WO2023288056A2

WO2023288056A2 - Compositions and methods for expansion of hla-e-restricted t cell populations

Info

Publication number: WO2023288056A2
Application number: PCT/US2022/037279
Authority: WO
Inventors: Adam PARKS; Daniel Bednarik; Mathias Oelke; Han MYINT; Charles Reed
Original assignee: Neximmune, Inc.
Priority date: 2021-07-15
Filing date: 2022-07-15
Publication date: 2023-01-19
Also published as: WO2023288056A8; WO2023288056A3

Abstract

In various aspects and embodiments, the invention provides recombinant HLA- E ligands engineered for presentation of peptide antigens to HLA-E-restricted T cells, and HLA-E ligands engineered to reduce or eliminate interaction with NKG2A/CD94. The HLA-E ligands described herein are useful for aAPC platforms for modulating T cell functions in vivo or ex vivo.

Description

COMPOSITIONS AND METHODS FOR EXPANSION OF HLA-E-RESTRICTED T

CELL POPULATIONS

BACKGROUND

Antigen Presenting Cells (APCs), including Artificial Antigen Presenting Cells (aAPC), are an important tool for immunotherapy, for modulating T cell function and/or activating and expanding T cells ex vivo or in vivo. However, there is a significant challenge in implementing APCs using human leukocyte antigen (HLA) ligands to modulate T cell responses in an antigen-specific fashion. Specifically, the three major HLA genes (e.g., HLA- A, HLA-B, and HLA-C) are highly polymorphic. Thus, APC platforms that employ these HLA ligands require the production and engineering of many different HLA ligands corresponding to the various alleles, as well as identification of corresponding antigen peptides. More universally applicable HLA ligands are desirable, including but not limited for use in APC platforms. Various aspects and embodiments of the invention meet these objectives.

Other aspects and embodiments will be apparent from the following detailed description.

SUMMARY OF THE DISCLOSURE

In various aspects and embodiments, the invention provides recombinant HLA- E ligands engineered for presentation of peptide antigens to HLA-E-restricted T cells, and HLA-E ligands engineered to reduce or eliminate interaction with NKG2A/CD94. The HLA-E ligands described herein are useful for aAPC platforms for modulating T cell functions in vivo or ex vivo.

In accordance with aspects and embodiments of this disclosure, recombinant HLA-E ligands are provided that are engineered for modulating CD8+ T cell function. In various embodiments, the invention decouples the NK cell deactivating function of HLA-E 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. These point mutations enable the redirection of HLA-E for modulating HLA-E-restricted T cells, while avoiding HLA-E exhaustion of Natural Killer cells. In accordance with other aspects and embodiments, 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. Disclosed herein is a recombinant HLA-E ligand comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 , and having a substitution to Cysteine at the amino acids corresponding to Y84 and A139 of SEQ ID NO: 1. SEQ ID NO: 1 is an HLA-E extracellular domain. These substitutions allow for the formation of a disulfide bond that stabilizes the peptide binding cleft.

Alternatively or in addition, the amino acid sequence has one or more amino acid modifications with respect to SEQ ID NO: 1 that reduce or eliminate interaction with NKG2A/CD94. In some embodiments, the amino acid modifications are selected from a substitution of D162 and a substitution of E166 with respect to SEQ ID NO: 1. In various embodiments, the substitutions do not include acidic side chains. For example, in some embodiments, the substitution at D 162 is selected from D162A, D162G, D162L, D162V, D162I, D162S, D162T, D162M, D162N, and D162Q; and the substitution at E166 is selected from E166A, E166G, E166L, E166V, E166I, E166S, E166T, E166M, E166N, and E166Q. In some embodiments, the recombinant HLA-E ligand comprises the substitutions D162A and E166A with respect to SEQ ID NO: 1. In some embodiments, the HLA-E ligand comprises the substitution to Cys at Y84 and A139, as well as substitution of D162 and El 66.

In various embodiments, the recombinant HLA-E ligand comprises an HLA-E amino acid sequence fused at its C-terminus to a second amino acid sequence, optionally through a linker. The second amino acid sequence can allow for conjugation to various supports, including particle supports useful for aAPC platforms. In some embodiments, the second amino acid sequence assembles as a multimer, such as, for example a dimer, allowing for engagement of multiple TCRs simultaneously. In some embodiments, the HLA-E ligands are monomeric, but their close association on a nanoparticle support is sufficient for avidity and activation. Dimeric HLA-E ligands can be constructed by fusion to immunoglobulin heavy chain sequences, which are then associated through one or more disulfide bonds (with or without associated light chains). HLA-E multimers can be created by direct tethering through peptide or chemical linkers, HLA-E ligands can be multimeric via association with streptavidin through biotin moieties. In various aspects and embodiments, the invention provides artificial antigen presenting cells (aAPC) comprising the HLA-E ligand. The aAPC is useful for modulating HLA-E-restricted T cell responses in vivo or ex vivo. In various embodiments, the aAPC carries additional ligands (or “signals”) to modulate T cell function. For example, the aAPC may present a “Signal 2”, which is generally a lymphocyte co-stimulatory ligand or a lymphocyte inhibitory ligand. In various embodiments, the aAPC may include a Signal 3, for example conjugated to the particle or encapsulated by the particle. Signal 3 ligands include cytokines that support T cell activation and expansion. In various embodiments, the aAPC is suitable for activating and/or expanding

HLA-E-restricted T cells in vitro or ex vivo. In these embodiments, the particle core may be polymeric or paramagnetic, or any other suitable material. In various embodiments, the particle has a size (e.g., average diameter) within about 10 to about 500 nm. Magnetic fields can be used ex vivo to manipulate cell-bound paramagnetic nanoparticles to drive activation via TCR clustering. For in vivo applications, nanosized particles, such as less than about 200 nm or less than about 150 nm, or less than about 100 nm are desired to allow the aAPCs to traffic to desired organs or to tumors, and to avoid occlusion of blood vessels.

In various embodiments, the one or more peptide antigens are tumor or cancer associated antigens, including tumor-derived, 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, particularly when using donor T lymphocytes. This is often a distinction observed between viral-specific and tumor antigen specific T cells. In some embodiments, the target peptide antigens include at least one that is associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen. 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. In some embodiments, the one or more target peptide antigens is an

“autoantigen”, or is associated with an autoimmune disease. In these embodiments, aAPCs carrying tolerogenic ligands induce tolerance to the target antigen. aAPCs carrying apoptotic signals (e.g., Fas ligand), will induce specific apoptosis of antigen- specific T cells. In some embodiments, the autoimmune disease is selected from asthma, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn’s disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture’s syndrome, Graves’ disease, pemphigus vulgaris, Addison’s disease, dermatitis herpetiformis, celiac disease, and Hashimoto’s thyroiditis.

