Injectable artificial antigen-presenting cells for immunotherapy

Abstract

In various aspects and embodiments, this disclosure provides artificial antigen-presenting cells (aAPCs) suitable for parenteral administration for immunotherapy. In various embodiments, aAPCs effectively activate or inhibit target T cells, including CD8+ or CD4+ T cells, in vivo. The aAPCs according to this disclosure provide a storage-stable nanoparticle platform for immunotherapy. In various embodiments, the storage-stable nanoparticle platform controls characteristics such as particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation tendency.

Description

[0001] priority This application claims priority and benefit to U.S. Provisional Application No. 63 / 454,361, filed March 24, 2023, which is hereby incorporated by reference in its entirety.

[0002] sequence list This application contains a sequence list, which is submitted in XML format via EFS-Web and is hereby incorporated in its entirety by reference. The XML copy created on March 25, 2024, is named NEX-013PC_107590-5013_Sequence_Listing and has a size of 61,440 bytes. Background Technology

[0003] Antigen-presenting cells (APCs) process antigenic peptides on their surface and display complexes of these peptides with major histocompatibility complexes (MHC) proteins. Effector cells, such as T cells, recognize these peptide-MHC (pMHC) complexes via cell surface receptors such as T cell receptors (TCRs). Dendritic cells (DCs) are an example of antigen-presenting cells that can be stimulated to efficiently present antigens and support the expansion of immune effector cells, thereby activating a cytotoxic response against the antigen. In some immunotherapies, DCs are harvested from a patient and transfected with antigen pulses or viral vectors. After being infused back into the patient, these activated cells present tumor antigens to effector lymphocytes (e.g., CD4+ T cells, CD8+ T cells, and B cells). When successful, the therapy initiates a cytotoxic response against cells expressing antigens, including tumor antigens. However, there remains a need for storage-stable pharmaceutical compositions capable of efficiently presenting peptide antigens to T cells in vivo (i.e., artificial antigen-presenting cells or aAPCs) to activate or inhibit T cells in an antigen-specific manner. This disclosure satisfies these and other objectives. Summary of the Invention

[0004] In various aspects and embodiments, this disclosure provides artificial antigen-presenting cells (aAPCs) suitable for parenteral administration for immunotherapy. In various embodiments, aAPCs effectively activate or inhibit target T cells, including CD8+ or CD4+ T cells, in vivo. The aAPCs according to this disclosure provide a storage-stable nanoparticle platform for immunotherapy. In various embodiments, the storage-stable nanoparticle platform controls characteristics such as particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation tendency.

[0005] In various aspects and embodiments, the present invention provides storage-stable compositions and methods for activating or inhibiting antigen-specific T cells in patients. In addition to storage stability, the nanoscale aAPCs described herein are designed to provide pharmacodynamic advantages, including those relating to circulatory properties, biodistribution, and degradation kinetics. These advantages may stem from physical parameters, including particle size, surface charge, polydispersity index, polymer composition, ligand conjugation chemistry, ligand density, and peptide loading. In some embodiments, aAPCs have peptide ligand densities that avoid aggregation potential and spatial constraints from a large number of ligands on the surface without loss of activity and / or potency. In some embodiments, aAPCs persist in peripheral blood circulation for a sufficient duration to allow distribution to target tissues, including transport to lymphatic organs (e.g., lymph nodes) and / or to tumors and / or to target organs via blood / lymph exchange. In some embodiments, aAPCs are suitable for subcutaneous administration.

[0006] In one aspect, this disclosure provides an aAPC comprising a poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymer. In these embodiments of the disclosure, the aAPC has advantages, for example, in terms of stability and ligand density. In these embodiments, the disclosure provides an aAPC suitable for parenteral administration (including subcutaneous administration in some embodiments), and said aAPC comprises a PLA-PEG or PLGA-PEG copolymer and one or more polypeptide ligands chemically conjugated to the PEG polymer via thioether bonds or other conjugation. The polypeptide ligand comprises an HLA ligand (human leukocyte antigen ligand) presenting a peptide antigen and optionally one or more signal 2 ligands. The signal 2 ligands include signal 2 ligands for T cell activation and / or amplification or T cell suppression. In various embodiments, about 40% by weight or less of the copolymer has functional groups for polypeptide ligand coupling.

[0007] In various embodiments, the HLA ligand can be an HLA class I and / or class II molecular complex, or a portion thereof containing an antigen-binding cleft. In some embodiments, the HLA molecular complex is a monomer or dimer and may contain additional heterologous sequences, such as immunoglobulin sequences. In some embodiments, HLA-fusions (e.g., HLA-immunoglobulin fusions) offer additional advantages in terms of stability, TCR binding affinity, and / or T cell activation or inhibition potency.

[0008] In various embodiments, aAPC comprises an HLA class I ligand for presenting peptide antigens to CD8+ T cells (e.g., for activating and / or expanding CD8+ cells, or for inhibiting CD8+ cells). In some embodiments, the HLA class I ligand comprises at least two fusion proteins. A first fusion protein comprises a first HLA class I α chain and a first immunoglobulin heavy chain, and a second fusion protein comprises a second HLA class I α chain and a second immunoglobulin heavy chain. The first and second immunoglobulin heavy chains associate to form an HLA class I molecular complex (e.g., via disulfide bonds). The HLA class I molecular complex comprises a first HLA class I peptide-binding cleft and a second HLA class I peptide-binding cleft.

[0009] Alternatively, aAPC includes an HLA class II ligand for presenting peptide antigens to CD4+ T cells (e.g., for activating and / or expanding CD4+ cells, or for suppressing CD4+ cells). In some embodiments, the HLA class II molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise (i) an extracellular domain of an immunoglobulin heavy chain and (ii) an extracellular domain of an HLA class II β chain. Two second fusion proteins comprise (i) an extracellular domain of an immunoglobulin light chain and (ii) an extracellular domain of an HLA class II α chain. The two first fusion proteins and the two second fusion proteins associate to form the HLA class II molecular complex. The extracellular domain of the HLA class II β chain of each first fusion protein and the extracellular domain of the HLA class II α chain of each second fusion protein form an HLA class II peptide-binding cleft.

[0010] The peptide antigen binds to the antigen-binding cleft of the antigen-presenting complex. This article describes peptide antigens for immunotherapies of oncological diseases, infectious diseases, and autoimmune diseases. In some embodiments, the peptide antigen does not induce aggregation of aAPCs. That is, peptide antigens with low aggregation tendency when loaded onto aAPCs are selected. In some embodiments, an aggregation potential score (APS) is used to select peptides with low aggregation tendency.

[0011] In some embodiments, the peptide ligand comprises a signal-2 ligand, which is a co-stimulatory ligand, for example, for activating and / or amplifying target T cells. Exemplary co-stimulatory ligands include agonists of any of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT. In various embodiments, the co-stimulatory ligand may induce activation and / or amplification of CTLs or Tregs. In other embodiments, and particularly where aAPC is intended to suppress target T cells, the peptide ligand does not contain any signal-2 ligand, or the peptide ligand comprises an inhibitory ligand that induces tolerance or apoptosis of target T cells. In various embodiments, the inhibitory ligand is an agonist of Fas, TGF-β, or PD-1. The agonist ligand may comprise a natural agonist ligand (or an engineered variant thereof, including immunoglobulin fusions as described), or in some embodiments, an antibody agonist. In some embodiments, the co-inhibitory ligand is PD-L1 (or an immunoglobulin fusion thereof) or FasL (or an immunoglobulin fusion thereof). Antibody agonists can be complete monoclonal antibodies or contain portions or fragments of antigen-binding sequences, such as Fab, Fab', F(ab')2, or scFv.

[0012] In various embodiments, signal 1 and signal 2 ligands can be combined in homodimeric or heterodimeric constructs. For example, an HLA ligand can comprise a fusion of an HLA extracellular domain with an immunoglobulin Fc region (such as the IgG4 Fc region), which can dimerize (e.g., via disulfide bonds) with a signal 2-immunoglobulin (Ig) fusion (i.e., a heterodimeric Ig fusion construct). In still other embodiments, the HLA ligand comprises a fusion with a signal 2 ligand. For example, the HLA extracellular domain can be fused at its C-terminus to an immunoglobulin Fc region and at its N-terminus to a signal 2 ligand to prepare a homodimeric ligand with both signals dimerized. In some embodiments, the signal 2 ligand comprises the activating portion of a single-chain antibody (e.g., scFv) or a native ligand.

[0013] In some embodiments, the co-inhibitory ligand is a Fas agonist antibody. In some embodiments, the Fas agonist antibody is an IgG4 antibody based on the CH11 clone. As demonstrated herein, the anti-Fas antibody is active when it is capable of crosslinking multiple Fas receptors, such as when the antibody is conjugated to nanoparticles.

[0014] In some embodiments, aAPC further comprises one or more cytokines that support T cell activation and / or expansion or T cell suppression. One or more cytokines or functional portions thereof may be conjugated to aAPC as peptide ligands. Alternatively, the cytokines or functional portions thereof may be fused with a signal 1 or signal 2 peptide ligand (which may optionally be presented in a homodimeric or heterodimeric Ig fusion construct as described herein). In some embodiments, the cytokines are encapsulated in copolymers and will be locally released in a targeted environment (e.g., in a lymphoid organ, tumor, or target tissue or organ). Examples of cytokines that may be used include IL-1β, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and interferon-gamma. For example, IL-2 may be used with a co-stimulatory signal 2 ligand. In some embodiments, aAPC comprises a tolerogenic cytokine as a peptide ligand.

[0015] In various embodiments, one or more peptide antigens are tumor or cancer-associated antigens, such as tumor-derived antigens, tumor-specific antigens, and neoantigens. In some embodiments, the target peptide antigen includes at least one antigen associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen. In some embodiments, one or more target peptide antigens are "autoantigens," meaning they are associated with autoimmune diseases or reactions.

[0016] In various embodiments, aAPC is included in a pharmaceutical composition suitable for administration to a subject. The pharmaceutical composition may have one or more excipients, such as buffers, surfactants, preservatives, polymers, fillers, and stabilizers.

[0017] In some aspects and embodiments, this disclosure provides aAPC suitable for parenteral (including subcutaneous) administration and exhibiting low aggregation tendency. In these aspects, the aAPC comprises polymeric or lipid nanoparticles containing a polyethylene glycol (PEG) sheath and one or more polypeptide ligands conjugated to PEG (e.g., the PEG terminus) via thioether bonds or other functional groups. The polypeptide ligand comprises an HLA class I or II ligand for presenting the peptide antigen and optionally one or more signal 2 ligands. The peptide antigen for presentation to T cells does not induce aggregation of the aAPC. For example, in some embodiments, the peptide antigen does not have exposed cysteine ​​residues, and / or the peptide antigen has one or more exposed glycine residues or exposed charged residues. In some embodiments, the peptide antigen does not have any cysteine ​​residues and contains one or more charged residues (e.g., 1, 2, or 3 charged residues). In some aspects and embodiments, the aggregation potential is estimated by computer simulation by determining the average aggregation potential score (APS) of the antigenic peptide residues within the HLA antigen-binding cleft.

[0018] In other aspects, the present invention provides a method for immunotherapy. The method includes administering an aAPC or pharmaceutical composition as described herein to a subject requiring treatment. In various embodiments, the subject has cancer or an infectious disease, and the aAPC contains a co-stimulatory ligand. In some embodiments, the peptide antigen is selected on a personalized basis for cancer patients based on analysis of the patient's tumor. In some embodiments, nano-aAPC is used as a booster vaccine following adoptive T-cell therapy, wherein naïve T cells, TILs, or T cells from an HLA-matched donor are expanded in vitro and administered to the patient. In some embodiments, the subject has an autoimmune disease, and the aAPC contains a co-inhibitory signal or does not contain a signal 2 ligand. In some embodiments, the autoimmune disease is type 1 diabetes.

[0019] Typically, aAPC or its pharmaceutical composition is administered parenterally. For example, aAPC or its pharmaceutical composition can be administered via intravenous, intra-arterial, subcutaneous, intradermal, intralymphatic, intramuscular, or intratumoral administration. In some embodiments, the aAPC composition is administered subcutaneously.

[0020] In other aspects of this disclosure, peptide ligands (including peptide ligands as aAPCs) for immunotherapy are disclosed. Such peptide ligands include anti-Fas agonist antibodies having an IgG isotype (e.g., IgG4), which can be conjugated to nanoparticles with an HLA ligand for a peptide-presenting antigen (e.g., associated with an autoimmune disease). In other embodiments, the peptide ligand is a dimer PD-L1 ligand comprising the activating portion of PD-L1, such as amino acid residues F19 to T239 of human PD-L1. Each PD-L1 activating fragment can be fused directly or indirectly via a linker to an IgG Fc region (e.g., IgG4) at its C-terminus, and the ligand can dimerize via disulfide bonds in the Fc region. The dimer PD-L1 ligand can be conjugated to nanoparticles with an HLA ligand for a peptide-presenting antigen and used to drive tolerance to the antigen. In other embodiments, the peptide ligand is a dimerized FasL ligand comprising an activating moiety of FasL (such as amino acids P132 to L279 of human FasL), the activating moiety of FasL being fused directly or indirectly at its N-terminus to a dimerized IgG-Fc region (e.g., IgG4). In some embodiments, the dimerized Fc region can be conjugated to nanoparticles via a Cys-containing linker. The nanoparticles can further present HLA peptide antigen ligands to drive apoptosis of antigen-specific T cells. In other embodiments, the peptide ligand is a tolerogenic ligand comprising an activating fragment of PD-L1 (such as amino acids F19-T239 of human PD-L1), the activating fragment of PD-L1 being fused directly or indirectly via a linker to an HLA-immunoglobulin fusion protein. Such ligands can be conjugated to nanoparticles as disclosed herein and used in immunotherapy to drive tolerance in targeted T cells. In other embodiments, the polypeptide ligand is a costimulatory ligand comprising an anti-CD28 agonist scFv conjugated to an HLA-immunoglobulin fusion protein, thereby providing a homodimeric ligand comprising signal 1 and costimulatory signal 2 ligands. The scFv can be fused to an HLA sequence via either the heavy chain or the light chain sequence.

