Injectable engineered antigen-presenting cells for immunotherapy

Abstract

In various embodiments, the Disclosure provides engineered antigen-presenting cells (aAPCs) suitable for parenteral administration for immunotherapy. In various embodiments, the aAPCs are effective in vivo for activating or inhibiting target T cells, including CD8+ T cells or CD4+ T cells. The aAPCs according to the Disclosure provide a storage-stable nanoparticle platform for immunotherapy. The storage-stable nanoparticle platform in various embodiments controls, among other properties, particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation tendency.

Description

Technical Field

[0001] Priority This application claims the benefit of, and priority to, U.S. Provisional Application No. 63 / 454,361, filed Mar. 24, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Array Listing This application is filed via EFS-Web in XML format and includes an array listing that is incorporated herein by reference in its entirety. The XML copy, created on Mar. 25, 2024, is named NEX-013PC_107590-5013_Sequence_Listing and is 61,440 bytes in size.

Background Art

[0003] Antigen-presenting cells (APCs) process and present antigenic peptides that are complexed with major histocompatibility complex (MHC) proteins on the surface of the MHC proteins. Effector cells such as T cells recognize these peptide-MHC (pMHC) complexes via cell surface receptors such as the T cell receptor (TCR). Dendritic cells (DCs) are an example of antigen-presenting cells, can effectively present antigens, and stimulate to support the proliferation of immune effector cells, thereby activating a cytotoxic response against the antigen. In some immunotherapies, DCs are harvested from a patient and pulsed with an antigen or transfected with a viral vector. When returned to the patient by infusion, these activated cells present tumor antigens to effector lymphocytes (e.g., CD4+ T cells, CD8+ T cells, and B cells). When this therapy is successful, a cytotoxic reaction against cells expressing the antigen (including tumor antigens) is initiated. However, there remains a need for a storable pharmaceutical composition that can effectively present peptide antigens to T cells in vivo (i.e., engineered antigen-presenting cells, or aAPCs) and activate or inhibit T cells in an antigen-specific manner. The present disclosure meets these and other objectives. [Overview of the Initiative]

[0004] In various embodiments, the Disclosure provides engineered antigen-presenting cells (aAPCs) suitable for parenteral administration for immunotherapy. In various embodiments, the aAPCs are effective in vivo for activating or inhibiting target T cells, including CD8+ T cells or CD4+ T cells. The aAPCs according to the Disclosure provide a storage-stable nanoparticle platform for immunotherapy. The storage-stable nanoparticle platform in various embodiments controls, among other properties, particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation tendency.

[0005] In various aspects and / or 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 nanometer-scale aAPCs described herein are designed to provide pharmacodynamic advantages, including circulatory properties, biodistribution, and degradation kinetics. These advantages may be due, in particular, to physical parameters such as particle size, surface charge, polydispersity index, polymer composition, ligand-conjugated chemistry, ligand density, and peptide loading. In some embodiments, aAPCs have polypeptide ligand density to avoid the possibility of aggregation, as well as steric constraints due to abundant ligands on the surface without loss of activity and / or potency. In some embodiments, aAPCs persist in peripheral blood circulation for a sufficient time to allow distribution to target tissues, including transport to lymphoid organs (e.g., lymph nodes) via blood / lymph exchange, and / or transport to tumors, and / or transport to target organs. In some embodiments, aAPCs are suitable for subcutaneous administration.

[0006] In one embodiment, the Disclosure provides 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), which comprises a PLA-PEG or PLGA-PEG copolymer and one or more polypeptide ligands conjugated to the PEG polymer via thioether linkage or other conjugation chemistry. The polypeptide ligands include an HLA ligand (human leukocyte antigen ligand) that presents 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 proliferation, or T cell inhibition. 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 may be an HLA class I and / or class II molecular complex, or a part thereof that constitutes an antigen-binding groove. In some embodiments, the HLA molecular complex may be a monomer or dimer and may contain additional heterogeneous sequences, such as immunoglobulin sequences. In some embodiments, the HLA fusion (e.g., an HLA-immunoglobulin fusion) offers further advantages in terms of stability, TCR binding affinity, and / or efficacy against T cell activation or inhibition.

[0008] In various embodiments, the aAPC comprises an HLA class I ligand for the presentation of a peptide antigen to CD8+ T cells (e.g., activation and / or proliferation of CD8+ cells, or inhibition of CD8+ cells). In some embodiments, the HLA class I ligand comprises at least two fusion proteins. The first fusion protein comprises a first HLA class I α chain and a first immunoglobulin heavy chain, and the 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 groove and a second HLA class I peptide binding groove.

[0009] Alternatively, aAPC contains an HLA class II ligand for the presentation of a peptide antigen to CD4+ T cells (e.g., activation and / or proliferation of CD4+ cells, or inhibition of CD4+ cells). In some embodiments, the HLA class II molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise (i) an immunoglobulin heavy chain and (ii) the extracellular domain of an HLA class II β chain. Two second fusion proteins comprise (i) an immunoglobulin light chain and (ii) the extracellular domain of an HLA class II α chain. The two first and two second fusion proteins associate to form an HLA class II molecular complex. The extracellular domains of the HLA class II β chains of each of the first fusion proteins and the extracellular domains of the HLA class II α chains of each of the second fusion proteins form an HLA class II peptide bond groove.

[0010] Peptide antigens bind to the antigen-binding groove of the antigen-presenting complex. Peptide antigens for immunotherapy of cancerous diseases, infectious diseases, and autoimmune diseases are described herein. In some embodiments, the peptide antigen does not induce aggregation of aAPC. That is, peptide antigens are selected that have a low tendency to aggregate when loaded onto aAPC. In some embodiments, peptides with a low tendency to aggregate are selected using an aggregation score (APS).

[0011] In some embodiments, the polypeptide ligand includes a signal-2 ligand, for example, a costimulatory ligand for activation and / or proliferation of target T cells. Exemplary costimulatory ligands include agonists for any one of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT. The costimulatory ligand can induce activation and / or proliferation of CTLs or Tregs in various embodiments. In other embodiments, and particularly when aAPC is intended to inhibit target T cells, the polypeptide ligand may not include a signal-2 ligand, or the polypeptide ligand may include an inhibitory ligand, which induces resistance or apoptosis of target T cells. In various embodiments, the inhibitory ligand is an agonist for Fas, TGF-β, or PD-1. The agonist ligand may include a native 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 its immunoglobulin fusion) or FasL (or its immunoglobulin fusion). 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.

[0012] In various embodiments, the signal 1 and signal 2 ligands can be combined in homodimer or heterodimer constructs. For example, the HLA ligand may include a signal 2-immunoglobulin (Ig) fusion (i.e., a heterodimer Ig fusion construct) and the fusion of an extracellular HLA domain to an immunoglobulin Fc region, such as an IgG4Fc region, which can be dimerized (e.g., via a disulfide bond). In yet another embodiment, the HLA ligand includes a fusion with the signal 2 ligand. For example, the extracellular HLA domain can be fused to an immunoglobulin Fc region at its C-terminus and to the signal 2 ligand at its N-terminus to prepare a homodimer ligand in which both signals are dimerized. In some embodiments, the signal 2 ligand includes a single-chain antibody (e.g., scFv) or the activating portion of a native ligand.

[0013] In some embodiments, the co-inhibitory ligand is an agonist antibody against Fas. In some embodiments, the agonist antibody against Fas is an IgG4 antibody based on clone CH11. As demonstrated herein, this anti-Fas antibody is active when it can crosslink multiple Fas receptors, such as when the antibody is bound to nanoparticles.

[0014] In some embodiments, aAPC further comprises one or more cytokines that support T cell activation and / or proliferation, or T cell inhibition. One or more cytokines or functional moieties may be bound to aAPC as polypeptide ligands. Alternatively, cytokines or functional moieties may be fused to signal 1 or signal 2 polypeptide ligands (which may optionally be presented in homodimeric or heterodimeric Ig fusion constructs as described herein). In some embodiments, cytokines are encapsulated by copolymers and locally released in a target environment (e.g., lymphoid organs, tumors, or target tissues or organs). Examples of cytokines that may be used include IL-1β, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and γ interferon. For example, IL-2 may be employed with a costimulatory signal 2 ligand. In some embodiments, aAPC comprises immunotolerogenic cytokines as polypeptide ligands.

[0015] In various embodiments, one or more peptide antigens are tumor or cancer-related antigens, such as tumor-derived antigens, tumor-specific antigens, and novel antigens. In some embodiments, the target peptide antigens include at least one that is 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 those associated with an autoimmune disease or reaction.

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

[0017] In some embodiments and aspects, the Disclosure provides aAPCs suitable for parenteral administration (including subcutaneous administration) and with a low tendency to aggregate. In these embodiments, the aAPC comprises polymer nanoparticles or lipid nanoparticles comprising a polyethylene glycol (PEG) sheath and one or more polypeptide ligands bonded to PEG (e.g., PEG terminus) via thioether links or other functional groups. The polypeptide ligand comprises an HLA class I or class II ligand that presents a 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 ​​and / or the peptide antigen has one or more exposed glycine residues or exposed charged residues. In some embodiments, the peptide antigen has no cysteine ​​residues and contains one or more charged residues (e.g., one, two, or three charged residues). In some aspects and embodiments, the aggregation ability is estimated using a computer by determining the average aggregation score (APS) of antigen peptide residues in the HLA antigen binding groove.

[0018] In other embodiments, the present invention provides a method for immunotherapy. This method comprises administering an aAPC or a pharmaceutical composition thereof as described herein to a subject in need of treatment. In various embodiments, the subject has cancer or an infection, and the aAPC comprises a co-stimulatory ligand. In some embodiments, the peptide antigen is selected on an individualized basis for cancer patients based on an analysis of the patient's tumor. In some embodiments, nano-aAPC is used as an add-on immunizing vaccine after adoptive T-cell therapy (in which unsensitized T cells, TILs, or T cells from an HLA-matched donor are proliferated exovivoically and administered to the patient). In some embodiments, the subject has an autoimmune disease, and the aAPC comprises a co-inhibitory signal or does not contain a signal 2 ligand. In some embodiments, the autoimmune disease is type 1 diabetes.

[0019] Generally, aAPC or its pharmaceutical composition is administered parenterally. For example, aAPC or its pharmaceutical composition may be administered intravenously, intra-arterially, subcutaneously, intradermally, lymphatically, intramuscularly, or intratumorally. In some embodiments, the aAPC composition is administered subcutaneously.

