Antigen presenting cell mimic scaffold and preparation and use methods thereof

Abstract

Embodiments described herein provide antigen presenting cell mimic scaffolds (APC-MS) and the use of such scaffolds for manipulating T cells. More specifically, the scaffolds can be used to promote the growth, division, differentiation, amplification, proliferation, activity, vitality, exhaustion, incompetence, dormancy, apoptosis or death of T cells in various situations (e.g., in vitro, ex vivo, or in vivo). Embodiments described herein further relate to pharmaceutical compositions, kits and packaging containing such scaffolds. Other embodiments relate to methods for preparing the scaffolds, compositions and kits / packaging. Methods for using the scaffolds, compositions and / or kits in the diagnosis or treatment of diseases (e.g., cancer, immunodeficiency disorders and / or autoimmune disorders) are also described herein.

Description

[0001] This application is a divisional application of invention patent application No. 201780054844.6, whose application date is July 13, 2017 and whose invention name is "Antigen presenting cell simulation scaffold and its preparation and use method".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 361,891, filed on July 13, 2016, the entire contents of which are expressly incorporated herein by reference. Background Art

[0004] Immunotherapy involving priming and expansion of T lymphocytes (T cells) still holds promise for the treatment of cancer and infectious diseases, particularly in humans (Melief et al., Immunol. Rev. 145:167-177 (1995); Riddell et al., Annu. Rev. Immunol. 13:545-586 (1995)). Current studies of adoptive transfer in patients with viral infections and / or cancer involve the infusion of T cells that have been stimulated, cloned, and expanded for multiple weeks in vitro on autologous dendritic cells (DCs), virally infected B cells, and / or allogeneic feeder cells (Riddell et al., Science 257:238-241 (1992); Yee et al., J. Exp. Med. 192:1637-1644 (2000), Brodie et al., Nat. Med. 5:34-41 (1999); Riddell et al., Hum. Gene Ther. 3:319-338 (1992), Riddell et al., J. Immunol. Methods 128:189-201 (1990)). However, since adoptive T cell immunotherapy clinical trials often require billions of cells (Riddell et al., 1995), existing in vitro T cell expansion protocols are often insufficient to meet the needs of such trials.

[0005] In addition, optimal transplantation needs to use functional T cells, rather than aging T cells, when reperfusion occurs. For clinical application, it is important to ensure that T cells have the required functionality, that is, they proliferate, perform effector functions and produce cytokines in a required manner (Liebowitz et al., Current Opinion Oncology, 10, 533-541, 1998). In the natural environment, T cell activation is started (Schwartz, Science 248: 1349) by the engagement of the T cell receptor / CD3 complex (TCR / CD3) of the peptide-antigen combined with the major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APC). Although this is the main signal in T cell activation, for complete activation, other receptor-ligand interactions between APC and T cell are also required. For example, in the absence of other molecular interactions, TCR stimulation can induce an anergy state, rendering these cells unable to respond to full activation signals upon restimulation (Schwartz, 1990; Harding et al., Nature 356:607, 1992; Dudley et al., Clinical Cancer Research., 16, 6122-6131, 2010; Rosenberg et al., Clinical Cancer Research., 17, 4550-4557, 2011). Alternatively, upon activation by TCR engagement alone, T cells may die by programmed cell death (apoptosis) (Webb et al., Cell 63:1249, 1990; Kawabe et al., Nature 349:245, 1991; Kabelitz et al., Int. Immunol. 4:1381, 1992; Groux et al., Eur J. Immunol. 23:1623, 1993).

[0006] Therefore, by using a second signal transduction molecule, for example, a secretory product of a membrane-bound protein or APC, optimal functionality can be given. In the case of membrane-bound proteins, such secondary interactions are generally adhesive in nature, strengthening the contact between the two cells (Springer et al., Ann. Rev. Immunol. 5: 223, 1987). Other signal transduction molecules may also be involved, such as other activation signals that are conducted from APC to T cells (Bierer et al., Adv. Cancer Res. 56: 49, 1991). For example, CD28 is a surface glycoprotein present on 80% of peripheral T cells in humans and is present on resting and activated T cells. CD28 binds to B7-1 (CD80) or B7-2 (CD86) and is one of the most effective costimulatory molecules known (June et al., Immunol. Today 15: 321 (1994), Linsley et al., Ann. Rev. Immunol. 11: 191 (1993)). Ligation of CD28 on T cells and engagement of the TCR induces the production of interleukin-2 (IL-2) (June et al., 1994; Jenkins et al., 1993; Schwartz, 1992). Secreted IL-2 is an important factor for ex vivo T cell expansion (Smith et al., Ann. NY Acad. Sci. 332:423-432 (1979); Gillis et al., Nature 268:154-156 (1977)).

[0007] Costimulation of T cells has been shown to affect various aspects of T cell activation (June et al., 1994). It reduces the concentration of anti-CD3 required to induce a proliferative response in culture (Gimmi et al., Proc. Natl. Acad. Sci. USA 88:6575 (1991)). CD28 costimulation also significantly enhances lymphokine production by helper T cells through transcriptional and post-transcriptional regulation of gene expression (Lindsten et al., Science 244:339 (1989); Fraser et al., Science 251:313 (1991)), and can activate the cytolytic potential of cytotoxic T cells. Inhibition of CD28 costimulation in vivo can block xenograft rejection and significantly delay allograft rejection (Lenschow et al., Science 257:789 (1992); Turka et al., Proc. Natl. Acad. Sci. USA 89:11102 (1992)).

[0008] More importantly, the above-mentioned effector for stimulating / co-stimulatory stimulation has been widely used in the case of in vitro T cell manipulation. In this regard, the combination of anti-CD3 monoclonal antibody (first signal) and anti-CD28 monoclonal antibody (second signal) is most commonly used to stimulate APC. When the antibody is fixed on a solid surface, the signal provided by anti-CD3 and anti-CD28 monoclonal antibodies is optimally delivered to T cells, and the solid surface is such as plastic plates (Baroja et al., Cellular Immunology, vol.120,205-217,1989; Damle et al., The Journal of Immunology, vol.143,1761-1767,1989) or agarose beads (Anderson et al., Cellular Immunology, vol.115,246-256,1988). Also referring to U.S. Patent No. 6,352,694 authorized to June et al.

[0009] Various surfaces and reagents containing anti-CD3 and anti-CD28 monoclonal antibodies have been developed for obtaining and expanding T cells for various applications. For example, Levine et al. (The Journal of Immunology, vol. 159, No. 12: pp. 5921-5930, 1997) disclosed tosyl-activated paramagnetic beads with a diameter of 4.5 micrometers (μM) containing anti-CD3 and anti-CD28 monoclonal antibodies, which can be used to stimulate and proliferate T cells and induce them to produce proinflammatory cytokines. It has also been demonstrated that T cells activated with these beads exhibit properties that make them potentially useful for adoptive immunotherapy, such as cytokine production (Garlie et al., J Immunother 22 (4): 336-45, 1999; Shibuya et al., Arch Otolaryngol Head Neck Surg, vol. 126, No. 4: 473-479, 2000). These beads are commercially available from Thermo-Fisher Scientific, Inc. under the trade name DYNABEADS CD3 / CD28 T-Cell Expansion.

[0010] In cell therapy, the use of paramagnetic beads with fixed monoclonal antibodies for T cell expansion needs to be separated and removed from T cells before patient infusion. This is a very laborious process and causes cell loss, cell damage, the risk of contamination and increased processing costs. Due to the tight binding of the monoclonal antibodies fixed on the beads and the corresponding ligands on the target T-cell surface, it is difficult to remove beads from T cells. Often by waiting until the target antigen is internalized by T cells and mechanical destruction technology is used to separate beads and T cells, thereby separating beads-cell conjugates. This technology can cause the damage of T cells and can also cause the antigen connected on the T cells to be removed from the cell surface (Rubbi et al., Journal of Immunology Methods, 166, 233-241, 1993). In addition, because activated T cells are often the most needed for cell therapy schemes, and lose ideal cell characteristics during the 24-72 hour waiting time, paramagnetic separation has limited use in adoptive cell therapy situations.

[0011] Techniques for isolating and purifying cells that adhere to paramagnetic beads are also unavailable in clinical settings. For example, methods for removing paramagnetic beads after separation from T cells require passing the cell / bead solution through a magnet. This method, while significantly reducing the number of beads remaining associated with the T cells, does not completely eliminate the beads. Implanting a composition containing beads into a patient can cause toxic effects. The bead removal process also reduces the number of T cells available for treatment, as many T cells remain associated with the paramagnetic beads even after mechanical dissociation. Some cell loss also occurs for T cells that were manipulated but not otherwise associated with the beads, as these cells are washed away prior to the internalization and / or mechanical removal steps.

[0012] Therefore, there is an unmet need for compositions and methods that allow for the isolation of T cells that can be readily used in the treatment of human diseases, such as immunodeficiency disorders, autoimmune disorders, and cancer. The embodiments of the present invention, described in detail below, address these needs. Summary of the Invention

[0013] The present invention provides compositions and methods for manipulating (e.g., activating, stimulating, amplifying, proliferating, or empowering) T cells. In this regard, embodiments of the present invention provide methods for producing a large number of activated T cells (or substantially pure subpopulations) that express certain markers and / or cell surface receptors or produce certain cytokines that are optimal for T cell-mediated immune responses. Such manipulated T cells can be used in the treatment and prevention of many diseases, such as cancer, infectious diseases, autoimmune diseases, allergies, immune dysfunction associated with aging, or any other disease state in which T cells are required for treatment. Additional embodiments described herein relate to methods and compositions for effectively treating any of the above-mentioned diseases by utilizing T cells with optimal responsiveness, which cells are selected or screened using the compositions and / or methods of the present invention. The compositions and methods of the present invention are more effective than existing compositions and methods, not only in terms of the ability to produce a large number of activated T cells, but also in terms of the significantly improved effectiveness of such T cells in an in vivo environment. Therefore, the compositions and methods of the present invention are useful for generating highly desirable human T lymphocytes for transplantation, autologous transfer, and therapeutic applications.

[0014] Thus, in one embodiment, the present invention provides an antigen presenting cell mimic scaffold (APC-MS) comprising a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a plurality of T cell activation molecules and T cell co-stimulatory molecules adsorbed on the scaffold; and a plurality of T cell homeostatic agents adsorbed on the scaffold.

[0015] In one embodiment, the present invention provides an antigen presenting cell mimetic scaffold (APC-MS) that sequesters T cells selected from natural killer (NK) cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs), or a combination thereof.

[0016] In one embodiment, the present invention provides an antigen presenting cell mimic scaffold (APC-MS) comprising a plurality of T cell homeostatic agents adsorbed onto a SLB layer.

[0017] In one embodiment, the present invention provides an antigen presenting cell mimic scaffold (APC-MS) comprising a plurality of T cell homeostatic agents adsorbed onto an MSR layer.

[0018] In one embodiment, the invention provides an antigen presenting cell mimic scaffold (APC-MS) containing multiple T cell steady state agents released from scaffold in a controlled release manner. In some embodiments, the T cell steady state agent is released from scaffold in a controlled release manner in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer time period.

[0019] In one embodiment, the invention provides the antigen presenting cell simulation support (APC-MS) containing multiple T cell steady-state agents that are released from support for up to 15 days in a sustained manner. In some embodiments, T cell steady-state agent is released from support for up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer in a sustained manner. In some embodiments, T cell steady-state agent is released from support for at least 30 days in a sustained manner. In some embodiments, the T cell homeostatic agent is released from the scaffold in a sustained manner for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or longer.

[0020] In one embodiment, the present invention provides an antigen presenting cell mimetic scaffold (APC-MS) containing multiple T cell homeostatic agents, wherein the T cell homeostatic agents are selected from IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 and transforming growth factor β (TGF-β), or agonists thereof, mimetics thereof, variants thereof, functional fragments thereof, or combinations thereof.

[0021] In one embodiment, the present invention provides an antigen presenting cell mimetic scaffold (APC-MS) containing multiple T cell homeostatic agents, wherein the T cell homeostatic agent is IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a composition thereof with a second homeostatic agent selected from IL-7, IL-21, IL-15, and an IL-15 superagonist. In one embodiment, the T cell homeostatic agent can be selected from an N-terminal IL-2 fragment comprising the first 30 amino acids (p1-30) of IL-2, an IL-2 superkine peptide, and an IL-2 partial agonist peptide, or a combination thereof.

[0022] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) containing a variety of activation and costimulatory molecules, wherein T cell activation molecules and T cell costimulatory molecules are each independently adsorbed on a lipid bilayer (SLB) supported by a fluid. In one embodiment, T cell activation molecules and T cell costimulatory molecules can be adsorbed by affinity pairing or chemical coupling. In some embodiments, chemical coupling includes click chemistry reagents (e.g., DBCO or azide). In one embodiment, T cell activation molecules and T cell costimulatory molecules can be adsorbed by affinity pairing, and the affinity pairing includes biotin-avidin protein streptavidin pairs, antibody-antigen pairs, antibody-hapten pairs, aptamer affinity pairs, capture protein pairs, Fc receptor-IgG pairs, metal-chelated lipid pairs, metal-chelated lipid-histidine (HIS) labeled protein pairs, or a combination thereof. In one embodiment, the T cell activating molecule and the T cell co-stimulatory molecule can be adsorbed by chemical coupling including azide-alkyne chemistry (AAC) reaction, dibenzo-cyclooctyne linkage (DCL) or tetrazine-olefin linkage (TAL).

[0023] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) containing multiple activation and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each independently coated on a fluid-supported lipid bilayer (SLB). Alternatively, in another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) containing multiple activation and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each independently partially embedded in a fluid-supported lipid bilayer (SLB).

[0024] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) containing multiple activation and costimulatory molecules, wherein T cell activation molecules and T cell costimulatory molecules are each independently adsorbed on mesoporous silica microrods (MSR).

[0025] In another embodiment, the present invention relates to an antigen presenting cell mimetic scaffold (APC-MS) comprising multiple activation and co-stimulatory molecules, wherein the T cell activation molecule and the T cell co-stimulatory molecule are each independently an antibody molecule or an antigen binding fragment thereof.

[0026] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) containing multiple activation and co-stimulatory molecules, wherein the T cell activation molecule is selected from anti-CD3 antibodies or antigen-binding fragments thereof, anti-CD2 antibodies or antigen-binding fragments thereof, anti-CD47 antibodies or antigen-binding fragments thereof, anti-macrophage scavenger receptor (MSR1) antibodies or antigen-binding fragments thereof, anti-T cell receptor (TCR) antibodies or antigen-binding fragments thereof, major histocompatibility complex (MHC) molecules or multimers thereof loaded with MHC peptides and MHC-immunoglobulin (Ig) conjugates or multimers thereof, or a combination thereof.

[0027] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) containing multiple activation and costimulatory molecules, wherein the T cell costimulatory molecule is an antibody or an antigen binding fragment thereof, which specifically binds to a costimulatory antigen selected from the group consisting of: CD28, 4.1BB (CD137), OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTβR (TNFRSF3), DR3 (TNFRSF25 ), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD 244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CD279 (PD1) and CRACC (CD319, BLAME).

[0028] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) comprising multiple activation and co-stimulatory molecules, wherein the T cell activation molecules and T cell co-stimulatory molecules comprise bispecific antibodies or antigen-binding fragments thereof.

[0029] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) comprising a plurality of activation and co-stimulatory molecules, wherein the T cell activation molecules and the T cell co-stimulatory molecules comprise pairs selected from CD3 / CD28, CD3 / ICOS optionally with CD28, CD3 / CD27 optionally with CD28, and CD3 / CD137 optionally with CD28, or a combination thereof.

[0030] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS) further comprising an immunoglobulin molecule that specifically binds to an Fc-fusion protein.

[0031] In another embodiment, the present invention relates to an antigen presenting cell mimicking scaffold (APC-MS), which further comprises a recruitment compound selected from the group consisting of granulocyte macrophage colony stimulating factor (GM-CSF), chemokine (CC motif) ligand 21 (CCL-21), chemokine (CC motif) ligand 19 (CCL-19), CXC motif chemokine ligand 12 (CXCL12), interferon gamma (IFNγ) or FMS-like tyrosine kinase (Flt-3) ligand, or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof. In one embodiment, the scaffold further comprises a recruitment compound, which is granulocyte macrophage colony stimulating factor (GM-CSF), or an agonist thereof, a mimetic thereof, a variant thereof, or a functional fragment thereof.

[0032] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS), which further comprises an antigen. In one embodiment, the antigen comprises a tumor antigen.Further in this embodiment, the tumor antigen is selected from MAGE-1, MAGE-2, MAGE-3, CEA, tyrosinase, midkin, BAGE, CASP-8, β-catenin, β-catenin, γ-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gp100, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Rα, IL13Rα2, AIM-2, AIM-3, NY-ESO-1, C9orf112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, , RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L, CYNL2, TPR, SOX2, GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, nestin, OLIG2, ART1, ART4, B-cyclin, Gli1, Cav-1, cathepsin B, CD74, E-cadherin, EphA2 / Eck, Fra-1 / Fosl1, GAGE-1, ganglioside / GD2, GnT-V, β1,6-N, Ki67, Ku70 / 80, PROX1, PSCA, SOX10, SOX11, survivin, UPAR, WT-1, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (AD Abp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, T-cell receptor / CD3-ζ chain, GAGE-family of tumor antigens, RAGE, LAGE-I, NAG, GnT-V, RCASl, α-fetoprotein, pl20ctn, Pmel117, PRAME, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5, SCP-I, CT- 7, cdc27, adenomatous polyposis coli protein (APC), fodrin, P1A, connexin 37, Ig-idiotype, p15, GM2, GD2 ganglioside, Smad family of tumor antigens, 1mp-1, EBV-encoded nuclear antigen (EBNA)-1, UL16-binding protein-like transcript 1 (Mult1), RAE-1 protein, H60, MICA, MICB, c-erbB-2, new antigens identified in a patient-specific manner, or immunogenic peptides thereof, or a combination thereof.

[0033] In a related embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS), comprising a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a plurality of T cell activation molecules and T cell co-stimulatory molecules adsorbed on the scaffold; and a plurality of T cell homeostatic agents adsorbed on the scaffold, wherein the weight ratio of the supported lipid bilayer (SLB) to the mesoporous silica microrods (MSR) is between about 10:1 and about 1:20. In one embodiment, the weight ratio reflects the ratio of SLB to MSR before loading. In another embodiment, the weight ratio is adjusted to obtain the desired scaffold composition. In one embodiment, the weight ratio of SLB to MSR can be between about 9:1 and about 1:15, between about 5:1 and about 1:10, between about 3:1 and about 1:5, including all ratios therebetween, for example, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, and about 1:10.

[0034] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS), comprising a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a variety of T cell activation molecules and T cell co-stimulatory molecules adsorbed on the scaffold; and a variety of T cell homeostatic agents adsorbed on the scaffold, wherein the continuous fluid-supported lipid bilayer (SLB) comprises a lipid comprising 14 to 23 carbon atoms. In one embodiment, the lipid is phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylserine (PS) or phosphoinositide, or a derivative thereof. In one embodiment, APC-MS includes a fluid-supported lipid bilayer (SLB) comprising a lipid selected from dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) or a combination thereof. In some embodiments, the lipid bilayer includes a lipid composition that simulates the lipid composition of a mammalian cell membrane (e.g., human cell plasma membrane). The lipid composition of many mammalian cell membranes has been characterized and is readily determined by those skilled in the art (see, e.g., Essaid et al., Biochim.Biophys.Acta 1858 (11): 2725-36 (2016), the entire contents of which are incorporated herein by reference). In some embodiments, the lipid bilayer includes cholesterol. In some embodiments, the lipid bilayer comprises a sphingolipid. In some embodiments, the lipid bilayer comprises a phospholipid. In some embodiments, the lipid is a phosphatidylethanolamine, a phosphatidylcholine, a phosphatidylserine, a phosphoinositide, a sphingomyelin with a saturated or unsaturated tail comprising 6-20 carbons, or a combination thereof.

[0035] In another embodiment, the present invention relates to an antigen presenting cell mimicking scaffold (APC-MS), comprising a substrate comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR substrate; a plurality of T cell activating molecules and T cell co-stimulatory molecules adsorbed on the scaffold; and a plurality of T cell homeostatic agents adsorbed on the scaffold, wherein the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold maintains a continuous, fluidic architecture for at least 14 days. In some embodiments, the MSR-SLB scaffold maintains a continuous, fluidic architecture for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 50 days or longer. In some embodiments, the MSR of the MSR-SLB scaffold degrades within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 50 days, or more. In some embodiments, the lipid bilayer of the MSR-SLB scaffold degrades within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 450 days, or more.

[0036] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS), including a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a variety of T cell activation molecules and T cell costimulatory molecules adsorbed on the scaffold; and a variety of T cell homeostatic agents adsorbed on the scaffold, wherein the dry weight ratio of the mesoporous silica microrods (MSR) to the T cell activation / costimulatory molecules is between about 1: 1 and about 50: 1. In one embodiment, the ratio of MSR to T cell activation / costimulatory molecules is a reflection of the weight of the MSR weight and the weight of the antibody used as the T cell activation / costimulatory molecule. In another embodiment, the MSR: antibody weight ratio is adjusted to obtain the desired scaffold composition. In one embodiment, the weight ratio of SLB to antibody composition is between about 2:1 and about 20:1, between about 3:1 and about 10:1, between about 4:1 and about 8:1, including all ratios therein, for example, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 40:1.

[0037] In another embodiment, the present invention relates to an antigen presenting cell mimic scaffold (APC-MS), comprising a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed on the scaffold; and a plurality of T cell homeostatic agents adsorbed on the scaffold, wherein the scaffold is stacked to selectively allow T cells to infiltrate the mesoporous silica microrods (MSR). In one embodiment, the present invention further provides an APC-MS wherein T cell activation and / or costimulatory molecules are present on the scaffold at a concentration sufficient to allow in situ manipulation of T cells.

[0038] In another aspect, the present invention relates to a pharmaceutical composition comprising an antigen presenting cell mimic scaffold (APC-MS) and a pharmaceutically acceptable carrier, wherein the antigen presenting cell mimic scaffold comprises a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a variety of T cell activation molecules and T cell costimulatory molecules adsorbed on the scaffold; and a variety of T cell homeostatic agents adsorbed on the scaffold. In one embodiment, the present invention further provides a pharmaceutical composition formulated for intravenous administration, subcutaneous administration, intraperitoneal administration, or intramuscular administration.

[0039] In another aspect, the present invention relates to a pharmaceutical composition comprising an antigen presenting cell mimic scaffold (APC-MS) and T cells clustered therein, wherein the antigen presenting cell mimic scaffold comprises a basal layer comprising high surface area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a variety of T cell activation molecules and T cell co-stimulatory molecules adsorbed on the scaffold; and a variety of T cell homeostatic agents adsorbed on the scaffold. In one embodiment, the present invention further provides a composition containing APC-MS and T cells selected from natural killer (NK) cells, CD3+T cells, CD4+T cells, CD8+T cells and regulatory T cells (Treg) or a combination thereof.

[0040] Still further, embodiments of the present invention relate to methods for treating a disease in a subject in need, including contacting a sample comprising a T cell population obtained from a subject with an antigen presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and maintaining the T cell population in a steady state; optionally amplifying the T cell population; and administering the activated, costimulated, maintained, and optionally amplified T cells to the subject, thereby treating the subject's disease. In one embodiment, the present invention further provides a method for treating a disease in a subject in need, wherein the method further includes restimulating the T cell population before the administration step. In one embodiment, the method includes amplifying the T cell population after a period of 2 to 5 days of contact with the scaffold.

[0041] In another therapeutic embodiment, the present invention relates to a method for treating a disease in a subject in need thereof, comprising contacting a sample with an antigen presenting cell mimicking scaffold (APC-MS), the sample being a blood sample, a bone marrow sample, a lymphoid sample, or a spleen sample comprising a T cell population obtained from the subject, thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally amplifying the T cell population; and administering the activated, costimulated, maintained, and optionally amplified T cells to the subject, thereby treating the disease in the subject. In one embodiment, the subject is a human subject. In one embodiment, the method provides for the treatment of cancer, and the scaffold includes at least one cytotoxic T cell-specific activating molecule and at least one cytotoxic T cell characteristic costimulatory molecule.

[0042] In another treatment embodiment, the present invention relates to a method for treating cancer in a subject in need thereof, comprising contacting a sample comprising a T cell population obtained from the subject with an antigen presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and maintaining the T cell population in a steady state; optionally amplifying the T cell population; and administering the activated, costimulated, maintained, and optionally amplified T cells to the subject, thereby treating the cancer in the subject. In one embodiment, the cancer is selected from head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, esophageal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, glioblastoma, leukemia, lymphoma, mantle cell lymphoma, preneoplastic lesions of the lung, colon cancer, melanoma, and bladder cancer. In one embodiment, the method may further include sorting and optionally enriching cytotoxic T cells from the sample and / or amplified cell population.

[0043] In yet another therapeutic embodiment, the present invention relates to a method for treating an immunodeficiency disorder in a subject in need thereof, comprising contacting a sample comprising a T cell population obtained from the subject with an antigen presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally amplifying the T cell population; and administering the activated, costimulated, maintained, and optionally amplified T cells to the subject, thereby treating the immunodeficiency disorder in the subject. In one embodiment, the scaffold includes at least one helper T cell (Th)-specific activating molecule and at least one helper T cell (Th)-specific costimulatory molecule. In one embodiment, the method can be used to treat an immunodeficiency disorder selected from primary immunodeficiency disorder and acquired immunodeficiency disorder. In one embodiment, the method can be used to treat acquired immune deficiency syndrome (AIDS) or a genetic disorder selected from DiGeorge syndrome (DGS), chromosome breakage syndrome (CBS), ataxia telangiectasia (AT), and Wiskott-Aldrich syndrome (WAS), or a combination thereof.

[0044] In another embodiment, the present invention relates to a method for treating a disease in a subject in need, including contacting a sample comprising a T cell colony obtained from a subject with an antigen presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and maintaining the T cell colony in a steady state; optionally expanding the T cell colony; further sorting and optionally enriching T cells from the sample and / or amplified cell colony; and administering activated, costimulated, maintained, and optionally amplified T cells to the subject, thereby treating the disease in the subject. In one embodiment, T cells can be selected from natural killer (NK) cells, CD3+T cells, CD4+T cells, CD8+T cells, and regulatory T cells (Treg), or a combination thereof.

[0045] In another embodiment, the present invention is directed to a method for treating an autoimmune disorder in a subject in need thereof, comprising contacting a sample comprising a T cell population obtained from the subject with an antigen presenting cell mimic scaffold (APC-MS), thereby activating, co-stimulating and homeostatically maintaining the T cell population; optionally expanding the T cell population; further optionally sorting and enriching T cells from the sample and / or the expanded cell population; and administering the activated, co-stimulated, maintained and optionally expanded T cells to the subject, thereby treating the autoimmune disorder in the subject.

[0046] In another embodiment, the present invention relates to a method for treating a disease in a subject in need, including contacting a sample comprising a T cell colony obtained from a subject with an antigen presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and maintaining the T cell colony in a steady state; optionally amplifying the T cell colony; further optionally sorting and enriching T cells from the sample and / or amplified cell colony; and administering activated, costimulated, maintained, and optionally amplified T cells subcutaneously or intravenously to the subject, thereby treating the disease in the subject. In one embodiment, T cells can be activated, costimulated, maintained, and optionally amplified by contacting the sample with a scaffold for a period of about 1 day to about 20 days.

[0047] In another embodiment, the present invention relates to a method for manipulating T cells, comprising contacting an antigen presenting cell simulation scaffold (APC-MS) with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state and optionally amplifying the T cell population present in the sample, thereby manipulating T cells. In one embodiment, the manipulation may include stimulation, activation, changes in vitality, promotion of growth, division, differentiation, amplification, proliferation, exhaustion, anemia, dormancy, apoptosis, death of T cells. In one embodiment, the manipulation preferably includes promoting the amplification or proliferation of T cells. In other embodiments, the manipulated T cells can be further transformed. In a specific embodiment, T cells can be transformed to express chimeric antigen receptors (CAR). CAR T- cell products can be further amplified by incubating with an antigen presenting cell simulation scaffold (APC-MS) containing an antigen specific to CAR T cells. In certain embodiments, CAR T cell-specific antigens are selected from CD19, CD22, or fragments thereof or variants thereof. In some embodiments, CAR T- cell-specific antigens are tumor antigens. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. In some embodiments, the CAR T-cell product can be polyclonally expanded after production to generate a larger population of CAR T-cells.

[0048] In another embodiment, the present invention relates to a method for manipulating T cells, including contact with antigen presenting cell mimic scaffold (APC-MS), wherein compared with a control scaffold, the method described makes it possible to give the increase of T cell colony expansion after about 1 week of contact scaffold, and the control scaffold includes a basal layer comprising high surface area mesoporous silica microrods (MSR) and a continuous fluid-supported lipid bilayer (SLB), but does not contain T cell activation molecules and T cell costimulatory molecules. In one embodiment, compared with a control scaffold, the method described makes it possible to increase the amplification of T cell colony after about 1 week of contact scaffold by about 50 times to 800 times, and the control scaffold includes a basal layer comprising high surface area mesoporous silica microrods (MSR) and a continuous fluid-supported lipid bilayer (SLB), but does not contain T cell activation molecules and T cell costimulatory molecules.

[0049] In another embodiment, the present invention relates to a method for manipulating T cells, including contact with antigen presenting cell mimic scaffold (APC-MS), wherein compared with superparamagnetic spherical polymer particles (DYNABEAD) including T cell activation molecules and T cell costimulatory molecules, the method described makes the increase of T cell colony expansion given after about 1 week of contact scaffold. In one embodiment, compared with superparamagnetic spherical polymer particles (DYNABEAD) including T cell activation molecules and T cell costimulatory molecules, the method described makes the increase of T cell colony expansion after about 1 week of contact scaffold about 5 times to 20 times.

