Antigen-presenting cell-mimicking scaffolds and methods for making and using same

Abstract

To provide a composition and a method in which isolation of a T-cell is made possible, and which can be easily used for treatment of human diseases (e.g., immunodeficiency disorder, autoimmune disorder and cancer).SOLUTION: Provided is a composition and a method for manipulating a T-cell, which is activating, stimulating, enlarging, propagating, or energizing, for example. In this state, an embodiment provides a method for expressing certain kinds of marker and / or cell surface receptor or for generating a large number of (or substantially pure partial population of) activation T-cells which generates certain kinds of cytokine optimum for T-cell-mediated immune response. Such a manipulated T-cell can be used in treatment and prevention of many diseases (e.g., cancer, infectious disease, autoimmune disorder, allergy, immune dysfunction associated with aging, or any other disease states for the treatment of which T-cell is desired).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 361,891, filed July 13, 2016, the entire contents of which are expressly incorporated herein by reference.

[0002] Statement of government support This invention was made with government support under Grant Nos. EB015498, EB014703 and DE013033 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Immunotherapy, which involves the priming and expansion of T lymphocytes (T cells), holds promise, particularly in humans, for the treatment of cancer and infectious diseases (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 autologous dendritic cells (DCs), virally infected B cells, and / or T cells stimulated, cloned, and expanded in vitro for many weeks on 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, clinical trials of adoptive T cell immunotherapy often require billions of cells (Riddell et al., 1995), and existing in vitro T cell expansion protocols are often insufficient to meet the demands of such trials.

[0004] Furthermore, optimal engraftment requires the use of functional, non-senescent T cells at the time of reinfusion. For clinical application, it is important to ensure that T cells have the desired functionality, i.e., they proliferate, exert effector functions, and produce cytokines in the desired manner (Liebowitz et al., Current Opinion Oncology, 10, 533-541, 1998). In the natural context, T cell activation is initiated by engagement of the T cell receptor / CD3 complex (TCR / CD3) with peptide antigens bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) (Schwartz, Science 248:1349 (1990)). While this is the primary signal in T cell activation, other receptor-ligand interactions between APCs and T cells are also required for full activation. For example, TCR stimulation in the absence of other molecular interactions can induce a state of anergy such that these cells are 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, T cells can die by programmed cell death (apoptosis) when activated by TCR engagement alone (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).

[0005] Thus, optimal functionality can be conferred through the use of a second signaling molecule, such as a membrane-bound protein or a secreted product of the APC. In the context of membrane-bound proteins, such a second interaction is usually adhesive in nature, strengthening the contact between the two cells (Springer et al., Ann. Rev. Immunol. 5:223, 1987). Other signaling molecules, such as those transmitting additional activation signals from the APC to the T cell, may also be involved (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, present on both resting and activated T cells. CD28 binds to B7-1 (CD80) or B7-2 (CD86) and is one of the most potent costimulatory molecules known (June et al., Immunol. Today 15:321(1994), Linsley et al., Ann. Rev. Immunol. 11:191(1993)). CD28 ligation on T cells, in conjunction with TCR engagement, 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)).

[0006] T cell costimulation has been shown to affect multiple 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 posttranscriptional 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 allograft rejection is significantly delayed (Lenschow et al., Science 257:789 (1992); Turka et al., Proc. Natl. Acad. Sci. USA 89:11102 (1992)).

[0007] More importantly, the aforementioned effectors for stimulatory / costimulatory stimulation have been widely applied in the context of in vitro T cell manipulation. In this context, a combination of anti-CD3 monoclonal antibody (first signal) and anti-CD28 monoclonal antibody (second signal) is most commonly used to stimulate APCs. The signals provided by anti-CD3 and anti-CD28 monoclonal antibodies are expressed by the fact that the antibodies adhere to a solid surface (e.g., a plastic plate (Baroja et al., Cellular Immunology, vol. 120, 205-217, 1989; Damle et al., The It is best delivered to T cells when immobilized on T cells (Journal of Immunology, vol. 143, 1761-1767, 1989) or Sepharose beads (Anderson et al., Cellular Immunology, vol. 115, 246-256, 1988). See also U.S. Patent No. 6,352,694 issued to June et al.

[0008] Various surfaces and reagents containing anti-CD3 and anti-CD28 monoclonal antibodies have been developed to obtain and expand 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 microns (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 shown that T cells activated with these beads exhibit properties, such as cytokine production, that make them potentially useful for adoptive immunotherapy (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.

[0009] The use of paramagnetic beads with immobilized monoclonal antibodies for the expansion of T cells in cell therapy requires the separation and removal of the beads from the T cells before infusion into patients. This is a very labor-intensive process, resulting in cell loss, cell damage, increased risk of contamination, and increased processing costs. Due to the tight association between the immobilized monoclonal antibodies on the beads and the corresponding ligands on the surface of the target T cells, it is difficult to remove the beads from the T cells. The bead-cell conjugates are often separated by waiting until the T cells internalize their target antigen and then separating the beads from the T cells using mechanical disruption techniques. This technique can cause damage to the T cells and can also remove the bound antigen on the T cells from their cell surface (Rubbi et al., Journal of Immunology Methods, 166, 233-241, 1993). Furthermore, paramagnetic separation has had limited use in adoptive cell therapy settings because activated T cells are often the most desirable for use in cell therapy protocols, and the desirable properties of the cells are lost during the 24-72 hour waiting period. Techniques for separating and purifying cells attached to paramagnetic beads also cannot be used in clinical settings. For example, the process of removing paramagnetic beads after separation from T cells requires passing the cell / bead solution over a magnet. This process greatly reduces the number of beads remaining with the T cells, but does not completely eliminate the beads. Transplanting a composition containing beads into a patient can cause toxic effects. The bead removal process also reduces the number of T cells available for therapy, as many T cells remain associated with paramagnetic beads even after mechanical dissociation. Cell loss can also occur with engineered but otherwise unbound T cells because these cells are washed away before the internalization and / or mechanical removal steps. Thus, there is an unmet need for compositions and methods that allow for the isolation of T cells that can be readily utilized in the treatment of human diseases (e.g., immunodeficiency disorders, autoimmune disorders, and cancer). Embodiments of the present invention, described in detail below, address these needs. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 6,352,694 [Non-patent literature]

[0011] [Non-Patent Document 1] Melief et al., Immunol. Rev. 145: 167-177(1995) [Non-patent document 2] Riddell et al., Annu. Rev. Immunol. 13:545-586 (1995) [Non-patent document 3] Riddell et al., Science 257:238-241 (1992) [Non-patent document 4] Yee et al., J. Exp. Med. 192:1637-1644 (2000) [Non-patent document 5] Brodie et al., Nat. Med. 5:34-41(1999) [Non-patent document 6] Riddell et al., Hum. Gene Ther. 3:319-338 (1992) [Non-Patent Document 7] Riddell et al., J. Immunol. Methods 128:189-201 (1990) [Non-patent document 8] Liebowitz et al., Current Opinion Oncology, 10, 533-541, 1998 [Non-Patent Document 9] Schwartz, Science 248:1349(1990) [Non-Patent Document 10] Harding, et al., Nature 356:607, 1992 [Non-Patent Document 11] Dudley et al., Clinical Cancer Research., 16, 6122-6131, 2010 [Non-Patent Document 12] Rosenberg et al., Clinical Cancer Research., 17, 4550-4557, 2011 [Non-Patent Document 13] Webb et al., Cell 63:1249, 1990 [Non-Patent Document 14] Kawabe et al., Nature 349:245, 1991 [Non-Patent Document 15] Kabelitz et al., Int. Immunol. 4:1381, 1992 [Non-Patent Document 16] Groux et al., Eur J. Immunol. 23:1623, 1993 [Non-Patent Document 17] Springer et al., Ann. Rev. Immunol. 5:223, 1987 [Non-Patent Document 18] Bierer et al., Adv. Cancer Res. 56:49, 1991 [Non-Patent Document 19] June et al., Immunol. Today 15:321(1994) [Non-Patent Document 20] Linsley et al., Ann. Rev. Immunol. 11:191 (1993) [Non-Patent Document 21] Smith et al., Ann. NY Acad. Sci. 332:423-432 (1979) [Non-Patent Document 22] Gillis et al., Nature 268:154-156 (1977) [Non-Patent Document 23] Gimmi et al., Proc. Natl. Acad. Sci. USA 88:6575 (1991) [Non-Patent Document 24] Lindsten et al., Science 244:339 (1989) [Non-Patent Document 25] Fraser et al., Science 251:313 (1991) [Non-Patent Document 26] Lenschow et al., Science 257:789 (1992) [Non-Patent Document 27] Turka et al., Proc. Natl. Acad. Sci. USA 89:11102 (1992) [Non-patent document 28] Baroja et al., Cellular Immunology, vol. 120, 205-217, 1989 [Non-Patent Document 29] Damle et al., The Journal of Immunology, vol. 143, 1761-1767, 1989) [Non-Patent Document 30] Anderson et al., Cellular Immunology, vol. 115, 246-256, 1988) [Non-Patent Document 31] Levine et al., The Journal of Immunology, vol. 159, No. 12: pp. 5921-5930, 1997 [Non-Patent Document 32] Garlie et al., J Immunother 22(4): 336-45, 1999 [Non-Patent Document 33] Shibuya et al., Arch Otolaryngol Head Neck Surg, vol. 126, No. 4: 473-479, 2000 [Non-Patent Document 34] Rubbi et al., Journal of Immunology Methods, 166, 233-241, 1993 Summary of the Invention [Means for solving the problem]

[0012] Summary of the Invention The present invention provides compositions and methods for engineering, e.g., activating, stimulating, expanding, proliferating, or energizing, T cells. In this context, embodiments of the present invention provide methods for generating large numbers (or substantially pure subpopulations) of activated T cells that express certain markers and / or cell surface receptors or produce certain cytokines optimal for T cell-mediated immune responses. Such engineered T cells can be used in the treatment and prevention of many diseases (e.g., cancer, infectious diseases, autoimmune diseases, allergies, immune dysfunction that may be associated with aging, or any other disease state for which T cells are desirable for treatment). Further embodiments described herein relate to methods and compositions for the effective treatment of any of the aforementioned diseases by utilizing optimally responsive T cells, 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 their ability to generate large numbers of activated T cells, but also in terms of the significantly improved efficacy of such T cells in an in vivo setting. Thus, the compositions and methods of the present invention are useful for generating human T lymphocytes that are highly desirable for engraftment, autologous transfer, and for therapeutic applications.

[0013] Thus, in one embodiment, the present invention provides an antigen-presenting cell-mimetic scaffold (APC-MS) comprising: a base layer comprising high surface area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto the scaffold.

[0014] In one embodiment, the present invention provides an antigen-presenting cell mimic scaffold (APC-MS) that sequester T cells selected from the group consisting of natural killer (NK) cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs), or a combination thereof.

[0015] In one embodiment, the present invention provides an antigen-presenting cell mimetic scaffold (APC-MS) comprising a plurality of T cell homeostasis factors adsorbed onto the SLB layer.

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

[0017] In one embodiment, the present invention provides an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of T cell homeostasis factors released from the scaffold in a controlled-release manner. In some embodiments, the T cell homeostasis factors are released from the scaffold in a controlled-release manner 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, 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.

[0018] In one embodiment, the present invention provides an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of T cell homeostasis factors that are released from the scaffold in a sustained manner over a period of up to 15 days. In some embodiments, the T cell homeostasis factors are released from the scaffold in a sustained manner over a period of 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 some embodiments, the T cell homeostasis factor is released from the scaffold in a sustained manner for at least 30 days. In some embodiments, the T cell homeostasis factor 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 more.

[0019] In one embodiment, the present invention provides an antigen-presenting cell-mimicking scaffold (APC-MS) comprising multiple T cell homeostasis factors 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, and transforming growth factor β (TGF-β), or an agonist, mimetic, variant, functional fragment, or combination thereof.

[0020] In one embodiment, the present invention provides an antigen-presenting cell-mimetic scaffold (APC-MS) comprising multiple T cell homeostasis factors, which are IL-2, its agonist, mimetic, variant, functional fragment, or a combination thereof, and a second homeostasis factor selected from the group consisting of IL-7, IL-21, IL-15, and an IL-15 superagonist. In one embodiment, the T cell homeostasis factor may be selected from the group consisting of 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.

[0021] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each independently adsorbed onto the fluid-supported lipid bilayer (SLB). In one embodiment, the T cell activation molecules and the T cell costimulatory molecules can be adsorbed via affinity pairing or chemical coupling. In some embodiments, the chemical coupling comprises a click chemistry reagent (e.g., DBCO or azide). In one embodiment, the T cell activation molecules and the T cell costimulatory molecules can be adsorbed via affinity pairing comprising a biotin-streptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an aptamer affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelating lipid pair, a metal-chelating lipid-histidine (HIS)-tagged protein pair, or a combination thereof. In one embodiment, the T cell activation molecule and the T cell costimulatory molecule can be adsorbed via chemical coupling, including azide-alkyne chemistry (AAC), dibenzo-cyclooctyne linkage (DCL), or tetrazine-alkene linkage (TAL).

[0022] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each independently coated on the fluid-supported lipid bilayer (SLB). Alternatively, in another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each independently partially embedded in the fluid-supported lipid bilayer (SLB).

[0023] In another embodiment, the present invention relates to an antigen-presenting cell mimetic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each independently adsorbed onto the mesoporous silica microrods (MSR).

[0024] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activation molecules and the T cell costimulatory molecules are each, independently, an antibody molecule or an antigen-binding fragment thereof.

[0025] In another embodiment, the present invention relates to an antigen-presenting cell-mimicking scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activating molecules are selected from the group consisting of an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD2 antibody or antigen-binding fragment thereof, an anti-CD47 antibody or antigen-binding fragment thereof, an anti-macrophage scavenger receptor (MSR1) antibody or antigen-binding fragment thereof, an anti-T cell receptor (TCR) antibody or antigen-binding fragment thereof, an MHC peptide-loaded major histocompatibility complex (MHC) molecule or multimer thereof, and an MHC-immunoglobulin (Ig) conjugate or multimer thereof, or a combination thereof.

[0026] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell costimulatory molecules are 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 (PD1), and CRACC (CD319, BLAME), or an antigen-binding fragment thereof.

[0027] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activation molecules and T cell costimulatory molecules comprise bispecific antibodies or antigen-binding fragments thereof.

[0028] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) comprising a plurality of activating and costimulatory molecules, wherein the T cell activating and T cell costimulatory molecules comprise pairs selected from the group consisting of CD3 / CD28, CD3 / ICOS optionally with CD28, CD3 / CD27 optionally with CD28, and CD3 / CD137 optionally with CD28, or combinations thereof.

[0029] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) that further comprises an immunoglobulin molecule that specifically binds to the Fc fusion protein.

[0030] In another embodiment, the present invention relates to an antigen-presenting cell-mimetic scaffold (APC-MS) further comprising 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), C-X-C motif chemokine ligand 12 (CXCL12), interferon-γ (IFNγ), or FMS-like tyrosine kinase 3 (Flt-3) ligand, or an agonist, mimetic, variant, functional fragment, or combination thereof. In one embodiment, the scaffold further comprises a recruitment compound that is granulocyte-macrophage colony-stimulating factor (GM-CSF), or an agonist, mimetic, variant, or functional fragment thereof.

[0031] In another embodiment, the present invention relates to an antigen-presenting cell mimic scaffold (APC-MS) further comprising an antigen. In one embodiment, the antigen comprises a tumor antigen. Still further under this embodiment, the tumor antigen may be selected from the group consisting of MAGE-1, MAGE-2, MAGE-3, CEA, tyrosinase, midkine, 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, and 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, 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-type glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5, SCP-I, CT-7, cdc27, adenomatous polyposis coli The antigen is selected from the group consisting of anti-cancer drug protease inhibitor (APC), fodrin, PIA, connexin 37, Ig-idiotype, P15, GM2, GD2 ganglioside, the Smad family of tumor antigens, IMP-1, EBV-encoded nuclear antigen (EBNA)-I, UL16-binding protein-like transcript 1 (Mult1), RAE-1 protein, H60, MICA, MICB, c-erbB-2, neoantigens identified in a patient-specific manner, or immunogenic peptides thereof, or combinations thereof.

[0032] In a related embodiment, the present invention relates to an antigen-presenting cell-mimetic scaffold (APC-MS) comprising: a base layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto 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 achieve a desired scaffold composition. In one embodiment, the weight ratio of the SLB to the 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, or about 1:10.

[0033] In another embodiment, the present invention relates to an antigen-presenting cell-mimetic scaffold (APC-MS) comprising: a base layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto the scaffold, wherein the continuous fluid-supported lipid bilayer (SLB) comprises a lipid containing 14 to 23 carbon atoms. In one embodiment, the lipid is phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylserine (PS), or a phosphoinositide, or a derivative thereof. In one embodiment, the APC-MS comprises a fluid-supported lipid bilayer (SLB) comprising a lipid selected from the group consisting of 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 comprises a lipid composition that mimics the lipid composition of a mammalian cell membrane (e.g., the plasma membrane of a human cell). The lipid composition of many mammalian cell membranes has been characterized and is readily ascertainable by one of skill 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 comprises 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 phosphosphingolipid with a saturated or unsaturated tail containing 6 to 20 carbons, or a combination thereof.

[0034] In another embodiment, the present invention relates to an antigen-presenting cell-mimetic scaffold (APC-MS) comprising: a base layer comprising high surface area mesoporous silica microrods (MSR); a continuous fluid-supporting lipid bilayer (SLB) layered on the MSR base layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed to the scaffold; and a plurality of T cell homeostasis factors adsorbed to the scaffold, wherein the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold maintains a continuous fluid system for at least 14 days. In some embodiments, the MSR-SLB scaffold maintains a continuous fluid system 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 in 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 than 50 days. In some embodiments, the lipid bilayer of the MSR-SLB scaffold degrades in 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 than 50 days.

[0035] In another embodiment, the present invention relates to an antigen-presenting cell-mimetic scaffold (APC-MS) comprising: a base layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto the scaffold, wherein the weight ratio of the mesoporous silica microrods (MSR) to the T cell activation molecules / costimulatory molecules is between about 1:1 and about 50:1. In one embodiment, the ratio of MSR to T cell activation molecules / costimulatory molecules reflects the weight of the MSR to the weight of the antibody used as the T cell activation molecule / costimulatory molecule. In another embodiment, the MSR:antibody weight ratio is adjusted to achieve a desired scaffold composition. In one embodiment, the weight ratio of the SLB to the 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 in between), 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, or about 40:1.

[0036] In another embodiment, the present invention relates to an antigen-presenting cell-mimetic scaffold (APC-MS) comprising: a base layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto the scaffold, wherein the scaffolds are stacked to selectively allow T cell infiltration into the mesoporous silica microrods (MSR). In one embodiment, the present invention further provides an APC-MS, wherein the T cell activation molecules and / or costimulatory molecules are present on the scaffold at a concentration sufficient to allow in situ manipulation of T cells.

[0037] In another aspect, the present invention relates to a pharmaceutical composition comprising: a base layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; an antigen-presenting cell-mimetic scaffold (APC-MS) comprising a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto the scaffold; and a pharmaceutically acceptable carrier. In one embodiment, the present invention further provides a pharmaceutical composition formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

[0038] In another aspect, the present invention relates to a composition comprising: a base layer comprising high-surface-area mesoporous silica microrods (MSR); a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer; an antigen-presenting cell-mimetic scaffold (APC-MS) comprising a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold; and a plurality of T cell homeostasis factors adsorbed onto the scaffold; and T cells clustered within the scaffold. In one embodiment, the present invention further provides a composition comprising APC-MS and T cells selected from the group consisting of natural killer (NK) cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs), or a combination thereof.

[0039] Still further, embodiments of the present invention relate to methods for treating a disease in a subject in need thereof, comprising contacting a sample containing 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 expanding the T cell population; and administering the activated, costimulated, maintained, and optionally expanded T cells to the subject, thereby treating the disease in the subject. In one embodiment, the present invention further relates to methods for treating a disease in a subject in need thereof, further comprising restimulating the T cell population prior to the administering step. In one embodiment, the method comprises expanding the T cell population after contact with the scaffold for a period of between two and five days.

[0040] In another therapeutic embodiment, the present invention relates to a method for treating a disease in a subject in need thereof, comprising the steps of contacting a sample containing a T cell population obtained from the subject, the sample being a blood sample, a bone marrow sample, a lymph sample, or a spleen sample, with the antigen-presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally expanding the T cell population; and administering the activated, costimulated, maintained, and optionally expanded 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 is provided for the treatment of cancer, and the scaffold comprises at least one cytotoxic T cell-specific activating molecule and at least one cytotoxic T cell-specific costimulating molecule.

[0041] In another therapeutic embodiment, the present invention relates to a method for treating cancer in a subject in need thereof, comprising contacting a sample containing a T cell population obtained from the subject with the antigen-presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally expanding the T cell population; and administering the activated, costimulated, maintained, and optionally expanded T cells to the subject, thereby treating the cancer in the subject. In one embodiment, the cancer is selected from the group consisting of 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, precancerous lesions in the lung, colon cancer, melanoma, and bladder cancer. In one embodiment, the method may further comprise sorting and optionally enriching cytotoxic T cells from the sample and / or the expanded cell population.

[0042] 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 containing a T cell population obtained from the subject with the antigen-presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally expanding the T cell population; and administering the activated, costimulated, maintained, and optionally expanded T cells to the subject, thereby treating the immunodeficiency disorder in the subject. In one embodiment, the scaffold comprises 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 the group consisting of primary immunodeficiency disorders and acquired immunodeficiency disorders. In one embodiment, the method may be used to treat acquired immune deficiency syndrome (AIDS), or a genetic disorder selected from the group consisting of DiGeorge syndrome (DGS), chromosomal breakage syndrome (CBS), ataxia-telangiectasia (AT) and Wiskott-Aldrich syndrome (WAS), or a combination thereof.

[0043] In another embodiment, the present invention relates to a method for treating a disease in a subject in need thereof, comprising contacting a sample containing a T cell population obtained from the subject with the antigen-presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally expanding the T cell population; further sorting and optionally enriching the T cells from the sample and / or the expanded cell population; and administering the activated, costimulated, maintained, and optionally expanded T cells to the subject, thereby treating the disease in the subject. In one embodiment, the T cells may be selected from the group consisting of natural killer (NK) cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs), or a combination thereof.

[0044] In another embodiment, the present invention relates 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 the antigen-presenting cell-mimicking scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the population of T cells; optionally expanding the population of T cells; optionally further sorting and enriching the T cells from the sample and / or the expanded cell population; and administering the activated, costimulated, maintained, and optionally expanded T cells to the subject, thereby treating the autoimmune disorder in the subject.

[0045] In another embodiment, the present invention relates to a method for treating a disease in a subject in need thereof, comprising contacting a sample containing a T cell population obtained from the subject with the antigen-presenting cell mimic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the T cell population; optionally expanding the T cell population; optionally further sorting and enriching the T cells from the sample and / or the expanded cell population; and subcutaneously or intravenously administering the activated, costimulated, maintained, and optionally expanded T cells to the subject, thereby treating the disease in the subject. In one embodiment, the T cells can be activated, costimulated, homeostatically maintained, and optionally expanded by contacting the sample with the scaffold for a period of between about 1 day and about 20 days.

[0046] In another embodiment, the present invention relates to a method for engineering T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS) with a biological sample from a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample, thereby engineering the T cells. In one embodiment, the engineering may include promoting T cell stimulation, activation, altered viability, growth, division, differentiation, expansion, proliferation, exhaustion, anergy, quiescence, apoptosis, or death. In one embodiment, the engineering preferably includes promoting T cell expansion or proliferation. In a further embodiment, the engineered T cells may be further transformed. In a specific embodiment, the T cells may be transformed to express a chimeric antigen receptor (CAR). The CAR T cell product may be further expanded by incubating with the antigen-presenting cell mimic scaffold (APC-MS) containing an antigen specific for the CAR T cells. In certain embodiments, the CAR T cell-specific antigen is selected from the group consisting of CD19, CD22, or a fragment or variant thereof. In some embodiments, the CAR T cell-specific antigen is a tumor antigen. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-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, prostate, 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 generation to generate a larger population of CAR T cells.

[0047] In another embodiment, the present invention relates to a method for manipulating T cells, comprising contacting the antigen-presenting cell-mimicking scaffold (APC-MS) with the high-surface-area mesoporous silica microrods (MSR) and the continuous fluid-supported lipid bilayer (SLB), wherein the method confers increased expansion of the T cell population after about one week of contact with the scaffold compared to a control scaffold comprising a base layer comprising the high-surface-area mesoporous silica microrods (MSR) and the continuous fluid-supported lipid bilayer (SLB), but not the T cell activation molecule and the T cell costimulatory molecule. In one embodiment, the method confers about a 50- to 800-fold increase in expansion of the T cell population after about one week of contact with the scaffold compared to a control scaffold comprising a base layer comprising the high-surface-area mesoporous silica microrods (MSR) and the continuous fluid-supported lipid bilayer (SLB), but not the T cell activation molecule and the T cell costimulatory molecule.