In various aspects and embodiments, the invention provides a method for enriching, activating and/or expanding HLA-E-restricted T cells, comprising contacting a population of source lymphocytes (e.g., CD8-enriched or CD4-depleted T cells) with the paramagnetic aAPCs described herein, and applying a magnetic field to simultaneously enrich and activate antigen-specific HLA-E-restricted T cells. The magnetic fraction can be recovered, and optionally expanded in culture. The external magnetic field is useful to enrich HLA-E-restricted antigen-specific T cells (including rare naive cells) and to drive aggregation of magnetic nano-aAPC bound to TCR, resulting in aggregation of TCR clusters and enhanced activation of naive T cells. Resulting CD8+ T cells may be phenotypically characterized. In various embodiments, the cells and will be predominately central and effector memory phenotype, and may comprise T memory stem cells (Tscm). After enrichment and expansion, antigen-specific T cell component of the sample will be at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25% antigen specific T cells, in various embodiments.

In various aspects and embodiments, HLA-E-restricted T cells are activated in a subject. In these embodiments, the method comprises administering to the subject a population of nanoparticles having conjugated thereto the HLA-E ligand (presenting a peptide antigen) described herein and a T cell costimulatory ligand. In some embodiments, the co-stimulatory signal is a monoclonal antibody agonist (e.g., an anti- CD28 agonistic antibody), or other co-stimulatory ligand as described herein. In some embodiments, the particles have a signal 3 cytokine conjugated thereto or encapsulated therein. In some embodiments, the peptide antigens are cancer-associated antigens, and the subject has or is at risk of developing cancer. In some embodiments, the peptide antigens are associated with one or more infectious agents (e.g., viral antigens), and the subject is infected or is at risk of infection by the infectious agent. For administration to a subject, the aAPCs may be constructed of polymeric particles, as described. In some embodiments, the particle comprises PLA-PEG and/or PLGA-PEG block co-polymers.

Other aspects and embodiments of the invention will be apparent to the skilled artisan.

DESCRIPTION OF THE FIGURES

FIGURE 1 shows the amino acid sequences of an HLA-E extracellular domain (SEQ ID NO: 1), an engineered HLA-E extracellular domain (mutations shown in bold and underline) (SEQ ID NO: 2), an IgG4 Fc sequence (SEQ ID NO: 3), a flexible linker sequence (SEQ ID NO: 4), an HLA-E-Fc fusion sequence (HLA-E mutations shown in bold and underline, IgG4 Fc shown in capital italics, flexible linker shown in lowercase italics (SEQ ID NO: 5), and a b2 microglobulin sequence (SEQ ID NO: 6).

FIGURE 2 is an image showing an Artificial Immune Modulation (AIM) platform for the generation and robust expansion of CD8+ antigen specific T cells. The HLA-IgG4 fusion protein (hinge dimer, signal 1) is loaded with peptides that target specific T-cell populations for activation and expansion. The anti-CD28 monoclonal antibody binds to CD28 on the surface of CD8(+) T-cells and supports robust activation and expansion of antigen-specific HLA-E-restricted T cells.

FIGURE 3 is an image showing phylogenetic analysis of HLA alleles. Phylogenetic analysis of class I HLA amino acid sequences represents the amount of amino acid similarity between HLA molecules. As shown in FIGURE 3, Class la HLAs (HLA-A and B allele groups) display a high degree of diversity, whereas Class lb HLA- E alleles have few differences in amino acids, and thus a relative low degree of diversity. Murine MHC-I alleles (H2-) are provided for contrast, and human MR-1 is provided as an out-group to ensure proper rooting of the phylogenetic tree.

FIGURE 4 is an image showing a structural representation of the engineered HLA-E ligand (referred to as bbHLA-E^DNK).

FIGURE 5A is an image showing results from an Octet biolayer light interferometry experiment comparing the NKG2A/CD94 interaction of HLA-E™¹ vs. bbHLA-E^DNK. FIGURE 5B is a graph showing the results of the association (K_on) and dissociation (Koff) steps in the experiment. Peptide antigen targets include HLA-A leader (SEQ ID NO: 7), CMV UL40 peptide (SEQ ID NO: 8), and EBV BZLF1 peptide (SEQ ID NO: 9).

FIGURE 6 shows T-Cell staining with wild-type bbHLA-E loaded with either CMV, EBV, or Survivin peptides. X-axis indicates CD8+ staining and the Y-axis indicates peptide specific bbHLA-E (“dimer”) staining.

FIGURE 7 shows T-Cell staining with NK mutant bbHLA-E^dnk loaded with either CMV, EBV, or Survivin peptides. The X-axis indicates CD8+ staining and the Y-axis indicates peptide specific bbHLA-E^dnk (“dimer”) staining. FIGURE 8 shows enrichment and expansion of T-cells using either bbHLA-E”¹ or bbHLA-E^dnk. Cells from the same donor were incubated with paramagnetic nanoparticles conjugated to aCD28 agonistic antibody and either bbHLA-E”¹ or bbHLA- E^dnk, enriched using a magnetic column, and the magnetic fraction grown under T-cell stimulating conditions for 14 days.

DETAILED DESCRIPTION

HLA-E, encoded by the HLA-E gene, is a non-classical MHC Class I molecule. HLA-E is represented by only two principle alleles. Given this low polymorphism, HLA- E ligands may be adaptable to create a nearly universal aAPC platform. However, 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 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). Notably, 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”).

In accordance with aspects and embodiments of this disclosure, recombinant HLA-E ligands are provided that are engineered for modulating CD8+ T cell function. In various embodiments, the invention decouples the NK cell deactivating function of HLA-E 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. These point mutations, as described in detail below, enable the redirection of HLA-E for modulating HLA-E-restricted T cells, while avoiding HLA-E exhaustion of Natural Killer cells. In accordance with other aspects and embodiments, 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.

Disclosed herein is a recombinant HLA-E ligand comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and having a substitution to Cysteine at the amino acids corresponding to Y84 and A139 of SEQ ID NO: 1. SEQ ID NO: 1 is an HLA-E extracellular domain. These substitutions allow for the formation of a disulfide bond that stabilizes the peptide binding cleft. In some embodiments, the amino acid sequence has at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

Alternatively or in addition, the amino acid sequence has one or more amino acid modifications with respect to SEQ ID NO: 1 that reduce or eliminate interaction with NKG2A/CD94. In some embodiments, the amino acid modifications are selected from a substitution of D162 and a substitution of E166 with respect to SEQ ID NO: 1. In various embodiments, the substitutions do not include acidic side chains. For example, in some embodiments, the substitution at D 162 is selected from D162A, D162G, D162L, D162V, D162I, D162S, D162T, D162M, D162N, and D162Q; and the substitution at E166 is selected from E166A, E166G, E166L, E166V, E166I, E166S, E166T, E166M, E166N, and E166Q. In some embodiments, the recombinant HLA-E ligand comprises the substitutions D162A and E166A with respect to SEQ ID NO: 1. In some embodiments, the HLA-E ligand comprises the substitution to Cys at Y84 and A139, as well as substitution of D162 and El 66. In various embodiments, the recombinant HLA-E ligand comprises an HLA-E amino acid sequence fused at its C-terminus to a second amino acid sequence, optionally through a linker. The second amino acid sequence can allow for conjugation to various supports, including particle supports useful for aAPC platforms. In some embodiments, the second amino acid sequence assembles as a multimer, such as, for example a dimer, allowing for engagement of multiple TCRs simultaneously. In some embodiments, the HLA-E ligands are monomeric, but their close association on a nanoparticle support is sufficient for avidity and activation. In some embodiments, the HLA-E ligand is dimeric. Dimeric HLA-E ligands can be constructed by fusion to immunoglobulin heavy chain sequences, which are then associated through one or more disulfide bonds (with or without associated light chains). HLA-E multimers can be created by direct tethering through peptide or chemical linkers, HLA-E ligands can be multimeric via association with streptavidin through biotin moieties.