[0050] In another embodiment, the present invention relates to a method for improving the metabolic activity of T cells, including contacting an antigen presenting cell simulation scaffold (APC-MS) with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state and optionally amplifying the T cell population present in the sample, thereby improving the metabolic activity of T cells. In one embodiment, compared with a control scaffold, the method described makes it possible to give the improvement of T cell population metabolic activity after about 1 week of contact scaffold, and the control scaffold includes the basal layer comprising high surface area mesoporous silica microrods (MSR) and the lipid bilayer (SLB) supported by a continuous fluid, but without T cell activation molecules and T cell costimulatory molecules. In one embodiment, compared with a control scaffold, the method described makes it possible to improve the metabolic activity of T cell population after about 1 week of contact scaffold by about 5 times to 20 times, and the control scaffold includes the basal layer comprising high surface area mesoporous silica microrods (MSR) and the lipid bilayer (SLB) supported by a continuous fluid, but without T cell activation molecules and T cell costimulatory molecules. In one embodiment, compared with the superparamagnetic spherical polymer particles (DYNABEAD) including T cell activation molecules and T cell costimulatory molecules, the method described makes the increase of T cell colony metabolic activity given after about 1 week of contact support. In one embodiment, compared with the superparamagnetic spherical polymer particles (DYNABEAD) including T cell activation molecules and T cell costimulatory molecules, the method described further makes the expansion of T cell colony increase about 1 times to 10 times after about 1 week of contact support.

[0051] In another embodiment, the present invention relates to a method for screening metabolically active T cells, comprising contacting an antigen presenting cell mimic scaffold (APC-MS) with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state, and optionally amplifying a T cell population present in the sample; identifying metabolically active cells in the activated, costimulated, steadily maintained, and optionally amplified T cell populations; thereby screening metabolically active T cells. In one embodiment, the amplified T cells maintain metabolic activity for at least about 7 days after contacting the scaffold. In one embodiment, the amplified T cells form aggregates for at least about 7 days after contacting the scaffold.

[0052] In another embodiment, the present invention relates to a method for producing a polyclonal T cell population, comprising contacting an antigen presenting cell mimic scaffold (APC-MS) with a biological sample of a subject, thereby activating, costimulating, homeostatically maintaining, and optionally amplifying a T cell population present in the sample; identifying a specific T cell population from the amplified T cell population based on the expression of multiple markers in the amplified T cells; optionally isolating or purifying the identified T cell population, thereby producing a polyclonal population of T cells. In one embodiment, the method can be used to produce a polyclonal population of CD4+ cells or CD8+ cells. In a related embodiment, the method can be used to produce a polyclonal population of CD4+ / FOXP3+ T cells. Further, the method can be adapted to produce a polyclonal population of CD44+ / CD62L- T cells (effector memory and / or effector T cells). In another embodiment, the method can be used to produce a polyclonal population of CD8+ / CD69+ T cells (activated T cells). In another embodiment, the method can be used to produce a polyclonal population of granzyme B+CD8+ T cells (T cells that secrete cytotoxicity). In yet another embodiment, the method can be used to generate a polyclonal population of IFNγ+T cells (T cells that secrete activation cytokines). In yet another embodiment, the method can be used to generate a polyclonal population of CD62L+ / CCR7+T cells (memory T cells).

[0053] In another embodiment, the present invention relates to a method for producing a polyclonal subpopulation of T cells, comprising contacting an antigen presenting cell mimic scaffold (APC-MS) with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state, and optionally amplifying the T cell population present in the sample; identifying a specific exhausted T cell population from the amplified T cell population based on the expression of multiple markers in the amplified T cells; optionally removing the identified T cell population, thereby producing a polyclonal subpopulation of T cells. In one embodiment, exhausted T cells are identified or separated based on the cell surface expression of CD8+ / PD-1+. In another embodiment, exhausted T cells are identified or separated based on the cell surface expression of LAG3+ / TIM3+.

[0054] In another embodiment, the present invention relates to a method for manipulating T cells in vitro, comprising contacting an antigen presenting cell simulated scaffold (APC-MS) with a biological sample of a subject in vitro, thereby activating, co-stimulating, homeostatically maintaining and optionally amplifying the T cell population present in the sample, thereby manipulating T cells in vitro. In one embodiment, the sample is contacted with the scaffold for a period of about 1 day to about 20 days. In one embodiment, the method may involve detecting the production of one or more cytokines produced by the manipulated T cells. In one embodiment, the method involves further detecting the production of cytokines selected from interferon gamma (IFNγ), tissue necrosis factor alpha (TNFα), IL-2, IL-1, IL-4, IL-5, IL-10 and IL-13, IL-17 or a combination thereof of the manipulated T cells.

[0055] In a related embodiment, the present invention relates to a method for manipulating T cells ex vivo according to the previous method, wherein the manipulated T cells are T helper 1 (Th1) cells, and the method comprises detecting the production of cytokines selected from IL-2, interferon gamma (IFNγ) and tissue necrosis factor alpha (TNFα), or a combination thereof. Alternatively, in a related embodiment, the present invention relates to a method for manipulating T cells ex vivo according to the previous method, wherein the manipulated T cells are T helper 2 (Th2) cells, and the method comprises detecting the production of cytokines selected from IL-4, IL-5, IL-10 and IL-13, or a combination thereof. Still further, in a related embodiment, the present invention relates to a method for manipulating T cells ex vivo according to the previous method, wherein the manipulated T cells are cytotoxic T (Tc) cells, and the method comprises detecting the production of cytokines selected from interferon gamma (IFNγ) and lymphotoxin alpha (LTα / TNFβ) or a combination thereof. In one embodiment, the manipulated T cells are cytotoxic (Tc) cells, and the method comprises detecting the secretion of a cytotoxin selected from granzymes or perforins or a combination thereof.

[0056] In a related embodiment, the present invention relates to a method for ex vivo manipulation of T cells according to the previous method, wherein the method further comprises detecting expression of cell surface markers in the manipulated T cells. In one embodiment, the cell surface markers are selected from CD69, CD4, CD8, CD25, CD62L, FOXP3, HLA-DR, CD28 and CD134, or a combination thereof. Alternatively or additionally, in one embodiment, the cell surface markers are non-T cell markers selected from CD36, CD40 and CD44, or a combination thereof.

[0057] In another related embodiment, the invention relates to a method for ex vivo manipulation of T cells according to the previous method, wherein the subject is a human subject.

[0058] In another related embodiment, the present invention relates to a method for manipulating T cells in vivo according to previous methods, wherein the scaffold is applied to a subject to allow a biological sample comprising T cells to contact the scaffold in vivo. In one embodiment, the scaffold can maintain a time period of about 3 days to about 15 days in a subject, preferably a time period of about 7 days to about 11 days. In some embodiments, the scaffold can maintain a time period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days or longer in a subject.

[0059] In yet another embodiment, the present invention relates to a method for preparing an antigen presenting cell mimic scaffold (APC-MS), comprising (a) providing a basal layer comprising high surface area mesoporous microrods (MSR); (b) optionally loading a T cell homeostatic agent onto the MSR; (c) layering a continuous fluid-supported lipid bilayer (SLB) onto the basal layer comprising the MSR, thereby producing an MSR-SLB scaffold; (d) if step (b) is not performed, loading a T cell homeostatic agent onto the MSR-SLB scaffold; (e) optionally blocking one or more nonspecific integration sites in the MSR-SLB scaffold with a blocking agent; and (f) loading T cell activation molecules and T cell co-stimulatory molecules onto the MSR-SLB scaffold, thereby producing an APC-MS. In one embodiment, the method may further involve assembling multiple scaffolds to produce a stack having sufficient porosity to allow T cell infiltration. In one embodiment, the method may include loading at least one additional agent selected from growth factors, cytokines, interleukins, adhesion signaling molecules, integrin signaling molecules, or fragments thereof, or combinations thereof.

[0060] Other features and advantages of the present invention will become apparent from the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Phase contrast and fluorescence microscopy images of lipids bound to mesoporous silica microrods (MSRs) are shown. The upper panel shows a merged image of lipids and mesoporous silica microrods at a lipid:MSR ratio of 1:20 (scale bar = 200 μm). The middle panel shows a merged image of lipids and mesoporous silica microrods at a lipid:MSR ratio of 1:4 (scale bar = 200 μm). The lower panel shows a merged phase contrast microscopy image of lipids bound to MSRs at a higher magnification (scale bar = 20 μm).

[0062] Figure 2A 、 2B , 2C, and 2D show that the assembly and characteristics of the antigen-presenting cell-mimicking scaffold (APC-MS) depend on the type and content of lipids. Figure 2A The chemical structures of various lipids are shown. Abbreviations: DOPC - dioleoylphosphatidylcholine; POPC - palmitoyl-oleoylphosphatidylcholine; and DSPC - distearoylphosphatidylcholine. Figure 2B The percentage of lipid retained in various compositions containing mesoporous silica microrods (MSR) and fluid-supported lipid bilayers (SLBs) is shown. In this experiment, a payload of 250 μg lipid was input into a 500 μg MSR composition. Figure 2C Shown are the relative fluorescence changes of various MSR-SLB compositions containing DOPC, POPC, or DSPC in phosphate-buffered saline (PBS) at 37°C over two weeks (14 days). Figure 2D Shown are the relative fluorescence changes of various MSR-SLB compositions containing DOPC, POPC, or DSPC in complete Roswell Park Memorial Institute medium (cRPMI) at 37°C over two weeks (14 days).

[0063] Figure 3 Shown are the stability of various MSR-SLB compositions in PBS at day 0, day 3, day 7, and day 14, as analyzed using phase contrast and fluorescence microscopy (lipid coating). The top panel shows the stability of DOPC in MSR-SLB compositions; the middle panel shows the stability of POPC in MSR-SLB compositions; and the bottom panel shows the stability of DSPC in MSR-SLB compositions.

[0064] Figure 4A 、 4B , 4C, 4D, and 4E show the changes in the assembly and characteristics of the MSR-SLB fluid structure over time. Figure 4A Shown are phase contrast and fluorescence microscopy images of lipids bound to mesoporous silica microrods (MSRs) taken at high magnification (scale bar = 2 μm) before bleaching of the lipid composition (pre), just after bleaching (t = 0), and 5 minutes after bleaching (t = 5 min). Figure 4B Shown are changes in fluorescence recovery over time after photobleaching (FRAP). The fluorescence "source" is depicted in region (2), the fluorescence "sink" is depicted in region (3), and the normalization point is represented by region (1). The differential distribution is best seen at early time points after seeding and reaches equilibrium at approximately 2 min (120 s). Figure 4CShown are smooth-fit curves depicting the average change in FRAP over time, as derived from normalized images. Figure 4D and 4E Two sets of high-resolution images of the MSR-SLB fluid structure before bleaching of the lipid composition (pre), just after bleaching (t=0), and 3 minutes after bleaching (t=3 min) are shown.

[0065] Figure 5A and 5B The structural and functional properties of MSR-S;B compositions containing various different moieties are shown. Based on experiments using the B3Z reporter T cell line, maximum functionality of the APC-MS scaffold was observed when all individual components were present in the scaffold. Figure 5A A schematic diagram of the structure of an APC-MS containing a POPC lipid bilayer containing phycoerythrin biotin (biotin PE) conjugated to a streptavidin molecule (e.g., a streptavidin dimer), which is then conjugated to a biotinylated antibody (e.g., a biotinylated anti-CD3 antibody or a biotinylated anti-CD28 antibody or another specific or non-specific antibody) is shown. Figure 5B Shown are spectrophotometric analyses of B3Z reporter cell β-galactosidase expression following treatment with MPS (silica), POPC (lipid), MPS-POPC complex, biotinylated MPS-POPC complex in the presence or absence of streptavidin, and combinations of MPS-POPC complex in the presence or absence of phycoerythrin biotin (biotin PE) and / or streptavidin together with a biotinylated antibody. A significant increase in absorbance was observed in MSR-SLB compositions containing all individual components—phosphoethanolamine biotin (biotin PE) conjugated to a biotinylated antibody via a streptavidin linker (dark bars; ** indicates statistical significance (p < 0.001, analyzed using one-way ANOVA followed by Tukey HSD post-hoc test; data represent mean ± sd of three experimental replicates and are representative of at least two independent experiments).

[0066] Figure 6A and 6B Controlled release of IL-2 from MSR-SLB compositions containing IL-2 was demonstrated. Figure 6A Electron micrographs of the porous structure of the MSR containing IL-2 are shown (scale bar = 100 nm). Figure 6B The curve shows the cumulative release level of IL-2 over a period of 15 days.

[0067] Figure 7A and 7BConfocal microscopy images showing T cells (spheroids) infiltrating into antigen-presenting cell-mimicking scaffolds containing MSR-SLB composites are shown. Figure 7A Shown are cells that have been stained with two different dyes. Figure 7B Cells that have been stained with a single dye (indicating viable cells) are shown.

[0068] Figure 8 Phase contrast microscopy and fluorescence images of lipids bound to mesoporous silica microrods (MSR) co-cultured with primary T cells are shown. It was observed that when T cell activation signals (cues) were connected to the surface of the material, primary T cells tended to form cell / material clusters. The figure below shows a merged image of lipids and mesoporous silica microrods in MSR-SLB composites containing conjugated antibodies, IL-2, or a combination of conjugated antibodies and IL-2. The image on the right shows an MSR-SLB composite containing both conjugated antibodies and IL-2 at high magnification (scale bar = 20 μm).

[0069] Figure 9A and 9B Shown are dose-response graphs of antibody-induced changes in mouse splenic T cells. Figure 9A Shown are polyclonal expansion of T cells after 3 days of stimulation with control scaffolds (mock; none; POPC lipid only; and a combination of POPC and IL-2) and experimental scaffolds (containing a combination of POPC and IL-2, and antibody). Three different doses of antibody were studied (MSR:antibody ratios of 1:50, 1:25, and 1:10). Figure 9B IFNγ secretion after 3 days of stimulation of T cells with control scaffolds (mock; none; POPC lipid only; and a combination of POPC and IL-2) and experimental scaffolds (containing a combination of POPC and IL-2, and antibody) is shown. Three different doses of antibody were studied (MSR:antibody ratios of 1:50, 1:25, and 1:10).

[0070] Figure 10 and 11 It was shown that the antigen presenting cell mimetic scaffold (APC-MS) of the present invention promoted the rapid expansion of metabolically active T cells. Figure 10 The fold expansion of primary T cells when incubated with controls (mock; none; SLB + IL-2; DYNABEAD + IL-2) or experimental compositions is shown. Compared to mock compositions or compositions without SLB, incubation of primary T cells with the compositions of the present invention significantly induced T cell expansion (with or without restimulation). More importantly, incubation of primary T cells with the scaffolds of the present invention resulted in measurably greater proliferation upon restimulation at day 7 compared to the compositions of DYNABEADS and IL-2. Figure 11Shown are bar graphs of cellular metabolic activity (as measured by relative fluorescence units (RFU) of Alamar blue reduction normalized to cell number) of T cells incubated with scaffolds of the invention loaded with IL-2 (SLB / IL2 / ABS) or DYNABEADS loaded with IL-2 (DYNABEADS-IL2).

[0071] Figure 12A and 12B It was shown that the scaffold of the present invention (APC-MS) confers polyclonal expansion of splenic T cells (mouse) and promotes the formation of T cell aggregates. Figure 12A Shown are micrographs (at 4x magnification) of aggregates of splenic T cells upon incubation with DYNABEADS or APC-MS on days 0, 3, and 7. Figure 12B Shown are micrographs (at 10× magnification) of aggregates of splenic T cells upon incubation with DYNABEADS or APC-MS on days 0, 3, and 7. (White scale bar = 100 μM).

[0072] Figure 13A and 13B Shown are polyclonal expansions of mouse splenic T cells when incubated with APC-MS or DYNABEADS. Figure 13A Shown are flow cytometry (FACS) scatter plots of T cell populations at various time points (t = 0, 5, 7, 11, and 13 days) after incubation with APC-MS or DYNABEADS (with restimulation or IL-2 treatment after 7 days of incubation), where the values on the X-axis depict the intensity of CD8+ staining and the values on the Y-axis depict the intensity of CD4+ staining. At each time point, flow data were gated relative to the Fluorescence Minus One (FMO) control for each sample. Data represent at least two independent experiments. Figure 13B Figure 2 is a line graph showing the percentage change in CD4+ vs. CD8+ T cell subsets after incubation with APC-MS (squares) or DYNABEADS (triangles) at various time points (t = 0, 5, 7, 11, and 13 days). After 7 days of incubation, cells were separated into two subsets, the first of which was restimulated (dashed line) and the second was treated with IL-2 (solid line). APC-MS was used for restimulation in the APC-MS condition, and DYNABEADS was used for restimulation in the DYNABEADS condition.

[0073] Figure 14The measurement of the polyclonal expansion of FoxP3+ mouse spleen T cell subsets when incubated with APC-MS or DYNABEADS is shown. The results are depicted in the form of flow cytometry (FACS) dot plots of T cell populations at various time points (t=0 days, 5 days, 7 days, 11 days and 13 days) after incubation with APC-MS or DYNABEADS (using restimulation or IL-2 treatment after 7 days of incubation), where the values on the X-axis depict the intensity of FoxP3+ staining, and the values on the Y-axis depict the intensity of CD4+ staining. Rectangular gating is used to count the number and / or ratio of FoxP3+ cells in each different part. As shown, there is limited or no expansion of FoxP3+ mouse spleen T cells using a specific formulation.

[0074] Figure 15 Shown is the polyclonal expansion of CD62L+ mouse spleen T cell subsets when incubated with APC-MS or DYNABEADS. At various time points (t=0 days, 5 days, 7 days, 11 days and 13 days) after incubation with APC-MS or DYNABEADS (using restimulation or IL-treatment after 7 days of incubation), the results are depicted in the form of flow cytometry (FACS) dot plots of T cell populations, wherein the values on the X-axis depict the intensity of CD62L+ staining, and the values on the Y-axis depict the intensity of CD44+ staining. CD62L+ cells appear on the right side of the dot plot (upper and lower right quadrants).

[0075] Figure 16 Shown is the polyclonal expansion of CD8+ / CD69+ mouse spleen T cell subsets when incubated with APC-MS or DYNABEADS. At various time points (t=0 days, 5 days, 7 days, 11 days and 13 days) after incubation with APC-MS or DYNABEADS (using restimulation or IL-treatment after 7 days of incubation), the results are depicted in the form of flow cytometry (FACS) dot plots of T cell populations, where the values on the X-axis depict the intensity of CD8+ staining, and the values on the Y-axis depict the intensity of CD69+ staining. CD8+ / CD69+ cells appear in the upper right quadrant of the dot plot.

[0076] Figure 17Shown is the polyclonal expansion of CD8+ / Granzyme B+ mouse spleen T cell subsets when incubated with APC-MS or DYNABEADS. The results are depicted in the form of flow cytometry (FACS) dot plots of T cell populations at various time points (t=0 days, 5 days, 7 days, 11 days, and 13 days) after incubation with APC-MS or DYNABEADS (using restimulation or IL-treatment after 7 days of incubation), where the values on the X-axis depict the intensity of CD8+ staining and the values on the Y-axis depict the intensity of granzyme B+ staining. CD8+ / Granzyme B+ cells appear in the upper right quadrant of the dot plot.

[0077] Figure 18 Shown are T cell secretion of IFNγ (pg / cell) at various time points (t = 0, 5, 7, 11, and 13 days) after treatment with APC-MS (squares) or DYNABEADs (triangles). After 7 days of incubation, cells were divided into two subpopulations, the first of which was restimulated (dashed line) and the second was treated with IL-2 (solid line). In this article, APC-MS was used to restimulate cell populations incubated with APC-MS and DYNABEADs.

[0078] Figure 19 The levels of PD-1+ mouse spleen T cells when incubated with APC-MS or DYNABEADS are shown. The results are depicted in the form of flow cytometry (FACS) dot plots of T cell populations at various time points (t=0, 5, 7, 11, and 13 days) after incubation with APC-MS or DYNABEADS (using restimulation or IL-treatment after 7 days of incubation), where the values on the X-axis depict the intensity of CD8+ staining and the values on the Y-axis depict the intensity of PD-1+ staining (a potential marker of exhaustion).

[0079] Figure 20A and 20B The effects of incubating human peripheral blood T cells with various compositions are shown. Figure 20A Line graphs showing polyclonal expansion of primary T cells incubated with control or experimental scaffolds at various time points (t = 0, 5, 7, 11, and 13 days). Control scaffolds included a mock ("Mock"; black line) composition and a composition without SLB ("None"; red line). Experimental scaffolds included (1) DYNABEADS (blue line) and (2) lipid bilayers (SLBs) of the present invention (green line). Figure 20BBar graphs are shown showing the metabolic activity (measured using standard Alamar blue staining assay) of primary T cells incubated with control or experimental scaffolds at various time points (t = 0, 5, 7, 11, and 13 days). Control scaffolds included a sham composition ("mock"; "m") and a composition without SLB ("none"; "f"). Experimental scaffolds included (1) DYNABEADS ("d") and (2) lipid bilayers (SLBs) of the present invention ("s").

[0080] Figure 21A and 21B The effect of incubating human peripheral blood T cells with various anti-CD3 antibodies is shown. Human peripheral blood T cells obtained from subject 1 ( ) were incubated with a control scaffold ("mock") or experimental scaffolds containing the listed anti-CD3 antibodies - muromonab (OKT3), an antibody that recognizes the 17-19 kD epsilon chain of CD3 within the CD3 antigen / T cell antigen receptor (TCR) complex (HIT3a), and a monoclonal antibody that recognizes the 20 kDa subunit of the TCR complex within CD3 e (UCHT1). Figure 21A ) and subject 2 ( Figure 21B ) human blood samples. Three different doses were studied - 5 μg (upper slide), 1 μg (lower slide for subject 2), and 0.5 μg (lower slide for subject 1). In each case, co-stimulation with anti-CD28 antibody was provided, wherein the ratio of anti-CD3 antibody: anti-CD28 antibody was maintained at 1:1. Fold expansion of T cells was measured at various time points (t = 0 days, 5 days, 7 days, 11 days, and 13 days).

[0081] Figure 22 The figure shows the polyclonal expansion of human T cells when incubated with a control scaffold ("mock") or experimental scaffolds containing the listed anti-CD3 antibodies—OKT3, HIT3a, and UCHT1. The lower panel shows flow cytometry (FACS) scatter plots of T cell populations at various time points (t = 8 days, 11 days, and 14 days) after incubation with APC-MS containing each of the anti-CD3 antibodies as stimulatory molecules and the anti-CD28 antibody as a co-stimulatory molecule. The values on the x-axis of the scatter plot depict the intensity of CD8+ staining, while the values on the y-axis depict the intensity of CD4+ staining. The curves are summarized in the line graph in the upper panel, which shows the percentage changes in CD4+ vs. CD8+ T cell subsets after incubation with APC-MS containing the above-mentioned anti-CD3 antibodies—OKT3 (circles), HIT3a (squares), and UCHT1 (triangles). Two different antibody doses were studied: 5 μg (1× dilution) and 0.5 μg (1:10× dilution).

[0082] Figure 23Shown is the expression of CD62L and CCR7 on live T cells expanded for 14 days using APC-MS containing IL-2 and a 1:1 ratio of the aforementioned anti-CD3 antibodies—OKT3 (left panel), HIT3a (middle panel), and UCHT1 (right panel)—to an anti-CD28 antibody at a 1× loading concentration (approximately 5 μg). The upper panel shows the expression of CD62L and CCR7 in total live cells, while the lower panel shows the expression of these markers in gated CD8+ cells. The majority of cells expanded using the APC-MS of the present invention remained CD62L+CCR7+ after 14 days of incubation, demonstrating that this is important for in vivo functionality in human patients. Furthermore, the APC-MS scaffold containing OKT3 was particularly effective at expanding and / or maintaining CD62L+CCR7+ T cells compared to scaffolds containing UCHT1 and / or HIT3a.

[0083] Figure 24 Representative protocols for preparing the scaffolds of the present invention are outlined.

[0084] Figure 25A and 25B The design of the antigen-presenting cell-mimicking scaffold (APC-MS) is depicted. Figure 25A An exemplary method for preparing APC-MS is described: 1) synthesizing mesoporous silica microrods (MSRs); 2) adsorbing IL-2 onto the MSRs; 3) coating the IL-2-adsorbed MSRs with liposomes to form MSR-SLBs; 4) attaching T cell activation signals to the surface of the MSR-SLBs; 5) culturing the MSR-SLBs with T cells; and 6) sedimenting and stacking the MSR-SLBs to form a scaffold permeable to T cells. The scaffold formed from MSR-SLBs loaded with IL-2 and surface-engineered with T cell activation signals is called APC-MS. Figure 25B Depict the exemplary structure and function of different APC-MS configurations. Over time, IL-2 is released from APC-MS, resulting in IL-2 paracrine delivery to local T cells. A predetermined amount of biotinylated phospholipids is incorporated into the liposome formulation to enable accurate surface adhesion of biotinylated T cell activation signals by avidin streptavidin-biotin interactions, simulating the signal cell surface presentation of T cells by natural APC. For polyclonal T cell expansion, adhesion anti-CD3 (αCD3) and CD28 (αCD28) activation antibodies (left). For antigen-specific T cell expansion, adhesion peptide-loaded MHC (pMHC) and αCD28.

[0085] Figure 26A and 26B Physical characterization of the components used to assemble MSR-SLBs is depicted. Figure 26A Representative bright-field microscopy images of MSR are shown. Scale bar = 100 μm. Figure 26B Depicted are the size distribution of POPC liposomes, as measured by dynamic light scattering (DLS). Figure 26B The data in represent the average size distribution of 3 samples.

[0086] Figure 27A and 27B is a microscopic image of lipid-coated MSR. Figure 27A Figure 2 is a microscopic image showing MSR aggregation at low lipid:MSR ratio. Representative microscopic images of lipid-coated MSR (lipid:MSR 1:20 w / w) show bright-field images of MSR (left), fluorophore-labeled phospholipids (1 mol% of total lipid; center), and co-localization of MSR and lipids (right). Scale bar = 200 μm. Figure 27B are microscopic images of lipid-coated MSR (lipid:MSR 1:4 w / w), showing bright-field images of MSR (left), fluorophore-labeled phospholipids (1 mol% of total lipids; center), and co-localization of MSR and lipids (right). Scale bar = 200 μm.

[0087] Figures 28A-28E The assembly and characterization of the APC-MS are depicted. Figure 28A Depicted is the retention of the lipid coating (containing 1 mol % fluorophore-labeled lipids) on MSRs maintained under cell culture conditions in PBS or RPMI medium containing 10% serum (cRPMI) over time. Figure 28B Representative overlay fluorescence microscopy images of lipid-coated MSRs maintained in cRPMI under standard cell culture conditions over time (MSR, bright field; lipids (1 mol% fluorophore-labeled lipids), green). Scale bar = 100 μm. Data represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments. Figure 28C is a graph depicting the quantification of IL-2 released from MSR-SLBs (500 μg MSR) in vitro over time (data points) with a one-phase exponential fit (dashed line; R 2 =0.98). Data represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments. Figure 28D Graph depicting the quantification of the adhesion of various inputs of biotinylated IgG to MSR coated with lipid formulations containing 0.01 mol%, 0.1 mol% or 1 mol% biotinylated lipids. The values above the bars represent the concentration (μg) of IgG adhered for each corresponding condition. The data represent the mean ± sd of four experimental replicates and are representative of at least two independent experiments. Figure 28E This is a SEM image showing the tight binding of primary human T cells to APC-MS. Scale bar = 10 μm.

[0088] Figure 29 Shown is the binding of T cells to APC-MS. Representative microscopic images of MSR-SLBs cultured with primary mouse T cells for one day, without presenting any surface signal (signal -), or presenting αCD3 and αCD28 (signal +) on the surface, at low (left) and high (right) magnifications. Cells and materials are visible in the bright field image (top), and the MSR-SLB lipid coating is visible in the green channel (1 mol% fluorophore-labeled lipids; middle). Merged images are shown at the bottom. Low magnification scale bar = 500 μm, high magnification scale bar = 100 μm.

[0089] Figure 30A 、 30B , 30C, 30D, 30E, 30F, and 30G show polyclonal expansion of primary mouse and human T cells. Figure 30A Representative bright-field microscopy images of primary mouse T cells cultured with DYNABEADS or APC-MS at various time points at low magnification (left) or high magnification using APC-MS (right). Scale bar = 100 μm. Figure 30B Shown is the expansion of primary mouse T cells cultured with either untreated (mock), free signal (110 nM αCD3, 110 nM αCD28, 1.3 μg / ml IL-2), commercial CD3 / CD28 mouse T cell expansion beads and exogenous IL-2 (DYNABEADS), IL-2-loaded MSR-SLB without T cell signal presented on the bilayer surface (MSR-SLB(Signal-)), or APC-MS (loaded with αCD3, αCD28, IL-2). The mock and free curves can be distinguished from the MSR-SLB(Signal-) curve. Figure 30C Figure 3. The frequency of CD4+ and CD8+ cells in live single cells cultured in APC-MS or Dynabeads over time, measured using FACS. Data were analyzed using two-way ANOVA followed by Tukey HDS post-hoc test. Figure 30DRepresentative bright-field microscopy images of primary human T cells cultured with DYNABEADS or APC-MS formulations at various time points. Scale bar = 100 μm. (F1) APC-MS saturated with 1 mol% biotinylated lipids presenting αCD3 and αCD28, infused into the initial culture at 333 μg / ml MSR, (F2) APC-MS saturated with 1 mol% biotinylated lipids presenting αCD3 and αCD28, infused into the initial culture at 33 μg / ml MSR, (F3) APC-MS saturated with 0.1 mol% biotinylated lipids presenting αCD3 and αCD28, infused into the initial culture at 333 μg / ml MSR, and (F4) APC-MS saturated with 0.1 mol% biotinylated lipids presenting αCD3 and αCD28, infused into the initial culture at 33 μg / ml MSR. Figure 30E Shown is the expansion of primary human T cells that were untreated (mock), cultured with commercial CD3 / CD28 human T cell expansion beads and exogenous IL-2 (DYNABEADS), or cultured using various APC-MS formulations. Figure 30F Depicted are FACS quantification of CD4 and CD8 single positive cells in live single CD3+ cells in samples expanded for 14 days with DYNABEADS or with various APC-MS preparations. Figure 30G Depicted are FACS quantification of cells co-expressing PD-1 and LAG-3 in live single cells from samples amplified with DYNABEADS or with various APC-MS preparations. Figure 30F and 30G The data in represent the mean±s.d. of three experimental replicates and are representative of at least two independent experiments. Figure 30E The data in represent the mean±s.d. of at least three different donor samples from two independent experiments. Figure 30F and 30G The data in represent the mean ± sd of three different donor samples and are representations of at least two independent experiments. **p < 0.01, ***p < 0.001.

