986, Immunology Today 7:241). Somatic hybridization includes fusion of two established hybridomas generating a quadroma (Milstein et al. (1983) Nature 305:537-540) or fusion of one established hybridoma with lymphocytes derived from a mouse immunized with a second antigen generating a trioma (Nolan et al. (1990) Biochem. Biophys. Ada 1040:1-11). Hybrid hybridomas are selected by making each hybridoma cell line resistant to a specific drug- resistant marker (De Lau et al. (1989) J. Immunol. Methods 117:1-8), or by labeling each hybridoma with a different fluorochrome and sorting out the heterofluorescent cells (Karawajew etα/. (1987) J Immunol. Methods 96:265-270).

[00115] Bispecific antibodies may also be constructed by chemical means using procedures such as those described by Staerz et al. (1985) Nature 314:628 and Perez et al. (1985) Nature 316:354. Chemical conjugation may be based, for example, on the use of homo- and heterobifunctional reagents with E-amino groups or hinge region thiol groups. Homobifunctional reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB) generate disulfide bonds between, the two Fabs, and O-phenylenedimaleimide (O-PDM) generate thioether bonds between the two Fabs (Brenner et al. (1985) Cell 40:183-190, Glennie et al. (1987) J Immunol. 139:2367-2375). Heterobifunctional reagents such as N-succinimidyl-3 -(2- pyridylditio) propionate (SPDP) combine exposed amino groups of antibodies and Fab fragments, regardless of class or isotype (Van Dijk et al. (1989) Int. J. Cancer 44:738-743).

[00116] Bifunctional antibodies may also be prepared by genetic engineering techniques. Genetic engineering involves the use of recombinant DNA based technology to ligate sequences of DNA encoding specific fragments of antibodies into plasmids, and expressing the recombinant protein. Bispecific antibodies can also be made as a single covalent structure by combining two single chains Fv (scFv) fragments using linkers (Winter et al. (1991) Nature 349:293-299); as leucine, zippers coexpressing sequences derived from the transcription factors fos and jun (Kostelny et al. (1992) J. Immunol. 148:1547-1553); as helix-turn-helix coexpressing an interaction domain of p53 (Rheinnecker et al. (1996) J. Immunol. 157:2989- 2997), or as diabodies (Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90-:6444-6448).

[00117] Antibodies against selected antigens on the surface of target cells and RBCs may also be obtained from commercial sources.

[0011§] A tetrameric immunological complex may be prepared by mixing a first monoclonal antibody which is capable of binding to at least one receptor on the surface of a target cell and a second monoclonal antibody which is capable of binding to a moiety on an RBC. The first and second monoclonal antibodies are from a first animal species. The first and second antibodies are reacted with monoclonal antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species. The first and second antibody may also be reacted with the F(ab')₂ fragments of monoclonal antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species. See, for example, U.S. Pat. No. 4,868,109. For example, the first and second antibody may be reacted with an about equimolar amounts of the monoclonal antibodies of the second animal species or of the F(ab')₂ fragments thereof.

[00119] An anti-target cell receptor antibody and anti-RBC moiety antibody may also be coupled through a biotin-(strept)avidin linkage as described herein. Additionally, an anti- target cell receptor antibody may be coupled to the surface of an RBC through a biotin- (strept)avidin linkage as described herein. Many alternative indirect ways to specifically link the antibodies and the RBCs in the composition for use in the invention would also be apparent to those skilled in the art. [00120] Antibodies may be selected for use in the antibody compositions of the invention based on their ability to stimulate the desired biological effect in the target cell. In some embodiments, anti-target cell antibodies include antibodies specific for the antigens CD3 and CD28 which are present on the surface of human CD4+ T cells. In some embodiments, an antibody that binds GPA is used to couple the RBC to the target cell directed moiety.

[00121] In some embodiments, an anti-CD3 or an anti-CD28 antibody are coupled to an anti-GPA antibody which in turn is bound to the surface of an RBC to form the target cell directed complex. Thus, in an embodiment, a composition for stimulating proliferation and differentiation of T cells from PBMCs comprises a) complexes comprising an anti-CD3 antibody, or CD3 binding portion thereof, coupled to an anti-GPA antibody, or GPA binding portion thereof, bound to an RBC and b) complexes comprising an anti-CD28 antibody, or CD28 binding portion thereof, coupled to an anti-GPA antibody, or GPA binding portion thereof, bound to an RBC. In some embodiments, the composition further comprises complexes comprising an anti-GPA antibody, or GPA binding portion thereof, coupled to an anti-GPA antibody, or GPA binding portion thereof.

