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WO2024206207A1 - Cellules présentatrices d'antigène artificiel injectables pour une thérapie immunitaire - Google Patents

Cellules présentatrices d'antigène artificiel injectables pour une thérapie immunitaire Download PDF

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Publication number
WO2024206207A1
WO2024206207A1 PCT/US2024/021277 US2024021277W WO2024206207A1 WO 2024206207 A1 WO2024206207 A1 WO 2024206207A1 US 2024021277 W US2024021277 W US 2024021277W WO 2024206207 A1 WO2024206207 A1 WO 2024206207A1
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WIPO (PCT)
Prior art keywords
aapc
ligand
hla
ligands
polypeptide
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PCT/US2024/021277
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English (en)
Inventor
Daniel P. Bednarik
Mathias Oelke
Adam PARKS
Aniket Wadajkar
Charles Reed
David Langan
Jack RAGHEB
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Neximmune Inc
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Neximmune Inc
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Priority to AU2024244921A priority Critical patent/AU2024244921A1/en
Publication of WO2024206207A1 publication Critical patent/WO2024206207A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/33—Antibodies; T-cell engagers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • An antigen-presenting cell processes and displays antigenic peptides in complexes with major histocompatibility complex (MHC) proteins on their surfaces. Effector cells, such as T-cells, recognize these peptide-MHC (pMHC) complexes through cell-surface receptors, such as T-cell receptors (TCRs).
  • TCRs T-cell receptors
  • DCs Dendritic cells
  • DCs are an example of an antigen presenting cell that can be stimulated to effectively present antigen and support expansion of immune effector cells, thereby activating a cytotoxic response towards an antigen.
  • DCs are harvested from a patient and either pulsed with an antigen or transfected with a viral vector.
  • tumor antigen to effector lymphocytes (e.g. CD4+ T cells, CD8+ T cells, and B cells).
  • effector lymphocytes e.g. CD4+ T cells, CD8+ T cells, and B cells.
  • this therapy initiates a cytotoxic response against cells expressing antigens (including tumor antigens).
  • aAPCs artificial antigen presenting cells
  • the present disclosure provides artificial antigen presenting cells (aAPCs) that are suitable for parenteral administration for immune therapy.
  • the aAPCs are effective to activate or inhibit target T cells in vivo, including CD8+ or CD4+ T cells.
  • the aAPCs according to this disclosure provide a shelf- stable nanoparticle platform for immune therapies.
  • the shelf-stable nanoparticle platform in various embodiments controls particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation propensity, among other features.
  • the present invention provides shelf-stable compositions and methods for activating or inhibiting antigen-specific T cells in a patient.
  • the nanoscale aAPCs described herein are designed to provide pharmacodynamic advantages, including with respect to circulating properties, biodistribution, and degradation kinetics. These advantages can result from physical parameters including particle size, surface charge, polydispersity index, polymer composition, ligand conjugation chemistry, ligand density, and peptide loading, among others.
  • the aAPCs have a polypeptide ligand density that avoids aggregation potential as well as steric constraints from an abundance of ligands on the surface, without loss of activity and/or potency.
  • the aAPCs persist in peripheral blood circulation sufficiently long to allow distribution to target tissues, including trafficking to lymphoid organs (e.g., lymph nodes) via blood/lymph exchange and/or trafficking to tumors and/or trafficking to target organs.
  • the aAPCs are suitable for subcutaneous administration.
  • the disclosure provides aAPCs comprising poly(lactic acid)- polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) co-polymers.
  • the aAPCs have advantages in stability and ligand density, for example.
  • the disclosure provides an aAPC suitable for parenteral administration (including subcutaneous administration in some embodiments), and which comprises PLA-PEG or PLGA-PEG DB1/ 145613893.1 2 Attorney docket: NEX-013PC/107578-5013 copolymers and one or more polypeptide ligands conjugated to PEG polymers through a thioether bond or other conjugation chemistry.
  • the polypeptide ligands comprise HLA ligands (Human Leukocyte Antigen ligands) presenting a peptide antigen, and optionally one or more signal 2 ligands.
  • Signal 2 ligands include signal 2 ligands for T cell activation and/or expansion, or T cell inhibition.
  • the copolymers have a functional group for polypeptide ligand coupling.
  • the HLA ligands in various embodiments may be HLA Class I and/or Class II molecular complexes, or portions thereof comprising an antigen-binding cleft.
  • the HLA molecular complexes are monomeric or dimeric, and may contain additional heterologous sequences, such as immunoglobulin sequences.
  • HLA-fusions e.g., HLA-Immunoglobulin fusions in some embodiments provide additional advantages in stability, TCR binding affinity, and/or potency for T cell activation or inhibition.
  • the aAPCs comprise HLA class I ligands for presentation of peptide antigens to CD8+ T cells (e.g., for activation and/or expansion of CD8+ cells, or inhibition of CD8+ cells).
  • the HLA class I ligand comprises at least two fusion proteins.
  • a first fusion protein comprises a first HLA class I ⁇ chain and a first immunoglobulin heavy chain
  • a second fusion protein comprises a second HLA class I ⁇ chain and a second immunoglobulin heavy chain.
  • the first and second immunoglobulin heavy chains associate to form the HLA class I molecular complex (e.g., associate through disulfide bonds).
  • the HLA class I molecular complex comprises a first HLA class I peptide binding cleft and a second HLA class I peptide binding cleft.
  • the aAPC comprises HLA class II ligands for presentation of peptide antigens to CD4+ T cells (e.g., for activation and/or expansion of CD4+ cells, or inhibition of CD4+ cells).
  • the HLA class II molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise (i) an immunoglobulin heavy chain and (ii) an extracellular domain of an HLA class II ⁇ chain. Two second fusion proteins comprise (i) an immunoglobulin light chain and (ii) an extracellular domain of an HLA class II ⁇ chain.
  • the two first and the two second fusion proteins associate to form the HLA class II molecular complex.
  • the extracellular domain of the HLA class II ⁇ chain of each first DB1/ 145613893.1 3 Attorney docket: NEX-013PC/107578-5013 fusion protein and the extracellular domain of the HLA class II ⁇ chain of each second fusion protein form an HLA class II peptide binding cleft.
  • Peptide antigens are bound to an antigen binding cleft of the antigen presenting complex. Peptide antigens for immune therapy of oncological disease, infectious diseases, and autoimmune diseases are described herein. In some embodiments, the peptide antigen does not induce aggregation of the aAPC.
  • the polypeptide ligands comprise a signal-2 ligand that is a co-stimulatory ligand, e.g., for activation and/or expansion of target T cells.
  • exemplary co-stimulatory ligands include agonists for any one of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT, among others.
  • Co-stimulatory ligands can induce activation and/or expansion of CTLs or Tregs in various embodiments.
  • the polypeptide ligands do not comprise any signal 2 ligand or the polypeptide ligands comprise inhibitory ligands, which induce tolerance or apoptosis of target T cells.
  • the inhibitory ligand is an agonist for Fas, TGF- ⁇ , or PD-1.
  • Agonistic ligands can include natural agonistic ligands (or engineered variants thereof, including immunoglobulin fusions as described), or in some embodiments, antibody agonists.
  • the co-inhibitor ligand is PD-L1 (or immunoglobulin fusion thereof) or FasL (or immunoglobulin fusion thereof).
  • Antibody agonists can be full monoclonal antibodies, or portions or fragments comprising antigen- binding sequences, such as Fab, Fab', F(ab') 2 or scFv.
  • the signal 1 and signal 2 ligands can be combined in homodimeric or heterodimeric constructs.
  • the HLA ligand can comprise fusion of HLA extracellular domains to an immunoglobulin Fc region, such as IgG4 Fc region, which can be dimerized (e.g., through disulfide bonds) with a signal 2-Immunoglobulin (Ig) fusion (i.e., a heterodimeric Ig fusion construct).
  • the HLA ligand comprises a fusion to a signal 2 ligand.
  • an HLA extracellular domain can be fused at its C-terminus to an immunoglobulin Fc region, and fused at its N-terminus to a DB1/ 145613893.1 4 Attorney docket: NEX-013PC/107578-5013 signal 2 ligand, to prepare homodimeric ligands with both signals dimerized.
  • the signal 2 ligand comprises a single chain antibody (e.g., scFv) or agonistic portion of a natural ligand.
  • the co-inhibitory ligand is an agonist antibody for Fas.
  • the agonist antibody for Fas is an IgG4 antibody based on clone CH11. As demonstrated herein, this anti-Fas antibody has activity when it is able to crosslink multiple Fas receptors, such as when the antibody is conjugated to a nanoparticle.
  • the aAPC further comprises one or more cytokines that support T cell activation and/or expansion or T cell inhibition. The one or more cytokines or functional portion thereof may be conjugated to the aAPC as a polypeptide ligand.
  • the cytokine or functional portion thereof may be fused to a signal 1 or signal 2 polypeptide ligand (which can optionally be presented in homodimeric or heterodimeric Ig fusion constructs as described herein).
  • the cytokine is encapsulated by the copolymers, and will release cytokine locally in targeted environments (e.g., in lymphoid organs, tumor, or target tissue or organ).
  • cytokines include IL-1 ⁇ , IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and gamma interferon.
  • IL-2 can be employed with a co-stimulatory signal 2 ligand.
  • the aAPC comprises a tolerogenic cytokine as a polypeptide ligand.
  • the one or more peptide antigens are tumor or cancer associated antigens, such as tumor-derived antigens, tumor-specific antigens, and neoantigens.
  • 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.
  • the one or more target peptide antigens is an “autoantigen”, meaning that it is associated with an autoimmune disease or reaction.
  • the aAPC is comprised in a pharmaceutical composition suitable for administration to a subject.
  • the pharmaceutical composition may have one or more excipients such as buffering agents, surfactants, preservative agents, polymers, bulking agents, and stabilizers.
  • the present disclosure provides aAPCs suitable for parenteral administration (including subcutaneous administration), and which have low aggregation propensity.