[0090] Figure 31 Depicts representative FACS plots of CD4 and CD8 expression on polyclonally expanded primary mouse T cells. Representative FACS plots show CD4 and CD8 expression on live single cells polyclonally expanded using APC-MS or DYNABEADS. At each time point, flow data were gated relative to the Fluorescence Minus One (FMO) control for each sample.

[0091] Figure 32A 、 32B, 32C, and 32D depict phenotypic characterization of polyclonally expanded primary mouse T cells. Figure 32A Depicted are FACS quantification of granzyme B-positive cells in live single CD8+ cells from samples amplified with DYNABEADS or with APC-MS (left), and representative FACS plots (right). Figure 32B Depicted are FACS quantification of FoxP3-positive cells in live single CD4+ cells in samples amplified with DYNABEADS or with APC-MS. Figure 32C and 32D Representative FACS plots showing PD-1 expression on live single cells as a function of CD8 expression are shown. At each time point, for each sample, flow data were gated relative to the Fluorescence Minus One (FMO) control. Data represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments.

[0092] Figure 33 Adhesion molecule expression on polyclonally expanded primary human T cells is shown. FACS quantification of live single cells co-expressing CD62L and CCR7 in samples expanded with DYNABEADS or using various APC-MS preparations. (F1) APC-MS presenting αCD3 and αCD28 saturated with 1 mol% biotinylated lipids, input to initial culture at 333 μg / ml MSR, (F2) APC-MS presenting αCD3 and αCD28 saturated with 1 mol% biotinylated lipids, input to initial culture at 33 μg / ml MSR, (F3) APC-MS presenting αCD3 and αCD28 saturated with 0.1 mol% biotinylated lipids, input to initial culture at 333 μg / ml MSR, and (F4) APC-MS presenting αCD3 and αCD28 saturated with 0.1 mol% biotinylated lipids, input to initial culture at 33 μg / ml MSR. Data represent mean ± sd of three different donor samples and are representative of at least two independent experiments.

[0093] Figure 34A 、 34B , 34C, 34D, and 34E depict antigen-specific expansion of primary mouse T cells. Figure 34A Representative bright-field microscopy images of primary CD8+ OT-I T cells cultured for two days with APC-MS presenting an irrelevant peptide (SVYDFFVWL (SEQ ID NO: 3); left) or a related peptide (SIINFEKL (SEQ ID NO: 4); right) in H-2K (b) are shown. Scale bar = 100 μm. Figure 34BShown are the expansion of primary CD8+ OT-I T cells that were untreated (mock) or cultured with various APC-MS preparations. (F1) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and αCD28 saturated with 1 mol% biotinylated lipids, input to initial culture at an MSR of 333 μg / ml, (F2) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and αCD28 saturated with 1 mol% biotinylated lipids, input to initial culture at an MSR of 33 μg / ml, (F3) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and αCD28 saturated with 0.1 mol% biotinylated lipids, input to initial culture at an MSR of 333 μg / ml, and (F4) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and αCD28 saturated with 0.1 mol% biotinylated lipids APC-MS of NO: 4) / H-2K (b) and αCD28, with 33 μg / ml of MSR input to the initial culture. Figure 34C Depicted are FACS quantification of IFNγ and TNFα expression by live single CD8+ OT-I T cells expanded for 13 days with various APC-MS preparations and subsequently co-cultured with B16-F10 cells that were mock-pulsed (-) or pulsed (+) with SIINFEKL (SEQ ID NO: 4) peptide. Figure 34D Depicted are quantifications of in vitro killing of mock-pulsed (-) or SIINFEKL (SEQ ID NO: 4)-pulsed (+) B16-F10 target cells by CD8+ OT-IT cells expanded for 13 days with various APC-MS preparations and subsequently co-cultured at various effector:target cell ratios. Figure 34E Depicted are quantification of IFNγ secretion by CD8+ OT-I T cells expanded for 13 days with various APC-MS preparations in response to co-culture with mock-pulsed (pep-) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (pep+) B16-F10 cells at various effector:target cell ratios. Figure 34B 、 34C The data in , 34D, and 34E represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments.

[0094] Figure 35A 、 35B , 35C, 35D, and 35E show expanded characterization of primary human T cells expanded with antigen-specific APC-MS preparations. Figure 35AShown are total expansion of primary human CD8+ T cells mock treated (30 U / ml IL-2) or cultured with APC-MS (loaded with pMHC, αCD28, IL-2) presenting CLG or GLC peptides in HLA-A2. For mock treated cells only data from day 0 and day 7 are available. Figure 35B 、 35C and 35D show the expression of unpulsed (peptide-)( Figure 35B ), (+CLG peptide) pulsed with CLG peptide ( Figure 35C ) or pulsed with GLC peptide (+GLC peptide) ( Figure 35D Quantification of IFNγ secretion by CD8+ T cell isolates mock-treated (30 U / ml IL-2) or cultured with APC-MS presenting CLG peptide (APC-MS CLG) or GLC peptide (APC-MS GLC) after co-culture with T2 cells. For mock-treated cells, only data on day 7 are available. Figure 35E Representative FACS plots showing IFNγ and TNFα expression by CD8+ T cell isolates cultured with APC-MS presenting CLG peptide (APC-MS / CLG) or GLC peptide (APC-MS / GLC) after co-culture with T2 cells that were unpulsed (no peptide; top), pulsed with CLG peptide (+CLG peptide; middle), or pulsed with GLC peptide (+GLC peptide; bottom). Figure 35A and 35B The data in represent the mean±s.d. of three experimental replicates and are representative of two experiments using two different donor samples.

[0095] Figure 36A 、 35B , 36C, 36D, 36E, 36F, 36G, 36H, 36I, 36J, 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells. Figure 36A 、 36B , 36C, 36D, 36E, 36F, 36G, 36H, 36I, and 36J depict antigen-specific expansion of primary human T cells from CD8+ T cell isolation. Figure 36A 、 36B and 36D depict the expression of the EBV-derived peptide CLGGLLTMV (SEQ ID NO: 1) (CLG; Figure 36A and 36B ) and GLCTLVAML (SEQ ID NO: 2) (GLC; Figure 36D and 36ERepresentative FACS plots of primary HLA-A2+ human CD8+ T cells cultured with APC-MS (loaded with pMHC, αCD28, IL-2) presenting CLG or GLC peptides in HLA-A2. The numbers in the gates indicate the percentage of live single CD8+ cells positive for the corresponding tetramer ( Figure 36A and 36D ), and quantification of FACS data at different time points ( Figure 36B and 36E ). For mock-treated cells, only data from day 0 and day 7 were available. Figure 36F Shown are the effects of APC-MS mock-treated or incubated with CLG or GLC peptides presented in HLA-A2 on CLG ( Figure 36C ) or GLC( Figure 36F ) Expansion of primary human CD8+ T cells specific for α-HRP-positive T cells. For mock-treated cells, only data from days 0 and 7 were available. Figure 36G 、 36H and 36I depict the difference between the unpulsed (peptide-)( Figure 36G ), pulsed with CLG peptide (+CLG peptide; Figure 36H ) or pulsed with GLC peptide (+GLC peptide; Figure 36I Frequency of TNFα+IFNγ+ cells in live single CD8+ T cells mock-treated (30 U / ml IL-2) or cultured with APC-MS presenting CLG or GLC peptides in HLA-A2 after co-culture with T2 cells. Data are available only on day 7 for mock-treated cells. Figure 36J Shown is the quantification of in vitro killing of T2 target cells mock-pulsed (no peptide) or pulsed with CLG peptide (+CLG) or GLC peptide (+GLC) by primary human CD8+ T cells expanded for 14 days with APC-MS presenting CLG or GLC peptides in HLA-A2. Figure 36K 、 36L , 36M, and 36N show antigen-specific expansion of primary human T cells from PBMCs. Figure 36K Depicted are the frequencies of GLC-specific cells in live single CD8+ T cells within PBMC cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting GLC peptides in HLA-A2. Figure 36L Shown are the numbers of GLC-specific CD8+ T cells in PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting GLC peptides in HLA-A2. Numbers above the bars indicate fold expansion (mean ± sd). Figure 36M and 36NFigure 2 shows the frequency of TNFα+IFNγ cells among live single CD8+ T cells from PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting GLC peptide in HLA-A2 after co-culture with T2 cells that were unpulsed (no peptide), pulsed with CLG peptide (+CLG), or pulsed with GLC peptide (+GLC peptide). Figure 36M ) and IFNγ secretion ( Figure 36N ). All data represent the mean ± sd of three experimental replicates and are representative of two experiments using two different donor samples.

[0096] Figure 37 The degradation of APC-MS scaffolds in vitro was depicted. Primary mouse T cells (25×10 4 T cells / 167 μg APC-MS) were cultured to present αCD3 / αCD28 (1% biotinylated lipids) and release IL-2 APC-MS (167 μg). At various time points, the culture was centrifuged at 700 rcf for 5 min, and the Si content in the precipitate was quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES; Galbraith Laboratories). Until 1 week after the start of culture, Si was undetectable in the culture precipitate.

[0097] Figure 38 Controlled release of diverse soluble immune-directed payloads from APC-MS was demonstrated. Four APC-MS were generated, each containing 2 μg of IL-2, IL-21, TGFβ, or IL-15SA loaded into 500 μg of APC-MS before lipid coating. Samples were thoroughly washed to remove unbound protein and then maintained at 37°C for up to 28 days. Payload release over time was assessed by ELISA.

[0098] Figure 39A and 39B Depicted are fluorescence recovery after photobleaching (FRAP) experiments using MSR-SLBs containing 10% carboxyfluorescein in headgroup-labeled lipids. Figure 39A Representative images of three independent FRAP events. Images show a fluorescently labeled MSR-SLB before photobleaching (left), immediately after photobleaching (center), and after fluorescence recovery (right). The photobleached region is indicated by a red arrow. Figure 39B Quantification of fluorescence recovery over time is shown. Fluorescence recovery from eight independent photobleaching events on different MSR-SLBs is shown as black dashed lines, and the average trend is shown as a solid line.

[0099] Figure 40A 、 40B, 40C and 40D depict the results of T cell expansion experiments using APC-MS compared to DYNABEAD, where the amount of DYNABEAD was normalized to contain the same amount of anti-CD3 and anti-CD28 antibodies as APC-MS. Figure 40A A bicinchoninic acid assay (BCA) for total protein quantification was performed to determine the amount of protein bound to the surface of commercial mouse or human CD3 / CD28 T cell activator DYNABEAD. The DYNABEAD stock solution was thoroughly washed and the DYNABEAD antibody loading was assessed by BCA analysis. DYNABEAD targeting mouse and human T cells was found to have similar antibody loading (~20 μg / ml). On a per-cell basis, a 5:1 ratio of DYNABEAD:cells (condition DB) corresponds to the same dose of anti-CD28 / anti-CD3 as APC-MS (condition MD) that presented a 0.1% T cell signal at 16.7 μg. Figure 40B A dose-dependent expansion of primary mouse T cells was observed with APC-MS over a 13-day culture period, but not with DYNABEAD within the dose range tested. Compared to DYNABEAD presenting the same amount of anti-CD3 and anti-CD28 antibodies, APC-MS significantly promoted enhanced T cell expansion (see conditions MD vs. DB). Figure 40C Despite higher expansion, cells expanded using APC-MS-conditioned MD did not show enhanced co-expression of the exhaustion markers PD-1 and LAG-3 compared to cells expanded using DYNABEAD (condition DB) presenting the same amounts of anti-CD3 and anti-CD28 antibodies. Figure 40D T cells expanded with low to moderate doses of DYNABEAD showed a predominantly CD4-biased skewing (conditions DA, DB). When very high doses of DYNABEAD were added, a moderate CD8-biased skewing was observed (condition DC). In contrast, APC-MS tended to show a severe CD8-biased skewing, with the degree of skewing depending on the APC-MS formulation. Figure 40B 、 40C Data in and 40D represent mean ± sd of samples from four different mice and are representative of at least two independent experiments. ***p < 0.001, (b) analyzed using two-way ANOVA followed by Tukey HSD post-hoc test.

[0100] Figure 41A and 41B Depicted are the results of experiments performed to evaluate the effects of IL-2 dose and sustained release from APC-MS compared to DYNABEAD on the expansion of primary mouse T cells. Figure 41AShown is the expansion of primary mouse T cells treated with IL-2-loaded APC-MS (MD), APC-MS and IL-2 added to the culture medium (MD bIL2); DYNABEAD (DB), or DYNABEAD and IL-2 added to the culture medium (DB bIL-2). DB: DYNABEAD 5:1; DB-bIL-2: DYNABEAD 5:1 + IL-2 bolus; MD: 0.1% T cell signal / 1:10× material / loaded IL-2; MS / bIL-2: 0.1% T cell signal / 1L10× material / IL-2 bolus. Figure 41B Co-expression of the exhaustion markers PD-1 and LAG-3 in primary mouse T cells expanded with IL-2-loaded APC-MS (MD), APC-MS and IL-2 added to the culture medium (MD bIL2); DYNABEAD (DB), or DYNABEAD and IL-2 added to the culture medium (D-BbIL-2) is shown. Data represent the mean ± SD of samples from four different mice and are a representation of at least two independent experiments. ***p < 0.001, analyzed using two-way ANOVA followed by Tukey HSD post-hoc test.

[0101] Figure 42A and 42B Depicted is the linkage of azide-labeled IgG to DBCO-presenting MSR-SLB via click chemistry conjugation. Figure 42A Different amounts of azide-modified IgG (as indicated) were incubated with MSR-SLB containing different amounts of DBCO-modified lipids (as indicated). The values above the bars represent the number of ug of azide-modified IgG attached to MSR-SLB. Figure 42B A broad dose titration of azide-modified IgG input to MSR-SLBs containing varying amounts of DBCO-modified lipids is shown. nIgG indicates non-azide-modified IgG. Values above the bars indicate μg of azide-modified IgG attached to MSR-SLBs. DETAILED DESCRIPTION

[0102] The present invention provides a solution to the problem of manipulating T cells. Specifically, the present invention provides an antigen presenting cell simulation scaffold (APC-MS), which is useful in such cell manipulation. The scaffold includes a mesoporous silica rod (MSR), which is combined with or covered with a continuous, fluid-supported lipid bilayer (SLB), thereby forming an MSR-SLB scaffold. The MSR-SLB scaffold further contains a variety of T cell activation and T cell costimulatory molecules, together with a variety of T cell homeostatic agents, which together constitute a structure that simulates antigen presenting cells (APC) and allows the scaffold to trigger various effector effects on target cells (e.g., T cells). In some embodiments, the scaffold mediates these effects by direct or indirect interactions between the various binding partners presented by the cell surface molecules and the scaffold in the target cell. Depending on the application using the scaffold, the scaffold regulates the survival and growth of the targeted cells by the physical or chemical characteristics of the scaffold itself. Depending on the application, the scaffold composition can be modified to contain specific activation and costimulatory signals, as well as homeostatic signaling molecules that act together to mediate various effector functions, such as activation, division, promotion of differentiation, growth, expansion, reprogramming, anergy, dormancy, aging, apoptosis, or death of target cells. In these applications, it was found that the scaffold surprisingly increased the cellular metabolic activity and growth of the targeted cells. In addition, the increase in growth and metabolic activity imparted by the scaffold of the present invention was unexpectedly superior to existing platforms, such as magnetic beads.

[0103] In order to allow the manipulation of specific cells (such as T cells), the permeability of the scaffold composition can be regulated, for example, by selecting or engineering materials for larger or smaller pore size, density, polymer crosslinking, rigidity, toughness, ductility or elasticity. The scaffold composition can contain physical channels or paths, and the cells of the target interact with the scaffold and / or move to the specific compartment or the region of the scaffold by it. In order to promote compartmentalization, the scaffold composition can be optionally organized into compartments or layers, each of which has different permeabilities so that cells are sorted or filtered to only allow specific cell subgroups to enter. The isolation of the target cell group in the scaffold can also be regulated by degraded, dehydration or rehydration, oxygenation, chemistry or pH changes or ongoing self-assembly of the scaffold composition. After capture, the cells of the target can be allowed to grow or increase in the scaffold by means of the stimulating molecules, cytokines and other cofactors present in the scaffold. In other cases, negative selection agents can be used to repel or remove the non-targeted cells that infiltrate the scaffold in addition.

[0104] The cells contained or isolated in the support of the present invention are mainly immune cells. In certain embodiments, the present invention relates to a support for isolating and / or manipulating T cells. In other embodiments, the present invention relates to a support permeable to other lymphocytes (e.g., B cells). In other embodiments, the present invention relates to a combination of supports, e.g., a combination of a T cell support and a B cell support. Immune cells (e.g., T cells) are optionally collected and analyzed to identify different subpopulations useful in the diagnosis or treatment of a disease. The collected cells can also be reprogrammed or amplified for the development of a composition or preparation for treatment.

[0105] The present invention is further described in more detail in the following subsections.

[0106] I. Antigen Presenting Cell-Mimetic Scaffold (APC-MS)

[0107] In one embodiment, the present invention provides an antigen presenting cell mimicking scaffold (APC-MS). The scaffold comprises a basal layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous, fluid-supported lipid bilayer (SLB) layered on the MSR basal layer; a plurality of T cell activation molecules and T cell co-stimulatory molecules adsorbed on the scaffold; and a plurality of T cell homeostatic agents adsorbed on the scaffold.

[0108] A. Mesoporous silica

[0109] In one embodiment, the components of the support of the present invention include mesoporous silica. Mesoporous silica is a porous body with hexagonal close-packed, cylindrical uniform pores. This material is synthesized by using rod-shaped micelles of surfactant as a template, which is formed in water by dissolving and hydrating a silica source (such as alkoxysilane, sodium silicate solution, kanemite, silica fine particles) in water or alcohol in the presence of an acid or base catalyst. See, U.S. Publication No. 2015-0072009 and Hoffmann et al., Angewandte Chemie International Edition, 45, 3216-3251, 2006. Many types of surfactants, such as cationic, anionic and nonionic surfactants, have been studied as surfactants, and it is known that in general, the alkyl trimethylammonium salts of cationic surfactants result in mesoporous silica with the largest specific surface area and pore volume. See, U.S. Publication No. 2013 / 0052117 and Katiyar et al. (Journal of Chromatography 1122(1–2):13–20). As used herein, the terms “mesoscale,” “mesopore,” “mesoporous,” and the like may refer to structures having characteristic dimensions in the range of 5 nm to 100 nm, particularly in the range of 2 nm to 50 nm. Thus, in some embodiments, the mesoporous material comprises pores having diameters in the range of 5 nm to 100 nm, which may be ordered or randomly distributed.

[0110] The mesoporous silica used in the scaffold of the present invention can be provided in various forms, for example, microspheres, irregular particles, rectangular rods, circular nanorods, etc., although structured rod forms (MSR) are particularly preferred. The particles can have various predetermined shapes, including, for example, spherical, ellipsoidal, rod-shaped or curved cylindrical shapes. Methods of assembling mesoporous silica to produce microrods are known in the art. See, Wang et al., Journal of Nanoparticle Research, 15:1501, 2013. In one embodiment, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate with a template made from micellar rods. The result is a collection of nanometer-sized spheres or rods filled with regularly arranged pores. The template can then be removed by washing with a solvent adjusted to an appropriate pH. In this example, after removing the surfactant template, hydrophilic silica nanoparticles characterized by uniform, ordered and connected mesoporosity are obtained, having, for example, about 600 m 2 / g to about 1200m 2 / g, especially about 800m 2 / g to about 1000m 2 / g, and especially about 850m 2 / g to about 950m 2 / g specific surface area. In another embodiment, a simple sol-gel method or spray drying method can be used to synthesize mesoporous particles. Tetraethyl orthosilicate is also used together with another polymer monomer (as a template). In yet another embodiment, one or more tetraalkoxy-silanes and one or more (3-cyanopropyl)trialkoxy-silanes can be co-condensed to provide mesoporous silicate particles as rods. See, U.S. Publication Nos. 2013-0145488, 2012-0264599 and 2012-0256336, which are incorporated by reference.

[0111] Mesoporous silica rods can include pores with a diameter of 2-50 nm, for example, 2-5 nm, 10-20 nm, 10-30 nm, 10-40 nm, 20-30 nm, 30-50 nm, 30-40 nm, or 40-50 nm. In a specific embodiment, microrods can include pores with a diameter of approximately 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, or 12 nm. The pore size can be changed depending on the type of application.

[0112] In another embodiment, the length of the microrods is in the micrometer range, ranging from about 5 μ to about 500 μm. In one example, the microrods include a length of 5-50 μm, for example, 10-20 μm, 10-30 μm, 10-40 μm, 20-30 μm, 30-50 μm, 30-40 μm, 40-50 μm. In other embodiments, the rods include a length of 50 μm to 250 μm, for example, about 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 225 μm or longer. For cell recruitment, it may be preferred to use an MSR composition with a higher aspect ratio, for example, using rods including a length of 50 μm to 200 μm, particularly a length of 80 μm to 120 μm, and especially a length of about 100 μm or longer.

[0113] In yet another embodiment, the MSR provides a high surface area for attachment and / or binding to target cells (e.g., T cells). Methods of obtaining high surface area mesoporous silicates are known in the art. See, for example, U.S. Patent No. 8,883,308 and U.S. Publication No. 2011-0253643, the entire contents of which are incorporated herein by reference. In one embodiment, the high surface area is due to the fibrous morphology of the nanoparticles, which allows for a high concentration of highly dispersed and easily accessible portions to be obtained on the surface. In certain embodiments, the high surface area MSR has a surface area of at least about 100 m 2 / g,150m 2 / g, or at least 300m 2 In other embodiments, the high surface area MSR has a surface area of about 100 m 2 / g to about 1000m 2 / g surface area, including all values or subranges therein, e.g., 50m 2 / g,100m 2 / g,200m 2 / g,300m 2 / g,400m 2 / g,600m 2 / g,800m 2 / g, 100-500m 2 / g, 100-300m 2 / g, 500-800m 2 / g or 500-1000m 2 / g.

[0114] B. lipids

[0115] The scaffold of the present invention comprises a continuous, fluid-supported lipid bilayer (SLB) on the MSR basal layer. The term "lipid" generally refers to a heterogeneous group of substances associated with a living system, which has the common property of being insoluble in water and can be extracted from cells by low-polarity organic solvents (such as chloroform and ether). In one embodiment, "lipid" refers to any substance comprising a long fatty acid chain, preferably containing 10-30 carbon units, particularly containing 14-23 carbon units, and especially containing 16-18 carbon units.

[0116] In one embodiment, the lipid is provided as a monolayer. In another embodiment, the lipid is provided as a bilayer. The lipid bilayer is a thin polar membrane formed by two layers of lipid molecules. Preferably, the lipid bilayer is a fluid in which individual lipid molecules can diffuse rapidly within the monolayer. The membrane lipid molecules are preferably amphoteric.

[0117] In one embodiment, the lipid layer is a continuous bilayer, for example, similar to those found in natural biological membranes (such as the plasma membrane). In another embodiment, lipids are provided in the form of a supported bilayer (SLB). SLB is a planar structure placed on a solid support (e.g., a mesoporous silica rod (MSR)). In such a configuration, the upper surface of the supported bilayer is exposed, while the inner surface of the supported bilayer contacts the support. The MSR-SLB scaffold is stable and remains intact to a large extent, even when subjected to high flow rates or vibrations, and can withstand cavities, for example, cavities aligned with the pores of the mesoporous silica substrate. Due to this stability, it is possible to continue experiments for weeks and even months using the supported bilayer. SLB is also easy to modify, derivatize, and chemically conjugate with many chemical and / or biological moieties.

[0118] In one embodiment, the SLBs can be fixed to the MSR substrate using any known method, including covalent and non-covalent interactions. Types of non-covalent interactions include, for example, electrostatic interactions, van der Waals' interactions, π-effects, hydrophobic interactions, etc. In one embodiment, the lipids are adsorbed on the MSR substrate. In another embodiment, the SLBs are linked or tethered to the MSR substrate via covalent interactions. Methods for attaching lipids to silicates are known in the art, for example, surface adsorption, physical fixation, for example, using phase transitions to capture substances in the scaffold material. In one embodiment, the lipid bilayer is layered on the MSR substrate. For example, a lipid film (containing, for example, a solution of DPPC / cholesterol / DSPE-PEG in a molar ratio of 77.5:20:2.5 in chloroform) can be spotted on the mesoporous silica and the solvent evaporated using a rotary evaporator. See Meng et al., ACS Nano, 9(4), 3540-3557, 2015. In one embodiment, for example, a lipid bilayer can be prepared using standard protocols by extruding a hydrated lipid membrane through a filter having, for example, a pore size of about 100 nm. The filtered lipid bilayer membrane can then be fused to the porous particle core, for example, by pipetting.

[0119] Alternatively, covalent coupling via an alkylating agent or acylating agent can be used to provide a stable, structured, and long-lasting SLB on the MSR layer. In such an embodiment, the lipid bilayer can be reversibly or irreversibly fixed to the MSR layer using known techniques. For example, the MSR substrate layer can be hydrophilic and can be further treated to provide a more hydrophilic surface, for example, using ammonium hydroxide or hydrogen peroxide. For example, using known coupling techniques, the lipid bilayer can be fused to the porous MSR substrate layer to form an MSR-SLB scaffold. The scaffold can be further processed and derivatized with other moieties to allow other secondary agents to be attached and / or fixed to the structure.

[0120] Therefore, in one embodiment, the present invention provides an MSR-SLB scaffold, wherein the SLB component is a phospholipid. Representative examples of such lipids include, but are not limited to, the amphoteric liposomes described in U.S. Patent Nos. 9,066,867 and 8,367,628. For example, the lipid bilayer can include lipids selected from dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE), or a combination thereof. In some embodiments, the lipid bilayer includes a lipid composition that mimics the lipid composition of a mammalian cell membrane (e.g., a human plasma membrane). The lipid composition of many mammalian cell membranes has been characterized and is readily available to those skilled in the art (see, e.g., Essaid et al., Biochim. Biophys. Acta 1858(11):2725-36 (2016), the entire contents of which are incorporated herein by reference). The composition of the lipid bilayer can be altered to change the charge or fluidity of the lipid bilayer. In some embodiments, the lipid bilayer comprises cholesterol. In some embodiments, the lipid bilayer comprises sphingolipids. In some embodiments, the lipid bilayer comprises phospholipids. In some embodiments, the lipid is phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphoinositide, a phospho-sphingolipid having a saturated or unsaturated tail comprising 6-20 carbons, or a combination thereof.

[0121] In another embodiment, the lipid is a DIYNE PC lipid. Representative examples of such lipids include, but are not limited to, 1-palmitoyl-2-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (16:0-23:2DIYNE PC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (23:2Diyne PC).

[0122] In one embodiment, the MSR-SLB scaffolds of the invention maintain a continuous, fluidic structure for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, or longer.

[0123] The architecture of the MSR-SLB scaffolds can be studied using any known technique, including microscopic visualization as described in the Examples below.

[0124] C. Functional molecules

[0125] In one embodiment of the present invention, the MSR-SLB scaffold may contain one or more functional molecules. The term "functional molecule" includes any molecule having biologically desirable properties. In the context of the present invention, examples of such functional molecules include proteins, peptides, antigens, antibodies, DNA, RNA, carbohydrates, haptens and other small molecules, for example, drugs. In one embodiment, the functional molecule is a T cell activating molecule. In one embodiment, the functional molecule is a T-cell co-stimulatory molecule. Further, in one embodiment, the functional molecule is a T cell homeostatic agent. In certain embodiments, the MSR-SLB scaffold includes a plurality of functional molecules, for example, at least one T cell activating molecule, at least one T cell co-stimulatory molecule and at least one T cell homeostatic agent.

[0126] T cell activating molecules

[0127] In one embodiment, the present invention provides an MSR-SLB scaffold containing multiple T cell activation molecules. These activation molecules can mediate direct, indirect, or semi-direct activation of target T cell populations. See, Benichou et al., Immunotherapy, 3(6):757-770, 2011. Preferably, the T cell activation molecules mediate direct activation of T cells.

[0128] In one embodiment, the present invention provides MSR-SLB scaffolds containing molecules that directly activate T cells, for example, by binding to cell surface receptors on target T cells. In particular, direct activation can be mediated by cluster of differentiation 3 (CD3), a T cell co-receptor that helps activate cytotoxic T cells. In another embodiment, T cells can be directly activated without the concomitant involvement of CD3, for example, in a CD3-independent manner.

[0129] In one embodiment, target T cells are activated in a CD3-dependent manner. It is generally believed that T cell activation requires T cell receptor (TCR) to recognize its cognate peptide in the context of MHC molecules. In addition, the combination of CD3 and TCR-peptide-MHC complex transmits the activation signal to the intracellular signal transduction molecule to start the signal transduction cascade in the T cell. See, Ryan et al., Nature Reviews Immunology 10, 7, 2010. The CD3 receptor complex found on T cells contains CD3γ chain, CD3δ chain and two CD3ε chains, which combine TCR and ζ chain (zeta-chain; CD247) to produce activation signals in T cells. TCR, ζ-chain and CD3 molecules together constitute the T cell receptor (TCR) complex. The combination of an activating molecule (e.g., antibody) and one or more members of the TCR complex can activate T cells.

[0130] In one embodiment, the T cell activating molecule is an antibody or an antigen binding fragment thereof. In the case where the T cell activating molecule acts in a CD3-dependent manner, the T cell activating molecule is preferably an anti-CD3 antibody or an antigen binding fragment thereof. In another embodiment, the T cell activating molecule may include, for example, an anti-CD2 antibody or an antigen binding fragment thereof, an anti-CD47 antibody or an antigen binding fragment thereof, an anti-macrophage scavenger receptor (MSR1) antibody or an antigen binding fragment thereof, an anti-T cell receptor (TCR) antibody or an antigen binding fragment thereof, etc. In another embodiment, the T cell activating molecule is a major histocompatibility complex (MHC) molecule or a multimer thereof optionally loaded with an MHC peptide. Further, the T cell activating molecule is a conjugate containing MHC and an immunoglobulin (Ig) or a multimer thereof.