In some embodiments, the HLA-E ligand is constructed as a fusion with immunoglobulin sequences. That is, the HLA-E molecular complexes may be formed in a conformationally intact fashion at the ends of immunoglobulin heavy chains. In various embodiments, the immunoglobulin heavy chain sequence is not full length, but comprises an Ig hinge region, and one or more of CHI, CH2, and/or CH3 domains. The immunoglobulin sequence may or may not comprise a variable region, but where variable region sequences are present, the variable region may be full or partial. The complex may further comprise immunoglobulin light chains. HLA-E-Ig lacking variable chain sequences (and lacking any light chain) may be employed with site-directed conjugation to particles (e.g., through an unpaired Cys), as described in US 10,435,668, US 10,632,193, and US 2016/0129133, which are hereby incorporated by reference in its entirety. In various embodiments, the immunoglobulin heavy chain sequences can be the heavy chain of an IgM, IgD, IgGl, IgG3, ¾ϋ2b, IgG2a, IgG4, IgE, or IgA. In some embodiments, the immunoglobulin sequences are IgG4 Fc sequences. If multivalent HLA-E molecular complexes are desired, IgM or IgA heavy chains can be used to provide pentavalent or tetravalent molecules, respectively.

In some embodiments, the second amino acid sequence is an immunoglobulin sequence, such as for example, an IgG Fc domain (e.g., an IgG4 Fc domain). In some embodiments, 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: 3. In some embodiments, 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: 3. In some embodiments, the recombinant HLA-E ligand comprises a linker between the HLA-E amino acid sequence, and the second amino acid sequence (e.g., IgG Fc domain, such as IgG4 Fc domain). In some embodiments, 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: 4. Alternatively, linkers can be selected from flexible and rigid peptide linkers.

Flexible linkers are predominately or entirely composed of small, non-polar or polar residues such as Gly, Ser and Thr. An exemplary flexible linker comprises (Gly_xSer)_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 unstructured. More rigid linkers include polyproline or poly Pro-Ala motifs and a-helical linkers. Generally, linkers of varying rigidity can be predominately composed of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences contain at least 5 amino acids, and may be in the range of 10 to 50 amino acids, or in the range of 10 to 30 amino acids. In various embodiments, the recombinant HLA-E ligand is associated with a b2 microglobulin protein. The amino acid sequence of b2 microglobulin is provided herein as SEQ ID NO: 6. In various embodiments, derivatives of b2 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. In various aspects and embodiments, the invention provides artificial antigen presenting cells (aAPC) comprising the HLA-E ligand. The aAPC is useful for modulating HLA-E-restricted T cell responses in vivo or ex vivo. In various embodiments, the aAPC carries additional ligands (or “signals”) to modulate T cell function. For example, the aAPC may present a “Signal 2”, which is generally a lymphocyte co-stimulatory ligand or a lymphocyte inhibitory ligand. T cell costimulatory molecules contribute to the activation of antigen-specific T cells. Such molecules include, but are not limited to, molecules that specifically bind to CD28 (including antibodies), CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, GITR, ICOS, antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, and antibodies that specifically bind to 0X40. In some embodiments, the costimulatory molecule is an antibody (e.g., a monoclonal antibody) or portion thereof, such as F(ab’)2, Fab, scFv, or single chain antibody, or other antigen binding fragment. In some embodiments, the antibody is a humanized monoclonal antibody or portion thereof having antigen-binding activity, or is a fully human antibody or portion thereof having antigen-binding activity. In some embodiments, the Signal 2 is a ligand that binds and activates through CD28. The CD28 agonist may be a monoclonal antibody agonist, as described for example in US 2016/0129133, which is hereby incorporated by reference.

In other embodiments, the Signal 2 ligand is a tolerogenic ligand. In some embodiments, the tolerogenic ligand is TGF-bI. In some embodiments, the Signal 2 ligand induces apoptosis (e.g., Fas ligand).

In various embodiments, the aAPC may include a Signal 3 conjugated to or encapsulated by the particle. Signal 3 ligands include cytokines that support T cell activation and expansion.

In various embodiments, the aAPC is suitable for activating and/or expanding HLA-E-restricted T cells in vitro or ex vivo. In these embodiments, the particle core may be polymeric or paramagnetic, or any other suitable material. Exemplary materials include metals such as iron, nickel, cobalt, gold, or alloy of rare earth metal. In some embodiments, the particle is paramagnetic, such as magnesium, molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beads suitable for enrichment of materials (including cells) are commercially available, and include iron dextran beads, such as dextran-coated iron oxide beads. In aspects of the invention where magnetic properties are not required, nanoparticles can also be materials such as cellulose, ceramics, glass, nylon, polystyrene, rubber, plastic, or latex. An exemplary polymeric material for preparation of nanoparticles is poly(lactic-co-glycolic acid) (PLGA) or polylactic acid (PLA) and copolymers thereof. Other polymers and co-polymers that may be employed include those described in US 2016/0129133, which is hereby incorporated by reference in its entirety.

In various embodiments, the particle has a size (e.g., average diameter) within about 10 to about 500 nm, or within about 40 to about 400 nm, or within about 40 nm to 200 nm. For magnetic clustering (i.e., driven by paramagnetic materials), it is preferred that the nanoparticles have a size (mean diameter) in the range of 10 to 250 nm, or 50 to 200 nm, or 80 to 200 nm, or 20 to 100 nm, or 60 to 120 nm. Nanoparticle binding and cellular activation are sensitive to membrane spatial organization, which is particularly important during T cell activation. Magnetic fields can be used ex vivo to manipulate cell-bound nanoparticles to drive activation via TCR clustering. For in vivo applications, nanosized particles, such as less than about 200 nm or less than about 150 nm, or less than about 100 nm are desired to allow the aAPCs to traffic to desired organs or to tumors, and to avoid occlusion of blood vessels.