[00122] In an embodiment, a composition for stimulating proliferation and differentiation of T eeϊls from PBMCs consists essentially of a) complexes comprising an anti-CD3 antibody, or CD3 binding portion thereof, coupled to an anti-GPA antibody bound to an RBC and b) complexes comprising an anti-CD28 antibody, or CD28 binding portion thereof, coupled to an anti-GPA antibody bound to an RBC.

[00123] In some embodiments, the anti-glycophorin A (anti-GPA) antibodies are used to couple the target cell directed moiety to an RBC and/or to couple an RBC to another RBC. Examples of monoclonal antibodies specific for glycophorin A are 10F7MN (U.S. Pat. No. 4,752,582, Cell lines: ATCC accession numbers HB-8473, HB-8474, and HB-8476), and D2.10 (Immunotech, Marseille, France). The concentration of anti-glycophorin A antibodies used in the antibody composition are generally less than the concentration that will cause agglutination (i.e. 3-10 μg/ml). Preferably the concentration of anti-glycophorin A antibodies used in the antibody composition is between about 0.5 to 5 μg/ml, preferably 1 to 2 μg/ml.

[00124] Monoclonal antibodies against CD3, and CD28, in the antibody composition of the invention are used to stimulate a biological effect in T cells. Examples of monoclonal antibodies specific for CD3 and CD28. are OKT3 and L293, respectively, and additional examples are in the art. Formulations and routes of administration

[00125] According to still another aspect of the invention, the compositions of the invention, including compositions comprising complexes of the invention and compositions comprising cells stimulated and/or generated using the methods of the invention, and mixtures thereof, are used in the preparation of medicaments, for treating the conditions described herein. These compositions of the invention are administered as pharmaceutically acceptable compositions. The compositions may be administered by any suitable means, including, but not limited to, intravenously, parenterally or locally. The compositions can be administered in a single dose by bolus injection or continuous infusion or in several doses over selected time intervals in order to titrate the dose.

[00126] The particular administration mode selected will depend upon the particular composition, treatment, cells involved, etc.. For the administration of cells, typically, about 10-10 cells can be administered in a volume of 50 μl to 1 liter, 1 ml to 1 liter, 10 ml to 250 ml, 50 ml to 150, and typically 100 ml. The volume will depend upon, for example, the type of cell administered, the disorder treated and the route of administration.

[00127] As used herein, "pharmaceutically acceptable excipient" includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and without causing disruptive reactions with the subject's immune system. Various pharmaceutically acceptable excipients are well known in the art.

[00128] Exemplary phaπnaceutically acceptable excipients include sterile aqueous or non- aqueous solutions and suspensions. Examples include, but are not limited to, any of the standard pharmaceutical excipients such as a phosphate buffered saline solution, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Compositions comprising such excipients are formulated by well known conventional methods (see: for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.).

[00129] The following Examples are provided to illustrate, but not limit, the invention. EXAMPLES [00130] T cells and receptors on their surface are used herein as examples of target cells and target cell receptors to demonstrate that interaction of target cell directed moieties coupled to RBCs can effect a biological response in the target cells.

EXAMPLE 1 Expansion of T cells [00131] To expand and differentiate T cells in culture, a cell population enriched for CD4+ T cells was stimulated with various complexes that included anti-CD3 and anti-CD28 monoclonal antibodies. The three different antibody complexes were tested. After culturing in the presence of the antibody complexes, the amount of T cell proliferation was determined.

Preparation of antibody complexes and cells

[00132] The antibody complexes A and B contained bispecific antibody complexes syntiiesized by StemCell Technologies, Inc.. These complexes were made by mixing affinity purified mouse anti-human CD3 antibody, mouse anti-human CD28 antibody and mouse anti- human glycophorin A (GPA) antibody together with a rat anti-mouse Ig antibody. The rat anti- mouse Ig antibody cross-linked the murine antibodies to form the bispecific complexes. The complexes were made using 30 micrograms/ml anti-CD3 and 30 micrograms/ml anti-CD28. For antibody complex A, the microgram ratio of anti-CD3/anti-CD28 : anti-GPA : anti-mouse Ig cross-linking antibody is 1:1:2. For antibody complex B, the microgram ratio of anti- CD3/anti-CD28 : anti-GPA : anti-mouse Ig cross-linking antibody is 1:3:4.

[00133] For a control complex, a standard operating protocol (SOP) preparation of human anti-CD3 antibody and human anti-CD28 antibody were coated on sheep anti-mouse paramagnetic beads (Dynal Corp.) and/or goat anti-mouse paramagnetic beads (Miltenyi Biotech). Such control complexes have been used to stimulate the generation of Thl cells from a population of CD4+ T cells as described in International Application WO 97/05239.

