SYNTAC polypeptides and their uses

Abstract

The present invention relates to SYNTAC polypeptides and their uses. The present invention provides methods and compositions for clonogenic suppression or clonogenic stimulation of T cells.

Description

[0001] Cross-references

[0002] This application is a divisional application of Chinese application No. 201580027510.0 corresponding to PCT / US2015 / 035777, filed on June 15, 2015, and entered the Chinese national phase on November 28, 2016. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 013,715, filed on June 18, 2014, which is incorporated herein by reference in its entirety.

[0003] Government Support Statement

[0004] This invention was made with government support under Grant Nos. 3U54GM094662-02 and 5U01GM094665-02 awarded by the National Institutes of Health (NIGMS). The government has certain rights in this invention.

[0005] Introduction

[0006] Various publications cited throughout this application are indicated in square brackets. Full citations for these references can be found at the end of this patent specification. The disclosures of these publications and all patents, patent application publications, and books cited herein are hereby incorporated by reference into this application in their entirety to more fully describe the field to which the present invention pertains.

[0007] The rapid progress of the past decade in the development of high-throughput technologies for the discovery of clinically relevant biomarkers has paralleled the progressive development and application of biologics (drugs in which the active substance is produced by or extracted from a biological source, such as monoclonal antibodies, therapeutic proteins, and peptides) and has revolutionized the treatment of immunogenic disorders. However, current biologic therapies are driving safety regulatory measures at double the rate of their synthetic counterparts (17% for biologics and 8.5% for synthetics) [1]. This is thought to be reflected in the mode of action of these immunomodulatory biologics: general immunosuppression in the context of autoimmunity (e.g., Humira [2]) and general immunostimulation in the context of cancer treatment (e.g., Yervoy [3]). These treatments do not adequately restrict immunomodulation to pathogenic cells, thereby predisposing patients to potentially fatal infections and a host of troublesome side effects [4-6]. Furthermore, the modest efficacy and safety profile of these drugs [7] has led to a recent trend toward targeted therapies. The first generation of “targeted” biologics directed their effects to more restricted T cell subsets (e.g., antibodies and protein therapeutics such as anti-4-1BB, anti-CD27, LAG-3, and TIM-3) [8-11]. However, like previous therapies, these “first generation” efforts were still unable to target only disease-associated cells.

[0008] At the heart of the molecular events that comprise the adaptive immune response is the engagement of the T cell receptor (TCR) with a small peptide antigen presented non-covalently by a major histocompatibility complex (MHC) molecule. This represents the targeting mechanism of the immune system and is an essential molecular interaction for T cell activation and effector function. Following epitope-specific cellular targeting, recruited T cells are activated by general engagement of co-stimulatory molecules found on antigen presenting cells. Both signals are necessary to drive T cell specificity and activation or inhibition. Importantly, during T cell development, the genome editing process results in the expression of a unique TCR on each T cell

[12] , whereas this co-stimulatory molecule is normally expressed on all T cells (or a large T cell “subpopulation”). Current approaches rely almost entirely on general engagement of this co-stimulatory molecule, resulting in “global therapies”. These global immunotherapies are very effective, but indiscriminately target T cells, leading to severe toxicity. If co-stimulatory molecules could preferentially bind to T cells with disease-associated TCRs, their efficacy would be transformed from a disadvantage to an advantage.

[0009] There are a variety of approaches to modulating T cells, including the use of soluble co-stimulatory molecules, usually expressed as Fc fusions, or antibodies against co-stimulatory molecules that can block co-stimulatory function [13, 14], antibody-drug conjugates (ADCs)

[15] , bispecific antibodies (BsAbs) [16, 17], and free peptide antigens

[18] . Notably, ADCs (often referred to as magic bullets) ensure targeted delivery of toxins (or other drug payloads) directly to pathological cells. However, ADCs currently suffer from a lack of preferred biomarkers for targeting antibodies and poor internalization rates, as only ~1.5% of the administered dose is found to be within tumor cells, which is generally required for cell killing. Bispecific antibodies offer an attractive opportunity to combine the additive and synergistic effects of multiple monoclonal antibodies (mAbs) and can be used to bridge tumor cells to T cells

[17] , thus eliminating the need for internalization to elicit a response. Although bispecific antibodies have been developed to have bivalent interactions with two different antigens

[19] , these constructs still lack modularity and have lower affinity compared to the parent mAb

[20] . Adoptive T cell (CAR-T) therapy has partially addressed these issues and is an attractive alternative to the above-mentioned traditional therapies

[21] . CAR-T utilizes genetically modified primary T cells with chimeric antigen receptors (CARs) on their surface: the patient's T cells are extracted, purified, and genetically modified to target tumor-specific antigens through the use of CARs. CARs typically have an external single-chain variable domain (antibody fragment) that targets pathological cells, but with a traditional co-stimulatory cytoplasmic domain. Once the engineered T cell binds to the target antigen, the internal stimulatory domain provides the necessary signals to make the T cell fully active. In this fully active state, the T cell can proliferate and attack cancer cells more effectively. Moderating this response to avoid cytokine release syndrome and related side effects, as well as scalability issues (such as the significant cost and difficulty associated with T cell extraction and modification) have currently prevented this technology from entering mainstream use

[22] .