  • the aAPC comprises a polymeric or lipid nanoparticle comprising a polyethylene glycol (PEG) sheath and one or more polypeptide ligands conjugated to PEG (e.g., the PEG terminus), which can be through a thioether bond or other functional group.
  • PEG polyethylene glycol
  • the polypeptide ligands comprise HLA Class I or Class II ligands presenting 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.
  • the peptide antigen does not have an exposed Cysteine and/or the peptide antigen has one or more exposed glycine residues or exposed charged residues.
  • the peptide antigen does not have any Cysteine residue, and contains one or more charged residues (e.g., 1, 2, or 3 charged residues).
  • aggregation potential is estimated in silico by determining the mean Aggregation Potential Score (APS) of antigen peptide residues within the HLA antigen binding cleft.
  • the invention provides a method for immunotherapy. The method comprises administering the aAPC or pharmaceutical composition thereof as described herein to a subject in need of treatment.
  • the subject has cancer or an infectious disease, and the aAPCs comprise a co-stimulatory ligand.
  • the peptide antigen is selected in a personalized basis for a cancer patient, based on an analysis of the patient's tumor.
  • the nano-aAPCs are used as a booster vaccine, after adoptive T cell therapy, in which naive T cells from the patient, TILs, or T cells from an HLA-matched donor are expanded ex vivo and administered to the patient.
  • the subject has an autoimmune condition, and the aAPCs comprise a co-inhibitory signal or do not contain a signal 2 ligand.
  • the autoimmune condition is type 1 diabetes.
  • the aAPC or pharmaceutical composition thereof is parenterally administered.
  • the aAPC or pharmaceutical composition thereof is administered by intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intra-lymphatic administration, intramuscular administration, or DB1/ 145613893.1 6 Attorney docket: NEX-013PC/107578-5013 intratumoral administration.
  • the aAPC composition is administered by subcutaneous administration.
  • polypeptide ligands for immune therapy including as polypeptide ligands for aAPCs are disclosed.
  • polypeptide ligands include an anti- Fas agonistic antibody having an IgG isotype (e.g., IgG4), which can be conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen (e.g., associated with an autoimmune disease).
  • the polypeptide ligand is a dimeric PD-L1 ligand comprising an activating portion of PD-L1, such as amino acids residues F19 to T239 of human PD-L1.
  • Each PD-L1 activating fragment may be fused directly or indirectly through a linker at its C-terminus to an IgG Fc region (e.g., IgG4), and the ligand may be dimerized by disulfide bonds in the Fc region.
  • the dimeric PD-L1 ligand can be conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen and used to drive tolerance to the antigen.
  • the polypeptide ligand is a dimeric FasL ligand comprising an activating portion of FasL, such as amino acids P132 to L279 of human FasL fused directly or indirectly through a linker at its N-terminus to a dimerized IgG-Fc region (e.g., IgG4).
  • the dimerized Fc region can be conjugated to nanoparticles through a Cys-containing linker in some embodiments.
  • the nanoparticles may further present HLA- peptide antigen ligands to drive apoptosis of antigen-specific T cells.
  • the polypeptide ligand is a tolerogenic ligand comprising an activating fragment of PD-L1, such as amino acids F19-T239 of human PD-L1, fused directly or through a linker to HLA- immunoglobulin fusion protein.
  • ligands can be conjugated to nanoparticles as disclosed herein and used for immunotherapy (to drive tolerance in targeted T cells).
  • the polypeptide ligand is a co-stimulatory ligand comprising an anti-CD28 agonistic scFv conjugated to an HLA-immunoglobulin fusion protein, providing homodimeric ligands comprising signal 1 and co-stimulatory signal 2 ligands.
  • FIGs. 1A and 1B show the size and surface charge of nanoparticles prepared according to this disclosure.
  • FIG. 1A shows TEM images of naked particles, protein- conjugated nanoparticles, and peptide loaded nanoparticles.
  • FIG. 1B shows (from left to right) the size distribution, average size of particles, polydispersity index (PDI), and surface charge.
  • PDI polydispersity index
  • FIG.2 is an illustration showing conjugation of thiolated ligands to PEG-maleimide functional groups on nanoparticles.
  • FIGs. 3A and 3B show the effect on ligand density (bars) and nanoparticle size (squares) upon altering %PEG-mal on the surface of the nanoparticles (FIG. 3A) or by altering the maleimide:thiol ratio during the coupling reaction (FIG.3B).
  • FIGs.4A and 4B show that use of PLGA-PEG or PLA-PEG do not lead to significant differences in size (bars) or PDI (dot) (FIG. 4A) or protein density (FIG.
  • FIGs.5A-D show that the use of PLGA-PEG or PLA-PEG do not lead to significant differences in antigen-specific CD8+ T cell stimulation.
  • FIG. 5A shows percent IFN ⁇ + CD8+ T cells.
  • FIG.5B shows percent TNF ⁇ + CD8+ T cells.
  • FIG.5C shows percent IL2+ CD8+ T cells.
  • FIG.5D shows percent CD107a+ CD8+ T cells.
  • FIG.6 shows that antigen-loaded aAPCs according to embodiments of this disclosure traffic to lymph nodes, spleen, and tumor.
  • FIGs 7A and 7B show that systemically administered aAPCs increased antigen- specific T cells in the spleen (A) and tumor (B) of tumor-bearing mice (B16-OVA, with implanted OT-1 T cells).
  • Splenocytes from mice that received peptide-loaded aAPC had greater killing potential as compared to peptide with Complete Freund’s Adjuvant (CFA).
  • FIG.7C. N 2 per arm.
  • In vitro killing assay performed with splenocytes harvested on day 22.
  • FIGs. 8A-C shows that T cells recovered from lymph nodes and tumors have a phenotype consistent with persistence and strong anti-tumor effect.
  • FIG.8A shows T cells (specific for ovalbumin antigen) recovered from lymph nodes in a non-disease model. These DB1/ 145613893.1 8 Attorney docket: NEX-013PC/107578-5013 T cells exhibited central memory and effector memory phenotype.
  • FIG. 8B quantifies antigen-specific T cells in tumors in a melanoma model (B16F10 cells), 63% of which had at least three effector functions.
  • FIG. 8C quantifies gp100-specific T cells in tumors in a melanoma model, and showing that antigen-specific CTLs recovered from the tumor exhibited a effector memory and central memory phenotype.
  • FIG.9 shows that aAPCs loaded with gp100 antigen extended survival in B16 mouse model (x-axis is days from tumor implantation).
  • FIG.9 shows that aAPC activate anti-tumor T cells in vivo, and in this model, clear lung metastases.
  • FIGs. 10A-C show the construction of an agonistic anti-Fas antibody ligand.
  • FIG. 10A shows construction of an IgG4 antibody based on the variable domain of CH11 clone (which is IgM isotype). The IgG4 antibody is crosslinked using an anti-IgG4 antibody for in vitro testing.
  • FIG. 10A shows construction of an IgG4 antibody based on the variable domain of CH11 clone (which is IgM isotype). The IgG4 antibody is crosslinked using an anti-IgG4 antibody for in vitro testing.
  • FIG. 10B and 10C shows that the anti-Fas agonistic ligand has little to no activity in the absence of crosslinking but shows robust activity upon crosslinking.
  • FIG.11 shows that aAPCs with PD-L1-Ig Signal 2 ligands rapidly inhibit antigen- specific killing of peptide-loaded target cells.
  • FIG.12 shows that aAPCs with anti-Fas Signal 2 ligands rapidly eliminate antigen- specific T cells.
  • FIG.13 shows that anti-Fas aAPCs eliminate up to 90% of MART-1-specific T cells by Day 13 in a mouse model, and that the effect is dose dependent.
  • FIG.11 shows that aAPCs with PD-L1-Ig Signal 2 ligands rapidly inhibit antigen- specific killing of peptide-loaded target cells.
  • FIG.12 shows that aAPCs with anti-Fas Signal 2 ligands rapidly eliminate antigen- specific T cells.
  • FIG.13 shows that anti-Fas a
  • FIG. 14 is a diagram showing a process for selecting antigens for presenting on aAPCs to mitigate aggregation driven by antigen loading.
  • FIG. 15A and 15B illustrate modelling and scoring of aggregating and non- aggregating peptides, respectively.
  • DETAILED DESCRIPTION the present disclosure provides artificial antigen presenting cells (aAPCs) that are suitable for parenteral administration for immune therapy.
  • the aAPCs are effective to activate or inhibit target T cells in vivo, including CD8+ or CD4+ T cells.
  • the aAPCs according to this disclosure provide a shelf- DB1/ 145613893.1 9 Attorney docket: NEX-013PC/107578-5013 stable nanoparticle platform for immune therapies.
  • the shelf-stable nanoparticle platform in various embodiments controls particle size and particle chemistry, ligand design and ligand density, and aAPC aggregation propensity, among other features.
  • the present invention provides shelf-stable compositions and methods for activating or inhibiting antigen-specific T cells in a patient.
  • the nanoscale aAPCs described herein are designed to provide pharmacodynamic advantages, including with respect to circulating properties, biodistribution, and degradation kinetics. These advantages result from physical parameters including particle size, surface charge, polydispersity index, polymer composition, ligand conjugation chemistry, ligand density, and peptide loading, among others.
  • the aAPCs have a polypeptide ligand density that avoids aggregation potential as well as steric constraints from an abundance of ligands on the surface, without loss of activity and/or potency.
  • the aAPCs persist in peripheral blood circulation sufficiently long to allow distribution to target tissues, including trafficking to lymphoid organs (e.g., lymph nodes) via blood/lymph exchange and/or trafficking to tumors and/or trafficking to target organs.
  • the aAPCs are suitable for subcutaneous administration.
  • the disclosure provides aAPCs (and pharmaceutical compositions thereof) comprising poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid- co-glycolic acid)-polyethylene glycol (PLGA-PEG) co-polymers.
  • PLA-PEG and PLGA- PEG nanoparticles can be prepared by nanoprecipitation using known processes.