In some embodiments, the particle is paramagnetic and is preferably biocompatible, such as dextran-coated iron oxide particles. Paramagnetic particles, and particularly paramagnetic nanoparticles, allow for magnetic capture or “enrichment” by application of a magnetic field, as well as simultaneous activation of antigen-specific lymphocytes. See US 10,098,939, US 10,987,412, and US 10,435,668, which are hereby incorporated by reference in their entireties. Additional materials for aAPCs that are useful for ex vivo applications are described in US 10,435,668.

In some embodiments, the aAPC is suitable for administration to a subject, to activate antigen-specific HLA-E-restricted T cells in vivo. While such aAPCs can also be constructed of dextran-coated iron oxide particles (or other biocompatible materials), in some embodiments the aAPCs are constructed from polymeric particles. Exemplary polymeric particles are described in US 2016/0129133 and US 10,632,193, which are hereby incorporated by reference in their entireties. Exemplary polymeric aAPCs include those constructed from PLA-PEG and/or PLGA-PEG block co-polymers. In some embodiments, the HLA-E ligand and co-stimulatory ligand (or signal 2 and/or signal 3 ligands) are conjugated to the particle through a functional groups at the PEG terminus.

Various activation chemistries can be used to provide the specific, stable attachment of molecules to the surface of nanoparticles. For example, the common cross- linker glutaraldehyde can be used to attach protein amine groups to an aminated nanoparticle surface in a two-step process. The resultant linkage is hydrolytically stable. Other methods include use of cross-linkers containing n-hydrosuccinimido (NHS) esters which react with amines on proteins, cross-linkers containing active halogens that react with amine-, sulfhydryl-, or histidine-containing proteins, cross-linkers containing epoxides that react with amines or sulfhydryl groups, conjugation between maleimide groups and sulfhydryl groups, and the formation of protein aldehyde groups by periodate oxidation of pendant sugar moieties followed by reductive amination.

In various embodiments, the one or more peptide antigens are tumor or cancer associated antigens, including tumor-derived, 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, particularly when using donor T lymphocytes. See, Quintarelli et ak, 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.

“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 sialy 1-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).

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 a 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); GP 100, MelanA/Mart-1, tyrosinase, gp75/brown, BAGE, and S-100 (expressed in melanoma); cytokeratins (expressed in various carcinomas); and CD19, CD20, and CD37 (expressed in lymphoma).

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).

Tumor-associated antigens are further disclosed in US 11,007,222, which is hereby incorporated by reference.

In some embodiments, the target peptide antigens include at least one that is associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen. For example, at least one peptide antigen may be associated with tuberculosis (TB), HIV (human immunodeficiency virus), hepatitis (e.g., A, B, C, or D) cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza, herpes virus (e.g., HSV 1 or 2, or varicella zoster), and Adenovirus. 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.

In some embodiments, the target peptide antigens include one or more tumor associated antigens, and one or more virus-associated antigens (such as TB, CMV, EBV, influenza, or Adenovirus), to provide an antitumor response while protecting against common pathogens that complicate recovery after HSCT.

In some embodiments, the one or more target peptide antigens is an “autoantigen”, or is associated with an autoimmune disease. In these embodiments, aAPCs carrying tolerogenic ligands induce tolerance to the target antigen. aAPCs carrying apoptotic signals (e.g., Fas ligand), will induce specific apoptosis of antigen- specific T cells. An “autoantigen” is an organism's own “self-antigen” to which the organism produces an immune response. Autoantigens are involved in autoimmune diseases such as Goodpasture's syndrome, multiple sclerosis, Graves' disease, myasthenia gravis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, rheumatoid arthritis, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, and Hashimoto's thyroiditis. In some embodiments, the autoimmune disease is selected from asthma, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn’s disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture’s syndrome, Graves’ disease, pemphigus vulgaris, Addison’s disease, dermatitis herpetiformis, celiac disease, and Hashimoto’s thyroiditis.

In some embodiments, peptide antigens for presentation by the HLA-E ligand 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. For example, sequencing data can provide information about both shared as well as personalized targets for cancer immunotherapy. In principle, mutant proteins are foreign to the immune system and are putative tumor-specific antigens. Indeed, 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. Mutation catalogues derived from whole exome sequencing provide a starting point for identifying such neo-epitopes. Using HLA binding prediction algorithms (Srivastava, PLoS One 4, e6094 (2009), it has been predicted that each cancer can have up 7-10 neo-epitopes. A similar approach estimated hundreds of tumor neo epitopes. Neoepitopes predicted from DNA or RNA sequencing of a patient’s tumor, can be tested for their activation potential in association with the HLA-E ligand, by determining whether (or to what extent) an aAPC carrying the predicted antigen in association with the HLA-E ligand is able to activate T cells from the subject.

In some embodiments, aAPCs carrying the HLA-E ligands are employed to enrich and/or activate, and/or expand (antigen-specific) HLA-E-restricted T cells ex vivo. See US 10,987,412 and US 2020/0291381, which are hereby incorporated by reference in their entireties. In some embodiments, T cells specific for one or more peptide antigens can be enriched and expanded in batch, allowing for rapid, parallel production of cell compositions. In some embodiments, the cell composition prepared contains HLA-E restricted T cells specific for from 2 to 15 or from 2 to 10 peptide antigens. HLA-E restricted T cell specificity toward a target peptide antigen in the composition is defined by HLA-E multimer staining (e.g., dimer or tetramer staining) as is well known in the art.

For example, a cocktail of nano-aAPCs comprising an HLA-E ligand, each aAPC presenting a different, distinct target antigen, is used to enrich T cells against multiple antigens simultaneously. For example, HLA-E-restricted T cells specific for from 2 to 10 antigens can be enriched simultaneously from a lymphocyte source. In this embodiment, a number of different nano-aAPC batches, each bearing a different MHC- peptide, would be combined and used to simultaneously enrich T cells against each of the antigens of interest. The resulting T cell pool would be activated against each of these antigens, and expanded together in culture. These antigens could be related to a single therapeutic intervention; for example, multiple antigens present on a single tumor or malignant cell.

In various aspects and embodiments, the invention provides a method for enriching, activating and/or expanding HLA-E-restricted T cells, comprising contacting a population of source lymphocytes (e.g., CD8-enriched or CD4-depleted T cells) with the paramagnetic aAPCs described herein, and applying a magnetic field to simultaneously enrich and activate antigen-specific HLA-E-restricted T cells. The magnetic fraction can be recovered, and optionally expanded in culture. Exemplary methods according to these embodiments are described in US 10,908,939, US 10,987,412, and US 2020/0291381, which are hereby incorporated by reference in its entirety.

The external magnetic field is useful to enrich HLA-E-restricted antigen-specific T cells (including rare naive cells) and to drive aggregation of magnetic nano-aAPC bound to TCR, resulting in aggregation of TCR clusters and enhanced activation of naive T cells. Magnetic fields can exert appropriately strong forces on paramagnetic particles, but are otherwise biologically inert. Nano-aAPC are themselves magnetized, and are attracted to both the field source and to nearby nanoparticles in the field, inducing bead and TCR aggregation to boost aAPC-mediated activation.