[0020] In other embodiments of this disclosure, polypeptide ligands for immunotherapy (including polypeptide ligands for aAPC) are disclosed. Such polypeptide ligands include an anti-Fas-agonist antibody having an IgG isotype (e.g., IgG4), which can be conjugated to nanoparticles together with an HLA ligand that presents a peptide antigen (e.g., associated with autoimmune diseases). In other embodiments, the polypeptide ligand is a dimeric PD-L1 ligand containing an activating moiety of PD-L1, such as amino acid residues F19-T239 of human PD-L1. Each PD-L1 activating fragment may be fused directly or indirectly to an IgG Fc region (e.g., IgG4) via its C-terminal linker, and the ligand may be dimerized by a disulfide bond in the Fc region. The dimeric PD-L1 ligand can be conjugated to nanoparticles together with an HLA ligand that presents a peptide antigen and can be used to induce resistance to the antigen. In other embodiments, the polypeptide ligand is a dimerized FasL ligand containing an activated moiety of FasL, such as amino acids P132-L279 of human FasL fused directly or indirectly to a dimerized IgG-Fc region (e.g., IgG4) via its N-terminal linker. In some embodiments, the dimerized Fc region can be conjugated to nanoparticles via a Cys-containing linker. The nanoparticles may further present an HLA-peptide antigen ligand to promote apoptosis of antigen-specific T cells. In other embodiments, the polypeptide ligand is an immunotolerogenic ligand containing an activated fragment of PD-L1, such as amino acids F19-T239 of human PD-L1, fused directly or via a linker to an HLA-immunoglobulin fusion protein. Such ligands can be conjugated to nanoparticles as disclosed herein and used in immunotherapy (to induce resistance in targeted T cells). In other embodiments, the polypeptide ligand is a costimulatory ligand containing an anti-CD28 operative scFv coupled to an HLA-immunoglobulin fusion protein, providing a homodimeric ligand containing a signal 1 ligand and a costimulatory signal 2 ligand. The scFv may be fused to the HLA sequence via a heavy chain sequence or a light chain sequence.

[0021] The present invention and various embodiments are further described through the following detailed description.

Brief Description of Drawings

[0022] [Figure 1A] Shows the particle size and surface charge of nanoparticles prepared according to the present disclosure. TEM images of bare particles, protein-bound nanoparticles, and peptide-bearing nanoparticles are shown. [Figure 1B] Shows the particle size and surface charge of nanoparticles prepared according to the present disclosure. (In order from left to right) Shows the particle size distribution, average particle size of the particles, polydispersity index (PDI), and surface charge. [Figure 2] It is a diagram showing the binding of thiolated ligands to PEG-maleimide functional groups on nanoparticles. [Figure 3] A shows the effects on ligand density (bars) and nanoparticle size (squares) when varying the %PEG-mal on the nanoparticle surface. B shows the effects on ligand density (bars) and nanoparticle size (squares) when varying the maleimide:thiol ratio during the coupling reaction. [Figure 4] A shows that the use of PLGA-PEG or PLA-PEG does not result in a significant difference in particle size (bars) or PDI (dots). B shows that the use of PLGA-PEG or PLA-PEG does not result in a significant difference in protein density. Ligands per NP are shown by bars, and μg protein at 10 OD are shown by dots. [Figure 5A] Shows that the use of PLGA-PEG or PLA-PEG does not result in a significant difference in the stimulation of antigen-specific CD8+ T cells. Shows the proportion of IFNγ+ CD8+ T cells. [Figure 5B] Shows that the use of PLGA-PEG or PLA-PEG does not result in a significant difference in the stimulation of antigen-specific CD8+ T cells. Shows the proportion of TNFα+ CD8+ T cells. [Figure 5C] Shows that the use of PLGA-PEG or PLA-PEG does not result in a significant difference in the stimulation of antigen-specific CD8+ T cells. Shows the proportion of IL2+ CD8+ T cells. [Figure 5D] The use of PLGA-PEG or PLA-PEG shows no significant difference in stimulating antigen-specific CD8+ T cells. The ratio of CD107a+ CD8+ T cells is shown. [Figure 6] It is shown that the antigen-loaded aAPCs according to embodiments of the present disclosure migrate to lymph nodes, spleen, and tumors. [Figure 7] A shows that systemically administered aAPCs increased antigen-specific T cells in the spleen of mice with tumors (B16-OVA, having transplanted OT-1 T cells). B shows that systemically administered aAPCs increased antigen-specific T cells in the tumors of mice with tumors (B16-OVA, having transplanted OT-1 T cells). Spleen cells of mice administered aAPCs with added peptide had higher killing ability compared to peptides added with complete Freund's adjuvant (CFA). C: N = 2 / treatment group. In vitro killing analysis performed using spleen cells collected on day 22. [Figure 8] It is shown that T cells recovered from lymph nodes and tumors have phenotypes consistent with a persistent and strong anti-tumor effect. A shows T cells (specific to ovalbumin antigen) recovered from lymph nodes of a non-disease model. These T cells showed central memory and effector memory phenotypes. B quantifies antigen-specific T cells in a melanoma model tumor (B16F10 cells), and 63% of them had at least three effector functions. C quantifies gp100-specific T cells in a melanoma model tumor, showing that antigen-specific CTLs recovered from the tumor showed effector memory and central memory phenotypes. [Figure 9] In a B16 mouse model, it is shown that aAPCs loaded with gp100 antigen extended the survival period (the x-axis is the number of days from tumor transplantation). It is shown that aAPCs activate anti-tumor T cells in vivo and eliminate lung metastases in this model. [Figure 10A]The construction of an operative anti-Fas antibody ligand is shown. The construction of an IgG4 antibody based on the variable domain of the CH11 clone (IgM isotype) is shown. The IgG4 antibody is crosslinked with an anti-IgG4 antibody for in vitro testing. [Figure 10B] This demonstrates the construction of an agonistic anti-Fas antibody ligand. The anti-Fas agonistic ligand shows little activity without crosslinking, but exhibits robust activity with crosslinking. [Figure 10C] This demonstrates the construction of an agonistic anti-Fas antibody ligand. The anti-Fas agonistic ligand shows little activity without crosslinking, but exhibits robust activity with crosslinking. [Figure 11] This study demonstrates that aAPC possessing the PD-L1-Ig signaling ligand rapidly inhibits antigen-specific killing of peptide-loaded target cells. [Figure 12] This study demonstrates that aAPCs possessing the anti-Fas signaling ligand rapidly eliminate antigen-specific T cells. [Figure 13] In a mouse model, anti-Fas aAPC eliminated up to 90% of MART-1 specific T cells by day 13, demonstrating that this effect is dose-dependent. [Figure 14] This diagram shows the process of selecting the antigen to present on the aAPC in order to mitigate aggregation caused by antigen loading. [Figure 15A] This paper demonstrates the modeling and scoring of aggregated peptides. [Figure 15B] This paper demonstrates the modeling and scoring of non-aggregating peptides. [Modes for carrying out the invention]

[0023] In various embodiments, the Disclosure provides engineered antigen-presenting cells (aAPCs) suitable for parenteral administration for immunotherapy. In various embodiments, the aAPCs are effective in vivo for activating or inhibiting target T cells, including CD8+ T cells or CD4+ T cells. The aAPCs according to the Disclosure provide a storage-stable nanoparticle platform for immunotherapy. The storage-stable nanoparticle platform in various embodiments controls, among other properties, particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation tendency.

[0024] In various aspects and / or 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 nanometer-scale aAPCs described herein are designed to provide pharmacodynamic advantages, including circulatory properties, biodistribution, and degradation kinetics. These advantages are due, in particular, to physical parameters such as particle size, surface charge, polydispersity index, polymer composition, ligand-conjugated chemistry, ligand density, and peptide loading. In some embodiments, aAPCs have polypeptide ligand density to avoid the possibility of aggregation, as well as steric constraints due to abundant ligands on the surface without loss of activity and / or potency. In some embodiments, aAPCs persist in peripheral blood circulation for a sufficient time to allow distribution to target tissues, including transport to lymphoid organs (e.g., lymph nodes) via blood / lymph exchange, and / or transport to tumors, and / or transport to target organs. In some embodiments, aAPCs are suitable for subcutaneous administration.

[0025] In one embodiment, the 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) copolymer. PLA-PEG nanoparticles and PLGA-PEG nanoparticles can be prepared by nanometer-scale precipitation using known processes. In these embodiments of the Disclosure, the aAPC has advantages, among other things, 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), which comprises a PLA-PEG or PLGA-PEG copolymer and one or more polypeptide ligands conjugated to the PEG polymer via thioether linkage or other conjugation chemistry (such as amine-mediated conjugation) (e.g., conjugated to the PEG terminus). The polypeptide ligands include an HLA ligand (human leukocyte antigen ligand) that presents a peptide antigen, and optionally, one or more signal 2 ligands. Signal 2 ligands are described elsewhere in this specification and include signal 2 ligands for T cell activation and / or proliferation, or T cell inhibition. In various embodiments, about 40% by weight or less of the copolymer has functional groups for polypeptide 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 polypeptide ligand coupling. In some embodiments, about 15% to about 35% by weight of the copolymer has functional groups for lipeptide ligand coupling. In various embodiments, PEG-Mal (by weight) or other PEG functional groups are less than about 10% or less than about 7% of the copolymer weight, for example, about 5%.

[0026] As shown in Figure 2, only a portion of the copolymer contains 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-terminated caps such as methyl ether-terminated caps). Terminally capped PEGs are sometimes referred to herein as mPEGs. Reactions between sulfhydryl groups and maleimides are well known in the art. See, for example, Joao MJM et al., Bioconjugation with Maleimides: A Useful Tool for Chemical Biology, Chemistry (August 2018). Other conjugation chemistry (including amine conjugation) can be used and are well known. Alternative conjugation chemistry is described, for example, in U.S. Patent No. 10,435,668, which is incorporated herein by reference in its entirety.

[0027] For example, molecules can be directly activated with various chemical functional groups, including nucleophiles, leaving groups, or electrophiles. Examples of activating functional groups include alkyl and acyl halides, amines, sulfhydryls, aldehydes, unsaturated bonds, hydrazides, isocyanates, isothiocyanates, ketones, and other groups known to be activated for chemical bonding. Alternatively, molecules can be bonded to nanoparticles using low-molecular-weight coupling reagents. Non-limiting examples of coupling reagents include carbodiimides, maleimides, N-hydroxysuccinimide esters, bischloroethylamine, bifunctional aldehydes such as glutaraldehyde, and anhydrides. In other embodiments, molecules can be bonded to nanoparticles via affinity bonds, such as biotin-streptavidin linkages or couplings.

[0028] In exemplary embodiments, the nanoparticles have a PLGA copolymer that can be adjusted to a specific biodegradation rate in vivo (by adjusting the LA:GA ratio and / or the molecular weight of the PLGA polymer). In exemplary embodiments, the PLGA is based on an LA:GA ratio of 20:1 to 1:20, comprising 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. The PLGA degrades by hydrolysis of its ester bonds. The time required for the degradation of the PLGA depends on the monomer ratio. The higher the glycoside unit content, the shorter the time required for degradation compared to when lactide units are dominant. In some embodiments, PLGA is 50 / 50 L / G.

[0029] In some embodiments, aAPC may further include a PLGA or PLA polymer (in addition to the copolymer). 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 exemplary embodiments, the PLGA or PLA polymer has a molecular weight of about 20 kDa. In various embodiments, the PLGA or PLA polymer is present in an amount 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.

[0030] In various embodiments, the ratio of PEG-maleimide groups to mPEG limits the density of polypeptide ligands, for example, conjugated via sulfhydryl groups. In various embodiments, aAPC has about 10 to about 500 polypeptide ligands per particle. In some embodiments, aAPC has about 50 to about 400 polypeptide ligands per particle. In some embodiments, aAPC has about 100 to about 300 polypeptide ligands per particle. In various embodiments, by refining the peptide density, aAPC particles with potent T-cell effector properties (activation or inhibition) can be prepared while avoiding undesirable properties, including but not limited to aAPC aggregation and steric limitation.

[0031] In some embodiments, other means may be employed to control the polypeptide ligand density on the particles, such as limiting the amount of thiolated polypeptide during the coupling reaction.