  • the aAPCs have advantages in stability and ligand density, among other things.
  • the disclosure provides an aAPC suitable for parenteral administration (including subcutaneous administration in some embodiments), and which comprises PLA-PEG or PLGA-PEG copolymers and one or more polypeptide ligands conjugated to PEG polymers (e.g., conjugated to the PEG terminus) through a thioether bond or other conjugation chemistry (such as conjugation through an amine).
  • the polypeptide ligands comprise HLA ligands (Human Leukocyte Antigen ligands) presenting a peptide antigen, and optionally one or more signal 2 ligands.
  • Signal 2 ligands are described elsewhere herein and include signal 2 ligands for T cell activation and/or expansion, or T DB1/ 145613893.1 10 Attorney docket: NEX-013PC/107578-5013 cell inhibition.
  • about 40% or less by weight of the copolymers have a functional group for polypeptide ligand coupling.
  • about 30% or less, or about 25% of less, or about 20% or less by weight of the copolymers have a functional group for polypeptide ligand coupling.
  • from about 15% to about 35% by weight of the copolymers have a functional group for polypeptide ligand coupling.
  • PEG-Mal (by weight) or other PEG-functional group is less than about 10%, or less than about 7%, such as about 5% of the co-polymer weight.
  • the functional group is a maleimide functional group at the PEG terminus, with other PEG groups being inert (e.g., comprising an alkyl ether end cap, such as a methyl ether end cap).
  • the end capped PEG may also be referred to herein as mPEG.
  • the reaction between sulfhydryl groups and maleimide is well known in the art.
  • Activating functional groups include alkyl and acyl halides, amines, sulfhydryls, aldehydes, unsaturated bonds, hydrazides, isocyanates, isothiocyanates, ketones, and other groups known to activate for chemical bonding.
  • a molecule can be bound to a nanoparticle through the use of a small molecule-coupling reagent.
  • Non-limiting examples of coupling reagents include carbodiimides, maleimides, N-hydroxysuccinimide esters, bischloroethylamines, bifunctional aldehydes such as glutaraldehyde, anyhydrides and the like.
  • a molecule can be coupled to a nanoparticle through affinity binding such as a biotin-streptavidin linkage or coupling.
  • the nanoparticles have a PLGA copolymer that can be tuned for a specific biodegradation rate in vivo (by adjusting the LA:GA ratio and/or DB1/ 145613893.1 11 Attorney docket: NEX-013PC/107578-5013 molecular weight of the PLGA polymer).
  • the PLGA is based on a LA:GA ratio of from 20:1 to 1:20, including compositions of L/G of: 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 50/50, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, or 95/5.
  • PLGA degrades by hydrolysis of its ester linkages.
  • the time required for degradation of PLGA is related to the ratio of monomers: the higher the content of glycolide units, the lower the time required for degradation as compared to predominantly lactide units.
  • the PLGA is 50/50 L/G.
  • the aAPCs may further comprise PLGA or PLA polymers (in addition to the copolymers).
  • the PLGA or PLA polymer has a molecular weight in the range of about 15 kDa to about 35 kDa, or from about 15 kDa to about 25 kDa. In an exemplary embodiment, the PLGA or PLA polymer has a molecular weight of about 20 kDa. In various embodiments, the PLGA or PLA polymer is present at about 5 wt% to about 40 wt%, or about 5 wt% to about 25 wt%, based on the total weight of the polymers and co-polymers.
  • the ratio of PEG-maleimide groups to mPEG limits the density of the polypeptide ligands conjugated through, for example, sulfhydryl groups.
  • the aAPC has about 10 to about 500 polypeptide ligands per particle. In some embodiments, the aAPC has about 50 to about 400 polypeptide ligands per particle. In some embodiments, the aAPC has about 100 to about 300 polypeptide ligands per particle.
  • aAPC particles can be prepared with potent T cell effector properties (activation or inhibition), while avoiding undesirable properties including but not limited to aAPC aggregation and steric limitations.
  • the PLA or PLGA portion of the copolymers have molecular weights of from about 15 kDa to about 50 kDa. In some embodiments, the PLA or PLGA portion of the copolymers have molecular weights in the range of about 15 kDa to DB1/ 145613893.1 12 Attorney docket: NEX-013PC/107578-5013 about 35 kDa, or from about 15 kDa to about 25 kDa.
  • the PLA or PLGA portion of the copolymers have a molecular weight of about 20 kDa.
  • the PEG portions of the copolymers have molecular weights in the range of about 2 kDa to about 10 kDa, or in the range of about 2 kDa to about 7 kDa.
  • the PEG portion of the copolymers have molecular weights in the range of about 2 kDa and about 5 kDa.
  • the PEG portions having a functional group for polypeptide ligand coupling have molecular weights of about 5 kDa, and the PEG portions without functional groups for ligand coupling have molecular weights of about 3 kDa.
  • the smaller mPEG moieties limit steric constraints on polypeptide ligand binding to targets and/or improve conjugation efficiency.
  • the PLA or PLGA portions of the copolymers have molecular weights of about 20 kDa
  • the PEG-functional group portions of the co-polymers have molecular weights of about 5 kDa
  • the mPEG portions of the co-polymers have molecular weights of about 3 kDa.
  • the aAPC has a diameter of from about 50 nm to about 150 nm. In some embodiments, the aAPC has a diameter of from about 50 nm to about 130 nm. In some embodiments, the aAPC has a diameter of from about 50 nm to about 120 nm.
  • the aAPC has a diameter of from about 50 nm to about 100 nm or from about 50 nm to about 75 nm. In exemplary embodiments, the aAPC has a diameter of about 60 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm. In some embodiments, the aAPC population has a size distribution with polydispersity index (PDI) of less than 0.2. In various embodiments, the aAPC has a surface charge of from about 0 to -15 mV, or from about 0 to about -10 mV.
  • PDI polydispersity index
  • the aAPC may have a surface charge of from about -2.5 mV to about -10 mV.
  • the aAPC size and surface charge allows for desired circulating and biodistribution properties, and in some embodiments provides advantages in particle stability.
  • the HLA ligands in various embodiments may be HLA Class I and/or Class II molecular complexes, or portions thereof comprising an antigen-binding cleft.
  • the HLA molecular complexes are monomeric or dimeric, and may contain additional heterologous sequences, such as immunoglobulin sequences.
  • heterologous sequences include dimerizing amino acid sequences such as c-fos or c-jun, or monomeric amino acid sequences such as albumin.
  • HLA-fusions e.g., HLA- Immunoglobulin fusions
  • the aAPCs comprise HLA class I ligands for presentation of peptide antigens to CD8+ T cells (e.g., for activation and/or expansion of CD8+ cells, or inhibition of CD8+ cells).
  • the HLA class I ligand comprises at least two fusion proteins.
  • a first fusion protein comprises a first HLA class I ⁇ chain and a first immunoglobulin heavy chain
  • a second fusion protein comprises a second HLA class I ⁇ chain and a second immunoglobulin heavy chain.
  • the first and second immunoglobulin heavy chains associate to form the HLA class I molecular complex (e.g., associate through disulfide bonds).
  • the HLA class I molecular complex comprises a first HLA class I peptide binding cleft and a second HLA class I peptide binding cleft.
  • the aAPC comprises HLA class II ligands for presentation of peptide antigens to CD4+ T cells (e.g., for activation and/or expansion of CD4+ cells, or inhibition of CD4+ cells).
  • the HLA class II molecular complex comprises at least four fusion proteins.
  • Two first fusion proteins comprise (i) an immunoglobulin heavy chain and (ii) an extracellular domain of an HLA class II ⁇ chain.
  • Two second fusion proteins comprise (i) an immunoglobulin light chain and (ii) an extracellular domain of an HLA class II ⁇ chain. The two first and the two second fusion proteins associate to form the HLA class II molecular complex.
  • the extracellular domain of the HLA class II ⁇ chain of each first fusion protein and the extracellular domain of the HLA class II ⁇ chain of each second fusion protein form an HLA class II peptide binding cleft.
  • the immunoglobulin sequence of the HLA polypeptide ligand is a partial heavy chain sequence comprising the hinge region to support dimerization.
  • the HLA-Ig fusion construct contains no variable region sequences.
  • the HLA extracellular domain sequences i.e., HLA DB1/ 145613893.1 14 Attorney docket: NEX-013PC/107578-5013 class I alpha chain extracellular domain
  • an HLA or antigen presenting portion thereof may be conjugated to a CH1 portion of each IgG heavy chain.
  • All IgG molecules consist of two identical heavy chains (constant and variable regions) joined together by disulfide bonds in the hinge region (upper and lower).
  • an HLA molecule or antigen presenting complex is fused to the CH1 (N- terminal end of the Ig heavy chain above the hinge region), thereby creating a dimeric fusion protein that is smaller than one created by fusion to the ends of full antibody heavy chains, due to lack of any VH and VL light chain sequences.
  • constructs would further include CH2 and CH3 domains.
  • Such a construct provides manufacturing advantages, as well as exhibits less potential for immunogenicity. In some embodiments, these constructs also display sufficient binding cooperativity for efficient T cell activation or inhibition.
  • the IgG sequence is an IgG4 sequence
  • the immunoglobulin sequence may comprise or consist of (or consist essentially of) the amino acid sequence of SEQ ID NO: 23.
  • the HLA ligands are HLA class I ligands, and are optionally HLA-A, HLA-B, HLA-C, or HLA-E ligands.
  • the HLA ligand comprises associated beta 2 microglobulin ( ⁇ 2M) polypeptide.
  • the HLA ligand (e.g., as presented by an 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.
  • the HLA ligand is modified with cysteines that form a disulfide bond across the alpha helices that make up the peptide binding groove, to increase complex stability with bound peptide.
  • Such Cysteines in some embodiments are substituted at positions 84 and 139 of the extracellular domain. These modifications bridge the F-pocket, where the C-terminus of the peptide binds.