[0048] In another embodiment, the present invention relates to a method for manipulating T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS), wherein the method confers increased expansion of the T cell population after about one week of contact with the scaffold compared to superparamagnetic spherical polymer particles (DYNABEAD) comprising the T cell activation molecule and the T cell costimulatory molecule. In one embodiment, the method confers about a 5- to 20-fold increase in expansion of the T cell population after about one week of contact with the scaffold compared to superparamagnetic spherical polymer particles (DYNABEAD) comprising the T cell activation molecule and the T cell costimulatory molecule.

[0049] In another embodiment, the present invention relates to a method for improving the metabolic activity of T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS) with a biological sample from a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample, thereby improving T cell metabolic activity. In one embodiment, the method confers improved metabolic activity of the population of T cells after about one week of contact with the scaffold, compared to a control scaffold comprising a base layer comprising the high surface area mesoporous silica microrods (MSR) and the continuous fluid-supported lipid bilayer (SLB), but not the T cell activation molecule and the T cell costimulatory molecule. In one embodiment, the method confers an approximately 5- to 20-fold improved metabolic activity of the T cell population after about one week of contact with the scaffold compared to a control scaffold comprising a base layer comprising the high surface area mesoporous silica microrods (MSR) and the continuous fluid-supported lipid bilayer (SLB), but not comprising the T cell activation molecule and the T cell costimulatory molecule. In one embodiment, the method confers an improved metabolic activity of the T cell population after about one week of contact with the scaffold compared to superparamagnetic spherical polymer particles (DYNABEAD) comprising the T cell activation molecule and the T cell costimulatory molecule. In one embodiment, the method further confers an approximately 1- to 10-fold increase in the expansion of the T cell population after about one week of contact with the scaffold compared to superparamagnetic spherical polymer particles (DYNABEAD) comprising the T cell activation molecule and the T cell costimulatory molecule.

[0050] In another embodiment, the present invention relates to a method for screening for metabolically active T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS) with a biological sample from a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample; identifying metabolically active cells in the activated, costimulated, homeostatically maintained, and optionally expanded population of T cells; thereby screening for metabolically active T cells. In one embodiment, the expanded T cells are metabolically active for at least about 7 days after contact with the scaffold. In one embodiment, the expanded T cells form aggregates for at least about 7 days after contact with the scaffold.

[0051] In yet another embodiment, the present invention relates to a method for generating a polyclonal population of T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS) with a biological sample from a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample; identifying a specific population of T cells from the expanded population of T cells based on expression of multiple markers on the expanded T cells; and optionally isolating or purifying the identified population of T cells, thereby generating a polyclonal population of T cells. In one embodiment, the method can be adapted for generating a polyclonal population of CD4+ or CD8+ cells. In a related embodiment, the method can be adapted for generating a polyclonal population of CD4+ / FOXP3+ T cells. Furthermore, the method can be adapted for generating a polyclonal population of CD44+ / CD62L- T cells (effector memory and / or effector T cells). In another embodiment, the method may be adapted for generating a polyclonal population of CD8+ / CD69+ T cells (activated T cells). In another embodiment, the method may be adapted for generating a polyclonal population of Granzyme B+ CD8+ T cells (cytotoxin-secreting T cells). In yet another embodiment, the method may be adapted for generating a polyclonal population of IFNγ+ T cells (activator cytokine-secreting T cells). In yet another embodiment, the method may be adapted for generating a polyclonal population of CD62L+ / CCR7+ T cells (memory T cells).

[0052] In another embodiment, the present invention relates to a method for generating a polyclonal subpopulation of T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS) with a biological sample from a subject, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample; identifying a specific population of exhausted T cells from the expanded population of T cells based on expression of multiple markers on the expanded T cells; and optionally removing the identified population of T cells, thereby generating a polyclonal subpopulation of T cells. In one embodiment, the exhausted T cells are identified or isolated based on cell surface expression of CD8+ / PD-1+. In another embodiment, the exhausted T cells are identified or isolated based on cell surface expression of LAG3+ / TIM3+.

[0053] In another embodiment, the present invention relates to a method for ex vivo engineering of T cells, comprising contacting the antigen-presenting cell mimic scaffold (APC-MS) with a biological sample from a subject ex vivo, thereby activating, costimulating, homeostatically maintaining, and optionally expanding a population of T cells present in the sample, thereby engineering the T cells ex vivo. 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 further comprise detecting production of one or more cytokines or cytotoxins by the engineered T cells. In one embodiment, the method further comprises detecting production by the engineered T cells of a cytokine selected from the group consisting of interferon-γ (IFNγ), tissue necrosis factor α (TNFα), IL-2, IL-1, IL-4, IL-5, IL-10, and IL-13, IL-17, or a combination thereof.

[0054] In a related embodiment, the invention relates to a method for ex vivo manipulation of T cells according to the aforementioned method, wherein the engineered T cells are T helper 1 (Th1) cells, and the method comprises detecting the production of a cytokine selected from the group consisting of IL-2, interferon gamma (IFNγ), and tissue necrosis factor alpha (TNFα), or a combination thereof. Alternatively, in a related embodiment, the invention relates to a method for ex vivo manipulation of T cells according to the aforementioned method, wherein the engineered T cells are T helper 2 (Th2) cells, and the method comprises detecting the production of a cytokine selected from the group consisting of IL-4, IL-5, IL-10, and IL-13, or a combination thereof. In yet a further related embodiment, the invention relates to a method for ex vivo manipulation of T cells according to the aforementioned method, wherein the engineered T cells are cytotoxic T (Tc) cells, and the method comprises detecting the production of a cytokine selected from the group consisting of interferon gamma (IFNγ) and lymphotoxin alpha (LTα / TNFβ), or a combination thereof. In one embodiment, the engineered T cells are cytotoxic T (Tc) cells, and the method includes detecting secretion of a cytotoxin selected from the group consisting of granzymes or perforin, or a combination thereof.

[0055] In a related embodiment, the invention relates to a method for ex vivo manipulation of T cells according to the aforementioned method, wherein the method further comprises detecting expression of a cell surface marker on the manipulated T cells. In one embodiment, the cell surface marker is selected from the group consisting of CD69, CD4, CD8, CD25, CD62L, FOXP3, HLA-DR, CD28, and CD134, or a combination thereof. Alternatively or additionally, in one embodiment, the cell surface marker is a non-T cell marker selected from the group consisting of CD36, CD40, and CD44, or a combination thereof.

[0056] In another related embodiment, the invention relates to a method for ex vivo manipulation of T cells according to the aforementioned method, wherein the subject is a human subject.

[0057] In another related embodiment, the invention relates to a method for in vivo manipulation of T cells according to the aforementioned method, wherein the scaffold is administered to the subject to allow a biological sample containing the T cells to contact the scaffold in vivo. In one embodiment, the scaffold can be maintained in the subject for a period of between about 3 days and about 15 days, preferably between about 7 days and about 11 days. In some embodiments, the scaffold can be maintained in the subject 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.

[0058] In yet another embodiment, the present invention relates to a method for producing the antigen-presenting cell-mimicking scaffold (APC-MS), comprising the steps of: (a) providing a base layer comprising high surface area mesoporous silica microrods (MSR); (b) optionally loading the T cell homeostasis factor onto the MSR; (c) layering a continuous fluid-supported lipid bilayer (SLB) onto the base layer comprising the MSR, thereby producing an MSR-SLB scaffold; (d) if step (b) was not performed, loading the T cell homeostasis factor 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 the T cell activation molecule and the T cell costimulatory molecule onto the MSR-SLB scaffold, thereby producing the APC-MS. In one embodiment, the method may further comprise assembling a plurality of scaffolds to produce a stack with sufficient porosity to allow T cell infiltration. In one embodiment, the method may comprise loading at least one additional agent selected from the group consisting of a growth factor, a cytokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, or a fragment thereof, or a combination thereof.

[0059] Other features and advantages of the present invention will become apparent from the following detailed description and drawings. [Brief explanation of the drawings]

[0060] [Figure 1]Figure 1 shows phase-contrast and fluorescence microscopy images of lipids associated with mesoporous silica microrods (MSR). The top panel shows a composite photograph of the lipids and mesoporous silica microrods at a lipid:MSR ratio of 1:20 (scale = 200 μm). The middle panel shows a composite photograph of lipids and mesoporous silica microrods at a lipid:MSR ratio of 1:4 (scale = 200 μm). The bottom panel shows a composite phase-contrast photograph of lipids associated with MSR at higher magnification (scale = 20 μm).

[0061] [Figure 2] Figures 2A, 2B, 2C, and 2D show that the assembly and properties of antigen-presenting cell-mimicking scaffolds (APC-MS) depend on the lipid type and lipid content. Figure 2A shows the chemical structures of various lipids. Abbreviations: DOPC-dioleoylphosphatidylcholine; POPC-palmitoyl-oleoylphosphatidylcholine; and DSPC-distearoylphosphatidylcholine. Figure 2B shows the percentage of lipid retained in various compositions, including mesoporous silica microrods (MSRs) and fluid-supported lipid bilayers (SLBs). In this experiment, a 250 μg lipid payload was loaded onto a 500 μg MSR composition. Figure 2C shows the change in relative fluorescence of various MSR-SLB compositions containing DOPC, POPC, or DSPC in phosphate-buffered saline (PBS) over a 2-week (14-day) period at 37 °C. FIG. 2D shows the change in relative fluorescence of various MSR-SLB compositions containing DOPC, POPC, or DSPC in complete Roswell Park Memorial Institute medium (cRPMI) over a 2-week (14-day) period at 37° C.

[0062] [Figure 3]Figure 3 shows the stability of various MSR-SLB compositions in PBS (lipid coated) on days 0, 3, 7, and 14, as analyzed by phase contrast and fluorescence microscopy. The top panel shows the stability of DOPC in the MSR-SLB composition; the middle panel shows the stability of POPC in the MSR-SLB composition; and the bottom panel shows the stability of DSPC in the MSR-SLB composition.

[0063] [Figure 4ABC] Figures 4A, 4B, 4C, 4D, and 4E show the change in assembly and properties of the MSR-SLB fluidic structure over time. Figure 4A shows phase-contrast and fluorescence microscopy images of lipids associated with mesoporous silica microrods (MSR) taken at high magnification before (pre), immediately after (t = 0), and 5 minutes after (t = 5 minutes) bleaching of the lipid composition (scale = 2 μM). Figure 4B shows the change in fluorescence recovery after photobleaching (FRAP) over time. The fluorescence "source" is shown in region (2), the fluorescence "sink" is shown in region (3), and the normalized point is shown in region (1). The differential distribution was most evident early after seeding, reaching equilibrium at approximately 2 minutes (120 seconds). Figure 4C shows a smooth-fitting curve showing the average change in FRAP over time as obtained from the normalized images. Figures 4D and 4E show two sets of high-resolution images of the MSR-SLB fluid structure before (pre), immediately after (t=0) and 3 minutes after (t=3 minutes) bleaching of the lipid composition. [Figure 4DE]Figures 4A, 4B, 4C, 4D, and 4E show the change in assembly and properties of the MSR-SLB fluidic structure over time. Figure 4A shows phase-contrast and fluorescence microscopy images of lipids associated with mesoporous silica microrods (MSR) taken at high magnification before (pre), immediately after (t = 0), and 5 minutes after (t = 5 minutes) bleaching of the lipid composition (scale = 2 μM). Figure 4B shows the change in fluorescence recovery after photobleaching (FRAP) over time. The fluorescence "source" is shown in region (2), the fluorescence "sink" is shown in region (3), and the normalized point is shown in region (1). The differential distribution was most evident early after seeding, reaching equilibrium at approximately 2 minutes (120 seconds). Figure 4C shows a smooth-fitting curve showing the average change in FRAP over time as obtained from the normalized images. Figures 4D and 4E show two sets of high-resolution images of the MSR-SLB fluid structure before (pre), immediately after (t=0) and 3 minutes after (t=3 minutes) bleaching of the lipid composition.

[0064] [Figure 5]Figures 5A and 5B show the structural and functional properties of MSR-SLB compositions containing various moieties. 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 shows a schematic diagram of the structure of an APC-MS comprising a lipid bilayer of POPC containing phycoerythrin-biotin (biotin-PE), which is conjugated to a streptavidin molecule (e.g., streptavidin dimer), which is in turn conjugated to a biotinylated antibody (e.g., biotinylated anti-CD3 antibody or biotinylated anti-CD28 antibody or another specific or nonspecific antibody). Figure 5B shows spectrophotometric analysis of B3Z reporter cell β-galactosidase expression after treatment with MPS (silica), POPC (lipid), MPS-POPC composite, biotinylated MPS-POPC composite (with or without streptavidin), and combinations of MPS-POPC composite with phycoerythrin-biotin (biotin-PE) and / or biotinylated antibody in the presence or absence of streptavidin. A significant increase in absorbance is observed in MSR-SLB compositions containing all individual components—phosphoethanolamine-biotin (biotin-PE) conjugated to biotinylated antibody via a streptavidin linker (dark bars; ** indicates statistical significance (p<0.001, analyzed by one-way ANOVA followed by Tukey's HSD post-hoc test; data represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments).

[0065] [Figure 6] Figures 6A and 6B show the controlled release of IL-2 from an MSR-SLB composition containing IL-2. Figure 6A shows an electron micrograph of the porous structure of an MSR containing IL-2 (scale bar = 100 nm). Figure 6B shows a plot of the cumulative release levels of IL-2 over a 15-day period.

[0066] [Figure 7]Figures 7A and 7B show confocal microscopy images showing the infiltration of T cells (spheres) into an antigen-presenting cell mimetic scaffold containing MSR-SLB composites. Figure 7A shows cells stained with two different dyes. Figure 7B shows cells stained with a single dye (showing live cells).

[0067] [Figure 8] Figure 8 shows phase-contrast and fluorescence images of lipids associated with mesoporous silica microrods (MSR) cocultured with primary T cells. We observed that primary T cells tended to form cell / material clusters when T cell activation cues were attached to the surface of the material. The bottom panel shows a composite photograph 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 a higher magnification of the MSR-SLB composite containing both conjugated antibodies and IL-2 (scale = 20 μm).

[0068] [Figure 9] Figures 9A and 9B show dose-response charts of antibody-induced changes in mouse splenic T cells. Figure 9A shows polyclonal expansion of T cells after 3 days of stimulation on 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 with antibody). Three different doses of antibody (MSR:antibody ratios of 1:50, 1:25, and 1:10) were studied. Figure 9B shows IFNγ secretion after 3 days of stimulation on 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 with antibody). Three different doses of antibody (MSR:antibody ratios of 1:50, 1:25, and 1:10) were studied.

[0069] [Figure 10]Figures 10 and 11 show that the antigen-presenting cell mimic scaffold (APC-MS) of the present invention promotes rapid expansion of metabolically active T cells. Figure 10 shows the fold expansion of primary T cells upon incubation with controls (mock; none; SLB + IL-2; DYNABEAD + IL-2) or experimental compositions. Incubation of primary T cells with the composition of the present invention significantly induced T cell expansion (with or without restimulation) compared with mock or no SLB compositions. More importantly, incubation of primary T cells with the scaffold of the present invention resulted in measurably stronger proliferation at day 7 upon restimulation compared with DYNABEADS and IL-2 compositions. FIG. 11 shows a bar graph of the cellular metabolic activity (as measured by relative fluorescence units (RFU) of Alamar Blue reduction normalized to cell number) of T cells incubated with a scaffold of the invention loaded with IL-2 (SLB / IL2 / ABS) or IL-2-loaded DYNABEADS (DYNABEADS-IL2). [Figure 11] Figures 10 and 11 show that the antigen-presenting cell mimic scaffold (APC-MS) of the present invention promotes rapid expansion of metabolically active T cells. Figure 10 shows the fold expansion of primary T cells upon incubation with controls (mock; none; SLB + IL-2; DYNABEAD + IL-2) or experimental compositions. Incubation of primary T cells with the composition of the present invention significantly induced T cell expansion (with or without restimulation) compared with mock or no SLB compositions. More importantly, incubation of primary T cells with the scaffold of the present invention resulted in measurably stronger proliferation at day 7 upon restimulation compared with DYNABEADS and IL-2 compositions. FIG. 11 shows a bar graph of the cellular metabolic activity (as measured by relative fluorescence units (RFU) of Alamar Blue reduction normalized to cell number) of T cells incubated with a scaffold of the invention loaded with IL-2 (SLB / IL2 / ABS) or IL-2-loaded DYNABEADS (DYNABEADS-IL2).

[0070] [Figure 12]Figures 12A and 12B show 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 shows photomicrographs (4x magnification) of splenic T cell aggregates upon incubation with DYNABEADS or APC-MS on days 0, 3, and 7. Figure 12B shows photomicrographs (10x magnification) of splenic T cell aggregates upon incubation with DYNABEADS or APC-MS on days 0, 3, and 7 (white scale bar = 100 μM).

[0071] [Figure 13A] Figures 13A and 13B show polyclonal expansion of mouse splenic T cells upon incubation with APC-MS or DYNABEADS. Figure 13A shows 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). The values ​​on the X-axis indicate the intensity of CD8+ staining, and the values ​​on the Y-axis indicate the intensity of CD4+ staining. Flow data were gated against the Fluorescence Minus ONE (FMO) control for each sample at each time point. Data are representative of at least two independent experiments. Figure 13B is a line graph showing the change in percentage of CD4+ vs. CD8+ T cell subpopulations 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, the cells were divided into two subpopulations, where the first subpopulation was restimulated (dashed line) and the second subpopulation 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. [Figure 13B]Figures 13A and 13B show polyclonal expansion of mouse splenic T cells upon incubation with APC-MS or DYNABEADS. Figure 13A shows 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). The values ​​on the X-axis indicate the intensity of CD8+ staining, and the values ​​on the Y-axis indicate the intensity of CD4+ staining. Flow data were gated against the Fluorescence Minus ONE (FMO) control for each sample at each time point. Data are representative of at least two independent experiments. Figure 13B is a line graph showing the change in percentage of CD4+ vs. CD8+ T cell subpopulations 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, the cells were divided into two subpopulations, where the first subpopulation was restimulated (dashed line) and the second subpopulation 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.

[0072] [Figure 14]Figure 14 shows the measurement of polyclonal expansion of a subset of FoxP3+ mouse splenic T cells upon incubation with APC-MS or DYNABEADS. Results are shown in the form of 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). The values ​​on the X-axis indicate the intensity of FoxP3+ staining, and the values ​​on the Y-axis indicate the intensity of CD4+ staining. Rectangular gates were applied to count the number and / or percentage of FoxP3+ cells in various fractions. As shown, expansion of FoxP3+ mouse splenic T cells was limited or absent with certain formulations.

[0073] [Figure 15] Figure 15 shows the polyclonal expansion of a subset of CD62L+ mouse splenic T cells upon incubation with APC-MS or DYNABEADS. Results are shown in the form of 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). Here, values ​​on the X-axis indicate the intensity of CD62L+ staining, and values ​​on the Y-axis indicate the intensity of CD44+ staining. CD62L+ cells appear on the right side of the scatter plot (upper and lower quadrants).

[0074] [Figure 16]Figure 16 shows the polyclonal expansion of a subset of CD8+ / CD69+ mouse splenic T cells upon incubation with APC-MS or DYNABEADS. The results are shown in the form of 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). Here, values ​​on the X-axis indicate the intensity of CD8+ staining, and values ​​on the Y-axis indicate the intensity of CD69+ staining. CD8+ / CD69+ cells appear in the upper right quadrant of the scatter plot.

[0075] [Figure 17] Figure 17 shows the polyclonal expansion of a subset of CD8+ / Granzyme B+ mouse splenic T cells upon incubation with APC-MS or DYNABEADS. The results are shown in the form of 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). Here, the values ​​on the X-axis indicate the intensity of CD8+ staining, and the values ​​on the Y-axis indicate the intensity of Granzyme B+ staining. CD8+ / Granzyme B+ cells appear in the upper right quadrant of the scatter plot.

[0076] [Figure 18] Figure 18 shows T cell secretion of IFNγ (pg / cell) at various time points (t = 0, 5, 7, 11, and 13 days) after incubation with APC-MS (squares) or DYNABEADS (triangles). After 7 days of incubation, the cells were divided into two subpopulations: the first subpopulation was restimulated (dashed line), and the second subpopulation was treated with IL-2 (solid line). Here, APC-MS was used in the restimulation of both the cell populations incubated with APC-MS and the cell populations incubated with DYNABEADS.

[0077] [Figure 19] Figure 19 shows the levels of PD-1+ mouse splenic T cells upon incubation with APC-MS or DYNABEADS. Results are shown in the form of 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). Values ​​on the X-axis indicate the intensity of CD8+ staining, and values ​​on the Y-axis indicate the intensity of PD-1+ staining (a potential marker of exhaustion).

[0078] [Figure 20] Figures 20A and 20B show the effect of incubating human peripheral blood T cells with various compositions. Figure 20A shows a line graph of polyclonal expansion at various time points (t = 0, 5, 7, 11, and 13 days) of primary T cells incubated with control or experimental scaffolds. Control scaffolds contain a mock ("mock"; black line) composition and a composition without an SLB ("none"; red line). Experimental scaffolds contain (1) DYNABEADS (blue line) and (2) a lipid bilayer (SLB) of the present invention (green line). Figure 20B shows a bar graph showing metabolic activity (measured by a 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 contain a mock ("mock"; "m") composition and a composition without an SLB ("none"; "f"). The experimental scaffolds include (1) DYNABEADS ("d") and (2) lipid bilayers (SLBs) of the present invention ("s").

[0079] [Figure 21]Figures 21A and 21B show the effect of incubating human peripheral blood T cells with various anti-CD3 antibodies. Human blood samples obtained from subject 1 (Figure 21A) and subject 2 (Figure 21B) were incubated with a control scaffold ("mock") or experimental scaffolds containing the listed anti-CD3 antibodies—muromonab (OKT3), an antibody recognizing the 17-19 kD ε chain of CD3 within the CD3 antigen / T cell antigen receptor (TCR) complex (HIT3a), and a monoclonal antibody recognizing the 20 kDa subunit of the TCR complex within CD3e (UCHT1). Three different doses were investigated: 5 μg (top slide), 1 μg (bottom slide of subject 2), and 0.5 μg (bottom slide of subject 1). In each case, costimulation was provided with anti-CD28 antibody, where the ratio of anti-CD3 antibody to anti-CD28 antibody was maintained at 1:1. The fold expansion of T cells was measured at various time points (t=0 days, 7 days, 11 days and 13 days).

[0080] [Figure 22] Figure 22 shows the polyclonal expansion of human T cells upon incubation with a control scaffold ("mock") or experimental scaffolds containing the listed anti-CD3 antibodies—OKT3, HIT3a, and UCHT1. The bottom panel shows flow cytometry (FACS) scatter plots of T cell populations at various time points (t = 8, 11, and 14 days) after incubation with APC-MS containing anti-CD3 antibody as a stimulatory molecule and anti-CD28 antibody as a costimulatory molecule. The values ​​on the x-axis of the scatter plot indicate the intensity of CD8+ staining, and the values ​​on the y-axis indicate the intensity of CD4+ staining. The scatter plots are summarized in the line graph in the top panel, which shows the change in the percentage of CD4+ vs. CD8+ T cell subpopulations after incubation with APC-MS containing the aforementioned anti-CD3 antibodies—OKT3 (circles), HIT3a (squares), and UCHT1 (triangles). Two different antibody doses were investigated - 5 μg (1× dilution) and 0.5 μg (1:10× dilution).

[0081] [Figure 23]Figure 23 shows the expression of CD62L and CCR7 on live T cells expanded for 14 days using APC-MS containing IL-2 and the aforementioned anti-CD3 antibodies OKT3 (left panel), HIT3a (middle panel), and UCHT1 (right panel), and an anti-CD28 antibody at a 1:1 ratio at a 1x loading concentration (approximately 5 μg). Expression of CD62L and CCR7 on total live cells is shown in the top panel, and expression of these markers on gated CD8+ cells is shown in the bottom panel. The majority of cells expanded with the APC-MS of the present invention retained CD62L+CCR7+ (which has been shown to be important for in vivo functionality in human patients) after 14 days of incubation. Furthermore, APC-MS scaffolds containing OKT3 were particularly effective at expanding and / or retaining CD62L+CCR7+ T cells compared to scaffolds containing UCHT1 and / or HIT3a.

[0082] [Figure 24] FIG. 24 outlines a representative scheme for fabricating a scaffold of the present invention.