[0032] 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 an exemplary embodiment, the PLA or PLGA portion of the copolymer has a molecular weight of about 20 kDa.

[0033] In various embodiments, 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. In exemplary embodiments, the PEG portion of the copolymer has a molecular weight in the range of about 2 kDa to about 5 kDa. In some embodiments, the PEG portion having a functional group for polypeptide 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. In such embodiments, the smaller mPEG portion (compared to the PEG-functional group) limits the steric constraints on the binding of the polypeptide ligand to the target and / or improves the conjugation efficiency.

[0034] Therefore, in the 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.

[0035] In various embodiments, aAPCs have a diameter of approximately 50 nm to approximately 150 nm. In some embodiments, aAPCs have a diameter of approximately 50 nm to approximately 130 nm. In some embodiments, aAPCs have a diameter of approximately 50 nm to approximately 120 nm. In some embodiments, aAPCs have a diameter of approximately 50 nm to approximately 100 nm, or approximately 50 nm to approximately 75 nm. In exemplary embodiments, aAPCs have a diameter of approximately 60 nm, approximately 80 nm, approximately 90 nm, approximately 100 nm, approximately 110 nm, or approximately 120 nm. In some embodiments, the aAPC population has a particle size distribution with a polydispersity index (PDI) of less than 0.2. In various embodiments, aAPCs have a surface charge of approximately 0 to approximately -15 mV, or approximately 0 to approximately -10 mV. For example, aAPCs may have a surface charge of approximately -2.5 mV to approximately -10 mV. The particle size and surface charge of aAPC enable desired circulation and biodistribution characteristics, and in some embodiments, offer advantages in particle stability.

[0036] In various embodiments, the HLA ligand may be an HLA class I and / or class II molecular complex, or a part thereof that constitutes an antigen-binding groove. In some embodiments, the HLA molecular complex may be monomeric or dimeric and may include additional heterologous sequences, such as immunoglobulin sequences. Alternative heterologous sequences include dimeric amino acid sequences such as c-fos or c-jun, or monomeric amino acid sequences such as albumin. In some embodiments, the HLA fusion (e.g., an HLA-immunoglobulin fusion) offers further advantages in terms of stability, TCR binding affinity, and / or efficacy against T cell activation or inhibition.

[0037] In various embodiments, the aAPC comprises an HLA class I ligand for the presentation of a peptide antigen to CD8+ T cells (e.g., activation and / or proliferation of CD8+ cells, or inhibition of CD8+ cells). In some embodiments, the HLA class I ligand comprises at least two fusion proteins. The first fusion protein comprises a first HLA class I α chain and a first immunoglobulin heavy chain, and the 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 groove and a second HLA class I peptide binding groove.

[0038] Alternatively, aAPC contains an HLA class II ligand for the presentation of a peptide antigen to CD4+ T cells (e.g., activation and / or proliferation of CD4+ cells, or inhibition of CD4+ cells). In some embodiments, the HLA class II molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise (i) an immunoglobulin heavy chain and (ii) the extracellular domain of an HLA class II β chain. Two second fusion proteins comprise (i) an immunoglobulin light chain and (ii) the extracellular domain of an HLA class II α chain. The two first and two second fusion proteins associate to form an HLA class II molecular complex. The extracellular domains of the HLA class II β chains of each of the first fusion proteins and the extracellular domains of the HLA class II α chains of each of the second fusion proteins form an HLA class II peptide bond groove.

[0039] 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 facilitate dimerization. In some embodiments, the HLA-Ig fusion construct does not include a variable region sequence. For example, the extracellular domain sequence of the HLA (i.e., the extracellular domain of the HLA class Iα chain) may be fused to the Ig constant region sequence above the hinge region to provide a dimeric HLA. For example, the HLA or its antigen-presenting portion may be bound to the CH1 portion of each IgG heavy chain. All IgG molecules consist of two identical heavy chains (constant region and variable region) linked by a disulfide bond in the hinge region (upper and lower). For example, in some embodiments, the HLA molecule or antigen-presenting complex is fused to CH1 (the N-terminus of the Ig heavy chain above the hinge region), thereby creating a dimeric fusion protein that is smaller than that created by fusion to the end of a complete antibody heavy chain due to the absence of VH and VL light chain sequences. Therefore, such constructs further include CH2 and CH3 domains. Such constructs not only offer manufacturing advantages but also exhibit lower immunogenicity potential. In some embodiments, these constructs also exhibit sufficient binding cooperativity for efficient T cell activation or inhibition. In some embodiments, the IgG sequence is an IgG4 sequence, and the immunoglobulin sequence may include or consist of (or essentially consist of) the amino acid sequence of SEQ ID NO: 23.

[0040] In some embodiments, the HLA ligand is an HLA class I ligand and is optionally an HLA-A, HLA-B, HLA-C, or HLA-E ligand. In embodiments, the HLA ligand contains the associated β2 microglobulin (β2M) polypeptide. In various embodiments, the HLA ligand (e.g., presented by HLA-Ig) corresponds to an allele 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*07:02. In some embodiments, the HLA ligand is modified with cysteine ​​that forms a disulfide bond across the α-helix constituting the peptide binding groove to enhance the stability of the complex with the bound peptide. In some embodiments, such cysteine ​​is substituted at positions 84 and 139 of the extracellular domain. These modifications crosslink the F-pocket to which the peptide's C-terminus binds.

[0041] In some embodiments, the HLA ligand is the HLA-A*02:01 ligand (IMGT acceptance number HLA00005). In some embodiments, the HLA ligand is modified with cysteine ​​that forms a disulfide bond across the α-helix constituting the peptide bond groove to enhance the stability of the complex with the bound peptide. For example, the HLA-A*02:01 ligand may be modified with cysteine ​​at positions 84 and 139, as shown in SEQ ID NO: 26, to form a disulfide bond across the α-helix constituting the peptide bond groove. Consequently, in some embodiments, the HLA-Ig ligand has 1 to 5 amino acid modifications selected from substitutions, deletions, and insertions, provided that it contains the sequence of SEQ ID NO: 26 and optionally, positions 84 and 139 in relation to SEQ ID NO: 26 are cysteine.

[0042] In other exemplary embodiments, HLA class I (e.g., HLA-A*02) has W51C and G175C substitutions (e.g., with respect to SEQ ID NO: 26) that form a disulfide bond at the end of the peptide groove to which the N-terminus of the peptide is bound. In other exemplary embodiments, HLA class I (e.g., HLA-A*02) has F22C and S71C substitutions (e.g., with respect to SEQ ID NO: 26) that form a disulfide bond within one of the α-helice adjacent to the peptide bond groove.

[0043] In various embodiments, recombinant HLA class I ligands associate with β2-microglobulin protein. The amino acid sequence of β2-microglobulin is provided herein as Sequence ID No. 8. In various embodiments, derivatives of β2-microglobulin may employ, for example, amino acid modifications independently selected from 1 to 10 or 1 to 5 substitutions, deletions, and insertions.

[0044] 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 to the antibody heavy and light chains, as already described, thereby forming a dimeric HLA class II antigen-presenting complex.

[0045] In some embodiments, aAPC includes 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. Due to its low polymorphism, the HLA-E ligand may be adaptable to create a nearly universal aAPC platform. However, HLA-E plays a dual role in both the innate and adaptive immune systems. In the innate immune response, HLA-E's role is to present peptides of other HLA class I molecules and inhibit natural killer (NK) cell-mediated lysis via recognition by NKG2A / CD94. NK cells sense the presence of the HLA-E-presented self-peptide and thereby receive an inhibitory signal via the NKG2A / CD94 complex (inhibiting NK-mediated lysis). HLA-E can also conjugate and present peptide sequences for recognition by T cells (e.g., CD8+ T cells) (adaptive immune response). In particular, HLA-E molecules bind to NKG2A / CD94 via a binding surface that overlaps with the binding surface for interacting with the T cell receptor ("TCR").

[0046] In various embodiments, the NK cell inactivation function of HLA-E is decoupled from its T cell activation function by introducing point mutations that affect only the binding of HLA-E to NKG2A / CD94, rather than its binding to the TCR. These point mutations allow for 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 embodiments, HLA-E amino acid substitutions are performed to provide a stable peptide binding groove for the presentation of binding antigens to HLA-E-restricted T cells.

[0047] In some embodiments, the recombinant HLA-E ligand has at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21 (HLA-E extracellular domain) and includes an amino acid sequence having cysteine ​​substitutions at amino acids corresponding to Y84 and A139 of SEQ ID NO: 21. These substitutions enable the formation of disulfide bonds that stabilize the peptide bond groove. In some embodiments, the amino acid sequence has at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21.

[0048] In some embodiments, the amino acid sequence has one or more amino acid modifications to SEQ ID NO: 21 that reduce or eliminate interaction with NKG2A / CD94. In some embodiments, the amino acid modifier is selected from substitutions at D162 and E166 with respect 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 includes substitutions D162A and E166A with respect to SEQ ID NO: 21. In some embodiments, the HLA-E ligand includes substitutions for Cys in Y84 and A139, as well as substitutions for D162 and E166.

[0049] In some embodiments, the engineered HLA-E polypeptide ligand is bound to nanoparticles having a co-stimulatory ligand to activate antigen-specific HLA-E restriction T cells in response to one or more tumor antigens or infectious antigens. In other embodiments, the engineered HLA-E polypeptide ligand is bound to nanoparticles having a co-inhibitory ligand to inhibit antigen-specific HLA-E restriction T cells in response to one or more autoantigens.

[0050] In various embodiments, the immunoglobulin heavy chain sequence fused to 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 IgG4Fc sequence. In some embodiments, the IgG4Fc domain includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgG4Fc domain includes an amino acid sequence having at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

[0051] In some embodiments, recombinant HLA ligands include a linker between the HLA amino acid sequence and the immunoglobulin sequence (e.g., IgG4Fc 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 includes the amino acid sequence of SEQ ID NO: 24. Alternatively, the linker may be selected from flexible peptide linkers and rigid peptide linkers. Flexible linkers are composed primarily of or entirely of small and / or polar residues such as Gly, Ser, and Thr. An exemplary flexible linker is (Gly x Ser) nLinkers are included, where x is 1 to 10 (e.g., 2 to 6) and n is 1 to about 10, and in some embodiments, 2 to about 6. In exemplary embodiments, x is 2 to 4 and n is 2 to 4. Due to their flexibility, these linkers are substantially unstructured. More rigid linkers include polyproline or polyPro-Ala motifs and α-helical linkers. Generally, linkers of varying stiffness can be composed 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.

[0052] Peptide antigens bind to the antigen-binding groove of the antigen-presenting complex. Optionally, the antigen peptide can be covalently bound to the peptide-binding groove. If desired, a peptide linking chain can be used to link the antigen peptide to the peptide-binding groove. For example, crystallographic analysis of several class I MHC molecules has shown that the amino terminus of β2M is very close, approximately 20.5 angstroms, to the carboxyl terminus of the antigenic peptide present in the MHC peptide-binding groove. Therefore, a peptide can be linked to the amino terminus of β2M using a relatively short linker sequence of approximately 13 amino acids. If the sequence is appropriate, the peptide will bind to the MHC-binding groove. Peptide antigens for immunotherapy of cancerous diseases, infectious diseases, and autoimmune diseases are described herein.