In various embodiments, the invention involves making cell compositions suitable for administration to a recipient, by enrichment and expansion of HLA-E restricted and antigen-specific CD8+ T cells. Precursor T cells can be obtained from the patient or from a suitable donor. Source T cells can be either fresh or frozen samples. Precursor T cells can be obtained from a number of sources that comprise WBCs, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, buffy coat fraction, and tumors (tumor infiltrating lymphocytes). In some embodiments, precursor T cells are obtained from a unit of blood collected from a subject using any number of techniques known to one or skill in the art. For example, precursor T cells from the circulating blood of an individual can be obtained by apheresis or leukapheresis.

Magnetic activation of T cells with the paramagnetic nano-aAPCs and a magnetic field may take place for from 2 minutes to 5 hours, or from 2 minutes to about 2 hours, or from about 2 minutes to about 30 minutes (e.g, about 5 or 10 minutes), followed by expansion in culture for at least 5 days, and up to 2 weeks or up to 3 weeks in some embodiments. In some embodiments, magnetic activation occurs for at least 2 minutes, but less than 30 minutes or less than 15 minutes (e.g., about 5 or 10 minutes). Resulting CD8+ T cells may be phenotypically characterized. In various embodiments, the cells and will be predominately central and effector memory phenotype, and will comprise T memory stem cells (Tscm).

In some embodiments, the recovered cells are expanded in culture for one to three weeks in the presence of growth factors, as described in US 11,007,222, which is hereby incorporated by reference in its entirety. The T cell growth factors affect proliferation and/or differentiation of T cells. Examples of T cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens. If desired, cytokines can be present in molecular complexes comprising fusion proteins, or can be encapsulated by or conjugated to the aAPC, or provided in soluble form. Particularly useful cytokines include MIR-Ib, IL-Ib, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-g, and CXCL10.

In some embodiments, the cells are expanded in culture in the presence of from 4 to 8 cytokines, to achieve a balance between T cell expansion (including antigen- specific T cell expansion), activation, and memory phenotype. In some embodiments, the cells are expanded in the presence of IL-4. In some embodiments, the cells are expanded in the presence of IL-4 and IL-6. In some embodiments, the cells are expanded in the presence of IL-4 and IL-Ib. In some embodiments, the cells are expanded in the presence of IL-4, IL-6, and IL-Ib. In some embodiments, the cells are expanded in the presence of IL-2, IL-4, and IL-6. In some embodiments, the cells are expanded in culture in the presence of IL-2, IL-4, IL-6, INF-g, and IL-Ib. In some embodiments, the cells are further expanded in the presence of IL-10. In various embodiments, these cytokines are used in conjunction with artificial or natural antigen presenting cells to expand antigen specific T cells. In some embodiments, the growth factors consist, or consist essentially of, IL-Ib, IL-2, IL-4, IL-6, and INF-g.

After enrichment and expansion, antigen-specific T cell component of the sample will be at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25% antigen specific T cells. From the original sample isolated from the patient or donor, the antigen-specific T cells in various embodiments are expanded (in about 7 days) from about 100-fold to about 10,000 fold, such as at least about 100-fold, or at least about 200-fold. After 2 weeks, antigen-specific T cells are expanded at least 1000-fold, or at least about 2000-fold, at least about 3,000 fold, at least about 4,000-fold, or at least about 5,000-fold in various embodiments. In some embodiments, antigen-specific T cells are expanded by greater than 5000-fold or greater than 10,000 fold after two weeks. After one or two weeks of expansion, at least about 10⁶, or at least about 10⁷, or at least about 10⁸, or at least about 10⁹ antigen-specific T cells are obtained. In some embodiments, the HLA-E-restricted and antigen-specific T cell composition further comprises a pharmaceutically acceptable carrier suitable for intravenous infusion, and which may be suitable as a cryoprotectant. An exemplary carrier is DMSO (e.g., about 10%). Cell compositions may be provided in unit vials or bags, and stored frozen until use. Unit doses may comprise from about 5 x 10⁵ to about 5 x 10⁶ cells per ml, in a volume of from 50 to 200 ml. In certain embodiments, the volume of the composition is <100 ml (e.g., from 50 to 100 ml).

In various embodiments, the cell composition (activated and expanded ex vivo using aAPCs carrying the HLA-E ligands) or the aAPCs themselves, may be administered to patients in need. These compositions may be administered in conjunction with other immunotherapies (including immune checkpoint inhibitors). In various embodiments, the checkpoint inhibitor(s) target one or more of CTLA-4 or PD-1/PD-L1, which may include antibodies against such targets, such as monoclonal antibodies, or portions thereof, or humanized or fully human versions thereof. In some embodiments, the checkpoint inhibitor therapy comprises ipilimumab or Keytruda (pembrolizumab), or comparable monoclonal antibody. In some embodiments, the patient previously received PD1 blockade therapy, and was refractory or only partially responsive to that treatment. In such embodiments, the cell composition described herein can restore a robust T cell response, optionally in combination with a second round of immunotherapy (e.g., anti- CTLA4 or PD-1 blockade therapy).

The cell or aAPC compositions can be administered to patients by any appropriate routes, including intravenous infusion, intra-arterial administration, intralymphatic administration, and intratumoral administration.

In some embodiments, the patient has a hematological cancer, which in some embodiments has relapsed after allogeneic stem cell transplantation. In some embodiments, the patient has acute myelogenous leukemia (AML) or myelodysplastic syndrome.

Other cancers that can be treated according to this disclosure include cancers that historically illicit poor immune responses or have a high rate of recurrence. Exemplary cancers include various types of solid tumors, including carcinomas, sarcomas, and lymphomas. In various embodiments the cancer is melanoma (including metastatic melanoma), colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma. In various embodiments, the cancer is stage I, stage II, stage III, or stage IV. In some embodiments, the cancer is metastatic and/or recurrent, and/or is nonresectable.

In some embodiments, the patient is refractory to chemotherapy and/or checkpoint inhibitor therapy.

In some embodiments, the cancer is a hematological malignancy, including leukemia, lymphoma, or myeloma. For example, the hematological malignancy may be acute myeloid leukemia, chronic myelogenous leukemia, childhood acute leukemia, non- Hodgkin’s lymphomas, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoids, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell rich cutaneous lymphoid hyperplasia. In an exemplary embodiment, the patient has a hematological cancer such as acute myelogenous leukemia (AML) or myelodysplastic syndrome, and in some embodiments the patient has relapsed after allogeneic stem cell transplantation. In some embodiments, the therapy does not induce GVHD.

In some embodiments, the patient, in addition to allogeneic stem cell transplantation, has also undergoes lympho-deleting therapy, cyto-reductive therapy, or immunomodulatory therapy (prior to administration of the cell therapy). In some embodiments, the cell therapy may be further provided with or without cytokine support post treatment.