[0021] The invention and various embodiments are further illustrated in the following detailed description. Attached Figure Description

[0022] Figure 1A and Figure 1B The size and surface charge of the nanoparticles prepared according to this disclosure are shown. Figure 1A TEM images of bare particles, protein-conjugated nanoparticles, and peptide-loaded nanoparticles are shown. Figure 1B The particle size distribution, average size, polydispersity index (PDI), and surface charge are shown (from left to right).

[0023] Figure 2This is a schematic diagram showing the conjugation of thiolized ligands with PEG-maleimide functional groups on nanoparticles.

[0024] Figures 3A and 3B show the effects of changing the PEG-mal % on the nanoparticle surface (Figure 3A) or by changing the maleimide:thiol ratio during the coupling reaction (Figure 3B) on the ligand density (bars) and nanoparticle size (squares).

[0025] Figures 4A and 4B show that using PLGA-PEG or PLA-PEG does not result in significant differences in size (bars) or PDI (dots) (Figure 4A) or protein density (Figure 4B) (ligands for each NP are shown by bars, and μg of protein at 10 OD is shown by dots).

[0026] Figures 5A-5D show that the use of PLGA-PEG or PLA-PEG does not result in significant differences in antigen-specific CD8+ T cell stimulation. Figure 5A shows the percentage of IFNγ+ CD8+ T cells. Figure 5B shows the percentage of TNFα+ CD8+ T cells. Figure 5C shows the percentage of IL2+ CD8+ T cells. Figure 5D shows the percentage of CD107a+ CD8+ T cells.

[0027] Figure 6 This demonstrates the transport of antigen-loaded aAPCs to lymph nodes, spleen, and tumors according to embodiments of this disclosure.

[0028] Figures 7A and 7B show that systemic administration of aAPC increased antigen-specific T cells in the spleen (A) and tumor (B) of tumor-bearing mice (B16-OVA, with implanted OT-1 T cells). Spleen cells from mice receiving peptide-loaded aAPC exhibited greater killing potential compared to peptides containing complete Freund's adjuvant (CFA). Figure 7C. N=2 / group. In vitro killing assays were performed using spleen cells harvested on day 22.

[0029] Figures 8A-8C show that T cells recovered from lymph nodes and tumors exhibit phenotypes consistent with persistence and strong antitumor effects. Figure 8A shows T cells recovered from lymph nodes in a non-disease model (specific to ovalbumin antigen). These T cells exhibit central and effector memory phenotypes. Figure 8B quantifies antigen-specific T cells in tumors of a melanoma model (B16F10 cells), of which 63% possess at least three effector functions. Figure 8C quantifies gp100-specific T cells in tumors of a melanoma model and shows that antigen-specific CTLs recovered from tumors exhibit effector and central memory phenotypes.

[0030] Figure 9The results showed that aAPC loaded with gp100 antigen prolonged survival in the B16 mouse model (x-axis is the number of days after tumor implantation). Figure 9 The results showed that aAPC activated anti-tumor T cells in vivo and cleared lung metastases in the model.

[0031] Figure 10A Figure 10C shows the construction of the agonist anti-Fas antibody ligand. Figure 10A The construction of an IgG4 antibody based on a variable domain of the CH11 clone (which is an IgM isotype) is shown. The IgG4 antibody was cross-linked with an anti-IgG4 antibody for in vitro assays. Figures 10B and 10C show that the anti-Fas agonist ligand exhibits almost no activity in the absence of cross-linking, but displays robust activity after cross-linking.

[0032] Figure 11 The results showed that aAPC with PD-L1-Ig signaling ligand 2 rapidly inhibited antigen-specific killing of target cells loaded with peptides.

[0033] Figure 12 The results showed that aAPCs with anti-Fas signaling 2 ligands rapidly eliminated antigen-specific T cells.

[0034] Figure 13 The results showed that anti-Fas aAPC eliminated up to 90% of MART-1-specific T cells by day 13 in a mouse model, and the effect was dose-dependent.

[0035] Figure 14 It is a graph showing the process of selecting antigens for presentation on aAPC to mitigate antigen-load-driven aggregation.

[0036] Figure 15A and Figure 15B The modeling and scoring of aggregated peptides and non-aggregated peptides are shown separately. Detailed Implementation

[0037] In various aspects and embodiments, this disclosure provides artificial antigen-presenting cells (aAPCs) suitable for parenteral administration for immunotherapy. In various embodiments, aAPCs effectively activate or inhibit target T cells, including CD8+ or CD4+ T cells, in vivo. The aAPCs according to this disclosure provide a storage-stable nanoparticle platform for immunotherapy. In various embodiments, the storage-stable nanoparticle platform controls characteristics such as particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation tendency.

[0038] In various aspects and embodiments, the present invention provides storage-stable compositions and methods for activating or inhibiting antigen-specific T cells in patients. In addition to storage stability, the nanoscale aAPCs described herein are designed to provide pharmacodynamic advantages, including those relating to circulatory properties, biodistribution, and degradation kinetics. These advantages stem from physical parameters, including particle size, surface charge, polydispersity index, polymer composition, ligand conjugation chemistry, ligand density, and peptide loading. In some embodiments, aAPCs have peptide ligand densities that avoid aggregation potential and spatial constraints from a large number of ligands on the surface without loss of activity and / or potency. In some embodiments, aAPCs persist in peripheral blood circulation for a sufficient duration to allow distribution to target tissues, including transport to lymphatic organs (e.g., lymph nodes) and / or to tumors and / or to target organs via blood / lymph exchange. In some embodiments, aAPCs are suitable for subcutaneous administration.

[0039] In one aspect, this disclosure provides aAPC (and pharmaceutical compositions thereof) comprising a poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymers. PLA-PEG and PLGA-PEG nanoparticles can be prepared by nanoprecipitation using known processes. In these embodiments of the disclosure, aAPC has advantages, particularly in terms of stability and ligand density. In these embodiments, the disclosure provides an aAPC suitable for parenteral administration (including subcutaneous administration in some embodiments), and said aAPC comprises a PLA-PEG or PLGA-PEG copolymer and one or more polypeptide ligands conjugated to the PEG polymer (e.g., conjugated to the PEG terminus) via thioether bonds or other conjugation chemistry (such as via amine conjugation). The polypeptide ligand comprises an HLA ligand (human leukocyte antigen ligand) presenting a peptide antigen and optionally one or more signal 2 ligands. Signal 2 ligands are described elsewhere herein and include signal 2 ligands for T cell activation and / or amplification or T cell suppression. In various embodiments, about 40% by weight or less of the copolymer has functional groups for peptide ligand coupling. In various embodiments, about 30% by weight or less, or about 25% by weight or less, or about 20% by weight or less of the copolymer has functional groups for peptide ligand coupling. In some embodiments, about 15% by weight to about 35% by weight of the copolymer has functional groups for peptide ligand coupling. In various embodiments, PEG-Mal (by weight) or other PEG functional groups are less than about 10% or less than about 7% by weight of the copolymer, such as about 5%.

[0040] like Figure 2As shown, only a portion of the copolymer contains the functional groups for ligand coupling. In some embodiments, the functional group is a maleimide functional group at the PEG terminus, and the other PEG groups are inert (e.g., including alkyl ether terminators, such as methyl ether terminators). The terminator-capped PEG may also be referred to herein as mPEG. The reaction between the thiol group and the maleimide is well known in the art. See, for example, João MJM et al. Bioconjugation with Maleimides: A Useful Tool for Chemical Biology , Chemistry (August 2018). Other conjugation chemistry (including amine conjugation) may be used, and such conjugation chemistry is well known. Alternative conjugation chemistry is described, for example, in U.S. Patent No. 10,435,668, which is hereby incorporated herein by reference in its entirety.

[0041] For example, molecules can be directly activated using a variety of chemical functional groups, including nucleophilic, leaving, or electrophilic groups. Activating functional groups include alkyl and acyl halides, amines, thiol groups, aldehydes, unsaturated bonds, hydrazides, isocyanates, isothiocyanates, ketones, and other groups known to activate chemical bonding. Alternatively, molecules can be bound to nanoparticles using small molecule coupling agents. Non-limiting examples of coupling agents include carbodiimides, maleimides, N-hydroxysuccinimides, dichloroethylamine, bifunctional aldehydes such as glutaraldehyde, anhydrides, etc. In other embodiments, molecules can be coupled to nanoparticles via affinity bonding, such as biotin-streptavidin bonding or coupling.

[0042] In exemplary embodiments, the nanoparticles have a PLGA copolymer that can be tuned for a specific in vivo biodegradation rate (by adjusting the LA:GA ratio and / or molecular weight of the PLGA polymer). In exemplary embodiments, the PLGA is based on an LA:GA ratio of 20:1 to 1:20, including L / G compositions of 5 / 95, 10 / 90, 15 / 85, 20 / 80, 25 / 75, 30 / 70, 35 / 65, 40 / 60, 45 / 55, 50 / 50, 55 / 45, 60 / 40, 65 / 35, 70 / 30, 75 / 25, 80 / 20, 85 / 15, 90 / 10, or 95 / 5. PLGA degrades via the hydrolysis of its ester bonds. The time required for PLGA degradation is related to the monomer ratio: the higher the content of glycolide units compared to predominantly lactide units, the shorter the degradation time. In some embodiments, the PLGA is 50 / 50 L / G.

[0043] In some embodiments, aAPC may also comprise a PLGA or PLA polymer (in addition to copolymers). In some embodiments, the PLGA or PLA polymer has a molecular weight in the range of about 15 kDa to about 35 kDa or about 15 kDa to about 25 kDa. In one exemplary embodiment, the PLGA or PLA polymer has a molecular weight of about 20 kDa. In various embodiments, the PLGA or PLA polymer is present in amounts of about 5 wt% to about 40 wt% or about 5 wt% to about 25 wt% based on the total weight of the polymer and copolymer.

[0044] In various embodiments, the ratio of PEG-maleimide groups to mPEG limits the density of peptide ligands, for example, conjugated with thiol groups. In various embodiments, each aAPC particle has about 10 to about 500 peptide ligands. In some embodiments, each aAPC particle has about 50 to about 400 peptide ligands. In some embodiments, each aAPC particle has about 100 to about 300 peptide ligands. In various embodiments, by improving peptide density, aAPC particles with effective T-cell effector properties (activation or inhibition) can be prepared while avoiding undesirable properties, including but not limited to aAPC aggregation and spatial confinement.

[0045] In some implementations, other methods can also be used to control the density of peptide ligands on the particles, such as by limiting the amount of thiolized peptides during the coupling reaction.

[0046] In various embodiments, the PLA or PLGA portion of the copolymer has a molecular weight of about 15 kDa to about 50 kDa. In some embodiments, the PLA or PLGA portion of the copolymer has a molecular weight in the range of about 15 kDa to about 35 kDa or about 15 kDa to about 25 kDa. In one exemplary embodiment, the PLA or PLGA portion of the copolymer has a molecular weight of about 20 kDa.

[0047] In various embodiments, the PEG moiety of the copolymer has a molecular weight in the range of about 2 kDa to about 10 kDa or in the range of about 2 kDa to about 7 kDa. In one exemplary embodiment, the PEG moiety of the copolymer has a molecular weight in the range of about 2 kDa to about 5 kDa. In some embodiments, the PEG moiety having functional groups for peptide ligand coupling has a molecular weight of about 5 kDa, and the PEG moiety without functional groups for ligand coupling has a molecular weight of about 3 kDa. In such embodiments, the smaller mPEG moiety (compared to the PEG functional groups) restricts the spatial constraint regarding peptide ligand binding to the target and / or improves conjugation efficiency.

[0048] Therefore, in an exemplary embodiment, the PLA or PLGA portion of the copolymer has a molecular weight of about 20 kDa, the PEG functional group portion of the copolymer has a molecular weight of about 5 kDa, and the mPEG portion of the copolymer has a molecular weight of about 3 kDa.

[0049] In various embodiments, aAPCs have a diameter of about 50 nm to about 150 nm. In some embodiments, aAPCs have a diameter of about 50 nm to about 130 nm. In some embodiments, aAPCs have a diameter of about 50 nm to about 120 nm. In some embodiments, aAPCs have a diameter of about 50 nm to about 100 nm or about 50 nm to about 75 nm. In exemplary embodiments, aAPCs have diameters of about 60 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm. In some embodiments, the aAPC population has a size distribution with a polydispersity index (PDI) of less than 0.2. In various embodiments, aAPCs have a surface charge of about 0 to -15 mV or about 0 to about -10 mV. For example, aAPCs may have a surface charge of about -2.5 mV to about -10 mV. The size and surface charge of aAPCs allow for desired cycling and biodistribution characteristics and provide advantages in particle stability in some embodiments.

[0050] In various embodiments, the HLA ligand may be an HLA class I and / or class II molecular complex, or a portion thereof containing an antigen-binding cleft. In some embodiments, the HLA molecular complex is a monomer or dimer and may contain additional heterologous sequences, such as immunoglobulin sequences. Alternative heterologous sequences include dimerized amino acid sequences such as c-fos or c-jun, or monomeric amino acid sequences such as albumin. In some embodiments, HLA-fusions (e.g., HLA-immunoglobulin fusions) offer additional advantages in terms of stability, TCR binding affinity, and / or T cell activation or inhibition potency.