[0010] Biologics (also known as biopharmaceuticals) are drugs in which the active substance is produced by or extracted from a biological source (as opposed to "small molecule" drugs). Biologics are relatively recent entrants into the global therapeutic market and are mostly recombinant proteins produced through genetic engineering; these biologics include monoclonal antibodies, therapeutic proteins, and peptides. Most currently marketed biologics are used to alleviate symptoms in patients with chronic diseases such as cancer, diabetes, cardiovascular disease, infertility, and cystic fibrosis. The global biologics market was valued at $163 billion in 2012 and is projected to reach $252 billion in 2017, supporting a five-year compound annual growth rate of 9%. This growth is driven by the demand for a larger drug pipeline, the identification of attractive targets for challenging diseases, and the pursuit of follow-on biologics (biosimilars, generic biologics), exemplified by the recently introduced shortened FDA approval process.

[0011] The present invention addresses the need for precision therapeutics for immuno-oncology and autoimmunity - tailored therapeutics that clonally target only disease-relevant T cells for upregulation (e.g., in the case of cancer) or suppression (e.g., in the case of autoimmunity), as opposed to the general and "pseudo-targeted" modulators currently on the market or in development.

[0012] Brief Description

[0013] The present invention provides a recombinant polypeptide as described below: it comprises an amino acid sequence identical to a first B2M leader sequence, the amino acid sequence identical to the first B2M leader sequence is adjacent to a candidate epitope peptide, the candidate epitope peptide is adjacent to a first amino acid linker sequence, the first amino acid linker sequence is adjacent to an amino acid sequence identical to a natural human B2M peptide sequence, the amino acid sequence identical to a natural human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a T cell regulatory domain peptide sequence, the T cell regulatory domain peptide sequence is adjacent to a third amino acid linker, the third amino acid linker is adjacent to a second B2M leader sequence, the second B2M leader sequence is adjacent to an amino acid sequence identical to an MHC heavy chain, the amino acid sequence identical to an MHC heavy chain is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain.

[0014] The present invention also provides a recombinant polypeptide as described below: it comprises an amino acid sequence identical to a first B2M leader sequence, the amino acid sequence identical to the first B2M leader sequence is adjacent to a candidate epitope peptide, the candidate epitope peptide is adjacent to a first amino acid linker sequence, the first amino acid linker sequence is adjacent to an amino acid sequence identical to a natural human B2M peptide sequence, the amino acid sequence identical to a natural human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a second B2M leader sequence, the second B2M leader sequence is adjacent to a T cell regulatory domain peptide sequence, the T cell regulatory domain peptide sequence is adjacent to a third amino acid linker, the third amino acid linker is adjacent to an amino acid sequence identical to an MHC heavy chain, and the amino acid sequence identical to an MHC heavy chain is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain.

[0015] The present invention also provides a method for inhibiting a T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cell with an amount of a recombinant peptide as described herein that is effective in inhibiting the T cell clone, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is an inhibitory domain.

[0016] The present invention also provides a method for treating an autoimmune disease by suppressing an autoreactive T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cells with an amount of a recombinant peptide as described herein that is effective for treating the autoimmune disease, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is an inhibitory domain.

[0017] The present invention also provides a method for stimulating a T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cell with an amount of a recombinant peptide as described herein that is effective to stimulate the T cell clone, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is a stimulatory domain.

[0018] The present invention also provides a method for treating cancer by stimulating a T cell clone that recognizes an epitope peptide on cancer, comprising contacting the cloned T cells with an amount of a recombinant peptide as described herein effective to treat the cancer, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is a stimulatory domain.

[0019] The present invention also provides a recombinant polypeptide construct comprising (i) a candidate epitope peptide bound to a first amino acid linker sequence, wherein the first amino acid linker sequence is adjacent to an amino acid sequence comprising a sequence identical to a natural human B2M peptide sequence, the amino acid sequence comprising a sequence identical to a natural human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a T cell regulatory domain peptide, wherein (i) is bound to (ii) an amino acid sequence having an MHC heavy chain sequence via one or more disulfide bonds, the amino acid sequence having an MHC heavy chain sequence is adjacent to a third amino acid linker sequence, the third amino acid linker sequence is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain.