[00134] From a human leukapheresis product, CD4+ T cells were isolated using positive selection with a CliniMACS system (Miltenyi Biotech). The positive selection used the anti- human CD4 antibody labeled with biotin and the anti-biotin labeled microbeads to separate CD4+ cells from cells that did not express CD4.

[00135] At the time of leukapheresis, 20 ml of whole blood was drawn in heparinized vacutainer tubes. The whole blood was subjected to a Ficoll Hypaque density gradient separation to separate the red blood cell (RBC) fraction from the non-RBC fraction. The RBC fraction was washed twice with HBSS (BioWhittaker) and finally suspended in 20 ml of XVIVO-15™ (BioWhittaker) supplemented with 1.5 ml of CPDA1 solution (Baxter). The RBCs were stored at 4-8°C.

Cell culture

[00136] Each of three sets of isolated CD4+ T cells were stimulated with a different antibody complex preparation and cultured as follows. On day 0, a pellet of 8 x 10^δ purified CD4+ T cells was resuspended with 1 ml RBC suspension followed by the addition of 0.25 ml of antibody complex. The mixture was incubated on ice for 20 minutes. Following the incubation, the cells were suspended at 1 x 10⁶/ml by adding fresh XVIVO-15™ medium and placed in a 5% CO₂ incubator at 37°C. The cultures were left undisturbed on days 1 and 2.

[00137] On day 3, cell density in the cultures was measured using Sysmax automated counter. The cell cultures were adjusted to 4 x 10⁶ cells/ml and were restimulated with 1.5 ml RBCs and 0.1 ml of antibody complex and incubated on ice for 20 minutes. Following the incubation, the cells were suspended at 1 x 10 /ml in XVIVO-15™ medium and placed in a 5% C0₂ incubator at 37°C.

[00138] On days 4 and 5, cell density in the cultures was measured and cell density was adjusted to 1 x 10⁶/ml by adding fresh XVIVO-15™ medium. On day 6, cell density in the cultures was measured. The cell cultures were adjusted to 4 x 10 /ml and were restimulated with 1.5 ml RBCs and 0.1 ml antibody complex and incubated on ice for 20 minutes.

Following the incubation, the cells were suspended at 1 x 10 /ml in XVIVO-15™ medium and placed in a 5% CO incubator at 37°C.

[00139] On day 7, cell density in the cultures was measured and adjusted to 1 x 10⁶/ml by adding fresh XVIVO-15™ medium. On day 8, cell density in the cultures was measured and the cells in culture were harvested.

[00140] The T cell growth results from this experiment are presented in Fig. 1. As depicted in Fig. 1, the T cell populations stimulated with RBC + antibody complex compositions expanded more rapidly than the population stimulated with the SOP composition.

[00141] Through day 6, the two T cell populations stimulated with RBC + antibody complexes expanded at approximately the same rate. The rate of expansion for the population stimulated with RBCs and antibody complex B continued to increase through day 8 and the population experienced a 47-fold expansion over the 8 day period. The rate of expansion for the population stimulated with RBCs and antibody complex A began to slow at day 6 and the population experienced a 17-fold expansion over the 8 day period. By day 8, the population of T cells stimulated with the SOP compositions experienced a 15 -fold expansion.

[00142] Thus, contacting T cells with a complex comprising an anti-CD3 antibody coupled to an RBC through an anti-GPA antibody and a complex comprising an anti-CD28 antibody coupled to an RBC through an anti-GPA antibody is very effective in stimulating expansion of T cells in culture.

EXAMPLE 2 Expansion of T cells

[00143] Another RBC + antibody complex preparation was tested in the expansion and differentiation of Thl cells in culture. A cell population enriched for CD4+ T cells was isolated as described in Example 1. Antibody complexes were prepared by mixing affinity purified mouse anti-human CD3 antibody, mouse anti-human CD28 antibody and mouse anti- human glycophorin A (GPA) antibody together with a rat anti-mouse Ig antibody. The complexes were made using 30 micrograms/ml anti-CD3 and 30 micrograms/ml anti-CD28. The microgram ratio of anti-CD3/anti-CD28 : anti-GPA : anti-mouse Ig cross-linking antibody is 1 :3:4.

[00144] The CD4+ T cells (starting with 5 l0⁶ cells) were stimulated on day 0, 3, 6 and 9 with RBCs and antibody complexes as described in Example 1. One set of cells were stimulated by adding the antibody complex composition directly as a mixture to the CD4+ T cells and the RBCs ("RBC-conj."). For a second set of cells, the antibody complex composition was first incubated with RBCs, the RBCs were washed of excess antibody complex and the coated RBCs were then incubated with the CD4+ T cells ("RBC-conj (W)").