[0051] In various embodiments, aAPC comprises an HLA class I ligand for presenting peptide antigens to CD8+ T cells (e.g., for activating and / or expanding CD8+ cells, or for inhibiting CD8+ cells). In some embodiments, the HLA class I ligand comprises at least two fusion proteins. A first fusion protein comprises a first HLA class I α chain and a first immunoglobulin heavy chain, and a second fusion protein comprises a second HLA class I α chain and a second immunoglobulin heavy chain. The first and second immunoglobulin heavy chains associate to form an HLA class I molecular complex (e.g., via disulfide bonds). The HLA class I molecular complex comprises a first HLA class I peptide-binding cleft and a second HLA class I peptide-binding cleft.

[0052] Alternatively, aAPC includes an HLA class II ligand for presenting peptide antigens to CD4+ T cells (e.g., for activating and / or expanding CD4+ cells, or for suppressing CD4+ cells). In some embodiments, the HLA class II molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise (i) an extracellular domain of an immunoglobulin heavy chain and (ii) an extracellular domain of an HLA class II β chain. Two second fusion proteins comprise (i) an extracellular domain of an immunoglobulin light chain and (ii) an extracellular domain of an HLA class II α chain. The two first fusion proteins and the two second fusion proteins associate to form the HLA class II molecular complex. The extracellular domain of the HLA class II β chain of each first fusion protein and the extracellular domain of the HLA class II α chain of each second fusion protein form an HLA class II peptide-binding cleft.

[0053] In various embodiments, the immunoglobulin sequence of the HLA polypeptide ligand (i.e., HLA-Ig) is a partial heavy chain sequence containing a hinge region to support dimerization. In some embodiments, the HLA-Ig fusion construct does not contain a variable region sequence. For example, an HLA extracellular domain sequence (i.e., the HLA class I α chain extracellular domain) may be fused to an Ig constant region sequence above the hinge region to provide a dimer HLA. For example, HLA or its antigen-presenting portion may be conjugated to the CH1 portion of each IgG heavy chain. All IgG molecules consist of two identical heavy chains (constant and variable regions) linked together by disulfide bonds in the hinge regions (upper and lower). For example, in some embodiments, an HLA molecule or antigen-presenting complex is fused to CH1 (the N-terminus of the Ig heavy chain above the hinge region) to produce a dimer fusion protein smaller than that produced by fusion with the end of an intact antibody heavy chain due to the lack of any VH and VL light chain sequences. Therefore, such constructs will further include CH2 and CH3 domains. This provides manufacturing advantages and exhibits low immunogenicity potential. In some embodiments, these constructs also demonstrate sufficient binding synergy to achieve effective T cell activation or inhibition. In some embodiments, the IgG sequence is the IgG4 sequence, and the immunoglobulin sequence may comprise or consist of (or substantially comprise) the amino acid sequence of SEQ ID NO: 23.

[0054] In some embodiments, the HLA ligand is an HLA class I ligand, and optionally an HLA-A, HLA-B, HLA-C, or HLA-E ligand. In some embodiments, the HLA ligand comprises an associated β2-microglobulin (β2M) polypeptide. In various embodiments, the HLA ligand (e.g., as presented by HLA-Ig) corresponds to a ligand selected from HLA-A. 02:01, HLA-A 01:01, HLA-A 02:05, HLA-A 02:06, HLA-A 02:12, HLA-A 03:01, HLA-A 11:01, HLA-A 24:02 and HLA-B The allele for 07:02. In some embodiments, the HLA ligand is modified with a cysteine ​​residue that forms a disulfide bond on the α-helix constituting the peptide-binding groove to increase the stability of the complex with the bound peptide. In some embodiments, this cysteine ​​residue is substituted at positions 84 and 139 in the extracellular domain. These modifications bridge the F-pocket, into which the C-terminus of the peptide binds.

[0055] In some implementations, the HLA ligand is HLA-A. 02:01 Ligand (IMGT Registry No. HLA00005). In some embodiments, the HLA ligand is modified with cysteine, which forms a disulfide bond on the α-helix constituting the peptide binding groove to increase the stability of the complex with the bound peptide. For example, HLA-A The 02:01 ligand may be modified with cysteine ​​residues at positions 84 and 139 as shown in SEQ ID NO: 26 to form disulfide bonds on the α-helix constituting the peptide binding groove. Therefore, in some embodiments, the HLA-Ig ligand comprises the sequence of SEQ ID NO: 26, optionally having one to five amino acid modifications selected from substitutions, deletions, and insertions, provided that positions 84 and 139 relative to SEQ ID NO: 26 are cysteine ​​residues.

[0056] In other exemplary embodiments, HLA class I (e.g., HLA-A) 02) It has W51C and G175C substitutions (e.g., relative to SEQ ID NO: 26), thereby forming disulfide bonds at the ends of the peptide groove, with the N-terminus of the peptide binding to said peptide groove. In other exemplary embodiments, HLA class I (e.g., HLA-A) 02) It has F22C and S71C substitutions (e.g., relative to SEQ ID NO: 26), thereby forming a disulfide bond within one of the α-helices adjacent to the peptide binding groove.

[0057] In various embodiments, the recombinant HLA class I ligand associates with β2 microglobulin. The amino acid sequence of β2 microglobulin is provided herein as SEQ ID NO: 8. In various embodiments, derivatives of β2 microglobulin may be used, for example, having 1 to 10 or 1 to 5 amino acid modifications independently selected from substitutions, deletions, and insertions.

[0058] In some embodiments, the HLA ligand is an HLA class II ligand. In various embodiments, the HLA ligand is HLA-DR, HLA-DP, or HLA-DQ. In some embodiments, the HLA class II ligand comprises an immunoglobulin fusion of HLA α and β chains with antibody heavy and light chains as already described, thereby generating a dimer HLA-II antigen-presenting complex.

[0059] In some embodiments, aAPC contains an HLA-E ligand, which is optionally HLA-E-Ig (e.g., as described). In some embodiments, the HLA-E ligand is engineered to reduce or eliminate interaction with NKG2A / CD94. HLA-E is a non-classical MHC class I molecule. HLA-E is represented by only two major alleles. Given this low polymorphism, HLA-E ligands can be adapted to generate a virtually universal aAPC platform. However, HLA-E has a dual role in both the innate and adaptive immune systems. In the innate immune response, HLA-E presents peptides of other HLA class I molecules to inhibit natural killer (NK) cell-mediated cleavage by recognition by NKG2A / CD94. NK cells sense the presence of HLA-E-presented peptides, thereby receiving an inhibitory signal (inhibition of NK-mediated cleavage) through the NKG2A / CD94 complex. HLA-E can also bind to and present peptide sequences for recognition by T cells (e.g., CD8+ T cells) (adaptive immune response). Notably, the HLA-E molecule binds to NKG2A / CD94 via a binding surface that overlaps with the binding surface that interacts with the T cell receptor (“TCR”).

[0060] In various implementations, point mutations that engineer HLA-E binding only to NKG2A / CD94 but not to TCR are used to decouple the NK cell inactivation function of HLA-E from its T cell activation function. These point mutations enable the redirection of HLA-E to regulate HLA-E-restricted T cells while avoiding HLA-E depletion in natural killer cells. According to other aspects and implementations, HLA-E amino acid substitutions are implemented to provide stable peptide-binding gaps for the presentation of bound antigens to HLA-E-restricted T cells.

[0061] In some embodiments, the recombinant HLA-E ligand comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 21 (HLA-E extracellular domain) and substituted with cysteine ​​residues at amino acids corresponding to Y84 and A139 of SEQ ID NO: 21. These substitutions allow the formation of disulfide bonds that stabilize peptide-binding clefts. In some embodiments, the amino acid sequence has at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 21.

[0062] In some embodiments, the amino acid sequence has one or more amino acid modifications relative to SEQ ID NO: 21, said one or more amino acid modifications reducing or eliminating the interaction with NKG2A / CD94. In some embodiments, the amino acid modifications are selected from substitutions at D162 and E166 relative to SEQ ID NO: 21. In various embodiments, the substitutions do not include acidic side chains. For example, in some embodiments, the substitution at D162 is selected from D162A, D162G, D162L, D162V, D162I, D162S, D162T, D162M, D162N, and D162Q; and the substitution at E166 is selected from E166A, E166G, E166L, E166V, E166I, E166S, E166T, E166M, E166N, and E166Q. In some embodiments, the recombinant HLA-E ligand comprises substitutions of D162A and E166A relative to SEQ ID NO: 21. In some embodiments, the HLA-E ligand comprises substitutions of Cys at Y84 and A139, and substitutions of D162 and E166.

[0063] In some embodiments, engineered HLA-E peptide ligands are coupled to nanoparticles together with co-stimulatory ligands to activate antigen-specific HLA-E-restricted T cells targeting one or more tumor or infectious disease antigens. In other embodiments, engineered HLA-E peptide ligands are coupled to nanoparticles together with co-inhibitory ligands to inhibit antigen-specific HLA-E-restricted T cells targeting one or more self-antigens.

[0064] In various embodiments, the immunoglobulin heavy chain sequence fused with HLA (class I or II) can be any isotype, and in some embodiments it is IgG. In some embodiments, the isotype is selected from IgG1, IgG3, IgG2β, IgG2α, and IgG4. In some embodiments, the immunoglobulin sequence is an IgG4 Fc sequence. In some embodiments, the IgG4 Fc domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgG4 Fc domain comprises an amino acid sequence having at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0065] In some embodiments, the recombinant HLA ligand comprises a linker between an HLA amino acid sequence and an immunoglobulin sequence (e.g., the IgG4 Fc domain). In some embodiments, the linker is a flexible linker, such as a linker primarily composed of glycine and serine amino acid residues. An exemplary flexible linker comprises the amino acid sequence of SEQ ID NO: 24. Alternatively, the linker may be selected from flexible and rigid peptide linkers. Flexible linkers are primarily or entirely composed of small and / or polar residues such as Gly, Ser, and Thr. An exemplary flexible linker comprises (Gly... x Ser) n The linker, wherein x is 1 to 10 (e.g., 2 to 6), and n is 1 to approximately 10, and in some embodiments, is 2 to approximately 6. In exemplary embodiments, x is 2 to 4, and n is 2 to 4. Due to their flexibility, these linkers are essentially unstructured. More rigid linkers include polyproline or polyPro-Ala motifs and α-helical linkers. Typically, linkers of varying rigidity may consist primarily of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences contain at least 5 amino acids and may range from 5 to 30 amino acids or from 5 to 20 amino acids.

[0066] The peptide antigen binds to the antigen-binding cleft of the antigen-presenting complex. Optionally, the antigenic peptide can be covalently bound to the peptide-binding cleft. If desired, a peptide chain can be used to tether the antigenic peptide to the peptide-binding cleft. For example, crystallographic analysis of several class I MHC molecules indicates that the N-terminus of β2M is very close to the C-terminus of the antigenic peptide residing in the MHC peptide-binding cleft, at a distance of approximately 20.5 Å. Therefore, a relatively short linker sequence of approximately 13 amino acids in length can be used to tether the peptide to the N-terminus of β2M. If the sequence is appropriate, the peptide will bind to the MHC binding groove. This article describes peptide antigens for immunotherapy in oncological diseases, infectious diseases, and autoimmune diseases.

[0067] In some embodiments, the peptide antigen does not induce aggregation of aAPCs. That is, peptide antigens with a low tendency to aggregate when loaded onto aAPCs are selected. In some embodiments, an aggregation potential score (APS) is used to select peptides with a low tendency to aggregate, which can be evaluated as described elsewhere herein. For example, peptides with low aggregation potential show an average APS of less than about 0.07 when loaded onto an HLA ligand (e.g., HLA-A ligand) and modeled by computer simulation. In some embodiments, the peptide antigen does not have exposed cysteine ​​residues, and / or the peptide has one or more exposed glycine residues and / or charged residues (e.g., amino acids Glu, Asp, Lys, Arg, and His). In some embodiments, the peptide antigen does not have more than 2, 3, or 4 hydrophobic residues, such as those selected from Phe, Val, Leu, and Ile. Furthermore, aggregation tendency can be limited by restricting the density of HLA ligands (as described). In some implementations, the HLA ligand is HLA-A (e.g., HLA-A 02:01), and positions 1 and 3-5 contain at least one or at least two of Gly and charged residues (Glu, Asp, Lys, Arg and His) independently and do not contain more than one exposed hydrophobic residue (e.g. Phe, Val, Leu and Ile).

[0068] In some embodiments, the peptide ligand comprises a signal-2 ligand, which is a co-stimulatory ligand, for example, for activating and / or amplifying target T cells. Exemplary co-stimulatory ligands include agonists of any of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT. In various embodiments, the co-stimulatory ligand may induce activation and / or amplification of CTLs or Tregs. In other embodiments, and particularly where aAPC is intended to suppress target T cells, the peptide ligand does not contain any signal-2 ligand, or the peptide ligand comprises a co-inhibitory ligand that induces tolerance or apoptosis of target T cells. In various embodiments, the co-inhibitory ligand is an agonist of Fas, TGF-β, or PD-1. The agonist ligand may comprise a natural agonist ligand (or an engineered variant thereof, including immunoglobulin fusions as described), or in some embodiments, an antibody agonist. In some embodiments, the co-inhibitory ligand is PD-L1 (or an immunoglobulin fusion thereof) or FasL (or an immunoglobulin fusion thereof). This document describes exemplary PD-L1-Ig and Fc-FasL fusion protein ligands (SEQ ID NO: 1 and SEQ ID NO: 4, respectively). In some embodiments, the immunoglobulin fusion sequence is IgG4 and its variants as described herein, and may contain the amino acid sequence of SEQ ID NO: 23. Embodiments of these constructs are described in more detail elsewhere herein. The antibody agonist may be a complete monoclonal antibody, or a portion or fragment containing an antigen-binding sequence, such as Fab, Fab', F(ab')2, or scFv.