[0083] [Figure 25]Figures 25A and 25B show the design of antigen-presenting cell-mimicking scaffolds (APC-MS). Figure 25A shows an exemplary process for preparing APC-MS: 1) synthesizing mesoporous silica microrods (MSR); 2) adsorbing IL-2 onto the MSR; 3) coating the IL-2-adsorbed MSR with liposomes to form MSR-SLBs; 4) attaching a T cell activation cue to the surface of the MSR-SLB; 5) culturing the MSR-SLBs with T cells; and 6) precipitating and stacking the MSR-SLBs to form a T cell-infiltrated scaffold. A scaffold formed from MSR-SLBs loaded with IL-2 and surface-functionalized with a T cell activation cue is referred to as an APC-MS. Figure 25B shows exemplary structures and functions of distinct APC-MS formulations. IL-2 is released from the APC-MS over time, resulting in paracrine delivery of IL-2 to local T cells. Incorporation of a predetermined amount of biotinylated phospholipid into the liposome formulation allows precise surface attachment of biotinylated T cell activation cues via streptavidin-biotin interactions, mimicking cell surface presentation of cues to T cells by natural APCs. For polyclonal T cell expansion, activating antibodies against CD3 (αCD3) and CD28 (αCD28) are attached (left). For antigen-specific T cell expansion, peptide-loaded MHC (pMHC) and αCD28 are attached (right).

[0084] [Figure 26] Figures 26A and 26B show the physical characterization of the components used to assemble the MSR-SLB. Figure 26A shows a representative bright-field microscope image of the MSR. Scale bar = 100 μm. Figure 26B shows the size distribution of POPC liposomes as measured by dynamic light scattering (DLS). The data in Figure 26B represent the average size distribution of three samples.

[0085] [Figure 27]Figures 27A and 27B show microscopy images of lipid-coated MSR. Figure 27A is a microscopy image showing aggregation of MSR at low lipid:MSR. Representative microscopy images of lipid-coated MSR (lipid:MSR 1:20 w / w) showing MSR (left), fluorophore-tagged phospholipid (1 mol% of total lipid; center), and brightfield images of co-localization of MSR and lipid (right). Scale bar = 200 μm. Figure 27B is a microscopy image of lipid-coated MSR (lipid:MSR 1:4 w / w) showing MSR (left), fluorophore-tagged phospholipid (1 mol% of total lipid; center), and brightfield images of co-localization of MSR and lipid (right). Scale bar = 200 μm.

[0086] [Figure 28AB] Figures 28A-28E show the assembly and characterization of the APC-MS. Figure 28A shows the retention of lipid coating (containing 1 mol% fluorophore-tagged lipid) on MSR over time in either PBS or RPMI medium containing 10% serum (cRPMI), maintaining cell culture conditions. Figure 28B shows representative overlaid fluorescence microscopy images of lipid-coated MSR (MSR, bright field; lipid (1 mol% fluorophore-tagged lipid), green) maintained in cRPMI over time under standard cell culture conditions. Scale bar = 100 μm. Data represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments. Figure 28C shows the quantification of IL-2 released from MSR-SLB (500 μg of MSR) over time (data points) in vitro, along with a one-phase exponential fit (dashed line; R2 = 0.98). Data represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments. Figure 28D is a graph showing quantification of the attachment of various input amounts of biotinylated IgG onto MSR coated with lipid formulations containing 0.01 mol%, 0.1 mol%, or 1 mol% biotinylated lipid. Values ​​above the bars indicate the concentration (μg) of attached IgG for each respective condition. Data represent the mean ± sd of four experimental replicates and are representative of at least two independent experiments. Figure 28E is an SEM image showing tight association of primary human T cells with APC-MS. Scale bar = 10 μm. [Figure 28CDE] Figures 28A-28E show the assembly and characterization of the APC-MS. Figure 28A shows the retention of lipid coating (containing 1 mol% fluorophore-tagged lipid) on MSR over time in either PBS or RPMI medium containing 10% serum (cRPMI), maintaining cell culture conditions. Figure 28B shows representative overlaid fluorescence microscopy images of lipid-coated MSR (MSR, bright field; lipid (1 mol% fluorophore-tagged lipid), green) maintained in cRPMI over time under standard cell culture conditions. Scale bar = 100 μm. Data represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments. Figure 28C shows the quantification of IL-2 released from MSR-SLB (500 μg of MSR) over time (data points) in vitro, along with a one-phase exponential fit (dashed line; R2 = 0.98). Data represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments. Figure 28D is a graph showing quantification of the attachment of various input amounts of biotinylated IgG onto MSR coated with lipid formulations containing 0.01 mol%, 0.1 mol%, or 1 mol% biotinylated lipid. Values ​​above the bars indicate the concentration (μg) of attached IgG for each respective condition. Data represent the mean ± sd of four experimental replicates and are representative of at least two independent experiments. Figure 28E is an SEM image showing tight association of primary human T cells with APC-MS. Scale bar = 10 μm.

[0087] [Figure 29] Figure 29 shows the association of T cells with APC-MS. Representative microscopy images at low magnification (left) and high magnification (right) of MSR-SLBs either not displaying any surface cues (cue-) or displaying αCD3 and αCD28 (cue+) that were cultured with primary mouse T cells for 1 day. 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-tagged lipid; center). A merged image is shown below. Scale bar at low magnification = 500 μm, scale bar at high magnification = 100 μm.

[0088] [Figure 30ABC]Figures 30A, 30B, 30C, 30D, 30E, 30F, and 30G show polyclonal expansion of primary mouse and human T cells. Figure 30A shows representative bright-field microscopy images of primary mouse T cells cultured with DYNABEADS or APC-MS at various time points at low (left) or high (right) magnification. Scale bar = 100 μm. Figure 30B shows the expansion of primary murine T cells that were either untreated (mock) or cultured with free cues (110 nM αCD3, 110 nM αCD28, 1.3 μg / ml IL-2), commercially available CD3 / CD28 murine T cell expansion beads and exogenous IL-2 (DYNABEADS), IL-2-loaded MSR-SLBs without T cell cues presented on the bilayer surface (MSR-SLB(cue-)), or APC-MS (loaded with αCD3, αCD28, and IL-2). The mock and free curves were indistinguishable from the MSR-SLB(cue-) curve. Figure 30C shows the frequency of CD4+ and CD8+ cells among viable single cells over time in APC-MS or Dynabead cultures, as measured by FACS. Data were analyzed using two-way ANOVA followed by Tukey's HSD post-hoc test. Figure 30D shows representative 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 presenting 1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 333 μg / ml MSR; (F2) APC-MS presenting 1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 33 μg / ml MSR; (F3) APC-MS presenting 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 333 μg / ml MSR; and (F4) APC-MS presenting 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 33 μg / ml MSR.Figure 30E shows the expansion of primary human T cells that were either untreated (mock) or cultured with commercially available CD3 / CD28 human T cell expansion beads and exogenous IL-2 (DYNABEADS) or various APC-MS formulations. Figure 30F shows FACS quantification of CD4 and CD8 single-positive cells among live, single CD3+ cells in samples expanded for 14 days with either DYNABEADS or various APC-MS formulations. Figure 30G shows FACS quantification of cells coexpressing PD-1 and LAG-3 among live, single cells in samples expanded with either DYNABEADS or various APC-MS formulations. Data in Figures 30F and 30G represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments. Data in Figure 30E represent the mean ± SD of at least three different donor samples from two independent experiments. Data in Figures 30F and 30G represent the mean ± sd of three different donor samples and are representative of at least two independent experiments. **p<0.01, ***p<0.001. [Figure 30DE]Figures 30A, 30B, 30C, 30D, 30E, 30F, and 30G show polyclonal expansion of primary mouse and human T cells. Figure 30A shows representative bright-field microscopy images of primary mouse T cells cultured with DYNABEADS or APC-MS at various time points at low (left) or high (right) magnification. Scale bar = 100 μm. Figure 30B shows the expansion of primary murine T cells that were either untreated (mock) or cultured with free cues (110 nM αCD3, 110 nM αCD28, 1.3 μg / ml IL-2), commercially available CD3 / CD28 murine T cell expansion beads and exogenous IL-2 (DYNABEADS), IL-2-loaded MSR-SLBs without T cell cues presented on the bilayer surface (MSR-SLB(cue-)), or APC-MS (loaded with αCD3, αCD28, and IL-2). The mock and free curves were indistinguishable from the MSR-SLB(cue-) curve. Figure 30C shows the frequency of CD4+ and CD8+ cells among viable single cells over time in APC-MS or Dynabead cultures, as measured by FACS. Data were analyzed using two-way ANOVA followed by Tukey's HSD post-hoc test. Figure 30D shows representative 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 presenting 1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 333 μg / ml MSR; (F2) APC-MS presenting 1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 33 μg / ml MSR; (F3) APC-MS presenting 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 333 μg / ml MSR; and (F4) APC-MS presenting 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 33 μg / ml MSR.Figure 30E shows the expansion of primary human T cells that were either untreated (mock) or cultured with commercially available CD3 / CD28 human T cell expansion beads and exogenous IL-2 (DYNABEADS) or various APC-MS formulations. Figure 30F shows FACS quantification of CD4 and CD8 single-positive cells among live, single CD3+ cells in samples expanded for 14 days with either DYNABEADS or various APC-MS formulations. Figure 30G shows FACS quantification of cells coexpressing PD-1 and LAG-3 among live, single cells in samples expanded with either DYNABEADS or various APC-MS formulations. Data in Figures 30F and 30G represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments. Data in Figure 30E represent the mean ± SD of at least three different donor samples from two independent experiments. Data in Figures 30F and 30G represent the mean ± sd of three different donor samples and are representative of at least two independent experiments. **p<0.01, ***p<0.001. [Figure 30FG]Figures 30A, 30B, 30C, 30D, 30E, 30F, and 30G show polyclonal expansion of primary mouse and human T cells. Figure 30A shows representative bright-field microscopy images of primary mouse T cells cultured with DYNABEADS or APC-MS at various time points at low (left) or high (right) magnification. Scale bar = 100 μm. Figure 30B shows the expansion of primary murine T cells that were either untreated (mock) or cultured with free cues (110 nM αCD3, 110 nM αCD28, 1.3 μg / ml IL-2), commercially available CD3 / CD28 murine T cell expansion beads and exogenous IL-2 (DYNABEADS), IL-2-loaded MSR-SLBs without T cell cues presented on the bilayer surface (MSR-SLB(cue-)), or APC-MS (loaded with αCD3, αCD28, and IL-2). The mock and free curves were indistinguishable from the MSR-SLB(cue-) curve. Figure 30C shows the frequency of CD4+ and CD8+ cells among viable single cells over time in APC-MS or Dynabead cultures, as measured by FACS. Data were analyzed using two-way ANOVA followed by Tukey's HSD post-hoc test. Figure 30D shows representative 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 presenting 1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 333 μg / ml MSR; (F2) APC-MS presenting 1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 33 μg / ml MSR; (F3) APC-MS presenting 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 333 μg / ml MSR; and (F4) APC-MS presenting 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 added to initial cultures in 33 μg / ml MSR.Figure 30E shows the expansion of primary human T cells that were either untreated (mock) or cultured with commercially available CD3 / CD28 human T cell expansion beads and exogenous IL-2 (DYNABEADS) or various APC-MS formulations. Figure 30F shows FACS quantification of CD4 and CD8 single-positive cells among live, single CD3+ cells in samples expanded for 14 days with either DYNABEADS or various APC-MS formulations. Figure 30G shows FACS quantification of cells coexpressing PD-1 and LAG-3 among live, single cells in samples expanded with either DYNABEADS or various APC-MS formulations. Data in Figures 30F and 30G represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments. Data in Figure 30E represent the mean ± SD of at least three different donor samples from two independent experiments. Data in Figures 30F and 30G represent the mean ± sd of three different donor samples and are representative of at least two independent experiments. **p<0.01, ***p<0.001.

[0089] [Figure 31] Figure 31 shows representative FACS plots of CD4 and CD8 expression on polyclonally expanded primary mouse T cells. Representative FACS plots showing CD4 and CD8 expression on live single cells polyclonally expanded with either APC-MS or DYNABEADS. Flow data were gated against fluorescence minus one (FMO) controls at each time point for each sample. Data are representative of at least two independent experiments.

[0090] [Figure 32AB]Figures 32A, 32B, 32C, and 32D show expanded phenotypic characterization of polyclonally expanded primary murine T cells. Figure 32A shows FACS quantification (left) and a representative FACS plot (right) of granzyme B-positive cells among live single CD8+ cells in samples expanded with either DYNABEADS or APC-MS. Figure 32B shows FACS quantification of FoxP3-positive cells among live single CD4+ cells in samples expanded with either DYNABEADS or APC-MS. Figures 32C and 32D show representative FACS plots showing PD-1 expression on live single cells as a function of CD8 expression. Flow data were gated against fluorescence minus one (FMO) controls for each sample at each time point. Data represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments. [Figure 32CD] Figures 32A, 32B, 32C, and 32D show expanded phenotypic characterization of polyclonally expanded primary murine T cells. Figure 32A shows FACS quantification (left) and a representative FACS plot (right) of granzyme B-positive cells among live single CD8+ cells in samples expanded with either DYNABEADS or APC-MS. Figure 32B shows FACS quantification of FoxP3-positive cells among live single CD4+ cells in samples expanded with either DYNABEADS or APC-MS. Figures 32C and 32D show representative FACS plots showing PD-1 expression on live single cells as a function of CD8 expression. Flow data were gated against fluorescence minus one (FMO) controls for each sample at each time point. Data represent the mean ± SD of three experimental replicates and are representative of at least two independent experiments.

[0091] [Figure 33]Figure 33 shows adhesion molecule expression on polyclonally expanded primary human T cells. FACS quantification of live single cells co-expressing CD62L and CCR7 in samples expanded with either DYNABEADS or various APC-MS formulations. (F1) APC-MS displaying 1 mol% biotinylated lipid saturated with αCD3 and αCD28 in initial cultures at 333 μg / ml MSR; (F2) APC-MS displaying 1 mol% biotinylated lipid saturated with αCD3 and αCD28 in initial cultures at 33 μg / ml MSR; (F3) APC-MS displaying 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 in initial cultures at 333 μg / ml MSR; and (F4) APC-MS displaying 0.1 mol% biotinylated lipid saturated with αCD3 and αCD28 in initial cultures at 33 μg / ml MSR. Data represent the mean ± sd of three different donor samples and are representative of at least two independent experiments.

[0092] [Figure 34ABCD]Figures 34A, 34B, 34C, 34D, and 34E show antigen-specific expansion of primary murine T cells. Figure 34A shows 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). Scale bar = 100 μm. Figure 34B shows the expansion of primary CD8+ OT-I T cells that were either untreated (mock) or cultured with various APC-MS formulations. (F1) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 333 μg / ml MSR; (F2) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 33 μg / ml MSR; (F3) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 0.1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 333 μg / ml MSR; and (F4) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 0.1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 33 μg / ml MSR. Figure 34C shows FACS quantification of IFNγ and TNFα expression by live single CD8+ OT-I T cells expanded for 13 days in various APC-MS formulations and then co-cultured with B16-F10 cells that were either mock-pulsed (−) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (+). Figure 34D shows quantification of in vitro killing of mock-pulsed (−) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (+) B16-F10 target cells by CD8+ OT-I T cells expanded for 13 days in various APC-MS formulations and then co-cultured at various effector:target cell ratios.Figure 34E shows quantification of IFNγ secretion by CD8+ OT-I T cells expanded for 13 days in various APC-MS formulations in response to co-culture with either mock-pulsed (pep-) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (pep+) B16-F10 cells at various effector:target cell ratios. Data in Figures 34B, 34C, 34D, and 34E represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments. [Figure 34E]Figures 34A, 34B, 34C, 34D, and 34E show antigen-specific expansion of primary murine T cells. Figure 34A shows 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). Scale bar = 100 μm. Figure 34B shows the expansion of primary CD8+ OT-I T cells that were either untreated (mock) or cultured with various APC-MS formulations. (F1) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 333 μg / ml MSR; (F2) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 33 μg / ml MSR; (F3) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 0.1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 333 μg / ml MSR; and (F4) APC-MS presenting SIINFEKL (SEQ ID NO: 4) / H-2K(b) and 0.1 mol% biotinylated lipid saturated with αCD28 added to initial cultures at 33 μg / ml MSR. Figure 34C shows FACS quantification of IFNγ and TNFα expression by live single CD8+ OT-I T cells expanded for 13 days in various APC-MS formulations and then co-cultured with B16-F10 cells that were either mock-pulsed (−) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (+). Figure 34D shows quantification of in vitro killing of mock-pulsed (−) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (+) B16-F10 target cells by CD8+ OT-I T cells expanded for 13 days in various APC-MS formulations and then co-cultured at various effector:target cell ratios.Figure 34E shows quantification of IFNγ secretion by CD8+ OT-I T cells expanded for 13 days in various APC-MS formulations in response to co-culture with either mock-pulsed (pep-) or SIINFEKL (SEQ ID NO: 4) peptide-pulsed (pep+) B16-F10 cells at various effector:target cell ratios. Data in Figures 34B, 34C, 34D, and 34E represent the mean ± sd of three experimental replicates and are representative of at least two independent experiments.

[0093] [Figure 35ABCD]Figures 35A, 35B, 35C, 35D, and 35E show the expanded characterization of primary human T cells expanded with antigen-specific APC-MS formulations. Figure 35A shows the total expansion of primary human CD8+ T cell isolates that were mock-treated (30 U / ml IL-2) or cultured with APC-MS (pMHC, αCD28, IL-2 loaded) presenting either the CLG or GLC peptide in HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figures 35B, 35C, and 35D show quantification of IFNγ secretion of CD8+ T cell isolates that were mock-treated (30 U / ml IL-2) or cultured with APC-MS presenting either the CLG peptide (APC-MS CLG) or the GLC peptide (APC-MS GLC) after coculture with T2 cells that were either not pulsed (peptide-) (Figure 35B), pulsed with the CLG peptide (+CLG peptide) (Figure 35C), or pulsed with the GLC peptide (+GLC peptide) (Figure 35D). Data for mock-treated cells are only available for day 7. Figure 35E shows representative FACS plots showing IFNγ and TNFα expression in CD8+ T cell isolates cultured with APC-MS presenting either the CLG peptide (APC-MS / CLG) or the GLC peptide (APC-MS / GLC) after coculture with T2 cells that were either unapplied (no peptide; top), applied with the CLG peptide (+CLG peptide; middle), or applied with the GLC peptide (+GLC peptide; bottom). Data in Figures 35A and 35B represent the mean ± sd of three experimental replicates and are representative of two experiments with two different donor samples. [Figure 35E]Figures 35A, 35B, 35C, 35D, and 35E show the expanded characterization of primary human T cells expanded with antigen-specific APC-MS formulations. Figure 35A shows the total expansion of primary human CD8+ T cell isolates that were mock-treated (30 U / ml IL-2) or cultured with APC-MS (pMHC, αCD28, IL-2 loaded) presenting either the CLG or GLC peptide in HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figures 35B, 35C, and 35D show quantification of IFNγ secretion of CD8+ T cell isolates that were mock-treated (30 U / ml IL-2) or cultured with APC-MS presenting either the CLG peptide (APC-MS CLG) or the GLC peptide (APC-MS GLC) after coculture with T2 cells that were either not pulsed (peptide-) (Figure 35B), pulsed with the CLG peptide (+CLG peptide) (Figure 35C), or pulsed with the GLC peptide (+GLC peptide) (Figure 35D). Data for mock-treated cells are only available for day 7. Figure 35E shows representative FACS plots showing IFNγ and TNFα expression in CD8+ T cell isolates cultured with APC-MS presenting either the CLG peptide (APC-MS / CLG) or the GLC peptide (APC-MS / GLC) after coculture with T2 cells that were either unapplied (no peptide; top), applied with the CLG peptide (+CLG peptide; middle), or applied with the GLC peptide (+GLC peptide; bottom). Data in Figures 35A and 35B represent the mean ± sd of three experimental replicates and are representative of two experiments with two different donor samples.

[0094] [Figure 36ABC]Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, 36J, 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells. Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, and 36J show antigen-specific expansion of primary human T cells from CD8+ T cell isolates. Figures 36A, 36B, and 36D show tetramer analysis of live CD8+ single cells specific for the EBV-derived peptides CLGGLLTMV (SEQ ID NO: 1) (CLG; Figures 36A and 36B) and GLCTLVAML (SEQ ID NO: 2) (GLC; Figures 36D and 36E). Representative FACS plots (Figures 36A and 36D) with counts within gates showing the % of viable single CD8+ cells specific for each tetramer, and quantification of FACS data (Figures 36B and 36E) at various time points for primary HLA-A2+ human CD8+ T cells that were mock-treated (30 U / ml IL-2) or cultured with APC-MS (pMHC, αCD28, loaded with IL-2) presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figure 36F shows the expansion of primary human CD8+ T cells specific for CLG (Figure 36C) or GLC (Figure 36F) that were either mock-treated or cultured with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figures 36G, 36H, and 36I show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells cultured with mock-treated or APC-MS presenting either CLG or GLC peptides in HLA-A2 after coculture with T2 cells that were either unpeptide (-; Figure 36G), pulsed with CLG peptide (+CLG peptide; Figure 36H), or pulsed with GLC peptide (+GLC peptide; Figure 36I). Data for mock-treated cells are only available for day 7.Figure 36J shows quantification of in vitro killing of T2 target cells that were mock-pulsed (no peptide) or pulsed with either CLG peptide (+CLG) or GLC peptide (+GLC) by primary human CD8+ T cells expanded for 14 days with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Figures 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells derived from PBMCs. Figure 36K shows the frequency of GLC-specific cells among viable single CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Figure 36L shows the number of GLC-specific CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Numbers above the bars indicate fold expansion (mean ± sd). Figures 36M and 36N show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells derived from PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2 after coculture with T2 either unapplied (no peptide), applied with CLG peptide (+CLG), or applied with GLC peptide (+GLC) (Figure 36M), as well as IFNγ secretion (Figure 36N). All data represent the mean ± sd of three experimental replicates and are representative of two experiments with two different donor samples. [Figure 36DEF]Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, 36J, 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells. Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, and 36J show antigen-specific expansion of primary human T cells from CD8+ T cell isolates. Figures 36A, 36B, and 36D show tetramer analysis of live CD8+ single cells specific for the EBV-derived peptides CLGGLLTMV (SEQ ID NO: 1) (CLG; Figures 36A and 36B) and GLCTLVAML (SEQ ID NO: 2) (GLC; Figures 36D and 36E). Representative FACS plots (Figures 36A and 36D) with counts within gates showing the % of viable single CD8+ cells specific for each tetramer, and quantification of FACS data (Figures 36B and 36E) at various time points for primary HLA-A2+ human CD8+ T cells that were mock-treated (30 U / ml IL-2) or cultured with APC-MS (pMHC, αCD28, loaded with IL-2) presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figure 36F shows the expansion of primary human CD8+ T cells specific for CLG (Figure 36C) or GLC (Figure 36F) that were either mock-treated or cultured with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figures 36G, 36H, and 36I show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells cultured with mock-treated or APC-MS presenting either CLG or GLC peptides in HLA-A2 after coculture with T2 cells that were either unpeptide (-; Figure 36G), pulsed with CLG peptide (+CLG peptide; Figure 36H), or pulsed with GLC peptide (+GLC peptide; Figure 36I). Data for mock-treated cells are only available for day 7.Figure 36J shows quantification of in vitro killing of T2 target cells that were mock-pulsed (no peptide) or pulsed with either CLG peptide (+CLG) or GLC peptide (+GLC) by primary human CD8+ T cells expanded for 14 days with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Figures 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells derived from PBMCs. Figure 36K shows the frequency of GLC-specific cells among viable single CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Figure 36L shows the number of GLC-specific CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Numbers above the bars indicate fold expansion (mean ± sd). Figures 36M and 36N show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells derived from PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2 after coculture with T2 either unapplied (no peptide), applied with CLG peptide (+CLG), or applied with GLC peptide (+GLC) (Figure 36M), as well as IFNγ secretion (Figure 36N). All data represent the mean ± sd of three experimental replicates and are representative of two experiments with two different donor samples. [Figure 36GHIJ]Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, 36J, 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells. Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, and 36J show antigen-specific expansion of primary human T cells from CD8+ T cell isolates. Figures 36A, 36B, and 36D show tetramer analysis of live CD8+ single cells specific for the EBV-derived peptides CLGGLLTMV (SEQ ID NO: 1) (CLG; Figures 36A and 36B) and GLCTLVAML (SEQ ID NO: 2) (GLC; Figures 36D and 36E). Representative FACS plots (Figures 36A and 36D) with counts within gates showing the % of viable single CD8+ cells specific for each tetramer, and quantification of FACS data (Figures 36B and 36E) at various time points for primary HLA-A2+ human CD8+ T cells that were mock-treated (30 U / ml IL-2) or cultured with APC-MS (pMHC, αCD28, loaded with IL-2) presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figure 36F shows the expansion of primary human CD8+ T cells specific for CLG (Figure 36C) or GLC (Figure 36F) that were either mock-treated or cultured with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figures 36G, 36H, and 36I show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells cultured with mock-treated or APC-MS presenting either CLG or GLC peptides in HLA-A2 after coculture with T2 cells that were either unpeptide (-; Figure 36G), pulsed with CLG peptide (+CLG peptide; Figure 36H), or pulsed with GLC peptide (+GLC peptide; Figure 36I). Data for mock-treated cells are only available for day 7.Figure 36J shows quantification of in vitro killing of T2 target cells that were mock-pulsed (no peptide) or pulsed with either CLG peptide (+CLG) or GLC peptide (+GLC) by primary human CD8+ T cells expanded for 14 days with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Figures 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells derived from PBMCs. Figure 36K shows the frequency of GLC-specific cells among viable single CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Figure 36L shows the number of GLC-specific CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Numbers above the bars indicate fold expansion (mean ± sd). Figures 36M and 36N show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells derived from PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2 after coculture with T2 either unapplied (no peptide), applied with CLG peptide (+CLG), or applied with GLC peptide (+GLC) (Figure 36M), as well as IFNγ secretion (Figure 36N). All data represent the mean ± sd of three experimental replicates and are representative of two experiments with two different donor samples. [Figure 36KLMN]Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, 36J, 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells. Figures 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, and 36J show antigen-specific expansion of primary human T cells from CD8+ T cell isolates. Figures 36A, 36B, and 36D show tetramer analysis of live CD8+ single cells specific for the EBV-derived peptides CLGGLLTMV (SEQ ID NO: 1) (CLG; Figures 36A and 36B) and GLCTLVAML (SEQ ID NO: 2) (GLC; Figures 36D and 36E). Representative FACS plots (Figures 36A and 36D) with counts within gates showing the % of viable single CD8+ cells specific for each tetramer, and quantification of FACS data (Figures 36B and 36E) at various time points for primary HLA-A2+ human CD8+ T cells that were mock-treated (30 U / ml IL-2) or cultured with APC-MS (pMHC, αCD28, loaded with IL-2) presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figure 36F shows the expansion of primary human CD8+ T cells specific for CLG (Figure 36C) or GLC (Figure 36F) that were either mock-treated or cultured with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Data for mock-treated cells are available only for days 0 and 7. Figures 36G, 36H, and 36I show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells cultured with mock-treated or APC-MS presenting either CLG or GLC peptides in HLA-A2 after coculture with T2 cells that were either unpeptide (-; Figure 36G), pulsed with CLG peptide (+CLG peptide; Figure 36H), or pulsed with GLC peptide (+GLC peptide; Figure 36I). Data for mock-treated cells are only available for day 7.Figure 36J shows quantification of in vitro killing of T2 target cells that were mock-pulsed (no peptide) or pulsed with either CLG peptide (+CLG) or GLC peptide (+GLC) by primary human CD8+ T cells expanded for 14 days with APC-MS presenting either the CLG or GLC peptide on HLA-A2. Figures 36K, 36L, 36M, and 36N show antigen-specific expansion of primary human T cells derived from PBMCs. Figure 36K shows the frequency of GLC-specific cells among viable single CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Figure 36L shows the number of GLC-specific CD8+ T cells within PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2. Numbers above the bars indicate fold expansion (mean ± sd). Figures 36M and 36N show the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells derived from PBMCs cultured for 7 days in 30 U / ml IL-2 (mock) or with APC-MS presenting the GLC peptide on HLA-A2 after coculture with T2 either unapplied (no peptide), applied with CLG peptide (+CLG), or applied with GLC peptide (+GLC) (Figure 36M), as well as IFNγ secretion (Figure 36N). All data represent the mean ± sd of three experimental replicates and are representative of two experiments with two different donor samples.