[0100] Control cell cultures included: CD4+ T cells only, CD4+ T cells stimulated with RBCs only, CD4+ T cells stimulated with antibody complex only, and CD4+ T cells stimulated with the SOP complex described in Example 1.

[0101] The cell density in each culture was determined on days 3, 6, 7, 8 and 9. The T cell growth results from this experiment are presented in Fig. 2. As depicted in Fig. 2, the T cell populations stimulated with antibody complex compositions expanded more rapidly than the population stimulated with the SOP composition. The cell cultures that received only RBCs or nothing did not expand. [0102] Through day 7, the three T cell populations stimulated with antibody complexes (with or without RBCs) expanded at approximately the same rate. The rate of expansion for the population stimulated with RBC-conj (W) continued to increase through day 9 and the population experienced a 76-fold expansion over the 9 day period. The rate of expansion for the population stimulated with RBC-conj also continued to increase through day 9 and the population experienced a 52-fold expansion over the 9 day period. The rate of expansion for the population stimulated with antibody complex alone appeared to stop at day 6 and the population experienced only a 13-fold expansion over the 9 day period. By day 9, the population of T cells stimulated with the SOP compositions experienced a 24-fold expansion.

[0103] Again, contacting T cells with a complex comprising an anti-CD3 antibody coupled to an RBC through an anti-GPA antibody and a complex comprising an anti-CD28 antibody coupled to an RBC through an anti-GPA antibody is very effective in stimulating expansion of Thl cells in culture.

EXAMPLE 3 Thl cell assay

[0104] The Thl or Th2 phenotype of CD4 T cells is characterized, in part, by the cell's production and or lack of production of specific cytokines. For example, Thl cells produce IFN-gamma and do not produce IL-4 whereas Th2 cells produce IL-4 but not IFN-gamma. Accordingly, to assess the differentiation state of the T cell population expanded using the RBCs + antibody complexes as described herein., IFN-gamma and IL-4 production from the expanded populations was assessed.

[0105] CD4+ T cells obtained from 2 healthy subjects were isolated and expanded using the RBCs + antibody complex as described in Example 2. Cells from one subject were mixed with complexes formed by mixing RBCs with the anti-CD3, anti-CD28 and anti-GPA antibodies (RBC-conj). Cells from the other subject were mixed with complexes that were formed by mixing RBCs with the anti-CD3, anti-CD28 and anti-GPA antibodies, and washed of excess complex before adding to the CD4+ T cells (RBC-conj (W)), as described in Example 2. The cells in culture were stimulated with the RBC + Ab complex on days 0, 3, 6 and 9 and were maintained at 1 x 10⁶/ml in XVIVO 15™ serum free culture medium. Cell culture supematants were collected on day 10 and stored at -80 °C until assayed.

[0106] The amount of IFN-gamma and IL-4 in the cell culture supematants was determined using ELISA kits. The ELISA kits (IFN-gamma: R&D Systems #DIF50; IL-4: Biosource #KHC0044C) were used according to the manufacturer's instructions. The standard curve range for detection of IFN-gamma was 1000-15.6 pg/ml and for detection of IL-4 was 27-7.8 pg/ml. Since the supematants were expected to contain different concentrations of the different cytokines, the culture supematants were diluted to 1 : 50 for IFN-gamma analysis and to 1 :1 for IL-4 analysis.

[0107] The expanded cell populations produced a mean value of 1513.6 ± 429 pg/10⁶ cells IFN-γ and undetectable amounts of IL-4 (<0.78 pg/10⁶ cells). Both culture conditions, RBC- conj and RBC-conj (W), generated similar cytokine profiles. The production of cytokine by the RBC protocol is comparable to that produced using the SOP composition (see Example 1) which resulted in production of 1253 pg /10⁶ cells of IFN-gamma on day 9 of culture.

[0108] The ability of the expanded cell population for produce IFN-gamma in large excess of IL-4 indicates that the RBC + antibody complex stimulation method described herein not only stimulates expansion of T cells but also lead to their differentiation to a Thl phenotype.

EXAMPLE 4 Streptavidinated RBCs

[§109] Heparinized whole blood (25 ml) was subjected to a Ficoll Hypaque density gradient separation to separate the red blood cell (RBC) fraction from the non-RBC fraction. The RBC fraction was washed twice with 50 ml of cold Hank's balanced salt solution (HBSS, Sigma Chemical Co.) and finally suspended in 50 ml of cold HBSS. The cell number was determined using Sysmex automated hematology analyzer. One billion RBCs were transferred to a 5 ml tube, washed with cold HBSS and collected by centrifugation (without braking).