[0131] As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant or derivative thereof that retains the important epitope binding characteristics of the Ig molecule. Such mutant, variant or derivative antibody forms are known in the art. Non-limiting embodiments thereof are discussed herein. In one embodiment, the T cell activating antibody used in the compositions and methods of the present disclosure is an anti-CD3 antibody selected from muromonab (OKT3), otelixizumab (TRX4), tilizumab (hOKT3γ1 (Ala-Ala)), visilizumab, an antibody that recognizes the 17-19 kD epsilon chain of CD3 within the CD3 antigen / T cell antigen receptor (TCR) complex (HIT3a), and an antibody that recognizes the 20 kDa subunit of the TCR complex (UCHT1) within CD3e, or an antigen-binding fragment thereof. Other anti-CD3 antibodies, including antigen-binding fragments thereof, are described in U.S. Patent Publication No. 2014-0088295, which is incorporated by reference.

[0132] Embodiments of the present invention include "full-length" antibodies. In full-length antibodies, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0133] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-13). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can also be bispecific, dual-specific, or multispecific; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment, consisting of the VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546, Winter et al., PCT Publication WO 90 / 05144A1, incorporated herein by reference), which includes a single variable domain; and (vi) isolated complement determining regions (CDRs). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and form two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)).

[0134] An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, and typically most or all, of the functions normally associated with the portion when present in an intact antibody. In one embodiment, an antibody fragment comprises the antigen binding site of an intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, e.g., comprising an Fc region, retains at least one biological function normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement fixation. In one embodiment, an antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence that imparts in vivo stability to the fragment.

[0135] As used herein, the term "antibody construct" refers to a polypeptide comprising one or more antigen-binding portions of the present disclosure connected to a linker polypeptide or an immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues connected by a peptide bond and is used to connect one or more antigen-binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994)). An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and are disclosed in Table 2 of U.S. Patent No. 7,915,388, the entire contents of which are incorporated herein by reference.

[0136] Further, antibody or its antigen-binding portion thereof can be a part for larger immunoadhesion molecule, and described larger immunoadhesion molecule is formed by the covalent or non-covalent combination of antibody or antibody portion and one or more other proteins or peptides.The example of such immunoadhesion molecule comprises using avidin protein streptavidin core region to prepare tetrameric scFv molecule (Kipriyanov etc., Human Antibodies and Hybridomas 6:93-101 (1995)) and using cysteine residues, marker peptide and C-terminal polyhistidine tag to prepare divalent and biotinylated scFv molecule (Kipriyanov etc., Mol.Immunol.31:1047-1058 (1994)).Can use respectively conventional technique such as complete antibody respectively papain or pepsin digestion to prepare antibody portion such as Fab and F (ab ') 2 fragment from complete antibody.In addition, can use standard recombinant DNA technology as described herein, obtain antibody, antibody portion and immunoadhesion molecule.

[0137] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds CD3 is substantially free of antibodies that specifically bind antigens other than CD3). However, an isolated antibody that specifically binds CD3 may have cross-reactivity with other antigens (e.g., CD3 molecules from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0138] As used herein, the term "human antibody" is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in CDRs, and particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0139] As used herein, the term "recombinant human antibody" is intended to include all human antibodies produced, expressed, formed or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (further described in U.S. Patent No. 7,915,388, the contents of which are incorporated herein by reference), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom et al., TIB Tech. 15:62-70 (1994); Azzazy et al., Clin. Biochem. 35:425-445 (2002); Gavilondo et al., BioTechniques 29:128-145 (2002); Hoogenboom et al., Immunology Today 21:371-378 (2000)), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al., Nucl. Acids Res. 20: 6287-6295 (1992); Kellermann et al., Current Opinion in Biotechnology 13: 593-597 (2002); Little et al., Immunology Today 21: 364-370 (2002)) or antibodies prepared, expressed, formed or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using an animal that is transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo. One embodiment provides fully human antibodies capable of binding to human CD3, which can be generated using techniques known in the art, such as, but not limited to, using human Ig phage libraries such as those disclosed in Jermutus et al., PCT Publication No. WO 2005 / 007699 A2.

[0140] The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having mouse heavy and light chain variable regions connected to human constant regions. Methods for producing chimeric antibodies are known in the art and are discussed in detail in Example 2.1. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al. (1989), J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567 and 4,816,397, all of which are incorporated herein by reference. In addition, "chimeric antibodies" can be produced by techniques known in the art. See, Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454, all of which are incorporated herein by reference.

[0141] As used herein, the term "specifically binds" or "specifically binds," with respect to the interaction of an antibody, protein, or peptide with a second chemical substance, means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than binding to proteins generally. If the antibody is specific for epitope "A," the presence of molecules containing epitope A (or free, unlabeled A) in a reaction comprising labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0142] The antibodies used in the scaffolds of the present invention can be "monospecific," "bispecific," or "multispecific." As used herein, the term "antibody" is intended to include monospecific antibodies (e.g., anti-CD3 antibodies) as well as bispecific antibodies comprising one arm that binds to a target antigen (e.g., a CD3-binding arm) and a second arm that binds to a second target antigen. The target antigen bound by the other arm of the CD3 bispecific antibody can be any antigen expressed on or near a cell, tissue, organ, microorganism, or virus, to which a targeted immune response is desired. In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T cell proliferation. Also included within the meaning of the term are antibodies that bind to different regions of the CD3 molecule, for example, an arm that binds to the 17-19 kD epsilon chain of CD3 within the CD3 antigen / T cell antigen receptor (TCR) complex (e.g., derived from HIT3a), and an arm that binds to the 20 kDa subunit of the CD3 epsilon TCR complex (e.g., derived from UCHT1). Preferably, the anti-CD3 antibody is OKT3 or a CD3-binding fragment thereof.

[0143] In one embodiment, the antibody molecule used in the support of the present invention is a bispecific antibody. Bispecific antibodies can be used in the context of the present invention to allow target cells (e.g., cancer cells or pathogens) to approach target effector cells of the present invention (e.g., cytotoxic T cells) so that the effector functions of the target effector cells are specifically mediated on the target cells. Therefore, in one embodiment, the invention provides a support containing a bispecific antibody, wherein one arm of the antibody binds to CD3, and the other arm binds to a target antigen, which is a tumor-associated antigen. Non-limiting examples of specific tumor-associated antigens include, for example, AFP, ALK, BAGE protein, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1 B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAG E-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Mud, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ES01, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLHI), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0144] In a specific embodiment, the cancer antigen is a member of the epidermal growth factor receptor (EGFR) family, for example, a receptor selected from EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4) or a mutant thereof.

[0145] In another embodiment, the present invention relates to a scaffold containing a bispecific T cell engager (BiTE) molecule. The BiTE molecule is particularly an antibody that recognizes at least one of the above-mentioned tumor antigens and at least one T cell cell surface molecule (e.g., CD3). Representative examples of such bispecific T cell engager molecules include, but are not limited to, solitomab (CD3×EpCAM), blinatumomab (CD3×CD19), MAB MT-111 (CD3×CEA), and BAY-2010112 (CD3×PSMA).

[0146] Bispecific antibodies can also be used in the context of the present invention to target effector cells, such as T cells or B cells, to directly or indirectly mediate the effects on pathogens, such as bacteria, viruses, fungi, protozoa, and other microorganisms. In one embodiment, the pathogen is a virus. In another embodiment, the pathogen is a bacterium. Bispecific antibodies have been used to treat bacterial infections, for example, drug-resistant Pseudomonas aeruginosa. See, DiGiandomenico et al., Sci Transl Med., 6(262), 2014; Kingwell et al., Nat Rev Drug Discov., 14(1):15, 2015. Other bispecifics have been developed to redirect cytotoxic T lymphocytes to kill HIV (Berg et al., Proc Natl Acad Sci., 88(11):4723-7, 1991), protect against HBV infection (Park et al., Mol Immunol., 37(18):1123-30, 2000) and other prototypical pathogens (Taylor et al., J Immunol., 159(8):4035-44, 1997).

[0147] Thus, in one embodiment, the present invention provides a scaffold comprising a bispecific antibody, wherein one arm of the antibody binds to CD3 and the other arm binds to a target antigen, which is an infectious disease associated antigen (e.g., a bacterial, protozoan, viral, or fungal antigen). Non-limiting examples of infectious disease associated antigens include, for example, antigens expressed on the surface of viral particles, or preferentially expressed on cells infected with a virus, wherein the virus is selected from HIV, hepatitis (hepatitis A, B, or C), herpes virus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. Alternatively, the target antigen can be an antigen expressed on the surface of a bacterium, or preferentially expressed on cells infected with a bacterium, wherein the bacterium is from a genus selected from the group consisting of Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumonococci, Meningococcus, Gonococcus, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Clostridium and Leptospira. In some embodiments, the bacterium causes cholera, tetanus, botulism, anthrax, plague or Lyme disease. In certain embodiments, the target antigen is an antigen expressed on the surface of a fungus, or preferentially expressed on cells infected with a fungus, wherein the fungus is selected from the genus Candida (e.g., Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g., Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (e.g., M. mucor, M. absidia, M. rhizopus, etc.), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. In certain embodiments, the target antigen is an antigen expressed on the surface of a parasite, or preferentially expressed on cells infected with a parasite, wherein the parasite is selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia parvum, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus braziliensis, Taenia crenata, and Brugia malayi. Non-limiting examples of specific pathogen-associated antigens include, for example, HIV gp120, HIV CD4, hepatitis B glycoprotein L, hepatitis B glycoprotein M, hepatitis B glycoprotein S, hepatitis C El, hepatitis C E2, hepatocyte-specific protein, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope protein.

[0148] In some embodiments, the scaffold of the present invention can be used to treat and / or prevent allergic reactions or allergic responses. For example, in some embodiments, the scaffold can be used to produce T cells (e.g., Treg) that inhibit allergic responses or reactions. For example, in some embodiments, the scaffold comprises anti-CD3 antibodies and TGF-β. In some embodiments, the scaffold comprises anti-CD3 antibodies and IL-10. In some embodiments, the scaffold comprises anti-CD3 antibodies and rapamycin. In some embodiments, the scaffold comprises anti-CD3 antibodies, TGF-β, IL-10, and rapamycin. In some embodiments, the scaffold comprises anti-CD3 antibodies, TGF-β, IL-10, and rapamycin. In some embodiments, the scaffold comprises anti-CD3 antibodies, TGF-β, and IL-10. In some embodiments, the scaffold comprises anti-CD3 antibodies, TGF-β, and rapamycin. In some embodiments, the scaffold comprises anti-CD3 antibodies, IL-10, and rapamycin.

[0149] In some embodiments, the scaffolds of the present invention can be used to selectively expand allergen-reactive T cells (e.g., Tregs). In some embodiments, the scaffold comprises a peptide derived from an allergen. In some embodiments, the peptide derived from the allergen is presented on (e.g., complexed with) an MHC molecule (e.g., class I MHC or class II MHC molecule). In some embodiments, the MHC molecule is a monomer. In some embodiments, the allergen is a food allergen (e.g., banana, milk, beans, shellfish, tree nuts, drupes, eggs, fish, soy, or wheat allergens). In one embodiment, the allergen is selected from food allergens, plant allergens, insect allergens, animal allergens, fungal allergens, viral allergens, latex allergens, and mold spore allergens. In one embodiment, the allergen polypeptide is an insect allergen. In one embodiment, the insect allergen is a dust mite allergen (e.g., an allergen from dust mite or house dust mite). In one embodiment, the allergen polypeptide is an ovalbumin polypeptide. In one embodiment, the allergen polypeptide is a food allergen polypeptide. In some embodiments, the scaffold comprises a peptide derived from an allergen and a Th1-skewing cytokine (e.g., IL-12 or IFNγ). In one embodiment, the allergen polypeptide is a food allergen polypeptide. In some embodiments, the scaffold comprises a peptide derived from an allergen presented on an MHC molecule and a Th1-skewing cytokine (e.g., IL-12 or IFNγ).

[0150] According to certain exemplary embodiments, the present invention includes bispecific antigen binding molecules that specifically bind to CD3 and CD28. Such molecules may be referred to herein as, for example, "anti-CD3 / anti-CD28," or "anti-CD3×CD28," or "CD3×CD28" bispecific molecules, or other similar terms.

[0151] As used herein, the term "CD28" refers to human CD28 protein, unless specifically indicated as being from a non-human species (e.g., "mouse CD28," "monkey CD28," etc.). Human CD28 protein has the amino acid sequence shown in GENBANK Accession No. NP_001230006.1, NP_001230007.1, or NP_006130.1. Mouse CD28 protein has the amino acid sequence shown in GENBANK Accession No. NP_031668.3. The various polypeptide sequences included in the above-mentioned accession numbers include corresponding mRNA and gene sequences, all of which are incorporated herein by reference. As used herein, the expression "antigen binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least one complementary determining region (CDR) or consisting of at least one complementary determining region (CDR), the CDR being individually or in combination with one or more additional CDRs and / or framework regions (FRs) to specifically bind to a specific antigen. In certain embodiments, the antigen binding molecule is an antibody or an antibody fragment, as defined elsewhere herein.

[0152] As used herein, the expression "bispecific antigen binding molecule" refers to a protein, polypeptide or molecular complex comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain within a bispecific antigen binding molecule includes at least one CDR, which specifically binds to a specific antigen, either alone or in combination with one or more additional CDRs and / or framework regions (FRs). In the context of the present invention, the first antigen binding domain specifically binds to a first antigen (e.g., CD3), while the second antigen binding domain specifically binds to a second, different antigen (e.g., CD28).

[0153] The first antigen-binding domain and the second antigen-binding domain of a bispecific antibody can be directly or indirectly bound to each other. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The combination of a multimerization domain and another multimerization domain promotes the combination between the two antigen-binding domains, thereby forming a bispecific antigen binding molecule. As used herein, "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid with the ability to be combined with the second multimerization domain of identical or similar structure or structure. For example, the multimerization domain can be a polypeptide comprising an immunoglobulin CH3 domain. The limiting examples of multimerization components are the Fc portion (comprising CH2-CH3 domains) of an immunoglobulin, for example, selected from any allotype IgG Fc domains of isotype IgG1, IgG2, IgG3 and IgG4 and each isotype group.

[0154] The bispecific antigen binding molecules of the present invention will generally comprise two multimerization domains, e.g., two Fc domains that are each individually part of an independent antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, such as, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0155] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0156] Bispecific antibody form or technology can be used to prepare the bispecific antigen binding molecules of the present invention.For example, the antibody or its fragment with the first antigen binding specificity can be functionally connected (for example, by chemical coupling, gene fusion, non-covalent binding or other) one or more other molecular entities, such as another antibody or antibody fragment with the second antigen binding specificity, to produce bispecific antigen binding molecules.The specific exemplary bispecific form that can be used for the situation of the present invention includes, but is not limited to, for example, based on the bispecific form of scFv or double antibody, IgG-scFv fusions, dual variable domains (DVD)-Ig, Quadroma, knob-into-hole (knobs-into-hole), common light chain (for example, with the common light chain of knob-into-hole etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, double action Fab (DAF)-IgG and Mab2 bispecific form (for the summary of aforesaid form, referring to, for example, Klein etc., mAbs 4:6,1-11,2012 and references cited therein).

[0157] Multispecific antibodies can be specific for different epitopes of a target polypeptide or can contain antigen-binding domains that are specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The anti-CD3 antibodies of the present invention can be linked to another functional molecule or co-expressed with another functional molecule, for example, another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity. A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Conventional techniques available in the art can be used to adapt any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, to the context of the antigen-binding fragments of the antibodies of the present invention. The multispecific antigen-binding molecules of the present invention are derived from chimeric, humanized, or fully human antibodies. Methods for preparing multispecific antibodies are well known in the art. For example, using VELOCIMMUNE TM One or more heavy chains and / or light chains of the bispecific antigen-binding molecules of the present invention can be prepared using VELOCIMMUNE technology. TM The invention relates to the isolation of high-affinity chimeric antibodies to specific antigens (e.g., CD3 or CD28) using the human variable region and mouse constant region technology (or any other human antibody generation technology). Antibodies are characterized and selected for their desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to produce fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the present invention.

[0158] In the case of the bispecific antigen binding molecules of the present invention, compared with the wild-type, naturally occurring Fc domain form, multimerization domain, for example, Fc domain, can include one or more amino acid changes (for example, insertion, deletion or replacement). For example, the present invention includes bispecific antigen binding molecules, which include one or more changes in the Fc domain, resulting in the modified Fc domain with the binding interaction between Fc and FcRn (for example, enhanced or eliminated). In one embodiment, the bispecific antigen binding molecules include the modification in CH2 or CH3 district, wherein the modification improves the affinity of the Fc domain to FcRn in an acidic environment (for example, in endosomes, wherein the pH range is about 5.5 to about 6.0). For example, non-limiting examples are provided in U.S. Publication No.2014-0088295. The present invention also includes bispecific antigen binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. In certain embodiments, the Fc domain can be a chimeric, combined Fc sequence derived from more than one immunoglobulin isotype.

[0159] In another embodiment, the T cell activating molecule is a major histocompatibility complex (MHC) molecule that binds CD3. Representative examples include, but are not limited to, type I MHC that binds TCR and CD8 or type II MHC that binds TCR and CD4. The MHC molecule can optionally be loaded with an antigen, for example, a biotinylated peptide. In other embodiments, the MHC molecule can be conjugated to an immunoglobulin, for example, the Fc portion of an immunoglobulin G (IgG) chain. In another embodiment, multiple MHC-peptide complexes can be used. In the latter case, multiple copies of the MHC-peptide complex can be covalently or non-covalently linked to a multimerization domain. Known examples of such MHC multimers include, but are not limited to, MHC-dimers (containing two copies of an MHC-peptide; IgG is used as the multimerization domain, and one of the domains of the MHC protein is covalently linked to the IgG); MHC-tetramers (containing four copies of an MHC-peptide, each of which is biotinylated and the MHC complex is held together in a complex by a streptavidin tetramer protein, which provides a non-covalent link between the streptavidin monomers and the MHC protein); MHC-pentamers (containing five copies of an MHC-peptide complex, multimerized by self-assembling coiled-coil domains); MHC dextramers (typically containing more than ten MHC complexes linked to a dextran polymer) and MHC streptamers (containing 8-12 MHC-peptide complexes linked to streptavidin). MHC tetramers are described in US Pat. No. 5,635,363; MHC pentamers are described in US Pat. No. 2004109295; MHC-dextramers are described in patent application WO 02 / 072631. MHC chain multimers are described in Knabel M et al., Nature Medicine 6, 631-637, 2002.

[0160] Target T cells can also be activated in a CD3-independent manner, for example, by binding and / or ligation of one or more cell surface receptors other than CD3. Representative examples of such cell surface molecules include, for example, CD2, CD47, CD81, MSR1, etc.

[0161] In this case, CD2 is found on virtually all T cells (and also natural killer (NK) cells) and is important in T lymphocyte function. CD2 is associated with several proteins, including CD3, CD5, and CD45. The CD2-CD58 interaction promotes cell-cell contact between T cells and APCs, thereby enhancing antigen recognition through the TCR / CD3 complex. CD2 also plays a signaling role. Co-stimulation blocking using antibodies against CD2 may be an effective immunosuppressive strategy in organ transplantation. Therefore, in one embodiment, T cells are activated by using antibodies or antigen-binding fragments thereof that specifically bind to CD2. Representative examples of anti-CD2 antibodies include, for example, siplizumab (MEDI-507) and LO-CD2b (ATCC Accession No. PTA-802; deposited on June 22, 1999).

[0162] CD47 (IAP) belongs to the immunoglobulin superfamily and is a membrane integrin chaperone, and also binds to the ligands thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα). See Barclay et al., Curr. Opin. Immunol. 21(1):47-52, 2009; Br. J. Pharmacol., 167(7):1415-30, 2012. CD47 interacts with signal regulatory protein alpha (SIRPα), a transmembrane receptor present on myeloid cells. The CD47 / SIRPα interaction results in bidirectional signaling, leading to different cell-cell responses, including inhibition of phagocytosis, stimulation of cell-cell fusion, and T cell activation. See, Reinhold et al., J Exp Med., 185(1):1-12, 1997. According to the present invention, in one embodiment, T cells are activated by using an antibody or antigen-binding fragment thereof that specifically binds to CD47. Representative examples of anti-CD47 antibodies include, for example, the monoclonal antibody Hu5F9-G4, which has been studied in various clinical trials against myeloid leukemia, and the monoclonal antibodies MABL-1 and MABL-2 (FERM Deposit Nos. BP-6100 and BP-6101). See, for example, WO 1999 / 12973, the disclosure of which is incorporated herein by reference.

[0163] CD81 is a member of the tetraspanin superfamily of proteins. It is expressed on a wide range of tissues, including T cells and hematopoietic cells. CD81 is known to play an immunomodulatory role. In particular, crosslinking of CD81 enhances CD3-mediated activation of αβ and γδ T-lymphocytes in vitro and induces TCR-independent cytokine production by γδ T cells. According to the present invention, in one embodiment, T cells are activated by using an antibody or antigen-binding fragment thereof that specifically binds to CD81. See, Menno et al., J. Clin. Invest., 4:1265, 2010. Representative examples of anti-CD81 antibodies include, for example, monoclonal antibody 5A6. See, for example, Maecker et al., BMC Immunol., 4:1, 2003., the disclosure of which is incorporated herein by reference.

[0164] MSR1 (CD204) belongs to the family of class A macrophage scavenger receptors, which include three different types (1, 2, 3) produced by alternative splicing of the MSR1 gene. These receptors or subtypes are trimeric integral membrane glycoproteins and are involved in many macrophage-related physiological and pathological processes, including atherosclerosis, Alzheimer's disease and host defense. See, Matsumoto et al., Proc. Natl. Acad. Sci. USA 87 (23): 9133-7, 1990. Recent studies have demonstrated that dendritic (DC) MSR1 affects the activation and proliferation of CD8 T cells, and that antibody-mediated blockade of MSR1 increases the proliferation and expansion of T cells in vitro. Lerret et al., PLoS One., 7 (7): e41240, 2012. According to the present invention, in one embodiment, T cells are activated by using an antibody or antigen-binding fragment thereof that specifically binds to MSR1. Representative examples of anti-MSR1 antibodies include, for example, rat anti-human CD204 antibody (Thermo Catalog No. MA5-16494) and goat anti-human CD204 / MSR1 antibody (Biorad Catalog No. AHP563).

[0165] In another embodiment, T cells are activated by connecting / binding to T cell receptor (TCR) molecules that are ubiquitously expressed in T cells. TCR is a heterodimer composed of two different protein chains. In humans, in 95% of T cells, TCR is composed of alpha (α) and beta (β) chains, while in 5% of T cells, TCR is composed of gamma and delta (γ / δ) chains. When TCR engages with antigenic peptides and MHC (peptide / MHC), T lymphocytes are activated by signal transduction. According to the present invention, in one embodiment, T cells are activated by using antibodies or antigen-binding fragments thereof that specifically bind to TCR. Representative examples of anti-TCR antibodies include, for example, mouse anti-human TCR monoclonal antibody IMMU510 (Immunotech, Beckman Coulter, Fullerton, CA) (described in Zhou et al., Cell Mol Immunol., 9(1):34-44, 2012) and the monoclonal antibody defining α / β TCR WT31 (described in Gupta et al., Cell Immunol., 132(1):26-44, 1991).

[0166] In another embodiment, the T cell activation molecule is a major histocompatibility complex (MHC) molecule that is optionally loaded with an MHC peptide. There are two major classes of MHC molecules. Class I MHC (pMHC) molecules are found on almost all cells and present peptides to cytotoxic T lymphocytes (CTLs). Class II MHC molecules are mainly found on antigen presenting immune cells (APCs), which take in polypeptide antigens (e.g., in microorganisms) and digest them into peptide fragments. Subsequently, MHC-II molecules present peptide fragments to helper T cells, which, after activation, generally provide the required auxiliary activity for the response of other cells of the immune system (e.g., CTLs or antibody-producing B cells). The interaction between the peptide bound in the binding cleft of the heavy chain of Class I MHC (pMHC) and the complementary determining region (CDR) of the T cell receptor determines the potential for T cell activation in the input and output stages of cellular immunity. The affinity between the TCR and the MHC peptide complex regulates T cell fate during the generation, initial activation, and execution of effector functions.

[0167] Thus, in one embodiment, the present invention relates to an MSR-SLB scaffold comprising a human MHC molecule, optionally loaded with a peptide. Representative examples of such MHC molecules include HLA-A, HLA-B, HLA-C, DP, DQ, and DR, or combinations thereof. The MHC molecules are monovalent or bivalent. In some embodiments, bivalency or multivalency of the MHC molecule is required for signal delivery (activation or inhibition) to T cells. Thus, in some embodiments, the MSR-SLB scaffold of the present invention comprises at least two identical MHC molecules connected to a linker.

[0168] The joint of the bivalent MHC molecule plays three roles. First, the joint gives the required bivalence or multivalence. Second, the joint increases the half-life of the entire fusion protein in vivo. Third, the joint determines whether the fusion protein will activate or inhibit T cells. T cell activation requires stimulation by TCR and another second signal usually delivered by APC. In the absence of the second signal, it may cause T cell response to be low. By constructing a fusion protein in which the joint allows the delivery of the second signal, T cell stimulation leads to enhanced T cell immunity. By constructing a fusion protein in which the joint does not provide the delivery of the second signal, T cell inhibition leads to immunosuppression. Fusion proteins with T cell stimulation properties can be constructed by using a joint that allows the second signal to be delivered to T cells (in addition to the signal delivered by TCR). This can be accomplished by using a joint with binding affinity to a cell surface structure on another cell, and the other cell is able to deliver the second signal to the T cell. Therefore, the joint is used to bridge T cells and other cells. By bringing other cells near the T cell, the other cells can deliver the second signal to the T cell.

[0169] Examples include linkers that can bind to Fc receptors on other cells, such as certain immunoglobulin chains or portions of immunoglobulin chains. Specific examples include IgG, IgA, IgD, IgE, and IgM. When using immunoglobulins, the entire protein is not required. For example, immunoglobulin genes can be split at the hinge region, and only genes encoding the hinge, CH2, and CH3 domains of the heavy chain are used to form a fusion protein. Linkers can bind to other cell surface structures. For example, linkers can include homologous portions for many cell surface antigens, which can serve as a bridge to bring a second cell to the vicinity of a T cell. Linkers can also independently deliver a second signal. For example, a linker with binding affinity for the T cell antigen CD28 can deliver a second signal. In addition, linkers can increase the half-life of the entire fusion protein in vivo. Fusion proteins with T cell inhibitory properties can be constructed by using linkers that do not result in the delivery of a second signal. Examples include Ig chains that do not bind to Fc receptors, Ig F(ab')2 fragments, zinc finger motifs, leucine zippers, and non-biological substances. Examples of non-biological substances include plastic microbeads, or even larger plastic elements such as plastic rods or tubes, and other physiologically acceptable carriers that can be implanted in the body.

[0170] In some embodiments, MHC molecules are not connected to joints. Without wishing to be bound by any particular theory, it is believed that the fluid properties of lipid bilayers allow T cell recognition membranes to form multivalent clusters. These clusters can be decomposed subsequently, and if signaling molecules are linked together with joints, then this is impossible. Failure to decompose these multivalent clusters may potentially lead to overstimulation and T cell exhaustion or anemia (see, for example, Lee KH et al., Science 302 (5648): 1218-22 (2003)).

[0171] In some embodiments, the lipid bilayer of the APC-MS includes a lipid composition that facilitates spontaneous partitioning of lipid species into liquid-ordered domains (see, e.g., Wang TY et al., Biochemistry 40(43):13031-40 (2001)).

[0172] Optionally, MHC molecules can be loaded with specific peptides (e.g., peptides derived from viral antigens, bacterial antigens, or allergens). After the fusion protein is prepared, the specific peptides of the fusion protein can be loaded into the MHC molecule. The peptide can also be covalently linked to MHC subsequently, for example, by UV cross-linking. Alternatively, the peptide sequence can be incorporated into the DNA sequence encoding the fusion protein so that the peptide is loaded into the MHC molecule during the fusion protein production process. In the latter case, the peptide can utilize a tether, such as a polylysine connection, which allows it to be compounded with the MHC portion of the fusion protein. The specific peptide to be loaded into the MHC molecule is actually unlimited and is determined based on the desired application. For example, in order to enhance T cell immunity, peptides from different sources such as viruses, fungi, and bacterial infections or tumors can be used. In order to suppress T cell immunity in autoimmunity, autoreactive peptides can be used. In order to suppress T cell immunity to transplanted tissues, self-peptides presented by alloantigens can be used.

[0173] Toxins, such as ricin and diphtheria toxin, and radioactive isotopes can be complexed with fusion proteins (e.g., using 5-methyl-2-iminothiolane) to kill specific T cell clones. These toxins can be chemically coupled to a linker or the MHC portion of the fusion protein, or they can be incorporated into the DNA sequence encoding the fusion protein so that the toxin is complexed with the fusion protein during its production.

[0174] MHC-peptide / immunoglobulin fusion proteins can be prepared by constructing genes encoding the production of fusion proteins. Alternatively, chemical conjugation methods can be used to assemble the components of the fusion protein. The sources of genes encoding MHC molecules and linkers can be obtained from various databases. In the case of class I MHC fusion proteins, the MHC fragment can be connected to the linker and can allow the β2 microglobulin to self-associate. Alternatively, the fusion protein gene can be constructed so that the β2 microglobulin is connected to the MHC fragment through an ether. In the case of class II MHC fusion proteins, either the α or β chain can be connected to the linker and can allow the other chain to self-associate. Alternatively, the fusion protein gene can be constructed so that the α and β chains are connected by a tether. Peptides can be prepared by encoding into fusion protein gene constructs, or alternatively, peptides can be prepared using a peptide synthesizer using standard methods available to those of ordinary skill in the art. The resulting complete fusion protein can be administered using conventional techniques.

[0175] T cell co-stimulatory molecules

[0176] In one embodiment, the present invention provides an MSR-SLB scaffold containing multiple T cell co-stimulatory molecules. These co-stimulatory molecules can mediate direct, indirect, or semi-direct stimulation of target T cell populations. Preferably, the co-stimulatory molecules mediate T cell activation in the presence of one or more T cell activation molecules.