  • the HLA ligands are HLA-A*02:01 ligands (IMGT Accession No. HLA00005).
  • the HLA ligand is modified with cysteines that form a disulfide bond across the alpha helices that make up the peptide binding groove, to increase complex stability with bound peptide.
  • the HLA-A*02:01 ligand may be modified with cysteines at positions 84 and 139 as shown in SEQ ID NO: DB1/ 145613893.1 15 Attorney docket: NEX-013PC/107578-5013 26, to form the disulfide bond across the alpha helixes that make up the peptide binding groove.
  • the HLA-Ig ligand comprises the sequence of SEQ ID NO: 26, optionally having from one to five amino acid modifications selected from substitutions, deletions, and insertions, with the proviso that the positions of 84 and 139 with respect to SEQ ID NO: 26 are Cysteine.
  • the HLA class I (e.g., HLA-A*02) have W51C and G175C substitutions (e.g., with respect to SEQ ID NO: 26) forming a disulfide bond at end of the peptide groove where the N-terminus of the peptide binds.
  • the HLA class I (e.g., HLA-A*02) have F22C and S71C substitutions (e.g., with respect to SEQ ID NO: 26) forming a disulfide bond within one of the alpha helices that borders the peptide binging groove.
  • the recombinant HLA class I ligand is associated with a ⁇ 2 microglobulin protein.
  • the amino acid sequence of ⁇ 2 microglobulin is provided herein as SEQ ID NO: 8.
  • derivatives of ⁇ 2 microglobulin may be employed, for example, having from 1 to 10 or from 1 to 5 amino acid modifications independently selected from substitutions, deletions, and insertions.
  • the HLA ligands are HLA class II ligands.
  • the HLA-ligands are HLA-DR, HLA-DP, or HLA-DQ.
  • the HLA class II ligands comprise immunoglobulin fusions of HLA alpha and beta chains to antibody heavy and light chains as already described, thereby creating a dimeric HLA-class II antigen presenting complex.
  • the aAPCs contain HLA-E ligands, which are optionally HLA-E-Ig (e.g., as described).
  • the HLA-E ligands are 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 principal alleles.
  • HLA-E ligands may be adaptable to create a nearly universal aAPC platform.
  • HLA-E has a dual role in both the innate and adaptive immune systems.
  • the role of HLA-E in the innate immune response is to present peptides of other HLA class I molecules to inhibit Natural Killer (NK) cell-mediated lysis via recognition by NKG2A/CD94.
  • NK cells sense DB1/ 145613893.1 16 Attorney docket: NEX-013PC/107578-5013 the presence of HLA-E presenting self-peptides, and thereby receive inhibitory signals through the NKG2A/CD94 complex (inhibiting NK-mediated lysis).
  • HLA-E can also bind and present peptide sequences for recognition by T-cells (e.g., CD8+ T cells) (the adaptive immune response).
  • T-cells e.g., CD8+ T cells
  • the HLA-E molecule binds NKG2A/CD94 through a binding surface that overlaps with the binding surface for interacting with the T-cell Receptor (“TCR”).
  • TCR T-cell Receptor
  • the NK cell deactivating function of HLA-E is decoupled from the T cell activating function, by engineering point mutations that affect only the HLA- E binding to NKG2A/CD94, but not the binding of HLA-E to the TCR.
  • HLA-E amino acid substitutions are implemented to provide a stable peptide- binding cleft for presentation of bound antigen to HLA-E-restricted T cells.
  • a recombinant HLA-E ligand comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 21 (an HLA-E extracellular domain), and having a substitution to Cysteine at the amino acids corresponding to Y84 and A139 of SEQ ID NO: 21.
  • 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 to the amino acid sequence of SEQ ID NO: 21.
  • the amino acid sequence has one or more amino acid modifications with respect to SEQ ID NO: 21 that reduce or eliminate interaction with NKG2A/CD94.
  • the amino acid modifications are selected from a substitution of D162 and a substitution of E166 with respect to SEQ ID NO: 21. In various embodiments, the substitutions do not include acidic side chains.
  • 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 DB1/ 145613893.1 17 Attorney docket: NEX-013PC/107578-5013 E166Q.
  • the recombinant HLA-E ligand comprises the substitutions D162A and E166A with respect to SEQ ID NO: 21.
  • the HLA-E ligand comprises the substitution to Cys at Y84 and A139, as well as the substitution of D162 and E166.
  • engineered HLA-E polypeptide ligands are coupled to nanoparticles with co-stimulatory ligands to activate antigen-specific HLA-E restricted T cells against one or more tumor or infectious disease antigens.
  • engineered HLA-E polypeptide ligands are coupled to nanoparticles with co-inhibitory ligands to inhibit antigen-specific HLA-E restricted T cells against one or more autoantigens.
  • the immunoglobulin heavy chain sequences fused to the HLA can be any isotype, and in some embodiments are IgG.
  • the isotype is selected from IgG1, IgG3, IgG2 ⁇ , IgG2 ⁇ , and IgG4.
  • the immunoglobulin sequences are IgG4 Fc sequences.
  • the IgG4 Fc domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 23.
  • the IgG4 Fc domain comprises an amino acid sequence that has at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.
  • the recombinant HLA ligand comprises a linker between the HLA amino acid sequence, and the immunoglobulin sequences (e.g., IgG4 Fc domain).
  • the linker is a flexible linker, such as a linker that is predominately glycine and serine amino acid residues.
  • An exemplary flexible linker comprises the amino acid sequence of SEQ ID NO: 24.
  • linkers can be selected from flexible and rigid peptide linkers. Flexible linkers are predominately or entirely composed of small and/or polar residues such as Gly, Ser, and Thr.
  • An exemplary flexible linker comprises (GlyxSer)n linkers, where x is from 1 to 10 (e.g., from 2 to 6), and n is from 1 to about 10, and in some embodiments, is from 2 to about 6. In exemplary embodiments, x is from 2 to 4, and n is from 2 to 4. Due to their flexibility, these linkers are substantially unstructured. More rigid linkers include polyproline or poly Pro-Ala motifs and ⁇ -helical linkers. Generally, linkers of varying rigidity can be predominately composed of amino acids selected from Gly, Ser, DB1/ 145613893.1 18 Attorney docket: NEX-013PC/107578-5013 Thr, Ala, and Pro.
  • Exemplary linker sequences contain at least 5 amino acids, and may be in the range of 5 to 30 amino acids or in the range of 5 to 20 amino acids.
  • Peptide antigens are bound to an antigen binding cleft of the antigen presenting complex.
  • an antigenic peptide can be covalently bound to a peptide binding cleft.
  • a peptide tether can be used to link an antigenic peptide to a peptide binding cleft.
  • crystallographic analyses of multiple class I MHC molecules indicate that the amino terminus of ⁇ 2M is very close, approximately 20.5 Angstroms away, from the carboxyl terminus of an antigenic peptide resident in the MHC peptide binding cleft.
  • peptide antigens for immune therapy of oncological disease, infectious diseases, and autoimmune diseases are described herein.
  • the peptide antigen does not induce aggregation of the aAPC. That is, peptide antigens are selected with low aggregation propensity when loaded onto aAPCs. Peptides with low aggregation propensity in some embodiments are selected using an Aggregation Potential Score (APS), which can be assessed as described elsewhere herein.
  • APS Aggregation Potential Score
  • a peptide with low aggregation potential when loaded onto HLA ligands shows a mean APS of peptide residues 1 and 3-5 that is less than about 0.07.
  • the peptide antigen does not have an exposed Cysteine residue, 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).
  • the peptide antigen does not have more than 2, 3, or 4 hydrophobic residues, such as those selected from Phe, Val, Leu, and Ile.
  • the HLA ligand is HLA-A (e.g., HLA-A*02:01), and positions 1 and 3-5 contain at least one or at least two independently selected from Gly and charged residues (Glu, Asp, Lys, Arg, and His) and do not contain more than one exposed hydrophobic residue (e.g., Phe, Val, Leu, and Ile).
  • HLA-A HLA-A*02:01
  • positions 1 and 3-5 contain at least one or at least two independently selected from Gly and charged residues (Glu, Asp, Lys, Arg, and His) and do not contain more than one exposed hydrophobic residue (e.g., Phe, Val, Leu, and Ile).
  • the polypeptide ligands comprise a signal-2 ligand that is a co-stimulatory ligand, e.g., for activation and/or expansion of target T cells.
  • exemplary co-stimulatory ligands include agonists for any one of CD28, 4-1BB, CD27, OX-40, CD30, ICOS, and LIGHT, among others.
  • Co-stimulatory ligands can induce activation and/or expansion of CTLs or Tregs in various embodiments.
  • the polypeptide ligands do not comprise any signal 2 ligand or the polypeptide ligands comprise co-inhibitory ligands, which induce tolerance or apoptosis of target T cells.
  • the co-inhibitory ligand is an agonist for Fas, TGF- ⁇ , or PD-1.
  • Agonistic ligands can include natural agonistic ligands (or engineered variants thereof, including immunoglobulin fusions as described), or in some embodiments, antibody agonists.
  • the co-inhibitor ligand is PD-L1 (or immunoglobulin fusion thereof) or FasL (or immunoglobulin fusion thereof).
  • PD-L1-Ig and Fc-FasL fusion protein ligands are described herein (SEQ ID NO: 1 and SEQ ID NO: 4, respectively).
  • the immunoglobulin fusion sequences are IgG4 and variants thereof as described herein, and may comprise the amino acid sequence of SEQ ID NO: 23. Embodiments of these constructs are described in more detail elsewhere herein.
  • Antibody agonists can be full monoclonal antibodies, or portions or fragments comprising antigen-binding sequences, such as Fab, Fab', F(ab')2 or scFv.
  • the signal 1 and signal 2 ligands can be combined in homodimeric or heterodimeric constructs (e.g., homodimeric or heterodimeric Ig fusion constructs).