[0110] After the supernatant was removed, the pellet of cells was thoroughly disturbed. 2 ml of 10 mM DTT (sterile-filtered) was added to the RBC cell pellet, the tube of cells was covered with aluminum foil to protect the contents from light and the tube was put in an end- to-end rotator at room temperature for 2 hours. The cells were then washed three times with cold HBSS and collected by centrifugation (without braking). The pellet of RBCs was resuspended in 0.5 ml HBSS at room temperature, the cell number determined using Sysmex and the RBC density adjusted to 2 x 10⁹ cells/ml with HBSS.

[0111] The streptavidin reagent was prepared as follows. Two mg streptavidin (SA) was dissolved at 1 mg/ml in HBSS and filtered through a 0.2 μm filter into a sterile 5 ml polypropylene tube. Ten mg of sulfo-SMCC was thoroughly dissolved in 10 ml of 37 °C HBSS. The solution was protected from light and rotated on an end-to-end rotator at room temperature for 2 hours. After 2 hours, the SA-SMCC solution was transferred to a Centricon filter and centrifuged at 3500 rpm for 30 minutes at room temperature. To the retentate in the upper chamber, 1 to 1.5 ml of sterile HBSS was added and the filter was centrifuged at 3500 rpm for 40 minutes at room temperature. The retentate was then transferred to a sterile 5 ml polypropylene tube and the final volume brought to 1 ml with HBSS. The SA-SMCC complex solution was sterile filtered through a 0.2 μm filter and stored at 4 °C until the DTT-reduced RBCs were ready.

[0112] The SA-SMCC complex was added to the DTT-reduced RBCs at a concentration of 1 mg per 1 x 10⁹ cells and the final reaction volume was 1 x 10⁹ RBCs/ml, e.g., 0.5 ml of 2 mg ml SA-SMCC + 0.5 ml of 2 x 10⁹ RBCs/ml. While protected from light, the cells + the complex were rotated at room temperature for 1 hour. The cells were collected by centrifugation, washed twice with cold HBSS and resuspended in 2 ml RBC storage buffer (10 ml HBSS, 1.4 ml CPDA-1 (citrate phosphate dextrose adenine solution; Baxter), 50 μg/ml gentamicin). The streptavidinated RBCs were counted and the cell concentration was adjusted to 2 x 10⁹ cells/ml with RBC storage buffer and stored at 4 °C.

[0113] The streptavidinated RBCs were assayed for the presence of streptavidin by FACS using a fluorescently labeled anti-SA antibody. The following 5 x 10⁶ RBCs were transferred to tubes containing 3 ml FACS buffer (Dulbecco's phosphate buffered saline without Ca/Mg (DPBS, Sigma Chemical Co.), 0.05% human Ig, 0.01% NaN₃; filter sterilized): DTT-reduced RBCs (two tubes) and streptavidinated RBCs (two tubes). The cells were pelleted and resuspended in 50 μl FACS buffer. To one of the tubes containing DTT-reduced RBCs was added 2 μl of anti-streptavidin FITC antibody (Vector Labs) and the other tube of DTT- reduced RBCs received no antibody (unstained control). To one of the tubes containing streptavidinated RBCs was added 2 μl of the anti-streptavidin FITC antibody and the other tube of streptavidinated RBCs received no antibody. After 20 minutes of incubation at 4 °C, 4 ml of FACS buffer was added to each tube and the cells were collected by centrifugation.

[0114] The cells were resuspended in 0.3 ml FACS buffer and subjected to FACS analysis. A typical FACS histogram following staining of streptavidinated RBCs with anti-streptavidin FITC antibody is shown in Fig. 4. EXAMPLE 5 T cell expansion/differentiation with RBC complexes and soluble SA complexes

[0115] Streptavidinated RBCs (SA-RBCs) were prepared as described in Example 4 and stored overnight at °4 C in RBC storage buffer or, if used the same day, stored in HBSS. At day 0, the desired stimulation ratio of CD4 celhRBC complex was 1 :5 and the number of SA- RBCs required for the complex preparation was determined as follows:

SA-RBCs required = (# CD4 T cells to be used for expansion x 10⁶) x 5 x 1.2. The 1.2 factor is used to account for cell losses during the procedure.

[0116] The number of SA-RBCs required to stimulate the CD4 T cells were transferred to a sterile polypropylene tube and washed twice with cold HBSS. The SA-RBCs were collected by centrifugation and resuspended in room temperature HBSS at 2 x 10⁹/ml.