[0095] [Figure 37]Figure 37 shows the degradation of APC-MS scaffolds in vitro. APC-MS (167 μg) presenting αCD3 / αCD28 (1% biotinylated lipid) and releasing IL-2 were cultured with primary mouse T cells (25 × 10 T cells / 167 μg APC-MS). At various time points, the cultures were centrifuged at 700 rcf for 5 minutes, and the Si content in the pellets was quantified via inductively coupled plasma optical emission spectroscopy (ICP-OES; Galbraith Laboratories). Si is undetectable in the culture pellets up to one week after initiation of culture.

[0096] [Figure 38] Figure 38 shows the controlled release of various soluble immunotropic payloads from APC-MS. Four APC-MS were generated, each containing 2 μg of either IL-2, IL-21, TGFβ, or IL-15SA loaded onto 500 μg APC-MS prior to lipid coating. Samples were washed extensively to remove unloaded protein and then maintained at 37°C for up to 28 days. Payload release over time was assessed via ELISA.

[0097] [Figure 39] Figures 39A and 39B show fluorescence recovery after photobleaching (FRAP) experiments using MSR-SLBs containing 10% carboxyfluorescein headgroup-tagged lipids. Figure 39A is a representative image of three independent FRAP events. The images show the fluorescently tagged MSR-SLB before photobleaching (left), immediately after photobleaching (center), and after fluorescence recovery (right). The photobleached area is indicated by a red arrow. Figure 39B shows quantification of fluorescence recovery over time. The fluorescence recovery of eight independent photobleaching runs for different MSR-SLBs is shown as dashed black lines, and the average trend is shown as a solid line.

[0098] [Figure 40AB]Figures 40A, 40B, 40C, and 40D show the results of T cell expansion experiments performed using APC-MS compared to DYNABEAD. The amount of DYNABEAD was normalized to contain the same amount of anti-CD3 and anti-CD28 antibodies as APC-MS. Figure 40A. Bichichoninic acid assay (BCA) analysis for total protein quantification was performed to determine the amount of protein bound to the surface of commercially available mouse or human CD3 / CD28 T cell activator DYNABEADS. DYNABEAD stock solutions were extensively washed, and DYNABEAD antibody loading was assessed via the BCA assay. DYNABEADs targeted to mouse and human T cells were found to have similar antibody loading (approximately 20 μg / ml). On a per-cell basis, a 5:1 DYNABEAD:cell ratio (Condition DB) corresponded to the same dose of anti-CD28 / anti-CD3 antibodies as APC-MS (Condition MD), presenting 0.1% T cell cue input at 16.7 μg. Figure 40B. Dose-dependent expansion of primary murine T cells was observed with APC-MS, but not with DYNABEAD, over a 13-day culture period within the dose range tested. APC-MS significantly promoted enhanced T cell expansion compared with DYNABEADS presenting the same amount of anti-CD3 and anti-CD28 antibodies (see condition MD vs. DB). Figure 40C. Despite greater expansion, cells expanded with APC-MS condition MD did not exhibit enhanced co-expression of the exhaustion markers PD-1 and LAG-3 compared with cells expanded with DYNABEAD presenting the same amount of anti-CD3 and anti-CD28 antibodies (condition DB). Figure 40D. T cells expanded with low to moderate doses of DYNABEAD showed asymmetry primarily toward CD4 (conditions DA, DB). When DYNABEAD were added at extremely high doses, a moderate asymmetry toward CD8 was observed (condition DC). In contrast, APC-MS tended to show asymmetry heavily biased toward CD8, with the degree of asymmetry depending on the APC-MS formulation. Data in Figures 40B, 40C, and 40D represent the 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. [Figure 40CD]Figures 40A, 40B, 40C, and 40D show the results of T cell expansion experiments performed using APC-MS compared to DYNABEAD. The amount of DYNABEAD was normalized to contain the same amount of anti-CD3 and anti-CD28 antibodies as APC-MS. Figure 40A. Bichichoninic acid assay (BCA) analysis for total protein quantification was performed to determine the amount of protein bound to the surface of commercially available mouse or human CD3 / CD28 T cell activator DYNABEADS. DYNABEAD stock solutions were extensively washed, and DYNABEAD antibody loading was assessed via the BCA assay. DYNABEADs targeted to mouse and human T cells were found to have similar antibody loading (approximately 20 μg / ml). On a per-cell basis, a 5:1 DYNABEAD:cell ratio (Condition DB) corresponded to the same dose of anti-CD28 / anti-CD3 antibodies as APC-MS (Condition MD), presenting 0.1% T cell cue input at 16.7 μg. Figure 40B. Dose-dependent expansion of primary murine T cells was observed with APC-MS, but not with DYNABEAD, over a 13-day culture period within the dose range tested. APC-MS significantly promoted enhanced T cell expansion compared with DYNABEADS presenting the same amount of anti-CD3 and anti-CD28 antibodies (see condition MD vs. DB). Figure 40C. Despite greater expansion, cells expanded with APC-MS condition MD did not exhibit enhanced co-expression of the exhaustion markers PD-1 and LAG-3 compared with cells expanded with DYNABEAD presenting the same amount of anti-CD3 and anti-CD28 antibodies (condition DB). Figure 40D. T cells expanded with low to moderate doses of DYNABEAD showed asymmetry primarily toward CD4 (conditions DA, DB). When DYNABEAD were added at extremely high doses, a moderate asymmetry toward CD8 was observed (condition DC). In contrast, APC-MS tended to show asymmetry heavily biased toward CD8, with the degree of asymmetry depending on the APC-MS formulation. Data in Figures 40B, 40C, and 40D represent the 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.

[0099] [Figure 41] Figures 41A and 41B show the results of experiments conducted to evaluate the effect of IL-2 dose and sustained release from APC-MS compared to DYNABEAD on primary murine T cell expansion. Figure 41A shows the expansion of primary murine T cells treated with either 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 cue / 1:10X material / loaded IL-2; MS / bIL-2: 0.1% T cell cue / 1L10X material / IL-2 bolus. Figure 41B shows co-expression of exhaustion markers PD-1 and LAG-3 in primary murine T cells expanded with either 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). Data represent the mean ± SD of samples from four different mice and are representative of at least two independent experiments. ***p<0.001, analyzed by two-way ANOVA followed by Tukey HSD post-hoc test.

[0100] [Figure 42]Figures 42A and 42B show the attachment of azide-labeled IgG to DBCO-presenting MSR-SLBs via click chemistry conjugation. Figure 42A. Varying amounts of azide-modified IgG (as indicated) were incubated with MSR-SLBs containing various amounts of DBCO-modified lipid (as indicated). The values ​​above the bars represent μg of azide-modified IgG attached to the MSR-SLBs. Figure 42B shows a broader dose titration of azide-modified IgG loaded onto MSR-SLBs containing various amounts of DBCO-modified lipid. nIgG represents IgG that was not azide-modified. The values ​​above the bars represent μg of azide-modified IgG attached to the MSR-SLBs. DETAILED DESCRIPTION OF THE INVENTION

[0101] Detailed Description of the Invention The present invention provides a solution to the problem of engineering T cells. Specifically, the present invention provides an antigen-presenting cell-mimicking scaffold (APC-MS), which is useful in engineering such cells. The scaffold comprises mesoporous silica rods (MSR), which incorporate or are coated with continuous fluid-supported lipid bilayers (SLBs), thereby forming an MSR-SLB scaffold. The MSR-SLB scaffold further comprises multiple T cell activation molecules and T cell costimulatory molecules, along with multiple T cell homeostasis factors, which together mimic antigen-presenting cells (APCs) and form a structure that enables the scaffold to elicit various effector functions on target cells (e.g., T cells). In some embodiments, the scaffold mediates these effects through direct or indirect interactions between cell surface molecules on the target cells and various binding partners presented by the scaffold. Depending on the application for which the scaffold is used, the scaffold regulates the survival and growth of targeted cells through its own physical or chemical properties. Depending on the application, the scaffold composition can be modified to include certain activating and costimulatory signals, as well as homeostatic signaling molecules, which act together to mediate various effector functions of target cells, such as activation, division, and promote differentiation, growth, expansion, reprogramming, anergy, quiescence, senescence, apoptosis, or death. In these applications, the scaffold has been found to surprisingly improve the cellular metabolic activity and growth of targeted cells. Furthermore, the improvements in growth and metabolic activity imparted by the scaffold of the present invention are unexpectedly superior to existing platforms (e.g., magnetic beads).

[0102] To enable manipulation of specific cells (e.g., T cells), the permeability of the scaffold composition can be adjusted by selecting or engineering materials for, for example, larger or smaller pore size, density, polymer crosslinking, stiffness, toughness, ductility, or elasticity. The scaffold composition can contain physical channels or pathways through which targeted cells interact with the scaffold and / or migrate to specific compartments or regions of the scaffold. To facilitate compartmentalization, the scaffold composition can be optionally organized into compartments or layers (each with different permeabilities), so that cells are sorted or filtered to allow access to only certain subpopulations of cells. Segregation of target cell populations in the scaffold can also be controlled by degradation, dehydration or rehydration, oxygenation, chemical or pH changes, or continued self-assembly of the scaffold composition. After their capture, the targeted cells can be allowed to grow or expand within the scaffold with the help of stimulatory molecules, cytokines, and other cofactors present in the scaffold. In another example, non-targeted cells that would otherwise infiltrate the scaffold can be rejected or removed using negative selection agents.

[0103] The cells contained or isolated within the scaffold of the present invention are primarily immune cells. In certain embodiments, the present invention relates to a scaffold for isolating and / or manipulating T cells. In other embodiments, the present invention relates to a scaffold that is permeable to other lymphocytes, such as B cells. In yet other embodiments, the present invention relates to a combination of scaffolds, such as a combination of a T cell scaffold and a B cell scaffold. Immune cells, such as T cells, are optionally harvested and analyzed to identify distinct subpopulations that are useful in the diagnosis or treatment of disease. The harvested cells can also be reprogrammed or expanded to develop compositions or formulations to be used in therapy.

[0104] The invention is further described in more detail in the following subsections.

[0105] I. Antigen-presenting cell-mimicking scaffold (APC-MS) In one embodiment, the present invention provides an antigen-presenting cell-mimetic scaffold (APC-MS), which comprises a base layer comprising high surface area mesoporous silica microrods (MSR), a continuous fluid-supported lipid bilayer (SLB) layered on the MSR base layer, a plurality of T cell activation molecules and T cell costimulatory molecules adsorbed onto the scaffold, and a plurality of T cell homeostasis factors adsorbed onto the scaffold.

[0106] A. Mesoporous silica In one embodiment, a component of the scaffold of the present invention comprises mesoporous silica. Mesoporous silica is a porous body with cubic close-packed, uniform cylindrical pores. This material is synthesized by using rod-like micelles of surfactants as templates, which are formed in water by dissolving and hydrolyzing a silica source (e.g., alkoxysilane, sodium silicate solution, kanemite, silica microparticles) 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 (e.g., cationic, anionic, and nonionic surfactants) have been tested as surfactants, and it has generally been found that alkyltrimethylammonium salts of cationic surfactants produce mesoporous silica with the highest 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). The terms "mesoscale," "mesopore," "mesoporous," and the like, as used herein, can refer to structures having feature sizes in the range of 5 nm to 100 nm, particularly in the range of 2 nm to 50 nm. Thus, in some embodiments, mesoporous materials contain pores, which may be regularly or randomly distributed, and have diameters in the range of 5 nm to 100 nm.

[0107] The mesoporous silica used in the scaffolds of the present invention can be provided in various morphologies (e.g., microspheres, irregular particles, rectangular rods, cylindrical nanorods, etc.), with structured rod morphology (MSR) being particularly preferred. The particles can have a variety of predetermined shapes (e.g., spheroidal, ellipsoidal, rod-like, or curved cylindrical). Methods for 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 nano-sized spheres or rods filled with a regular arrangement of pores. The template can then be removed by washing with a solvent adjusted to the appropriate pH. In this example, after removal of the surfactant template, hydrophilic silica nanoparticles characterized by homogeneous, ordered, and continuous mesoporosity were obtained, e.g., about 600 m 2 / g~approx. 1200m 2 / g, especially at about 800m 2 / g~about 1000m 2 / g and especially about 850m 2 / g ~ approx. 950m 2 / g specific surface area. In another embodiment, the mesoporous particles can be synthesized using a simple sol-gel method or spray drying method. Tetraethyl orthosilicate can also be used with an additional polymer monomer (as a template). In yet another embodiment, one or more tetraalkoxy-silanes and one or more (3-cyanopropyl)trialkoxy-silanes can co-condense to provide mesoporous silicate particles as rods. See U.S. Publication Nos. 2013-0145488, 2012-0264599, and 2012-0256336, which are incorporated by reference.

[0108] The mesoporous silica rods can contain pores between 2 and 50 nm in diameter, e.g., between 2 and 5 nm, 10 and 20 nm, 10 and 30 nm, 10 and 40 nm, 20 and 30 nm, 30 and 50 nm, 30 and 40 nm, and 40 and 50 nm. In certain embodiments, the microrods contain pores approximately 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, or larger in diameter. The pore size can be varied depending on the type of application.

[0109] In another embodiment, the length of the microrods is in the micrometer range, ranging from about 5 μm to about 500 μm. In one example, the microrods comprise lengths of 5 to 50 μm, e.g., 10 to 20 μm, 10 to 30 μm, 10 to 40 μm, 20 to 30 μm, 30 to 50 μm, 30 to 40 μm, or 40 to 50 μm. In other embodiments, the rods comprise lengths of 50 μm to 250 μm, e.g., about 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 225 μm, or greater than 225 μm. For cell recruitment, it may be preferable to use MSR compositions with higher aspect ratios (e.g., rods comprising lengths of 50 μm to 200 μm, particularly 80 μm to 120 μm, and particularly about 100 μm or longer).

[0110] In yet another embodiment, the MSR provides a high surface area for attachment and / or binding to target cells, e.g., T cells. Methods for 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 obtaining a high concentration of highly dispersed and easily accessible moieties on the surface. In certain embodiments, the high surface area MSR has a surface area of ​​at least about 100 m 2 / g, at least 150m 2 / g, or at least 300m2 In another embodiment, the high surface area MSR has a surface area of ​​about 100 m 2 / g~about 1000m 2 / g (including any value or subrange 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) surface area.

[0111] B. Lipids The scaffold of the present invention comprises a continuous, fluid-supporting lipid bilayer (SLB) on an MSR base layer. The term "lipid" generally refers to a heterogeneous group of substances associated with biological systems that share the common property of being insoluble in water and can be extracted from cells with low-polarity organic solvents (e.g., chloroform and ether). In one embodiment, "lipid" refers to any substance containing long fatty acid chains (preferably containing 10-30 carbon units, particularly 14-23 carbon units, and especially 16-18 carbon units).

[0112] In one embodiment, lipid is provided as a monolayer.In another embodiment, lipid is provided as a bilayer.Lipid bilayer is a thin polar membrane made of two layers of lipid molecules.Preferably, the lipid bilayer is fluid, and individual lipid molecules can rapidly diffuse within the monolayer.The membrane lipid molecules are preferably amphiphilic.

[0113] In one embodiment, the lipid layer is a continuous bilayer, resembling those found in natural biological membranes, such as cell plasma membranes. In another embodiment, the lipids are provided in the form of supported bilayers (SLBs). SLBs are planar structures that rest on a solid support, such as mesoporous silica rods (MSRs). In this configuration, the upper surface of the supported bilayer is exposed, while the inner surface of the supported bilayer is in contact with the support. The MSR-SLB scaffold is stable and remains sufficiently intact even when subjected to high flow rates or vibration, and can tolerate holes, e.g., holes that are aligned with the pores of the mesoporous silica base layer. Due to this stability, experiments lasting for weeks and even months are possible using supported bilayers. SLBs are also amenable to modification, derivatization, and chemical conjugation with many chemical and / or biological moieties.

[0114] In one embodiment, SLBs can be immobilized on the MSR base layer using any known method, including covalent and non-covalent interactions. Non-covalent interactions include, for example, electrostatic interactions, van der Waals interactions, π-effects, hydrophobic interactions, etc. In one embodiment, lipids are adsorbed onto the MSR base layer. In another embodiment, SLBs are attached or tethered to the MSR base layer via covalent interactions. Methods for attaching lipids to silicates are known in the art (e.g., surface adsorption, physical immobilization, using phase changes to capture substances within the scaffold). In one embodiment, a lipid bilayer is layered on the MSR base layer. For example, a lipid film (e.g., comprising a solution of DPPC / cholesterol / DSPE-PEG in a molar ratio of 77.5:20:2.5 in chloroform) can be spotted onto mesoporous silica, and the solvent can be evaporated using a rotary evaporator. See Meng et al., ACS Nano, 9(4), 3540-3557, 2015. In one embodiment, the lipid bilayer can be prepared, for example, by extruding a hydrated lipid film through a filter with a pore size of, for example, about 100 nm using a standard protocol. The filtered lipid bilayer film can then be fused with the porous particle core, for example, by mixing with a pipette.

[0115] Alternatively, covalent coupling via alkylating or acylating agents can be used to provide stable, structured, and long-term retention of SLBs on the MSR layer. In such embodiments, the lipid bilayer can be reversibly or irreversibly immobilized on the MSR layer using known techniques. For example, the MSR base layer can be hydrophilic and further treated with, for example, ammonium hydroxide and hydrogen peroxide to provide a more hydrophilic surface. The lipid bilayer can be fused onto the porous MSR base layer, for example, using known coupling techniques, to form an MSR-SLB scaffold. The scaffold can be further processed and derivatized with additional moieties to allow attachment and / or immobilization of other secondary agents onto the structure.

[0116] Thus, 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 8,3676,28. For example, the lipid bilayer may comprise a lipid selected from dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), dimyristoyl-phosphatidylethanolamine (DMPE), and dipalmitoyl-phosphatidylethanolamine (DPPE), or a combination thereof. In some embodiments, the lipid bilayer comprises a lipid composition that mimics the lipid composition of a mammalian cell membrane (e.g., a human cell plasma membrane). The lipid composition of many mammalian cell membranes has been characterized and can be readily ascertained by one of skill 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 modify the charge or fluidity of the lipid bilayer. In some embodiments, the lipid bilayer comprises 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 phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphoinositides, phosphosphingolipids with saturated or unsaturated tails containing 6 to 20 carbons, or combinations thereof.

[0117] 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 (16:0-23:2 DIYNE PC) and 1,2-bis(10,12-tricosadiynoyl)-SN-glycero-3-phosphocholine (23:2 DIYNE PC).

[0118] In one embodiment, the MSR-SLB scaffold of the invention maintains a continuous fluid system 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 more than 50 days.

[0119] The organization of the MSR-SLB scaffold can be studied by any known technique, including microscopic visualization techniques exemplified in the Examples below.

[0120] C. Functional molecules In one embodiment of the present invention, the MSR-SLB scaffold can comprise one or more functional molecules. The term "functional molecule" includes any molecule having a desired biological property. 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, such as drugs. In one embodiment, the functional molecule is a T cell activation molecule. In another embodiment, the functional molecule is a T cell costimulatory molecule. Furthermore, in one embodiment, the functional molecule is a T cell homeostasis factor. In certain embodiments, the MSR-SLB scaffold comprises multiple functional molecules, for example, at least one T cell activation molecule, at least one T cell costimulatory molecule, and at least one T cell homeostasis factor.

[0121] T cell activation molecule In one embodiment, the present invention provides an MSR-SLB scaffold comprising multiple T cell activation molecules. These activation molecules can mediate direct, indirect, or semi-direct activation of a target population of T cells. See Benichou et al., Immunotherapy, 3(6): 757-770, 2011. Preferably, the T cell activation molecules mediate direct activation of T cells.

[0122] In one embodiment, the present invention provides an MSR-SLB scaffold comprising a molecule that directly activates T cells, e.g., via binding to a cell surface receptor on target T cells. In particular, the direct activation can be mediated via cluster of differentiation-3 (CD3), a T cell coreceptor that helps activate cytotoxic T cells. In another embodiment, T cells can be directly activated without the simultaneous involvement of CD3, e.g., in a CD3-independent manner.

[0123] In one embodiment, target T cells are activated in a CD3-dependent manner. T cell activation is generally believed to require the T cell receptor (TCR) to recognize its cognate peptide in the context of an MHC molecule. Furthermore, the association of CD3 with the TCR-peptide-MHC complex transmits an activation signal to intracellular signaling molecules, initiating a 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 the CD3γ chain, the CD3δ chain, and two CD3ε chains, which associate with the TCR and the ζ chain (zeta chain; CD247) to generate an activation signal in the T cell. The TCR, ζ chain, and CD3 molecule together constitute the T cell receptor (TCR) complex. Binding of an activation molecule, e.g., an antibody, to one or more members of the TCR complex can activate the T cell.

[0124] In one embodiment, the T cell activation molecule is an antibody or an antigen-binding fragment thereof. When the T cell activation molecule acts in a CD3-dependent manner, the T cell activation molecule is preferably an anti-CD3 antibody or an antigen-binding fragment thereof. In another embodiment, the T cell activation 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 activation molecule is a major histocompatibility complex (MHC) molecule or a multimer thereof optionally loaded with an MHC peptide. Furthermore, the T cell activation molecule is a conjugate or a multimer comprising an MHC and an immunoglobulin (Ig).