In some embodiments, the patient has an infectious disease or is at risk for an infectious disease. For example, patients that have undergone HSCT are at particular risk for infectious disease, given the immunocompromised state. Infectious diseases that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc. Such diseases include AIDS, hepatitis B/C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpes virus infection (including shingles), and adenovirus infection. 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. In these embodiments, the patient may receive adoptive immunotherapy comprising T cells specific for pathogen antigens. The method can entail generation of virus-specific CTL derived from the patient or from an appropriate donor before initiation of the transplant procedure.

Post-transplant lymphoproliferative disorders (PTLD) occur in a significant fraction of transplant patients and results from Epstein-Barr virus (EBV) infection. 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. Other aspects and embodiments of the invention will be apparent to the skilled artisan.

EXAMPLES

Example 1: Design of HLA-E-based nanoparticles This example demonstrates the engineering of HLA-E ligands for activation of

HLA-E-restricted and antigen-specific CD8+ T-cells (CTLs).

To construct the HLA-E-based nanoparticles, the HLA-E ligands were engineered to decouple the Natural Killer cell deactivating function of an HLA-E ligand from the T-Cell activating function. Point mutations were introduced into the HLA-E ligands (FIGURE 1, FIGURE 4). In FIGURE 1, mutations are provided in bold and underlined, and the flexible linker is shown in lower-case, and the Fc portion of IgG4 is provided in italicized text. The flexible linker provides space and mobility for the HLA- E domain to engage TCRs without hinderance from the Fc fusion. The Fc portion of the molecule aids in stability of the protein, purification of the protein, and conjugation to nanoparticles. As shown below, these mutations enabled the redirection of HLA-E for expansion of antigen-specific T-Cell using HLA-E-based nanoparticles, while preventing HLA-E involvement in exhaustion of Natural Killer cells.

FIGURE 2 shows the engineering of HLA-E-based nanoparticles. As shown in FIGURE 2, the nanoparticles have dimeric HLA-E ligands conjugated to the surface (presenting the target peptide antigen) that can incorporate multiple tumor specific antigenic peptides. While HLA proteins represent a highly diverse group of proteins, HLA-E is a monomorphic HLA, represented by two principle alleles spanning the global patient population. See FIGURE 3. The dimeric HLA-E ligand contains two HLA-E domains, comprising the peptide binding clefts, each fused to an arm of the Ig hinge region. Dimeric HLA-E-Ig are co-expressed with b2 microglobulin. A dimeric HLA-E ligand, including an HLA-E-IgG4 hinge dimer, provides direct engagement with target T cells.

Co-stimulatory or inhibitory ligands, such as an anti-CD28 monoclonal antibody, were also conjugated to the nanoparticle, as shown in FIGURE 2. The co-stimulatory or inhibitory ligands provide specific instructions ( e.g activation, suppression) to target T cells (i.e., naive T cells or memory T cells) relative to the therapeutic goal. Ligands and aAPC constructs are disclosed in WO 2016/044530 and WO 2016/105542, which are hereby incorporated by reference in their entirety.

FIGURE 4 shows a structural representation of bbHLA-E^DNK. The image on the left represents the view of HLA-E from the top of the molecule. The Y84C and A139C mutations and the D162A and E166A mutations are shown (left image). The overall structure of the HLA-E-IgG4 fusion protein is shown in the right image. bbHLA-E^DNK is a fusion between the extracellular portion of HLA-E*01:03 allele and the Fc portion of a human IgG4 heavy chain. The HLA-E of this molecule contains four amino acid mutations that make it distinct from the wild-type version of this protein. These mutations are Y84C, A139C, D162A, and E166A. The Y84C and A139C mutations result in a disulfide bond that stabilizes the peptide binding pocket of the molecule (FIGURE 5A and FIGURE 5B), and enhances peptide loading. Stabilizing the peptide binding pocket further stabilizes the interaction of HLA-E with b2M and prevents aggregation and/or degradation. The D162A and E166A mutations disrupt interaction with NKG2A/CD94 (FIGURE 5A and FIGURE 5B), while maintaining specific interaction with T-cell receptors (TCR). These mutations are essential for preventing down-regulation of NK cell populations in vivo. HLA-E structural analysis is described in Sullivan, L. C. el al. The heterodimeric assembly of the CD94-NKG2 receptor family and implications for human leukocyte antigen-E recognition. Immunity 27, 900-911 (2007); Sullivan, L. C. et al. A conserved energetic footprint underpins recognition of human leukocyte antigen- E by two distinct ab T cell receptors. J Biol Chem 292, 21149-21158 (2017)).

FIGURE 5A is an image showing results from an Octet biolayer light interferometry experiment comparing the NKG2A/CD94 interaction of HLA-E™¹ vs. bbHLA-E^DNK. In this experiment, a baseline for streptavidin biosensors was established by immersion in a buffer, and the biotinylated NKG2A/CD94 was then loaded onto streptavidin biosensors at a concentration of 4 pg/ml. A new baseline for the NKG2A/CD94-loaded biosensors was determined by immersion in the buffer. NKG2A/CD94-loaded biosensors were then immersed in peptide/HLA-E samples, and protein-protein association were measured. Biosensors were then immersed in buffer to measure dissociation of peptide/HLA-E complexes fromNKG2A/CD94. FIGURE 5B is a graph showing the results of the association (K_on) and dissociation (K_off) steps in the experiment. HLA-E™¹ loaded with HLA-A2 leader or CMV peptides show robust interaction with NKG2A/CD94, while bbHLA-E^DNK mutant shows substantially reduced interaction. Both proteins loaded with EBV peptide show very little interaction with NKG2A/CD94. By disrupting interaction between HLA-E and NKG2A/CD94, the risk of HLA-E triggering NK cell inactivation is reduced, thereby allowing NK cells to continue to perform their innate immune function. By preserving the TCR interacting surface of HLA-E, the molecules selectively expand HLA-E-restricted T-cells.

Example 2: Staining and ex vivo T cell expansion with HLA-E

T-cells were stained using the wild-type bbHLA-E"¹ or bbHLA-E^dnk loaded with either CMV, EBV, or Survivin peptides. CMV (UL40, sequence VMARTLIL, SEQ ID NO: 8) and EBV (BZLF 39-47), sequence VMARTLIL, SEQ ID NO: 9) are derived from viral antigens from CMV and EBV respectively. Survivin (sequence LDRERAKNKI, SEQ ID NO: 10) is an irrelevant peptide that does not bind HLA-E. T- cells were derived from five unique patients as indicated: L175, L178, L185, L278, and L327. In FIGURES 6 (bbHLA-E^wt) and 7 (bbHLA-E^dnk) , the X-axis indicates CD8 (+) staining and the Y-axis indicates peptide specific bbHLA-E (“dimer”) staining. The boxes in the upper left comer provide the percent of peptide-specific dimer/CD8+ cells. As shown in FIGURES 6 and 7, both bbHLA-E”¹ and bbHLA-E^dnk were able to detect CMV- and EBV-peptide specific T-cells.