[0117] In a different tube, biotinylated anti-CD3 antibody and biotinylated anti-CD28 antibody were mixed in room temperature HBSS in a final volume of 200 μl. For each 100 x 10⁶ SA-RBCs, 25 μg of each antibody was used. The 200 μl anti-CD3 + anti-CD28+ mixture was added to the SA-RBCs. The volume was adjusted to a final volume with room temperature HBSS. For example, for 0.2 x 10⁹ SA-RBCs, the final volume was 500 μl and for 0.5 x 10 the final volume was 1.0 ml. The antibody and RBC mixture was mixed on an end- to-end rotator at room temperature for 30 minutes and then washed twice with room temperature HBSS. The RBC-3/28 were collected by centrifugation and resuspended in room temperature HBSS at a density of 2 x 107μl.

[0118] CD4+ T cells were prepared as described in Example 1. 20 x 10⁶ purified CD4+ T cells were collected by centrifugation and resuspended in 0.8 ml of XVIVO-15™ + gentamicin (i.e., 25 x 10⁶/ml). The RBC-3/28 suspension was added to the T cells at a ratio of 1 :5 CD4+ T cells:RBC-3/28 (50 μl of RBC-3/28 = 100 x 10⁶ RBCs). After gentle mixing, the cell mixture was incubated on ice for 20 minutes and gently mixed every 5 minutes. After incubation, the CD4+ T cells were suspended at 1 x 107ml in XVIVO-15™ + gentamicin and distributed into two 25 cm² flasks. The cells were incubated in a 5% CO₂/37 °C humidified incubator.

[0119] At day 3, the cells and media were collected from the flasks, placed in 50 ml tubes and the tubes were returned to the incubator. From a sample of the cells, the total cell number and cell viability was determined using the Sysmex. The total number of cells (viable and dead) was used to determine the amount of soluble SA-CD3/CD28 complex needed. Each 1 x 10⁶ CD4+ T cells is stimulated with a mixture of 100 ng of biotinylated anti-CD3 antibody, 100 ng of biotinylated anti-CD28 antibody and 80 ng streptavidin on an equimolar basis. For 100 x 10⁶ CD4+ T cells, 500 μl of a SA-CD3/CD28 complex solution containing.20 ng/μl of each antibody and 16 ng/μl SA was needed.

[0120] For the preparation of the soluble SA-CD3/CD28 complex, streptavidin (SA) was dissolved in HBSS at 1 mg/ml and the solution was filtered through a 0.2 μm filter. An amount of sterile HBSS was added to a polypropylene tube appropriate for the final volume at the concentration described above. The biotinylated anti-CD3 antibody and the biotinylated anti-CD28 antibody were added to the HBSS and the solution was mixed well. Finally, the SA was added to the solution and the mixture was rotated end-to-end for 30 minutes at room temperature.

[0121] The CD4+ T cells were collected by centrifugation, the conditioned culture medium was transferred to a sterile tube and the cells were resuspended in the conditioned medium at a concentration of 25 x 10⁶ cells/ml. Based on the total cell count (viable and dead cells), the soluble SA-CD3/CD28 complex was added to the CD4 + T cells at 5 μl per 1 x 10⁶ cells and the suspension was gently mixed. The suspension was incubated at room temperature for 20 minutes, being gently mixed every 5 minutes. Following the incubation, conditioned medium and fresh XVIVO-15™ was added to the cells in an amount to bring the concentration to 1 x 10⁶ cells/ml. For this, conditioned medium was added to the incubated cells to 25 % of the final volume and the remaining volume was fresh XVIVO-15™ + gentamicin. When the cell count was 10 x 10⁶ cells, the cells were incubated in a volume of 0.4 ml, 2.1 ml of conditioned medium was added to the cells to bring the volume to 2.5 ml, and 7.5 ml of fresh XVIVO-15™ + gentamicin was added. The cells were transferred to cell culture flasks and incubated in a 5% C0₂/37 °C humidified incubator.

[0122] At day 4 and at day 5, an equal volume of fresh XVIVO-15™ + gentamicin was added to the cells and the cells were transferred to larger flasks accordingly. The cells continued incubation in a 5% C0₂/37 °C humidified incubator.

[0123] At day 6, the cells in the flasks were collected and the cell number and cell viability determined. The steps of day 3 were repeated for a second round of soluble SA-CD3/CD28 complex stimulation of the cells.