[0177] The term "costimulatory molecules" is used herein according to its meaning in immune T cell activation recognized in the art. Specifically, "costimulatory molecules" refers to a group of immune cell surface receptors / ligands, which engage between T cells and antigen presenting cells, and produce stimulatory signals in T cells, which are combined with the stimulating signal in the T cells produced by the T cell receptor (" TCR ") of the antigen on antigen presenting cells (that is, " costimulation "). As used herein, the soluble form of the costimulatory molecules "derived from APC" refers to the costimulatory molecules expressed generally by B cells, macrophages, monocytes, dendritic cells and other APCs. See, Huppa et al., Nature Reviews Immunology.3, 973-983 (2003). " costimulators for T cell activation " refers to the ability of costimulatory ligands to bind and activate T cells, and the T cells have been activated by any of the above-mentioned mechanisms or approaches, for example, by CD3 dependency or non-CD3 dependency T cell activation. Costimulatory activation of T cells can be measured as known by cytokine production and by known proliferation assays (eg, CFSE staining) and / or as described in the Examples below.

[0178] In one embodiment, the present invention provides an MSR-SLB scaffold comprising a molecule that specifically binds a co-stimulatory antigen. In particular, the MSR-SLB scaffold contains multiple T cell co-stimulatory molecules that specifically bind to CD28, 4.1BB (CD137), OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTβR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CD279 (PD-1) and / or CRACC (CD319, BLAME).

[0179] In one embodiment, costimulatory molecules are antibodies or their antigen binding fragments specifically in conjunction with one or more of the above-mentioned costimulatory antigens. In this regard, CD28 is a prototype T cell costimulatory antigen and in conjunction with the molecules of the B7 family expressed on APC (such as dendritic cells and activated B cells). Human CD28 is found on all CD4+T cells and about half of the CD8+T cells. The T cell activity due to CD28 includes the blocking of energy, the induction of cytokine gene transcription, the stabilization of cytokine mRNA and the activation of CD8+ cytotoxic T lymphocytes. The CD28 ligands identifiable as CD80 (B7-1) and CD86 (B7-2) are immunoglobulin superfamily monomeric transmembrane glycoproteins of 60kd and 80kd respectively.

[0180] In one embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD28. Representative examples of anti-CD28 antibodies include, for example, lulizumab pegol and TGN1412. See also U.S. Patent No. 8,785,604.

[0181] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds ICOS (CD278). ICOS is a co-stimulatory molecule of the CD28 superfamily expressed on activated T cells. It is believed to be particularly important for Th2 cells. Representative examples of anti-ICOS antibodies include, for example, monoclonal antibody 2C7, which recognizes ICOS molecules expressed on activated T cells and induces activation and proliferation of T cells pre-stimulated with anti-human CD3 monoclonal antibodies. See, Deng et al., Hybrid Hybridomics., 23(3):176-82, 2004.

[0182] In another embodiment, the present invention provides an MSR-SLB containing an antibody or an antigen-binding fragment thereof that specifically binds to CD152 (CTLA4). The antibody is preferably a neutralizing antibody or a blocking antibody. CD152 is expressed on activated CD4+ and CD8+ T cells and on regulatory T cells (Treg). It plays a role in T cell biology, in the immune response process for infection, and as a target for cancer immunotherapy, it has been fully described (Egen et al., Nat. Immunol., 3 (7): 611-618, 2002). CTLA-4 is a homologous counterpart of CD28, both of which bind to CD80 and CD86 on APCs. The importance of CTLA-4 for immune tolerance is clear (Waterhouse et al., Science, 270 (5238): 985-988, 1995). These include a significantly lower affinity for ligand binding than the CD28 molecule (out-competing) to minimize T cell co-stimulation, recruitment of inhibitory phosphatases to the TCR complex (to disrupt the forward signaling cascade), and removal of CD80 and CD86 from the surface of APCs by reverse endocytosis, thereby eliminating the ability of APCs to correctly activate otherwise responsive T cells. Therefore, utilization of the CTLA-4 receptor / pathway is an attractive strategy for regulating T cell immunity. In fact, anti-CTLA-4 is the first FDA-approved monoclonal antibody (ipilimumab) for checkpoint blockade therapy of cancer patients. Other examples of CTLA-4 antibodies that can be used according to the present invention include tremelimumab and its antigen-binding fragment.

[0183] In another embodiment, the present invention provides an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds programmed death-1 (PD-1; CD279). PD-1 is a member of the same receptor family as CD28 and CTLA-4 and is widely expressed on lymphoid and myeloid cells. PD-1 uniquely binds to the B7 ligands PD-L1 and PD-L2 on APCs and other peripheral tissues, significantly influencing the fate of CD8+ T cells responding in chronic infection settings. On T cells, PD-1 is expressed after antigen encounter but acts almost immediately to prevent T cell activation by recruiting the phosphatases SHP-1 and SHP-2 via signaling motifs in the PD-1 cytoplasmic tail. This reduces Akt phosphorylation and decreases T cell metabolism, proliferation, and survival. Therefore, the antibody is preferably a neutralizing or blocking antibody. Representative examples of such anti-PD-1 antibodies include, for example, nivolumab, lambrolizumab (MK-3475), pidilizumab (CT-011), and AMP-224.

[0184] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD81. Engagement of CD81 reduces the signaling threshold required to initiate T cell / CD3-mediated proviral DNA in CD4+ T cells (Tardif et al., J. Virol. 79(7):4316-28, 2005). Representative examples of anti-CD81 antibodies include, for example, monoclonal antibody 5A6. See, for example, Maecker et al., BMC Immunol., 4:1, 2003, the disclosure of which is incorporated herein by reference.

[0185] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD137. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In addition, it can enhance immune activity to eliminate tumors in vivo. Therefore, the antibody that binds to CD137 is preferably an agonist antibody. Representative examples of anti-CD137 antibodies include, for example, the monoclonal antibody utomilumab, which is a human IgG currently being studied in clinical trials. See National Clinical Trials ID: NCT01307267.

[0186] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to OX40 (CD134). OX40L binds to the OX40 receptor on T cells, thereby preventing their death and subsequently increasing cytokine production. OX40 plays a key role in maintaining the immune response beyond the first few days and contributes to memory responses due to its ability to enhance survival. OX40 also plays a key role in both Th1 and Th2-mediated responses in the body. Therefore, the antibody that binds to OX40 is preferably an agonist antibody. Representative examples of anti-OX40 antibodies include, for example, the anti-OX40 monoclonal antibody utomilumab, which is being studied in various clinical trials (see National Clinical Trials IDs: NCT01644968, NCT01303705, and NCT01862900).

[0187] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD27 (TNFRSF7). CD27 is a member of the TNF-receptor superfamily and is required for the generation and long-term maintenance of T cell immunity. It binds to its ligand CD70 and plays a key role in regulating immunoglobulin synthesis. CD27 supports natural ( ) antigen-specific expansion of T cells (but without effector cell maturation), independent of the cell cycle-promoting activity of CD28 and IL2 (Hendriks et al., Nature Immunology 1, 433-440, 2000). Therefore, the MSR-SLB scaffold of the present invention preferably includes an agonistic antibody that binds CD27. Representative examples of anti-CD27 antibodies include, for example, the monoclonal antibody varlilumab. See, Ramakrishna et al., Journal for ImmunoTherapy of Cancer, 3:37, 2015.

[0188] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to a gene regulated by the glucocorticoid-induced TNF receptor family (GITR or CD357). GITR is a 25kD member of the TNF receptor superfamily that is expressed on activated lymphocytes. GITR is upregulated by T cell receptor engagement. The cytoplasmic domain of GITR is homologous to CD40, 4-1BB, and CD27. GITR signaling has been shown to regulate T cell proliferation and TCR-mediated apoptosis, and to disrupt immune self-tolerance. GITR further binds to GITRL and is involved in regulating the development of T cells and regulating the activity of Th1 subsets. Regulating GITR with agonistic antibodies has been shown to amplify anti-tumor immune responses in animal models through multiple mechanisms. Anti-GITR antibodies are designed to activate GITR receptors, thereby increasing the proliferation and function of effector T cells. At the same time, GITR connection on the surface of Tregs can eliminate the inhibitory function of these cells on tumor-specific effector T cells, thereby further enhancing the T cell immune response. Representative examples of anti-GITR antibodies include, for example, the humanized, Fc-disabled anti-human GITR monoclonal antibody TRX518, which induces activation of tumor antigen-specific T effector cells and eliminates suppression induced by inappropriately activated T regulatory cells. TRX518 is being studied in various clinical trials (see National Clinical Trial ID: NCT01239314).

[0189] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD30 (TNFRSF8). The CD30 antigen is a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily that, when stimulated, exerts pleiotropic effects on cell growth and survival. In normal or inflamed tissues, CD30 expression is restricted to medium / large activated B and / or T lymphocytes. It is expressed by activated, but not resting, T and B cells (Guo et al., Infect. Immun., 81(10), 3923-3934, 2013). In vivo administration of an agonistic anti-CD30 monoclonal antibody (MAb) to stimulate CD30L / CD30 signaling restored IL-17A production by Vγ1-Vγ4-γδ T cells in CD30L knockout mice. Representative examples of anti-CD30 antibodies include, for example, brentuximab vedotin (Adcetris).

[0190] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to HVEM (CD270). CD270 is a member of the TNF-receptor superfamily. This receptor has been identified as a cellular mediator of herpes simplex virus (HSV) entry. Mutations in this gene have been repeatedly associated with cases of diffuse large B-cell lymphoma. Representative examples of anti-CD270 antibodies include, for example, the monoclonal antibody HVEM-122. See, Cheung et al., J. Immunol., 185:1949, 2010; Hobo et al., J Immunol., 189:39, 2012.

[0191] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to the lymphotoxin beta receptor (LTβR; TNFRSF3). LTβR is involved in CD4+ T cell activation (Summers de Luca et al., J Exp Med., 204(5):1071-81, 2007). Representative examples of anti-LTβR antibodies include, for example, the monoclonal antibody BBF6. See also WO 2010 / 078526, which is incorporated by reference.

[0192] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds DR3 (TNFRSF25). DR3 is believed to be involved in controlling lymphocyte proliferation induced by T cell activation. Specifically, DR3 activation is dependent on prior engagement of the T cell receptor. Upon binding to TL1A, DR3 signaling increases T cell sensitivity to endogenous IL-2 via the IL-2 receptor and enhances T cell proliferation. Because receptor activation is T cell receptor-dependent, in vivo DR3 activity is specific to those T cells that encounter the cognate antigen. At rest, and in individuals without underlying autoimmunity, the majority of T cells that regularly encounter the cognate antigen are FoxP3+ regulatory T cells. TNFRSF25 stimulation, in the absence of any other exogenous signals, stimulates significant and highly specific proliferation of FoxP3+ regulatory T cells, from 8-10% of all CD4+ T cells to 35-40% of all CD4+ T cells within 5 days. Representative examples of DR3 agonists include, for example, antibodies that specifically bind to DR3 (Reddy et al., J. Virol., 86(19):10606-10620, 2012) and the agonist 4C12 (Wolf et al., Transplantation, 27;94(6):569-74, 2012).

[0193] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD26 (DNAM1). CD226 is a ~65 kDa glycoprotein expressed on the surface of natural killer cells, platelets, monocytes, and T cell subsets. It is a member of the immunoglobulin superfamily and mediates cell adhesion to other cells bearing its ligands CD112 and CD155. Crosslinking of CD226 with the antibody causes cell activation, and ligation of CD226 and LFA-1 with their respective ligands cooperates in eliciting cytotoxicity and cytokine secretion in T and NK cells (Tahara et al., Int. Immunol. 16(4):533-8, 2004).

[0194] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CRTAM (CD355). CTRAM is a class I MHC-restricted T cell-associated molecule that regulates late cell polarity in some CD4+ T cells. CTRAM also regulates interferon-γ (IFNγ) and interleukin-22 (IL-22) production. In one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CTRAM antibody. Representative examples of CTRAM antibodies include, for example, the mouse anti-human CTRAM antibody 21A9 (GENTEX Inc. USA, Irvine, CA).

[0195] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to TIM1 (HAVCR1, KIM1). The TIM gene belongs to a type I cell surface glycoprotein, which includes an N-terminal immunoglobulin (Ig)-like domain, a mucin domain of varying lengths, a single transmembrane domain, and a C-terminal short cytoplasmic tail. The localization and function of the TIM gene are divergent between each member. TIM-1 is preferably expressed on Th2 cells and has been identified as a stimulatory molecule for T cell activation (Umetsu et al., Nat. Immunol. 6(5):447-54, 2005). In one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-TIM1 antibody. Representative examples of TIM1 antibodies include, for example, rabbit anti-human TIM1 antibody ab47635 (ABCAM, Cambridge, MA).

[0196] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to SLAM (CD150, SLAMF1). SLAM (CD150) is a self-ligand and cell surface receptor that acts as a co-stimulatory molecule and is also a microbial sensor that controls macrophage killing of Gram-negative bacteria. In particular, SLAM regulates the activity of the NADPH oxidase NOX2 complex and the maturation of phagolysosomes after entry into the phagosome (after interaction with bacterial outer membrane proteins) (Berger et al., Nature Immunology 11, 920-927, 2010). Slamf1 is expressed on the surface of activated and memory T cells, as well as on activated B cells, dendritic cells, macrophages, and platelets (Calpe et al., Adv. Immunol. 2008; 97: 177). In one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-SLAM1 antibody or an antigen-binding fragment thereof. Representative examples of SLAM1 antibodies include, for example, rabbit anti-human SLAM1 antibody 600-401-EN3 (Rockland Antibodies, Limerick, PA).

[0197] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to 2B4 (CD244, SLAMF4). CD244 is a cell surface receptor expressed on natural killer cells (NK cells) (and some T cells) that mediates non-major histocompatibility complex (MHC) restricted killing. It is believed that the interaction between NK cells and target cells through this receptor mediates NK cell cytolytic activity. CD244 is a co-inhibitory SLAM family member that, in the presence of immunomodulatory selective CD28 blockade, impairs primary antigen-specific CD8(+) T cell responses. Recent studies have revealed that 2B4 specifically upregulates antigen-specific CD8(+) T cells in animals in which CD28 signaling is blocked (Liu et al., J Exp Med. 2014 Feb 10; 211(2): 297-311). In one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD244 antibody or an antigen-binding fragment thereof. Representative examples of CD244 antibodies include, for example, the anti-2B4 antibody C1.7 or PE-conjugated anti-2B4 (C1.7), which has been characterized in Sandusky et al. (Eur J Immunol. 2006 Dec;36(12):3268-76).

[0198] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to Ly108 (NTBA, CD352, SLAMF6). SLAMF6 is a type I transmembrane protein belonging to the CD2 subfamily of the immunoglobulin superfamily, which is expressed on natural killer (NK), T and B lymphocytes. Compared with the standard CD28 pathway, T lymphocytes mediate a more effective effect on IL-17A expression through costimulation of the SLAMF3 / SLAMF6 pathway. SLAMF3 / SLAMF6 signaling mediates increased nuclear abundance and recruits RORγt to the nearby IL17A promoter, resulting in increased transactivation and gene expression (Chatterjee et al., J Biol Chem., 287(45):38168-38177, 2012). In one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD244 antibody or an antigen-binding fragment thereof. Representative examples of CD244 antibodies include, for example, the anti-NTB-A antibodies characterized in Flaig et al. (J. Immunol. 2004. 172: 6524-6527) and Stark et al. (J. Immunol. Methods 2005. 296: 149-158).

[0199] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to CD84 (SLAMF5). CD84 is a member of the CD2 subgroup of the immunoglobulin receptor superfamily. Members of this family are involved in the activation of T cells and NK cells. CD84 enhances the proliferative response of activated T cells, and homophilic interactions enhance interferon gamma secretion in lymphocytes. CD84 can also serve as a marker for hematopoietic progenitor cells. See the disclosures in references with PUBMED ID Nos. 11564780, 12115647, 12928397, 12962726, and 16037392, which indicate that it is required for prolonged T cell:B cell contact, optimal Th function, and germinal center formation. In one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD84 antibody or an antigen-binding fragment thereof. Representative examples of CD84 antibodies include, for example, PE anti-human CD84 antibody CD84.1.21, which can enhance CD3-induced IFN-γ production and partially block the binding of CD84-Ig to lymphocytes (BioLegend, San Diego, CA; Catalog No. 326008).

[0200] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to Ly9 (CD229, SLAMF3). CD229 participates in the adhesion reaction between T lymphocytes and accessory cells through homophilic interactions. It also promotes T cell differentiation into the helper T cell Th17 phenotype, resulting in increased IL-17 secretion; SH2D1A is required for co-stimulatory activity (Chatterjee et al., J Biol Chem., 287(45):38168-38177, 2012). In particular, simultaneous attachment of CD229 and TCR to immobilized CD229-His protein and anti-CD3 antibody significantly enhanced cell proliferation and IFN-γ secretion in mouse CD3+ splenocytes in a dose-dependent manner (Wang et al., The Journal of Immunology, 188(Suppl 1)176.7, May 2012). Therefore, in one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD229 antibody or an antigen-binding fragment thereof. Representative examples of CD229 antibodies include, for example, PE anti-human CD229 antibody HLy-9.1.25 (BIOLEGEND, San Diego, CA; Catalog No. 326108) or mouse anti-human CD229 antibody (R&D Systems Catalog No. AF1898).

[0201] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CD279 (PD-1). PD-1 functions as an immune checkpoint and plays an important role in downregulating the immune system by preventing the activation of T cells, which subsequently reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved through a dual mechanism of promoting apoptosis (programmed cell death) of antigen-specific T cells in the lymph nodes while reducing apoptosis in regulatory T cells (suppressor T cells). Representative examples of CD229 antibodies include, for example, nivolumab, pembrolizumab, pidilizumab (CT-011, Cure Tech), BMS936559, and atezolizumab.

[0202] In another embodiment, the present invention relates to an MSR-SLB scaffold containing an antibody or antigen-binding fragment thereof that specifically binds to CRACC (CD319, BLAME). CD319 mediates NK cell activation through an ERK-mediated pathway regulated by extracellular signals that is independent of SH2D1A (Bouchon et al., J Immunol. 2001 Nov 15; 167(10): 5517-21). CD319 also positively regulates NK cell function and may contribute to NK cell activation. Therefore, in one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD319 antibody or an antigen-binding fragment thereof. Representative examples of CD319 antibodies include, for example, elotuzumab or an antigen-binding fragment thereof.

[0203] In certain embodiments, the present invention provides an MSR-SLB scaffold comprising a binding pair comprising at least one T cell activating molecule and at least one T cell co-stimulatory molecule. Representative examples of such pairs include, but are not limited to, for example, antibodies that bind CD3 / CD28, CD3 / ICOS, CD3 / CD27, and CD3 / CD137, or a combination thereof. In this regard, depending on the desired modulation of the activity of the co-stimulatory molecule, it may be desirable to use an agonistic antibody for the first component (CD3) and an agonistic or antagonistic antibody for the second component.

[0204] In certain embodiments, the present invention provides an MSR-SLB scaffold containing a binding pair containing at least one T cell activating molecule (which is an antibody that binds to CD3) and at least one T cell co-stimulatory molecule (which is an antibody that binds to CD28), optionally together with a second co-stimulatory molecule (which is an antibody that binds to an antigen selected from ICOS, CD27, and CD137). In one embodiment, the MSR-SLB scaffold contains a combination of functional molecules selected from the following combinations: (a) antibodies that bind to CD3, CD28, and ICOS, (b) antibodies that bind to CD3, CD28, and CD27, (c) antibodies that bind to CD3, CD28, and CD137, (d) antibodies that bind to CD3, CD28, ICOS, and CD27. In this regard, experimental data indicate that stimulation of these second T cell co-stimulators can stimulate the differentiation of certain types of T cells when administered together with suitable activating stimuli (such as CD3+CD28). For example, ICOS stimulation, when combined with CD3+CD28+ stimulation, promotes the differentiation of Th effector cells, while it supports the differentiation of regulatory T cells when costimulatory signals are insufficient. See, Mesturini et al., Eur J Immunol., 36(10):2601-12, 2006. Similarly, anti-CD27 antibodies can be used to fine-tune this system. In this regard, the anti-CD27 antibody 1F5 (when used with anti-CD3 antibodies) did not potentially trigger dangerous polyclonal T cell activation - a phenomenon observed using costimulatory CD28-specific superagonist antibodies. See, Thomas et al., Oncoimmunology, 3:e27255, 2014.

[0205] In one embodiment, the binding pair comprises a monospecific antibody, wherein a first antibody binds to a first member of the pair (e.g., CD3) and a second antibody binds to a second member of the pair (e.g., CD28). In another embodiment, the pair comprises a bispecific antibody, wherein a single antibody binds to a single pair member, e.g., a bispecific antibody that binds to CD3 and CD28. In this regard, bispecific antibodies are preferred due to their ability to provide enhanced T cell activation. See, Willems et al., Cancer Immunol Immunother. 2005 Nov; 54(11): 1059-71.

[0206] Alternatively, the binding pair comprises monospecific antibodies, wherein the first antibody binds CD3 and the second antibody binds ICOS. In the case of antibodies that bind ICOS, an antagonistic antibody that neutralizes ICOS may be preferred, as the molecule has been implicated in the etiology of graft-versus-host disease (see, Sato et al., Transplantation, 96(1):34-41, 2013). Bispecific antibodies comprising an agonistic CD3-binding antibody fragment and an antagonistic ICOS-binding antibody fragment may be used.

[0207] Alternatively, the binding pair comprises a monospecific antibody, wherein the first antibody binds to CD3 and the second antibody binds to CD27. In this embodiment, both antibodies are preferably stimulating or exciting antibodies. It has been reported that CD27 co-stimulation enhances the survival and anti-tumor activity of redirected human T cells in vivo (Song et al., Blood, 119(3):696-706, 2012). Bispecific antibodies containing an exciting CD3 binding antibody fragment and an exciting CD27 binding antibody fragment can also be used.

[0208] Alternatively, the binding pair comprises monospecific antibodies, wherein the first antibody binds CD3 and the second antibody binds CD137. In this embodiment, both antibodies are preferably stimulatory or agonist antibodies. CD137 co-stimulation has been reported to increase the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T cell therapy (Chacon et al., PLoS One. 2013; 8(4): e60031, 2013). Bispecific antibodies containing an agonist CD3-binding antibody fragment and an agonist CD27-binding antibody fragment can also be used.

[0209] T-cell homeostatic agents

[0210] In one embodiment, the MSR-SLB scaffold and / or the antigen presenting cell mimicking scaffold contains a stabilizer selected from IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 and transforming growth factor β, or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof. In some embodiments, the MSR-SLB scaffold and / or the antigen presenting cell mimicking scaffold contains a plurality of stabilizers selected from IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 and transforming growth factor β, or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof. Functional fragments of these stabilizers can also be used, characterized by their ability to regulate target cell activity. Representative types of stabilizers are provided in Table 1, including the NCBI accession numbers of their human and / or mouse homologs.

[0211] Table 1. Types of T cell homeostatic agents that can be used in scaffolds

[0212]

[0213]

[0214] Fragments and variants of the above-mentioned T cell homeostatic agents are known in the art. For example, the UNIPROT database entry for each of the above-mentioned homeostatic agents lists "natural variants," including the structural relationship between the variant and the wild-type biomarker. Purely as a representative example, the human IL-1β protein (UNIPROT: P01584) includes a natural variant (VAR_073951) with an E→N amino acid substitution at amino acid residue 141 of the putative human IL-1β protein sequence. If known, the fragments are similarly listed under this section.

[0215] Preferably, the T cell homeostatic agent is interleukin-2 (IL-2) or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof with one or more T cell homeostatic agents listed in Table 1. Examples of IL-2 agonists include, for example, BAY 50-4798 (Margolin et al., Clin Cancer Res. 2007 Jun 1; 13(11): 3312-9). Examples of IL-2 mimetics include, for example, peptide 1-30 (P1-30), which acts synergistically with IL-2 (Eckenberg et al., J Immunol 2000; 165: 4312-4318). Examples of IL-2 fragments include, for example, a ballast portion containing the first 100 amino acids of IL-2 (see, U.S. Patent No. 5,496,924). Examples of IL-2 variants include, for example, natural variants VAR_003967 and natural variants VAR_003968. Also included are fusion proteins containing IL-2, for example, F16-IL2, which is an scFv against the extra domain A1 of tenascin-C, fused to a recombinant form of human IL-2 via a short 5-amino acid linker. The monoclonal antibody portion of the F16-IL2 fusion protein binds to tumor cells expressing the tumor-associated antigen (TAA) tenascin-C. Subsequently, the IL-2 portion of the fusion protein stimulates natural killer (NK) cells, macrophages, and neutrophils, and induces a T cell anti-tumor cell immune response. Other IL-2 mimetics that can be used according to the present invention include, for example, IL-2 superkine peptides (Levin et al., Nature 484, 529-533, 2012) and IL-2 partial agonist peptides (Zurawski et al., EMBO Journal, 9(12):3899-3905, 1990 and U.S. Patent No. 6,955,807), or combinations thereof.

[0216] Embodiments of the present invention further include MSR-SLB scaffolds, including APC-MS scaffolds made from such scaffolds, which further include a variety of the above-mentioned T cell homeostatic agents. Therefore, in one embodiment, the present invention provides an MSR-SLB scaffold containing a first T cell homeostatic agent that is IL-2 and a second T cell homeostatic agent that is IL-7, IL-21, IL-15 or an IL-15 superagonist. In this regard, an IL-15 superagonist (IL-15SA) is a combination of IL-15 and a soluble IL-15 receptor-α that has a biological activity higher than that of IL-15 alone. Due to its ability to selectively amplify NK and memory CD8+T (mCD8+T) lymphocytes, IL-15SA is considered to be an attractive anti-tumor and antiviral agent. See, Guo et al., J Immunol. 2015 Sep 1; 195(5): 2353-64.

[0217] Embodiments of the present invention further relate to a scaffold comprising a plurality of T cell stimulatory molecules, T cell costimulatory molecules and T cell homeostatic agents. Typical scaffolds can include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or more of the above-mentioned T cell stimulatory molecules, T cell costimulatory molecules and T cell homeostatic agents.

[0218] In the scaffolds of the present invention, any functional molecule, such as an antigen, antibody, protein, enzyme, including fragments thereof, can be directly or indirectly immobilized on the MSR basal layer and / or SLB using conventional techniques. In certain embodiments, the functional molecule can be provided in an organelle (e.g., Golgi membrane or plasma membrane), cell, cell cluster, tissue, microorganism, animal, plant, or extract thereof, and then immobilized on the MSR layer or SLB layer. Functional molecules can also be synthesized by genetic engineering or chemical reaction at the desired location (e.g., the outer surface of the SLB layer).

[0219] The scaffold described herein includes and releases signaling molecules, for example, T cell homeostatic agents, to induce functional T cell responses. In one embodiment, the released T cell homeostatic agent is a polypeptide isolated from an endogenous source or synthesized in vivo or in vitro. For example, endogenous IL-2 polypeptides can be isolated from healthy human tissue. Alternatively, the synthetic functional molecules can be synthesized by transfecting or transforming the template DNA into a host organism or cell (for example, a cultured human cell line or a mammal (for example, a humanized mouse or rabbit). Alternatively, the synthetic functional molecules (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), incorporated herein by reference) in the form of protein can be synthesized in vitro by polymerase chain reaction (PCR) or other methods known in the art.

[0220] Functional molecules can be modified to improve protein stability in vivo. Alternatively, functional molecules can be engineered to be more or less immunogenic. For example, where the structures of various functional molecules are known, the sequence can be modified at one or more amino acid residues (e.g., glycosylation sites) to produce immunogenic variants.

[0221] In one embodiment, the functional molecule is recombinant. Alternatively, the functional molecule is a humanized derivative of a mammalian counterpart. Exemplary mammalian species from which the functional molecule is derived include, but are not limited to, mice, rats, hamsters, guinea pigs, ferrets, cats, dogs, monkeys, or primates. In a preferred embodiment, the functional molecule is a human or humanized form of the above-mentioned functional molecules.

[0222] Each of the above-mentioned functional molecules, for example, T cell stimulatory molecules, T cell costimulatory molecules and T cell homeostatic agents, can be adsorbed or integrated into the MSR basal layer or the SLB basal layer independently of each other. Therefore, in one embodiment, an APC-MS is provided, wherein the T cell stimulatory molecules are adsorbed or integrated into the MSR basal layer. Preferably, an APC-MS is provided, wherein the T cell stimulatory molecules are adsorbed or integrated into the SLB layer. In another embodiment, an APC-MS is provided, wherein the T cell stimulatory molecules are adsorbed or integrated into the MSR basal layer and the SLB layer. In another embodiment, an APC-MS is provided, wherein the T cell costimulatory molecules are adsorbed or integrated into the MSR basal layer. Preferably, an APC-MS is provided, wherein the T cell costimulatory molecules are adsorbed or integrated into the SLB layer. In another embodiment, an APC-MS is provided, wherein the T cell costimulatory molecules are adsorbed or integrated into the MSR basal layer and the SLB layer. In another embodiment, an APC-MS is provided, wherein the T cell homeostatic agent is adsorbed or integrated into the MSR basal layer. In another embodiment, an APC-MS is provided, wherein a T cell homeostatic agent is adsorbed or integrated into the SLB layer. In yet another embodiment, an APC-MS is provided, wherein a T cell homeostatic agent is adsorbed or integrated into the MSR basal layer and the SLB layer.

[0223] Typically, the functional molecules and the MSR substrate layer and / or SLB layer can be linked together using reactive groups, which are typically converted into new functional groups or non-reactive species through the linking process. The reactive functional groups can be located in any of the above components. Reactive groups and reaction classes useful in practicing the present invention are generally those well known in the field of bioconjugate chemistry. Currently advantageous reaction classes that can be used in conjunction with reactive chelates are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic displacements (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic displacements (e.g., enamine reactions), and carbon-carbon and carbon-heteroatom multiple bond additions (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed in, for example, March, Advanced Organic Chemistry, 3rd ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; vol. 198, American Chemical Society, Washington, DC, 1982.