Enrichment and Expansion (E+E) of CD8+-enriched T-cells was conducted using either bbHLA-E”¹ or bbHLA-E^dnk conjugated to paramagnetic nanoparticles with an anti- CD28 agonistic antibody, essentially as described in US Patent No. 10,987,412 and US Patent No. 11,007,222, each of which are hereby incorporated by reference in their entireties. Specifically, cells from the same donor were incubated with the paramagnetic nanoparticles conjugated with aCD28 and either bbHLA-E”¹ or bbHLA-E^dnk, the mixture was passed over a magnetic column, and the magnetic fraction grown under T-cell stimulating conditions for 14 days. As shown in FIGURE 7, both bbHLA-E”¹ and bbHLA-E^dnkwere able to enrich for peptide-specific T cells and support their expansion ex vivo.

Claims

CLAIMS:

1. A recombinant HLA-E ligand comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and having a substitution to Cysteine at the amino acids corresponding to Y84 and A139 of SEQ ID NO: 1.

2. The recombinant HLA-E ligand of claim 1, wherein the amino acid sequence has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.

3. The recombinant HLA-E ligand of claim 1, wherein the amino acid sequence has at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 1.

4. The recombinant HLA-E ligand of any one of claims 1 to 3, wherein the amino acid sequence has one or more amino acid modifications with respect to SEQ ID NO: 1 that reduce or eliminate interaction with NKG2A/CD94.

5. The recombinant HLA-E ligand of claim 4, wherein the amino acid modifications are selected from a substitution of D 162 and a substitution of El 66 with respect to SEQ ID NO: 1.

6. The recombinant HLA-E ligand of claim 5, wherein the substitution at D162 is selected from D 162 A, D162G, D162L, D162V, D162I, D162S, D162T, D162M, D162N, and D162Q; and the substitution at E166 is selected from E166A, E166G, E166L, El 66V, El 661, E166S, E166T, E166M, E166N, and E166Q.

7. The recombinant HLA-E ligand of claim 6, comprising the substitutions D162A and E166A with respect to SEQ ID NO: 1.

8. The recombinant HLA-E ligand of claim 7, wherein the HLA-E ligand comprises the amino acid sequence of SEQ ID NO: 2.

9. The recombinant HLA-E ligand of any one of claims 1 to 8, wherein the HLA-E amino acid sequence is fused at its C-terminus to a second amino acid sequence, optionally through a linker.

10. The recombinant HLA-E ligand of claim 9, wherein the second amino acid sequence assembles as a multimer.

11. The recombinant HLA-E ligand of claim 10, wherein the second amino acid sequence assembles as a dimer.

12. The recombinant HLA-E ligand of claim 11, wherein the second amino acid sequence is an immunoglobulin sequence.

13. The recombinant HLA-E ligand of claim 12, wherein the immunoglobulin sequence is an IgG Fc domain.

14. The recombinant HLA-E ligand of claim 13, wherein the IgG Fc domain is an IgG4 Fc domain.

15. The recombinant HLA-E ligand of claim 14, wherein 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: 3.

16. The recombinant HLA-E ligand of any one of claims 9 to 15, comprising a flexible linker that is predominately glycine and serine amino acid residues.

17. The recombinant HLA-E ligand of claim 16, comprising the amino acid sequence of SEQ ID NO: 4.

18. The recombinant HLA-E ligand of any one of claims 1 to 17, further comprising an associated b2 microglobulin protein.

19. The recombinant HLA-E ligand of any one of claims 1 to 18, further comprising a peptide antigen.

20. A recombinant HLA-E ligand comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence has one or more amino acid modifications with respect to SEQ ID NO: 1 that reduce or eliminate interaction with NKG2A/CD94.

21. The recombinant HLA-E ligand of claim 20, wherein the amino acid modifications are selected from a substitution of D 162 and a substitution of El 66 with respect to SEQ ID NO: 1.

22. The recombinant HLA-E ligand of claim 21, wherein the substitution at D 162 is selected from D 162 A, D162G, D162L, D162V, D162I, D162S, D162T, D162M, D162N, and D162Q; and the substitution at E166 is selected from E166A, E166G, E166L, El 66V, El 661, E166S, E166T, E166M, E166N, and E166Q.

23. The recombinant HLA-E ligand of claim 22, comprising the substitutions D162A and E166A with respect to SEQ ID NO: 1.

24. The recombinant HLA-E ligand of any one of claims 20 to 23, wherein the amino acid sequence has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.

25. The recombinant HLA-E ligand of claim 24, wherein the amino acid sequence has at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 1.

26. The recombinant HLA-E ligand of any one of claims 20 to 25, having a substitution to Cysteine at the amino acids corresponding to Y84 and A139 of SEQ ID NO: 1.

27. The recombinant HLA-E ligand of claim 26, wherein the HLA-E ligand comprises the amino acid sequence of SEQ ID NO: 2.

28. The recombinant HLA-E ligand of any one of claims 20 to 27, wherein the HLA- E amino acid sequence is fused at its C-terminus to a second amino acid sequence optionally through a linker.

29. The recombinant HLA-E ligand of claim 28, wherein the second amino acid sequence assembles as a multimer.

30. The recombinant HLA-E ligand of claim 29, wherein the second amino acid sequence assembles as a dimer.

31. The recombinant HLA-E ligand of claim 30, wherein the second amino acid sequence is an immunoglobulin sequence.

32. The recombinant HLA-E ligand of claim 31, wherein the immunoglobulin sequence is an IgG Fc domain.

33. The recombinant HLA-E ligand of claim 32, wherein the IgG Fc domain is an IgG4 Fc domain.

34. The recombinant HLA-E ligand of claim 33, wherein 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: 3.

35. The recombinant HLA-E ligand of any one of claims 28 to 34, comprising a flexible linker that is predominately glycine and serine amino acid residues.

36. The recombinant HLA-E ligand of claim 35, comprising the amino acid sequence of SEQ ID NO: 5.

37. The recombinant HLA-E ligand of any one of claims 20 to 36, further comprising an associated b2 microglobulin protein.

38. The recombinant HLA-E ligand of any one of claims 20 to 37, further comprising a peptide antigen.

39. An artificial antigen presenting cell (aAPC) comprising the HLA-E ligand of any one of claims 1 to 38 conjugated to a particle support.

40. The aAPC of claim 39, wherein the aAPC further comprises a lymphocyte co stimulatory signal conjugated to the particle.

41. The aAPC of claim 40, wherein the lymphocyte co-stimulatory signal is selected from an agonist of one or more of CD28, CD80 (B7-1), CD86 (B7-2), 4-1BB, CD27, CD30, 0X40, B7h, GITR, and ICOS.