[0124] At day 7 and at day 8, an equal volume of fresh XVIVO- 15™ + gentamicin was added to the cells and the cells were transferred to larger flasks accordingly. The cells continued incubation in a 5% CO₂/37 °C humidified incubator. [0125] At day 9, the cells in the flasks were collected, the cell number and cell viability determined and the cells returned to flasks. The amount of soluble SA-CD3/CD28 complex for the number of cells was calculated and prepared as on day 3. The soluble SA-CD3/CD28 complex was added directly to the flasks and the cells were incubated in a 5% CO₂/37 °C humidified incubator for 20 minutes. After 20 minutes, fresh XVIVO-15™ + gentamicin was added to bring the cell density to 1 x 10 cells/ml and the cells were incubated in a 5% CO₂/37 °C humidified incubator.

[0126] At day 10, the cells in the flasks were collected and the cell number and cell viability determined. Culture supernatant was collected for quantitation of IFN-γ using ELISA assay and intracellular cytokine (ICC) staining and cell surface staining was performed. The expanded T cell population was assayed for expression of various cell phenotype markers, activation markers, adhesion molecules, chemokine receptors and TCR Vβ repertoire.

[0127] The T cell growth results from this experiment are presented in Fig. 4. As depicted in Fig. 4, the T cell populations stimulated with RBC-3/28 complex and with soluble SA- CD3/CD28 complexes expanded 32 to 118 fold in the 10 days of culture. As depicted in Figs. 5A and 5B, the expanded T cells are CD4+, CD3+, CD45RO+ and CD45RA+ and the cells express the activation markers CD25 and CD44. With regard to adhesion molecules, the expanded T cells are CD62L^{low high}, LFA-1+, CD49D+, CD162+, CLA+ and integrin α4β7-, as shown in Fig. 6. The expression of chemokine receptors on the T cell population was determined to be: CCR1-, CCR2-, CCR3^low, CCR4^low, CCR5-, CCR6-, CCR7^low, CCR9^low, CXCR3+, CXCR5^Iow and CXCR6-, as shown in Fig. 7. Thus, stimulation of CD4+ T cells with both RBC-3/28 complex and with soluble SA-CD3/CD28 leads not only to expansion of the T cells but also to their differentiation to an activated memory Thl phenotype characterized by expression of CD25, CD44, CD45RO and CXCR3 receptor.

[0128] The TCR Vβ repertoire of the T cell population was determined both before expansion and after expansion and the results are depicted in Fig. 8. The TCR Vβ repertoire of the T cell population after expansion was very similar to the repertoire before expansion. Thus, this method of T cell expansion did not appear to result in a change in the TCR Vβ repertoire of the population of T cells.

[0129] Supematants harvested on day 10 were examined for the production of IFN-γ and IL-4 to determine the Thl/Th2 pattern of the expanded population. Aliquots of the supematants were stored at -80 °C until assayed to avoid repeated freezing and thawing and the amount of IFN-γ and IL-4 in the cell culture supematants was determined by ELISA as described in Example 3. The sensitivity of the ELISA assay for detection of IFN-γ was 1000- 15.6 pg/ml and for the detection of IL-4 was 16-0.25 pg/ml. Since they were expected to contain different cytokines at different concentrations, the culture supematants were diluted to 1 : 100 for IFN-γ analysis and to 1 : 1 for IL-4 analysis. The expanded cell population produced a mean value of 7290 ± 1560 pg/10⁶ cells of IFN-γ and a mean value of 8.5 ± 1.8 pg/10⁶ cells of IL-4.

[0130] The expanded cell population was also assessed for the production of IFN-γ and IL- 4. The number of cells staining for intracellular IFN-γ and IL-4 was determined using ICC analysis as follows. A portion of the cell population was cultured with 25 μg/ml PMA and 0.75 mg/ml ionomycin for 2 hours and an additional 4 hours in the presence of the intracellular transport inhibitors brefeldin A and monensin. Another portion of the cell population was cultured without the PMA, ionomycin, brefeldin A and monensin for a control. The cells were washed, permeabilized and stained with the appropriate anti-cytokine antibodies for 30 minutes. Cells were washed and fixed with PFA and data was acquired on the flow cytometer 24 hours later. With no stimulation, about 38% of the expanded T cell population produced a high level of IFN-γ and little, if any, IL-4 (see Fig. 9) and upon stimulation with PMA and ionomycin, about 96% of the expanded population produced a high level of IFN-γ and little, if any, IL-4.

[0131] Thus, stimulation of CD4+ T cells with both RBC-3/28 complex and with soluble SA-CD3/CD28 leads not only to expansion of the T cells but also to their differentiation to a Thl phenotype.

Claims

CLAIMS We claim:

1. A method for stimulating a biological effect in a target cell, comprising contacting a target cell with a first complex comprising a first moiety coupled to the surface of a red blood cell (RBC), wherein the first moiety interacts with a receptor on the surface of the target cell and wherein the interaction of the first moiety with the receptor stimulates a biological effect in the target cell.