[0020] The present invention also provides a recombinant polypeptide construct comprising (i) a candidate epitope peptide bound to a first amino acid linker sequence, wherein the first amino acid linker sequence is adjacent to an amino acid sequence comprising a sequence identical to a natural human B2M peptide sequence, wherein (i) is bound to (ii) a T cell regulatory domain peptide via one or more disulfide bonds, wherein the T cell regulatory domain peptide is adjacent to a second amino acid linker sequence, wherein the second amino acid linker sequence is adjacent to an amino acid sequence having an MHC heavy chain sequence, wherein the amino acid sequence having an MHC heavy chain sequence is adjacent to a third amino acid linker sequence, wherein the third amino acid linker sequence is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain.

[0021] The present invention also provides a protein comprising two recombinant polypeptide constructs described herein, wherein the two constructs are linked by one or more disulfide bonds between their corresponding immunoglobulin Fc domains.

[0022] The present invention also provides a protein comprising two recombinant polypeptide constructs described herein, wherein the two constructs are linked by one or more disulfide bonds between their corresponding immunoglobulin Fc domains.

[0023] The present invention provides isolated suspension adapted cells transduced or transfected with a heterologous nucleic acid comprising, in 5' to 3' order, a sequence encoding a recombinant polypeptide as described herein.

[0024] The present disclosure provides a recombinant polypeptide as described below: it comprises an amino acid sequence identical to a first B2M leader sequence, the amino acid sequence identical to the first B2M leader sequence is adjacent to a candidate epitope peptide, the candidate epitope peptide is adjacent to a first amino acid linker sequence, the first amino acid linker sequence is adjacent to an amino acid sequence identical to a native human B2M peptide sequence, the amino acid sequence identical to a native human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a T cell regulatory domain peptide sequence, the T cell regulatory domain peptide sequence is adjacent to a third amino acid linker sequence, the third amino acid linker sequence is adjacent to a second B2M leader sequence, the second B2M leader sequence is adjacent to an amino acid sequence identical to an MHC heavy chain, the amino acid sequence identical to an MHC heavy chain is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain. In some cases, the candidate epitope comprises 7 to 20 amino acids. In some cases, the third amino acid linker is self-cleavable. In some cases, the second amino acid linker is self-cleavable. In some cases, the self-cleaving peptide is a viral 2A peptide or has the sequence thereof. In some cases, the first and / or second B2M leader sequences have the sequence of a human B2M leader sequence. In some cases, the MHC heavy chain is a human MHC heavy chain. In some cases, the MHC heavy chain is an MHC I molecule. In some cases, the MHC heavy chain is HLA-A02:01. In some cases, the MHC heavy chain is an MHC II molecule. In some cases, the immunoglobulin Fc domain is an IgG Fc domain. In some cases, the immunoglobulin Fc domain is an IgFc domain. In some cases, the immunoglobulin Fc domain is an IgM Fc domain. In some cases, the immunoglobulin Fc domain is a human immunoglobulin Fc domain. In some cases, the immunoglobulin Fc domain is an IgG1 Fc domain. In some cases, the recombinant polypeptide comprises a His-8 tag adjacent to its C-terminus. In some cases, the T cell regulatory domain is an inhibitory domain. In some cases, the T cell regulatory domain is a stimulatory domain. In some cases, the T cell regulatory domain is an antibody and antibody fragment, a peptide ligand, a T cell costimulatory peptide, a cytokine or a toxin. In some cases, the T cell regulatory domain includes a PD-L1 peptide, an Ig variable domain of a PD-L1 peptide, and the T cell regulatory domain includes 4-1BBL, and the T cell regulatory domain includes B7-1W88A or the T cell regulatory domain includes an anti-CD28 single-chain Fv. In some cases, the recombinant polypeptide includes a mutation in its human natural B2M peptide sequence and its heavy chain sequence to achieve a disulfide bond between the B2M peptide sequence and the heavy chain sequence.