[0125] The term "antibody," as used herein, 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, which retains the essential epitope-binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, non-limiting embodiments of which are discussed herein. In one embodiment, the T cell activating antibody used in the compositions and methods of the present disclosure is selected from the group consisting of muromonab (OKT3), otelixizumab (TRX4), teplizumab (hOKT3γ1(Ala-Ala)), visilizumab, and the 17-19 kDa domain of CD3 in the CD3 antigen / T cell antigen receptor (TCR) complex. The anti-CD3 antibody is selected from the group consisting of an antibody that recognizes the ε chain (HIT3a) and an antibody that recognizes the 20 kD subunit of the TCR complex within CD3e (UCHT1), or antigen-binding fragments 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.

[0126] Embodiments of the present invention include "full-length" antibodies. In full-length antibodies, each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity-determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0127] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, 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. Embodiments of such antibodies may 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 in the hinge region); (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of one arm of an antibody; (v) a dAb fragment comprising a single variable domain (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT Publication WO 90 / 05144 A1, incorporated herein by reference); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker, which allows the VL and VH regions to be produced as a single protein chain (known as a 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) pairing to form a monovalent molecule. 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 (e.g., diabodies) are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating 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 moieties are known in the art (Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)).

[0128] An "antibody fragment" includes only a portion of an intact antibody, where that portion preferably retains at least one, and typically most or all, of the functions normally associated with that portion when present in the intact antibody. In one embodiment, an antibody fragment includes the antigen-binding site of an intact antibody and thus retains the ability to bind to antigen. In another embodiment, an antibody fragment (e.g., one including the Fc region) retains at least one of the biological functions normally associated with the Fc region when present in the intact antibody (e.g., FcRn binding, antibody half-life regulation, ADCC function, and complement fixation). In one embodiment, an antibody fragment is a monovalent antibody with an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may include an antigen-binding arm linked to an Fc sequence, which may confer in vivo stability to the fragment.

[0129] The term "antibody construct," as used herein, refers to a polypeptide comprising one or more antigen-binding moieties of the present disclosure linked to a linker polypeptide or immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues connected by a peptide bond and is used to link one or more antigen-binding moieties. 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 the constant domain of a heavy or light chain. The amino acid sequences of the constant domains of human IgG heavy and light chains are known in the art and are disclosed in Table 2 of U.S. Pat. No. 7,915,388, the entire contents of which are incorporated herein by reference.

[0130] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to generate tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Antibody portions (e.g., Fab and F(ab')2 fragments) can be prepared from whole antibodies using conventional techniques (e.g., digestion of whole antibodies with papain or pepsin, respectively). Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0131] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CD3 is substantially free of antibodies that specifically bind to antigens other than CD3). However, an isolated antibody that specifically binds to CD3 may have cross-reactivity to other antigens (e.g., CD3 molecules from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0132] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, and particularly in CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody," as used herein, 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.

[0133] The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, generated, or isolated by recombinant means (e.g., an antibody expressed using a recombinant expression vector that is introduced into a host cell (further described in U.S. Pat. No. 7,915,388, the contents of which are incorporated herein by reference)), an antibody 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 animals (e.g., mice) that are 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, generated, 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 animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within 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 well known in the art (e.g., but not limited to, using a human Ig phage library such as that disclosed in Jermutus et al., PCT Publication No. WO 2005 / 007699 A2).

[0134] The term "chimeric antibody" refers to an antibody containing heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species (e.g., an antibody having murine heavy and light chain variable regions linked 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 (which are incorporated by reference in their entireties). Furthermore, "chimeric antibodies" can be produced by techniques known in the art. Morrison et al., 1984, Proc. Natl. See Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454, which are incorporated herein by reference in their entireties.

[0135] The terms "specific binding" or "specifically binding," as used herein, refer to the interaction of an antibody, protein, or peptide with a second chemical species, and mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on that chemical species; for example, an antibody recognizes and binds to a particular protein structure, rather than to proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction involving labeled "A" and that antibody can reduce the amount of labeled A bound to the antibody.

[0136] Antibodies used in the scaffolds of the present invention may be "monospecific," "bispecific," or "multispecific." As used herein, the expression "antibody" is intended to include both monospecific antibodies (e.g., anti-CD3 antibodies) and bispecific antibodies that include an arm that binds to an antigen of interest (e.g., a CD3-binding arm) and a second arm that binds to a second target antigen. The target antigen to which the other arm of a CD3 bispecific antibody binds can be any antigen expressed on or near a cell, tissue, organ, microorganism, or virus against which a targeted immune response is desired. In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T-cell proliferation. Antibodies that bind to different regions of the CD3 molecule, such as an arm that binds to the 17-19 kD ε 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 TCR complex within CD3e (e.g., derived from UCHT1), are also included within the meaning of this term. Preferably, the anti-CD3 antibody is OKT3 or a CD3-binding fragment thereof.

[0137] In one embodiment, the antibody molecule used in the scaffold of the present invention is a bispecific antibody. In the context of the present invention, a bispecific antibody can be used to bring a cell of interest (e.g., a cancer cell or a pathogen) into close proximity with a target effector cell of the present invention (e.g., a cytotoxic T cell), so that the effector function of the target effector cell is specifically mediated on the cell of interest. 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 that 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, EGFRvl1, 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, and 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, p 53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA(FOLHI), RAGE protein, 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.

[0138] In one specific embodiment, the cancer antigen is a member of the epidermal growth factor receptor (EGFR) family, e.g., a receptor selected from the group consisting of EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4), or variants thereof.

[0139] In another embodiment, the present invention provides a bispecific T cell engager. The present invention relates to a scaffold comprising a bispecific T-cell engager (BiTE) molecule. The BiTE molecule is specifically an antibody that recognizes at least one of the aforementioned tumor antigens and at least one T cell surface molecule (e.g., CD3). Representative examples of such bispecific T cell engager molecules include, but are not limited to, solitomab (CD3 x EpCAM), blinatumomab (CD3 x CD19), MAB MT-111 (CD3 x CEA), and BAY-2010112 (CD3 x PSMA).

[0140] Bispecific antibodies can also be used in the context of the present invention to target effector cells (e.g., T cells or B cells) to mediate, either directly or indirectly, an effect on a pathogen (e.g., 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 (e.g., 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 bispecific antibodies 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 protect against other prototypic pathogens (Taylor et al., J Immunol., 159(8):4035-44, 1997).

[0141] Thus, in one embodiment, the 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 that is an antigen associated with an infectious disease (e.g., a bacterial, protozoan, viral, or fungal antigen). Non-limiting examples of antigens associated with infectious diseases include, for example, antigens expressed on the surface of viral particles or preferentially expressed on cells infected with a virus, such as HIV, hepatitis virus (A, B, or C), herpesvirus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, RA virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum contagiosum 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, Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococci, Gonococcci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, 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 group consisting of Candida (e.g., C. albicans, C. krusei, C. glabrata, C. tropicalis, etc.), Crytococcus neoformans, Aspergillus (e.g., A. fumigatus, A. niger, etc.), Mucorales (e.g., M. mucor, M. absidia, M. rhizopus, etc.), Sporothrix schenkii, 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 sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, Taenia crassiceps, and Brugia malayi. Non-limiting examples of antigens associated with specific pathogens include, for example, HIV. gp120, HIV CD4, hepatitis B glycoprotein L, hepatitis B glycoprotein M, hepatitis B glycoprotein S, hepatitis C E1, hepatitis C E2, hepatocyte-specific protein, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope protein.

[0142] In some embodiments, the scaffolds of the present invention can be used for the treatment and / or prevention of allergic reactions or allergic responses. For example, in some embodiments, the scaffolds can be used to generate T cells (e.g., Tregs) that suppress allergic reactions or allergic responses. For example, in some embodiments, the scaffolds comprise an anti-CD3 antibody and TGF-β. In some embodiments, the scaffolds comprise an anti-CD3 antibody and IL-10. In some embodiments, the scaffolds comprise an anti-CD3 antibody and rapamycin. In some embodiments, the scaffolds comprise an anti-CD3 antibody, TGF-β, IL-10, and rapamycin. In some embodiments, the scaffolds comprise an anti-CD3 antibody, TGF-β, and IL-10. In some embodiments, the scaffolds comprise an anti-CD3 antibody, TGF-β, and rapamycin. In some embodiments, the scaffolds comprise an anti-CD3 antibody, TGF-β, and rapamycin. In some embodiments, the scaffolds comprise an anti-CD3 antibody, IL-10, and rapamycin.

[0143] In some embodiments, scaffolds of the 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., an MHC class I or MHC class II molecule). In some embodiments, the MHC molecule is monomeric. In some embodiments, the allergen is a food allergen (e.g., a banana, milk, legume, shellfish, nut, stone fruit, egg, fish, soy, or wheat allergen). In one embodiment, the allergen is selected from the group consisting of a food allergen, a plant allergen, an insect allergen, an animal allergen, a fungal allergen, a viral allergen, a latex allergen, and a mold spore allergen. 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 Dermatophagoides farina or Dermatophagoides pteronyssinus). 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 asymmetric 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 asymmetric cytokine (e.g., IL-12 or IFNγ). According to certain exemplary embodiments, the present invention comprises a bispecific antigen binding molecule that specifically binds to CD3 and CD28. Such molecules may be referred to herein as, for example, "anti-CD3 / anti-CD28," or "anti-CD3xCD28" or "CD3xCD28" bispecific molecules (or other similar terminology).

[0144] The term "CD28," as used herein, refers to human CD28 protein unless specified as being derived from a non-human species (e.g., "mouse CD28," monkey "CD28," etc.). Human CD28 protein has the amino acid sequence set forth in GENBANK Accession Nos. NP_001230006.1, NP_001230007.1, or NP_006130.1. Mouse CD28 protein has the amino acid sequence set forth in GENBANK Accession No. NP_031668.3. The various polypeptide sequences encompassed by the foregoing accession numbers, including their corresponding mRNA and gene sequences, are incorporated herein by reference in their entirety. As used herein, the phrase "antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising or consisting of at least one complementarity-determining region (CDR) that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen-binding molecule is an antibody or a fragment of an antibody (as those terms are defined elsewhere herein).

[0145] As used herein, the expression "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a first and a second antigen-binding domain. Each antigen-binding domain in the bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds to a second, distinct antigen (e.g., CD28).

[0146] The first and second antigen-binding domains of a bispecific antibody can be directly or indirectly connected to each other. Alternatively, the first and second antigen-binding domains can each be connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" refers to any polymer, protein, polypeptide, peptide, or amino acid capable of associating with a second multimerization domain of the same or similar structure or composition. For example, the multimerization domain can be a polypeptide containing an immunoglobulin CH3 domain. Non-limiting examples of multimerization components include the Fc portion of an immunoglobulin (containing the CH2-CH3 domain), such as the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0147] Bispecific antigen-binding molecules of the present invention typically comprise two multimerization domains, e.g., two Fc domains that are each part of separate antibody heavy chains. The first and second multimerization domains may be of the same IgG isotype (e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4). Alternatively, the first and second multimerization domains may be of different IgG isotypes (e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.).

[0148] 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 that contain or consist of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0149] Any bispecific antibody format or technology can be used to generate the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof with a first antigen-binding specificity can be operatively linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) to one or more other molecular entities (e.g., another antibody or antibody fragment with a second antigen specificity) to generate the bispecific antigen-binding molecule. Specific exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (such as common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a review of the aforementioned formats see, e.g., Klein et al., mAbs 4:6, 1-11, 2012 and references cited therein).

[0150] Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains 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 may be linked to or coexpressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof may be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other methods) to one or more other molecular entities (e.g., another antibody or antibody fragment) to generate a bispecific or multispecific antibody having a second binding specificity. A multispecific antigen-binding fragment of an antibody typically contains at least two different variable domains, each capable of specifically binding to a distinct antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody format disclosed herein, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art. The multispecific antigen-binding molecules of the present invention are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for producing multispecific antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the present invention may be VELOCIMMUNE TM It can be prepared using techniques such as: TM Using this technique (or any other human antibody generation technique), high-affinity chimeric antibodies to a particular antigen (e.g., CD3 or CD28) having human variable regions and mouse constant regions are first isolated. The antibodies are characterized and selected for desirable properties (including affinity, selectivity, epitope, etc.). The mouse constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules of the invention.

[0151] In the context of bispecific antigen-binding molecules of the invention, the multimerization domain, e.g., the Fc domain, can contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain, which result in a modified Fc domain with an altered binding interaction (e.g., enhanced or decreased) between the Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule contains a modification in the CH2 or CH3 region, which increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes, where the pH ranges from about 5.5 to about 6.0). Non-limiting examples are provided, for example, 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 that amino acid difference. In certain embodiments, the Fc domain may be chimeric, combining Fc sequences from more than one immunoglobulin isotype.

[0152] In another embodiment, the T cell activation molecule is a major histocompatibility complex (MHC) molecule that binds to CD3. Representative examples include, but are not limited to, MHC type I that binds to TCR and CD8 or MHC type II that binds to TCR and CD4. The MHC molecule can be optionally loaded with an antigen, for example, a biotinylated peptide. In other embodiments, the MHC molecule can be conjugated to the Fc portion of an immunoglobulin, for example, 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 noncovalently attached to the multimerization domain. Known examples of such MHC multimers include, but are not limited to, MHC-dimers (containing two copies of 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 MHC-peptide, each of which is biotinylated, and the MHC complex is held together in the complex by streptavidin tetramer protein, providing a non-covalent link between the streptavidin monomer and the MHC protein); MHC pentamers (containing five copies of MHC-peptide complexes, multimerized by self-assembling coiled-coil domains), MHC dextramers (typically containing more than 10 MHC complexes attached to a dextran polymer), and MHC streptamers (containing 8-12 MHC-peptide complexes attached to streptactin). MHC tetramers are described in U.S. Patent No. 5,635,363; MHC pentamers are described in U.S. Patent No. 2004209295; MHC-dextramers are described in patent application WO 02 / 072631. MHC streptamers are described in Knabel M et al., Nature Medicine 6. 631-637, 2002.

[0153] Target T cells can also be activated in a CD3-independent manner, e.g., through binding and / or ligation of one or more cell surface receptors other than CD3. Representative examples of such cell surface molecules include, e.g., CD2, CD47, CD81, MSR1, etc.

[0154] In this context, CD2 is found on virtually all T cells (and also on natural killer (NK) cells) and is important in T lymphocyte function. CD2 associates with several proteins, including CD3, CD5, and CD45. 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. Costimulatory blockade using antibodies directed against CD2 can be a powerful immunosuppressive strategy in organ transplantation. Thus, in one embodiment, the T cells are activated through the use of an antibody or antigen-binding fragment thereof that specifically binds to CD2. Representative examples of anti-CD2 antibodies include, for example, siplizumab (MEDI-507) and LO-CD2b (ATCC Accession No. PTA-802; deposited June 22, 1999).

[0155] CD47 (IAP) belongs to the immunoglobulin superfamily, is a partner of membrane integrins, 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), an inhibitory transmembrane receptor present on myeloid cells. The CD47 / SIRPα interaction results in bidirectional signaling, resulting in distinct intercellular 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 through the use of an antibody or antigen-binding fragment thereof that specifically binds to CD47. Representative examples of anti-CD47 antibodies include, for example, monoclonal antibody Hu5F9-G4 (which is under investigation in various clinical trials for myeloid leukemia), and monoclonal antibodies MABL-1 and MABL-2 (FERM accession numbers BP-6100 and BP-6101). See, for example, WO1999 / 12973 (the disclosure of which is incorporated herein by reference).

[0156] 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 immunoregulatory role. In particular, cross-linking of CD81 enhances CD3-mediated activation of αβ and γδ T lymphocytes and induces TCR-independent production of cytokines by γδ T cells in vitro. According to the present invention, in one embodiment, T cells are activated through the use of 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).

[0157] MSR1 (CD204) belongs to the class A macrophage scavenger receptor family, which includes three distinct types (1, 2, and 3) generated by alternative splicing of the MSR1 gene. These receptors, or isoforms, are trimeric integral membrane glycoproteins and have been implicated in many macrophage-related physiological and pathological processes, including atherosclerosis, Alzheimer's disease, and host defense. Matsumoto et al., Proc. Natl. Acad. Sci. USA 87(23): 9133-7, 1990. Recent studies have shown that dendritic cell (DC) MSR1 affects the activation and proliferation of CD8 T cells, and antibody-mediated blockade of MSR1 increased T cell proliferation and expansion in vitro. See Lerret et al., PLoS One., 7(7):e41240, 2012. According to the present invention, in one embodiment, T cells are activated through the use of 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 number MA5-16494) and goat anti-human CD204 / MSR1 antibody (Biorad catalog number AHP563).

[0158] In another embodiment, T cells are activated by ligation / binding to a T cell receptor (TCR) molecule, which is ubiquitously expressed on T cells. TCRs are heterodimers composed of two different protein chains. In humans, in 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain, whereas in 5% of T cells, the TCR consists of a gamma and delta (γ / δ) chain. When the TCR engages with an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction. According to the present invention, in one embodiment, T cells are activated through the use of an antibody or antigen-binding fragment thereof that specifically binds to the TCR. Representative examples of anti-TCR antibodies include, for example, the 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 the alpha / beta TCR WT31 (described in Gupta et al., Cell Immunol., 132(1):26-44, 1991).

[0159] In another embodiment, the T cell activation molecule is a major histocompatibility complex (MHC) molecule optionally loaded with an MHC peptide. There are two general 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 found primarily on antigen-presenting immune cells (APCs), which ingest polypeptide antigens (e.g., in microorganisms) and digest them into peptide fragments. The MHC-II molecules then present the peptide fragments to helper T cells, which, after activation, provide the helper activity generally required 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 groove of the heavy chain of MHC class I (pMHC) and the complementarity-determining region (CDR) of the T cell receptor (TCR) determines the potential for T cell activation during the afferent and efferent stages of cellular immunity. The affinity that exists between the TCR and the MHC-peptide complex determines the fate of the T cell during development, initial activation, and exertion of effector functions.

[0160] Thus, in one embodiment, the present invention relates to an MSR-SLB scaffold comprising human MHC molecules, optionally loaded with peptides. Representative examples of such MHC molecules include HLA-A, HLA-B, HLA-C, DP, DQ, and DR, or combinations thereof. The MHC molecules can be monovalent or bivalent. In some embodiments, bivalency or multivalency of MHC molecules is desirable for signal delivery (either activating or inhibitory signals) to T cells. Thus, in some embodiments, the MSR-SLB scaffold of the present invention comprises at least two identical MHC molecules attached to a linker.

[0161] The linker in a bivalent MHC molecule serves three functions. First, the linker provides the required bivalency or multivalency. Second, the linker increases the half-life of the overall fusion protein in vivo. Third, the linker determines whether the fusion protein activates or suppresses T cells. T cell priming requires stimulation via the TCR and an additional second signal, typically delivered by an APC. In the absence of a second signal, T cell hyporesponsiveness can occur. By constructing a fusion protein in which the linker allows delivery of a second signal, T cell stimulation results in enhanced T cell immunity. By constructing a fusion protein in which the linker does not provide for delivery of a second signal, T cell suppression results in immunosuppression. Fusion proteins with T cell stimulatory properties can be constructed by using a linker that allows delivery of a second signal to T cells in addition to the signal delivered via the TCR. This can be achieved by using a linker that has binding affinity for a cell surface structure on another cell that can deliver a second signal to T cells. Thus, the linker serves to bridge the T cell and the other cell. By bridging the other cell in close proximity to the T cell, the other cell can deliver a second signal to the T cell.

[0162] Examples include linkers (e.g., certain immunoglobulin chains or portions of immunoglobulin chains) that can bind to Fc receptors on other cells. Specific examples include IgG, IgA, IgD, IgE, and IgM. When immunoglobulins are used, the entire protein is not required. For example, the immunoglobulin gene can be truncated at the hinge region, and only the gene encoding the hinge, CH2, and CH3 domains of the heavy chain is used to form the fusion protein. The linker can bind to other cell surface structures. For example, the linker can contain the cognate moiety for many cell surface antigens that can act as a bridge to bring the second cell into close proximity with the T cell. The linker 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. Furthermore, the linker 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 materials. Examples of non-biological materials include plastic microbeads or even larger plastic members (e.g., plastic rods or tubes), as well as other physiologically acceptable carriers that can be implanted in vivo.

[0163] In some embodiments, the MHC molecule is not attached to a linker. Without wishing to be bound by any particular theory, it is believed that the fluid nature of the lipid bilayer allows T cells to recognize the membrane and form multivalent clusters. These clusters can then be disassembled. This is not possible when signaling molecules are attached together with the linker. The inability to disassemble these multivalent clusters can potentially lead to overstimulation and T cell exhaustion or anergy (see, for example, Lee KH et al. Science 302(5648): 1218-22(2003)).

[0164] In some embodiments, the lipid bilayer of an APC-MS comprises a lipid composition that promotes spontaneous partitioning of lipid species into liquid-ordered domains (see, e.g., Wang TY et al. Biochemistry 40(43):13031-40(2001)).

[0165] If desired, MHC molecules can be loaded with specific peptides (e.g., peptides derived from viral antigens, bacterial antigens, or allergens). The specific peptides of the fusion protein can be loaded onto MHC molecules after the fusion protein is created. The peptides can also be subsequently covalently attached to MHC, for example, by UV crosslinking. Alternatively, the peptide sequence can be incorporated into the DNA sequence encoding the fusion protein so that the peptide is loaded onto the MHC molecule during production. In the latter case, the peptide can be attached with a tether (e.g., a polypeptide) that allows it to complex with the MHC portion of the fusion protein. The specific peptide to be loaded onto the MHC molecule is virtually limitless and is determined based on the desired application. For example, peptides from various sources (e.g., viral, fungal, and bacterial infections) or tumors can be used to enhance T-cell immunity. In autoimmunity, self-reactive peptides can be used to suppress T-cell immunity. Self-peptides presented by alloantigens can be used to suppress T-cell immunity against transplanted tissue.

[0166] Toxins (e.g., ricin and diphtheria toxin) and radioisotopes can be conjugated to the 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 to the MHC portion of the fusion protein, or they can be incorporated into the DNA sequence encoding the fusion protein such that the toxin is conjugated to the fusion protein during its production.

[0167] MHC-peptide / immunoglobulin fusion proteins can be prepared by constructing a gene encoding the production of the fusion protein. Alternatively, the components of the fusion protein can be assembled using chemical conjugation methods. Sources of genes encoding MHC molecules and linkers can be obtained from various databases. In the case of MHC class I fusion proteins, the MHC fragment can be attached to a linker and β2 microglobulin can be allowed to self-associate. Alternatively, the fusion protein gene can be constructed so that β2 microglobulin is attached to the MHC fragment by an ether. In the case of MHC class II fusion proteins, either the α or β chain can be attached to a linker and the other chain can be allowed 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 them into the fusion protein gene construct, or alternatively, using standard methodologies available to those skilled in the art on a peptide synthesizer. The resulting complete fusion protein can be administered using conventional techniques.

[0168] T cell costimulatory molecule In one embodiment, the present invention provides an MSR-SLB scaffold comprising multiple T cell costimulatory molecules. These costimulatory molecules can mediate direct, indirect, or semi-direct stimulation of a target population of T cells. Preferably, the costimulatory molecules mediate T cell activation in the presence of one or more T cell activation molecules.

[0169] The term "co-stimulatory molecule" is used herein according to its art-recognized meaning in immune T cell activation. Specifically, "co-stimulatory molecule" refers to a group of immune cell surface receptors / ligands that engage between T cells and antigen-presenting cells and generate a stimulatory signal in T cells in conjunction with the stimulatory signal in T cells (i.e., "co-stimulation") resulting from T cell receptor ("TCR") recognition of an antigen on an antigen-presenting cell. As used herein, soluble forms of costimulatory molecules "derived from an APC" refer to costimulatory molecules normally expressed by B cells, macrophages, monocytes, dendritic cells, and other APCs. See Huppa et al., Nature Reviews Immunology. 3, 973-983 (2003). A "co-stimulator of T cell activation" refers to the ability of a costimulatory ligand to bind to and activate T cells that have been activated via any of the mechanisms or pathways described above, e.g., via CD3-dependent or CD3-independent T cell activation. Costimulatory activation can be measured for T cells by cytokine production, as is well known, and by proliferation assays, as is well known (e.g., CFSE staining) and / or as described in the Examples below.

[0170] In one embodiment, the present invention provides an MSR-SLB scaffold comprising a molecule that specifically binds to a costimulatory antigen. In particular, the MSR-SLB scaffold comprises a molecule that specifically binds to a costimulatory antigen, such as 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, These include multiple T cell costimulatory molecules that specifically bind to 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).

[0171] In one embodiment, the costimulatory molecule is an antibody or antigen-binding fragment thereof that specifically binds to one or more of the aforementioned costimulatory antigens. In this context, CD28 is a prototypical T cell costimulatory antigen and binds to molecules of the B7 family expressed on APCs (e.g., dendritic cells and activated B cells). Human CD28 binds to all CD4+ It is found on T cells and on approximately half of CD8+ T cells. T cell functions attributed to CD28 include prevention of anergy, induction of cytokine gene transcription, stabilization of cytokine mRNA, and activation of CD8+ cytotoxic T lymphocytes. The ligands for CD28, identified as CD80 (B7-1) and CD86 (B7-2), are 60-kd and 80-kd immunoglobulin superfamily monomeric transmembrane glycoproteins, respectively.