[0053] In some embodiments, the peptide antigen does not induce aggregation of aAPC. That is, the peptide antigen is selected to have a low tendency to aggregate when loaded onto aAPC. In some embodiments, peptides with a low tendency to aggregate are selected using an aggregation score (APS), which can be evaluated as described elsewhere in this specification. For example, a peptide that has a low aggregation ability when loaded onto an HLA ligand (e.g., an HLA-A ligand) and has been modeled using a computer exhibits an average APS of less than approximately 0.07 for peptide residues 1 and 3-5. 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 certain embodiments, the peptide antigen does not have more than two, three, or four hydrophobic residues, such as those selected from Phe, Val, Leu, and Ile. Furthermore, the tendency to aggregate can be limited by limiting the density of HLA ligands (as already mentioned). In some embodiments, the HLA ligand is HLA-A (e.g., HLA-A*02:01), and positions 1 and 3-5 include at least one or at least two residues independently selected from Gly and charged residues (Glu, Asp, Lys, Arg, and His), and do not contain two or more exposed hydrophobic residues (e.g., Phe, Val, Leu, and Ile).

[0054] In some embodiments, the polypeptide ligand includes a signal-2 ligand, for example, a co-stimulatory ligand for activation and / or proliferation of target T cells. Exemplary co-stimulatory ligands include agonists for any one of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT. The co-stimulatory ligand can induce activation and / or proliferation of CTLs or Tregs in various embodiments. In other embodiments, and particularly when aAPC is intended to inhibit target T cells, the polypeptide ligand may not include a signal-2 ligand, or the polypeptide ligand may include a co-inhibitory ligand to induce resistance or apoptosis of target T cells. In various embodiments, the co-inhibitory ligand is an agonist for Fas, TGF-β, or PD-1. The agonist ligand may include a native 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 its immunoglobulin fusion) or FasL (or its immunoglobulin fusion). Exemplary PD-L1-Ig and Fc-FasL fusion protein ligands are described herein (SEQ ID NO: 1 and SEQ ID NO: 4, respectively). In certain embodiments, the immunoglobulin fusion sequence is IgG4 and its variants as described herein, and may include 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.

[0055] In various embodiments, the signal 1 and signal 2 ligands can be combined in a homodimer or heterodimer construct (e.g., a homodimer or heterodimer Ig fusion construct). For example, the HLA ligand may include a signal 2-immunoglobulin (Ig) fusion (i.e., a heterodimer Ig fusion construct) and the fusion of the HLA extracellular domain to an immunoglobulin Fc region, such as an IgG4Fc region, which can be dimerized (e.g., via a disulfide bond). In yet another embodiment, the HLA ligand includes a fusion with the signal 2 ligand. For example, the HLA extracellular domain can be fused at its C-terminus to an immunoglobulin Fc region (e.g., IgG4Fc as already described) and at its N-terminus to the signal 2 ligand, preparing a homodimer ligand in which both signals are dimerized. In some embodiments, the signal 2 ligand includes a single-chain antibody (e.g., scFv) or the activating portion of a native ligand. See SEQ ID NOs: 6 and 7, which contain an anti-CD28 scFv fused to the N-terminus of HLA-Ig. SEQ ID NO: 6 employs a VH-linker-VL oriented scFv, and SEQ ID NO: 7 employs a VL-linker-VH oriented scFv. See also SEQ ID NO: 5, which shows the fusion of a PD-L1 activating fragment to the N-terminus of HLA-A-IgG4.

[0056] In some embodiments, the co-stimulatory ligand is an activating antibody against CD28, which is 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), or 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 mouse 9.3 mAb (Tan et al. J. Exp. Med. 1993 177:165). In some embodiments, the antibody has a set of 9.3 mAb heavy-chain CDRs and / or a set of light-chain CDRs. For example, in some embodiments, the heavy chain variable region includes one, two, or three of the following CDRs, each optionally modified by one, two, or three amino acid substitutions: 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 includes one, two, or three of the following CDRs, each possibly modified by one, two, or three amino acid substitutions: CDR1(RASESVEYYVTSLMQ, SEQ ID NO: 12), CDR2(AASNVES, SEQ ID NO: 13), and CDR3(QQSRKVPYT, SEQ ID NO: 14). Exemplary heavy chain variable amino acid sequences and light chain amino acid sequences of humanized anti-CD28 agonist antibodies are provided herein as SEQ ID NOs: 15-20.

[0057] In some embodiments, the anti-CD28 antibody (or a portion thereof) binds to an epitope identical or overlapping with that of 9.3 mAb, or to an epitope identical or overlapping with that of the antibody having CDR1, CDR2, and CDR3 of 9.3 mAb. Antibodies having identical or overlapping epitopes can be selected by any suitable technique, including, for example, competitive immunoassays using surface plasmon resonance (Biacore).

[0058] Alternative CDR sequences, variable regions, or CD28-binding ligands may be employed in various embodiments. Alternative ligands, CD28 epitopes, and anti-CD28 antibodies are described, for example, in U.S. Patents 7,612,170, 6,987,171, and 6,887,466, the disclosures of which are incorporated herein by reference in their entirety.

[0059] In some embodiments, the co-inhibitory ligand is an agonist antibody against Fas. In some embodiments, the agonist antibody against Fas is an IgG4 antibody based on clone CH11. Sequence IDs 2 and 3 exemplify the heavy chain variable region and light chain sequence of an IgG4 anti-Fas agonist antibody. As demonstrated herein, this anti-Fas antibody is active when it can crosslink multiple Fas receptors, such as when the antibody is bound to nanoparticles. In some embodiments, the heavy and light chain sequences of Sequence IDs 2 and 3 are humanized and include the CDRs of Sequence IDs 2 and 3 shown in Table 1 below: [Table 1]

[0060] The antibody polypeptide ligands according to this disclosure include a constant region, which 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 (e.g., S241P) that may be introduced into the CH chain. In some embodiments, the antibody ligand includes a constant region, which includes one or more mutations suitable for chemically coupling the antibody to a solid support. One or more mutations suitable for coupling produce unpaired cysteine. An exemplary mutation in the constant region of IgG4 is S473C. Other mutations to the constant region include modifications to reduce Fc gamma receptor binding. For example, the CH chain may be modified with L248, e.g., L248E.

[0061] In some embodiments, the antibody-based polypeptide ligand may be minimized to better suit the ligand's functional attachment to the nanoparticles. For example, the antibody may be an antibody fragment such as F(ab')2 or Fab, or a single-chain antibody (scFv), or another antigen-binding antibody fragment. For example, the antibody fragment may be the scFv of the humanized mAb described herein, or another operative anti-CD28 antibody.

[0062] In some embodiments, the antibody (signal 2 ligand) is an scFv containing, or essentially 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 contain one or more (two, three, four, or five) VH and VL hypervariable region chains (each chain portion forming a three-dimensional antigen epitope-binding pocket) linked in a head-to-head or head-to-tail configuration by a short peptide linker. In some embodiments, these constructs are fused to an HLA-Ig sequence as described to produce a homodimer construct.

[0063] In some embodiments, other ligand-binding forms, including peptides, aptamers, and AdNectins, are used to produce co-stimulatory or inhibitory ligands. Various forms for binding to targets include single-domain antibodies, recombinant heavy chain-only antibodies (VHH), single-chain antibodies (scFv), shark heavy chain-only antibodies (VNAR), microproteins (cysteine ​​node protein, Notchin), DARPin, Tetranectin, afibody, Transbody, antikalin, affin, Microbody, peptide aptamers, Phylomer, Stradobody, Maxibody, Evibody, Fynomer, armadillo repeat protein, Kunitz domain, avimer, Atrimer, Probody, Immunobody, Triomab, Troybody, Pepbody, UniBody, DuoBody, Fv, Fab, Fab', F(ab')2, peptide mimetic molecules, or combinations. Component, or U.S. Patent No. or Publication No., U.S. Patent No. 7,417,130, U.S. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 6,004,746, U.S. Patent No. 5 Examples include those listed in US Nos. 475,096, US2004 / 146938, US2004 / 157209, US Nos. 6,994,982, 6,794,144, US2010 / 239633, US Nos. 7,803,907, US2010 / 119446, and / or US Nos. 7,166,697, the contents of which are incorporated herein by reference in their entirety. See also Storz MAbs. 2011 May-June;3(3):310-317.

[0064] In some embodiments, aAPC further comprises one or more cytokines that assist in the activation and / or proliferation of T cells, or in the inhibition of T cells. One or more cytokines or functional moieties may be bound to aAPC as polypeptide ligands. Alternatively, cytokines or functional moieties may be fused to signal 1 or signal 2 polypeptide ligands (which may optionally be presented in homodimeric or heterodimeric Ig fusion constructs as described herein). In some embodiments, cytokines are encapsulated by copolymers and locally released in a target environment (e.g., lymphoid organs, tumors, or target tissues or organs). Examples of cytokines that may be used include IL-1β, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and γ interferon. For example, IL-2 may be employed with a co-stimulatory signal 2 ligand. In some embodiments, aAPC comprises immunotolerogenic cytokines as polypeptide ligands. An exemplary immunotolerogenic cytokine is IL-10.

[0065] In some embodiments, the ligand further comprises one or more homing ligands for lymphoid organs. For example, an exemplary homing ligand is CD62L. In some embodiments, ligands are included to target aAPC to a tissue or organ of interest, such as the pancreas, intestines, lungs, liver, muscles, or skin (these may or may not be polypeptide ligands). Suitable peptide ligands or other ligands may be selected based on information in the Art.

[0066] In various embodiments, one or more peptide antigens are tumor or cancer-associated antigens, such as tumor-derived antigens, tumor-specific antigens, and novel antigens. Tumor-associated antigen-specific T cells are very rare in the peripheral blood of healthy individuals and are often undetectable. Furthermore, these cells often exhibit an unsensitized 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 a distinction observed between virus-specific T cells and tumor antigen-specific T cells. According to these embodiments, the injectable aAPCs of this disclosure can activate and / or proliferate such T cells in vivo, thereby inducing an antitumor immune response.

[0067] "Tumor-associated antigens" or "cancer-specific antigens" include unique tumor antigens or cancer antigens expressed only by tumors or malignant cells from which they originate, co-tumor antigens (tumor-fetal antigens) expressed in many tumors but not in normal adult tissues, and tissue-specific antigens expressed in normal tissues in which tumors have developed. Tumor-associated antigens may include, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressors, fusion proteins, or oncoviral proteins.

[0068] Various tumor-associated antigens are known in this field. Tumor-fetal antigens and embryonic antigens include carcinoembryonic antigen and α-fetal protein (usually highly expressed only in developing embryos, but often highly expressed in liver and colon tumors, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-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 carcinomas), pancreatic tumor-fetal antigen, 5T4 (expressed in gastric cancer), α-fetal protein receptor (expressed in multiple tumor types, especially breast tumors), and M2A (expressed in germ cell neoplasms).

[0069] Mutant oncogenes or tumor suppressor gene products include Ras and p53, which are expressed in many tumor types, Her-2 / neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene product, and myc (associated with lung cancer). Fusion proteins include BCR-ABL, which is expressed in chromogenic myeloid leukemia. Oncoviral proteins include HPV types 16, E6, and E7, which are found in cervical cancer.