[0069] In various embodiments, signal 1 and signal 2 ligands can be combined in homodimeric or heterodimeric constructs (e.g., homodimeric or heterodimeric Ig fusion constructs). For example, an HLA ligand can comprise a fusion of an HLA extracellular domain with an immunoglobulin Fc region (such as the IgG4 Fc region), which can dimerize (e.g., via disulfide bonds) with a signal 2-immunoglobulin (Ig) fusion (i.e., a heterodimeric Ig fusion construct). In still other embodiments, the HLA ligand comprises a fusion with a signal 2 ligand. For example, the HLA extracellular domain can be fused at its C-terminus with an immunoglobulin Fc region (e.g., IgG4 Fc as described) and at its N-terminus with a signal 2 ligand to prepare a homodimeric ligand with both signals dimerized. In some embodiments, the signal 2 ligand comprises the activating portion of a single-chain antibody (e.g., scFv) or a native ligand. See SEQ ID NO: 6 and 7, which comprise anti-CD28 scFv fused to the N-terminus of HLA-Ig. SEQ ID NO: 6 employs a VH-connector-VL oriented scFv, and SEQ ID NO: 7 employs a VL-connector-VH oriented scFv. Also see SEQ ID NO: 5, demonstrating a fusion of a PD-L1 activating fragment with the N-terminus of HLA-A-IgG4.

[0070] In some embodiments, the co-stimulatory ligand is an agonistic antibody against CD28, optionally a humanized or human monoclonal antibody or a scFv based thereon. For example, the anti-CD28 antibody may be an IgG isotype (e.g., IgG4) and may be as described in U.S. Patent No. 10,632,193, which is incorporated herein by reference in its entirety. In some embodiments, one, two, three, or more complementarity-determining regions (CDRs) are based on 9.3 mAb of mouse CDRs (Tan et al., J. Exp. Med. 1993 177:165). In some embodiments, the antibody has a full set of heavy chain and / or light chain CDRs of 9.3 mAb. For example, in some embodiments, the heavy chain variable region contains one, two, or three of the following CDRs, which may optionally be modified by substitution of one, two, or three amino acids respectively: CDR1 (DYGVH, SEQ ID NO: 9), CDR2 (VIWAGGGTNYNSALMS, SEQ ID NO: 10), and CDR3 (DKGYSYYYSMDY, SEQ ID NO: 11). In some embodiments, the light chain contains one, two, or three of the following CDRs, which may be modified by substitution of one, two, or three amino acids respectively: CDR1 (RASESVEYYVTSLMQ, SEQ ID NO: 12), CDR2 (AASNVES, SEQ ID NO: 13), and CDR3 (QQSRKVPYT, SEQ ID NO: 14). Exemplary heavy chain variable and light chain amino acid sequences of humanized anti-CD28 agonist antibodies are provided herein as SEQ ID NOs: 15 to 20.

[0071] In some embodiments, the anti-CD28 antibody (or a portion thereof) binds to the same or overlapping epitopes with 9.3 mAb, or binds to the same or overlapping epitopes with antibodies of CDR1, CDR2, and CDR3 having 9.3 mAb. Antibodies with the same or overlapping epitopes can be selected using, for example, surface plasmon resonance (Biacore) by any suitable technique, including competitive immunoassay.

[0072] In various embodiments, alternative CDR sequences, variable regions, or CD28-binding ligands may be employed. Alternative ligands, CD28 epitopes, and anti-CD28 antibodies are described, for example, in U.S. Patent Nos. 7,612,170, 6,987,171, and 6,887,466, the entire contents of which are hereby incorporated by reference.

[0073] In some embodiments, the co-inhibitory ligand is a Fas agonist antibody. In some embodiments, the Fas agonist antibody is an IgG4 antibody based on clone CH11. SEQ ID NO: 2 and 3 illustrate the heavy chain variable region and light chain sequences of the IgG4 anti-Fas agonist antibody. As demonstrated herein, the anti-Fas antibody is active when it is capable of crosslinking multiple Fas receptors, such as when the antibody is conjugated with nanoparticles. In some embodiments, the heavy chain and light chain sequences of SEQ ID NO: 2 and 3 are humanized and contain the CDRs of SEQ ID NO: 2 and 3, as shown in Table 1 below: Table 1: Anti-Fas IgG4

[0074] The antibody peptide ligands according to this disclosure may include a constant region, and said constant region may be any isotype. In some embodiments, the antibody constant region is human IgG4 or a variant thereof. In some embodiments, the constant region includes one or more hinge-stabilizing mutations that may be introduced into the CH chain (e.g., S241P). In some embodiments, the antibody ligand includes a constant region, and said constant region includes one or more mutations suitable for chemically coupling the antibody to a solid support. The one or more mutations suitable for coupling produce unpaired cysteine ​​residues. An exemplary mutation in the IgG4 constant region is S473C. Other variations of the constant region include modifications that reduce Fcγ receptor binding. For example, the CH chain may be modified at L248, such as L248E.

[0075] In some implementations, the antibody-based peptide ligand can be minimized to make it more suitable for functional attachment to nanoparticles. For example, the antibody can be an antibody fragment, such as F(ab')2 or Fab, or a single-chain antibody (scFv), or other antigen-binding antibody fragment. For example, the antibody fragment can be an scFv of the humanized mAb or other agonistic anti-CD28 antibody described herein.

[0076] In some embodiments, the antibody (signal 2 ligand) is an scFv that comprises or is substantially composed of an antigen-binding loop formed by the VH and VL chains of a monoclonal antibody (e.g., an anti-CD28 antibody). The scFv antibody construct may comprise one or more (2, 3, 4, or 5) VH and VL hypervariable chains linked together in a head-to-head or head-to-tail configuration via short peptide linkers (each chain together forming a portion of a 3-D antigen epitope binding pocket). In some embodiments, these constructs are fused with an HLA-Ig sequence as described to produce a homodimeric construct.

[0077] In some implementations, other ligand-binding forms are used to generate co-stimulatory or inhibitory ligands, including peptides, aptamers, and AdNectin. Various forms used for target binding include single-domain antibodies, recombinant heavy-chain-only antibodies (VHH), single-chain antibodies (scFv), shark heavy-chain-only antibodies (VNAR), microbial proteins (cysteine ​​knottin), DARPin, tetratranectin, affibody, transmembrane antibody, anticarrier protein, affilin, microbody, peptide aptamer, phylomer, stradobody, macrobody, evibody, fynomer, armadillo repeat sequence protein, and Kunitz domain. domain), high-affinity multimers (avimer), atrimer, probody, immunobody, triomab, troybody, pepbody, unibody, dualbody, Fv, Fab, Fab', F(ab')2, peptide mimics or synthetic molecules, or as in US patent numbers or publication numbers: US Patent No. 7,417,130, US2004 / 132094, US Patent No. 5,831,012, US 2004 / 023334, US Patent No. 7,250,297, 6,818,418, US 2004 / 209243, US Patent No. 7,838,629, 7,186,524, 6,004,746, 5,475,096, US The contents of the patents described in US Patent Nos. 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446 and / or 7,166,697 are hereby incorporated by reference in their entirety. See also Storz MAbs. May-June 2011; 3(3): 310-317.

[0078] In some embodiments, the aAPC further comprises one or more cytokines that support T cell activation and / or expansion or T cell suppression. One or more cytokines or functional portions thereof may be conjugated to the aAPC as peptide ligands. Alternatively, the cytokines or functional portions thereof may be fused with a signal 1 or signal 2 peptide ligand (which may optionally be presented in a homodimeric or heterodimeric Ig fusion construct as described herein). In some embodiments, the cytokines are encapsulated in copolymers and will be locally released in a targeted environment (e.g., in a lymphoid organ, tumor, or target tissue or organ). Examples of cytokines that may be used include IL-1β, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and interferon-gamma. For example, IL-2 may be used with a co-stimulatory signal 2 ligand. In some embodiments, the aAPC comprises a tolerogenic cytokine as a peptide ligand. An exemplary tolerogenic cytokine is IL-10.

[0079] In some embodiments, the ligand further comprises one or more homing ligands targeting lymphoid organs. For example, an exemplary homing ligand is CD62L. In some embodiments, a ligand (which may or may not be a peptide ligand) is included to target aAPC to a tissue or organ of interest, such as the pancreas, intestine, lung, liver, muscle, skin, etc. Suitable peptide ligands or other ligands can be selected based on information in the art.

[0080] In various implementations, one or more peptide antigens are tumor or cancer-associated antigens, such as tumor-derived antigens, tumor-specific antigens, and neoantigens. T cells specific to tumor-associated antigens are often very rare and, in many cases, undetectable in the peripheral blood of healthy individuals. Furthermore, these cells typically possess the primary phenotype. See Quintarelli et al., Cytotoxic T lymphocytes directed to the preferentially expressed antigens of melanoma (PRAME) target chronic myeloid leukemia . Blood 2008; 112: 1876-1885. This is often the distinction observed between virus-specific T cells and tumor antigen-specific T cells. According to these embodiments, the injectable aAPC of this disclosure can activate and / or expand such T cells in vivo to generate an anti-tumor immune response.

[0081] "Tumor-associated antigens" or "cancer-specific antigens" include unique tumor or cancer antigens specifically expressed by the tumor or malignant cells from which they originate, common tumor antigens (carcinoembryonic antigens) expressed in many tumors but not in normal adult tissues, and tissue-specific antigens also expressed by normal tissues that produce tumors. Tumor-associated antigens can be, for example, embryonic antigens, antigens with aberrant post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressor genes, fusion proteins, or oncoviral proteins.

[0082] Multiple tumor-associated antigens are known in this field. Carcinoembryonic antigens (CEA) and embryonic antigens include CEA and alpha-fetoprotein (AFP) (which are usually highly expressed only in developing embryos, but are often highly expressed by tumors of the liver and colon, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialylation-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal tumors, liver tumors, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many cancers), pancreatic carcinoembryonic antigen, 5T4 (expressed in gastric cancer), alpha-fetoprotein receptor (expressed in various tumor types, particularly breast tumors), and M2A (expressed in germ cell tumor formation).

[0083] Mutated oncogenes or tumor-suppressor gene products include Ras and p53 (both expressed in many tumor types), Her-2 / neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene products, and myc (associated with lung cancer). Fusion proteins include BCR-ABL, expressed in chronic myeloid leukemia. Tumor viral proteins include HPV types 16, E6, and E7, found in cervical cancer.

[0084] Tissue-specific antigens include melanin transferrin and MUC1 (expressed in pancreatic and breast cancer); CD10 (previously known as a common acute lymphoblastic leukemia antigen or CALLA) or surface immunoglobulin (expressed in B-cell leukemia and lymphoma); IL-2 receptor, T-cell receptor, CD45R, and the α chain of CD4+ / CD8+ (expressed in T-cell leukemia and lymphoma); prostate-specific antigen and prostate acid phosphatase (expressed in prostate cancer); GP100, MelanA / Mart-1, tyrosinase, gp75 / brown, BAGE, and S-100 (expressed in melanoma); cytokeratin (expressed in various cancers); and CD19, CD20, and CD37 (expressed in lymphoma).

[0085] Tumor-associated antigens also include altered glycolipid and glycoprotein antigens, such as glycosphingolipids containing neuraminic acid (e.g., GM2 and GD2 expressed in melanoma and some brain tumors); blood group antigens that can be aberrantly expressed in cancer, particularly T and sialylated Tn antigens; and mucins such as CA-125 and CA-19-9 (expressed in ovarian cancer) or hypoglycosylated MUC-1 (expressed in breast and pancreatic cancer).

[0086] Tumor-associated antigens are further disclosed in U.S. Patent 11,007,222, which is hereby incorporated by reference.

[0087] In some embodiments, the target peptide antigen includes at least one antigen associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen. For example, at least one peptide antigen may be associated with tuberculosis (TB), HIV (human immunodeficiency virus), HTLV (human T-lymphotropic virus) type 1, hepatitis (e.g., hepatitis A, B, C, or D), cytomegalovirus (CMV), Epstein-Barr virus (EBV), HPV, influenza, herpesviruses (e.g., HSV 1 or 2, or varicella-zoster virus), and adenoviruses. For example, CMV is the most common viral pathogen found in organ transplant patients and is a leading cause of morbidity and death in patients undergoing bone marrow or peripheral blood stem cell transplantation.

[0088] In some embodiments, one or more target peptide antigens are “autoantigens,” meaning they are associated with autoimmune diseases or responses. In some embodiments, aAPCs carrying tolerance-inducing ligands induce tolerance of antigen-specific T cells to the target antigen. In some embodiments, aAPCs carrying apoptosis signals (e.g., Fas ligands or agonist anti-Fas antibodies) will induce specific apoptosis of antigen-specific T cells. Autoantigens can be associated with autoimmune diseases such as type 1 diabetes, Goodpasser syndrome, multiple sclerosis, Graves' disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pemphigus vulgaris, Addison's disease, herpetic dermatitis, celiac disease, Crohn's disease, Hashimoto's thyroiditis, vitiligo, etc.

[0089] In some implementations, the peptide antigens presented by HLA ligands are determined in a personalized manner, as described in US 10,098,939 and US 2020 / 0291381, which are hereby incorporated in their entirety by reference. For example, sequencing data can provide information on shared targets and personalized targets for immunotherapies, such as those for cancer. In principle, mutant proteins are foreign substances to the immune system and are presumed tumor-specific antigens. In practice, sequencing work has defined hundreds, even thousands, of potentially relevant immune targets. Studies have demonstrated that T-cell responses against these novel epitopes can be found in cancer patients or induced by cancer vaccines. Catalogues of mutations derived from whole-exome sequencing provide a starting point for identifying such novel epitopes. Using HLA binding prediction algorithms (Srivastava, PLoS One 4, e6094 (2009)), it is predicted that each cancer may have up to 7 to 10 novel epitopes. Similar methods have estimated hundreds of novel tumor epitopes. The activation potential of novel epitopes predicted from DNA or RNA sequencing of a patient's tumor can be tested by determining whether (or to what extent) an aAPC carrying a predicted antigen associated with an HLA ligand can activate T cells from the subject. Similar assays can be used to identify associated autoantigens.