2. The method of claim 1 , wherein said first moiety is coupled to said RBC through a first linker.

3. The method according to claim 1, further comprising contacting the target cell with a second complex comprising a second moiety coupled to the surface of an RBC, wherein the second moiety interacts with a second receptor on the surface of the target cell.

4. The method of claim 3, wherein said second moiety is coupled to said RBC through a second linker.

5. The method according to claim 2, wherein the first linker comprises an antibody or antigen-binding portion thereof that binds to a molecule comprising the first moiety.

6. The method according to claim 4, wherein the second linker comprises an antibody or antigen-binding portion thereof that binds to a molecule comprising the second moiety.

7. The method according to claim 1 , wherein the first moiety comprises an antibody or antigen-binding portion thereof that binds the receptor on the target cell.

8. The method according to claim 3, wherein the second moiety comprises an antibody or antigen-binding portion thereof that binds the receptor on the target cell.

9. The method according to claim 1, wherein the first moiety comprises a ligand that interacts with the receptor on the target cell.

10. The method according to claim 3, wherein the second moiety comprises a ligand that interacts with the receptor on the target cell.

11. The method according to claim 4, wherein the target cell is a T cell.

12. The method according to claim 11, wherein the first moiety interacts with CD3 on the T cell surface and the second moiety interacts with CD28 on the T cell surface.

13. The method according to claim 12, wherein the biological effect stimulated is T cell proliferation, or wherein the biological effect stimulated is T cell differentiation.

14. The method according to claim 1, wherein the target cell is a tumor cell and wherein the biological effect stimulated is apoptosis.

15. The method according to claim 14, wherein the first receptor on the surface of the target cell is a Fas receptor or a TNF receptor.

16. The method according to claim 1, wherein the RBC is allogeneic to the target cell.

17. The method according to claim 1, wherein the RBC is autologous to the target cell.

18. The method according to claim 1, wherein the contacting occurs in an individual.

19. The method according to claim 3, wherein the contacting occurs in an individual.

20. The method according to claim 19, wherein the RBC is allogeneic to the target cell.

21. The method according to claim 19, wherein the RBC is autologous to the target cell.

22. The method according to claim 1 , further comprising contacting the target cell with a soluble streptavidin complex comprising the first moiety.

23. The method according to claim 3, further comprising contacting the target cell with a soluble streptavidin complex comprising the first moiety and the second moiety.

24. The method according to claim 1 , wherein the biological effect stimulated is cytokine production.

25. The method according to claim 24, further comprising contacting the target cell with a soluble streptavidin complex comprising the first moiety.

26. The method according to claim 3, wherein the biological effect stimulated is cytokine production.

27. The method according to claim 26, further comprising contacting the target cell with a soluble streptavidin complex comprising the first moiety and the second moiety.

28. A method for stimulating T cell proliferation in an individual, comprising administering a composition comprising a first complex comprising an antibody or antigen-binding portion tliereof that binds a first receptor on the surface of a T cell coupled to the surface of a red blood cell (RBC).

29. The method of claim 28, further comprising administering a second complex comprising a second antibody or antigen-binding portion thereof that binds a second receptor on the surface of a T cell coupled to a moiety on the surface of an RBC.

30. The method according to claim 28, wherein the RBC is allogeneic to the target cell.

31. The method according to claim 28, wherein the RBC is autologous to the target cell.

32. A composition comprising red blood cells (RBCs), a first antibody complex and a second antibody complex, wherein the first antibody complex comprises a first antibody or antigen-binding portion thereof that binds CD3 coupled to the surface of an RBC, and wherein the second antibody complex comprises a second antibody or antigen-binding portion thereof that binds CD28, coupled to the surface of an RBC.

33. A composition comprising a red blood cell (RBC) and an antigen peptide, wherein the antigen peptide is associated with major histocompatability complex (MHC) molecule and wherein the MHC molecule is coupled to the surface of the RBC.

34. The composition according to claim 32, wherein the MHC molecule is a class I MHC molecule.

35. The composition according to claim 32, wherein the MHC molecule is a class II MHC molecule.

36. The composition according to claim 32, wherein the antigen peptide is a tumor antigen peptide or a viral antigen.

37. The composition according to claim 33, wherein the RBC is loaded with an agent selected from the group consisting of cytokine, chemokine and hormone.

38. The composition according to claim 37, wherein the agent is a cytokine selected from the group consisting of IL-2, IL-4, IL-5, IL-15, IL-18, IL-27, TNF-alpha, FasL and TRAIL.