[0070] Tissue-specific antigens include melanotransferrin and MUC1 (expressed in pancreatic and breast cancer); CD10 (formerly known as common acute lymphoblastic leukemia antigen (CALLA)) or surface immunoglobulin (expressed in B-cell leukemia and lymphoma); IL-2 receptor α chain, T cell receptor, CD45R, CD4+ / CD8+ (expressed in T-cell leukemia and lymphoma); prostate-specific antigen and prostatic acid phosphatase (expressed in prostate cancer); GP100, MelanA / Mart-1, tyrosinase, gp75 / brown, BAGE, and S-100 (expressed in melanoma); cytokeratin (expressed in various carcinomas); and CD19, CD20, and CD37 (expressed in lymphoma).

[0071] Tumor-associated antigens include modified glycolipid and glycoprotein antigens such as neuraminic acid-containing sphingoglycolipids (e.g., GM2 and GD2 (expressed in melanoma and some brain tumors); blood group antigens, particularly T antigen and sialylated Tn antigen (which may be abnormally expressed in carcinomas); and mucins (e.g., CA-125 and CA-19-9 (expressed in ovarian cancer) or less glycosylated MUC-1 (expressed in breast and pancreatic cancer)).

[0072] Tumor-associated antigens are further disclosed in U.S. Patent 11,007,222, which is incorporated herein by reference.

[0073] In some embodiments, the target peptide antigen comprises at least one that is 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., A, B, C, or D), cytomegalovirus (CMV), Epstein-Barr virus (EBV), HPV, influenza, herpesvirus (e.g., HSV1 or 2, or varicella-zoster), and adenovirus. For example, CMV is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation.

[0074] In some embodiments, one or more target peptide antigens are “autoantigens,” meaning those associated with an autoimmune disease or reaction. In some embodiments, aAPCs with immunotolerogenic ligands induce resistance of antigen-specific T cells to the target antigen. In some embodiments, aAPCs with apoptotic signals (e.g., Fas ligand or agonist anti-Fas antibody) induce specific apoptosis of antigen-specific T cells. Autoantigens may be involved in autoimmune diseases such as type 1 diabetes, Goodpasture syndrome, multiple sclerosis, Graves' disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, Crohn's disease, and Hashimoto's thyroiditis, vitiligo, etc.

[0075] In some embodiments, peptide antigens for presentation by HLA ligands are determined in an individualized manner, as described in US10,098,939 and US2020 / 0291381, and these are incorporated herein by reference in their entirety. For example, sequencing data can provide information about both shared and individualized targets in immunotherapies such as cancer. In principle, mutant proteins are foreign to the immune system and are presumed to be tumor-specific antigens. Indeed, sequencing efforts have defined hundreds, if not thousands, of potential relevant immune targets. Studies have shown that T cell responses to these novel epitopes are induced in cancer patients or by cancer vaccines. A mutation catalog obtained from whole-exome sequencing provides a starting point for identifying such novel epitopes. Using the HLA binding prediction algorithm (Srivastava, PLoS One 4, e6094 (2009)), it is predicted that each cancer has up to 7–10 novel epitopes. Using a similar approach, hundreds of new tumor epitopes were estimated. New epitopes predicted from DNA or RNA sequencing of a patient's tumor can be tested for activation potential in association with HLA ligands by determining whether (or to what extent) aaAPCs with the predicted antigen associated with the HLA ligand can activate the target T cells. Similar analyses can be used to identify relevant autoantigens.

[0076] In some embodiments, peptide antigens for the treatment of type 1 diabetes may be antigens 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: 379-392.

[0077] Non-specific examples of peptide antigens are shown in Table 2 below. [Table 2]

[0078] In various embodiments, aAPC is contained in a pharmaceutical composition suitable for administration to a target. 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 assist in 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 freeze-drying to increase volume. Hydrophilic polymers such as dextran, hydroxyethyl starch, polyethylene glycol, and gelatin may be used to stabilize proteins. Polymers with non-polar moieties, such as polyethylene glycol, may also be used as surfactants. Examples of protein stabilizers include polyols, sugars, amino acids, amines, and salts. Preferred sugars include sucrose and trehalose. Examples of 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 to the surface, inhibiting the aggregation of active agents. Certain formulation components can serve multiple purposes. For example, histidine can act as a buffer and antioxidant. Glycine can be used as a buffer and a bulking agent. In some embodiments, the pharmaceutical composition is freeze-dried.

[0079] In some aspects and embodiments, the Disclosure provides aAPCs suitable for parenteral administration (including subcutaneous administration) and with a low tendency to aggregate. In these aspects, the aAPC comprises polymer nanoparticles or lipid nanoparticles comprising a polyethylene glycol (PEG) sheath and one or more polypeptide ligands bonded to PEG (e.g., PEG-terminus) via thioether links or other functional groups. The polypeptide ligands include HLA class I or class II ligands that present peptide antigens and optionally one or more signal 2 ligands (as previously described). Peptide antigens for presentation to T cells do not induce aggregation of aAPCs. Certain peptides, as disclosed herein, induce aggregation when loaded onto aAPCs due to the properties of their exposed surface. Analyzing the peptide sequence alone cannot clearly demonstrate the aggregation ability in relation to HLA. Aggregation ability occurs at the level of peptide-supported nanoparticles, as opposed to protein complexes in solution alone.

[0080] In some embodiments, the peptide antigen has no exposed cysteine ​​and / or has one or more exposed glycine residues or exposed charged residues. In some embodiments, the peptide antigen has no cysteine ​​residues and contains one or more charged residues (e.g., one, two, or three charged residues). In various embodiments, the peptide antigen in the HLA binding groove has one or more exposed amino acids selected from glycine, aspartic acid, glutamic acid, lysine, arginine, and histidine; and also has no exposed cysteine. In some embodiments, positions 1 and 3-5 of the peptide satisfy these criteria. In this context, the term “exposed” means that the side chains of the amino acids are exposed to the surface. In some embodiments, the HLA is HLA-A, and in some embodiments, it is HLA-A2 such as HLA-A*02:01.

[0081] In some embodiments, aggregation ability is estimated using a computer by determining the average aggregation score (APS) of antigen peptide residues within the HLA antigen-binding groove. In some embodiments, for example with respect to HLA-A (e.g., HLA-A2 such as HLA-A*02:01), positions 1 and 3-5 of the peptide are evaluated for APS, and average scores are determined for these positions. For example, a score of 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 Systemes BIOVIA, Discovery Studio Modeling Environment, Release 2021, San Diego, CA). For example, the MODELLER program or a similar program 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 according to available tools, which are included in the DS2021 package or otherwise commercially available.

[0082] The spatial aggregation tendency (SAP) score is synthesized by combining the solvent-penetrating region of the residue side chain with a hydrophobic score based on a scale developed by Black and Mould (1991) (where the value for glycine is zero, and other residues are estimated as positive if highly hydrophobic and negative if less hydrophobic). By combining this score with the characteristics of specific peptides already described, it is possible to identify peptides that, when complexed with HLA ligands, are likely to promote the aggregation of proteins, and consequently, HLA-decorated nanoparticles.

[0083] The use of computer models of aggregation tendencies (and in conjunction with experimentally determined structures) is a known methodology. However, these tools have traditionally focused on antibody screening to find antibodies with superior drug-like properties or to engineer biophysical susceptibility. While comparative modeling and APS tools are commonly used, their application in terms of peptide-HLA complexes, particularly nanoparticle coating and the aggregation of these particles, has neither been described nor suggested. Furthermore, the significant influence of peptides within the HLA-binding pocket on particle aggregation was surprising, given that peptides occupy a relatively small portion of the aAPC structure and overall exposed surface.

[0084] In some embodiments, this process is initiated by generating a molecular model of a candidate related peptide-HLA complex. The protein model includes the related peptide placed 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 in the modeling stage, and manual sequence alignment is performed for each model. PDB acceptances of template structures used to model HLA-A*02:01 include 4L29, 1I7R, 5EUO, 1TVB, 6OPD, 6TRO, 6AMT, 6AM5, 2GT9, and 3OXS.

[0085] For example, a static model can be used to calculate the aggregation score. In some embodiments, a radius of 5 or 10 angstroms is selected for the score calculation. This parameter determines how many neighboring amino acid residues are included in the aggregation ability calculation. This is similar to the draw window in protein sequence parameter calculation. Depending on the particle size of the model being analyzed, the above radius can provide a well-smoothed surface map that is not excessively noisy. This protocol calculates the exposed surface of each residue and calculates the aggregation ability score based on the average of each residue and the 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 based on the solvent-penetrating surface of the model. This, coupled with the scored aggregation ability, provides a simple visualization of the surface shape. Sometimes, a manual examination of these results is sufficient to classify peptides as aggregates or non-aggregates.

[0086] According to several embodiments, the APS of residues 1 and 3-5 of the HLA-A peptide complex is averaged. If this score exceeds the current threshold of 0.07, it is considered a factor in aggregation. The presence of exposed Cys residues (determined by examination of the model structure) is considered another factor in aggregation. The presence of charged residues and Gly residues is considered a factor that counteracts aggregation. The total number of factors favoring or unfavoring aggregation can be used to determine whether the peptide-HLA complex is considered a potential aggregation factor.

[0087] In some embodiments, the nanoparticles are polymer nanoparticles comprising a polylactic acid-polyethylene glycol (PLA-PEG) or polylactic acid-co-glycolic acid (PLGA-PEG) copolymer and one or more polypeptide ligands bonded to PEG via thioether links. Such polymer nanoparticles can be as described above.

[0088] Alternative polymers that can be used in connection with the aAPC platform described herein include cyclodextrin-containing polymers, cationic cyclodextrin-containing polymers, poly(D,L-lactic acid-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 acid-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,Polyalkylenes such as L-lactide, polyalkylcyanoacrylic acid, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, poly(orthoester), poly(esteramide), polyamide, poly(ester ether), polycarbonate, polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl esters such as poly(vinyl acetate), polyhalogenated vinyls such as polyvinyl chloride (PVC), polyvinylpyrrolidone, polysiloxane, polystyrene (PS), polyurethane, alkylcellulose, hydroxyalkylcellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose Polymers of acrylic acid such as poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyacrylic acid, and copolymers thereof, as well as polymers of acrylic acid. Examples include and mixtures, polydioxanone and its copolymers, polyhydroxyalkyl carboxylic acids, poly(propylene fumarate), polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes, and polyphosphate esters, dendrimers and their derivatives, as well as mixtures and / or block copolymers of two or more such polymers.

[0089] In other embodiments, the nanoparticles are lipid nanoparticles containing PEG-conjugated lipids. Exemplary PEG lipids are selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The 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.

[0090] In some embodiments, 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 cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, 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 moiety bonded to the sn-2 carbon of the glycerol backbone), mixed acylglycerophospholipids, and symmetric acylglycerophospholipids. Examples of acylglycerophospholipid head groups include phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositide, and phosphatidylserine.Exemplary phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-diundecano Il-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 Selected from PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin.

[0091] In some embodiments, the nanoparticles are lipid nanoparticles further comprising polynucleotides for expression in target T cells. In some embodiments, the polynucleotide (e.g., mRNA) encodes a cytokine, including a cytokine described herein (e.g., IL-2 or IL-10).