[0090] In some implementations, for the treatment of type 1 diabetes, the peptide antigen may be an antigen derived from ICA, insulin, G6, GAD2, GAD65, insulinoma antigen-2, HSP, IGRP, imogen-38, PDX1, ZnT8, CHGA, and IAAP. See, for example, Han, S et al. Novel autoantigens in type 1 diabetes . Am J Transl Res . 2013; 5(4): 379–392.

[0091] Non-limiting examples of peptide antigens include those shown in Table 2 below.

[0092] Table 2: Peptide Antigens

[0093] In various embodiments, aAPC is included in a pharmaceutical composition suitable for administration to a subject. The pharmaceutical composition may have one or more excipients, such as buffers, surfactants, preservatives, polymers, fillers, and stabilizers. Buffers are used to control the pH of the composition. Surfactants are used to stabilize proteins, inhibit protein aggregation, inhibit protein adsorption to surfaces, and facilitate protein refolding. Exemplary surfactants include Tween 80, Tween 20, Brij 35, Triton X-10, Pluronic F127, and sodium dodecyl sulfate. Preservatives are used to prevent microbial growth. Examples of preservatives include benzyl alcohol, m-cresol, and phenol. Fillers are used during lyophilization to increase volume. Hydrophilic polymers such as dextran, hydroxyethyl starch, polyethylene glycol, and gelatin can be used to stabilize proteins. Polymers with nonpolar moieties, such as polyethylene glycol, can also be used as surfactants. Protein stabilizers may include polyols, sugars, amino acids, amines, and salts. Suitable sugars include sucrose and trehalose. Amino acid stabilizers include histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, and mixtures thereof. Proteins such as human serum albumin can also competitively adsorb onto the surface and reduce the aggregation of active agents. Specific formulation components can be used for a variety of purposes. For example, histidine can act as a buffer and antioxidant. Glycine can be used as a buffer and filler. In some embodiments, the pharmaceutical composition is lyophilized.

[0094] In some aspects and embodiments, this disclosure provides aAPCs suitable for parenteral (including subcutaneous) administration and exhibiting low aggregation tendency. In these aspects, the aAPC comprises polymeric or lipid nanoparticles containing a polyethylene glycol (PEG) sheath and one or more polypeptide ligands conjugated to PEG (e.g., the PEG terminus) via thioether bonds or other functional groups. The polypeptide ligand comprises an HLA class I or II ligand for presenting the peptide antigen and optionally one or more signal 2 ligands (as described). The peptide antigen for presentation to T cells does not induce aggregation of the aAPC. As disclosed herein, certain peptides, when loaded onto aAPC, will induce aggregation due to the properties of the exposed surface. Analysis of the peptide sequence alone does not provide a clear indication of aggregation potential in the HLA context. Aggregation potential occurs at the level of the nanoparticles loaded with the peptide, rather than at the individual protein complexes in solution.

[0095] In some embodiments, the peptide antigen does not have an exposed cysteine ​​residue, and / or the peptide antigen has one or more exposed glycine residues or exposed charged residues. In some embodiments, the peptide antigen does not have any cysteine ​​residues and contains one or more charged residues (e.g., 1, 2, or 3 charged residues). In various embodiments, the peptide antigen in the HLA-binding cleft has one or more exposed amino acids selected from glycine, aspartic acid, glutamic acid, lysine, arginine, and histidine; and does not have an exposed cysteine ​​residue. In some embodiments, positions 1 and 3 to 5 of the peptide meet these criteria. In this context, the term "exposed" means that the side chain of the amino acid is surface-exposed. In some embodiments, the HLA is HLA-A, and in some embodiments it is HLA-A2, such as HLA-A. 02:01.

[0096] In some aspects and embodiments, aggregation potential is estimated by computer simulation by determining the average aggregation potential score (APS) of antigenic peptide residues within the HLA antigen-binding cleft. In some embodiments, such as for HLA-A (e.g., HLA-A2, etc.), aggregation potential is estimated by computer simulation. (02:01) APS assessment is performed on peptide positions 1 and 3-5, and the average score for these positions is determined. For example, a score less than approximately 0.07 indicates a non-aggregating peptide. Protein modeling and aggregation tendency analysis can be performed using Discovery Studio 2021 (DS2021, Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release2021, San Diego CA). For example, the MODELLER program or similar programs can be used for homology or comparative modeling of protein three-dimensional structures (e.g., HLA with binding peptides). Spatial aggregation tendency (SAP) can be calculated using available tools, which are included in the DS2021 package and are also commercially available through other means.

[0097] The Spatial Aggregation Propensity (SAP) score integrates the solvent-accessible region of the residue side chains with a hydrophobicity score based on a scale developed by Black and Mould (1991), where glycine has a value of zero, and other residues have positive values ​​if the hydrophobicity is stronger and negative if the hydrophobicity is weaker. This score, combined with previously described specific peptide characteristics, identifies peptides that may drive protein aggregation by extending HLA-modified nanoparticles when complexed with HLA ligands.

[0098] Using aggregation propensity in computer simulation models (and experimentally determined structures) is a known approach. However, these tools are typically focused on antibody screening to discover antibodies with favorable drug-like properties or to engineer out biophysical defects. While comparative modeling and APS tools are commonly used, their application to peptide-HLA complexes, particularly in the context of nanoparticle coatings and the aggregation of these particles, has not been described or proposed. Furthermore, considering that peptides constitute only a relatively small portion of the structure of aAPC and the entire exposed surface, the significant influence of peptides in the HLA-binding pocket on particle aggregation is surprising.

[0099] In some implementations, the process is initiated by generating molecular models of candidate related peptide-HLA complexes. The protein models include the related peptides located in the HLA binding groove. The MODELLER algorithm, implemented in Discovery Studio 2021, or other suitable software can be used. Multiple template structures can be used for the modeling step, and each model undergoes manual sequence alignment. HLA-A The PDB entries for the template structures used in the 02:01 modeling include: 4L29, 1I7R, 5EUO, 1TVB, 6OPD, 6TRO, 6AMT, 6AM5, 2GT9, and 3OXS.

[0100] For example, using a static model, an aggregation score can be calculated. In some implementations, a radius of 5 or 10 angstroms is chosen for the score calculation. This parameter determines how many nearby amino acid residues are included in the aggregation potential calculation. This is similar to a sliding window in protein sequence parameter calculations. Depending on the size of the model being analyzed, the radius can provide a reasonably smooth and not overly noisy surface map. The protocol calculates the exposed surface for each residue and calculates the aggregation potential score based on the average of each residue and residues within a defined radius surrounding it. The numerical score (APS) is recorded along with sequence and structural information and used to generate a surface map of the model-based solvent-accessible surface. This provides an easy visualization of the surface shape in conjunction with the scored aggregation potential. In some cases, manual examination of these results is sufficient to classify the peptide as an aggregator or a non-aggregator.

[0101] According to some implementation schemes, the average APS (average percentage of plasma concentrations) of residues 1 and 3-5 of the HLA-A peptide complex is calculated. If this score is higher than the current threshold of 0.07, it is considered a factor in aggregation. The presence of exposed Cys residues (as determined by examining model structures) is considered another factor in aggregation. The presence of charged residues and glycine residues is considered a factor preventing aggregation. The total number of factors that favor or prevent aggregation can be used to determine whether the peptide-HLA complex is considered a potential aggregater.

[0102] In some embodiments, the nanoparticles are polymer nanoparticles comprising a poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymer and one or more polypeptide ligands conjugated to PEG via thioether bonds. Such polymer nanoparticles may be as described above.

[0103] Alternative polymers that can be used in conjunction with the aAPC platform described herein include one or more of the following: cyclodextrin-containing polymers, cyclodextrin-containing cationic polymers, poly(D,L-lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), PLGA-b-poly(ethylene glycol)-PLGA (PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG-maleimide (PLGA-PEG-mal), PLA-PEG-maleimide, poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), alkyl cyanoacrylates, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(ester amide), polyamide, poly(ester ether), polycarbonate, polyolefins such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol (PEG), polyepoxide (PEO), polyalkylene terephthalates such as polyethylene terephthalate, polyvinyl alcohol (PVA), polyethylene ether, polyethylene esters such as polyvinyl acetate, polyethylene halides such as polyvinyl chloride. Polyvinylpyrrolidone (PVC), polysiloxane, polystyrene (PS), polyurethane, derived cellulose such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic polymers such as polymethyl methacrylate (PMA), poly((meth)acrylate), poly(((meth)acrylate), poly(((meth)acrylate),) isobutyl methacrylate, poly(((meth)acrylate),) hexyl methacrylate, poly(((meth)acrylate),) isodecyl methacrylate, poly(((meth)acrylate),) lauryl methacrylate, poly(((meth)acrylate),) poly((meth)acrylate), poly(methacrylic acid), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate). Polyacrylic acid and its copolymers and blends, polydioxane and its copolymers, polyhydroxyalkanoates, polyfuranes, polyoxymethylene, poloxamer, poly(orthocyanins), poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthocyanins, polyphosphazenes and polyphosphates, dendritic polymers and their derivatives, and blends and / or block copolymers of two or more such polymers.

[0104] In other embodiments, the nanoparticles are lipid nanoparticles comprising PEG-conjugated lipids. Exemplary PEG lipids are selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. PEG lipids may be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-cholesterol, PEG-tocopherol, or PEG-DSPE lipids.

[0105] In some embodiments, the lipid nanoparticles further comprise cationic or ionizable lipids, neutral lipids or phospholipids, and structural lipids. Exemplary structural lipids may be selected from one or more of the following: cholesterol, cod sterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, and tocopherol (e.g., α-tocopherol). In some embodiments, the structural lipid is cholesterol. In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from the group consisting of: cardiolipin, sterol-modified lipids (modified with a cholesterol portion attached to the sn-2 carbon of the glycerol backbone), mixed acylglycerol phospholipids, and symmetrical acylglycerol phospholipids. The head groups of acylglycerol phospholipids include, for example, phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, inositol phosphophosphate, and phosphatidylserine. Exemplary phospholipids are selected from 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:O diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), and 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso). PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin.

[0106] In some embodiments, the nanoparticles are lipid nanoparticles that also contain a polynucleotide for expression in target T cells. In some embodiments, the polynucleotide (e.g., mRNA) encodes cytokines, including those described herein (e.g., IL-2 or IL-10).

[0107] In some embodiments, the thiol groups on the peptide ligands are coupled to the PEG-maleimide functional groups of the lipid nanoparticles. In various embodiments, the lipid nanoparticles aAPC have about 10 to about 500 peptide ligands, about 50 to about 400 peptide ligands, or about 100 to about 300 peptide ligands. The peptide ligand density can typically be controlled by reducing the proportion of PEG moieties having maleimide groups for conjugation as described previously, or by varying the level of PEG lipids in the composition.

[0108] The lipid nanoparticles aAPC have a diameter of about 50 nm to about 150 nm, or about 60 nm to about 130 nm, or about 80 nm to about 120 nm. In various embodiments, aAPC has a diameter of about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm. The lipid nanoparticles aAPC have a surface charge of about 0 to -15 mV, or about 0 to about -10 mV, or about -2.5 mV to about -10 mV. The aAPC population has a size distribution with a polydispersity index (PDI) of less than 0.2.

[0109] In various embodiments, aAPC is contained in the pharmaceutical composition as described and may optionally be lyophilized. In various embodiments, the aAPC and pharmaceutical composition described herein may be used to treat patients with infectious diseases, cancer, or autoimmune diseases, or to provide prophylactic protection for immunosuppressed patients.

[0110] In other aspects, the present invention provides a method for immunotherapy. The method includes administering an aAPC or pharmaceutical composition as described herein to a subject requiring treatment. According to this aspect, the aAPC and its compositions described herein are used for immunotherapy.

[0111] In various implementation schemes, the subject has cancer or an infectious disease, and the aAPC contains a co-stimulatory ligand. Treatable infectious diseases include those caused by bacteria, viruses, prions, fungi, parasites, worms, etc. Such diseases include human papillomavirus (HPV) (and related cancers), AIDS, adult T-cell leukemia / lymphoma (ATL), hepatitis, CMV infection, and post-transplant lymphoproliferative disorder (PTLD). For example, CMV is the most common viral pathogen found in organ transplant patients and is a leading cause of morbidity and death in patients undergoing bone marrow or peripheral blood stem cell transplantation. This is due to the compromised immune status of these patients, which allows for the reactivation of latent viruses in seropositive patients or opportunistic infections in seronegative individuals. Current treatment focuses on the use of antiviral compounds such as ganciclovir, which has drawbacks, most notably the development of drug-resistant CMV.

[0112] PTLD occurs in a significant proportion of transplant patients and is caused by Epstein-Barr virus (EBV) infection. It is believed that approximately 90% of the adult population in the United States is infected with EBV. Active viral replication and infection are controlled by the immune system, but as with CMV, individuals whose immune systems are compromised due to transplantation lose control of their T-cell populations, allowing viral reactivation. This represents a significant obstacle to transplantation. EBV may also be involved in tumor promotion in a variety of hematologic and non-hematologic cancers. There is a strong association between EBV and nasopharyngeal carcinoma. Therefore, prophylactic therapy using EBV-specific T cells offers an excellent alternative to current therapies.