[0025] The present disclosure provides a recombinant polypeptide as described below: providing an amino acid sequence identical to a first B2M leader sequence, the amino acid sequence identical to the first B2M leader sequence is adjacent to a candidate epitope peptide, the candidate epitope peptide is adjacent to a first amino acid linker sequence, the first amino acid linker sequence is adjacent to an amino acid sequence identical to a native human B2M peptide sequence, the amino acid sequence identical to a native human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a second B2M leader sequence, the second B2M leader sequence is adjacent to a T cell regulatory domain peptide sequence, the T cell regulatory domain peptide sequence is adjacent to a third amino acid linker, the third amino acid linker is adjacent to an amino acid sequence identical to an MHC heavy chain, the amino acid sequence identical to an MHC heavy chain is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain. In some cases, the candidate epitope comprises 7 to 20 amino acids. In some cases, the third amino acid linker is self-cleavable. In some cases, the second amino acid linker is self-cleavable. In some cases, the self-cleaving peptide is a viral 2A peptide or has the sequence thereof. In some cases, the first and / or second B2M leader sequences have the sequence of a human B2M leader sequence. In some cases, the MHC heavy chain is a human MHC heavy chain. In some cases, the MHC heavy chain is an MHC I molecule. In some cases, the MHC heavy chain is HLA-A02:01. In some cases, the MHC heavy chain is an MHC II molecule. In some cases, the immunoglobulin Fc domain is an IgG Fc domain. In some cases, the immunoglobulin Fc domain is an IgFc domain. In some cases, the immunoglobulin Fc domain is an IgM Fc domain. In some cases, the immunoglobulin Fc domain is a human immunoglobulin Fc domain. In some cases, the immunoglobulin Fc domain is an IgG1 Fc domain. In some cases, the recombinant polypeptide comprises a His-8 tag adjacent to its C-terminus. In some cases, the T cell regulatory domain is an inhibitory domain. In some cases, the T cell regulatory domain is a stimulatory domain. In some cases, the T cell regulatory domain is an antibody and antibody fragment, a peptide ligand, a T cell costimulatory peptide, a cytokine or a toxin. In some cases, the T cell regulatory domain includes a PD-L1 peptide, an Ig variable domain of a PD-L1 peptide, and the T cell regulatory domain includes 4-1BBL, and the T cell regulatory domain includes B7-1W88A or the T cell regulatory domain includes an anti-CD28 single-chain Fv. In some cases, the recombinant polypeptide includes a mutation in its human natural B2M peptide sequence and its heavy chain sequence to achieve a disulfide bond between the B2M peptide sequence and the heavy chain sequence.

[0026] In some cases, the recombinant polypeptide comprises mutations in its human native B2M peptide sequence and its heavy chain sequence to achieve a disulfide bond between the B2M peptide sequence and the heavy chain sequence. In some cases, the heavy chain sequence is HLA and wherein the disulfide bond connects one of the following residue pairs: B2M residue 12, HLA residue 236; B2M residue 12, HLA residue 237; B2M residue 8, HLA residue 234; B2M residue 10, HLA residue 235; B2M residue 24, HLA residue 236; B2M residue 28, HLA residue 232; B2M residue 98, HLA residue 192; B2M residue 99, HLA residue 234; B2M residue 3, HLA residue 120; B2M residue 31, HLA residue 96; B2M residue 53, HLA residue 35; B2M residue 60, HLA residue 96; B2M residue 60, HLA residue 122; B2M residue 63, HLA residue 27; B2M residue Arg3, HLA residue Gly120; B2M residue residue His31, HLA residue Gln96; B2M residue Asp53, HLA residue Arg35; B2M residue Trp60, HLA residue Gln96; B2M residue Trp60, HLA residue Asp122; B2M residue Tyr63, HLA residue Tyr27; B2M residue Lys6, HLA residue Glu232; B2M residue Gln8, HLA residue Arg234; B2M residue Tyr10, HLA residue Pro235; B2M residue Ser11, HLA residue Gln242; B2M residue Asn24, HLA residue Ala236; B2M residue Ser28, HLA residue Glu232; B2M residue Asp98, HLA residue His192; and B2M residue Met99, HLA residue Arg234.

[0027] In some cases, the recombinant polypeptide comprises mutations in its native human B2M peptide sequence and its heavy chain sequence to achieve a disulfide bond between the B2M peptide sequence and the heavy chain sequence. In some cases, the heavy chain sequence is HLA and the disulfide bond connects one of the following residue pairs: first linker position Gly 2, heavy chain (HLA) position Tyr 84; light chain (B2M) position Arg 12, HLA A1a236; and / or B2M residue Arg 12, HLA residue Gly237.

[0028] In some cases, the T cell regulatory domain is an inhibitory domain. In some cases, the T cell regulatory domain is a stimulatory domain. In some cases, the T cell regulatory domain is an antibody and antibody fragment, a peptide ligand, a T cell costimulatory peptide, a cytokine or a toxin. In some cases, the T cell regulatory domain comprises a PD-L1 peptide, an Ig variable domain of a PD-L1 peptide, the T cell regulatory domain comprises 4-1BBL, the T cell regulatory domain comprises B7-1W88A or the T cell regulatory domain comprises an anti-CD28 single-chain Fv.