  • the HLA ligand can comprise fusion of HLA extracellular domains to an immunoglobulin Fc region, such as IgG4 Fc region, which can be dimerized (e.g., through disulfide bonds) with a signal 2-Immunoglobulin (Ig) fusion (i.e., a heterodimeric Ig fusion construct).
  • an immunoglobulin Fc region such as IgG4 Fc region
  • Ig 2-Immunoglobulin
  • the HLA ligand comprises a fusion to a signal 2 ligand.
  • an HLA extracellular domain can be fused at its C-terminus to an immunoglobulin Fc region (e.g., IgG4 Fc as already described), and fused at its N- terminus to a signal 2 ligand, to prepare homodimeric ligands with both signals dimerized.
  • the signal 2 ligand comprises a single chain antibody (e.g., scFv) or agonistic portion of a natural ligand. See SEQ ID NOS: 6 and 7, which comprise an anti- CD28 scFv fused to the N-terminus of HLA-Ig.
  • SEQ ID NO: 6 employs the scFv in VH- Linker-VL orientation
  • SEQ ID NO: 7 employs the scFv in VL-Linker-VH orientation.
  • DB1/ 145613893.1 20 Attorney docket: NEX-013PC/107578-5013 See also SEQ ID NO: 5, demonstrating fusion of PD-L1 activating fragment to the N- terminus of HLA-A-IgG4.
  • the co-stimulatory ligand is an agonistic antibody against CD28, which is optionally a humanized or human monoclonal antibody or a scFv based thereon.
  • the anti-CD28 antibody may be an IgG isotype (e.g., IgG4), and may be as described in US Patent No.10,632,193, which is hereby incorporated by reference in its entirety.
  • one, two, three, or more complementarity determining regions (CDRs) are based on mouse 9.3 mAb (Tan et al. J. Exp. Med. 1993177:165).
  • the antibody has the full set of heavy chain and/or full set of light chain CDRs of 9.3 mAb.
  • the heavy chain variable region contains one, two or three of the following CDRs, which optionally may each be 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).
  • the light chain contains one, two, or three of the following CDRs, which each may be 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).
  • CDR1 RASESVEYYVTSLMQ, SEQ ID NO: 12
  • CDR2 AASNVES, SEQ ID NO: 13
  • CDR3 QQSRKVPYT, SEQ ID NO: 14
  • Heavy chain variable and Light Chain amino acid sequences for exemplary humanized anti-CD28 agonistic antibodies are provided herein as SEQ ID NOS: 15 to 20.
  • the anti-CD28 antibody (or portion thereof) binds to the same or overlapping epitope as 9.3 mAb, or binds the same or overlapping epitope as an antibody having CDR1, CDR2, and CDR3 of 9.3 mAb.
  • Antibodies with the same or overlapping epitope can be selected by any suitable technique, including competitive immunoassays, using, for example, Surface Plasmon Resonance (Biacore).
  • Biacore Surface Plasmon Resonance
  • 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 in U.S. Pat.
  • the co-inhibitory ligand is an agonist antibody for Fas.
  • the agonist antibody for Fas is an IgG4 antibody based on clone CH11. SEQ ID NOS: 2 and 3 exemplify heavy chain variable region and light chain sequences for an IgG4 anti-Fas agonistic antibody.
  • the heavy and light chain sequences of SEQ ID NOS: 2 and 3 are humanized and comprise the CDRs of SEQ ID NOS: 2 and 3, shown below in Table 1: Table 1: Anti-Fas IgG4 Variable Light (SEQ ID NO: 3) Variable Heavy (SEQ ID NO: 2) DVVMTQSPLSLPVSLGDQASISC EVQLQQSGPELVKPGASVKISCKASGYT Y ant region and the constant region may be any isotype.
  • the antibody constant region is human IgG4 or variant thereof.
  • the constant region comprises one or more hinge stabilizing mutations, which may be introduced in the CH chain (e.g., S241P).
  • the antibody ligand comprises a constant region and the constant region comprises one or more mutations suitable for chemically coupling the antibody to a solid support.
  • the one or more mutations suitable for coupling create an unpaired cysteine.
  • An exemplary mutation in an IgG4 constant region is S473C.
  • Other changes to the constant region include those modifications to reduce Fc gamma receptor binding.
  • the CH chain may be modified at L248, e.g., L248E.
  • polypeptide ligands based on antibodies may be minimized such that the ligand is more suitable for functional attachment to nanoparticles.
  • the antibody may be an antibody fragment, such as F(ab') 2 or Fab, or is a single chain antibody (scFv), or other antigen-binding antibody fragment.
  • the antibody fragment can be a scFv of the humanized mAb described herein or other agonistic anti-CD28 antibody.
  • the antibody is a scFv comprising or consisting essentially of the antigen binding loops formed by the VH and VL chains of the monoclonal antibody (e.g., anti-CD28).
  • scFv antibody constructs may comprise one or several (2, 3, 4, or 5) VH and VL hypervariable region chains (the portion of each chain that together form the 3-D antigenic epitope binding pockets) linked together in head-head or head-tail configurations by short peptide linkers.
  • these constructs are fused to an HLA-Ig sequence as described, to create homodimeric constructs.
  • other ligand-binding formats are used to produce the co- stimulatory or inhibitory ligand, including peptides, aptamers, and AdNectins.
  • the various formats for target binding include a single-domain antibody, a recombinant heavy-chain- only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody; a Transbody, an Anticalin, an Affilin, a Microbody, a peptide aptamer, a phylomer, a stradobody, a maxibody, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troy
  • the aAPC further comprises one or more cytokines that support T cell activation and/or expansion or T cell inhibition.
  • the one or more cytokines or functional portion thereof may be conjugated to the aAPC as a polypeptide ligand.
  • the cytokine or functional portion thereof may be fused to a signal 1 or signal 2 polypeptide ligand (which can optionally be presented in homodimeric or heterodimeric Ig fusion constructs as described herein).
  • the cytokine is encapsulated by the copolymers, and will release cytokine locally in targeted environments (e.g., in lymphoid organs, tumor, or target tissue or organ).
  • cytokines include IL-1 ⁇ , IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and gamma interferon.
  • IL-2 can be employed with a co-stimulatory signal 2 ligand.
  • the aAPC comprises a tolerogenic cytokine as a polypeptide ligand.
  • An exemplary tolerogenic cytokine is IL-10.
  • the ligands further comprise one or more homing ligands for lymphoid organs.
  • an exemplary homing ligand is CD62L.
  • ligands (which may or may not be polypeptide ligands) are included to target the aAPCs to a tissue or organ or interest, such as the pancreas, intestine, lungs, liver, muscle, skin, etc. Suitable peptide ligands or other ligands can be selected based on information in the art.
  • the one or more peptide antigens are tumor or cancer associated antigens, such as tumor-derived antigens, tumor-specific antigens, and neoantigens.
  • T cells specific for tumor associated antigens are often very rare, and in many cases undetectable, in the peripheral blood of healthy individuals. Further, the cells are often of a naive 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 viral-specific and tumor antigen specific T cells.
  • the injectable aAPC of this disclosure can activate and/or expand such T cells in vivo, to produce an anti-tumor immune response.
  • DB1/ 145613893.1 24 Attorney docket: NEX-013PC/107578-5013 “Tumor-associated antigens” or “cancer specific antigens” include unique tumor or cancer antigens expressed exclusively by the tumor or malignant cells from which they are derived, shared tumor antigens expressed in many tumors but not in normal adult tissues (oncofetal antigens), and tissue-specific antigens expressed also by the normal tissue from which the tumor arose.
  • Tumor associated antigens can be, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressors, fusion proteins, or oncoviral proteins.
  • a variety of tumor-associated antigens are known in the art.
  • Oncofetal and embryonic antigens include carcinoembryonic antigen and alpha-fetoprotein (usually only highly expressed in developing embryos but frequently highly expressed by tumors of the liver and colon, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal, hepatic, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many carcinomas), pancreatic oncofetal antigen, 5T4 (expressed in gastriccarcinoma), alphafetoprotein receptor (expressed in multiple tumor types, particularly mammary tumors), and M2A (expressed in germ cell neoplasia).
  • carcinoembryonic antigen and alpha-fetoprotein usually
  • Mutated oncogene or tumor-suppressor gene products include Ras and p53, both of 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, myc (associated with lung cancer). Fusion proteins include BCR-ABL, which is expressed in chromic myeloid leukemia. Oncoviral proteins include HPV type 16, E6, and E7, which are found in cervical carcinoma.
  • Tissue-specific antigens include melanotransferrin and MUC1 (expressed in pancreatic and breast cancers); CD10 (previously known as common acute lymphoblastic leukemia antigen, or CALLA) or surface immunoglobulin (expressed in B cell leukemias and lymphomas); the ⁇ chain of the IL-2 receptor, T cell receptor, CD45R, CD4+/CD8+ (expressed in T cell leukemias and lymphomas); prostate specific antigen and prostatic acid- phosphatase (expressed in prostate carcinoma); GP100, MelanA/Mart-1, tyrosinase, DB1/ 145613893.1 25 Attorney docket: NEX-013PC/107578-5013 gp75/brown, BAGE, and S-100 (expressed in melanoma); cytokeratins (expressed in various carcinomas); and CD19, CD20, and CD37 (expressed in lymphoma).
  • CALLA common acute lymphoblastic
  • Tumor-associated antigens also include altered glycolipid and glycoprotein antigens, such as neuraminic acid-containing glycosphingolipids (e.g., GM2 and GD2, expressed in melanomas and some brain tumors); blood group antigens, particularly T and sialylated Tn antigens, which can be aberrantly expressed in carcinomas; and mucins, such as CA-125 and CA-19-9 (expressed on ovarian carcinomas) or the underglycosylated MUC-1 (expressed on breast and pancreatic carcinomas).
  • neuraminic acid-containing glycosphingolipids e.g., GM2 and GD2, expressed in melanomas and some brain tumors
  • blood group antigens particularly T and sialylated Tn antigens, which can be aberrantly expressed in carcinomas
  • mucins such as CA-125 and CA-19-9 (expressed on ovarian carcinomas) or the underglycosylated MUC-1 (expressed on breast and pancreatic carcinoma
  • 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.