[0172] In one embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or its antigen-binding fragment that specifically binds to CD28. Representative examples of anti-CD28 antibodies include, for example, lurizumab pegol and TGN1412. See also U.S. Patent No. 8,785,604.

[0173] In another embodiment, the present invention relates to an SR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to ICOS (CD278). ICOS is a CD28 superfamily costimulatory molecule expressed on activated T cells. It is thought to be particularly important for Th2 cells. Representative examples of anti-ICOS antibodies include monoclonal antibody 2C7, which recognizes the ICOS molecule expressed on activated T cells and induces the activation and proliferation of T cells pre-stimulated with anti-human CD3 monoclonal antibody. See Deng et al., Hybridomics., 23(3):176-82, 2004.

[0174] In another embodiment, the present invention provides an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to CD152 (CTLA4). The antibody is preferably a neutralizing or blocking antibody. CD152 is expressed on activated CD4+ and CD8+ T cells and on regulatory T cells (Tregs). Its function in T cell biology, during the immune response to infection and as a target for cancer immunotherapy, has been well described (Egen et al., Nat. Immunol., 3(7):611-618, 2002). CTLA-4 is the homologous counterpart of CD28, which both 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 outcompeting the low-affinity CD28 molecule for ligand binding to minimize T cell costimulation, recruiting inhibitory phosphatases to the TCR complex to disrupt positive signaling cascades, and removing CD80 and CD86 from the surface of APCs by transendocytosis, thereby reducing the ability of APCs to properly activate or otherwise render T cells responsive. Thus, exploiting the CTLA-4 receptor / pathway is an attractive strategy for modulating T cell immunity. In fact, anti-CTLA-4 was the first monoclonal antibody (ipilimumab) approved by the FDA for checkpoint blockade treatment in cancer patients. Other examples of CTLA-4 antibodies that can be used in accordance with the present invention include tremelimumab and its antigen-binding fragments.

[0175] In another embodiment, the present invention provides an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to 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 specifically binds to the B7 ligands, PD-L1 and PD-L2, on APCs and other surrounding tissues, profoundly influencing the outcome of responding CD8+ T cells in the setting of chronic infection. On T cells, PD-1 is expressed after antigen encounter and acts almost immediately to disrupt T cell activation by recruiting the phosphatases SHP-1 and SHP-2 through signaling motifs in the PD-1 cytoplasmic tail, which reduces Akt phosphorylation and decreases T cell metabolism, proliferation, and survival. Thus, 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.

[0176] In another embodiment, the present invention provides an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to CD81. Engagement of CD81 lowers the signaling threshold required for T cell / CD3-mediated proviral DNA induction in CD4+ T cells (Tardif et al., J. Virol. 79(7): 4316-28, 2005). Representative examples of anti-CD81 antibodies include monoclonal antibody 5A6. See, for example, Maecker et al., BMC Immunol., 4:1, 2003 (the disclosure of which is incorporated herein by reference).

[0177] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising 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. Furthermore, it can enhance immune activity and eliminate tumors in vivo. Therefore, the antibody that binds to CD137 is preferably an agonistic antibody. Representative examples of anti-CD137 antibodies include the monoclonal antibody utomilumab, which is a human IgG currently being investigated in clinical trials. See National Clinical Trials ID: NCT01307267.

[0178] In another embodiment, the present invention provides an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to OX40 (CD134). OX40L binds to the OX40 receptor on T cells, preventing them from dying and subsequently increasing cytokine secretion. Due to its ability to enhance survival, OX40 plays a crucial role in maintaining immune responses beyond the first few days and progressing to memory responses. OX40 also plays a crucial role in both Th1- and Th2-mediated responses in vivo. Therefore, antibodies that bind to OX40 are preferably agonistic antibodies. Representative examples of anti-OX40 antibodies include, for example, the anti-OX40 monoclonal antibody utomilumab, which is currently being investigated in various clinical trials (see National Clinical Trials IDs: NCT01644968, NCT01303705, and NCT01862900).

[0179] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising 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 the ligand CD70 and plays an important role in regulating immunoglobulin synthesis. CD27 supports antigen-specific expansion (but not effector cell maturation) of naive T cells, independently 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 comprises an agonistic antibody that binds to CD27. Representative examples of anti-CD27 antibodies include the monoclonal antibody varlilumab. Ramakrishna et al., Journal for ImmunoTherapy of Cancer, 3:37, 2015.

[0180] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to the glucocorticoid-inducible TNF receptor family regulatory gene (GITR or CD357). GITR is a 25 kD TNF receptor superfamily member 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 break immunological self-tolerance. GITR further binds to GITRL, which is involved in the development of regulatory T cells and regulates the activity of the Th1 subset. Modulation of 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 the GITR receptor, thereby increasing the proliferation and function of effector T cells. At the same time, ligation of GITR on the surface of Tregs can eliminate the suppressive function of these cells against tumor-specific effector T cells, thus further enhancing the T cell immune response. Representative examples of anti-GITR antibodies include the humanized Fc-disabled anti-human GITR monoclonal antibody TRX518, which induces both the activation of tumor antigen-specific T effector cells and eliminates the suppression induced by inappropriately activated T regulatory cells. TRX518 is currently being investigated in various clinical trials (see National Clinical Trials ID: NCT01239134).

[0181] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising 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, which exerts pleiotropic effects on cell growth and survival when stimulated. In normal or inflamed tissues, CD30 expression is restricted to activated medium / large B lymphocytes and / or T lymphocytes. It is expressed by activated T cells and B cells, but not by resting T cells and B cells (Guo et al., Infect. Immun., 81(10), 3923-3934, 2013). Stimulation of CD30L / CD30 signaling by in vivo administration of an agonistic anti-CD30 monoclonal antibody (MAb) restored IL-17A production by Vγ1- Vγ4- γδ T cells in CD30L knockout mice. Representative examples of anti-CD30 antibodies include brentuximab vedotin (Adcetris).

[0182] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to HVEM (CD270). CD270 is a member of the TNF receptor superfamily. This receptor was identified as a cellular mediator of herpes simplex virus (HSV) entry. Mutations in this gene have repeatedly been associated with cases of diffuse large B-cell lymphoma. Representative examples of anti-CD270 antibodies include the monoclonal antibody HVEM-122. Cheung et al., J. Immunol., 185:1949, 2010; See Hobo et al., J Immunol., 189:39, 2012.

[0183] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to lymphotoxin β receptor (LTβR; TNFRSF3). LTβR is involved in CD4+ T cell priming (Summers deLuca et al., J Exp Med., 204(5):1071-81, 2007). Representative examples of anti-LTβR antibodies include the monoclonal antibody BBF6. See also WO2010 / 078526 (incorporated by reference).

[0184] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to DR3 (TNFRSF25). DR3 is thought to be involved in the regulation of lymphocyte proliferation induced by T cell activation. Specifically, DR3 activation depends on prior engagement of the T cell receptor. After binding to TL1A, DR3 signaling increases T cell sensitivity to endogenous IL-2 via the IL-2 receptor, enhancing T cell proliferation. Because receptor activation is T cell receptor-dependent, the in vivo activity of DR3 is specific to those T cells that encounter their cognate antigen. At rest, and in individuals without underlying autoimmunity, the majority of T cells that regularly encounter their cognate antigen are FoxP3+ regulatory T cells. Stimulation with TNFRSF25 stimulates a significant and highly specific proliferation of FoxP3+ regulatory T cells, increasing their representation from 8-10% of total CD4+ T cells to 35-40% of total CD4+ T cells within 5 days in the absence of any other exogenous signals. Representative examples of DR3 agonists include 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).

[0185] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to CD226 (DNAM1). CD226 is an approximately 65 kDa glycoprotein expressed on the surface of natural killer cells, platelets, monocytes, and a subset of T cells. It is a member of the immunoglobulin superfamily and mediates cell adhesion to other cells bearing its ligands, CD112 and CD155. Cross-linking of CD226 with an antibody triggers cell activation, and ligation of CD226 and LFA-1 with their respective ligands cooperates to induce cytotoxicity and cytokine secretion by T cells and NK cells (Tahara et al., Int. Immunol. 16(4): 533-8, 2004).

[0186] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to CRTAM (CD355). CTRAM is an MHC class I-restricted T cell-associated molecule that regulates the late phase of 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).

[0187] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to TIM1 (HAVCR1, KIM1). TIM genes belong to type I cell surface glycoproteins, which contain an N-terminal immunoglobulin (Ig)-like domain, a mucin domain of varying length, a single transmembrane domain, and a short C-terminal cytoplasmic tail. The location and function of TIM genes vary among members. TIM-1 is preferentially 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 the rabbit anti-human TIM1 antibody ab47635 (ABCAM, Cambridge, MA).

[0188] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to SLAM (CD150, SLAMF1). SLAM (CD150) is an autoligand and cell surface receptor that functions as a costimulatory molecule and is also a microbial sensor that controlled the killing of Gram-negative bacteria by macrophages. In particular, SLAM regulated the activity of the NADPH oxidase-NOX2 complex and phagolysosomal maturation after phagosome entry after interacting 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 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 antigen-binding fragment thereof. Representative examples of SLAM1 antibodies include rabbit anti-human SLAM1 antibody 600-401-EN3 (Rockland Antibodies, Limerick, PA).

[0189] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising 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. Interaction between NK cells and target cells via this receptor is thought to regulate the cytolytic activity of NK cells. CD244 is a co-inhibitory SLAM family member that attenuates primary antigen-specific CD8(+) T cell responses in the presence of immunomodulation associated with selective CD28 blockade. Recent studies have demonstrated the specific upregulation of 2B4 on 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 are characterized in Sandusky et al. (Eur J Immunol. 2006 Dec;36(12):3268-76).

[0190] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising 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 and is expressed on natural killer (NK) cells, T lymphocytes, and B lymphocytes. Costimulation of T lymphocytes through the SLAMF3 / SLAMF6 pathway mediates a more potent effect on IL-17A expression compared to the canonical CD28 pathway. SLAMF3 / SLAMF6 signaling mediates increased nuclear abundance and recruitment of 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 those described, for example, in Flaig et al. (J. Immunol. 2004. 172: 6524-6527) and Stark et al. (J. Immunol. Methods 2005. 296: 149-158).

[0191] 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 have been implicated in the activation of T cells and NK cells. CD84 enhances the proliferative response of activated T cells, and homophilic interactions enhance interferon-γ secretion in lymphocytes. CD84 can also serve as a marker for hematopoietic precursor cells. See the disclosures in references with PUBMED ID numbers 11564780, 12115647, 12928397, 12962726, and 16037392, which indicate that it is required for long-term 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 antigen-binding fragment thereof. Representative examples of CD84 antibodies include, for example, the PE anti-human CD84 antibody CD84.1.21, which can enhance CD3-induced IFN-γ production and partially block CD84-Ig binding to lymphocytes (BioLegend, San Diego, CA; catalog number 326008).

[0192] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to Ly9 (CD229, SLAMF3). CD229 is involved in the adhesion reaction between T lymphocytes and accessory cells through homophilic interactions. It also promotes T cell differentiation toward the helper T cell Th17 phenotype, resulting in increased IL-17 secretion; costimulatory activity requires SH2D1A (Chatterjee et al., J Biol Chem., 287(45): 38168-38177, 2012). In particular, simultaneous ligation of CD229 and TCR with 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(sup. 1) 176.7, May 2012). Thus, in one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD229 antibody or antigen-binding fragment thereof. Representative examples of CD229 antibodies include, for example, the PE anti-human CD229 antibody Hly-9.1.25 (BIOLEGEND, San Diego, CA; catalog number 326108) or the mouse anti-human CD229 antibody (R&D Systems catalog number AF1898).

[0193] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising 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 T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved through a dual mechanism that promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). Representative examples of CD229 antibodies include, for example, nivolumab, pembrolizumab, pidilizumab (CT-011, CureTech), BMS936559, and atezolizumab.

[0194] In another embodiment, the present invention relates to an MSR-SLB scaffold comprising an antibody or antigen-binding fragment thereof that specifically binds to CRACC (CD319, BLAME). CD319 mediates NK cell activation through an SH2D1A-independent extracellular signal-regulated ERK-mediated pathway (Bouchon et al., J Immunol. 2001 Nov 15;167(10):5517-21). CD319 may also positively regulate NK cell function and contribute to NK cell activation. Thus, in one embodiment, the MSR-SLB scaffold comprises a monoclonal anti-CD319 antibody or antigen-binding fragment thereof. Representative examples of CD319 antibodies include, for example, elotuzumab or its antigen-binding fragment.

[0195] In certain embodiments, the present invention provides an MSR-SLB scaffold comprising a binding pair comprising at least one T cell activation molecule and at least one T cell costimulatory molecule. Representative examples of such pairs include, but are not limited to, antibodies that bind to CD3 / CD28, CD3 / ICOS, CD3 / CD27, and CD3 / CD137, or combinations thereof. In this context, depending on the desired modulation of the costimulatory molecule activity, it may be desirable to use an agonistic antibody directed against the first component (CD3) and an agonistic or antagonistic antibody directed against the second component.

[0196] In certain embodiments, the present invention provides an MSR-SLB scaffold comprising a binding pair including at least one T cell activation molecule that is an antibody that binds to CD3 and at least one T cell costimulatory molecule that is an antibody that binds to CD28, optionally together with a second costimulatory molecule that is an antibody that binds to an antigen selected from the group consisting of ICOS, CD27, and CD137. In one embodiment, the MSR-SLB scaffold comprises 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, or (d) antibodies that bind to CD3, CD28, ICOS, and CD27. In this regard, experimental data suggest that stimulation with these secondary T cell costimulators, when applied in conjunction with an appropriate activation stimulus (e.g., CD3 + CD28), can stimulate certain types of differentiation in T cells. For example, ICOS stimulation, when combined with CD3+CD28+ stimulation, promotes the differentiation of Th effector cells, whereas 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 the system. In this context, the anti-CD27 antibody 1F5 (when used with anti-CD3 antibodies) did not induce the phenomenon observed with potentially dangerous polyclonal T cell activation-costimulatory CD28-specific superagonistic antibodies. See Thomas et al., Oncoimmunology, 3: e27255, 2014.

[0197] In one embodiment, the binding pair is a monospecific antibody, where 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 is a bispecific antibody, where a single antibody binds to each member of the pair (e.g., a bispecific antibody that binds to CD3 and CD28). In this context, bispecific antibodies are preferred due to their ability to confer enhanced T cell activation. See Willems et al., Cancer Immunol Immunother. 2005 Nov;54(11):1059-71.

[0198] Alternatively, the binding pair may be monospecific antibodies, where one antibody binds to CD3 and the second antibody binds to ICOS. In the context of antibodies that bind to ICOS, it may be preferable to use antagonistic antibodies that neutralize ICOS, insofar as this molecule is involved in the pathogenesis 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 also be used.

[0199] Alternatively, the binding pair may comprise monospecific antibodies, where the first antibody binds to CD3 and the second antibody binds to CD27. In this embodiment, both antibodies are preferably stimulatory or agonistic antibodies. CD27 costimulation has been reported to enhance the survival and antitumor activity of redirected human T cells in vivo (Song et al., Blood, 119(3):696-706, 2012). Bispecific antibodies comprising an agonistic CD3-binding antibody fragment and an agonistic CD27-binding antibody fragment may also be used.

[0200] Alternatively, the binding pair may comprise monospecific antibodies, where one antibody binds to CD3 and the second antibody binds to CD137. In this embodiment, both antibodies are preferably stimulatory or agonistic antibodies. CD137 costimulation has been reported to improve 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 comprising an agonistic CD3-binding antibody fragment and an agonistic CD27-binding antibody fragment may also be used.

[0201] T cell homeostasis factor In one embodiment, the MSR-SLB scaffold and / or antigen-presenting cell-mimicking scaffold comprises a homeostatic factor 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, and transforming growth factor β (TGF-β), or an agonist, mimetic, variant, functional fragment, or combination thereof. In some embodiments, the MSR-SLB scaffold and / or antigen-presenting cell-mimicking scaffold comprises a plurality of homeostatic factors 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, and transforming growth factor β (TGF-β), or an agonist, mimetic, variant, functional fragment, or combination thereof. Functional fragments of these homeostatic factors, characterized by their ability to regulate target cell activity, can also be used. Representative types of homeostatic factors (including the NCBI accession numbers of their human and / or mouse homologs) are provided in Table 1. [Table 1-1] [Table 1-2]

[0202] Fragments and variants of the aforementioned T cell homeostasis factors are known in the art. For example, the UNIPROT database entry for each of the aforementioned homeostasis factors lists "natural variants," including the structural relationship between the variant and the wild-type biomarker. Purely by way of representation, the human IL-1β protein (UNIPROT: P01584) contains a natural variant (VAR_073951) with an E→N amino acid substitution at amino acid residue 141 of the predicted human IL-1β protein sequence. Fragments, if known, are also listed under this section.

[0203] Preferably, the T cell homeostasis factor is interleukin-2 (IL-2) or an agonist, mimetic, variant, or functional fragment thereof, or a combination thereof with one or more T cell homeostasis factors listed in Table 1. An example of an IL-2 agonist includes, for example, BAY 50-4798 (Margolin et al., Clin Cancer Res. 2007 Jun 1;13(11):3312-9). An example of an IL-2 mimetic includes, for example, peptide 1-30 (P1-30), which acts synergistically with IL-2 (Eckenberg et al., J Immunol 2000;165:4312-4318). An example of an IL-2 fragment includes, for example, the ballast portion comprising the first 100 amino acids of IL-2 (see U.S. Patent No. 5,496,924). Examples of IL-2 variants include, for example, the natural variant VAR_003967 and the natural variant VAR_003968. Also included are IL-2-containing fusion proteins, such as 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 five-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. In turn, the IL-2 portion of the fusion protein stimulates natural killer (NK) cells, macrophages, and neutrophils, inducing T cell antitumor cellular immune responses. Other IL-2 mimetics that can be used in accordance with 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 a combination thereof.

[0204] Embodiments of the present invention further include MSR-SLB scaffolds (including APC-MS scaffolds made from such scaffolds), which further comprise a plurality of the aforementioned T cell homeostasis factors. Thus, in one embodiment, the present invention provides an MSR-SLB scaffold comprising a first T cell homeostasis factor that is IL-2 and a second T cell homeostasis factor that is IL-7, IL-21, IL-15, or an IL-15 superagonist. In this context, the IL-15 superagonist (IL-15 SA) is a combination of IL-15 and soluble IL-15 receptor-α, which has greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent due to its ability to selectively expand NK and memory CD8+ T (mCD8+ T) lymphocytes. See Guo et al., J Immunol. 2015 Sep 1;195(5):2353-64.

[0205] Embodiments of the present invention further relate to scaffolds comprising multiple T cell stimulatory molecules, T cell costimulatory molecules, and T cell homeostasis factors. Exemplary scaffolds may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or more than eleven of each of the aforementioned T cell stimulatory molecules, T cell costimulatory molecules, and T cell homeostasis factors.

[0206] In the scaffold of the present invention, any functional molecule, such as an antigen, antibody, protein, or enzyme (including fragments thereof), can be directly or indirectly immobilized on the MSR base layer and / or SLB using conventional techniques. In certain embodiments, the functional molecule can be provided in an organelle (e.g., a Golgi membrane or plasma membrane), a cell, a cell cluster, a tissue, a microorganism, an animal, a plant, or an extract thereof, which is then immobilized on the MSR layer or SLB layer. The functional molecule can also be synthesized by genetic engineering or chemical reaction at the desired location (e.g., the outer surface of the SLB layer).

[0207] The scaffolds described herein contain and release signaling molecules, such as T cell homeostasis factors, to induce functional T cell responses. In one embodiment, the released T cell homeostasis factors are polypeptides isolated from endogenous sources or synthesized in vivo or in vitro. For example, endogenous IL-2 polypeptides can be isolated from healthy human tissue. Alternatively, synthetic functional molecules can be synthesized via transfection or transformation of template DNA into a host organism or cell (e.g., a cultured human cell line or a mammal (e.g., a humanized mouse or rabbit)). Alternatively, synthetic functional molecules in protein form can be synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods (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)).

[0208] Functional molecules can be modified to increase in vivo protein stability. Alternatively, functional molecules can be engineered to be more or less immunogenic. For example, to the extent that the structures of various functional molecules are known, their sequences can be modified at one or more amino acid residues, such as at glycosylation sites, to generate immunogenic variants.

[0209] In one embodiment, the functional molecule is recombinant. Alternatively, the functional molecule is a humanized derivative of its mammalian counterpart. Exemplary mammalian species from which the functional molecule is derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In a preferred embodiment, the functional molecule is a human or humanized version of the aforementioned functional molecule.

[0210] The aforementioned functional molecules, e.g., T cell stimulatory molecules, T cell costimulatory molecules, and T cell homeostasis factors, can be adsorbed or incorporated into the MSR base layer or the SLB base layer independently of one another. Thus, in one embodiment, an APC-MS is provided in which a T cell stimulatory molecule is adsorbed or incorporated into the MSR base layer. Preferably, an APC-MS is provided in which a T cell stimulatory molecule is adsorbed or incorporated into the SLB layer. In another embodiment, an APC-MS is provided in which a T cell stimulatory molecule is adsorbed or incorporated into both the MSR base layer and the SLB layer. In another embodiment, an APC-MS is provided in which a T cell costimulatory molecule is adsorbed or incorporated into the MSR base layer. Preferably, an APC-MS is provided in which a T cell costimulatory molecule is adsorbed or incorporated into the SLB layer. In yet another embodiment, an APC-MS is provided in which a T cell costimulatory molecule is adsorbed or incorporated into both the MSR base layer and the SLB layer. In another embodiment, an APC-MS is provided in which a T cell homeostasis factor is adsorbed or incorporated into the MSR base layer. In another embodiment, an APC-MS is provided in which a T cell homeostasis factor is adsorbed or incorporated into the SLB layer. In yet another embodiment, an APC-MS is provided in which a T cell homeostasis factor is adsorbed or incorporated into both the MSR base layer and the SLB layer.

[0211] Generally, functional molecules and the MSR base layer and / or SLB layer can be linked together through the use of reactive groups, which are typically converted by the linking process into new organic functional groups or non-reactive species. The reactive functional groups can be located on any of the aforementioned components. The reactive groups and reaction classes useful in the practice of the present invention are generally well known in the field of bioconjugation chemistry. Currently convenient classes of reactions available with reactive chelating compounds are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitution (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are described, for example, in 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, Discussed in American Chemical Society, Washington, DC, 1982.

[0212] Useful reactive pendant functional groups include, for example, the following: (a) carboxyl groups and various derivatives thereof (including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides (e.g., I, Br, Cl), acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters); (b) hydroxyl groups (which can be converted, for example, to esters, ethers, aldehydes, etc.) (c) haloalkyl groups (wherein the halide can be later displaced with a nucleophilic group (such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion), thereby resulting in the covalent attachment of a new group at the halogen atom functionality); (d) dienophile groups (which can participate in Diels-Alder reactions (e.g., maleimide groups)); (e) aldehyde or ketone groups (subsequent derivatization is possible via formation of carbonyl derivatives (such as, for example, imines, hydrazones, semicarbazones, or oximes) or via mechanisms such as Grignard addition or alkyllithium addition); (f) sulfonyl halide groups for subsequent reaction with amines to form, for example, sulfonamides; (g) thiol groups (which can be converted, for example, to disulfides or reacted with acyl halides); (h) amine or sulfhydryl groups (which may, for example, be acylated, alkylated, or oxidized); (i) alkenes (which can undergo, for example, cycloaddition, acylation, Michael addition, etc.); (j) epoxides (which can react with, for example, amines and hydroxyl compounds); and (k) Phosphoramidites and other standard functional groups useful in nucleic acid synthesis.

[0213] The reactive functional group can be selected so that it does not participate in or interfere with the reactions required to assemble the reactive chelating compound. Alternatively, the reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those skilled in the art will understand how to protect a particular functional group so that it does 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.

[0214] In one embodiment, functional molecules are loaded / adsorbed onto the MSR base layer or the SLB, or onto both the MSR layer and the SLB, via affinity pairing or chemical coupling.

[0215] The term "affinity pair" as used herein refers to antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, hybridizing sequences of nucleic acids (RNA or DNA), Fc receptor or mouse IgG-Protein A, avidin-biotin, streptavidin-biotin, biotin / biotin-binding agent, Ni 2+ or Cu 2+ A variety of other specific binding pairs are contemplated for use in practicing the methods of the present invention, including a / His tag (6 x histidine) and virus-receptor interactions.