[0113] Cancers that can be treated according to this disclosure include melanoma, cancers such as colon cancer, head and neck cancer, duodenal cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, ductal carcinoma, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, gastric cancer, developmental dysplastic oral mucosal cancer, polyposis, invasive oral cancer, non-small cell lung cancer, urethral transitional cell carcinoma, and squamous cell carcinoma; malignant tumors of the nervous system, such as neuroblastoma and glioma; hematologic malignancies, such as chronic myeloid leukemia, childhood acute leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, malignant cutaneous T-cell tumors, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell-rich cutaneous lymphoproliferative disorders, bullous pemphigoid, discoid lupus erythematosus, lichen planus, and so on. See, for example, Mackensen et al., Int. J. Cancer 86, 385-92, 2000; Jonuleit et al., Int. J. Cancer 93, 243-51, 2001; Lan et al., J. Immunotherapy 24, 66-78, 2001; Meidenbauer et al., J. Immunol. 170(4), 2161-69, 2003. In some embodiments, the subject has a solid tumor, which may be stage I, II, III, or IV cancer. In some embodiments, the cancer is metastatic and / or recurrent, and / or unresectable. In some embodiments, the patient is refractory to chemotherapy and / or has only a partial response to immune checkpoint inhibitor therapy.

[0114] In some embodiments, the present invention provides a method of treating cancer (including those cancers identified above) by administering the pharmaceutical composition described herein to activate T cells with anti-tumor activity. In some embodiments, the therapy is provided in conjunction with one or more immune checkpoint inhibitors, such as nivolumab, pembrolizumab, and ipilimumab. In some embodiments, an additional therapy is anti-CTLA4, anti-PD1, or anti-PD-L1. The additional therapy or checkpoint inhibitor may be administered alone using its conventional regimen, or may be administered as an additional ligand for the nanoparticles described herein, or conjugated to a separate group of nanoparticles. In some embodiments, one or more immune checkpoint inhibitors are provided as an initial therapy, and subsequently, for example, after about 1 to about 8 weeks of checkpoint inhibitor therapy or after about 2 to about 4 weeks of checkpoint inhibitor therapy, the aAPC therapy described herein is initiated. In some embodiments, one or more checkpoint inhibitors are provided concurrently with the nanoparticle therapy, for example, at the start of therapy and approximately every two weeks, or at the start of therapy and approximately every two weeks for one or more checkpoint inhibitors and approximately every four weeks for the nanoparticle therapy. In some embodiments, patients are resistant to checkpoint inhibitor therapy or show only a partial or transient response, and the aAPC described herein enhances tumor regression in these patients. In still other embodiments, for cancers that are typically resistant to immune checkpoint inhibitor therapy, the compositions described herein extend the successful use of checkpoint inhibitors to such cancers.

[0115] In some implementations, peptide antigens are selected on a personalized basis, based on analysis of the patient's tumor. For example, peptide antigens derived from Ionov Y., A high throughput method for identifying personalized tumor-associated antigens , Oncotarget Methods described in 1(2):148-155 (2010) (which is hereby incorporated by reference) or other methods. In these embodiments, nanoparticles may be provided (on a "pre-existing" basis) and selected and loaded with tumor antigens on a personalized basis. Other methods for selecting peptides based on the ability of peptides to activate and amplify relevant T cells (including in a personalized manner in some embodiments) are described in U.S. Patents 10,987,412 and 10,098,939, which are hereby incorporated by reference in their entirety.

[0116] In some embodiments, nano-aAPC is used as a booster vaccine following adoptive T-cell therapy, wherein initial T cells from the patient or T cells from an HLA-matched donor are expanded in vitro and administered to the patient. In these embodiments, the nano-aAPC composition can be administered 1 to 10 times over a period of 4 months to 1 year to enhance cancer immunity.

[0117] In some implementations, the subject has an autoimmune disease, and the aAPC contains a co-inhibitory signal or does not contain a signal 2-ligand. In some implementations, the autoimmune disease is type 1 diabetes. In some implementations, the autoimmune disease is vitiligo. Treatable autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasser syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, herpetic dermatitis, celiac disease, Sjögren's disease, Hashimoto's thyroiditis, alopecia, ankylosing spondylitis, scleroderma, and HTLV-1-associated myelopathy (HAM) / tropical spastic paraplegia (TSP), etc.

[0118] Typically, aAPC or its pharmaceutical composition is administered parenterally. For example, aAPC or its pharmaceutical composition can be administered via intravenous, intra-arterial, subcutaneous, intradermal, intralymphatic, intramuscular, or intratumoral administration. In some embodiments, the aAPC composition is administered subcutaneously.

[0119] In other aspects of this disclosure, peptide ligands for immunotherapy (including peptide ligands as aAPC) are disclosed.

[0120] Such peptide ligands include anti-Fas agonist antibodies having an IgG isotype (e.g., IgG4), which can be conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen (e.g., associated with an autoimmune disease). In some embodiments, the anti-Fas antibody comprises the heavy chain of SEQ ID NO: 2, optionally having one to ten or one to five amino acid substitutions. The amino acid substitutions are optionally located in the Fc domain and may include amino acid substitutions having known pharmacological or stability advantages or advantages in eliminating potential immunogenicity. In some embodiments, the amino acid sequence of SEQ ID NO: 2 is humanized and comprises the same complementarity-determining region (CDR) of SEQ ID NO: 2 (see Table 1), having no more than one, two, or three amino acid substitutions. In some embodiments, the anti-Fas antibody comprises the light chain of SEQ ID NO: 3 (see Table 1), optionally having one to ten or one to five amino acid substitutions. In some embodiments, the amino acid sequence of SEQ ID NO: 3 is humanized and contains the same CDR as SEQ ID NO: 3, having no more than 1, 2 or 3 amino acid substitutions.

[0121] In other embodiments, the peptide ligand is a dimer PD-L1 ligand comprising the activating portion of PD-L1, such as amino acid residues F19 to T239 of human PD-L1. Alternatively, it may include an additional 20 residues (commonly at one or both ends), or delete up to 10 amino acids from F19 to T239. Each PD-L1 activating fragment may be fused directly or indirectly to the Fc region of IgG (e.g., IgG4) at its C-terminus, and the ligand may dimerize via disulfide bonds in the Fc region. Suitable linkers are described elsewhere herein and include flexible linkers such as Gly Ser linkers. The dimer PD-L1 ligand may be conjugated to nanoparticles together with an HLA ligand presenting the peptide antigen and used to drive tolerance to the antigen. An exemplary PD-L1-IgG4 is shown in SEQ ID NO: 1.

[0122] In other embodiments, the peptide ligand is a dimerized FasL ligand comprising the activating moiety of FasL (such as amino acids P132 to L279 of human FasL), the activating moiety of FasL being fused directly or indirectly to the dimerized IgG-Fc region (e.g., IgG4) at its N-terminus. Alternatively, it may include an additional 20 residues (commonly at one or both ends), or up to 10 amino acids may be deleted from P132 to L279. In some embodiments, the dimerized Fc region may be conjugated to the nanoparticle via a Cys-containing linker. Exemplary constructs according to these embodiments are provided by SEQ ID NO: 4. The nanoparticles may further present HLA-peptide antigen ligands to drive apoptosis of antigen-specific T cells.

[0123] In other embodiments, the peptide ligand is a tolerogenic ligand comprising an activating fragment of PD-L1 (such as amino acids F19-T239 of human PD-L1), said PD-L1 activating fragment fused directly or via a linker to an HLA-immunoglobulin fusion protein. Alternatively, it may comprise an additional 20 residues (commonly at one or both ends), or delete up to 10 amino acids from F19 to T239 of human PD-L1. These embodiments provide homodimeric constructs that are homodimers for both signal 1 and inhibitory signal 2 ligands. See SEQ ID NO: 5. Such ligands can be conjugated to nanoparticles as disclosed herein and used in immunotherapy (to drive tolerance in targeted T cells).

[0124] In other embodiments, the polypeptide ligand is a costimulatory ligand comprising an anti-CD28 agonist scFv conjugated to an HLA-immunoglobulin fusion protein, thereby providing a homodimeric ligand comprising signal 1 and costimulatory signal 2 ligands. See SEQ ID NO: 6 and 7. The scFv can be fused to an HLA sequence via either a heavy chain or a light chain sequence. For example, SEQ ID NO: 6 employs a VH-connector-VL oriented scFv, and SEQ ID NO: 7 employs a VL-connector-VH oriented scFv.

[0125] The singular forms “a / an” and “the” include plural indicators unless otherwise explicitly stated in the context.

[0126] As used in the specification and claims, open transitional phrases such as “comprising,” “including,” “having,” “containing,” and variations thereof require the presence of the specified feature / step and allow for the presence of other features / steps. These phrases should also be interpreted as disclosing closed phrases such as “consisting of” or “consisting substantially of”, which allow only the specified feature / step and unavoidable impurities and exclude other features / steps.

[0127] As used herein, unless the context otherwise requires, the term “about” means ±10% of the index value.

[0128] The term "identity" refers to the similarity between a pair of sequences (nucleotides or amino acids). Identity is measured as a percentage by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Therefore, two copies of an identical sequence have 100% identity, but sequences that are less conserved and have deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those employing algorithms like BLAST, can be used to determine sequence identity. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, with the default parameters of the respective programs.

[0129] The term CDR stands for complementarity-determining region. A CDR is a portion of the variable chain in an immunoglobulin (antibody). A set of CDRs constitutes a complementation site.

[0130] The invention is further illustrated by the following non-limiting embodiments.

[0131] Example Example 1: Construction of injectable artificial antigen-presenting cells Ligands of aAPC, including signal 1 ligand (HLA) and signal 2 ligand, are constructed with free thiol groups for coupling to particles. See US 10,632,193, which is hereby incorporated herein by reference in its entirety. Naked polymer nanoparticles were prepared from PLGA-PEG or PLA-PEG copolymers by nanoprecipitation using known methods. The PLGA and PLA moieties of the block copolymers are approximately 20 kDa. The PEG moieties of the block copolymers are 3000 Da or 5000 Da. Specifically, PEG polymers with functional groups for ligand conjugation are designed to be longer (5000 Da), while inert PEG polymers are shorter (3000 Da) to reduce steric effects. The experiments shown below employed NANOASSEMBLR IGNITE (Precision Nanosystems) for nanoprecipitation of particles. The nanoparticles comprise groups of copolymers with terminal maleimide functional groups (PEG-maleimide) for ligand coupling. Thiolized ligands are conjugated to naked particles via terminal maleimide functional groups and purified using known techniques. See US 10,632,193. HLA ligands on nanoparticles are loaded by incubation with an excess of peptide antigen. For example, nanoparticles loaded with peptides can be separated using an SEC column.

[0132] Using these methods, particles with a size and surface charge suitable for parenteral administration in immunotherapy can be obtained. Figure 1A TEM images of bare particles, protein-conjugated nanoparticles, and peptide-loaded nanoparticles are shown. Figure 1B As shown, the particles have an average diameter of approximately 100 nm. Those with conjugated ligands and loaded peptides are slightly larger. The polydispersity index is less than 0.2. The surface charge is shown in the range of 0 to -10 mV.

[0133] The target range for ligand density was 100 to 400 ligands per particle. Experiments were conducted to vary the density of PEG-maleimide functional groups on the particle surface within a range determined to allow for stable particles (1 wt% to 10 wt%). Specifically, particles were prepared using PLGA-PEG, wherein a portion of the PEG terminus contained maleimide functional groups. The maleimide:thiol ratio (Mal:thiol) in the conjugation reaction was kept constant at 1:1. These data are summarized in Table 3 below.

[0134] Table 3

[0135] In separate experiments, the ligand density was controlled by varying the ratio (Mal:thiol) of maleimide functional groups on the nanoparticles to thiol groups on the ligands during the coupling reaction. PEG-Mal was kept constant at 5%. These data are summarized in Table 4 below.

[0136] Table 4

[0137] In terms of stability, particle size, and ligand density, 5% PEG-maleimide polymers were selected as good candidates for nanoparticle design. Data from Tables 3 and 4 are plotted in Figures 3A and 3B, showing trends in particle density and size. In these examples, the 5% PEG-maleimide beads for PLGA-PEG correspond to a 3:1 ratio (by weight) of PLGA-mPEG:PLGA-PEG-maleimide. For PLA-PEG, 5% PEG-maleimide corresponds to approximately a 5.67:1 ratio (by weight) of PLA-mPEG:PLA-PEG-maleimide.

[0138] Nanoparticles were constructed using PLGA-PEG and PLA-PEG nanoparticles for comparison. As shown in Figures 4A and 4B, the differences were not significant. The size and protein density of PLA-PEG were slightly reduced. The ability of PLGA-PEG and PLA-PEG nanoparticles with signal 1 ligand (an HLA-Ig ligand loaded with the MART-1 antigen) and signal 2 ligand (an antibody agonist against CD28) to stimulate antigen-specific CD8+ T cells was determined. As shown in Figures 5A-5D, no significant differences were observed between PLGA-PEG and PLA-PEG in T cells positive for INFγ (Figure 5A), TNFα (Figure 5B), IL-2 (Figure 5C), and CD107a (Figure 5D). T cells prepared by enrichment and amplification (“AIM-ACT”) were used as a reference. See U.S. Patent Nos. 10,987,412 and 11,007,222, which are hereby incorporated herein by reference in their entirety.

[0139] The ability of PLGA-PEG and PLA-PEG aAPC loaded with CMV peptide antigen (D14) to drive the expansion of antigen-specific CD8+ T cells was tested. As shown in Table 5 below (using HLA_CMV tetramer staining), there was no significant difference between PLGA-PEG and PLA-PEG nanoparticles.

[0140] Table 5

[0141] The ability of PLGA-PEG and PLA-PEG aAPC loaded with CMV peptide antigens to induce T cell memory phenotypes was examined as follows: PBMCs were isolated from whole blood, and CD8+ T cells were isolated using a commercially available kit. Cells were loaded at 1 x 102 6 The cells were suspended in a cytokine mixture at a concentration of / mL (see U.S. Patent No. 11,007,222, which is hereby incorporated by reference). Cells (1 x 10⁶) were then suspended in the mixture. 6 (Number of cells) were resuspended together with aAPC (approximately 10 µg) in a total volume of 16 mL. 160 µL of the cell / aAPC suspension was seeded into each well of a 96-well round-bottom plate. On day 4, cells were fed with a cytokine mixture. On day 7, cells were collected, counted, and their phenotype was evaluated.