  • a pathogen such as a viral, bacterial, fungal, or parasitic pathogen.
  • 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, herpes virus (e.g., HSV 1 or 2, or varicella zoster), and Adenovirus.
  • TB tuberculosis
  • HIV human immunodeficiency virus
  • HTLV human T-lymphotropic virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • HPV influenza
  • herpes virus
  • CMV for example, 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.
  • the one or more target peptide antigens is an “autoantigen”, meaning that it is associated with an autoimmune disease or reaction.
  • aAPCs carrying tolerogenic ligands induce tolerance in antigen-specific T cells to the target antigen.
  • aAPCs carrying apoptotic signals e.g., Fas ligand or agonist anti-Fas antibody
  • Autoantigens can be involved in autoimmune diseases such as type 1 diabetes, Goodpasture's 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, among others.
  • autoimmune diseases such as type 1 diabetes, Goodpasture's 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, among others.
  • peptide antigens for presentation by the HLA ligands are determined in a personalized manner, as described in US 10,098,939 and US 2020/0291381, which are hereby incorporated by reference in their entireties.
  • sequencing data can provide information about both shared as well as personalized targets for immunotherapy, such as for cancer.
  • mutant proteins are foreign to the immune system and are putative tumor-specific antigens.
  • sequencing efforts have defined hundred if not thousands of potentially relevant immune targets. Studies have shown that T cell responses against these neo-epitopes can be found in cancer patients or induced by cancer vaccines.
  • Neoepitopes predicted from DNA or RNA sequencing of a patient’s tumor can be tested for their activation potential in association with the HLA ligand, by determining whether (or to what extent) an aAPC carrying the predicted antigen in association with the HLA ligand is able to activate T cells from the subject. Similar assays can be employed to identity relevant autoantigens.
  • peptide antigens for treatment of type 1 diabetes, can be antigens from ICA, insulin, G6, GAD2, GAD65, insulinoma antigen-2, HSP, IGRP, imogen- 38, PDX1, ZnT8, CHGA, and IAAP. See, e.g., Han, S, et al. Novel autoantigens in type 1 diabetes. Am J Transl Res.2013; 5(4): 379–392.
  • Non-limiting examples of peptide antigens include those shown in the following Table 2.
  • utical composition suitable for administration to a subject.
  • the pharmaceutical composition may have one or more excipients such as buffering agents, surfactants, preservative agents, polymers, bulking agents, and stabilizers. Buffering agents 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.
  • Bulking agents are used during lyophilization to add DB1/ 145613893.1 28 Attorney docket: NEX-013PC/107578-5013 bulk.
  • Hydrophilic polymers such as dextran, hydroxyl ethyl starch, polyethylene glycols, and gelatin can be used to stabilize proteins. Polymers with nonpolar moieties such as polyethylene glycol can also be used as surfactants.
  • Protein stabilizers can include polyols, sugars, amino acids, amines, and salts. Suitable sugars include sucrose and trehalose. Amino acid stabilizers include histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, and mixtures thereof. Proteins like human serum albumin can also competitively adsorb to surfaces and reduce aggregation of the active agent. Particular formulation components can serve multiple purposes. For example, histidine can act as a buffering agent and an antioxidant. Glycine can be used as a buffering agent and as a bulking agent. In some embodiments, the pharmaceutical composition is lyophilized.
  • the present disclosure provides aAPCs suitable for parenteral administration (including subcutaneous administration), and which have low aggregation propensity.
  • the aAPC comprises a polymeric or lipid nanoparticle comprising a polyethylene glycol (PEG) sheath and one or more polypeptide ligands conjugated to PEG (e.g., the PEG terminus), which can be through a thioether bond or other functional group.
  • the polypeptide ligands comprise HLA Class I or Class II ligands presenting a peptide antigen and optionally one or more signal 2 ligands (as already described). The peptide antigen for presentation to T cells does not induce aggregation of the aAPC.
  • the peptide antigen does not have an exposed Cysteine and/or the peptide antigen has one or more exposed glycine residues or exposed charged residues. In some embodiments, the peptide antigen does not have any Cysteine residue, and contains one or more charged residues (e.g., 1, 2, or 3 charged residues).
  • the peptide antigen in the HLA binding cleft has one or more exposed amino acids selected from glycine, aspartic acid, glutamic acid, lysine, arginine, and histidine; and does not have an exposed cysteine.
  • positions 1 and 3 to 5 of the peptide satisfy these DB1/ 145613893.1 29 Attorney docket: NEX-013PC/107578-5013 criteria.
  • the term “exposed” means that the side chain of the amino acid is surface exposed.
  • the HLA is HLA-A, and in some embodiments is HLA-A2, such as HLA-A*02:01.
  • aggregation potential is estimated in silico by determining the mean Aggregation Potential Score (APS) of antigen peptide residues within the HLA antigen binding cleft.
  • APS mean Aggregation Potential Score
  • peptide positions 1 and 3-5 are evaluated for APS and an average score determined for these positions. For example, a score of less than about 0.07 is indicative of a non-aggregating peptide.
  • Protein modeling and aggregation propensity analyses can be performed using Discovery Studio 2021 (DS2021, Dassault Systdiags BIOVIA, Discovery Studio Modeling Environment, Release 2021, San Diego CA).
  • the MODELLER program can be used for homology or comparative modeling of protein three-dimensional structures (e.g., HLA with bound peptide).
  • Spatial aggregation propensity can be calculated according to available tools, and which are included in the DS2021 package and otherwise commercially available.
  • the spatial aggregation propensity (SAP) score synthesizes the solvent accessible area of a residue side chain with a hydrophobicity score based on a scale developed by Black and Mould (1991) where glycine has a value of zero and other residues are scaled positively if more hydrophobic and negatively if less hydrophobic.
  • the process is initiated by generating molecular models of candidate relevant peptide-HLA complexes.
  • the protein model includes the relevant peptides positioned in the HLA binding groove.
  • the MODELLER algorithm as implemented in Discovery Studio 2021 can be used, or other suitable software. Multiple template structures can be used for the modeling steps, and manual sequence alignments are performed on each model.
  • PDB accessions for template structures used in HLA-A*02:01 modeling include: 4L29, 1I7R, 5EUO, 1TVB, 6OPD, 6TRO, 6AMT, 6AM5, 2GT9, and 3OXS.
  • Aggregation Scores can be calculated.
  • radii of 5 or 10 Angstroms are selected for calculating scores. This parameter determines how many nearby amino acid residues to include in the aggregation potential calculation. This is similar to a sliding window for protein sequence parameter calculations.
  • the radius can give an appropriately smoothed surface map that is not overly noisy.
  • the protocol calculates exposed surfaces for each residue and calculates an aggregation potential score based on the averaging of each residue and those in the defined radius surrounding the residue.
  • the numerical score (APS) is recorded with the sequence and structure information and is used to generate a surface map based on the solvent-accessible surface of the model. This provides an easy visualization of the shape of the surface coupled with the scored aggregation potential. In some instances, manual inspection of these results is sufficient to bin a peptide as an aggregator or non-aggregator.
  • the APS of residues 1 and 3-5 of HLA-A peptide complex are averaged. If this score is above a current threshold of 0.07, this is considered a factor for aggregation.
  • the nanoparticles are polymeric nanoparticles comprising poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)- polyethylene glycol (PLGA-PEG) copolymers and one or more polypeptide ligands conjugated to PEG through a thioether bond.
  • PVA-PEG poly(lactic acid)-polyethylene glycol
  • PLGA-PEG poly(lactic acid-co-glycolic acid)- polyethylene glycol
  • Alternative polymers that can be used in connection with the aAPC platforms described herein include one or more of 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-maieimide (PLGA-PEG-mal), PLA-PEG-maleimide, poly(D,L-lactide-co-caprolact
  • the nanoparticles are lipid nanoparticles comprising a PEG- conjugated lipid.
  • Exemplary PEG lipids are selected from one or more of a PEG-modified phosphatidylethanolamine, a PEG- modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, and a PEG-modified dialkylglycerol.
  • a PEG lipid may be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-Cholesterol, PEG tocopherol, or a PEG- DSPE lipid.
  • the lipid nanoparticles further comprise a cationic or ionizable lipid, a neutral lipid or phospholipid, and a structural lipid.
  • exemplary structural lipids can be selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and tocopherols (e.g., alpha tocopherol).
  • the structural lipid is cholesterol.
  • the LNP comprises one or more phospholipids.
  • Exemplary phospholipids are selected from the group consisting of cardiolipins, sterol modified lipids (modified with a cholesterol moiety attached at the sn-2 carbon of the glycerol backbone), mixed-acyl glycerophospholipids, and symmetrical acyl glycerophospholipids.
  • Head groups for acyl glycerophospholipids include, for example, phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidylserine.
  • Exemplary phospholipids are selected from 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), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether DB1/ 145613893.1 33 Attorney docket: NEX-013PC/107578-5013 PC
  • the nanoparticle is a lipid nanoparticle that further comprises a polynucleotide for expression in target T cells.
  • the polynucleotide e.g., mRNA
  • encodes a cytokine including a cytokine described herein (e.g., IL-2 or IL- 10).
  • sulfhydryl groups on the polypeptide ligands are coupled to PEG-maleimide functional groups of the lipid nanoparticles.
  • the lipid nanoparticle aAPC has 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 by reducing the proportion of PEG moieties having a maleimide group for conjugation as already described, or by modifying the level of PEG lipid in the composition.
  • the lipid nanoparticle aAPC has a diameter of from about 50 nm to about 150 nm, or a diameter of from about 60 nm to about 130 nm, or a diameter of from about 80 nm to about 120 nm. In various embodiments, the aAPC has a diameter of about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm.
  • the lipid nanoparticle aAPC has a surface charge of from about 0 to -15 mV, or from about 0 to about -10 mV, or a surface charge of from about -2.5 mV to about -10 mV.