[0092] In some embodiments, sulfhydryl groups on polypeptide ligands are bonded to PEG-maleimide functional groups of lipid nanoparticles. In various embodiments, lipid nanoparticles aAPC have about 10 to about 500 polypeptide ligands, or about 50 to about 400 polypeptide ligands, or about 100 to about 300 polypeptide ligands. Polypeptide ligand density can generally be controlled, as already described, by reducing the proportion of PEG moieties having maleimide groups for conjugation, or by changing the level of PEG lipids in the composition.

[0093] Lipid nanoparticles aAPC have diameters of approximately 50 nm to 150 nm, or approximately 60 nm to 130 nm, or approximately 80 nm to 120 nm. In various embodiments, aAPC has diameters of approximately 80 nm, 90 nm, 100 nm, 110 nm, or 120 nm. Lipid nanoparticles aAPC have surface charges of approximately 0 to -15 mV, or approximately 0 to -10 mV, or approximately -2.5 mV to -10 mV. The aAPC population has a particle size distribution with a polydispersity index (PDI) of less than 0.2.

[0094] In various embodiments, aAPC may be contained in a pharmaceutical composition as described herein and may be optionally lyophilized. In various embodiments, the aAPC and pharmaceutical compositions described herein may be useful for treating patients with infectious diseases, cancer, or autoimmune diseases, or for providing prophylactic protection to immunocompromised patients.

[0095] In another embodiment, the present invention provides a method for immunotherapy. This method comprises administering aAPC or a pharmaceutical composition thereof described herein to a subject in need of treatment. In this embodiment, aAPC and its compositions described herein are used for immunotherapy.

[0096] In various embodiments, the subject has cancer or an infection, and aAPC contains a costimulatory ligand. Treatable infections include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc. Such diseases include human papillomavirus (HPV) (and associated 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 major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplants. This is because these patients are immunocompromised, allowing latent viruses to reactivate in seropositive patients or opportunistic infections to occur in seronegative patients. Current treatments center on the use of antiviral agents such as ganciclovir, but this has drawbacks, the most significant of which is the development of drug-resistant CMV.

[0097] Post-transplant lung disease (PTLD) occurs in a significant proportion of transplant patients and is caused by Epstein-Barr virus (EBV) infection. EBV infection is thought to be present in approximately 90% of the adult population in the United States. While active viral replication and infection are suppressed by the immune system, as with CMV, in immunocompromised patients due to transplant therapy, the T cell population that enables viral reactivation becomes uncontrolled. This poses a significant obstacle to transplant protocols. EBV may also be involved in tumorigenesis of various hematological and non-hematological cancers. There is also a strong association between EBV and nasopharyngeal carcinoma. Therefore, prophylactic treatment with EBV-specific T cells is a superior alternative to current treatments.

[0098] Cancers that can be treated in accordance with this disclosure include melanoma, carcinoma (e.g., of the colon), head and neck cancer, duodenal cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, ductal cancer, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, gastric cancer, dysplastic oral mucosal cancer, polyposis cancer, invasive oral cancer, non-small cell lung cancer, transitional and squamous cell urinary tract cancer, etc.; neurological malignancies, e.g., neuroblastoma, glioma, etc.; hematological malignancies, e.g., chronic myeloid leukemia, childhood acute leukemia, non-Hodgkin lymphoma, chronic lymphocytic leukemia, malignant cutaneous T-cell lymphoma, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell-rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc. 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, stage II, stage III, or stage IV cancer. In some embodiments, the cancer is metastatic and / or recurrent and / or unresectable. In some embodiments, the patient is refractory to or partially responds to chemotherapy and / or immune checkpoint inhibitor therapy.

[0099] In some embodiments, the present invention provides a method for treating cancers, including those identified above, by administering a pharmaceutical composition described herein to activate T cells having antitumor activity. In some embodiments, the therapy is provided with one or more immune checkpoint inhibitors, such as nivolumab, pembrolizumab, and ipilimumab. In some embodiments, the additional therapy is anti-CTLA4 or anti-PD1, or anti-PD-L1. The additional therapy or checkpoint inhibitor may be administered separately through its conventional regimen, or as an additional ligand to the nanoparticles described herein, or attached to a separate population of nanoparticles. In some embodiments, one or more immune checkpoint inhibitors are provided as initial therapy, and therapy with aAPC described herein is subsequently initiated, for example, about 1 to 8 weeks after checkpoint inhibitor therapy, or about 2 to 4 weeks after checkpoint inhibitor therapy. In some embodiments, one or more checkpoint inhibitors are provided, for example, at the start of treatment and approximately every two weeks, or at the start of treatment and approximately every two weeks for one or more checkpoint inhibitors, and approximately every four weeks for nanoparticle therapy, concurrently with 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 yet 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.

[0100] In some embodiments, peptide antigens are selected on a patient-individualized basis based on an analysis of the patient's tumor. For example, a process described by Ionov Y., A high throughput method for identifying personalized tumor-associated antigens, Oncotarget 1(2):148-155 (2010) (which is incorporated herein by reference), or other processes may be used. In these embodiments, nanoparticles may be provided (on a “off-the-shelf” basis), and tumor antigens may be selected and loaded on an individualized basis. Other processes for selecting peptides based on their ability to activate and proliferate associated T cells (including, in some embodiments, individualized methods) are described in U.S. Patents 10,987,412 and 10,098,939, which are incorporated herein by reference in their entirety.

[0101] In some embodiments, nano-aAPCs are used as booster immunization vaccines after adoptive T-cell therapy, in which patient-derived unsensitized T cells or HLA-matched donor-derived T cells are proliferated in exovivo and administered to the patient. In these embodiments, the nano-aAPC composition may be administered one to about ten times over a period of four months to about one year to enhance cancer immunity.

[0102] In some embodiments, the subject has an autoimmune disease, and aAPC contains a co-inhibitory signal or does not contain a signal 2 ligand. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the autoimmune disease is vitiligo. Treatable autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, Sjögren's disease, Hashimoto's thyroiditis, alopecia, ankylosing spondylitis, scleroderma, and HTLV-1-associated myelopathy (HAM) / tropical spastic paraplegia (TSP).

[0103] Generally, aAPC or its pharmaceutical composition is administered parenterally. For example, aAPC or its pharmaceutical composition may be administered intravenously, intra-arterially, subcutaneously, intradermally, lymphatically, intramuscularly, or intratumorally. In some embodiments, the aAPC composition is administered subcutaneously.

[0104] Other aspects of this disclosure disclose polypeptide ligands for immunotherapy (including polypeptide ligands for aAPC).

[0105] Such polypeptide ligands include an anti-Fas-agonist antibody having an IgG isotype (e.g., IgG4), which can be conjugated to nanoparticles together with an HLA ligand that presents a peptide antigen (e.g., associated with autoimmune diseases). In some embodiments, the anti-Fas antibody comprises the heavy chain of SEQ ID NO: 2, having optionally 1 to 10 or 1 to 5 amino acid substitutions. The amino acid substitutions may optionally be located in the Fc domain and include those with known pharmacological or stability advantages, or those with the advantage of eliminating potential immunogenicity. In some embodiments, the amino acid sequence of SEQ ID NO: 2 is humanized and includes the same complementarity-determining region (CDR) as SEQ ID NO: 2 (see Table 1), having 1, 2, or 3 or fewer amino acid substitutions. In some embodiments, the anti-Fas antibody comprises the light chain of SEQ ID NO: 3 (see Table 1), having optionally 1 to 10 or 1 to 5 amino acid substitutions. In some embodiments, the amino acid sequence of SEQ ID NO: 3 is humanized and includes the same CDR as SEQ ID NO: 3, having 1, 2, or 3 or fewer amino acid substitutions.

[0106] In other embodiments, the polypeptide ligand is a dimeric PD-L1 ligand containing the activating moiety of PD-L1, such as the amino acid residues F19–T239 of human PD-L1. Alternatively, it may contain an additional 20 residues (collected at one or both ends), or up to 10 amino acids may be deleted from F19–T239. Each PD-L1 activating fragment may be fused directly or indirectly to an IgG Fc region (e.g., IgG4) via its C-terminal linker, and the ligand may be dimerized by a disulfide bond in the Fc region. Preferred linkers are described elsewhere herein and include flexible linkers such as the Gly Ser linker. The dimeric PD-L1 ligand can be conjugated to nanoparticles together with an HLA ligand that presents a peptide antigen and used to induce resistance to the antigen. An exemplary PD-L1-IgG4 is shown in SEQ ID NO: 1.

[0107] In other embodiments, the polypeptide ligand is a dimerized FasL ligand containing the active moiety of human FasL, such as amino acids P132-L279 of human FasL, which are fused directly or indirectly to a dimerized IgG-Fc region (e.g., IgG4) via its N-terminal linker. Alternatively, an additional 20 residues may be included (together at one or both ends), or up to 10 amino acids may be deleted from P132-L279. In some embodiments, the dimerized Fc region can be conjugated to nanoparticles via a Cys-containing linker. Exemplary constructs according to these embodiments are provided by SEQ ID NO: 4. The nanoparticles may further present an HLA-peptide antigen ligand to promote apoptosis of antigen-specific T cells.

[0108] In other embodiments, the polypeptide ligand is an immunotolerogenic ligand containing an activating fragment of human PD-L1, such as amino acids F19-T239 of human PD-L1, fused directly or via a linker to an HLA-immunoglobulin fusion protein. Alternatively, it may contain an additional 20 residues (collected at one or both ends), or up to 10 amino acids may be deleted from F19-T239 of human PD-L1. These embodiments provide constructs that are homodimers for both signal 1 and inhibitory signal 2 ligands. See SEQ ID NO: 5. Such ligands may be conjugated to nanoparticles as disclosed herein and used in immunotherapy (to induce resistance in targeted T cells).

[0109] In other embodiments, the polypeptide ligand is a costimulatory ligand containing an anti-CD28 operative scFv coupled to an HLA-immunoglobulin fusion protein, providing a homodimer ligand containing a signal 1 ligand and a costimulatory signal 2 ligand. See SEQ ID NOs: 6 and 7. The scFv may be fused to the HLA sequence via a heavy chain or light chain sequence. For example, SEQ ID NO: 6 employs a VH-linker-VL oriented scFv, and SEQ ID NO: 7 employs a VL-linker-VH oriented scFv.

[0110] The singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise.

[0111] As used herein and in the claims, “comprise(s),” “include(s),” “having(s),” “contain(s),” and their variations, the unrestricted transitional expressions, require the presence of named features / stages and permit the presence of other features / stages. These phrases should also be interpreted as disclosing selective expressions that permit only named features / stages and unavoidable impurities, and exclude other features / stages, such as “composed of” or “essentially composed of.”

[0112] In this specification, the term "approximately" means ±10% of a number unless otherwise specified in the context.

[0113] The term "identity" refers to the similarity between a pair of sequences (nucleotides or amino acids). Identity is measured by dividing the number of identical residues by the total number of residues, multiplying the product by 100, and calculating the percentage. Therefore, two copies of exactly the same sequence have 100% identity, but sequences that are not highly 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 are available to determine sequence identity, such as those employing algorithms like BLAST. BLAST nucleotide searches are performed by the NBLAST program, and BLAST protein searches are performed by the BLASTP program, using the initial settings parameters of each program.

[0114] The term CDR refers to the complementarity-determining region. CDRs are part of the variable chain of immunoglobulins (antibodies). A set of CDRs constitutes the antigen-binding site.