[0224] Useful reactive pendant functional groups include, for example:

[0225] (a) carboxyl and its various derivatives, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides (e.g., I, Br, Cl), acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters;

[0226] (b) Hydroxyl groups, which can be converted into, for example, esters, ethers, aldehydes, and the like.

[0227] (c) haloalkyl groups, where the halide can subsequently be displaced by a nucleophilic group such as, for example, an amine, a carboxylate cation, a thiol anion, a carbanion or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the functional group of the halogen atom;

[0228] (d) a dienophile group capable of participating in a Diels-Alder reaction, such as, for example, a maleimide group;

[0229] (e) an aldehyde or ketone group, so that subsequent derivatization is possible via the formation of carbonyl derivatives (e.g., imines, hydrazones, semicarbazones or oximes) or via mechanisms such as Grignard addition or alkyllithium addition;

[0230] (f) a sulfonyl halide group for subsequent reaction with an amine, e.g., to form a sulfonamide;

[0231] (g) thiol groups, which can, for example, be converted into disulfides or reacted with acyl halides;

[0232] (h) amine or thiol groups, which may, for example, be acylated, alkylated or oxidized;

[0233] (i) Olefins, which can undergo, for example, cycloadditions, acylations, Michael additions, etc.;

[0234] (j) epoxides, which can react with, for example, amines or hydroxy compounds; and

[0235] (k) Phosphoramidites and other standard functional groups useful in nucleic acid synthesis.

[0236] Reactive functional groups can be selected so that they do not participate in or interfere with the reactions required to assemble the reactive chelate. Alternatively, reactive functional groups can be protected to prevent them from participating in the reaction by the presence of a protecting group. Those skilled in the art understand how to protect specific functional groups so that they do not interfere with a selected set of reaction conditions. See, for example, Greene et al., Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0237] In one embodiment, the functional molecules are loaded / adsorbed onto the MSR substrate layer or SLB or both the MSR layer and SLB via affinity pairing or chemical coupling.

[0238] As used herein, the term "affinity pair" includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridization sequence, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin, biotin / biotin binder, Ni2+ or Cu2+ / HisTag (6×histidine), and virus-receptor interactions. Various other specific binding pairs are contemplated for use in practicing the methods of the present invention.

[0239] As used herein, "biotin binders" include avidin, streptavidin and other avidin analogs, such as streptavidin or avidin conjugates, highly purified and fractionated materials of avidin or streptavidin, and non- or partial amino acid variants, recombinant or chemically synthesized avidin analogs with amino acid or chemical substitutions (which are still suitable for biotin binding). Preferably, each biotin binder molecule binds at least two biotin moieties, and more preferably binds at least four biotin moieties. As used herein, "biotin" includes biotin and other biotin analogs other than biocytin, such as biotinamide hexanoate N-hydroxysuccinimide ester, biotin 4-amidobenzoic acid, biotinamide hexanoyl hydrazide and other biotin derivatives and conjugates. Other derivatives include biotin-dextran, biotin-disulfide-N-hydroxysuccinimide ester, biotin-6-amidoquinoline, biotin hydrazine, d-biotin-N-hydroxysuccinimide ester, biotin maleimide, d-biotin p-nitrophenyl ester, biotinylated nucleotides, and biotinylated amino acids such as Nε-biotinyl-l-lysine.

[0240] Ligands that can be functionalized by affinity pairing include, but are not limited to, receptors prepared or isolated from natural or synthetic sources, monoclonal or polyclonal antibodies, viruses, chemotherapeutic agents, receptor agonists and antagonists, antibody fragments, lectins, albumin, peptides, proteins, hormones, amino sugars, lipids, fatty acids, nucleic acids, and cells. In short, any site-specific ligand for any molecular epitope or receptor to be detected by the practice of the present invention can be utilized. Preferably, the ligand is a membrane-anchored protein. The ligand can also be a derivative of a membrane-anchored protein, such as a soluble extracellular domain. The ligand can be a receptor involved in receptor-receptor cell interactions, such as a TCR that binds to an MHC receptor.

[0241] The ligands of the present invention can be expressed and purified by any method known in the art. In certain embodiments, the protein can be expressed by a baculovirus-based insect expression system or a mammalian expression system. The fifteen-residue AVITAG TM The peptide was added to the C-terminus of all molecules. TM (Avidity, CO) can be specifically biotinylated on lysine residues by the enzyme BirA (Avidity, CO). Proteins can also be designed to be secreted into the supernatant of cell cultures.

[0242] As described above, the functional molecule can be any protein or peptide. Preferably, the protein is involved in a ligand-receptor interaction. For example, an important event in T cell activation is the result of membrane-membrane contact between T cells and APCs, wherein various ligand-receptor interactions occur between two opposing membranes, including MHC-peptide and TCR, LFA-1 and ICAM-1, CD2 and CD48, and B7 or CTLA-4 and CD28. Understanding the valence requirements of these interactions will facilitate the design of therapeutic agents that enhance or suppress the immune response to certain antigens. The present invention can also be used as a tool to study subtle differences in T cell intracellular signaling pathways induced by agonist or antagonist antigens. The scaffold provides a clear physiological environment to test the subtle differences without the need to use natural antigen-presenting cells that often complicate biochemical analysis.

[0243] Although streptavidin-biotin interactions are illustrated throughout the specification and examples, the specific binding pair members described above can be used in place of streptavidin and biotin in the methods of the present invention. In addition, more than one specific binding pair can be used, particularly when more than one ligand is attached to the membrane surface. In this regard, traditional pep-MHC-streptavidin tetramer technology can also be used to screen T cells with specific pep-MHC specificity. However, T cells with the same specificity can be activated or not activated by stimulation with the same antigen. In order to study immune responses (e.g., responses to vaccination [viral or cancer vaccines], immune tolerance, autoimmunity), it is important to distinguish T cells based on their reactivity to antigens. Calcium flux observed by microscopy is used as an indicator of T cell activation, and the present invention also provides a screening assay for quantifying primary T cells reactive to specific antigens. Alternatively, biotinylated pep-MHC and costimulatory molecules can be coupled to a chip covered with streptavidin, and the chip is paired with the scaffold of the present invention.

[0244] In another embodiment, the functional molecule is chemically coupled to the MSR substrate and / or SLB layer. In certain embodiments, the chemical coupling includes click chemistry reagents, for example, azide-alkyne chemistry (AAC) reaction, dibenzo-cyclone alkyne connection (DCL) or tetrazine-olefin connection (TAL). For example, in the case of AAC, the MSR or SLB contains a variety of single click chemistry functionalities, and often contains two, three or more such functionalities. Preferably, one or two such functionalities per molecule. In one embodiment, a clickable reagent, such as 3-azidopropylamine or 10-undecynoic acid, can be amide-bonded to the carboxyl or amino terminus of a peptide or protein, respectively, by a click reaction using the corresponding alkyne or azide compound and a suitable catalyst to form a 1,2,3-triazole ring linking group. See, for example, U.S. Publication No. 2007 / 0060658. To further expand the library of bioorthogonal copper-free click reagents, compounds containing aza-dibenzocyclooctyne (ADIBO) for azide-coupling reactions can be used for site-specific covalent anchoring of protein functional molecules (e.g., antibodies), interleukins, and cytokines. The same metal-free click reaction is used for PEGylation of unfunctionalized areas of the surface. Such treatment allows for a significant reduction or complete elimination of nonspecific binding. The copper-free click fixation method can be used to prepare various types of arrays, as well as the derivatization of microbeads and nanoparticles. See, for example, U.S. Patent No. 8,912,322. In some embodiments, the functional molecule is coupled to the MSR basal layer and / or SLB layer using a click reagent selected from azide, dibenzocyclooctyne (DBCO), trans-cyclooctene, tetrazine, and norbornene and variants thereof. In some embodiments, the functional molecule comprises an azide, and the lipid of the lipid bilayer of the MSR-SLB comprises DBCO.

[0245] The term "click chemistry" refers to a chemical concept introduced by K. Barry Sharpless of The Scripps Research Institute, describing a chemical action tailored for the rapid and reliable production of covalent bonds by linking together small units comprising reactive groups. Click chemistry does not refer to a specific reaction, but rather to the concept of reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular, wide-ranging, give high chemical yields, produce harmless by-products, are stereospecific, exhibit a large thermodynamic driving force of >84 kJ / mol to favor reactions with a single reaction product, and / or can be performed under physiological conditions. The significant exothermic reaction causes the reactants to be "spring loaded". In some embodiments, click chemistry reactions exhibit high atom economy, can be performed under simple reaction conditions, use readily available starting materials and reagents, use non-toxic solvents or use benign or easily removable solvents (preferably water), and / or provide simple product separation by non-chromatographic methods (crystallization or distillation).

[0246] As used herein, the term "click chemistry handle" refers to a reactant, or reactive group, that can participate in a click chemistry reaction. For example, a strained alkyne, such as cyclooctyne, is a click chemistry handle because it can participate in strain-promoted cycloadditions. Typically, a click chemistry reaction requires at least two molecules comprising click chemistry handles that can react. Such mutually reactive click chemistry handle pairs are sometimes referred to herein as partner click chemistry handles. For example, azide is a partner click chemistry handle for cyclooctyne or any other alkyne. Exemplary click chemistry handles suitable for use according to aspects of the present invention are described herein, for example, US2014 / 0249296. Other suitable click chemistry handles are known to those skilled in the art.

[0247] In one embodiment, the present invention provides an APC-MS comprising a plurality of T cell activating molecules and T cell costimulatory molecules, optionally in combination with a T cell homeostatic agent (which is adsorbed onto a support via a metal chelating lipid end group). See, Maloney et al., Chem Biol., 3(3): 185-92, 1996. Several methods using chelated metal ions have been reported that allow histidine-tagged proteins to be fixed on several types of interfaces, such as lipid interfaces and lipid monolayers with metal chelating lipids, gold surfaces with self-assembled monolayers formed with metal chelating alkanols, and oxide surfaces with metal chelating silanes. For example, Peterson et al. (US 5,674,677) describe a method for connecting two amino acid sequences and loading the chelator with metal ions by coupling an organic chelating agent to a protein (e.g., an enzyme). This complex is then mixed with any protein containing a histidine tag to couple the complex to the histidine-tagged protein. See also US 6,087,452, which is incorporated herein by reference in its entirety.

[0248] The functional molecules of the present invention are preferably proteins. The terms "protein", "peptide" and "polypeptide" are used interchangeably and refer to polymers of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides or polypeptides of any size, structure or function. Typically, a protein, peptide or polypeptide is at least three amino acids long. A protein, peptide or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide or polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, linkers, for conjugation, functionalization or other modifications, etc. A protein, peptide or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide or polypeptide may be merely a fragment of a naturally occurring protein or peptide. A protein, peptide or polypeptide may be naturally occurring, recombinant or synthetic, or any combination thereof.

[0249] The term "conjugated" or "conjugation" refers to the binding of two molecules (e.g., two proteins) to each other in a manner that is connected by direct or indirect covalent or non-covalent interactions. In the case of conjugation by click chemistry, the conjugation is a covalent bond formed by the reaction of a click chemistry handle. In certain embodiments, the binding is covalent, and the entities are referred to as being "conjugated" to each other. In some embodiments, the protein is post-translationally conjugated to another molecule, e.g., a second protein, by forming a covalent bond between the protein after translation of the protein, and in some embodiments, after separation of the protein from other molecules. In some embodiments, post-translational conjugation of a protein and a second molecule (e.g., a second protein) is achieved by placing a click chemistry handle on the protein and placing a second click chemistry handle that can react with the first click chemistry handle on the second molecule, and performing a click chemistry reaction in which the click chemistry handle reacts and forms a covalent bond between the protein and the second molecule, thereby producing a chimeric protein. In some embodiments, the two proteins are conjugated at their respective C-termini to produce a CC-conjugated chimeric protein. In some embodiments, the two proteins are conjugated at their respective N-termini to produce a NN-conjugated chimeric protein.

[0250] In certain embodiments, a variety of detectable labels can be used to analyze and / or study the conjugation process. As used herein, "detectable label" refers to a portion having at least one element, isotope, or functional group incorporated into the portion that enables detection of a molecule to which the label is attached, such as a protein or polypeptide, or other entity. The label can be directly connected (e.g., by a bond) or can be connected by tethering (e.g., an optionally substituted alkylene; an optionally substituted alkene; an optionally substituted alkynylene; an optionally substituted heteroalkylene; an optionally substituted heteroalkene; an optionally substituted heteroalkynylene; an optionally substituted arylene; an optionally substituted heteroarylene; or an optionally substituted acylene, or any combination thereof, which can constitute a tethering). It will be recognized that the label can be connected or incorporated into a molecule, such as a protein, polypeptide, or other entity, at any position.

[0251] In general, labels may fall into any one (or more) of the following five categories: a) Labels containing an isotopic moiety, which may be a radioactive or heavy isotope, including, but not limited to, 2 H. 3 H. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 67 Ga, 99 mTc (Tc-99m), 111 In,125 I, 131 I, 153 Gd, 169 Yb sum 186Re; b) a label containing an immune portion, which can be an antibody or an antigen, which can be bound to an enzyme (e.g., such as horseradish peroxidase); c) a label that is a colored, luminescent, phosphorescent, or fluorescent portion (e.g., such as the fluorescent label fluorescein isothiocyanate (FITC) or carboxyfluorescein); d) a label with one or more photoaffinity portions; and e) a label that is a ligand with one or more known binding partners (e.g., biotin-streptavidin, FK506-FKBP). In certain embodiments, the label comprises a radioactive isotope, preferably an isotope that emits a detectable particle. In certain embodiments, the label comprises a fluorescent portion. In certain embodiments, the label is the fluorescent label fluorescein isothiocyanate (FITC). In certain embodiments, the label comprises a ligand portion with one or more known binding partners. In certain embodiments, the label comprises biotin. In some embodiments, the label is a fluorescent polypeptide (e.g., GFP or a derivative thereof, such as enhanced GFP (EGFP) or a luciferase (e.g., firefly, Renilla, or Gaussia luciferase). It will be appreciated that in certain embodiments, the label can be reacted with a suitable substrate (e.g., luciferin) to produce a detectable signal. Non-limiting examples of fluorescent proteins include GFP and its derivatives, proteins that include chromophores that emit light of different colors, such as red, yellow, and cyan fluorescent proteins, etc. Exemplary fluorescent proteins include, for example, Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1 , mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. For a discussion of GFP and many other fluorescent or luminescent proteins, see, e.g., Chalfie, M. and Kain, SR (eds.) Green fluorescent protein: properties, applications, and protocols (Methods of Biochemical Analysis, v. 47). Wiley-Interscience, Hoboken, NJ, 2006, and / or Chudakov et al., Physiol Rev. 90(3): 1103-63, 2010.In some embodiments, a label includes a dark quencher, eg, a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.

[0252] In another embodiment, functional molecules can be loaded onto mesoporous silica and / or lipid bilayers using covalent or non-covalent loading techniques known in the art. In one embodiment, the functional molecules are non-covalently loaded. For example, Lei et al. (U.S. Publication No. 2011-0256184) describe mesoporous silicates that provide enhanced spontaneous loading of antibodies (such as IgG) within natural or functionalized structures by non-covalent bonding. Therefore, such silicates can be used to prepare the support of the present invention.

[0253] In another embodiment, the functional molecule can be chemically coupled to the MSR. In such an embodiment, coupling can be performed by utilizing one or more of the following molecules and the reactive groups contained therein: cysteine (thiol group), serine or threonine (hydroxyl group), lysine (amino group), aspartic acid or glutamic acid (carboxyl group). Alternatively, the functional molecule can be conjugated to the MSR by utilizing a polyhistidine-tag (His-tag), a peptide containing a polyhistidine tag, or an antibody containing a polyhistidine-tag. In this context, the polyhistidine-tag consists of at least four, five, six, or seven histidine (His) residues.

[0254] In one embodiment, an anchor is used to connect the functional molecule to the pore wall. However, the anchor is not an essential component. In certain embodiments, each pore of the mesoporous silica accommodates at least one functional molecule. Therefore, the pore must have a size suitable for fixing biological substances. The pore size depends on the size of the functional molecule to be fixed. When the functional molecule is fixed in the pore, the functional molecule can be adsorbed on the inner surface of the pore by electrostatic bonding. The functional molecule can also be retained in the pore by non-covalent bonding (such as van der Waals forces, hydrogen bonding or ionic bonding).

[0255] In the above-described embodiments where the MSR includes an anchoring moiety, the anchor can function to mitigate large structural changes in the functional molecule, thereby stably retaining it. Preferably, the anchor is composed of substantially the same components as the mesoporous material. The anchor can include one or more functional groups to allow for the binding of the desired functional molecule: hydroxyl, amide, amino, pyridyl, urea, carbamate, carboxyl, phenolic, azo, hydroxyl, maleimide, silane derivatives, or aminoalkylene groups.

[0256] Embodiments of the present invention further relate to the MSR-SLB scaffolds of the present invention, including scaffolds comprising such scaffolds, said scaffolds comprising a plurality of the above-described functional molecules adsorbed in a lipid matrix.

[0257] In one embodiment, the functional molecule is adsorbed into the supporting lipid bilayer by physical insertion. The techniques for inserting proteins into the bilayer of amphiphilic molecules are known in the art. In one embodiment, the protein in the environment of the bilayer, for example, in a hydrophobic medium and / or a hydrophilic body and / or a hydrated support, can be spontaneously inserted into the bilayer. Alternatively, the protein can be driven into the bilayer by applying a voltage and / or fusing a protein-loaded vesicle with the bilayer. The vesicle can be contained in a hydrophilic body or introduced into a hydrophilic body. In one case, the protein can be introduced into the membrane by using the probe method disclosed in PCT Publication No. WO2009 / 024775. The inserted protein can be a known membrane-associated protein, for example, one or more of the above-mentioned T cell activation molecules and / or T cell co-stimulatory molecules.

[0258] In another embodiment, the functional molecule can be an antigen for use in T cell expansion. Representative examples of antigens useful in T cell expansion include full-length CD19 or a fragment thereof or a variant thereof. CD19 is a prototype antigen used in the expansion of chimeric antigen receptor (CAR) T cells. See, Turtle et al., Blood, 126: 184, 2015; Turtle et al., J Clin Invest., 126, 2123-38, 2016. In another embodiment, the antigen is full-length CD22 or a fragment thereof or a variant thereof, which is also useful in the expansion of CAR T cells. See, Haso et al., Blood, 121 (7): 1165–1174, 2013; Qin et al., Blood, 122: 1431, 2013.

[0259] In an alternative embodiment, the functional molecule can be a membrane-associated protein that is directly or indirectly anchored to the bilayer. Other functional molecules, such as selective or non-selective membrane transporters, ion channels, pore-forming proteins, or membrane-resident receptors, can also be inserted into SLBs using this method.

[0260] In another embodiment, the functional molecule can be conjugated to a membrane-associated protein that can bind to and / or insert into SLBs, e.g., gramicidin; an α-helical bundle, e.g., a bacteriophage opsonin or a K+ channel; and a β-barrel structure, e.g., α-hemolysin, leukocidin, or an E. coliporin; or a combination thereof.

[0261] In certain embodiments, the manufactured SLB (containing one or more functional molecules) can be stabilized by compounds such as ionic or nonionic surfactants. Suitable surfactants include, but are not limited to, synthetic phospholipids, their hydrogenated derivatives, and mixtures thereof, sphingolipids and glycosphingolipids, saturated or unsaturated fatty acids, fatty alcohols, polyoxyethylene-polyoxypropylene copolymers, ethoxylated fatty acids and their esters or ethers, dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, or combinations of two or more of the foregoing. A preferred surfactant according to the present invention is dimyristoylphosphatidylglycerol.

[0262] The manufactured SLBs can optionally be stabilized by at least one co-surfactant selected from the group consisting of butanol, butyric acid, hexanoic acid, sodium cholate, sodium taurocholate and sodium glycocholate, more particularly sodium cholate, or consisting thereof.

[0263] The manufactured SLBs may also include other excipients such as polymers having bioadhesive or adsorption enhancing properties and selected from the group consisting of acrylic polymers ( Polycarbophil, ), medium chain fatty acids and polyethylene glycol or a mixture thereof. The preferred excipient is the acrylic polymer mentioned above.

[0264] SLBs can be modified with reagents for detecting membrane-associated proteins. Preferably, the membrane-associated proteins are ion channel proteins and / or pore-forming proteins. Preferably, the membrane-associated proteins diffuse into and / or bind to the bilayer, causing a detectable change in a bilayer property. The altered property can be physical, optical, electrical, or biochemical.

[0265] In some embodiments, the MSR-SLB scaffold and / or the antigen presenting cell mimicking scaffold comprises a small molecule drug. In some embodiments, the MSR-SLB scaffold and / or the antigen presenting cell mimicking scaffold comprises a thalidomide analog. In some embodiments, the MSR-SLB scaffold and / or the antigen presenting cell mimicking scaffold comprises an IDO / MEK inhibitor. In some embodiments, the MSR-SLB scaffold and / or the antigen presenting cell mimicking scaffold comprises a small molecule drug with an immunomodulatory effect. Small molecules with immunomodulatory effects are known in the art (see, e.g., Murphy et al., Hum. Vaccin. Immunother. 11(10):2463-8 (2015), the entire contents of which are expressly incorporated herein by reference).

[0266] In certain embodiments, MSR-SLB scaffolds containing functional molecules can be used to detect cells that can interact with amphiphilic molecules in the bilayer and / or functional molecules in the bilayer. The interaction can be specific or nonspecific in nature. Alternatively, the cell can interact with the functional molecule or with the lipid bilayer to induce physical, optical, electrical, or biochemical changes. Such interactions can be detected in many different ways, including, but not limited to, by visual changes, by activation of fluorescently labeled lipids or proteins in the SLB, or by changes in the capacitance of the SLB.

[0267] Biosolvable Stent

[0268] Embodiments of the present invention further relate to biodegradable scaffold.In one embodiment, when exposed to a biological environment, the scaffold structure can be substantially degraded.In one embodiment, the biological environment is a tissue culture condition, for example, optionally applicable to a tissue culture medium for cultivating lymphocytes (such as T cells).In another embodiment, the biological environment is a biological fluid, for example, blood, lymph, CSF, ascites, etc. In another embodiment again, the biological environment is the tissue environment at the implantation site (for example, blood vessel, lymphatic system, adipose tissue, etc.).

[0269] In certain embodiments, the biodegradable scaffold is substantially degraded after being exposed to a biological environment in vivo for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60 days, 90 days or more. In certain embodiments, the biodegradable scaffold is substantially degraded after being exposed to a biological environment in vivo for less than 1 week. In certain embodiments, the biodegradable scaffold is substantially degraded after being exposed to a biological environment in vitro for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60 days, 90 days or more. In certain embodiments, the biodegradable scaffold is substantially degraded after being exposed to a biological environment in vitro for less than 1 week. Substantially degraded means that at least 30%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95% or more of the scaffold composition degrades when the scaffold composition is exposed to a biological environment.

[0270] In certain embodiments, biodegradable scaffolds can be advantageously used. For example, by manufacturing the scaffold composition so that it substantially degrades during incubation (e.g., while allowing T cells to expand), it may be possible to utilize the expanded T cells without subjecting them to additional purification and / or formulation steps. Avoiding downstream purification and / or formulation steps will ensure that the T cells are healthy and have the desired functionality for the desired application.

[0271] Therefore, in certain embodiments, the degradation kinetics of the scaffold composition can be advantageously tailored by varying the properties of the mesoporous dioxide rods, such as size, geometry, and porosity. Alternatively, the degradation kinetics of the scaffold composition can be altered by varying the culture conditions (e.g., by adjusting the pH of the culture medium).

[0272] In accordance with the above objectives, embodiments of the present invention relate to MSR-SLB scaffolds comprising a variety of functional molecules, which are optionally biodegradable. In one embodiment, the scaffolds of the present invention can be encapsulated in other biodegradable scaffolds. Reagents and techniques useful in preparing such composite biodegradable scaffold compositions are known in the art. See, Liao et al., J. Biomed. Mater. Res. B. Appl. Biomater., 102(2): 293-302, 2014. In one embodiment, the scaffold is made of a physiologically compatible and optionally biodegradable polymer. Examples of polymers that can be used in the scaffold are known in the art. See, for example, U.S. Publication No. 2011 / 0020216, the entire contents of which are incorporated herein by reference. Representative examples of such polymers include, but are not limited to, poly(lactide), poly(glycolide), poly(lactic acid), poly(glycolic acid), polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone, polyamide esters, polycarbonates, polycyanoacrylates, polyurethanes, polyacrylates, and mixtures or copolymers thereof. Biodegradable scaffolds can include biodegradable materials, for example, collagen, alginate, polysaccharides, polyethylene glycol (PEG), poly (glycolide) (PGA), poly (L-lactide) (PLA) or poly (lactide-co-glycolide) (PLGA) or silk. Methods for making scaffold compositions are known in the art. See, for example, Martinsen et al. (Biotech. & Bioeng., 33 (1989) 79-89), (Matthew et al. (Biomaterials, 16 (1995) 265-274), Atala et al. (J Urology, 152 (1994) 641-643) and Smidsrod (TIBTECH 8 (1990) 71-78), the disclosures of which are incorporated herein by reference.

[0273] Exemplary stents utilize glycolide or alginate of relatively low molecular weight, preferably dissolved after the size of the kidney clearance threshold of people, for example, alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 daltons. Preferably, the molecular weight is 1000 to 60,000 daltons, particularly preferably 1000 to 50,000 daltons. It is also useful to use an alginate material with high guluronic acid (guluronate) content because guluronic acid monomers (as opposed to mannuronic acid units) provide sites for making polymer gelation by ionic crosslinking of divalent cations. For example, U.S. Patent No. 6,642,363, which is incorporated herein by reference, discloses a method for preparing and using a polymer containing polysaccharides (such as alginate).

[0274] The scaffolds of the present invention can be porous so that the scaffold can maintain antigen presentation and attract and manipulate immune cells. In one embodiment, the scaffold comprises a porous matrix wherein the pores have a diameter of 10 nm to 500 μm, particularly 100 nm to 100 μm. In these embodiments, the present invention utilizes scaffolds comprising mesoporous scaffolds. Methods for preparing polymer matrices having desired pore sizes and pore arrangements are described in the art, for example, U.S. Publication No. 2011 / 0020216 and U.S. Patent No. 6,511,650, which are incorporated herein by reference.

[0275] Mesoporous silica rods can be modified into multifunctional delivery platforms for delivering drugs such as chemotherapeutics and DNA / siRNA, antibodies and protein biologics, cells, etc. (Lee et al., Adv. Funct. Mater., 215-222, 2009; Liong et al., ACS Nano, 889-896, 2008; Meng et al., ACS Nano, 4539-4550, 2010; Meng et al., J. Am. Chem. Soc., 12690-12697, 2010; Xia et al., ACS Nano, 3273-3286, 2009; Radu et al., J. Am. Chem. Soc., 13216-13217, 2004; Slowing et al., J. Am. Chem. Soc., 8845-8849, 2007). This delivery platform allows for efficient and protective packaging of hydrophobic and charged anticancer drugs for controlled and on-demand delivery, with the added ability to image the site of delivery (Liong et al., ACS Nano, vol. 2, pp. 889-896, 2008). The key challenge now is to optimize the design features for effective and safe drug delivery in vivo (He et al., Small, vol. 7, pp. 271-280, 2011; Lee et al., Angew. Chem. Int. Ed., vol. 49, pp. 8214-8219, 2010; Liu et al., Biomaterials, vol. 32, pp. 1657-1668, 2011; Al Shamsi et al., Chem. Res. Toxicol., vol. 23, pp. 1796-1805, 2010), which can be evaluated using human xenograft tumors in nude mice (Lu et al., Small, vol. 6, pp. 1794-1805, 2010).

[0276] Embodiments described herein further relate to MSR-SLB scaffolds, including scaffolds containing such scaffolds, wherein the dry weight ratio of mesoporous silica microrods (MSR) to T cell activation / co-stimulatory molecules is from about 1:1 to about 100:1, preferably from about 10:1 to about 50:1, and particularly from about 20:1 to about 50:1. In some embodiments, the dry weight ratio of mesoporous silica microrods (MSR) to T cell activation / co-stimulatory molecules of the MSR-SLB scaffold is from about 10,000:1 to about 1:1. In some embodiments, the dry weight ratio of mesoporous silica microrods (MSR) to T cell activation / co-stimulatory molecules of the MSR-SLB scaffold is from about 5,000:1 to about 1:1, from about 1,000:1 to about 1:1, from about 500:1 to about 1:1, or from about 100:1 to about 1:1. In some embodiments, the dry weight ratio of mesoporous silica microrods (MSR) to T cell activation / co-stimulatory molecules of the MSR-SLB scaffold is about 10,000:1, about 5,000:1, about 2,500:1, about 1,000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 10:1 or about 1:1.

[0277] The embodiments described herein further relate to compositions and devices containing the above-mentioned scaffolds, wherein the scaffolds contain MSR-SLB scaffolds and functional molecules (e.g., T cell activation molecules and T cell co-stimulatory molecules and T cell homeostatic agents), optionally with one or more other reagents (listed below). In one embodiment, the invention provides compositions comprising a scaffold and clustered T cells therein. In one embodiment, the T cells are selected from natural killer (NK) cells, CD3+T cells, CD4+T cells, CD8+T cells and regulatory T cells (Treg), or a combination thereof. In other embodiments, the composition can be a pharmaceutical composition, which can be produced using methods well known in the art. For example, pharmaceutical compositions can be produced by those skilled in the art using generally recognized pharmaceutical chemistry principles. The compositions, scaffolds and devices can provide one or more reagents for selecting, culturing, amplifying, maintaining and / or transplanting target cells. Representative examples of cell selection kits, culture kits, amplification kits, and transplantation kits for T cells, B cells and antigen presenting cells are known in the art. For example, where the target cells of interest are T cells, they can be initially sorted using DYNABEADS, MACS-beads (Miltenyi Biosciences), maintained in STEMXVIVO Human T Cell Basal Medium (R&D Systems), and expanded with OPTIMIZER medium (Thermo Fisher Scientific). Cells can be isolated by centrifugation techniques known to those skilled in the art, including, for example, Gradients can be used to enrich cells in a sample. Cells can also be enriched in a sample based on the expression of certain markers by using positive selection, negative selection, or a combination thereof.