  • the aAPC population has a size distribution with polydispersity index (PDI) of less than 0.2.
  • the aAPC is comprised in a pharmaceutical composition as described and may be optionally lyophilized.
  • the aAPCs and DB1/ 145613893.1 34 Attorney docket: NEX-013PC/107578-5013 pharmaceutical compositions described herein are useful for treating patients with infectious diseases, cancer, or autoimmune diseases, or to provide prophylactic protection to immunosuppressed patients.
  • the invention provides a method for immunotherapy.
  • the method comprises administering the aAPC or pharmaceutical composition thereof as described herein to a subject in need of treatment.
  • the aAPC and compositions thereof described herein are used for immunotherapy.
  • the subject has cancer or an infectious disease
  • the aAPCs comprise a co-stimulatory ligand.
  • Infectious diseases that can be treated include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc.
  • Such diseases include human papilloma virus (HPV) (and related cancers), AIDS, adult T-cell leukaemia/lymphoma (ATL), hepatitis, CMV infection, and post-transplant lymphoproliferative disorder (PTLD).
  • HPV human papilloma virus
  • ATL adult T-cell leukaemia/lymphoma
  • hepatitis CMV infection
  • PTLD post-transplant lymphoproliferative disorder
  • CMV for example, 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 due to the immunocompromised status of these patients, which permits reactivation of latent virus in seropositive patients or opportunistic infection in seronegative individuals.
  • EBV infection is believed to be present in approximately 90% of the adult population in the United States. Active viral replication and infection is kept in check by the immune system, but, as in cases of CMV, individuals immunocompromised by transplantation therapies lose the controlling T cell populations, which permits viral reactivation. This represents a serious impediment to transplant protocols. EBV may also be involved in tumor promotion in a variety of hematological and non-hematological cancers.
  • Cancers that can be treated according to this disclosure include melanoma, carcinomas, e.g., colon, head and neck cancer, duodenal, prostate, breast, lung, ovarian, ductal, hepatic, pancreatic, renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma etc.; neurological malignancies, e.g., neuroblastoma, gliomas, etc.; hematological malignancies, e.g., chronic myelogenous leukemia, childhood acute leukemia, non- Hodgkin's lymphomas, chronic lymphocytic leukemia
  • the subject has a solid tumor, which can be Stage I, Stage II, Stage III, or Stage IV cancer.
  • the cancer is metastatic and/or recurrent, and/or is nonresectable.
  • the patient is refractory or only partially responsive to chemotherapy and/or immune checkpoint inhibitor therapy.
  • the invention provides a method for treating cancer, including those cancers identified above, through administration of the pharmaceutical composition described herein to activate T-cells having anti-tumor activity.
  • the therapy is provided together with one or more immune checkpoint inhibitors, such as Nivolumab, Pembrolizumab, and Ipilimumab.
  • 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 may be administered as an additional ligand to the nanoparticles described herein, or attached to a separate population of nanoparticles.
  • the one or more immune checkpoint inhibitors are provided as initial therapy, and therapy with the aAPCs described herein initiated subsequently, for example, after from about 1 to about 8 weeks of checkpoint inhibitor therapy, or after about 2 to about 4 weeks of checkpoint inhibitor therapy.
  • the one or more checkpoint inhibitors are provided concomitantly with the nanoparticle therapy, for example at initiation of therapy and about every two weeks, or at initiation of therapy and about every two weeks for the one or more checkpoint inhibitors DB1/ 145613893.1 36 Attorney docket: NEX-013PC/107578-5013 and about every four weeks for the nanoparticle therapy.
  • the patient is resistant or shows only a partial or transient response to checkpoint inhibitor therapy, and the aAPCs described herein enhance tumor regression in these patients.
  • the compositions described herein expand the successful use of checkpoint inhibitors to such cancers.
  • the peptide antigen is selected in a personalized basis for the patient, 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 hereby incorporated by reference) may be used, or other process.
  • the nanoparticles can be provided (on an “off-the shelf” basis), and tumor antigens selected and loaded in a personalized basis.
  • Other processes for selecting peptides based on their ability to activate and expand relevant T cells are described in US Patents 10,987,412 and 10,098,939, which are hereby incorporated by reference in their entireties.
  • the nano-aAPCs are used as a booster vaccine, after adoptive T cell therapy, in which naive T cells from the patient or T cells from an HLA-matched donor are expanded ex vivo and administered to the patient.
  • the nano aAPC composition may be administered from 1 to about 10 times over the course of from 4 months to about 1 year to enhance cancer immunity in these embodiments.
  • the subject has an autoimmune condition, and the aAPCs comprise a co-inhibitory signal or do not contain a signal 2 ligand.
  • the autoimmune condition is type 1 diabetes. In some embodiments, the autoimmune condition is vitiligo.
  • Autoimmune diseases that can be treated include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture's syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, Sjogren’s Disease, Hashimoto's thyroiditis, alopecia, ankylosing spondylitis, scleroderma, and HTLV-1-Associated Myelopathy (HAM)/Tropical Spastic Paraparesis (TSP), among others.
  • HAM HTLV-1-Associated Myelopathy
  • TSP HTLV-1-Associated Myelopathy
  • the aAPC or pharmaceutical composition thereof is parenterally administered.
  • the aAPC or pharmaceutical composition thereof is administered by intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intra-lymphatic administration, intramuscular administration, or intratumoral administration.
  • the aAPC composition is administered by subcutaneous administration.
  • polypeptide ligands for immune therapy are disclosed.
  • Such polypeptide ligands include an anti-Fas agonistic antibody having an IgG isotype (e.g., IgG4), which can be conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen (e.g., associated with an autoimmune disease).
  • the anti-Fas antibody comprises a heavy chain of SEQ ID NO: 2, optionally having from one to ten, or from one to five, amino acid substitutions. Amino acid substitutions are optionally in the Fc domain, and may include amino acid substitutions with known pharmacological or stability advantages, or advantages in removing potential immunogenicity.
  • the amino acid sequence of SEQ ID NO: 2 is humanized and comprises the same complementarity determining regions (CDRs) of SEQ ID NO: 2 (see Table 1) with no more than 1, 2, or 3 amino acid substitutions.
  • the anti-Fas antibody comprises a light chain of SEQ ID NO: 3 (see Table 1), optionally having from one to ten, or from one to five, amino acid substitutions.
  • the amino acid sequence of SEQ ID NO: 3 is humanized and comprises the same CDRs of SEQ ID NO: 3, with no more than 1, 2, or 3 amino acid substitutions.
  • the polypeptide ligand is a dimeric PD-L1 ligand comprising an activating portion of PD-L1, such as amino acids residues F19 to T239 of human PD-L1.
  • an additional 20 residues can be included (collectively on one or both termini), or up to 10 amino acids deleted from F19 to T239.
  • Each PD-L1 activating fragment may be fused directly or indirectly through a linker at its C-terminus to an IgG Fc region (e.g., IgG4), and the ligand may be dimerized by disulfide bonds in the Fc region.
  • Suitable linkers are described elsewhere herein, and include flexible linkers such as Gly Ser linkers.
  • the dimeric DB1/ 145613893.1 38 Attorney docket: NEX-013PC/107578-5013 PD-L1 ligand can be conjugated to nanoparticles together with an HLA ligand presenting a peptide antigen and used to drive tolerance to the antigen.
  • An exemplary PD-L1-IgG4 is shown in SEQ ID NO: 1.
  • the polypeptide ligand is a dimeric FasL ligand comprising an activating portion of FasL, such as amino acids P132 to L279 of human FasL fused directly or indirectly through a linker at its N-terminus to a dimerized IgG-Fc region (e.g., IgG4).
  • an additional 20 residues can be included (collectively on one or both termini), or up to 10 amino acids deleted from P132 to L279.
  • the dimerized Fc region can be conjugated to nanoparticles through a Cys-containing linker in some embodiments.
  • An exemplary construct according to these embodiments is provided by SEQ ID NO: 4.
  • the nanoparticles may further present HLA-peptide antigen ligands to drive apoptosis of antigen-specific T cells.
  • the polypeptide ligand is a tolerogenic ligand comprising an activating fragment of PD-L1, such as amino acids F19-T239 of human PD-L1, fused directly or through a linker to HLA-immunoglobulin fusion protein.
  • an additional 20 residues can be included (collectively on one or both termini), or up to 10 amino acids deleted from F19 to T239 of human PD-L1.
  • These embodiments provide a homodimeric construct that is homodimeric for both signal 1 and inhibitory signal 2 ligands. See SEQ ID NO: 5.
  • Such ligands can be conjugated to nanoparticles as disclosed herein and used for immunotherapy (to drive tolerance in targeted T cells).
  • the polypeptide ligand is a co-stimulatory ligand comprising an anti-CD28 agonistic scFv conjugated to an HLA-immunoglobulin fusion protein, providing homodimeric ligands comprising signal 1 and co-stimulatory signal 2 ligands.
  • the scFv may be fused to the HLA sequence through the heavy or light chain sequence.
  • SEQ ID NO: 6 employs the scFv in VH-Linker-VL orientation
  • SEQ ID NO: 7 employs the scFv in VL-Linker-VH orientation.
  • identity refers to the similarity between a pair of sequences (nucleotide or amino acid). Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST, are available for determining sequence identity.
  • CDR refers to a complementarity-determining region. CDRs are part of the variable chains in immunoglobulins (antibodies). A set of CDRs constitutes a paratope.
  • This invention is further illustrated by the following non-limiting examples.
  • Example 1 Construction of Injectable Artificial Antigen Presenting Cells Ligands for aAPCs, including signal 1 ligands (HLA) and signal 2 ligands are constructed with free sulfhydryl groups for coupling to particles. See US 10,632,193, which is hereby incorporated by reference in its entirety.
  • Naked polymer nanoparticles were prepared from PLGA-PEG or PLA-PEG copolymers by nanoprecipitation using known DB1/ 145613893.1 40 Attorney docket: NEX-013PC/107578-5013 processes.