42. The aAPC of claim 41, wherein the co-stimulatory signal is a monoclonal antibody agonist.

43. The aAPC of claim 42, wherein the co-stimulatory signal is an anti-CD28 agonistic antibody.

44. The aAPC of claim 39, wherein the aAPC further comprises a tolerogenic ligand.

45. The aAPC of claim 44, wherein the tolerogenic ligand is TGF-bI.

46. The aAPC of any one of claims 39 to 45, where the particle has a signal 3 cytokine conjugated to the particle.

47. The aAPC of any one of claims 39 to 46, wherein the particle has an average diameter of from 20 nm to about 200 nm.

48. The aAPC of claim 47, wherein the particle has an average diameter of from about 60 nm to about 120 nm.

49. The aAPC of any one of claims 39 to 48, wherein the particle is a paramagnetic particle.

50. The aAPC of claim 49, wherein the paramagnetic particle is a dextran-coated iron oxide particle.

51. The aAPC of any one of claims 39 to 48, wherein the particle is a polymeric particle.

52. The aAPC of claim 51, wherein the particle comprises PLA-PEG and/or PLGA- PEG block co-polymers.

53. The aAPC of claim 52, wherein the HLA-E ligand is conjugated to the PEG terminus.

54. A method for enriching, activating and/or expanding HLA-E-restricted T cells, comprising: contacting a population of cells comprising HLA-E restricted T cells with a population of paramagnetic particles comprising the HLA-E ligand of any one of claims 1 to 38 conjugated thereto, and enriching for the HLA-E restricted T cells by applying a magnetic field and recovering the fraction associated with the paramagnetic particles, and optionally expanding the recovered fraction in culture.

55. The method of claim 54, wherein the paramagnetic particles further comprise a lymphocyte co-stimulatory signal conjugated thereto.

56. The method of claim 55, wherein the lymphocyte co-stimulatory signal is selected from an agonist of one or more of CD28, CD80 (B7-1), CD86 (B7-2), 4-1BB, CD27, CD30, 0X40, B7h, GITR, and ICOS.

57. The method of claim 56, wherein the co-stimulatory signal is a monoclonal antibody agonist.

58. The method of claim 57, wherein the co-stimulatory signal is an anti-CD28 agonistic antibody.

59. The method of any one of claims 54 to 58, where the particle has a signal 3 cytokine conjugated to the particle.

60. The method of any one of claims 54 to 59, wherein the particle has an average diameter of from 20 nm to about 500 nm.

61. The method of claim 60, wherein the particle has an average diameter of from about 50 nm to about 250 nm.

62. The method of any one of claims 54 to 61 , wherein the paramagnetic particles are dextran-coated iron oxide particles.

63. The method of any one of claims 54 to 62, wherein the HLA-E ligands in the population of particles present one or more peptide antigens.

64. The method of claim 63, wherein the HLA-E ligands in the population of particles present from two to ten peptide antigens.

65. The method of claim 63 or 64, wherein the peptide antigens are tumor or cancer- associated antigens.

66. The method of claim 65, wherein the tumor or cancer-associated antigen is a tumor-derived antigen, tumor-specific antigen, or a neoantigen.

67. The method of claim 63 or 64, wherein the peptide antigens are associated with or derived from a pathogen.

68. The method of claim 67, wherein the peptide antigen is selected from a viral, bacterial, fungal, or parasitic pathogen.

69. The method of claim 68, wherein the peptide antigen is associated with or derived from tuberculosis (TB), human immunodeficiency virus, or cytomegalovirus.

70. The method of claim 63 or 64, wherein the peptide antigens are associated with an autoimmune disease.

71. The method of claim 70, wherein the autoimmune disease is selected from asthma, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn’s disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture’s syndrome, Graves’ disease, pemphigus vulgaris, Addison’s disease, dermatitis herpetiformis, celiac disease, and Hashimoto’s thyroiditis.

72. The method of claim 63 or 64, wherein the peptide antigens are associated with one or more infectious agents.

73. The method of any one of claims 54 to 72, wherein the recovered cells are expanded in culture for one to three weeks in the presence of growth factors.

74. The method of claim 73, wherein the growth factors are selected from MIR-Ib, IL-Ib, IL-2, IL-4, IL-6, IL-7, IL-10, IL-21, and INF-g.

75. The method of claim 74, wherein the growth factors comprise IL-Ib, IL-2, IL-4, IL-6, and INF-g.

76. The method of any one of claims 73 to 75, wherein the recovered cells are expanded in the presence of particles comprising the HLA-E ligand and a co-stimulatory signal conjugated thereto.

77. The method of any one of claims 73 to 76, wherein recovered cells predominately have the phenotypes of T memory stem cell, central memory, and effector memory.

78. A method for treating a subject in need of adoptive transfer of T cells, comprising administering the T cells of any one of claims 54 to 77.

79. The method of claim 78, wherein the subject has or is at risk of cancer.

80. The method of claim 78, wherein the subject has or is at risk of infection by an infectious agent.

81. The method of any one of claims 78 to 80, wherein the population of cells are from the subject.

82. The method of any one of claims 78 to 80, wherein the population of cells are from a donor.

83. A method for activating HLA-E restricted T cells in a subject, comprising administering to the subject a population of nanoparticles having conjugated thereto the HLA-E ligand of any one of claims 1 to 38 and a T cell costimulatory ligand.

84. The method of claim 83, wherein the T cell co-stimulatory signal is selected from an agonist of one or more of CD28, CD80 (B7-1), CD86 (B7-2), 4-1BB, CD27, CD30, 0X40, B7h, GITR, and ICOS.

85. The method of claim 84, wherein the co-stimulatory signal is a monoclonal antibody agonist.

86. The method of claim 85, wherein the co-stimulatory signal is an anti-CD28 agonistic antibody.

87. The method of any one of claims 83 to 86, where the particles have a signal 3 cytokine conjugated thereto or encapsulated therein.

88. The method of any one of claims 83 to 87, wherein the particles have an average diameter of from 20 nm to about 200 nm.

89. The method of claim 88, wherein the particles have an average diameter of from about 60 nm to about 120 nm.

90. The method of any one of claims 83 to 89, wherein the HLA-E ligands in the population of particles present one or more peptide antigens.

91. The method of claim 90, wherein the HLA-E ligands in the population of particles present from two to ten peptide antigens.

92. The method of claim 90 or 91 , wherein the peptide antigens are cancer-associated antigens, and the subject has or is at risk of developing cancer.

93. The aAPC of claim 90 or 91, wherein the peptide antigens are associated with one or more infectious agents, and the subject is infected or is at risk of infection by the infectious agent.

94. The method of any one of claims 83 to 93, wherein the particle is a polymeric particle.

95. The method of claim 94, wherein the particle comprises PLA-PEG and/or PLGA- PEG block co-polymers.

96. The method of claim 95, wherein the HLA-E ligand is conjugated to the PEG terminus.

97. The method of any one of claims 83 to 96, wherein the population of particles is administered parenterally.

98. The method of claim 97, wherein the subject is further administered an immune checkpoint inhibitor therapy.