[0115] The present invention can be further described by the following non-limiting embodiments. [Examples]

[0116] Example 1: Construction of injectable manipulated antigen-presenting cells Ligands for aAPC, including signal 1 ligand (HLA) and signal 2 ligand, have a structure with free sulfhydryl groups for coupling with particles. See US10,632,193 (the whole is incorporated herein by reference). Naked polymer nanoparticles were prepared from PLGA-PEG or PLA-PEG copolymers by nanoprecipitation using known processes. The PLGA and PLA portions of the block copolymers were approximately 20 kDa. The PEG portions of the block copolymers were 3000 Da or 5000 Da. Specifically, PEG polymers with functional groups for ligand binding were designed to be long (5000 Da), and inert PEG polymers were designed to be short (3000 Da) to reduce steric effects. In the experiments shown below, NANOASSEMBLR IGNITE (Precision Nanosystems) was used for nanoprecipitation of particles. The nanoparticles consist of a group of copolymers having terminal maleimide functional groups (PEG-maleimide) for ligand binding. The thiolated ligands are bound to bare particles via terminal maleimide functional groups and purified by known techniques. See US10,632,193. The HLA ligands on the nanoparticles are immobilized by culturing with excess peptide antigen. The peptide-immobilized nanoparticles can be separated, for example, using an SEC column.

[0117] These processes can be used to obtain particles with particle size and surface charge suitable for parenteral administration for immunotherapy. Figure 1A shows TEM images of bare particles, protein-bound nanoparticles, and peptide-supported nanoparticles. As shown in Figure 1B, the average diameter of the particles is approximately 100 nm. Those with ligands and those with peptides are slightly larger. The polydispersity index was shown to be less than 0.2. The surface charge was shown to be in the range of 0 to -10 mV.

[0118] The target ligand density range was 100–400 ligands per particle. Experiments were conducted to vary the density of PEG-maleimide functional groups on the particle surface within a range (1%–10% by weight) that was deemed to yield stable particles. Specifically, particles were prepared together with PLGA-PEG, in which a portion of the PEG terminus contains maleimide functional groups. The maleimide:thiol (Mal:Thiol) ratio in the conjugation reaction was kept constant at 1:1. These data are summarized in Table 3 below. [Table 3]

[0119] In another experiment, the ligand density was controlled during the coupling reaction by changing the ratio (Mal:Thiol) of maleimide functional groups on the nanoparticles to thiol groups on the ligands. PEG-Mal was kept constant at 5%. These data are summarized in Table 4 below. [Table 4]

[0120] 5% PEG-maleimide polymer was selected as a good candidate for nanoparticle design in terms of stability, particle size, and ligand density. The data from Tables 3 and 4 are plotted in Figures 3A and 3B to show the trends in particle density and particle 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 5.67:1 (by weight) of PLA-mPEG:PLA-PEG-maleimide.

[0121] For comparison, nanoparticles were constructed using PLGA-PEG and PLA-PEG nanoparticles. As shown in Figures 4A and 4B, the difference was not significant. PLA-PEG showed a small decrease in particle size and protein density. PLGA-PEG and PLA-PEG nanoparticles with signal 1 ligand (HLA-Ig ligand loaded with MART-1 antigen) and signal 2 ligand (antibody agonist against CD28) were analyzed for their ability to stimulate antigen-specific CD8+ T cells. As shown in Figures 5A-5D, no significant difference was observed between PLGA-PEG and PLA-PEG in terms of INFγ-positive (Figure 5A), TNFα-positive (Figure 5B), IL-2-positive (Figure 5C), and CD107a-positive (Figure 5D) T cells. T cells prepared by enrichment and proliferation ("AIM-ACT") were used as a reference. See U.S. Patent No. 10,987,412 and U.S. Patent No. 11,007,222. These patents are incorporated herein by reference in their entirety.

[0122] PLGA-PEG and PLA-PEG aAPC loaded with CMV peptide antigen (D14) were tested for their ability to promote the proliferation of antigen-specific CD8+ T cells. As shown in Table 5 below (using HLA_CMV tetramer staining), there was no significant difference between PLGA-PEG nanoparticles and PLA-PEG nanoparticles. [Table 5]

[0123] The ability of PLGA-PEG and PLA-PEG aAPC, which carry CMV peptide antigens, to generate T cell memory phenotypes was investigated basically as follows: PBMCs were isolated from whole blood, and CD8+ T cells were isolated using a commercially available kit. The cells were then subjected to a cytokine mixture (see U.S. Patent No. 11,007,222 (incorporated here by reference)) at a rate of 1 × 10⁶ cells per 1 mL. 6 The cells were suspended. 6The cells were resuspended in a total volume of 16 mL with aAPC (approximately 10 μg). The cell / aAPC suspension was plated in 96-well round-bottom plates, 160 μL per well. On day 4, the cytokine mixture was added to the cells. On day 7, the cells were harvested, counted, and their phenotype was evaluated.

[0124] As shown in Table 6 below, there was no significant difference in the T memory cell generation ability of PLGA-PEG and PLA-PEG nanoparticles. CMV-supported nanoparticles proliferated CMV-specific CD8+ T cells with a memory phenotype in over 90% of cases. [Table 6]

[0125] As shown in Figure 6, when aAPCs loaded with antigens essentially prepared according to this embodiment were systemically administered to tumor-bearing mice, migration to lymph nodes, spleen, and tumors (right) was observed compared to bare nanoparticles (left). The aAPCs contained MHC class I dimers (immunoglobulin fusions) and anti-CD28 agonist antibody ligands. Furthermore, systemic administration of aAPCs increased antigen-specific T cells in the spleen and tumors, and these T cells exhibited higher killing capacity compared to peptides and CFAs (Figures 7A-7C). As shown in Figures 8A-C, the phenotype of T cells recovered from lymph nodes and tumors (after aAPC administration) was consistent with a persistent and strong antitumor effect. In addition, aAPC administration (loaded with GP100 antigen) extended overall survival in the B16 model (see Figure 9).

[0126] Example 2: Construction of an anti-Fas-mediated antibody ligand CH11 is an IgM-activated antibody against human Fas. This antibody exhibits cytolytic activity against human cells expressing Fas. The variable domain of CH11 was conjugated to the IgG4 framework described in U.S. Patent 10,632,193. See Figure 10A. The heavy and light chain amino acid sequences are provided as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The activity of the anti-Fas antibody (with and without dimerization by anti-IgG4 antibody) was measured in CD8+ T cells (10 per well). 5 The induction of apoptosis in cells was analyzed in vitro. As shown in Figure 10B, pentameric CH11 strongly induced apoptotic effects in CD8+ T cells, while the effect of uncrosslinked anti-Fas IgG4 was negligible. However, once crosslinked (in this case, by dimerization with anti-IgG4 antibody), the apoptotic effect was significant.

[0127] These results suggest that anti-Fas antibodies (such as those derived from clone CH11) may be effective in specifically inducing apoptosis in antigen-specific T cells by binding them to nanoparticles containing a selected signal 1 ligand (for example, targeting autoreactive T cells).

[0128] Example 3: Injectable aAPC having PD-L1 or anti-Fas signaling 2 ligand Injectable aAPCs were prepared using the PD-L1 signaling 2 ligand (SEQ ID NO: 1) as described in Example 1. As shown in Figure 11, these aAPCs rapidly inhibit antigen-specific killing of peptide-loaded target cells. Specifically, MART-1 specific CD8+ T cells were cultured with 50 μg / mL of aAPC for 90 minutes. After washing, the cells were cultured with peptide-loaded target cells for 4 hours, and antigen-specific killing was evaluated by caspase 3 / 7 analysis. A 50% reduction in antigen-specific cytotoxic killing was observed.

[0129] Injectable aAPCs were prepared using an anti-Fas signaling 2 ligand (as described in Example 2), as described in Example 1. As shown in Figure 12, these aAPCs rapidly eliminate antigen-specific T cells. Figure 12 shows 50% elimination of MART-1 specific T cells after 4 hours of in vitro culture. Non-target T cells were unaffected. For example, aAPCs displaying survivin peptide did not affect the number of MART-1 specific T cells. As shown in Figure 13, anti-Fas aAPCs eliminate up to 90% of MART-1 specific T cells by day 13 in a mouse model. This effect is dose-dependent.

[0130] Example 4: Selection of peptide antigen for aAPC It was observed that certain peptide antigens, when supported on nanoparticles (aAPCs), induce particle aggregation. This effect was evaluated, and the characteristics of peptide antigens suitable for support on nano-aAPCs without inducing aggregation were determined.

[0131] Protein modeling and aggregation tendency analysis were performed using Discovery Studio 2021 (DS2021), a software product of BIOVIA (Dassault Systemes BIOVIA, Discovery Studio Modeling Environment, 2021, San Diego, CA). DS2021 includes a function for calculating aggregation tendency scores based on Trout's method. The MODELLER program (UCSF, San Francisco, CA) was used for homology and comparative modeling of protein three-dimensional structures.

[0132] Figure 14 schematically illustrates the process for identifying peptide antigens that exhibit aggregation tendencies when presented by HLA ligands on nanoparticles.

[0133] First, molecular models of the relevant peptide-HLA complexes are generated. The HLA-A:0201 structural models include 4L29, 1I7R, 5EUO, 1TVB, 6OPD, 6TRO, 6AMT, 6AM5, 2GT9, and 3OXS. The modeling of the complexes is based on the generation of standard comparative protein models of the relevant peptides located in the HLA binding groove. The MODELER algorithm, implemented in Discovery Studio 2021, was employed. Multiple template structures were used in the modeling stage, and manual sequence alignment was performed for each model.

[0134] Next, the "Calculate Aggregation Scores" protocol implemented in Discovery Studio 2021 is executed using the completed static model with the highest score. Radii of 5 and 10 angstroms were selected for score calculation. This parameter determines how many neighboring amino acid residues are included in the aggregation ability calculation. Depending on the particle size of the model being analyzed, the above radii can provide a well-smoothed surface map without excessive noise. This protocol calculates the exposed surface of each residue of the peptide antigen and calculates the aggregation ability score based on the average of each residue and the residues within the defined radius surrounding it. The numerical aggregation ability score (APS) is recorded along with sequence and structural information and used to generate a surface map based on the solvent-penetrating surface of the model. This, combined with the scored aggregation ability, provides a simple visualization of the surface shape. Peptides can be classified into agglutinative and non-aggregative peptides by visual inspection or computer processing.

[0135] By evaluating numerous peptide antigens, inferences were prepared to guide the evaluation of candidate peptides. Here, the APS of residues 1 and 3-5 were averaged. A threshold of 0.07 or higher is considered a factor in aggregation. The presence of exposed Cys residues (determined by model structure analysis) is considered another factor in aggregation. The presence of charged residues and Gly residues is considered a factor that counteracts aggregation. The total number of factors favoring or unfavoring aggregation can determine whether a peptide-HLA complex is classified as a potential aggregation factor.