  • PLGA and PLA portions of the block copolymer were about 20 kDa.
  • PEG portions of the block co-polymers were either 3000 Da or 5000 Da. Specifically, PEG polymers having functional groups for ligand conjugation were designed to be longer (5000 Da), while the inert PEG polymers were shorter (3000 Da) to reduce steric effects.
  • NANOASSEMBLR IGNITE Precision Nanosystems
  • Nanoparticles include a population of copolymers having a terminal maleimide functional group (PEG-maleimide) for ligand coupling.
  • Thiolated ligands are conjugated to naked particles through the terminal maleimide functional groups and purified by known techniques. See US 10,632,193.
  • HLA ligands on the nanoparticles are loaded by incubating with excess peptide antigen.
  • Peptide-loaded nanoparticles can be separated using an SEC column for example. Using these processes, particles can be obtained having a size and surface charge suitable for parenteral administration for immunotherapy.
  • FIG. 1A shows TEM images of naked particles, protein-conjugated nanoparticles, and peptide loaded nanoparticles. As shown in FIG.
  • the particles have an average diameter of about 100 nm.
  • the size with ligands conjugated and peptide loaded are slightly larger.
  • the polydispersity index was shown to be less than 0.2.
  • the surface charge was shown to be within 0 and -10 mV.
  • the targeted range of ligand density was 100 to 400 ligands per particle.
  • Experiments were conducted to vary the density of PEG-maleimide functional groups on the surface of the particle, within the ranges determined to allow for stable particles (1% to 10% by weight). Specifically, particles were prepared with PLGA-PEG where a portion of the PEG termini contained maleimide functional groups. Maleimide:Thiol (Mal:Thiol) in the conjugation reaction was kept constant at 1:1.
  • 5% PEG- maleimide beads for PLGA-PEG in these examples corresponds to a 3:1 ratio (by weight) of PLGA-mPEG : PLGA-PEG-maleimide.
  • 5% PEG-maleimide corresponds to a ratio of about 5.67 : 1 (by weight) of PLA-mPEG : PLA-PEG-maleimide.
  • Nanoparticles were constructed with PLGA-PEG and PLA-PEG nanoparticles for comparison. As shown in FIGs. 4A and 4B, differences were not significant. PLA-PEG showed a small reduction in size and protein density.
  • PLGA-PEG and PLA-PEG nanoparticles having signal 1 ligands HLA-Ig ligands loaded with MART-1 antigen
  • signal 2 ligands antibody agonist against CD28
  • FIGs. 5A-5D no significant differences were seen between PLGA-PEG and PLA-PEG in terms of T cells positive for INF ⁇ (FIG. 5A), TNF ⁇ (FIG. 5B), IL-2 (FIG. 5C), and CD107a (FIG. 5D).
  • T cells prepared by enrichment and expansion (“AIM-ACT”) were used as a reference.
  • Table 6 D14 Memory Phenotypes Tcm% Tn/Tscm% Tem% Temra% As shown in FIG. 6, antigen-loaded aAPCs prepared essentially according to this example, when administered systemically to tumor-bearing mice, traffic to lymph nodes, spleen, and tumor (right panel), as compared to naked nanoparticles (left panel).
  • FIGS. 7A-7C As shown in FIG. 8A-C, the phenotype of T cells recovered from lymph nodes and tumors (after aAPC administration) have a phenotype consistent with persistence and strong anti-tumor effect. Further, aAPC administration (loaded with GP100 antigen) extended overall survival in B16 model (see FIG.9).
  • Example 2 Construction of anti-Fas Agonistic Antibody Ligand CH11 is an IgM activating antibody against human Fas. The antibody demonstrates cytolytic activity on human cells that express Fas.
  • variable domains from CH11 were grafted to an IgG4 framework described in US Patent 10,632,193. See FIG.10A.
  • the heavy chain 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 an anti-IgG4 antibody) were assayed in vitro for induction of apoptosis of CD8+ T cells (10 5 cells per well).
  • FIG.10B while the pentameric CH11 induces a strong apoptotic effect on CD8+ T cells, non-crosslinked anti-Fas IgG4 has negligible effects.
  • Example 3 Injectable aAPC bearing PD-L1 or anti-Fas Signal 2 ligands Injectable aAPCs were created as described in Example 1, and with PD-L1 Signal 2 ligands (SEQ ID NO: 1).
  • these aAPCs rapidly inhibit antigen-specific killing of peptide-loaded target cells.
  • MART-1-specific CD8+ T cells were incubated for 90 minutes with 50 ⁇ g/mL of aAPCs. After washing, cells were incubated with peptide-loaded target cells for 4 hours and antigen-specific killing was assessed by caspase 3/7 assay. A 50% reduction of antigen-specific cytotoxic killing was observed.
  • DB1/ 145613893.1 44 Attorney docket: NEX-013PC/107578-5013 Injectable aAPCs were created as described in Example 1, and with anti-Fas Signal 2 ligands (as described in Example 2).
  • FIG.12 shows 50% elimination of MART-1 specific T cells after a 4-hour incubation in vitro. Non-target T cells were not affected. For example, aAPC presenting a survivin peptide did not impact the number of MART-1 specific T cells. As shown in FIG.13, the 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.
  • Example 4 Selection of Peptide Antigens for aAPCs It was observed that certain peptide antigens, when loaded onto nanoparticles (aAPCs), would induce aggregation of the particles.
  • Protein modeling and aggregation propensity analyses were performed using Discovery Studio 2021 (DS2021), a software product from BIOVIA (Dassault Systemes BIOVIA, Discovery Studio Modeling Environment, 2021, San Diego CA). Calculation of aggregation propensity scores based on Trout’s method is included in DS2021.
  • the MODELLER program (UCSF, San Francisco CA) was used for homology and comparative modeling of protein three-dimensional structures. The process for identifying peptide antigens with an aggregation propensity when presented by HLA ligands on nanoparticles is shown diagrammatically in FIG.14.
  • HLA- A:0201 structure models include: 4L29, 1I7R, 5EUO, 1TVB, 6OPD, 6TRO, 6AMT, 6AM5, 2GT9, and 3OXS. Modeling of the complexes is based on generating a standard comparative protein model of the relevant peptides positioned in the HLA binding groove.
  • the MODELER algorithm as implemented in Discovery Studio 2021 was employed. Multiple template structures are used for the modeling steps, and manual sequence alignments are performed on each model.
  • the ‘Calculate Aggregation Scores’ protocol as implemented in Discovery Studio 2021 is run.
  • Radii of 5 and 10 DB1/ 145613893.1 45 Attorney docket: NEX-013PC/107578-5013 Angstroms was selected as radii for calculating scores. This parameter determines how many nearby amino acid residues to include in the aggregation potential calculation. Depending on the size of the model being analyzed, the radius can give an appropriately smoothed surface map that is not overly noisy.
  • the protocol calculates exposed surface for each residue of the peptide antigen and calculates an aggregation potential score based on the averaging of each residue and those in the defined radius surrounding the residue.
  • the numerical aggregation potential score (APS) is recorded with the sequence and structure information and is used to generate a surface map based on the solvent-accessible surface of the model.
  • Peptides can be binned as aggregators or non-aggregators by manual inspection or computationally. 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. If above a threshold of 0.07, this is considered a factor for aggregation. The presence of an exposed Cys residue (as determined by inspection of the model structures) is considered another factor for aggregation. Presence of charged residues and Gly residues are considered factors against aggregation.
  • FIG. 15A and 15B illustrate modelling and scoring of aggregating and non- aggregating peptides, respectively. This process was used for a set of known aggregators and non-aggregators as summarized in Table 7 below.
  • Table 7 Aggregation Analysis Peptide Name Known Predicted DB1/ 145613893.1 46 Attorney docket: NEX-013PC/107578-5013 CMTWNQMNL WT1.235.M0010 x x (SEQ ID NO: 47) FLDRFLSCM CYCLIN.227.M0013 REFERENCES B. Webb, A. Sali.
  • T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface.
  • 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.
  • 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.

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Abstract

Dans divers aspects et modes de réalisation, la présente divulgation concerne des cellules présentatrices d'antigène artificielles (aAPC) qui sont appropriées pour une administration parentérale pour une thérapie immunitaire. Dans divers modes de réalisation, les aAPC sont efficaces pour activer ou inhiber des lymphocytes T cibles in vivo, comprenant des lymphocytes T CD8+ ou CD4+. Les aAPC selon la présente divulgation fournissent une plate-forme de nanoparticules à longue durée de conservation pour des thérapies immunitaires. La plate-forme de nanoparticules à longue durée de conservation dans divers modes de réalisation régule la taille des particules et la chimie des particules, la conception des ligands et la densité des ligands, et la propension à l'agrégation des aAPC, entre autres caractéristiques.
PCT/US2024/021277 2023-03-24 2024-03-25 Cellules présentatrices d'antigène artificiel injectables pour une thérapie immunitaire Pending WO2024206207A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120115223A1 (en) * 2006-10-04 2012-05-10 Zeling Cai Preparation of inactivated artificial antigen presenting cells and their use in cell therapies
WO2022015880A2 (fr) * 2020-07-14 2022-01-20 Cue Biopharma, Inc. Polypeptides modulateurs des lymphocytes t ayant des sites de conjugaison et procédés d'utilisation associés
US11510981B2 (en) * 2014-12-24 2022-11-29 Neximmune, Inc. Nanoparticle compositions and methods for immunotherapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120115223A1 (en) * 2006-10-04 2012-05-10 Zeling Cai Preparation of inactivated artificial antigen presenting cells and their use in cell therapies
US11510981B2 (en) * 2014-12-24 2022-11-29 Neximmune, Inc. Nanoparticle compositions and methods for immunotherapy
WO2022015880A2 (fr) * 2020-07-14 2022-01-20 Cue Biopharma, Inc. Polypeptides modulateurs des lymphocytes t ayant des sites de conjugaison et procédés d'utilisation associés

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