[0142] As shown in Table 6 below, there was no significant difference in the ability of PLGA-PEG and PLA-PEG nanoparticles to generate T memory cells. CMV-loaded nanoparticles expanded CMV-specific CD8+ T cells, exhibiting a memory phenotype of >90%.

[0143] Table 6: D14 memory phenotype

[0144] like Figure 6 As shown, compared to bare nanoparticles (left panel), the antigen-loaded aAPC prepared essentially according to this embodiment was transported to lymph nodes, spleen, and tumors upon systemic administration to tumor-bearing mice (right panel). aAPC contains an MHC class I dimer (immunoglobulin fusion compound) and an anti-CD28 agonist antibody ligand. Furthermore, systemic administration of aAPC resulted in an increase in antigen-specific T cells in the spleen and tumors, and these T cells exhibited greater killing potential compared to peptides and CFAs. Figures 7A-7C. As shown in Figures 8A-8C, the phenotype of T cells recovered from lymph nodes and tumors (after aAPC administration) was consistent with persistent and potent antitumor effects. Furthermore, aAPC administration (loaded with GP100 antigen) prolonged overall survival in the B16 model (see Figure 8A-8C). Figure 9 ).

[0145] Example 2: Construction of anti-Fas agonist antibody ligands CH11 is an IgM-activating antibody against human Fas. This antibody exhibits cytolytic activity against human cells expressing Fas. A variable domain from CH11 was grafted onto the IgG4 framework described in U.S. Patent 10,632,193. See also... Figure 10AThe heavy chain and light chain amino acid sequences are provided as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In vitro assays were performed on CD8+ T cells induced by anti-Fas antibody (with and without anti-IgG4 antibody dimerization) (10 cells per well). 5 The activity of apoptosis in CD8+ T cells was evaluated. As shown in Figure 10B, although pentamer CH11 induced a strong apoptotic effect on CD8+ T cells, non-crosslinked anti-Fas IgG4 had a negligible effect. However, once crosslinked (here, by dimerization with anti-IgG4 antibody), the apoptotic effect was significant.

[0146] These results demonstrate that anti-Fas antibodies (such as those derived from clone CH11) can effectively and specifically induce apoptosis of antigen-specific T cells by conjugating nanoparticles with selected signal 1 ligands (e.g., targeting autoreactive T cells).

[0147] Example 3: Injectable aAPC carrying PD-L1 or anti-Fas signaling 2 ligand Injectable aAPC was created as described in Example 1 and has a PD-L1 signaling ligand 2 (SEQ ID NO: 1). Figure 11 As shown, these aAPCs rapidly inhibited antigen-specific killing of target cells loaded with the peptide. Specifically, MART-1-specific CD8+ T cells were incubated with 50 µg / mL aAPCs for 90 minutes. After washing, the cells were incubated with target cells loaded with the peptide for 4 hours, and antigen-specific killing was assessed by a caspase 3 / 7 assay. A 50% reduction in antigen-specific cytotoxic killing was observed.

[0148] Injectable aAPCs were created as described in Example 1 and have an anti-Fas signaling 2 ligand (as described in Example 2). Figure 12 As shown, these aAPCs rapidly eliminate antigen-specific T cells. Figure 12 The study showed that 50% of MART-1-specific T cells were eliminated after 4 hours of in vitro incubation. Non-target T cells were unaffected. For example, aAPCs that present survivin peptides did not affect the number of MART-1-specific T cells. Figure 13 As shown, in a mouse model, anti-Fas aAPC eliminated up to 90% of MART-1-specific T cells by day 13. This effect was dose-dependent.

[0149] Example 4: Selection of peptide antigens for aAPC It has been observed that certain peptide antigens induce particle aggregation when loaded onto nanoparticles (aAPCs). This effect was evaluated to determine the properties of peptide antigens suitable for loading onto nano-aAPCs without inducing aggregation.

[0150] Protein modeling and aggregation tendency analysis were performed using Discovery Studio 2021 (DS2021) (a software product from BIOVIA) (Dassault Systems BIOVIA, Discovery Studio Modeling Environment, 2021, San Diego CA). DS2021 includes aggregation tendency score calculation based on the Trout method. The MODELLER program (UCSF, San Francisco CA) was used for homology and comparative modeling of protein 3D structures.

[0151] Figure 14 The diagram schematically illustrates a process for identifying peptide antigens that are prone to aggregation when presented by HLA ligands on nanoparticles.

[0152] First, molecular models of the relevant peptide-HLA complex were generated. The HLA-A:0201 structural model included: 4L29, 1I7R, 5EUO, 1TVB, 6OPD, 6TRO, 6AMT, 6AM5, 2GT9, and 3OXS. Complex modeling was based on standard comparative protein models that generated the relevant peptides located in the HLA binding groove. The MODELER algorithm, implemented in Discovery Studio 2021, was used. Multiple template structures were used in the modeling steps, and each model underwent manual sequence alignment.

[0153] Next, using the highest-scoring completed static model, the 'Calculate Aggregation Score' protocol implemented in Discovery Studio 2021 was run. Radiuses of 5 and 10 Å were chosen as the calculation radii. This parameter determines how many nearby amino acid residues are included in the aggregation potential calculation. Depending on the size of the model being analyzed, the radius provides a reasonably smooth surface map that is not overly noisy. The protocol calculates the exposed surface of each residue of the peptide antigen and calculates the aggregation potential score based on each residue and the average of residues within a defined radius surrounding the residue. The numerical aggregation potential score (APS) is recorded along with sequence and structural information and used to generate a surface map based on the model's solvent-accessible surface. This provides a visualization of the surface shape in conjunction with the scored aggregation potential. Peptides can be classified as aggregators or non-aggregators by manual inspection or calculation.

[0154] By evaluating multiple peptide antigens, inferences can be drawn to guide the evaluation of candidate peptides. Here, the APS of residues 1 and 3-5 are averaged. If it exceeds a threshold of 0.07, it is considered a factor for aggregation. The presence of exposed Cys residues (as determined by examining model structures) is considered another factor for aggregation. The presence of charged residues and glycine residues is considered a factor preventing aggregation. The total number of factors that favor or prevent aggregation can determine whether the peptide-HLA complex is classified as a potential aggregater.

[0155] Figure 15A and Figure 15B The modeling and scoring of aggregated and non-aggregated peptides are shown separately. This process was used for a known set of aggregaters and non-aggregators, summarized in Table 7 below.

[0156] Table 7: Cluster Analysis

[0157] References

[0158]

[0159] sequence SEQ ID NO: 1 – PD-L1- connector -IgG4 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT GG GGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 2 – CH11 heavy chain-IgG4 EVQLQQSGPELVKPGASVKISCKASGYTFTdynmhWVKQSHGKSLEWIGyiypynggtgynqkfksKATLTVDNSSSTAYMELRSLTSEDSAVYYCARsyyamdyWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 3 – CH11 light chain DVVMTQSPLSLPVSLGDQASISCrsskslvhsngntylhWYLQKPGQSPKLLIYkvsnrfSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCsqsthvppaFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 4 – Cys connector -IgG4Fc- connector -FasL DNSLCLSLGGGGS ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGS PSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL SEQ ID NO: 5 –PD-L1- connector -HLA-A 02:01(C84,C139)- connector -IgG4 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT GG GGSGGGGSGGGGS GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRW GGGGSGGGGSGGGGS ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 6 –αCD28scFv (VH- connector -VL)- connector -HLA-A 02:01(C84, C139)- catch head -IgG4 EVKLQQSGPGLVKPSETLSLTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVSLKMSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS GGGGSGGGGSGGGGS DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIKR GGGGSGGGGSGGGGS HSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRW GGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 7 –αCD28scFv (VL- connector -VH)- connector -HLA-A 02:01(C84, C139)- catch head -IgG4 DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIKR GGGGSGGGGSGGGGS EVKLQQSGPGLVKPSETLSLTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVSLKMSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVS GGGGSGGGGSGGGG SHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRW GGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 8 – β-2-microglobulin IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM SEQ ID NO: 9 – Anti-CD28 antibody HC CDR1 DYGVH SEQ ID NO: 10 – Anti-CD28 antibody HC CDR2 VIWAGGGTNYNSALMS SEQ ID NO: 11 – Anti-CD28 antibody HC CDR3 DKGYSYYYSMDY SEQ ID NO: 12 – Anti-CD28 antibody LC CDR1 RASESVEYYVTSLMQ SEQ ID NO: 13 – Anti-CD28 antibody LC CDR2 AASNVES SEQ ID NO: 14 – Anti-CD28 antibody LC CDR3 QQSRKVPYT SEQ ID NO: 15 – Humanized anti-CD28 heavy chain variable region EVKLQQSGPGLVKPSETLSLTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVFLKMNSLTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS SEQ ID NO: 16 – Humanized anti-CD28 heavy chain variable region EVKLQQSGPGLVKPSETLSLTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVSLKMSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS SEQ ID NO: 17 – Humanized anti-CD28 heavy chain variable region EVKLQQSGPGLVKPSETLSLTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRVTISKDNSKSQVSLKLSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS SEQ ID NO: 18 – Humanized anti-CD28 light chain DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTNFTLTISSLQEEDVAMYFCQQSRKVPYTFGGGTKVEIK SEQ ID NO: 19 – Humanized anti-CD28 light chain DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTNFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIK SEQ ID NO: 20 – Humanized anti-CD28 light chain DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIK SEQ ID NO: 21 – HLA-E extracellular domain GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW SEQ ID NO: 22 – Engineered HLA-E extracellular domain (mutations are shown in bold and underlined) GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRG C YNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDT C AQISEQKSNDASEAEHQRAYLE A TCV A WLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW SEQ ID NO: 23 – IgG4 Fc ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 24 – Flexible Joint GGGGSGGGGSGGGGS SEQ ID NO: 25 – HLA-E-Fc fusion (HLA-E mutation is shown in bold and underline, IgG4 Fc is shown in underline) (Uppercase italics are shown; flexible joints are shown in lowercase italics) GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRG C YNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDT C AQISEQKSNDASEAEHQRAYLE A TCV AWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW ggggsggggsggggsESKYGPP CPPCPAPEFEGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK SEQ ID NO: 26 -- HLA-A 02:01( C84 , C139 )- connector - IgG4 GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG C YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADM C AQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRW GGGGSGGGGSGGGGS ESKYGPPCPPC PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK

Claims

1. An artificial antigen-presenting cell (aAPC) suitable for parenteral administration, comprising: Poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymers and one or more polypeptide ligands conjugated to PEG via thioether bonds; in: The polypeptide ligand comprises an HLA ligand that presents the peptide antigen and optionally one or more signal 2 ligands, and The copolymer contains approximately 40% by weight or less of functional groups for peptide ligand coupling.

2. The aAPC of claim 1, wherein about 15% to about 35% by weight of the copolymer has functional groups for peptide ligand coupling.

3. The aAPC of any one of claims 1 or 2, wherein the PLA or PLGA portion of the copolymer has a molecular weight of about 15 kDa to about 50 kDa, or about 15 kDa to about 35 kDa, or about 15 kDa to about 25 kDa.

4. The aAPC of claim 3, wherein the PLA or PLGA portion of the copolymer has a molecular weight of about 20 kDa.

5. The aAPC of claim 3 or 4, wherein the PEG portion of the copolymer has a molecular weight in the range of about 2 kDa to about 10 kDa or in the range of about 2 kDa to about 7 kDa.

6. The aAPC of claim 5, wherein the PEG portion of the copolymer has a molecular weight in the range of about 2 kDa to about 5 kDa.

7. The aAPC of claim 6, wherein the PEG portion having a functional group for peptide ligand coupling has a molecular weight of about 5 kDa, and the PEG portion not having a functional group for ligand coupling has a molecular weight of about 3 kDa.

8. The aAPC according to any one of claims 1 to 7, wherein the thiol group on the polypeptide ligand is coupled to a PEG-maleimide functional group.

9. The aAPC according to any one of claims 1 to 8, wherein the aAPC has about 10 to about 500 polypeptide ligands.

10. The aAPC of claim 9, wherein the aAPC has about 50 to about 400 polypeptide ligands.

11. The aAPC of claim 10, wherein the aAPC has about 100 to about 300 polypeptide ligands.

12. The aAPC according to any one of claims 1 to 11, wherein the aAPC has a diameter of about 50 nm to about 150 nm.

13. The aAPC of claim 12, wherein the aAPC has a diameter of about 60 nm to about 130 nm.

14. The aAPC of claim 13, wherein the aAPC has a diameter of about 80 nm to about 120 nm.

15. The aAPC of claim 13, wherein the aAPC has a diameter of about 60 nm, 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm.

16. The aAPC as claimed in any one of claims 1 to 15, wherein the aAPC has a surface charge of about 0 to -15 mV or about 0 to about -10 mV.

17. The aAPC of claim 16, wherein the aAPC has a surface charge of about -2.5 mV to about -10 mV.

18. The aAPC as claimed in any one of claims 1 to 17, wherein the aAPC is a population of aAPCs having a size distribution with a polydispersity index (PDI) of less than 0.

2.

19. The aAPC of any one of claims 1 to 18, wherein the HLA ligand is an HLA class I ligand, and optionally an HLA-A, HLA-B, HLA-C, or HLA-E ligand.

20. The aAPC of claim 19, wherein the HLA ligand comprises β2 microglobulin (β2M).

21. The aAPC of claim 19 or 20, wherein the HLA ligand is an HLA-A ligand, and optionally HLA-A 02:01 Ligand.

22. The aAPC of any one of claims 19 to 21, wherein the HLA ligand has a peptide-binding cleft stabilized by a disulfide bond.

23. The aAPC according to any one of claims 19 to 22, wherein the peptide antigen does not induce aggregation of the aAPC.

24. The aAPC of claim 23, wherein the average aggregation potential score (APS) of peptide residues 1 and 3-5 is less than about 0.