[0278] More embodiments of the present invention relate to T cell manipulation devices. The devices contain a scaffold of the present invention and a plurality of molecules that attract / bind to target T cells. In one embodiment, the present invention relates to a device containing a scaffold stacked to selectively allow T cells to infiltrate mesoporous silica microrods (MSRs). Selective permeability means that due to selective permissivity / permeability, binding specificity, selective elimination (undesirable cells) and / or expansion (desired cells), after a period of incubation time, the scaffold contains at least 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 300%, more than 400%, more than 500%, more than 600%, more than 800%, more than 1000%, or more target T cells than those present in whole blood. In certain embodiments, the incubation period is 1-30 days, preferably 4-15 days, and particularly 7-12 days. In other embodiments, selective permeation involves the retention and / or expansion of T cells compared to other blood cells present in whole blood (e.g., B cells, dendritic cells, macrophages, red blood cells, or platelets).

[0279] In other embodiments, the scaffold of the present invention allows the selective penetration of specific T cell subsets, such as natural killer (NK) cells, CD3+T cells, CD4+T cells, CD8+T cells or regulatory T cells (Treg). In this article, after 4-14 days of incubation, compared with what exists in whole blood, the scaffold contains at least 10% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500% more, 600% more, 800% more, 1000% more, or more target T cells of more quantity. The percentages and ranges of various types of lymphocytes in human whole blood are as follows: NK cells 7% (range: 2-13%); helper T cells 46% (range: 28-59%); cytotoxic T cells 19% (range: 13-32%); γδ T cells 5% (range: 2%-8%); B cells 23% (range: 18-47%) (Berrington et al., Clin Exp Immunol 140(2):289-292, 2005).

[0280] Other reagents

[0281] The scaffolds of the present invention include one or more agents, which can be naturally occurring, synthetically produced, or recombinant compounds, such as peptides, polypeptides, proteins, nucleic acids, small molecules, haptens, carbohydrates, or other agents, including fragments thereof or combinations thereof. In one embodiment, the agent is an antigen. In one embodiment, the antigen is a peptide or protein or an immunologically active fragment thereof. In one embodiment, the antigens described herein are purified. The purified compound contains at least 60% by weight (dry weight) of the target compound. In particular, the antigen is at least 75% pure, preferably at least 90% pure, and more preferably at least 99% pure. Purity is measured by any suitable standard method, for example, by column chromatography, gel electrophoresis, or HPLC analysis. The antigen can be an autoantigen or a non-autoantigen.

[0282] Representative examples of non-self antigens include, for example, antigens derived from pathogens selected from viruses, bacteria, protozoa, parasites, and fungi. The antigens can optionally be loaded onto MHC molecules, such as HLA-A, HLA-B, HLA-C, DP, DQ, and DR, which are then incorporated into the scaffold.

[0283] Alternatively, the scaffold comprises a plurality of autoantigens, which are optionally associated or related to a disease or disorder. Preferably, the autoantigens are particularly associated with a human disease or disorder. In one embodiment, the autoantigens are associated with an autoimmune disorder selected from the group consisting of rheumatoid arthritis, lupus, celiac disease, inflammatory bowel disease or Crohn's disease, Syndrome polymyalgia rheumatica, multiple sclerosis, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, etc. Specific types of antigens associated with type 1 diabetes, multiple sclerosis, Crohn's disease and rheumatoid arthritis, including fragments thereof, have been identified in the literature. For example, the antigen associated with rheumatoid arthritis is a 47 kDa protein (RA-A47). See Hattori et al., J Bone Miner Metab., 18(6): 328-34 (2000). In Crohn's disease, the antigen may be bacterial flagellin. See Lodes et al., J Clin Invest. 113(9): 1296-306 (2004). Similarly, major myelin proteins, such as myelin basic protein (MBP) and proteolipid protein (PLP), are likely to be important in the course of multiple sclerosis (MS). See, deRosbo et al., J Clin Invest. 92(6):2602-260 (1993). In the case of type 1 diabetes, multiple autoantigens may be involved, such as preproinsulin (PPI), islet-specific glucose-6-phosphatase (IGRP), glutamic acid decarboxylase (GAD65), insulinoma antigen-2 (IA-2), chromogranin A, and heat shock protein 60. See, Roep et al., Cold Spring Harb Perspect Med. 2(4), 2012 (PMID: 22474615).

[0284] In another embodiment, the autoantigen is associated with cancer. Representative types of cancer antigens include, for example, MAGE-1, MAGE-2, MAGE-3, CEA, tyrosinase, midkin, BAGE, CASP-8, β-catenin, β-catenin, γ-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13α, IL13α2, AIM-2, AIM-3, NY-ESO-1, C9orf112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L, CYNL2, TPR, SOX2, GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, nestin, OLIG2, ART1, ART4, B-cyclin, Gli1, Cav-1, cathepsin B, CD74, E-cadherin, EphA2 / Eck, Fra-1 / Fosl1, GAGE-1, ganglioside / GD2, GnT-V, β1,6-N, Ki67, Ku70 / 80, PROX1, PSCA, SOX10, SOX11, survivin, UPAR, WT-1, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (AD-binding protein Abp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, T-cell receptor / CD3-ζ chain, GAGE family of tumor antigens, RAGE, LAGE-I, NAG, GnT-V, RCASl, α-fetoprotein, pl20ctn, Pmel117, PRAME, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5, SCP -1, CT-7, cdc27, adenomatous polyposis coli protein (APC), fodrin, PlA, connexin 37, Ig-idiotype, pl5, GM2, GD2 ganglioside, Smad family of tumor antigens, lmp-1, EBV-encoded nuclear antigen (EBNA)-1, UL16-binding protein-like transcript 1 (Mult1), RAE-1 protein, H60, MICA, MICB and c-erbB-2, or immunogenic peptides thereof, and combinations thereof.

[0285] In another embodiment, the antigen is a target of a modified T cell (e.g., CAR T cell described above). In such an embodiment, the antigen is CD19 or a fragment thereof or a variant thereof. In another embodiment, the antigen is CD22 or a fragment thereof or a variant thereof.

[0286] Any known method, including covalent and non-covalent interactions, can be used to bind the above-mentioned antigens to the scaffold composition. Some of these methods have been listed in the above section related to the manufacture of MSR-SLBs with functional molecules of the present invention. Examples of non-covalent interactions include, for example, electrostatic interactions, van der Waals' interactions, π-effects, hydrophobic interactions, physical insertions, etc. For example, conventional methods can be used to bind full-length transmembrane protein antigens to the lipid bilayer by physical insertion. See, Cymer et al., Journal of Molecular Biology, 427.5:999-1022, 2015 and U.S. Patent No. 7,569,850, which are incorporated herein by reference.

[0287] Antigens can also be connected or tethered to the scaffold composition by covalent interactions. Methods for connecting antigens to scaffolds / surfaces are known in the art, for example, surface adsorption, physical fixation, for example, using phase change to capture substances in the scaffold material. Alternatively, covalent coupling by alkylating or acylating agents can be used to provide a stable, long-term presentation of antigens in a defined conformation on the scaffold. Exemplary reagents and methods for covalently coupling peptides / proteins to polymers are known in the art. See, for example, U.S. Patent No. 6,001,395, which is incorporated herein by reference. In other embodiments, the antigen is encapsulated in the scaffold. Methods for encapsulating antigens into suitable scaffolds (e.g., PLGA microspheres) are known in the art. See, for example, U.S. Patent No. 6,913,767 and International Publication No. WO 1995 / 011010, the disclosures of each of which are incorporated herein by reference.

[0288] Antigen can be formulated into by directly binding or indirectly binding and interacting with immune cells.The type of direct binding includes, for example, the engagement or coupling of antigen and cognate receptor (for example, T cell receptor).Indirect binding can be carried out by the intermediary of one or more secondary reagents or cell types.For example, antigen can first be combined with B cell or antigen presenting cell (APC), obtain processing (for example, degradation) and presentation on the cell surface major histocompatibility complex (MHC) that target cell colony (for example, T cell) is combined with it.Or, antigen can raise other intermediate cells that secrete various cytokines, growth factors, chemokines etc., and it in turn attracts target immune cell colony.No matter how the mechanism, described component synergy is manipulated or modified immune cell.

[0289] Antigen can be derived from cell lysates, cell lysates of classification, freshly collected cells, biological fluids (comprising blood, serum, ascites), tissue extracts etc. In one embodiment, antigen is derived from the lysate of the target cell of required immune cell (for example, T cell) and its combination. In these embodiments, before loading support, first the antigen in the fractionated cell lysate. Lysate can be derived from required target tissue, for example, available from the autoimmune disease specific cells of primary tissue. Or lysate can be derived from cancerous cell, for example, the individual cell that the tumor sample or tissue culture or tumor cell that obtain from biopsy histology obtains.

[0290] The scaffold of the present invention may further contain one or more recruitment agents, which may be selected from the group consisting of T cell recruitment agents, B cell recruitment agents, dendritic cell recruitment agents, and macrophage recruitment agents.

[0291] In one embodiment, the scaffold contains a T cell recruiting agent. Non-limiting examples of T cell recruiting agents include, for example, granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine (CC motif) ligand 21 (CCL-21), chemokine (CC motif) ligand 19 (CCL-19) or FMS-like tyrosine kinase 3 (Flt-3) ligand, granulocyte colony stimulating factor (G-CSF), IFNγ, CXX motif chemokine ligand (CXCL) (selected from CXCL12 and CXCR4), or its fragment, its variant, or its combination. Other types of T cell recruiting agents include, for recruiting the ligands of CCR5 and CXCR3 receptors of type 1 T helper (Th1) subsets. CCR5 ligands, CCL5 and macrophage inflammatory protein (MIP-1α) are known. Alternatively, the ligands of CCR3, CCR4, CCR8 and CXCR4 can be used for the specific recruitment of Th2 subsets. A combination of ligands can also be used.

[0292] Various homologs of the above-mentioned T cell recruiting agents, including functional fragments thereof, or variants thereof, are known in the art. Representative examples of homologs include related proteins from flies, mice, rats, pigs, cattle, monkeys, humans, etc. Homologs preferably include human or mouse homologs of the above-mentioned recruiting agents.

[0293] The support of the present invention is applicable to preferentially raise cells of a single type or a single subtype, for example, preferentially raise T cells and particularly a subset of Treg cells or NK cells.Preferentially raise a feature that compared with other types of immune cells in the device (or a control support lacking a raising agent), one or more specific types of immune cells (e.g., T cells, B cells, DC / macrophages) in the device increase by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2 times, at least 5 times, at least 8 times, at least 10 times or higher accumulation. In the support applicable to raising immune cell combinations (e.g., a combination of T cells and DC / macrophages), preferentially raise a feature that the total percentage of the cells raised is compared with other types of immune cells in the device (or in a control support) by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2 times (i.e., 200%), at least 5 times, at least 8 times, at least 10 times or higher. In particular, preferential recruitment is characterized by a 1- to 10-fold increase in the number of target cells compared to other immune cells.

[0294] In one embodiment, the present invention relates to an MSR-SLB scaffold further comprising a recruitment agent, which is GM-CSF, an agonist thereof, a mimetic thereof, a fragment thereof, a variant thereof, or a combination thereof. Preferably, the recruitment agent is GM-CSF, bound to at least one of CCL-21, CCL-19, Flt-3, or GCSF. Representative examples of such recruitment agents include, for example, human GM-CSF (NCBI Accession No. NP_000749.2) and mouse GM-CSF (NCBI Accession No. NP_034099.2). In another embodiment, the present invention relates to an MSR-SLB scaffold comprising a GM-CSF fragment, for example, a polypeptide comprising amino acids 18-144 of the hGM-CSF sequence. In yet another embodiment, the present invention relates to a scaffold comprising GM-CSF variants, such as VAR_013089 and VAR_001975, whose sequences have been registered in UNIPROT (Accession No. P04141). In another embodiment, the invention relates to MSR-SLB scaffolds containing GM-CSF mimetics, including, for example, antibodies that bind to the GM-CSF receptor, such as those described in Monfardini et al., Curr Pharm Des., 8(24):2185-99, 2002.

[0295] Embodiments of the present invention further provide scaffolds for manipulating immune cells, comprising a plurality of other agents. In such embodiments, the other agents may comprise growth factors, cytokines, chemokines, interleukins, adhesion signaling molecules, integrin signaling molecules, or fragments thereof, or combinations thereof.

[0296] Representative examples of growth factors / cytokines include, but are not limited to, adrenomedullin (AM), angiopoietin (Ang), autotaxin, bone morphogenic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), fetal bovine growth hormone (FBS), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), growth differentiation factor 9 (GDF9), hepatocyte growth factor ( HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), migration-stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophic factor, platelet-derived growth factor (PDGF), thrombopoietin (TPO), T cell growth factor (TCGF), transforming growth factor (TGF-α or TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), Wnt, placental growth factor (PGF), or a functional fragment thereof, or a combination thereof.

[0297] Representative types of interleukins include, but are not limited to, IL-1 (activates T cells, B cells, NK cells and macrophages), IL-2 (activates B cells and NK cells), IL-3 (stimulates non-lymphoid cells), IL-4 (growth factor for activated B cells, resting T cells and mast cells), IL-5 (differentiation of activated B cells), IL-6 (growth factor for plasma cells and T cells), IL-7 (growth factor for pre-B cells / pre-T cells and NK cells), IL-10 (activates macrophages, B cells, mast cells, Th1 / Th2 cells), IL-12 (activates T cells and NK cells), IL-17 (activates Th cells). Functional fragments of interleukins can also be used, which are characterized by the ability to modulate target cell activity.

[0298] Optionally, the scaffold may contain adhesion molecules, which may also serve as signaling agents. Representative examples of adhesion signaling molecules include, but are not limited to, fibronectin, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, aggrecan, bone sialoprotein, cartilage matrix protein, fibrinogen, fibrin, fibrin, mucin, entactin, osteopontin, plasminogen, restritin, serglycin, SPARC / osteonectin, versican, von Willebrand factor, the polysaccharide heparan sulfate, connexin, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides and cyclic peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), chondroitin 6-sulfate, integrin ligands, selectins, cadherins, and members of the immunoglobulin superfamily. Other examples include neural cell adhesion molecule (NCAM), intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM-1), platelet-endothelial cell adhesion molecule (PECAM-1), L1 and CHL1. Functional fragments of adhesion molecules can also be used, characterized in that they regulate the ability of target cells to bind to the scaffold of the present invention. In particular, adhesion molecules include peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD), which are called cell adhesion ligands and are found in various natural extracellular matrix molecules. In another embodiment, the adhesion peptide is a collagen mimetic. Representative examples include peptides with the structure GGYGGGPC (GPP) 5GFOGER (GPP) 5GPC, where O is hydroxyproline. Such peptides can be collectively referred to as GFOGER peptides. GFOGER peptides have previously been shown to be particularly good for T cell adhesion. See, Stephan et al., Nature Biotechnology 33, 2015.

[0299] The polymer matrix with such modifications provides cell adhesion properties to the scaffold of the present invention and maintains the long-term survival of mammalian cell systems, as well as supporting cell growth and differentiation. Adhesion molecules can be coupled to the polymer matrix using synthetic methods generally known to those of ordinary skill in the art and described in the examples. See, for example, Hirano et al., Advanced Materials, 17-25, 2004; Hermanson et al., Bioconjugate Techniques, p. 152-185, 1996; Massia and Hubbell, J. Cell Biol. 114: 1089-1100, 1991; Mooney et al., J. Cell Phys. 151: 497-505, 1992; and Hansen et al., Mol. Biol. Cell 5: 967-975, 1994, the disclosures of which are incorporated by reference.

[0300] Depending on the target cell type, it may be preferred to use target cell-specific adhesion signaling molecules. Thus, in one embodiment, the scaffold contains adhesion receptors useful in the binding / sequestration of T cells. In these embodiments, the scaffold may contain T cell-specific adhesion molecules, for example, receptors selected from class II MHC (for CD4+ cells), class I MHC (for CD8+ cells), LFA-3 (a ligand for CD2), ICAM1 (a ligand for LFA-1), or variants, fragments thereof, or combinations thereof.

[0301] Depending on the needs, the scaffold can be specially formulated to contain a subset of recruitment agents and adhesion molecules so as to manipulate a specific subset of immune cells, for example, a specific T cell subset. In these embodiments, the scaffold can be formulated / manufactured using reagents that specifically bind to cell surface markers expressed in target cells. For example, in the case of T cells, the scaffold can be suitable for use in helper T cells (T cells). H cells; their differential expression CD4+), cytotoxic T cells (T c cells; their differential expression CD8+), memory T cells (T mcells; which differentially express CD45RO), suppressor T cells (Ts, whose cells), regulatory T cells (Treg; further characterized as FOXP3+Treg cells and FOXP3-Treg), natural killer T cells (NK cells; differentially express CD1d+), mucosal associated invariants (MAIT; differentially express MR1), gamma delta T cells (γδT cells; including TCRs containing one γ chain and one δ chain). Such reagents that bind to cell surface markers may include, for example, haptens, peptides, ligands, antibodies, etc. Other conventional techniques for enriching isolates having one or more cell subtypes can be optionally used in situ or ex situ

[0302] The scaffold can also be applicable to the immune cells that raise disease specificity.For example, a variety of T cells specific for a particular type of autoimmune disease can be raised. Therefore, in one embodiment, the scaffold useful in the diagnosis of autoimmune diseases can be formulated into a specific recruitment agent containing the immune cells involved in the obstacle. Such a recruitment agent can, for example, be specific for regulatory T cells (Treg), suppressor T cells (Ts) or a combination thereof. In a related embodiment, the scaffold useful in cancer diagnosis can be formulated into a recruitment agent containing a cancer-specific T cell type (for example, cytotoxic T cells (Tc), natural killer cells (NK) or a combination thereof) for preferentially raising cancer.

[0303] In certain embodiments, the scaffold is used to select disease-specific cells. This can include, for example, cells that directly promote disease progression. In the case of many autoimmune diseases, the disease can be mediated and promoted by the targeted killing of specific cell populations, for example, the beta cells of the pancreas in T1D and the neuronal cells in multiple sclerosis. In other autoimmune diseases, the disease can be facilitated by the targeted attack of specific epitopes, such as, for example, rheumatoid factor (RF) and citrullinated peptides in the case of rheumatoid arthritis, and antigens present in the intestinal flora in the case of Crohn's disease. The targeted destruction of cells generally involves immune cells of a specific type or subset. Therefore, based on the properties and characteristics of the cell target, the scaffold of the present invention can be used to preferentially manipulate immune cells specific to it.

[0304] In the above-mentioned embodiment, the scaffold is provided with an antigen to which disease-specific immune cells (e.g., T cells) bind. These autoimmune cells can be manipulated and optionally reprogrammed to a non-autoimmune phenotype. Methods for reprogramming T cells to pluripotency are known in the art. See, Nishimura et al., Stem Cell 12, 114-126 (2013); Themeli et al., Nature Biotechnology 31, 928-933 (2013). In some cases, particularly in the case of cancer-specific T cells, the reprogrammed cells can be updated to target cancer. Alternatively, in the case of T cells specific for autoimmune diseases, the cells can be eliminated.

[0305] In certain embodiments, the scaffolds of the present invention are fabricated into porous structures that have been engineered to maintain antigen presentation. Methods for fabricating porous scaffolds have been described in the art. See, for example, U.S. Publication Nos. 2011 / 0020216, 2013 / 0202707, 2011 / 0020216, and U.S. Patent No. 8,067,237, the disclosures of which are incorporated herein by reference.

[0306] Embodiments of the present invention further provide a scaffold containing an MSR-SLB scaffold having the desired stability for various ex vivo and in vivo applications. For example, the scaffold is stable in tissue culture applications, cell growth experiments, or as a transplant material to be applied to tissue (collected or engineered) and a subject. In one embodiment, the present invention relates to a mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold that maintains a continuous fluid architecture for at least 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days or longer. Conventional techniques, such as the microscopic visualization techniques described in the following examples, can be used to monitor the stability and / or fluid architecture of the scaffold.

[0307] II. Method for preparing the stent of the present invention

[0308] Embodiments of the present invention further relate to a method for preparing an antigen presenting cell mimic scaffold (APC-MS) of the present invention. The method includes providing a basal layer comprising high surface area mesoporous silica microrods (MSR); optionally loading a T cell homeostatic agent onto the MSR; layering a continuous fluid-supported lipid bilayer (SLB) onto a basal layer comprising the MSR, thereby producing an MSR-SLB scaffold; if step (b) is not performed, loading a T cell homeostatic agent onto the MSR-SLB scaffold; optionally blocking one or more nonspecific integration sites in the MSR-SLB scaffold with a blocking agent; and loading T cell activation molecules and T cell co-stimulatory molecules onto the MSR-SLB scaffold to produce an APC-MS. In these embodiments, the method may include further loading at least one other reagent into the scaffold, the other reagent being a growth factor, a cytokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, or a fragment thereof, or a combination thereof. Methods for loading other components have been previously described in the device manufacturing section. Figure 24 Representative methods for preparing the scaffolds of the present invention are provided in .

[0309] In one embodiment, a functional molecule mixture containing a 1:1 mixture of T cell activating molecules and T cell co-stimulatory molecules (e.g., anti-CD3 antibodies and anti-CD28 antibodies) is mixed with an MSR-SLB scaffold such that the weight ratio of functional molecules to MSR-SLB scaffold is from about 1:2 to about 1:20, preferably from about 1:4 to about 1:15, and particularly from about 1:5 to about 1:10. The weight ratio of T cell activating molecules to T cell co-stimulatory molecules can be adjusted, for example, from about 5:1 to about 1:5, while maintaining the same dry weight ratio between the functional molecules and the MSR-SLB scaffold.

[0310] Furthermore, embodiments of the present invention further relate to methods for preparing APC-MS by assembling multiple scaffolds to create a stack with sufficient porosity to allow penetration of T cells, more particularly, different subsets of helper T cells or cytotoxic T cells.

[0311] III. Methods of using the stents of the present invention

[0312] The scaffolds of the present invention can be used in various applications, including, but not limited to, manipulation of target effector cells (e.g., T cells), isolation of specific effector cell populations (e.g., subpopulations of CD8+ T cells), diagnosis and treatment of diseases, and production of compositions and kits for diagnosis and treatment of diseases.

[0313] Methods for manipulating target cells

[0314] In one embodiment, the present invention provides a method for manipulating a target effector cell or a subpopulation thereof (e.g., a helper T cell or a cytotoxic T cell). In this regard, the term "manipulation" includes, for example, activation, division, differentiation, growth, expansion, reprogramming, anergy, dormancy, aging, apoptosis, or death of the target effector cell.

[0315] In one embodiment, target effector cells, such as T cells, are manipulated (e.g., activated) in situ by providing a scaffold of the present invention so that the target effector cells contact the scaffold. To facilitate contact, the scaffold can be implanted at a suitable site in the subject's body, for example, subcutaneously or intravenously. In other embodiments, target cells are manipulated ex situ by culturing a sample containing target effector cells with the scaffold of the present invention.

[0316] A variety of target effector cells can be manipulated, including using fresh samples from subjects, primary cultured cells, immortalized cells, cell lines, and hybridomas. The manipulated cells can be used in various immunotherapy applications as well as in research.

[0317] The manipulation site of the target effector cells can be in situ or ex situ. Thus, in one embodiment, the cells are manipulated in situ (e.g., within a scaffold). In this regard, it is not necessary to physically remove the cells from the scaffold for manipulation. In another embodiment, the cells are manipulated ex situ (e.g., by first removing the cells from the scaffold and manipulating the removed cells). When the scaffold is implanted in the subject, the cells can be manipulated at or near the implantation site. In other embodiments, the implanted scaffold can first be removed from the implantation site and the effector cells manipulated in situ or ex situ as previously described.

[0318] In certain embodiments, the scaffold used in manipulating effector cells can have antigen presenting cells (APCs) and / or various antigens derived from these APCs. These secondary agents (e.g., APCs or antigens derived from APCs) can be provided within the scaffold structure or provided externally (e.g., in culture medium). In certain embodiments, the scaffold can have various antigens that attract and / or recruit APCs. Representative examples of such attracting and / or recruiting molecules have been provided in the previous section.

[0319] In certain embodiments, the antigen-containing scaffold can be used to manipulate target effector cells in vivo. For such applications, the scaffold can be implanted inside a blood vessel, in lymphoid tissue, at a tumor site, at a disease site (e.g., an area surrounding a tissue affected by rheumatoid arthritis), or subcutaneously so that the target effector cells contact the scaffold. Alternatively, the scaffold can be injected in a minimally invasive manner, for example, by a needle, catheter, or the like. The implanted scaffold can be allowed to remain at the implant site for about 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 7 months, 8 months, 9 months, 1 year, 2 years, or longer. Periodically, the scaffold can be explanted to study, molecule, or even further manipulate effector cells.

[0320] In a related embodiment, the present invention relates to manipulating antigen-specific effector cells in situ. In this regard, the scaffold of the present invention can contain the target antigen, and the target antigen is adsorbed onto the scaffold using the same strategy for adsorbing functional molecules. Alternatively, the scaffold of the present invention can be incubated with a sample containing antigen-specific effector T cells in a culture medium and an APC displaying the target antigen. Subsequently, the target effector cells are allowed to contact the scaffold and the functional molecules contained in the scaffold act together to promote the manipulation of the effector cells. Simply as a representative embodiment, as described in the Examples section, a sample containing T cells is incubated with the scaffold of the present invention, which activates, co-stimulates, and maintains the target effector cells in a stable state. The sample can be incubated with the scaffold for about 1 day to 30 days, about 1 to 15 days, or about 4 to 13 days, for example, about 7-8 days, resulting in selective manipulation of the effector cell population. Antigen-specific effector cells can be manipulated in addition by selecting cells based on the expression of certain gene products (for example, T cell receptors (TCR) that recognize target antigens or antigen presenting cells).

[0321] Embodiments described herein further relate to methods for ex situ manipulation of antigen-specific effector cells, wherein the scaffold has APCs expressing the antigen of interest or the antigen itself. The manipulation step can be performed ex situ or in situ.

[0322] In another embodiment, the effector target cells specific for the antigen or APC can be selectively manipulated relative to other effector cells (e.g., biased towards CD8+T cells relative to CD4+T cells). For example, a sample containing CD8+T cells (together with CD4+T cells) can be incubated with the scaffold of the present invention, and the scaffold of the present invention is mechanically or chemically manufactured to allow CD8+T cells to infiltrate and / or isolate. Techniques known in the art can be used to further expand, activate, proliferate or grow the infiltrated and / or isolated CD8+T cells. Representative methods have been described previously.

[0323] In another embodiment, the effector target cells specific for the antigen or APC may be undesirable (e.g., regulatory / inhibitory T cells), and upon contact with the scaffold of the present invention, they are induced to undergo apoptosis, anergy, or death. For example, a sample containing regulatory T cells (along with other T cells) can be incubated with the scaffold of the present invention, which is mechanically or chemically manufactured to allow regulatory / inhibitory T cells to infiltrate and / or isolate. Techniques known in the art can be used to eliminate infiltrated and / or isolated T cells.

[0324] In this regard, the properties of the cells that have been infiltrated and / or isolated in the support of the present invention can be further determined using techniques known in the art. Therefore, in one embodiment, the gene product used to identify or select activated T cells can be a cell surface marker or cytokine, or a combination thereof. Cell surface markers used to identify activated T cells include, but are not limited to, CD69, CD4, CD8, CD25, HLA-DR, CD28, and CD134. CD69 is an early activation marker found on B and T lymphocytes, NK cells, and granulocytes. CD25 is an IL-2 receptor and a marker for activated T cells and B cells. CD4 is a TCR co-receptor and a marker for thymic lymphocytes, TH1- and TH2-type T cells, monocytes, and macrophages. CD8 is also a TCR co-receptor and a marker for cytotoxic T cells. CD134 is only expressed in activated CD4+ T cells.

[0325] Cell surface markers for selecting activated T cells include, but are not limited to, CD36, CD40, and CD44. CD28 is a stimulatory T cell activation pathway unrelated to the T cell receptor pathway and is expressed on CD4+ and CD8+ cells. CD36 is a membrane glycoprotein and is a marker for platelets, monocytes, and endothelial cells. CD40 is a marker for B cells, macrophages, and dendritic cells. CD44 is a marker for macrophages and other phagocytes. T cell subsets can be isolated by positive selection, negative selection, or a combination thereof using the expression of cell surface gene products of helper T cells or cytotoxic T cells (e.g., CD4 vs. CD8). Cytokines for identifying activated T cells of the present invention include, but are not limited to, cytokines produced by TH1 type T cells (cell-mediated response) and TH2 type T cells (antibody response). Cytokines for identifying activated TH1 type T cells include, but are not limited to, IL-2, gamma interferon (γIFN), and tissue necrosis factor alpha (TNFα). Cytokines used to identify activated TH2 T cells include, but are not limited to, IL-4, IL-5, IL-10, and IL- 13. T cell subsets can also be isolated by positive selection, negative selection, or a combination thereof, for the expression of cytokine products of helper T cells or cytotoxic T cells (e.g., γIFN vs. IL4).

[0326] Activated TH1 T cells specific for the target antigen can be isolated by identifying cells expressing CD69, CD4, CD25, IL-2, IFNγ, TNFα, or a combination thereof. Activated TH1 T cells specific for the target antigen can also be isolated by identifying cells expressing CD69 and CD4 together with IFNγ or TNFα. Activated TH2 T cells specific for the target antigen can be isolated by identifying cells expressing CD69, CD4, IL-4, IL-5, IL-10, IL-13, or a combination thereof. Activated TH1 T cells specific for the target antigen and a combination of TH2 T cells can be isolated by identifying cells expressing CD69, CD4, CD25, IL-2, IFNγ, TNFα, or a combination thereof and cells expressing CD69, CD4, IL-4, IL-5, IL-10, IL-13, or a combination thereof.

[0327] The gene product of the positive or negative selection of the activated T cells of the present invention can be identified by immune selection techniques utilizing antibodies known to those skilled in the art, including but not limited to fluorescence activated cell sorting (FACS), magnetic cell sorting, elutriation, and chromatography. The identification of two or more markers on the activated T cells can be performed in one or more steps. When using FACS to perform immune selection of two or more markers in one step, two or more different antibodies are labeled with different fluorophores. Alternatively, as described above, microbeads can be used to sort cells.