[0136] Figures 15A and 15B show the models and scores for aggregated and non-aggregated peptides, respectively. This process was applied to a known set of aggregated and non-aggregated factors, as summarized in Table 7 below. [Table 7]

[0137] References B.Webb,A.Sali.Comparative Protein Structure Modeling Using Modeller.Current Protocols in Bioinformatics 54,John Wiley&Sons,Inc.,5.6.1-5.6.37,2016 MAMarti-Renom,et al.Comparative protein structure modeling of genes and genomes.Annu.Rev.Biophys.Biomol.Struct.29,291-325,2000 A.Sali&T.L.Blundell.Comparative protein modeling by satisfaction of spatial restraints.J.Mol.Biol.234,779-815,1993 A.Fiser,R.K.Do,&A.Sali.Modeling of loops in protein structures.Protein Science 9.1753-1773,2000 Chennamsetty,N.,et al.Design of therapeutic proteins with enhanced stability.Proc.Natl.Acad.Sci.USA 2009,106(29),11937-11942 Chennamsetty,N.,et al.Aggregation-Prone Motifs in Human Immunoglobulin G.J.Mol.Biol.2009,391,404-413 Chennamsetty,N.,et al.Prediction of Aggregation Prone Regions of Therapeutic Proteins.J.Phys.Chem.B 2010,114,6614-6624 Schutz C,et al.Antigen-specific T cell Redirectors:a nanoparticle based approach for redirecting T cells.Oncotarget.2016 Oct 18;7(42):68503-68512 Schappert A,et al.Soluble MHC class I complexes for targeted immunotherapy.Life Sci.2018 Sep 15;209:255-258 Turtle CJ,Riddell SR.Artificial antigen-presenting cells for use in adoptive immunotherapy.Cancer J.2010;16(4):374-381 Black SD,Mould DR.Development of hydrophobicity parameters to analyze proteins which bear post- or cotranslational modifications.Anal Biochem.1991 Feb 15;193(1):72-82 Riley TP,et al.Structure Based Prediction of Neoantigen Immunogenicity.Front Immunol.2019 Aug 28;10:2047 Liu J,et al.Structural insights into the binding of hepatitis B virus core peptide to HLA-A2 alleles: towards designing better vaccines.Eur J Immunol.2011 Jul;41(7):2097-106 Halabelian L et al.Class I major histocompatibility complex,the trojan horse for secretion of amyloidogenic β2-microglobulin.J Biol Chem.2014 Feb 7;289(6):3318-27 Coles CH,et al.T cell receptor interactions with human leukocyte antigen govern indirect peptide selectivity for the cancer testis antigen MAGE-A4.J Biol Chem.2020 Aug 14;295(33):11486-11494 Buslepp J, et al. T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface. J Biol Chem. 2001 Dec 14;276(50):47320-8 Borbulevych OY, et al. Structures of MART-126 / 27-35 Peptide / HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition. J Mol Biol. 2007 Oct 5;372(5):1123-36 Borbulevych OY, et al. Increased immunogenicity of an anchor-modified tumor-associated antigen is due to the enhanced stability of the peptide / MHC complex: implications for vaccine design. J Immunol. 2005 Apr 15;174(8):4812-20

[0138] Sequence [Chemical formula] SEQ ID NO: 3 - CH11 light chain DVVMTQSPLSLPVSLGDQASISCrsskslvhsngntylhWYLQKPGQSPKLLIYkvsnrfSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCsqsthvppaFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [ka] [ka] 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 Sequence ID No. 11 - Anti-CD28 antibody HC CDR3 DKGYSYYYSMDY Sequence ID No. 12 - Anti-CD28 antibody LC CDR1 RASESVEYYVTSLMQ Sequence ID No. 13 - Anti-CD28 antibody LC CDR2 AASNVES SEQ ID NO: 14 - Anti-CD28 antibody LC CDR3 QQSRKVPYT Sequence ID 15 - Humanized anti-CD28 heavy chain variable region EVKLQQSGPGLVKPSETLSLTTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVFLKMNSLTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS Sequence ID 16 - Humanized anti-CD28 heavy chain variable region EVKLQQSGPGLVKPSETLSLTTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVSLKMSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS Sequence ID 17 - Humanized anti-CD28 heavy chain variable region EVKLQQSGPGLVKPSETLSLTTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRVTISKDNSKSQVSLKLSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS Sequence ID 18 - Humanized anti-CD28 light chain DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTNFTLTISSLQEEDVAMYFCQQSRKVPYTFGGGTKVEIK Sequence ID 19 - Humanized anti-CD28 light chain DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTNFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIK Sequence ID 20 - Humanized anti-CD28 light chain DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIK Sequence ID 21-HLA-E extracellular domain GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDREETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVD TAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW [ka] SEQ ID NO: 23-IgG4 Fc ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLCLSLGK Sequence ID 24 - Flexible Linker GGGGSGGGGSGGGGS [ka]

Claims

1. A modified antigen-presenting cell (aAPC) suitable for parenteral administration, The material comprises 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 a thioether bond. The polypeptide ligand comprises an HLA ligand that presents a peptide antigen, and optionally one or more signal 2 ligands, Approximately 40% by weight or less of the copolymer has functional groups for polypeptide ligand coupling. The aforementioned manipulated antigen-presenting cells (aAPCs).

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

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

5. aAPC according to 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 according to 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. aAPC according to claim 6, wherein the molecular weight of the PEG portion having a functional group for polypeptide ligand coupling is about 5 kDa, and the molecular weight of the PEG portion not having a functional group for ligand coupling is about 3 kDa.

8. The aAPC according to any one of claims 1 to 7, wherein a sulfhydryl group on the polypeptide ligand is bonded 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 according to claim 9, wherein the aAPC has about 50 to about 400 polypeptide ligands.

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

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

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

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

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

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

2.

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

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

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

22. The aAPC according to any one of claims 19 to 21, wherein the HLA ligand has a peptide bond groove 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. aAPC according to claim 23, wherein the average aggregation point number (APS) of peptide residues 1 and 3-5 is less than approximately 0.

07.

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

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

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

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

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

30. The aAPC according to claim 29, wherein the signal 2 ligand optionally comprises a single-chain antibody which is scFv.

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

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

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

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

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

36. The aAPC according to claim 35, further comprising one or more cytokines that support T cell activation and / or proliferation, wherein the aAPC is bound to the aAPC as a polypeptide ligand, fused to a signal 1 or signal 2 polypeptide ligand, or encapsulated by the copolymer.

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

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

39. The aAPC according to claim 38, wherein the co-inhibitory ligand is an activator for Fas, TGF-β, or PD-1.

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

41. aAPC according to claim 40, wherein the activator antibody against Fas is an IgG4 antibody based on clone CH11.

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

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

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

45. The aAPC according to any one of claims 41 to 44, further comprising an immunotolerogenic cytokine in which the aAPC is fused to a signal 1 polypeptide ligand as a polypeptide ligand or encapsulated by the copolymer, wherein the immunotolerogenic cytokine is optionally IL-10.

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

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

48. The aAPC according to claim 47, wherein the pharmaceutical composition is freeze-dried.

49. Modified antigen-presenting cells (aAPCs) suitable for parenteral administration, The polymer nanoparticles or lipid nanoparticles comprising a polyethylene glycol (PEG) sheath and one or more polypeptide ligands bonded to the PEG via a thioether linkage; The polypeptide ligand comprises an HLA class I or class II ligand that presents a peptide antigen, and optionally one or more signal 2 ligands, The peptide antigen does not induce aggregation of the aAPC. The aforementioned manipulated antigen-presenting cells (aAPCs).

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

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

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

01.

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

54. aAPC according to any one of claims 49 to 53, wherein the average aggregation ability number (APS) of peptide residues 1 and 3 to 5 is less than approximately 0.

07.

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

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

57. aAPC according to any one of claims 49 to 56, wherein 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 bonded to PEG via thioether bonds.

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

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

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

62. aAPC according to 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 according to 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. aAPC according to claim 63, wherein the molecular weight of the PEG portion having a functional group for polypeptide ligand bonding is about 5 kDa, and the molecular weight of the PEG portion not having a functional group for ligand bonding is about 3 kDa.

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

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

67. aAPC according to any one of claims 49 to 66, wherein a sulfhydryl group on the polypeptide ligand is bonded 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 according to claim 68, wherein the aAPC has about 50 to about 400 polypeptide ligands.

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

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

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

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

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

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

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

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

2.

78. aAPC according to any one of claims 49 to 77, comprising two fusions of the HLA ligand being a dimer and an immunoglobulin Fc which is optionally an IgG4 isotype.

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

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

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

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

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

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

85. The aAPC according to any one of claims 49 to 84, further comprising one or more cytokines that support T cell activation and / or proliferation, wherein the aAPC is bound to the aAPC as a polypeptide ligand, fused to a signal 1 or signal 2 polypeptide ligand, or encapsulated by the copolymer.

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

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

88. The aAPC according to claim 87, wherein the co-inhibitory ligand is an activator for Fas, TGF-β, or PD-1.

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

90. The aAPC according to claim 89, wherein the activating agent antibody is an IgG4 antibody based on clone CH11.

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

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

93. The aAPC according to claim 92, wherein the activating fragment of PD-L1 is fused with an HLA class I ligand which is optionally dimerized by fusion with immunoglobulin Fc.

94. The aAPC according to any one of claims 86 to 93, further comprising an immunotolerogenic cytokine encapsulated by the copolymer, wherein the aAPC is optionally IL-10.

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

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

97. The aAPC according to claim 96, wherein the pharmaceutical composition is freeze-dried.

98. A method for immunotherapy comprising administering aAPC or a pharmaceutical composition thereof according to any one of claims 1 to 97 to a subject in need of treatment.

99. The method according to claim 98, wherein the subject is suffering from cancer or an infectious disease, and the aAPC includes a co-stimulatory ligand.

100. The method according to claim 99, wherein the subject has a hematological malignancy.

101. The method according to claim 99, wherein the subject has a solid tumor which is arbitrarily stage I, stage II, stage III, or stage IV.

102. The method according to any one of claims 99 to 101, wherein the subject is suffering from human papillomavirus (HPV)-related cancer.

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

104. The method according to claim 98, wherein the subject suffers from an autoimmune disease, and the aAPC includes a co-inhibitory signal or does not include a signal 2 ligand.

105. The method according to claim 104, wherein the autoimmune disease is type 1 diabetes.

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

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

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

109. The method according to claim 108, wherein the APC or its pharmaceutical composition is administered subcutaneously.

110. A method for producing manipulated antigen-presenting cells, To provide a computer-aided HLA ligand including an antigen-binding groove, wherein the antigen-binding groove contains a candidate peptide antigen; Creating a surface map of the antigen-binding groove containing the candidate peptide antigen, wherein the surface map provides the number of aggregation points for each residue of the candidate peptide antigen and the surrounding amino acid residues; Selecting candidate peptides with aggregation ability below the threshold; and The selected peptide is loaded onto aAPC. The method, including the method described above.

111. An anti-Fas-agonist antibody possessing an IgG isotype and bound to nanoparticles along with an HLA ligand that presents a peptide antigen.

112. A dimerized PD-L1 ligand comprising the amino acid residues F19 to T239 of human PD-L1 directly or indirectly fused to the dimerized IgG Fc region via a linker at the C-terminus, wherein the dimerized PD-L1 ligand is bound to a nanoparticle together with an HLA ligand that presents a peptide antigen.

113. A dimerized FasL ligand comprising amino acids P132 to L279 of human FasL directly or indirectly fused to a dimerized IgG-Fc region via a linker at the N-terminus, wherein the dimerized Fc region is bound to a nanoparticle via a Cys-containing linker, and the nanoparticle further presents an HLA-peptide antigen ligand.

114. An immunotolerative ligand containing amino acids F19-T239 of human PD-L1, used directly or via a linker to an HLA-immunoglobulin fusion protein.

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

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