[0216] As used herein, "biotin binding agent" refers to a The term "biotin-binding agent" encompasses avidin, streptavidin, and other avidin analogs (e.g., streptavidin or avidin conjugates, highly purified and fractionated species of avidin or streptavidin, as well as non- or partial amino acid variants, recombinant or chemically synthesized avidin analogs with amino acid substitutions or chemical substitutions that are still compatible with biotin binding. Preferably, each biotin-binding agent molecule binds at least two biotin moieties, and more preferably at least four biotin moieties. As used herein, "biotin" encompasses, in addition to biotin, biocytin and other biotin analogs (e.g., biotin aminocaproate N-hydroxysuccinimide ester, biotin 4-amidobenzoic acid, biotinamidocaproyl hydrazide, and other biotin derivatives and conjugates). Other derivatives include biotin-dextran, biotin-disulfide-N-hydroxysuccinimide ester, biotin-6 These include amidoquinoline, biotin hydrazide, d-biotin-N hydroxysuccinimide ester, biotin maleimide, d-biotin p-nitrophenyl ester, biotinylated nucleotides and biotinylated amino acids (eg, Nε-biotinyl-l-lysine).

[0217] Ligands that can be functionalized through affinity pairing include, but are not limited to, receptors, 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 prepared or isolated from natural or synthetic sources. Briefly, any site-specific ligand of any molecular epitope or receptor to be detected through 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 (e.g., a soluble extracellular domain). The ligand can also be a receptor involved in receptor-receptor cell interactions (e.g., TCR binding to an MHC receptor).

[0218] The ligands of the present invention can be expressed and purified by any method known in the art. In certain embodiments, the proteins are expressed in a baculovirus-based insect or mammalian expression system. TM 15 residues of the peptide can be added to any C-terminus of the molecule. TM (Avidity, CO) can be specifically biotinylated by the BirA enzyme (Avidity, CO). The protein can also be designed to be secreted into the cell culture supernatant.

[0219] The functional molecule can be any protein or peptide, as specifically mentioned hereinabove. Preferably, the protein is involved in ligand-receptor interactions. For example, a key event in T cell activation is the result of membrane-membrane contact between a T cell and an APC, where 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 valency requirements of these interactions will facilitate the design of therapeutic agents that enhance or inhibit immune responses 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 and antagonist antigens. The scaffold provides a robust physiological context in which to examine subtle differences without the need for natural antigen-presenting cells, which often complicates biochemical analysis.

[0220] While streptavidin-biotin interactions are illustrated throughout the present specification and examples, specific binding pair members as described hereinabove can be used in place of streptavidin and biotin in the methods of the present invention. Furthermore, more than one set of specific binding pairs can be used, especially when more than one ligand is attached to the membrane surface. In this situation, the traditional pep-MHC-streptavidin tetramer technique can also be used to screen T cells of a certain pep-MHC specificity. However, T cells with the same specificity may or may not be activated by the same antigenic stimulus. 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 responsiveness to antigens. Using calcium flux by microscopy as an indicator of T cell activation, the present invention also provides a screening assay to quantify primary T cells responding to a specific antigen. Alternatively, biotinylated pep-MHC and costimulatory molecules can be coupled onto a streptavidin-coated chip, which is paired with a scaffold of the invention.

[0221] In another embodiment, functional molecules are chemically coupled to the MSR base layer and / or SLB layer. In certain embodiments, chemical coupling includes click chemistry, such as azide-alkyne chemistry (AAC), dibenzo-cyclooctyne linkage (DCL), or tetrazine-alkene linkage (TAL). For example, in the context of AAC, either the MSR or SLB contains multiple single click chemistry functional groups, and often contains two, three, or more such functional groups. One or two such functional groups per molecule are preferred. In one embodiment, a clickable reagent (e.g., 3-azidopropylamine or 10-undecynoic acid) can be amide-linked to the carboxy or amino terminus of a peptide or protein via a click reaction with the corresponding alkyne or azide compound and an appropriate catalyst, respectively, to form a 1,2,3-triazole ring linker. See, e.g., U.S. Publication No. 2007 / 0060658. To further expand the arsenal of bioorthogonal copper-free click reagents, aza-dibenzocyclooctyne (ADIBO)-containing compounds of the azide coupling reaction 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 non-functionalized regions of a surface. Such treatment allows for a dramatic reduction or complete elimination of nonspecific binding. The copper-free click immobilization method can be applied to the preparation of various types of arrays and the derivatization of microbeads and nanoparticles. See, for example, U.S. Patent No. 8,912,322. In some embodiments, functional molecules are coupled to the MSR base layer and / or SLB layer using a click reagent selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine, and norbornene, and their variants.In some embodiments, the functional molecule comprises an azide and the lipid of the lipid bilayer of the MSR-SLB comprises DBCO.

[0222] The term "click chemistry" refers to a chemical principle introduced by K. Barry Sharpless of The Scripps Research Institute that describes chemical phenomena tailored to rapidly and reliably form covalent bonds by linking small units containing reactive groups together. Click chemistry does not refer to a specific reaction, but rather to a concept that includes reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular, broad in scope, give high chemical yields, generate benign by-products, are stereospecific, exhibit large thermodynamic driving forces favoring reactions of >84 kJ / mol with a single reaction product, and / or can be performed under physiological conditions. The well-defined exothermic reaction makes reactants "dramatically efficient." In some embodiments, click chemistry reactions exhibit high atom efficiency, can be performed under simple reaction conditions, use readily available starting materials and reagents, use no toxic solvents or mild or easily removable solvents (preferably water), and / or provide simple product isolation by non-chromatographic methods (crystallization or distillation).

[0223] The term "click chemistry handle," as used herein, 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 a strain-promoted cycloaddition. Generally, a click chemistry reaction requires at least two molecules containing click chemistry handles that can react with each other. Such pairs of click chemistry handles that are reactive with each other 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 in accordance with some aspects of the present invention are described herein, for example, in U.S. Pat. 2014 / 0249296. Other suitable click chemistry handles will be known to those skilled in the art.

[0224] In one embodiment, the present invention provides an APC-MS comprising multiple T cell activation molecules and T cell costimulatory molecules, optionally together with T cell homeostasis factors, adsorbed to the scaffold via metal-chelating lipid head groups. See Maloney et al., Chem Biol., 3(3):185-92, 1996. Several approaches using chelated metal ions have been reported to enable histidine-tagged proteins to be immobilized at several types of interfaces (e.g., lipid interfaces and lipid monolayers with metal-chelating lipids, gold surfaces with self-assembly monolayers formed with metal-chelating alkanethiols, and oxide surfaces with metal-chelating silanes). For example, Peterson et al. (U.S. Pat. No. 5,674,677) describe a method for linking two amino acid sequences by coupling an organic chelator to a protein (e.g., an enzyme) and charging the chelator with a metal ion. This complex is then mixed with any protein containing a histidine tag to couple the complex to the histidine-tagged protein. See also U.S. Patent No. 6,087,452, which is incorporated herein by reference in its entirety.

[0225] The functional molecules of the present invention are preferably proteins. The terms "protein," "peptide," and "polypeptide" are used interchangeably and refer to a polymer 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, proteins, peptides, or polypeptides are at least three amino acids in length. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity (e.g., a carboxylate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.). A protein, peptide, or polypeptide may also be a single molecule or a complex of multiple molecules. A protein, peptide, or polypeptide may simply be a fragment of a naturally occurring protein or peptide. The protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination of these.

[0226] The terms "conjugated" or "conjugation" refer to the association of two molecules, e.g., two proteins, with one another in such a way that they are linked by direct or indirect, covalent or non-covalent interactions. In the context of conjugation via click chemistry, the conjugation can be through a covalent bond formed by reaction of click chemistry handles. In certain embodiments, the association is covalent, and the entities are said to be "conjugated" to one another. In some embodiments, a protein is post-translationally conjugated to another molecule, e.g., a second protein, by forming a covalent bond between the protein and the other molecule after the protein has been translated, and in some embodiments, after the protein has been isolated. In some embodiments, post-translational conjugation of the protein and a second molecule, e.g., a second protein, is achieved by attaching a click chemistry handle on the protein and a second click chemistry handle on the second molecule (which can react with the first click chemistry handle), and performing a click chemistry reaction in which the click chemistry handles react to form a covalent bond between the protein and the second molecule, thus generating a chimeric protein. In some embodiments, two proteins are conjugated at their respective C-termini to generate a CC-conjugated chimeric protein. In some embodiments, two proteins are conjugated at their respective N-termini to generate an NN-conjugated chimeric protein.

[0227] In certain embodiments, multiple detectable labels can be used to analyze and / or study the conjugation process. As used herein, a "detectable label" refers to a moiety having at least one element, isotope, or functional group incorporated into the molecule (e.g., protein or polypeptide) or other entity to which the label is attached, allowing for detection. The label can be attached directly (i.e., via a bond) or by a tether (e.g., an optionally substituted alkylene; an optionally substituted alkenylene; an optionally substituted alkynylene; an optionally substituted heteroalkylene; an optionally substituted heteroalkenylene; an optionally substituted heteroalkynylene; an optionally substituted arylene; an optionally substituted heteroarylene; or an optionally substituted acylene, or any combination thereof, which can create a tether). It is recognized that the label can be attached to or incorporated into a molecule, such as a protein, polypeptide, or other entity, at any position.

[0228] Generally, labels can fall into any one (or more) of five classes: a) labels containing an isotopic moiety (which can be a radioactive or heavy isotope) ( 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, and 186Re); b) labels that include an immunological moiety (which may be an antibody or antigen, or may be conjugated to an enzyme (such as horseradish peroxidase); c) labels that are colored, luminescent, phosphorescent, or fluorescent moieties (such as the fluorescent labels fluorescein isothiocyanate (FITC) or carboxyfluorescein); d) labels that have one or more photoaffinity moieties; and e) labels that are ligands of one or more known binding partners (e.g., biotin-streptavidin, FK506-FKBP). In certain embodiments, the label includes a radioactive isotope, preferably an isotope that emits a detectable particle. In certain embodiments, the label includes a fluorescent moiety. In certain embodiments, the label is the fluorescent label, fluorescein isothiocyanate (FITC). In certain embodiments, the label includes a ligand moiety with one or more known binding partners. In certain embodiments, the label includes biotin. In some embodiments, the label is a fluorescent polypeptide (e.g., GFP or a derivative thereof (e.g., enhanced GFP (EGFP))) or a luciferase (e.g., firefly, Renilla, or Gaussia luciferase). It is understood that in certain embodiments, the label can react with an appropriate substrate (e.g., luciferin) to produce a detectable signal. Non-limiting examples of fluorescent proteins include GFP and its derivatives, proteins containing chromophores that emit light of various colors (e.g., 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, and mKeima. 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, for a discussion of GFP and many other fluorescent or luminescent proteins. In some embodiments, the label comprises a dark quencher (e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat).

[0229] 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, functional molecules are loaded non-covalently. For example, Lei et al. (US Publication No. 2011-0256184) describe mesoporous silicates that provide enhanced spontaneous loading of antibodies such as IgG through non-covalent bonds within their native or functionalized structures. Thus, the scaffolds of the present invention can be formulated with such silicates.

[0230] In another embodiment, the functional molecule is chemically coupled to the MSR. In such an embodiment, the coupling can be carried out 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 through the use of a polyhistidine tag (His tag), a peptide containing a polyhistidine tag, or an antibody containing a polyhistidine tag. As used herein, the polyhistidine tag consists of at least 4, 5, 6, or 7 histidine (His) residues.

[0231] In one embodiment, anchors are used to connect functional molecules to the pore walls. However, anchors are not a required component. In certain embodiments, each pore of mesoporous silica can accommodate at least one functional molecule. Therefore, the pores must be of a size suitable for immobilizing biological materials. The size of the pores depends on the size of the functional molecules to be immobilized. When functional molecules are immobilized in the pores, they can be adsorbed to the inner surface of the pores by electrostatic bonds. Functional molecules can also be held within the pores by non-covalent bonds (e.g., van der Waals forces, hydrogen bonds, or ionic bonds).

[0232] In the aforementioned embodiment in which the MSR includes a moiety fixed by an anchor, the anchor can have the effect of reducing large structural changes of the functional molecule and maintaining it stable. Preferably, the anchor is composed of substantially the same components as the mesoporous material. The anchor can include one or more functional groups that enable binding to the desired functional molecule: hydroxyl group, amide group, amino group, pyridine group, urea group, urethane group, carboxyl group, phenol group, azo group, hydroxyl group, maleimide group, silane derivative, or aminoalkylene group.

[0233] Embodiments of the present invention further relate to MSR-SLB scaffolds of the present invention, including scaffolds comprising such scaffolds comprising a plurality of the aforementioned functional molecules adsorbed within a lipid matrix.

[0234] In one embodiment, functional molecules are adsorbed into the supported lipid bilayer through physical insertion. Techniques for inserting proteins into bilayers of amphiphilic molecules are known in the art. In one embodiment, proteins in the bilayer environment, for example, in a hydrophobic medium and / or a hydrophilic body and / or a hydrated support, can spontaneously insert into the bilayer. Alternatively, proteins can be forced into the bilayer by application of voltage and / or by fusion of protein-loaded vesicles with the bilayer. The vesicles can be contained within or introduced into the hydrophilic body. In one example, proteins can be introduced into the membrane using the probe method disclosed in PCT Publication No. WO 2009 / 024775. The inserted protein can be a known membrane-associated protein (e.g., one or more of the aforementioned T cell activation molecules and / or T cell costimulatory molecules).

[0235] In another embodiment, the functional molecule can be an antigen used in the expansion of T cells. Representative examples of such antigens that can be used in T cell expansion include full-length CD19 or its fragments or variants. CD19 is a prototype antigen used in the expansion of chimeric antigen receptor (CAR) T cells. Turtle et al. See 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 or variant thereof, which are also useful in expanding CAR T cells. See Haso et al., Blood, 121(7): 1165-1174, 2013; Qin et al., Blood, 122:1431, 2013.

[0236] In an alternative embodiment, the functional molecule can be a membrane-associated protein that is anchored directly or indirectly to the bilayer. Other functional molecules, such as selective or nonselective membrane transport proteins, ion channels, pore-forming proteins, or membrane-resident receptors, can also be inserted into SLBs via this method.

[0237] In another embodiment, the functional molecule may be conjugated to a membrane-associated protein (e.g., gramicidin) that associates with and / or inserts into an SLB; an α-helical bundle (e.g., bacteriorhodopsin or a K+ channel); and a β-barrel (e.g., α-hemolysin, leukocidin, or E. coli porin); or a combination thereof.

[0238] In certain embodiments, the fabricated SLBs (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, sphingolipids and glycosphingolipids, saturated or unsaturated fatty acids, fatty alcohols, polyoxyethylene-polyoxypropylene copolymers, ethoxylated fatty acids and their esters or ethers, dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or a combination of two or more of those mentioned above. A preferred surfactant according to the present invention is dimyristoyl phosphatidylglycerol.

[0239] The fabricated SLBs may be optionally stabilized with at least one co-surfactant selected from the group including or consisting of butanol, butyric acid, hexanoic acid, sodium cholate, sodium taurocholate, and sodium glycocholate, more particularly sodium cholate.

[0240] The fabricated SLBs may also include other excipients (e.g., polymers with bioadhesive or absorption-enhancing properties), which may be selected from the group including or consisting of acrylic polymers (CARBOPOL®, Polycarbophil, NOVEON®), medium-chain fatty acids, and polyethylene glycol. Preferred excipients are the acrylic polymers mentioned above.

[0241] The SLB can be modified with a reagent 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 associate with the bilayer, causing a detectable change in the bilayer's properties. The changed properties can be physical, optical, electrical, or biochemical.

[0242] In some embodiments, the MSR-SLB scaffold and / or antigen-presenting cell mimicking scaffold comprises a small molecule drug. In some embodiments, the MSR-SLB scaffold and / or antigen-presenting cell mimicking scaffold comprises a thalomid analog. In some embodiments, the MSR-SLB scaffold and / or antigen-presenting cell mimicking scaffold comprises an IDO / MEK inhibitor. In some embodiments, the MSR-SLB scaffold and / or antigen-presenting cell mimicking scaffold comprises a small molecule drug having immunomodulatory effects. Small molecule drugs having 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)).

[0243] In certain embodiments, MSR-SLB scaffolds containing functional molecules can be used to detect cells that can interact with the amphiphilic molecules in the bilayer and / or with the functional molecules in the bilayer. The interaction can be specific or nonspecific in nature. Alternatively, the cells may interact with the functional molecules or with the lipid bilayer to cause a physical, optical, electrical, or biochemical change. Such interactions can be detected in many different ways, including, but not limited to, by a visual change, activation of fluorescently labeled lipids or proteins in the SLB, or via a change in the capacitance of the SLB.

[0244] Biodegradable scaffolds Embodiments of the present invention further relate to biodegradable scaffolds. In one embodiment, the scaffold structure can substantially degrade when exposed to a biological environment. In one embodiment, the biological environment is tissue culture conditions, e.g., tissue culture medium optionally adapted for culturing lymphocytes (e.g., T cells). In another embodiment, the biological environment is a biological fluid, e.g., blood, lymph, CSF, ascites, etc. In yet another embodiment, the biological environment is the tissue environment at the implantation site (e.g., blood vessels, lymphatic system, adipose tissue, etc.).

[0245] In certain embodiments, the biodegradable scaffold is substantially degraded after contact with 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 than 90 days. In certain embodiments, the biodegradable scaffold is substantially degraded after contact with a biological environment in vivo in less than 1 week. In certain embodiments, the biodegradable scaffold is substantially degraded after contact with 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 than 90 days. In certain embodiments, the biodegradable scaffold is substantially degraded after contact with an in vitro biological environment in less than one week. Substantial degradation means that at least 30%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or more than 95% of the scaffold composition degrades when the scaffold composition is in contact with a biological environment.

[0246] In certain embodiments, it may be advantageous to use a biodegradable scaffold. For example, by fabricating the scaffold composition so that it substantially degrades during the incubation period (e.g., when the T cells are allowed to expand), it may be possible to use the expanded T cells without having to be subjected to further purification and / or formulation steps. Avoiding downstream purification and / or formulation steps ensures that the T cells are compatible with and have the desired functionality for the desired application.

[0247] Thus, in certain embodiments, it may be advantageous to tailor the degradation kinetics of the scaffold composition by modifying the properties of the mesoporous silica rods (e.g., size, geometry, porosity). Alternatively, the degradation kinetics of the scaffold composition may be modified by changing the culture conditions (e.g., by adjusting the pH of the medium).

[0248] In accordance with the foregoing objectives, embodiments of the present invention relate to an MSR-SLB scaffold comprising multiple functional molecules, which is optionally biodegradable. In one embodiment, the scaffold of the present invention can be encapsulated into another biodegradable scaffold. Reagents and techniques useful for making 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 from a physiologically compatible and optionally biodegradable polymer. Examples of polymers that can be used in scaffolds 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, polyesteramides, polycarbonates, polycyanoacrylates, polyurethanes, polyacrylates, and blends or copolymers thereof. Biodegradable scaffolds can include biodegradable materials such as collagen, alginate, polysaccharides, polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA) or silk. Methods for fabricating scaffold compositions are known in the art. 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.

[0249] Exemplary scaffolds utilize glycolide or alginate of relatively low molecular weight, preferably at the renal threshold for human clearance after dissolution; for example, the alginate or polysaccharide is reduced to a molecular weight of 1,000 to 80,000 daltons. Preferably, the molecular mass is 1,000 to 60,000 daltons, with 1,000 to 50,000 daltons being particularly preferred. It is also useful to use alginate materials with a high guluronate content, because guluronate units, in contrast to mannuronate units, provide sites for ionic crosslinking via divalent cations to gel the polymer. For example, U.S. Patent No. 6,642,363 (incorporated herein by reference) discloses methods for making and using polymers containing polysaccharides (e.g., alginate).

[0250] The scaffolds of the present invention can be porous so that they can maintain antigen presentation and attract and manipulate immune cells. In one embodiment, the scaffolds comprise a porous matrix, wherein the pores have diameters between 10 nm and 500 μm, particularly between 100 nm and 100 μm. In these embodiments, the present invention utilizes scaffolds, including mesoporous scaffolds. Methods for producing polymer matrices with desired pore sizes and alignments are described in the art, for example, in U.S. Publication No. 2011 / 0020216 and U.S. Patent No. 6,511,650, which are incorporated herein by reference.

[0251] Mesoporous silica rods can be modified into multifunctional delivery platforms for delivering drugs (e.g., chemotherapeutic agents and DNA / siRNA, antibody 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 effective and protective packaging of hydrophobic and charged anticancer drugs with the added ability to image the delivery site for controlled and on-demand delivery (Liong et al., ACS Nano, vol. 2, pp. 889-896, 2008). A significant current challenge is optimizing the design features for efficient and safe in vivo drug delivery (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), these can be assessed through the use of human xenograft tumors in nude mice (Lu et al., Small, vol. 6, pp. 1794-1805, 2010).

[0252] Embodiments described herein further relate to MSR-SLB scaffolds (including scaffolds comprising such scaffolds) in which the dry weight ratio of mesoporous silica microrods (MSR) to T cell activating / co-stimulatory molecules is between about 1:1 and about 100:1, preferably between about 10:1 and about 50:1, and particularly between about 20:1 and about 50:1. In some embodiments, the dry weight ratio of mesoporous silica microrods (MSR) to T cell activating / co-stimulatory molecules in the MSR-SLB scaffold is between about 10,000:1 and about 1:1. In some embodiments, the dry weight ratio of mesoporous silica microrods (MSR) to T cell activating / co-stimulatory molecules in the MSR-SLB scaffold is between about 5,000:1 and about 1:1, between about 1,000:1 and about 1:1, between about 500:1 and about 1:1, or between about 100:1 and about 1:1. In some embodiments, the dry weight ratio of mesoporous silica microrods (MSR) to T cell activating / co-stimulatory molecules in 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.

[0253] The embodiments described herein further relate to compositions and devices comprising the MSR-SLB scaffold, together with functional molecules (e.g., T cell activation molecules, T cell costimulatory molecules, and T cell homeostasis factors), optionally together with one or more additional agents (listed below). In one embodiment, the present invention provides a composition comprising a scaffold and T cells clustered therein. In one embodiment, the T cells are selected from the group consisting of natural killer (NK) cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs), or a combination thereof. In other embodiments, the composition may be a pharmaceutical composition, which may be produced using methods well known in the art. For example, pharmaceutical compositions may be produced by those skilled in the art using accepted principles of medical science. The compositions, scaffolds, and devices may be provided with one or more reagents for selecting, culturing, expanding, maintaining, and / or transplanting cells of interest. Representative examples of cell selection kits, culture kits, expansion kits, and transplantation kits for T cells, B cells, and antigen-presenting cells are known in the art. For example, if the target cells of interest are T cells, such cells can be first sorted using DYNABEADS or MACS-beads (Miltenyi Biosciences), maintained in STEMXVIVO human T cell basal medium (R&D Systems), and expanded in OPTIMIZER culture medium (Thermo Fisher Scientific). The cells can be enriched in the sample by using centrifugation techniques known to those skilled in the art, including, for example, FICOLL® gradients. Cells can also be enriched in the sample by using positive selection, negative selection, or a combination thereof based on the expression of certain markers.

[0254] A further embodiment of the present invention relates to a T cell manipulation device. The device comprises a scaffold of the present invention together with a plurality of molecules that attract / bind target T cells. In one embodiment, the present invention relates to a device comprising a scaffold stacked to selectively allow T cell infiltration into mesoporous silica microrods (MSR). Selective infiltration means that, due to selective permeability / permeability, specificity of binding, selective exclusion (of undesired cells) and / or expansion (of desired cells), the scaffold contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 800%, 1000% or more target T cells after an incubation period compared to target T cells present in whole blood. In certain embodiments, the incubation period is between 1 and 30 days, preferably between 4 and 15 days, and particularly between 7 and 12 days. In other embodiments, selective infiltration relates to the retention and / or expansion of T cells relative to other blood cells (e.g., B cells, dendritic cells, macrophages, red blood cells, or platelets present in whole blood).

[0255] In other embodiments, the scaffolds of the invention allow for the selective infiltration of specific subpopulations of T cells, e.g., natural killer (NK) cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, or regulatory T cells (Tregs). Herein, the scaffolds contain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 800%, 1000% or more target T cells after 4-14 days of incubation compared to target T cells present in whole blood. The percentages and ranges of the 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%); and B cells 23% (range: 18-47%) (Berrington et al., Clin Exp Immunol 140(2): 289-292, 2005).

[0256] Further drugs The scaffolds of the invention comprise one or more agents, which can be naturally occurring, synthetically produced, or recombinant compounds (e.g., 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. A purified compound contains at least 60% by weight (dry weight) of the compound of interest. 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 appropriate method, for example, by column chromatography, gel electrophoresis, or HPLC analysis. The antigen can be an autoantigen or a non-autoantigen.

[0257] Representative examples of non-self antigens include, for example, antigens derived from pathogens selected from the group consisting of viruses, bacteria, protozoa, parasites, and fungi. The antigens can be optionally loaded onto MHC molecules (e.g., HLA-A, HLA-B, HLA-C, DP, DQ, and DR), which are then incorporated into the scaffold.