[0328] For the gene product of cell surface expression, antibody can directly bind gene product and can be used for cell selection.For the cell surface gene product expressed with low concentration, magnetic fluorescence (magnetofluorescent) liposome can be used for cell selection.Under low expression level, conventional fluorescent-labeled antibody may not be sensitive enough to detect the existence of the gene product of cell surface expression.Liposome containing fluorophore can be puted together with the antibody with target specificity, thus allowing to detect cell surface markers.

[0329] For intracellular gene products, such as cytokines, antibodies can be used after infiltrating the cells. Alternatively, in order to avoid cells being killed by osmotic ... For forms of cytokines expressed on the cell surface, conventional fluorescently labeled antibodies or liposomes containing fluorophores can be used to detect the target cytokine. One of ordinary skill in the art will recognize other techniques for detecting and selecting extracellular and intracellular gene products specific for activated T cells.

[0330] T cells isolated by the methods of the present invention can be enriched from whole blood by at least 40-90%. T cells can also be enriched from whole blood by at least 95%. T cells can also be enriched from whole blood by at least 98%. T cells can also be enriched from whole blood by at least 99.5%. Similar methods can be used for in situ or ex situ manipulation of B cells. In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour before activation.

[0331] Depending on the application, the dry weight ratio of the scaffold to the cell sample can be adjusted. For example, the scaffold:cell dry weight ratio can range from 1:500 to 500:1, and any integer value therein can be used to manipulate effector cells. As one of ordinary skill in the art will readily appreciate, the scaffold:cell ratio may depend on the size of the scaffold relative to the target cells.

[0332] Expansion of T cell populations

[0333] In a related embodiment, the present invention further relates to a method for amplifying T cells from an immune cell population, for example, amplifying T cells contained in a sample containing B cells, dendritic cells, macrophages, plasma cells, etc. In another embodiment, the present invention further relates to a method for amplifying a specific T cell population, for example, amplifying cytotoxic T cells from a sample containing helper T cells, natural killer T cells, regulatory / suppressor T cells, etc. Specific T cell subsets can then be used in various immunotherapy applications. Without wishing to be bound by any particular theory, it is believed that the APC-MS of the present invention is particularly effective for the amplification of T cells because the relatively large size and high aspect ratio of the mesoporous silica rods allow the T cells interacting with each rod to form large clusters, which promotes the effective amplification of T cells by allowing T cell / T cell interactions and / or paracrine signaling.

[0334] In one embodiment, by providing the scaffold of the present invention, the target effector cells are contacted with the scaffold, and the target effector cells, for example, T cells, are amplified in situ (e.g., grown or differentiated). In order to promote contact, the scaffold can be implanted in a suitable part in the subject's body, for example, subcutaneously or intravenously. In other embodiments, the target cells can be amplified non-in situ by culturing a sample containing target effector cells with the scaffold of the present invention. In one embodiment, the T cells can be first isolated from the sample and then stimulated by contacting the scaffold of the present invention so that the effector T cells are activated, co-stimulated, and maintained in a steady state, thereby performing non-in situ T cell amplification.

[0335] In one embodiment of the invention, T cells are primary T cells obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from infection site, spleen tissue, and tumors. In certain embodiments of the present invention, any number of primary T cells and / or T cell lines available in this area can be used.

[0336] The study of complete blood counts reveals that the T cell number in whole blood is very low. For example, according to the disclosed product catalog of StemCellTechnologies, Vancouver, BC, CANADA (Document#23629, VERSION 2.1.0), the white blood cell population in whole blood is about 0.1-0.2% (due to the advantage of red blood cells), wherein T cells constitute the about 7-24% of all white blood cell populations. In T cells, CD4+T cells constitute the about 4-20% of all white blood cell populations (converted to less than 0.04% of all cell populations in whole blood) and CD8+T cells constitute the about 2-11% of all white blood cell populations (converted to less than 0.022% of all cell populations in whole blood). Therefore, in certain embodiments of the present invention, the method of the present invention can be combined with other technologies known in the art for target cell enrichment. Enrichment steps can be carried out before sample is contacted with the support of the present invention. In another embodiment, enrichment steps can be carried out after sample is contacted with the support of the present invention.

[0337] In one embodiment, FICOLL can be used to separate and enrich effector cell colonies. In one embodiment, cells from individual circulating blood are obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes (including T cells), monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. The cells collected by apheresis can be washed to remove the plasma portion and the cells are placed in a suitable buffer or medium for subsequent processing steps. The cells are then washed with phosphate buffered saline (PBS). Alternatively, the washing solution lacks calcium and can lack magnesium or can lack many divalent cations (if not all). Semi-automatic "circulation" centrifugation can also be used according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, without Ca, without Mg-PBS. Alternatively, the undesirable components of the apheresis sample can be removed and the cells can be directly suspended in culture medium.

[0338] In another embodiment, the monocytes can be depleted by lysing the red blood cells, e.g., by PERCOLL TM Gradient centrifugation is used to enrich T cells from peripheral blood or whole blood. Specific T cell subsets, such as CD28+, CD4+, CD8+, CD45RA+, and CD45ro+ T cells, can be further isolated using positive or negative selection techniques.

[0339] According to the present invention, various sorting techniques can be optionally used. For example, a combination of antibodies directed against cell-unique surface markers can be used to further sort amplified or manipulated T cell populations. A preferred method is to use magnetic immunoadhesion or flow cytometry cell sorting and / or selection using a mixture of monoclonal antibodies directed against cell surface markers present on selected cells. For example, in order to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or negatively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.

[0340] For the separation of required cell colonies, the concentration of cells and the support surface can be changed. In certain embodiments, it may be desirable to significantly reduce the volume in which support and cells are mixed together (i.e., improve the concentration of cells) to ensure the maximum contact of cells and support. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, a cell concentration higher than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In another embodiment again, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / ml is used. In a further embodiment, a concentration of 125 or 150 million cells / ml can be used. Using high concentrations can result in improved cell yield, cell activation and cell amplification. In addition, using high cell concentrations allows more effectively capturing cells that may weakly express target antigens, such as CD28 negative T cells, or capturing (i.e., leukemia blood, tumor tissue, etc.) from samples in which there are many tumor cells. Such cell populations may be of therapeutic value and desirable to obtain. For example, using high concentrations of cells allows for more efficient selection of CD8+ T cells, which typically have weaker CD28 expression.

[0341] In a related embodiment, it may be desirable to use a lower concentration of cells. This can be achieved by reducing the scaffold:cell ratio, minimizing interactions between the scaffold and the cells. This approach selects cells that express a high amount of the desired antigen to be bound to the scaffold. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells at dilute concentrations. In one embodiment, the cell concentration used is 5×10 6 In other embodiments, the concentration used is about 1×10 5 / ml to 1×10 9 / ml, and any integer value therein, for example, 1×10 5 / ml to 1×10 8 / ml, 1×10 6 / ml to 1×10 7 / ml, 1×10 7 / ml to 1×10 9 / ml.

[0342] In one embodiment, the present invention may include procedures known in the art for sample preparation. For example, T cells can be frozen after a washing step and thawed before use. Freezing and subsequent thawing provide a more uniform product by removing granulocytes and a certain degree of monocytes in the cell population. After the washing steps to remove plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be useful in this regard, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media containing, for example, HESPAN and PLASMALYTE A, followed by freezing the cells to -80°C at a rate of 1°C / minute and storing them in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled freezing that is immediately frozen at -20°C or in liquid nitrogen.

[0343] In the context of the present invention, it is also contemplated that a blood sample or leukapheresis product is collected from a subject some time before the amplified cells described herein may be needed. Thus, the cell source to be amplified can be collected at any time point as needed, and the desired cells, such as T cells, are separated and frozen for subsequent T cell therapy for various diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, the blood sample or leukapheresis is taken from a generally healthy subject. In certain embodiments, the blood sample or leukapheresis is taken from a generally healthy subject who is at risk of developing the disease but has not yet developed the disease, and the target cells are separated and frozen for subsequent use. In certain embodiments, the T cells can be amplified, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a specific disease as described herein but before any treatment. In further embodiments, cells are isolated from a blood sample or leukapheresis of a subject prior to various related treatments, including but not limited to treatment with agents such as antivirals, chemotherapy, radiation therapy, immunosuppressants such as cyclophosphamide, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, FR901228, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi (supra)). In a further embodiment, cells are isolated from a patient and frozen for subsequent use in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablative therapy using a chemotherapeutic agent (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or an antibody (e.g., OKT3 or CAMPATH). In another embodiment, cells are isolated and can be frozen for subsequent treatment following B cell ablative therapy (e.g., an agent reactive with CD20, e.g., Rituxan).

[0344] In another embodiment of the present invention, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, particularly treatments using drugs that destroy the immune system, shortly after treatment, during the time period when the patient normally recovers from treatment, the T cell quality obtained may be optimal or improved for the ability of its ex vivo expansion. Similarly, after using the method described herein to manipulate in vitro, these cells are in a preferred state for enhanced transplantation and in vivo expansion. Therefore, within the context of the present invention, it is considered that blood cells are collected during this convalescence period, including other cells of T cells, dendritic cells or hematopoietic lineages. In addition, in some embodiments, mobilization (for example, using GM-CSF mobilization) and conditioning regimens can be used to form conditions in the experimenter that are conducive to the re-clustering, recycling, regeneration and / or expansion of specific cell types, especially during the limited time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0345] According to the present invention, a scaffold containing any ratio of T cell activating molecules: T cell co-stimulatory molecules can be used. In one embodiment, where both the T cell activating molecules and the T cell co-stimulatory molecules are antibodies, a 1:1 ratio of each antibody can be used. In one embodiment, the ratio of CD3:CD28 antibodies bound to the scaffold ranges from 100:1 to 1:100 and all integer values therein. In one aspect of the invention, the anti-CD28 antibodies bound to the scaffold are more than the anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti-CD28 antibodies bound to the scaffold to the anti-CD3 antibodies is higher than 2:1. In a particular embodiment, a scaffold-bound antibody having a ratio of 1:100 CD3:CD28 can be used. In another embodiment, a scaffold-bound antibody having a ratio of 1:75 CD3:CD28 can be used. In yet another embodiment, a scaffold-bound antibody having a ratio of 1:50 CD3:CD28 is used. In another embodiment, a scaffold-bound antibody having a ratio of 1:30 CD3:CD28 is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio of the antibody binding scaffold is used. In another embodiment, a 1:3 CD3:CD28 ratio of the antibody binding scaffold is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of the antibody binding scaffold is used.

[0346] One aspect of the present invention arises from the surprising discovery that wherein, after about one week of contact with the scaffold, the method provides an increase in T cell population expansion compared to a control scaffold containing a basal layer containing high surface area mesoporous silica microrods (MSR) and a continuous fluid-supported lipid bilayer (SLB) but without T cell activation molecules and T cell costimulatory molecules. In one embodiment, according to the method of the present invention, after about one week of contact with the scaffold, an increase in T cell population expansion of about 10-fold to 1000-fold, preferably about 50-fold to 500-fold, or more, is observed compared to a control scaffold containing a basal layer containing high surface area mesoporous silica microrods (MSR) and a continuous fluid-supported lipid bilayer (SLB) but without T cell activation molecules and T cell costimulatory molecules.

[0347] Another aspect of the present invention is derived from surprising discovery: wherein compared with the superparamagnetic spherical polymer particles (DYNABEAD) containing T cell activation molecules and T cell costimulatory molecules, after contacting the support for about 1 week, the method gives the increase of T cell colony expansion. In one embodiment, according to the method of the present invention, compared with the superparamagnetic spherical polymer particles (DYNABEAD) containing T cell activation molecules and T cell costimulatory molecules, after contacting the support for about 1 week, it is observed that the expansion of T cell populations increases by about 2 times to 100 times, preferably about 5 times to 20 times, or higher.

[0348] Another aspect of the present invention is derived from surprising discovery: manipulation of T cells according to the above method improves the metabolic activity of T cells. In particular, compared with a control scaffold containing a basal layer (which contains high surface area mesoporous silica microrods (MSR)) and a continuous fluid-supported lipid bilayer (SLB) but not containing T cell activation molecules and T cell costimulatory molecules, after contacting the scaffold for about 1 week, the metabolic activity of the T cells increased was observed. In one embodiment, according to the method of the present invention, compared with a control scaffold containing a basal layer (which contains high surface area mesoporous silica microrods (MSR)) and a continuous fluid-supported lipid bilayer (SLB) but not containing T cell activation molecules and T cell costimulatory molecules, after contacting the scaffold for about 1 week, it was observed that the metabolic activity of the T cell population increased by about 2 times to 100 times, preferably about 5 times to 20 times, or larger.

[0349] Another aspect of the present invention is derived from a surprising discovery: compared to superparamagnetic spherical polymer particles (DYNABEAD) containing T cell activation molecules and T cell costimulatory molecules, after contacting the scaffold for about 1 week, the method gives better metabolic activity of T cell populations. In one embodiment, according to the method of the present invention, compared to superparamagnetic spherical polymer particles (DYNABEAD) containing T cell activation molecules and T cell costimulatory molecules, after contacting the scaffold for about 1 week, it is observed that the expansion of T cell populations increases by about 1 times (for example, 100%) to 20 times, preferably 2 times to 10 times, or more.

[0350] In addition, according to the method of the present invention, after finding contact with the support, the T cells of amplification maintain metabolic activity for at least about 7 days. By conventional techniques, for example, the level of cytokine production or monitoring cell doubling are analyzed to measure T cell metabolic activity. In addition, according to the method of the present invention, the T cells of amplification form larger and more stable aggregates (for example, continue longer) than the control support. For example, in an experiment, after contacting the support, the T cells of amplification form stable aggregates for at least about 7 days, and the aggregates in the sample incubated with the control support containing only MSR basal layer and SLB layer are significantly different.

[0351] Further embodiments of the present invention relate to methods for obtaining a polyclonal population of CD8+ cells, comprising contacting a scaffold of the present invention with a biological sample of a subject, thereby activating, co-stimulating, homeostatically maintaining, and optionally expanding a T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD8+ cells; and isolating the detected CD8+ T cell subpopulation from the sample.

[0352] In a related embodiment, the present invention relates to a method for obtaining a polyclonal population of CD4+ cells, comprising contacting a scaffold of the present invention with a biological sample of a subject, thereby activating, co-stimulating, homeostatically maintaining, and optionally expanding a T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD4+ cells; and isolating the detected CD4+ T cell subpopulation from the sample.

[0353] In a related embodiment, the present invention relates to a method for obtaining a polyclonal population of CD4+ / FOXP3+ or CD4+ / FOXP3- cells. The method includes contacting the scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state, and optionally amplifying the T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD4+ cells; further contacting the T cells with a reagent for detecting FOXP3+ cells; and separating the detected CD4+ / FOXP3+ or CD4+ / FOXP3-T cell subsets from the sample. In these embodiments, the reagent for detection and / or separation of CD4+ and / or FOXP3+ T cells is preferably an antibody or its antigen-binding fragment that specifically binds to CD4+ and FOXP3 markers. In this regard, in the range of FOXP3 being identified as a major regulator of regulatory pathways in the development and function of regulatory T cells (which turn down immune responses), it may be desirable to separate FOXP3+ cells for certain applications, and FOXP3- cells for other applications. For example, in cancer treatment applications, it may be desirable to eliminate or reduce regulatory T cell activity in a T cell pharmaceutical composition. Thus, the method may be suitable for screening for FOXP3- cells. Alternatively, in the case of autoimmune disease treatment, it may be desirable to increase regulatory T cell activity in a T cell pharmaceutical composition (because reduced regulatory T cell activity may cause an autoimmune condition in the body). Therefore, in such cases, the formulation method may be modified to positively select for and include FOXP3+ cells.

[0354] In yet another embodiment, the present invention relates to a method for obtaining a polyclonal population of effector memory and / or effector T cells. The method comprises contacting the scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, homeostatically maintaining, and optionally amplifying the T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD44+ cells; further contacting the T cells with a reagent for detecting CD62L; and separating and detecting CD4+ / / CD62L+ or CD4+ / CD62L-T cell subsets from the sample. In these embodiments, the effector memory and / or effector T cells are preferably CD4+ / / CD62L-.

[0355] In yet another embodiment, the present invention relates to a method for obtaining a polyclonal population of activated CD8+T cells. The method includes contacting the scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state, and optionally amplifying the T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD8+ cells; further contacting the T cells with a reagent for detecting CD69+; and isolating the detected CD8+ / / CD69+ or CD8+ / / CD69-T cell subsets from the sample. In these embodiments, the activated T cells are preferably CD8+ / CD69+.

[0356] In yet another embodiment, the present invention relates to a method for obtaining a polyclonal population of cytotoxin-secreting T cells. The method comprises contacting a scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD8+ cells; further contacting the T cells with a reagent for detecting granzyme B; and isolating the detected CD8+ / / granzyme B+ or CD8+ / / granzyme B T cell subpopulation from the sample. In these embodiments, the cytotoxin-secreting T cells are preferably CD8+ / granzyme B+.

[0357] In yet another embodiment, the present invention relates to a method for obtaining a polyclonal population of activated cytokine-secreting T cells. The method comprises contacting a scaffold of the present invention with a biological sample from a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample; contacting the T cells in the sample with a reagent for detecting IFNγ+ cells; and isolating the detected IFNγ+ T cell subpopulation from the sample. In these embodiments, the T cells are preferably IFNγ-secreting cells.

[0358] In yet another embodiment, the present invention relates to a method for obtaining a polyclonal population of memory T cells. The method comprises contacting the scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state, and optionally amplifying the T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD62L+CCR7+T cells; and separating the detected CD62L+CCR7+T cell subpopulation from the sample. In these embodiments, the T cells are preferably CD62L+CCR7+CD4+ central memory T cells. See, Okada et al., Int Immunol., 20(9):1189-99, 2008. In another embodiment, the present invention relates to a method for obtaining a polyclonal population of memory T cells, comprising contacting the scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, maintaining in a steady state, and optionally amplifying the T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD62L+CCR7+T cells; and separating the detected CD62L-CCR7-T cell subpopulation from the sample. In these embodiments, the CD62L-CCR7- T cells are effector memory T cells. See, Sallusto et al., Nature 401:708-712, 1999.

[0359] In yet another embodiment, the present invention relates to a method for detecting and / or removing a polyclonal population of exhausted T cells from a sample. The method comprises contacting the scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, homeostatically maintaining, and optionally amplifying the T cell population present in the sample; contacting the T cells in the sample with a reagent for detecting CD8+T cells; further contacting the T cells with a reagent for detecting PD-1+T cells; and isolating the detected CD8+ / PD-1+T cell subpopulation from the sample. CD8+ / PD-1+T cells can optionally be eliminated from the sample, which represents exhausted cells.

[0360] In another embodiment for detecting and / or removing T cells from a sample, the present invention provides a method comprising contacting a scaffold of the present invention with a biological sample of a subject, thereby activating, costimulating, homeostatically maintaining, and optionally amplifying a population of T cells present in the sample; contacting the T cells in the sample with a reagent for detecting a co-inhibitory receptor on the T cells; and isolating a T cell subpopulation expressing the co-inhibitory receptor from the sample. The expression of the co-inhibitory receptor generally indicates exhausted cells, which can be optionally eliminated from the sample. In these embodiments, the co-inhibitory receptor is a receptor selected from CTLA-4, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG...

Claims

1. An antigen presenting cell mimic scaffold (APC-MS), comprising high surface area mesoporous silica microrods (MSRs) as the substrate layer; a fluid-supported lipid bilayer (SLB) layered on the MSR; and A T cell activating molecule and a T cell co-stimulatory molecule, wherein the T cell activating molecule and the T cell co-stimulatory molecule are present on the SLB; and The scaffold comprises a stacked MSR-SLB structure containing spaces that allow T cell infiltration.

2. The scaffold of claim 1, wherein (i) the T cell activating molecule, (ii) the T cell co-stimulatory molecule, or (iii) both the T cell activating molecule and the T cell co-stimulatory molecule are present on the SLB by affinity pairing or chemical coupling.

3. The scaffold of claim 1 or 2, further comprising a T cell homeostatic agent. The scaffold of claim 3 , wherein the T cell homeostatic agent is loaded on the MSR. The scaffold of claim 3 , wherein the T cell homeostatic agent is loaded on the SLB.

6. The scaffold of claim 3, wherein the T cell homeostatic agent is loaded on the MSR and is present in the culture medium in which the scaffold is suspended.

7. The scaffold of claim 3, wherein the T cell homeostatic agent is present in the culture medium in which the scaffold is suspended.

8. The scaffold of claim 1 or 2, wherein the T cells are selected from the group consisting of natural killer T cells, γδ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), and combinations thereof.

9. The scaffold of claim 8, wherein the regulatory T cells (Tregs) are selected from the group consisting of FOXP3+ Treg cells, FOXP3- Treg cells, and combinations thereof.

10. The scaffold of claim 1 or 2, wherein the T cell activating molecule is selected from the group consisting of an antibody or antigen-binding fragment thereof, a major histocompatibility complex (MHC) molecule loaded with an MHC peptide, an MHC-immunoglobulin (Ig) conjugate, and combinations thereof.

11. The scaffold of claim 1 or 2, wherein the T cell activating molecule is a multimer of major histocompatibility complex (MHC) molecules loaded with MHC peptides or a multimer of MHC-immunoglobulin (Ig) conjugates.

12. The scaffold of claim 1 or 2, wherein the T cell co-stimulatory molecule is an antibody or an antigen-binding fragment thereof.

13. The scaffold of claim 12, wherein the antibody or antigen-binding fragment thereof specifically binds to a co-stimulatory antigen.

14. The scaffold of claim 13, wherein the co-stimulatory antigen is optionally selected from CD28, 4.1BB (CD137), OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTβR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CRACC (CD319, BLAME), and combinations thereof.

15. The scaffold of claim 3, wherein the T cell homeostatic agent is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, transforming growth factor beta (TGF-β), and combinations thereof.

16. The scaffold of claim 1 or 2, comprising an immunoglobulin molecule that specifically binds to an Fc fusion protein, wherein the immunoglobulin molecule is present on the SLB.

17. The scaffold of claim 1 or 2, wherein the scaffold comprises a recruitment compound selected from the group consisting of granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine (CC motif) ligand 21 (CCL-21), chemokine (CC motif) ligand 19 (CCL-19), chemokine (CXC motif) ligand 12 (CXCL12), interferon gamma (IFNγ), FMS-like tyrosine kinase (Flt-3) ligand, and combinations thereof.

18. The scaffold of claim 17, wherein the recruitment compound comprises GM-CSF.

19. The scaffold of claim 1 or 2, wherein the scaffold comprises an antigen.

20. The scaffold of claim 19, wherein the antigen comprises a tumor antigen.

21. The scaffold of claim 20, wherein the tumor antigen is selected from the group consisting of MAGE-1, MAGE-2, MAGE-3, CEA, tyrosinase, midkin, BAGE, CASP-8, β-catenin, γ-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-2, IL13Rα, IL13Rα2, AIM-2, AIM-3, NY-ESO-1, C9orf 112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L, CYNL2, TPR, SOX2, GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, nestin, OLIG2, ART1, ART4, B-cyclin, Gli1, Cav-1, cathepsin B, CD74, E-cadherin, EphA2 / Eck, Fra-1 / Fosl 1, GAGE-1, Ki67, Ku70 / 80, PROX1, PSCA, SOX10, SOX11, survivin, UPAR, WT-1, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (AD Abp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, T-cell receptor / CD3-ζ chain, GAGE-family of tumor antigens, RAGE, LAGE-I, NAG, GnT-V, RCASl, alpha-fetoprotein, pl20ctn, Pmel117, PRAME, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SCP-I, CT-7, cdc27, adenomatous polyposis coli protein (APC), fodrin, P1A, connexin 37, Ig-idiotype, p15, GM2, GD2 ganglioside, Smad family of tumor antigens, 1mp-1, EBV-encoded nuclear antigen (EBNA)-1, UL16-binding protein-like transcript 1 (Mult1), RAE-1 protein, H60, MICA, MICB and c-erbB-2, patient-specific neoantigens, or immunogenic peptides thereof, and combinations thereof.

22. The stent of claim 1 or 2, wherein the weight ratio of the SLB to the MSR is 10:1 to 1:

20.

23. The stent of claim 22, wherein the weight ratio of the SLB to the MSR is 9:1 to 1:

15.

24. The scaffold of claim 1 or 2, wherein the SLB comprises a lipid selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and combinations thereof.

25. The scaffold of claim 1 or 2, wherein the SLB comprises a lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), and combinations thereof.

26. The scaffold of claim 1 or 2, wherein the dry weight ratio of the MSR to the T cell activating molecule and the T cell co-stimulatory molecule is 500:1 to 1:

1.

27. The stent of claim 1 or 2, wherein the stent is encapsulated within another biodegradable stent.

28. The scaffold of claim 1 or 2, wherein the scaffold allows for the infiltration and manipulation of T cells.

29. The scaffold of claim 28, wherein the manipulation of T cells comprises activation and / or expansion of T cells.

30. The stent of claim 1 or 2, wherein the MSR comprises a length of 5 μm to 500 μm.

31. The stent of claim 1 or 2, wherein the MSR comprises a length of 50 μm to 200 μm.

32. The stent of claim 1 or 2, wherein the MSR comprises a length of 80 μm to 120 μm.

33. The stent of claim 1 or 2, wherein the MSR comprises an average length of 100 μm.

34. The stent of claim 1 or 2, wherein the MSR comprises an average length of 88 μm.

35. The stent of claim 34, wherein the MSR comprises an average diameter of 4.5 μm.

36. The stent of claim 1 or 2, wherein the MSR comprises an aspect ratio of 20.

37. A device comprising a plurality of the scaffolds of any one of claims 1-36, wherein the MSR scaffolds are stacked to selectively allow infiltration of T cells.

38. A pharmaceutical composition comprising the stent according to any one of claims 1 to 36 and a pharmaceutically acceptable carrier.

39. The pharmaceutical composition of claim 38, wherein the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

40. A composition comprising the scaffold of any one of claims 1-36 and T cells clustered therein.

41. A method for preparing the stent according to any one of claims 1 to 36, comprising (a) providing the MSR; (b) layering the SLB on the MSR, thereby generating an MSR-SLB complex; and (c) loading the T cell activating molecule and the T cell co-stimulatory molecule onto the MSR-SLB complex, thereby preparing the scaffold according to any one of claims 1 to 36.

42. The method of claim 41, further comprising blocking one or more non-specific integration sites in the MSR-SLB complex with a blocking agent.

43. The method of claim 41, further comprising loading a T cell homeostatic agent onto the MSR after step (a) or onto the MSR-SLB complex after step (c).

44. An in vitro or ex vivo method for manipulating T cells, comprising contacting the antigen presenting cell mimic scaffold (APC-MS) of any one of claims 1-36 with a biological sample containing T cells.

45. The method of claim 44, wherein the T cells in the sample comprise exhausted T cells.

46. The method of claim 45, wherein the exhausted T cells are CD8+PD-1+ and / or LAG-3+TIM-3+.

47. The method of claim 44, further comprising detecting expression of one or more cell surface markers in the manipulated T cells.

48. The method of claim 47, wherein at least one of the cell surface markers is selected from the group consisting of CD4, CD8, CD25, CD28, CD36, CD40, CD44, CD45, CD62L, CD69, CD134, FOXP3, 4-1BB, LAG-3, TIM-3, and PD-1.

49. The method of claim 44, wherein the biological sample is obtained from a subject and the scaffold is contacted with the biological sample ex vivo.

50. The method of claim 44, wherein the T cells are selected from the group consisting of natural killer T cells, γδ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), tumor infiltrating lymphocytes, and combinations thereof.

51. The method of claim 44, wherein the T cells comprise Tregs selected from the group consisting of FOXP3+ Tregs, FOXP3- Tregs, and combinations thereof.

52. The method of claim 44, wherein the T cells are manipulated to produce an expanded population of effector memory and / or effector T cells.

53. The method of claim 44, wherein the manipulated T cells comprise CD8+ cells, CD4+ cells and / or CD44+ / CD62L- T cells.

54. The method of claim 44, wherein the manipulated T cells comprise CD4+ / FOXP3- T cells, CD8+ / CD69+ T cells and / or Granzyme B+CD8+ T cells.

55. The method of claim 44, wherein the manipulated T cells comprise IFN-γ-producing T cells.

56. The method of claim 44, wherein the scaffold comprises a T cell homeostatic agent.

57. The method of claim 56, wherein the T cell homeostatic agent is loaded onto the MSR.

58. The method of claim 44, wherein the scaffold comprises an immunoglobulin molecule that specifically binds to an Fc fusion protein, wherein the immunoglobulin molecule is present on the SLB.

59. The method of claim 44, wherein the scaffold comprises a recruitment compound selected from the group consisting of granulocyte macrophage colony stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL 19), chemokine (C-X-C motif) ligand 12 (CXCL12), interferon gamma (IFNγ), FMS-like tyrosine kinase 3 (Flt-3) ligand, and combinations thereof.

60. The method of claim 59, wherein the recruitment compound comprises GM-CSF.

61. The method of claim 44, wherein the scaffold comprises an antigen present on the SLB.

62. The method of claim 44, wherein the dry weight ratio of the MSR to the T cell activating molecule and the T cell co-stimulatory molecule is 500:1 to 1:

1.

63. The method of claim 44, wherein the MSR comprises a length of 50 μm to 200 μm.

64. The method of claim 44, wherein the MSR comprises a length of 80 μm to 120 μm.

65. The method of claim 44, wherein the MSR comprises an average length of 100 μm.

66. The method of claim 44, wherein the MSR comprises an average length of 88 μm.

67. The method of claim 65, wherein the MSR comprises an average diameter of 4.5 μm.

68. The method of claim 44, wherein the MSR comprises an aspect ratio of 20.

69. The method of claim 44, wherein the weight ratio of the SLB to the MSR is 9:1 to 1:

15.

70. The method of claim 44, wherein the biological sample is a blood sample, a bone marrow sample, a lymph sample, or a spleen sample obtained from a human.

71. The method of claim 44, wherein said contacting results in improved differentiation, expansion, or activity; and / or reduced exhaustion, anergy, or death of said T cells.

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