07.

25. The aAPC of claim 23 or 24, wherein the peptide antigen does not have exposed cysteine.

26. The aAPC of any one of claims 23 to 25, wherein the peptide antigen has one or more exposed glycine residues or exposed charged residues.

27. The aAPC of any one of claims 19 to 26, wherein the HLA ligand is a dimer and comprises a fusion with an immunoglobulin Fc region, wherein the immunoglobulin Fc region is optionally an IgG4 isotype.

28. The aAPC of any one of claims 19 to 26, wherein the HLA ligand comprises a fusion with immunoglobulin Fc, said immunoglobulin Fc optionally being an IgG4 isotype, and optionally dimerized with signal 2-Fc fusion.

29. The aAPC of any one of claims 19 to 27, wherein the HLA ligand comprises a fusion with the signal 2 ligand.

30. The aAPC of claim 29, wherein the signal 2 ligand comprises a single-chain antibody, the single-chain antibody optionally being scFv.

31. The aAPC of any one of claims 1 to 18, wherein the HLA ligand is an HLA class II ligand, and optionally is HLA-DR, HLA-DP, or HLA-DQ.

32. The aAPC of claim 31, wherein the HLA class II ligand comprises an immunoglobulin fusion of HLA α and β chains with antibody heavy and light chains.

33. The aAPC of any one of claims 1 to 32, wherein the polypeptide ligand comprises a co-stimulatory ligand.

34. The aAPC of claim 33, wherein the co-stimulatory ligand is an agonist of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT.

35. The aAPC of claim 34, wherein the co-stimulatory ligand is an agonist antibody against CD28.

36. The aAPC of claim 35, wherein the aAPC further comprises one or more cytokines that support T cell activation and / or expansion, the cytokines being conjugated to the aAPC as peptide ligands, fused to signal 1 or signal 2 peptide ligands, or encapsulated by the copolymer.

37. The aAPC of any one of claims 1 to 32, wherein the polypeptide ligand does not contain any signal 2 ligand.

38. The aAPC of any one of claims 1 to 32, wherein the polypeptide ligand comprises a co-inhibitory ligand.

39. The aAPC of claim 38, wherein the co-inhibitory ligand is an agonist of Fas, TGF-β, or PD-1.

40. The aAPC of claim 39, wherein the co-inhibitory ligand is an agonist antibody of Fas.

41. The aAPC of claim 40, wherein the agonist antibody of Fas is an IgG4 antibody based on clone CH11.

42. The aAPC of claim 39, wherein the co-inhibitory ligand is FasL, optionally an immunoglobulin Fc fusion.

43. The aAPC of claim 39, wherein the co-inhibitory ligand is PD-L1, optionally an immunoglobulin Fc fusion.

44. The aAPC of claim 43, wherein the activating fragment of PD-L1 is fused with an HLA class I ligand, said HLA class I ligand optionally dimerizing by fusion with immunoglobulin Fc.

45. The aAPC of any one of claims 41 to 44, wherein the aAPC further comprises a tolerogenic cytokine as a polypeptide ligand, the tolerogenic cytokine being fused to or encapsulated by the copolymer, wherein the tolerogenic cytokine is optionally IL-10.

46. ​​The aAPC of any one of claims 1 to 45, wherein the polypeptide ligand further comprises one or more homing ligands targeting a tissue or organ.

47. The aAPC of any one of claims 1 to 46, wherein the aAPC is contained in a pharmaceutical composition suitable for administration to a subject.

48. The aAPC of claim 47, wherein the pharmaceutical composition is lyophilized.

49. An artificial antigen-presenting cell (aAPC) suitable for parenteral administration, comprising: Polymer or lipid nanoparticles comprising a polyethylene glycol (PEG) sheath and one or more polypeptide ligands conjugated to PEG via thioether bonds; in: The polypeptide ligand comprises an HLA class I or II ligand that presents the peptide antigen and optionally one or more signal 2 ligands, and The peptide antigen does not induce the aggregation of the aAPC.

50. The aAPC of claim 49, wherein the HLA ligand is an HLA class I ligand, and optionally an HLA-A, HLA-B, HLA-C, or HLA-E ligand.

51. The aAPC of claim 50, wherein the HLA ligand comprises β2 microglobulin (β2M).

52. The aAPC of claim 51, wherein the HLA ligand is an HLA-A ligand, and optionally HLA-A 02:

01.

53. The aAPC of any one of claims 49 to 52, wherein the HLA ligand has a peptide-binding cleft stabilized by a disulfide bond.

54. The aAPC of any one of claims 49 to 53, wherein the average aggregation potential score (APS) of peptide residues 1 and 3-5 is less than about 0.

07.

55. The aAPC of any one of claims 49 to 54, wherein the peptide antigen does not have exposed cysteine.

56. The aAPC of any one of claims 49 to 55, wherein the peptide antigen has one or more exposed glycine residues or exposed charged residues.

57. The aAPC of any one of claims 49 to 56, wherein the nanoparticles are polymer nanoparticles comprising poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymers and one or more polypeptide ligands conjugated to PEG via thioether bonds.

58. The aAPC of claim 57, wherein about 40% by weight or less of the copolymer has functional groups for peptide ligand coupling.

59. The aAPC of claim 58, wherein about 15% to about 35% by weight of the copolymer has functional groups for peptide ligand coupling.

60. The aAPC of any one of claims 57 to 59, wherein the PLA or PLGA portion of the copolymer has a molecular weight of about 15 kDa to about 50 kDa, or about 15 kDa to about 35 kDa, or about 15 kDa to about 25 kDa.

61. The aAPC of claim 60, wherein the PLA or PLGA portion of the copolymer has a molecular weight of about 20 kDa.

62. The aAPC of claim 60 or 61, wherein the PEG portion of the copolymer has a molecular weight in the range of about 2 kDa to about 10 kDa or in the range of about 2 kDa to about 7 kDa.

63. The aAPC of claim 62, wherein the PEG portion of the copolymer has a molecular weight in the range of about 2 kDa to about 5 kDa.

64. The aAPC of claim 63, wherein the PEG portion having a functional group for peptide ligand coupling has a molecular weight of about 5 kDa, and the PEG portion not having a functional group for ligand coupling has a molecular weight of about 3 kDa.

65. The aAPC of any one of claims 49 to 56, wherein the nanoparticles are lipid nanoparticles comprising PEG-conjugated lipids.

66. The aAPC of claim 65, wherein the lipid nanoparticles further comprise cationic or ionizable lipids, neutral lipids or phospholipids and structural lipids such as cholesterol or cholesterol fractions.

67. The aAPC of any one of claims 49 to 66, wherein the thiol group on the polypeptide ligand is coupled to a PEG-maleimide functional group.

68. The aAPC according to any one of claims 49 to 67, wherein the aAPC has about 10 to about 500 polypeptide ligands.

69. The aAPC of claim 68, wherein the aAPC has about 50 to about 400 polypeptide ligands.

70. The aAPC of claim 69, wherein the aAPC has about 100 to about 300 polypeptide ligands.

71. The aAPC as claimed in any one of claims 49 to 70, wherein the aAPC has a diameter of about 50 nm to about 150 nm.

72. The aAPC of claim 71, wherein the aAPC has a diameter of about 60 nm to about 130 nm.

73. The aAPC of claim 72, wherein the aAPC has a diameter of about 80 nm to about 120 nm.

74. The aAPC of claim 71, wherein the aAPC has a diameter of about 60 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm.

75. The aAPC as claimed in any one of claims 49 to 74, wherein the aAPC has a surface charge of about 0 to -15 mV or about 0 to about -10 mV.

76. The aAPC of claim 75, wherein the aAPC has a surface charge of about -2.5 mV to about -10 mV.

77. The aAPC as claimed in any one of claims 49 to 76, wherein the aAPC is a group of aAPCs having a size distribution with a polydispersity index (PDI) of less than 0.

2.

78. The aAPC of any one of claims 49 to 77, wherein the HLA ligand is a dimer and comprises two fusions with immunoglobulin Fc, wherein the immunoglobulin Fc is optionally an IgG4 isotype.

79. The aAPC of any one of claims 49 to 77, wherein the HLA ligand comprises a fusion with immunoglobulin Fc, said immunoglobulin Fc optionally being an IgG4 isotype, and optionally dimerized with signal 2-Fc fusion.

80. The aAPC of any one of claims 49 to 78, wherein the HLA ligand comprises a fusion with the signal 2 ligand.

81. The aAPC of claim 80, wherein the signal 2 ligand comprises a single-chain antibody, wherein the single-chain antibody is optionally scFv.

82. The aAPC of any one of claims 49 to 81, wherein the polypeptide ligand comprises a co-stimulatory ligand.

83. The aAPC of claim 82, wherein the co-stimulatory ligand is an agonist of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT.

84. The aAPC of claim 83, wherein the co-stimulatory ligand is an agonist antibody against CD28.

85. The aAPC of any one of claims 49 to 84, wherein the aAPC further comprises one or more cytokines that support T cell activation and / or expansion, the cytokines being conjugated to the aAPC as peptide ligands, fused to signal 1 or signal 2 peptide ligands, or encapsulated by the copolymer.

86. The aAPC of any one of claims 49 to 78, wherein the polypeptide ligand does not contain any signal 2 ligand.

87. The aAPC of any one of claims 49 to 81, wherein the polypeptide ligand comprises a co-inhibitory ligand.

88. The aAPC of claim 87, wherein the co-inhibitory ligand is an agonist of Fas, TGF-β, or PD-1.

89. The aAPC of claim 88, wherein the co-inhibitory ligand is an agonist antibody of Fas.

90. The aAPC of claim 89, wherein the agonist antibody is an IgG4 antibody based on clone CH11.

91. The aAPC of claim 88, wherein the co-inhibitory ligand is FasL, optionally an immunoglobulin Fc fusion.

92. The aAPC of claim 88, wherein the co-inhibitory ligand is PD-L1, optionally an immunoglobulin Fc fusion.

93. The aAPC of claim 92, wherein the PD-L1 activation fragment is fused with an HLA class I ligand, said HLA class I ligand optionally dimerizing by fusion with immunoglobulin Fc.

94. The aAPC of any one of claims 86 to 93, wherein the aAPC further comprises a tolerogenic cytokine as a polypeptide ligand, as a fusion with a signal 1 or signal 2 polypeptide ligand, or encapsulated by the copolymer, wherein the tolerogenic cytokine is optionally IL-10.

95. The aAPC of any one of claims 49 to 94, wherein the polypeptide ligand further comprises one or more homing ligands.

96. The aAPC of any one of claims 49 to 95, wherein the aAPC is contained in a pharmaceutical composition suitable for administration to a subject.

97. The aAPC of claim 96, wherein the pharmaceutical composition is lyophilized.

98. A method for immunotherapy, the method comprising administering aaAPC or a pharmaceutical composition thereof, as described in any one of claims 1 to 97, to a subject requiring treatment.

99. The method of claim 98, wherein the subject has cancer or an infectious disease, and the aAPC comprises a co-stimulatory ligand.

100. The method of claim 99, wherein the subject suffers from a hematologic malignancy.

101. The method of claim 99, wherein the subject has a solid tumor, optionally stage I, II, III or IV.

102. The method of any one of claims 99 to 101, wherein the subject has human papillomavirus (HPV)-associated cancer.

103. The method of any one of claims 98 to 102, wherein the subject is given immune checkpoint inhibitor therapy before, during, or after administration of the aAPC or its pharmaceutical composition.

104. The method of claim 98, wherein the subject suffers from an autoimmune disease, and the aAPC contains a co-inhibitory signal or does not contain a signal 2 ligand.

105. The method of claim 104, wherein the autoimmune disorder is type 1 diabetes.

106. The method of claim 104, wherein the autoimmune disease is selected from vitiligo, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasser syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, herpetic dermatitis, celiac disease, Sjögren's disease, Hashimoto's thyroiditis, alopecia, ankylosing spondylitis, scleroderma, and HTLV-1-associated myelopathy (HAM) / tropical spastic paraplegia (TSP), etc.

107. The method of any one of claims 98 to 106, wherein the aAPC or its pharmaceutical composition is administered parenterally.

108. The method of claim 107, wherein the aAPC or its pharmaceutical composition is administered via intravenous, subcutaneous, or intramuscular administration.

109. The method of claim 108, wherein the aAPC or its pharmaceutical composition is administered subcutaneously.

110. A method for preparing artificial antigen-presenting cells, the method comprising: A computer-simulated HLA ligand comprising an antigen-binding cleft containing a candidate peptide antigen is provided; Prepare a surface map containing the antigen-binding cleft of the candidate peptide antigen, the surface map providing an aggregation potential score for each residue of the candidate peptide antigen and surrounding amino acid residues; Select candidate peptides with aggregation potential below a threshold; as well as The selected peptide is loaded onto aAPC.

111. An anti-Fas agonist antibody having an IgG isotype and conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen.

112. A dimer PD-L1 ligand comprising amino acid residues F19 to T239 of human PD-L1, wherein the amino acid residues F19 to T239 of human PD-L1 are fused directly or indirectly to the Fc region of a dimerized IgG at their C-terminus, and wherein the dimer PD-L1 ligand is conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen.

113. A dimerized FasL ligand comprising amino acids P132 to L279 of human FasL, wherein the amino acids P132 to L279 of human FasL are fused directly or indirectly to a dimerized IgG-Fc region at their N-terminus, the dimerized Fc region being conjugated to nanoparticles via a Cys-containing linker, the nanoparticles further presenting an HLA-peptide antigen ligand.

114. A tolerogenic ligand comprising amino acid F19-T239 of human PD-L1, wherein the amino acid F19-T239 of human PD-L1 is fused directly or via a linker to an HLA-immunoglobulin fusion protein.

115. A co-stimulatory ligand comprising an anti-CD28 agonist scFv conjugated to an HLA-immunoglobulin fusion protein.

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