[0258] Alternatively, the scaffold comprises multiple autoantigens, which are optionally linked to or associated with a disease or disorder. Preferably, the autoantigens are specifically associated with a human disease or disorder. In one embodiment, the autoantigen is associated with an autoimmune disorder selected from the group consisting of rheumatoid arthritis, lupus, celiac disease, inflammatory bowel disease or Crohn's disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis, ankylosing spondylitis, type I diabetes, alopecia areata, vasculitis, temporal arteritis, etc. Specific types of antigens (including fragments thereof) associated with type I diabetes, multiple sclerosis, Crohn's disease, rheumatoid arthritis, etc. have been characterized in the literature. For example, a rheumatoid arthritis-associated antigen 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 (e.g., myelin basic protein (MBP) and proteolipid protein (PLP)) appear to be important in the course of multiple sclerosis (MS). See deRosbo et al., J Clin Invest. 92(6):2602-260(1993). In the setting of type 1 diabetes, multiple autoantigens may be involved (e.g., preproinsulin (PPI), islet-specific glucose-6-phosphatase (IGRP), glutamate 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).

[0259] 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, midkine, 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, and 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, 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-type glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5, SCP-I, CT-7, c dc27, 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)-I, UL16-binding protein-like transcript 1 (Mult1), RAE-1 protein, H60, MICA, MICB, and c-erbB-2, or immunogenic peptides thereof, and combinations thereof.

[0260] In another embodiment, the antigen is delivered to an engineered T cell, e.g., a CAR as described above. In one embodiment, the antigen is CD19 or a fragment or variant thereof. In another embodiment, the antigen is CD22 or a fragment or variant thereof.

[0261] The antigens can be combined with the scaffold composition using any known method, including covalent and non-covalent interactions. Some of these methods are outlined above in the section relating to fabricating MSR-SLB scaffolds with the functional molecules of the present invention. Examples of non-covalent interactions include electrostatic interactions, van der Waals interactions, π-effects, hydrophobic interactions, physical insertion, and the like. For example, full-length transmembrane protein antigens can be incorporated into the lipid bilayer via physical insertion using conventional methods. 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.

[0262] Antigens can also be attached or tethered to the scaffold composition via covalent interactions. Methods for attaching antigens to scaffolds / surfaces are known in the art (e.g., surface adsorption, physical immobilization using, e.g., phase change to capture substances within the scaffold material). Alternatively, covalent coupling via alkylating or acylating agents can be used to provide stable, long-term presentation of antigens on the scaffold in a defined conformation. Exemplary reagents and methods for covalently coupling peptides / proteins to polymers are known in the art. See, e.g., U.S. Pat. No. 6,001,395, which is incorporated herein by reference. In other embodiments, antigens are encapsulated within the scaffold. Methods for encapsulating antigens within suitable scaffolds (e.g., PLGA microspheres) are known in the art. See, for example, US Pat. No. 6,913,767 and International Publication No. WO 1995 / 011010, the disclosures of each of which are incorporated herein by reference.

[0263] Antigens can be formulated to interact with immune cells through direct or indirect binding. Direct binding types include, for example, engagement or coupling of the antigen with a cognate receptor (e.g., a T cell receptor). Indirect binding can occur through the mediation of one or more secondary agents or cell types. For example, the antigen can first be bound by a B cell or antigen-presenting cell (APC), processed (e.g., degraded), and presented on a cell surface major histocompatibility complex (MHC) to which a target cell population (e.g., T cells) binds. Alternatively, the antigen can recruit other intermediary cells that secrete various cytokines, growth factors, chemokines, etc., which in turn attract the target immune cell population. Whatever the mechanism, the recruited components act in concert to manipulate or modify the immune cell.

[0264] Antigens can be derived from cell lysates, fractionated cell lysates, freshly harvested cells, biological fluids (including blood, serum, ascites), tissue extracts, etc. In one embodiment, the antigen is derived from the lysate of target cells to which the desired immune cells (e.g., T cells) bind. In these embodiments, the antigen is first fractionated in the cell lysate before loading onto the scaffold. The lysate can be derived from the desired target tissue (e.g., autoimmune disease-specific cells obtained from primary tissue). Alternatively, the lysate can be derived from cancer cells (e.g., individual cells obtained from a tumor sample or tissue culture, or tumor cells obtained from a biopsy tissue structure).

[0265] The scaffolds of the invention may also include one or more recruitment agents, which may be an agent selected from the group consisting of a T cell recruitment agent, a B cell recruitment agent, a dendritic cell recruitment agent, and a macrophage recruitment agent.

[0266] In one embodiment, the scaffold comprises a T cell-recruiting agent. Non-limiting examples of T cell-recruiting agents include, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), or FMS-like tyrosine kinase 3 (Flt-3) ligand, granulocyte colony-stimulating factor (G-CSF), IFNγ, a C-X-C motif chemokine ligand (CXCL) selected from the group consisting of CXCL12 and CXCR4, or fragments, variants, or combinations thereof. Other types of T cell-recruiting agents include ligands for the CCR5 and CXCR3 receptors to recruit the T helper type 1 (Th1) subset. CCR5 ligands, CCL5 and macrophage inflammatory protein (MIP-1α), are known. Alternatively, ligands for CCR3, CCR4, CCR8 and CXCR4 can be used for specific recruitment of the Th2 subset. Combinations of ligands can also be used.

[0267] Various homologs (including functional fragments or variants thereof) of the aforementioned T cell-recruiting agents are known in the art. Representative examples of homologs include related proteins derived from flies, mice, rats, pigs, cows, monkeys, humans, etc. The homologs preferably include human or mouse homologs of the aforementioned T cell-recruiting agents.

[0268] The scaffolds of the invention are adapted for preferential recruitment of a single type or subtype of cells (e.g., preferential recruitment of T cells, and in particular, a subset of Treg cells or NK cells). Preferential recruitment is characterized by an increased accumulation of at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, or more in one or more particular types of immune cells (e.g., T cells, B cells, DCs / macrophages) in the device compared to other types of immune cells in the device (or in a control scaffold lacking the recruitment agent). In scaffolds adapted to recruit a combination of immune cells (e.g., a combination of T cells and DC / macrophages), preferential recruitment is characterized when the overall percentage of recruited cells is at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2-fold (i.e., 200%), at least 5-fold, at least 8-fold, at least 10-fold, or more than other types of immune cells in the device (or control scaffold). In particular, preferential recruitment is characterized by a 1- to 10-fold increase in the number of cells of interest compared to other immune cells.

[0269] In one embodiment, the present invention relates to an MSR-SLB scaffold further comprising a recruitment agent that 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 in combination with at least one of CCL-21, CCL-19, Flt-3, or GCSF. Representative examples of such recruitment agents include 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 fragment of GM-CSF (e.g., a polypeptide comprising amino acids 18-144 of the hGM-CSF sequence). In yet another embodiment, the present invention relates to a scaffold comprising a GM-CSF variant (e.g., VAR_013089 and VAR_001975, the sequences of which have been accepted into UNIPROT (Accession No. P04141)). In another embodiment, the present invention relates to a scaffold comprising a GM-CSF mimetic (e.g., an antibody that binds to the GM-CSF receptor, e.g., Monfardini et al., Curr Pharm Des., 8(24): 2185-99, The present invention relates to MSR-SLB scaffolds comprising MSR-SLB scaffolds, including those described by Friedrich and Weiss, 2002.

[0270] Embodiments of the present invention further provide a scaffold for engineering immune cells that comprises a plurality of additional agents, which may include growth factors, cytokines, chemokines, interleukins, adhesion signaling molecules, integrin signaling molecules, or fragments thereof, or combinations thereof.

[0271] Representative examples of growth factors / cytokines include adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor (AM), bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), fetal bovine somatotrophin (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), and migration-stimulating factor (MGF). These include, but are not limited to, myostatin (GDF-8), nerve growth factor (NGF), neurotrophin, 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 functional fragments thereof, or combinations thereof.

[0272] 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 (for 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), and IL-17 (activates Th cells). Functional fragments of interleukins, which are characterized by their ability to modulate the activity of target cells, can also be used.

[0273] Optionally, the scaffold may comprise adhesion molecules, which may also act as signaling factors. Representative examples of adhesion signaling molecules include, but are not limited to, fibronectin, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibrinogen, fibrin, mucin, entactin, osteopontin, plasminogen, restrictin, serglycin, and the like. Examples of adhesion molecules include cycin, SPARC / osteonectin, versican, von Willebrand factor, polysaccharide heparin sulfate, connexin, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides, as well as cyclic peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), chondroitin-6-sulfate, integrin ligands, selectins, cadherins, and members of the immunoglobulin superfamily. Other examples include nervous cell adhesion molecule (NCAM), intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM-1), platelet-endothelial cell adhesion molecule (PECAM-1), L1, and CHL1. Functional fragments of adhesion molecules, characterized by their ability to modulate target cell binding to the scaffolds of the present invention, can also be used. In particular, adhesion molecules include peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD), which is known as a cell adhesion ligand and is found in various natural extracellular matrix molecules. In another embodiment, the adhesion peptide is a collagen mimetic. A representative example is a peptide having the structure GGYGGGPC(GPP)5GFOGER(GPP)5GPC (where O is hydroxyproline). Such peptides may be collectively referred to as GFOGER peptides. The GFOGER peptide has previously been shown to be particularly effective for T cell adhesion. See Stephan et al., Nature Biotechnology 33, 2015.

[0274] Polymer matrices with such modifications provide cell adhesion properties to the scaffolds of the present invention, maintaining long-term survival of mammalian cell lines and supporting cell proliferation and differentiation. Adhesion molecules can be coupled to the polymer matrices using synthetic methods commonly known to those skilled in the art and achieved using methods described in the Examples. See, for example, Hirano et al., Advanced Materials, 17-25, 2004; Hermanson et al., See Bioconjugate Techniques, pp. 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).

[0275] Depending on the target cell type, it may be preferable to use adhesion signaling molecules specific for the target cell. Thus, in one embodiment, the scaffold comprises an adhesion receptor useful in binding / sequestration of T cells. In these embodiments, the scaffold may comprise a T cell-specific adhesion molecule (e.g., a receptor selected from the group consisting of MHC class II (for CD4+ cells), MHC class I (for CD8+ cells), LFA-3 (a CD2 ligand), ICAM1 (a ligand for LFA-1), or variants, fragments, or combinations thereof).

[0276] Depending on the need, the scaffold can be specifically formulated to include a recruitment agent and a subset of adhesion molecules to manipulate a specific subset of immune cells (e.g., a specific subpopulation of T cells). In these embodiments, the scaffold can be formulated / fabricated with an agent that specifically binds to a cell surface marker expressed on the target cells. For example, in the context of T cells, the scaffold can be formulated / fabricated with an agent that specifically binds to a cell surface marker expressed on the target cells. For example, in the context of T cells, the scaffold can be formulated / fabricated with an agent that specifically binds to a cell surface marker expressed on the target cells. Hcells; which differentially express CD4+), cytotoxic T cells (T c cells; which differentially express CD8+), memory T cells (T m cells, which differentially express CD45RO), suppressor T cells (T s These antibodies may be adapted for preferential recruitment of T cells (NK cells; differentially expressing CD1d+), regulatory T cells (Tregs; further characterized as FOXP3+ Treg cells and FOXP3- Treg cells), natural killer T cells (NK cells; differentially expressing CD1d+), mucosal-associated invariant T cells (MAIT; differentially expressing MR1), and gamma delta T cells (γδ T cells; comprising a TCR containing one γ chain and one δ chain). Such agents that bind to cell surface markers may include, for example, haptens, peptides, ligands, antibodies, and the like. Other conventional techniques for enriching isolates with one or more cell subtypes may be used in situ or ex situ, as appropriate.

[0277] The scaffold can also be adapted to recruit disease-specific immune cells. For example, multiple T cells specific for a particular type of autoimmune disease can be recruited. Thus, in one embodiment, a scaffold useful in diagnosing an autoimmune disorder can be formulated to include a recruitment agent specific for immune cells involved in the disorder. Such a recruitment agent can be specific for, for example, regulatory T cells (Tregs), suppressor T cells (Ts), or a combination thereof. In a related embodiment, a scaffold useful in diagnosing cancer can be formulated to include a recruitment agent to preferentially recruit cancer-specific T cell types, such as cytotoxic T cells (Tc), natural killer cells (NK), or a combination thereof.

[0278] In certain embodiments, the scaffold is useful for targeting disease-specific cells. These may include, for example, cells that directly promote disease progression. In many autoimmune disease situations, the disease can be mediated and promoted through targeted killing of specific populations of cells, such as pancreatic beta cells in T1D and neuronal cells in multiple sclerosis. In other autoimmune diseases, the disease can be promoted by targeted attack of specific epitopes, such as rheumatoid factor (RF) and citrullinated peptides (ACPA) in rheumatoid arthritis, and antigens present in the intestinal flora in Crohn's disease. Targeted destruction of cells generally requires a specific type or subset of immune cells. Therefore, based on the nature and characteristics of the cellular target, specific immune cells can be preferentially engineered using the scaffold of the present invention.

[0279] In the aforementioned embodiments, the scaffold is provided with an antigen to which disease-specific immune cells (e.g., T cells) bind. These autoimmune cells can be engineered and optionally reprogrammed into 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 context of cancer-specific T cells, the reprogrammed cells can be rejuvenated to target cancer. Alternatively, in the context of autoimmune disease-specific T cells, the cells can be eliminated.

[0280] In some embodiments, the scaffold of the present invention is fabricated as a porous structure that is engineered to maintain antigen presentation.Methods for fabricating porous scaffolds have been described in the art.See, for example, U.S. Publication No. 2011 / 0020216, U.S. Publication No. 2013 / 0202707, U.S. Publication No. 2011 / 0020216 and U.S. Patent No. 8,067,237 (the disclosures of which are incorporated herein by reference).

[0281]

[0010] Embodiments of the present invention further provide scaffolds, including MSR-SLB scaffolds, that have desirable stability for various ex vivo and in vivo applications. For example, the scaffolds are stable in tissue culture applications, cell growth experiments, or as implantation materials for tissue (harvested or engineered) and also administered to subjects. In one embodiment, the present invention relates to mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffolds that maintain a continuous fluid system 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 more than 50 days. Scaffold stability and / or fluid systems can be monitored using conventional techniques (eg, microscopic visualization techniques as illustrated in the Examples below).

[0282] II. Methods for Making the Scaffolds of the Invention

[0013] Embodiments of the present invention further relate to methods for producing the antigen-presenting cell-mimicking scaffolds (APC-MS) of the present invention. The methods include providing a base layer comprising high-surface-area mesoporous silica microrods (MSR); optionally loading a T cell homeostasis factor onto the MSR; layering a continuous fluid-supported lipid bilayer (SLB) on the base layer comprising the MSR, thereby producing an MSR-SLB scaffold; if step (b) is not performed, loading a T cell homeostasis factor onto the MSR-SLB scaffold; optionally blocking one or more nonspecific interactions in the MSR-SLB scaffold with a blocking agent; and loading a T cell activation molecule and a T cell costimulatory molecule onto the MSR-SLB scaffold, thereby producing an APC-MS. In these embodiments, the methods may further include loading at least one additional agent into the scaffold, the agent being a growth factor, a cytokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, or a fragment or combination thereof. Methods for loading the additional components are described above in the device fabrication section. An exemplary method for making a scaffold of the present invention is provided in Figure 24.

[0283] In one embodiment, a mixture of functional molecules, including a 1:1 mixture of T cell activation molecules and T cell costimulatory molecules (e.g., anti-CD3 and anti-CD28 antibodies), is combined with an MSR-SLB scaffold at a weight ratio of functional molecules to MSR-SLB scaffold of about 1:2 to about 1:20, preferably about 1:4 to about 1:15, and particularly about 1:5 to about 1:10. The weight ratio of T cell activation molecules to T cell costimulatory molecules can be adjusted, for example, to about 5:1 to about 1:5, while maintaining the same dry weight ratio between functional molecules and MSR-SLB scaffold.

[0284] Moreover, embodiments of the present invention further relate to methods for making APC-MS by assembling multiple scaffolds to generate stacks with sufficient porosity to allow infiltration of distinct subpopulations of T cells, more specifically helper T cells or cytotoxic T cells.

[0285] III. Methods for Using the Scaffolds of the Invention The scaffolds of the present invention can be used for a variety of applications, including, but not limited to, engineering target effector cells, e.g., T cells, isolating specific populations of effector cells (e.g., subpopulations of CD8+ T cells), diagnosing and treating disease, and generating compositions and kits for diagnosing and treating disease.

[0286] Methods for manipulating target cells In one embodiment, the invention provides a method for manipulating target effector cells or subpopulations thereof (e.g., helper T cells or cytotoxic T cells). In this context, the term "manipulation" includes, for example, activation, division, differentiation, growth, expansion, reprogramming, anergy, quiescence, senescence, apoptosis, or death of target effector cells.

[0287] In one embodiment, target effector cells (e.g., T cells) are manipulated (e.g., activated) in situ by providing a scaffold of the invention so that the target effector cells come into contact with the scaffold. To promote contact, the scaffold can be implanted at an appropriate site in a subject, for example, subcutaneously or intravenously. In another embodiment, target cells are manipulated ex vivo by culturing a sample containing target effector cells with a scaffold of the invention.

[0288] A variety of target effector cells can be engineered, including fresh samples provided by a subject, primary cultured cells, immortalized cells, cell lines, hybridomas, etc. The engineered cells can be used for a variety of immunotherapeutic applications and studies.

[0289] The site of manipulation of target effector cells can be in situ or ex situ. Thus, in one embodiment, cells are manipulated in situ (e.g., within a scaffold). In this situation, cells do not need to be physically removed from the scaffold to be manipulated. In another embodiment, cells are manipulated ex situ (e.g., by first removing cells from the scaffold and manipulating the removed cells). When the scaffold is implanted into a subject, 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 can be manipulated in situ or ex situ, as previously described.

[0290] In certain embodiments, the scaffold used in engineering effector cells may be provided with antigen-presenting cells (APCs) and / or various antigens derived from such APCs. These secondary agents (e.g., APCs or antigens derived from APCs) may be provided in the scaffold structure or may be provided exogenously, e.g., in the culture medium. In certain embodiments, the scaffold may be provided with various antigens that attract and / or recruit APCs. Representative examples of such attracting and / or recruiting molecules are provided in the preceding section.

[0291] In certain embodiments, antigen-containing scaffolds can be used to manipulate target effector cells in vivo. For such applications, the scaffolds can be implanted inside blood vessels, lymphatic tissues, tumor sites, disease sites (e.g., areas surrounding tissues affected by rheumatoid arthritis), or subcutaneously so that target effector cells come into contact with the scaffold. Alternatively, the scaffolds can be injected in a minimally invasive manner, for example, via needles, catheters, etc. The implanted scaffolds can be allowed to remain at the implantation site for approximately 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 scaffolds can be explanted to study, analyze, or further manipulate effector cells.

[0292] In a related embodiment, the present invention relates to the in situ manipulation of antigen-specific effector cells. In this context, the scaffold of the present invention can include an antigen of interest adsorbed thereon, 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 culture medium, along with APCs presenting the antigen of interest. The target effector cells are then brought into contact with the scaffold, and the functional molecules contained in the scaffold act together to promote the manipulation of the effector cells. As a purely representative embodiment, as described in the Examples section, a sample containing T cells is incubated with the scaffold of the present invention, which activates, costimulates, and homeostatically maintains the target effector cells. The sample can be incubated with the scaffold for about 1 to 30 days, about 1 to 15 days, or about 4 to 13 days, e.g., about 7 to 8 days, resulting in the selective manipulation of the effector cell population. The antigen-specific effector cells can be further engineered by selecting cells based on the expression of certain gene products, such as T cell receptors (TCRs) that recognize the antigen or antigen-presenting cells of interest.

[0293]

[0010] Embodiments described herein further relate to methods for ex situ engineering of antigen-specific effector cells, in which a scaffold is provided with APCs expressing an antigen of interest or the antigen itself. The engineering step can be performed ex situ or in situ.

[0294] In another embodiment, effector target cells specific for an antigen or APC can be engineered to be selective over other effector cells (e.g., CD8+ T cells are preferred over CD4+ T cells). For example, a sample containing CD8+ T cells (along with CD4+ T cells) can be incubated with a scaffold of the invention that has been mechanically or chemically engineered to allow infiltration and / or sequestration of CD8+ T cells. The infiltrated and / or sequestered CD8+ T cells can be further expanded, activated, propagated, or grown using techniques known in the art. Representative methods have been described above.

[0295] In another embodiment, effector target cells specific for antigens or APCs may be unwanted (e.g., regulatory / suppressor T cells), which are induced to undergo apoptosis, anergy, or death after contact with the scaffold of the present invention. For example, a sample containing regulatory T cells (along with other T cells) can be incubated with a scaffold of the present invention that has been mechanically or chemically engineered to allow infiltration and / or sequestration of regulatory / suppressor T cells. The infiltrated and / or sequestered T cells can be eliminated using techniques known in the art.

[0296] In this situation, the identity of infiltrated and / or sequestered cells in the scaffold of the present invention can be further determined using techniques known in the art. Thus, in one embodiment, the gene product for identifying or selecting activated T cells can be a cell surface marker or cytokine, or a combination thereof. Cell surface markers for identifying 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 and B cells. CD4 is a TCR coreceptor and a marker for thymocytes, TH1 and TH2 T cells, monocytes, and macrophages. CD8 is also a TCR coreceptor and a marker for cytotoxic T cells. CD134 is expressed only on activated CD4+ T cells.

[0297] Cell surface markers for selecting activated T cells include, but are not limited to, CD36, CD40, and CD44. CD28 acts as a stimulatory T cell activation pathway independent of the T cell receptor pathway and is expressed on CD4+ and CD8+ cells. CD36 is a membrane glycoprotein and 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. A subset of T cells can be isolated by using positive selection, negative selection, or a combination thereof for 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 for identifying activated TH2-type T cells include, but are not limited to, IL-4, IL-5, IL-10, and IL-13. Subsets of T cells can also be isolated by using positive selection, negative selection, or a combination thereof for the expression of cytokine gene products of helper T cells or cytotoxi...

Claims

1. 1. An antigen-presenting cell mimic scaffold (APC-MS), comprising: High surface area mesoporous silica microrods (MSR); a fluid-supported lipid bilayer (SLB) layered on the MSR; an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof, wherein the anti-CD3 antibody or an antigen-binding fragment thereof and the anti-CD28 antibody or an antigen-binding fragment thereof are presented on the SLB via affinity pairing or chemical coupling; Including, the APC-MS comprises spaces between the MSRs that allow infiltration of T cells; APC-MS.

2. 2. The APC-MS of claim 1, wherein the affinity pairing or chemical coupling is via the pairing of biotin and streptavidin.

3. the SLB comprises biotin conjugated to streptavidin, and the anti-CD3 antibody or antigen-binding fragment thereof and the anti-CD28 antibody or antigen-binding fragment thereof are biotinylated and bind to the streptavidin; The APC-MS according to claim 2.

4. The APC-MS according to any one of claims 1 to 3, wherein the SLB comprises 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

5. The APC-MS according to claim 4, wherein the SLB further comprises 0.01 to 1 mol % of a biotinylated lipid.

6. The APC-MS of claim 5, wherein the biotinylated lipid is biotinylated phosphoethanolamine.

7. The APC-MS of any one of claims 1 to 5, wherein the weight ratio of the SLB to the MSR is between 3:1 and 1:

5.

8. The APC-MS of claim 7, wherein the weight ratio of the SLB to the MSR is 1:

2.

9. The APC-MS of claim 6, wherein the MSR comprises an average length of 50 μm to 200 μm.

10. The APC-MS according to any one of claims 1 to 9, comprising T cells.

11. The APC-MS of claim 10, wherein the T cells express a T cell receptor.

12. The APC-MS of claim 10, wherein the T cells express a chimeric antigen receptor.

13. 1. A method for stimulating T cells in vitro or ex vivo, comprising: Culturing the T cells in the presence of the APC-MS according to any one of claims 1 to 12. A method comprising:

14. 14. The method of claim 13, wherein the culturing of T cells includes one or more of IL-2, IL-7, IL-15, and IL-21.

15. Use of the APC-MS of any one of claims 1 to 12 for the manufacture of a medicament for treating cancer in a subject in need thereof.

16. A pharmaceutical composition comprising the APC-MS of any one of claims 1 to 12 and a pharmaceutically acceptable carrier for use in treating cancer in a subject in need thereof.

17. A composition comprising the APC-MS of any one of claims 1 to 12 for treating cancer in a subject in need thereof.

18. The use of claim 15, wherein the APC-MS comprises T cells derived from the subject.

19. The pharmaceutical composition for use according to claim 16, wherein the APC-MS comprises T cells derived from the subject.

20. The composition of claim 17, wherein the APC-MS comprises T cells derived from the subject.

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