[0029] The present disclosure provides nucleic acids encoding any of the recombinant polypeptides described above or elsewhere herein. The present disclosure provides cells transformed with nucleic acids encoding any of the recombinant polypeptides described above or elsewhere herein.

[0030] The present disclosure provides a method for inhibiting a T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cell with any recombinant peptide described above or elsewhere herein in an amount effective to inhibit the T cell clone, wherein the recombinant peptide comprises the epitope peptide and comprises a T cell regulatory domain that is an inhibitory domain.

[0031] The present disclosure provides a method for treating an autoimmune disorder by inhibiting an autoreactive T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cells with an amount of a recombinant peptide described above or elsewhere herein effective to treat the autoimmune disorder, wherein the recombinant peptide comprises the epitope peptide and comprises a T cell regulatory domain that is an inhibitory domain.

[0032] The present disclosure provides a method for stimulating a T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cell with any recombinant peptide described above or elsewhere herein in an amount effective to stimulate the T cell clone, wherein the recombinant peptide comprises the epitope peptide and comprises a T cell regulatory domain that is a stimulatory domain.

[0033] The present disclosure provides a method for treating cancer by stimulating a T cell clone that recognizes an epitope peptide on the cancer, comprising contacting the cloned T cells with an amount of a recombinant peptide described above or elsewhere herein effective to treat the cancer, wherein the recombinant peptide comprises the epitope peptide and comprises a T cell regulatory domain that is a stimulatory domain.

[0034] The present disclosure provides a recombinant polypeptide construct comprising (i) a candidate epitope peptide bound to a first amino acid linker sequence, the first amino acid linker sequence being adjacent to an amino acid sequence comprising a sequence identical to a native human B2M peptide sequence, the amino acid sequence comprising a sequence identical to a native human B2M peptide sequence being adjacent to a second amino acid linker sequence, the second amino acid linker sequence being adjacent to a T cell regulatory domain peptide, wherein (i) is bound by one or more disulfide bonds to (ii) an amino acid sequence having an MHC heavy chain sequence, the amino acid sequence having an MHC heavy chain sequence being adjacent to a third amino acid linker sequence, the third amino acid linker sequence being adjacent to an amino acid sequence identical to an immunoglobulin Fc domain. The present disclosure provides a protein comprising two recombinant polypeptide constructs, the two recombinant polypeptide constructs being linked by one or more disulfide bonds between their corresponding immunoglobulin Fc domains.

[0035] The present disclosure provides a recombinant polypeptide construct comprising (i) a candidate epitope peptide bound to a first amino acid linker sequence, the first amino acid linker sequence being adjacent to an amino acid sequence comprising a sequence identical to a native human B2M peptide sequence, wherein (i) is bound by one or more disulfide bonds to (ii) a T cell regulatory domain peptide, the T cell regulatory domain peptide being adjacent to a second amino acid linker sequence, the second amino acid linker sequence being adjacent to an amino acid sequence having an MHC heavy chain sequence, the amino acid sequence having an MHC heavy chain sequence being adjacent to a third amino acid linker sequence, the third amino acid linker sequence being adjacent to an amino acid sequence identical to an immunoglobulin Fc domain. The present disclosure provides a protein comprising two recombinant polypeptide constructs, the two recombinant polypeptide constructs being linked by one or more disulfide bonds between their corresponding immunoglobulin Fc domains.

[0036] The present disclosure provides a multimeric polypeptide comprising at least a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, in order from N-terminus to C-terminus: i) an epitope; and ii) a first major histocompatibility complex (MHC) polypeptide; and wherein the second polypeptide comprises, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide, wherein the multimeric polypeptide comprises an immunomodulatory domain at the C-terminus of the first polypeptide or at the N-terminus of the second polypeptide. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the multimeric polypeptide. The present disclosure provides a recombinant expression vector comprising the nucleic acid. The present disclosure provides a genetically modified host cell, wherein the genetically modified host cell is genetically modified by a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure. The present disclosure provides a composition comprising the multimeric polypeptide, including a pharmaceutical composition. The present disclosure provides a method for regulating T cell activity, the method involving contacting T cells with a multimeric polypeptide of the present disclosure. The present disclosure provides a method for treating a subject in need thereof, the method involving administering an effective amount of a multimeric polypeptide of the present disclosure to a subject in need thereof. The present disclosure provides containers for holding the multimeric polypeptides of the present disclosure or compositions (eg, pharmaceutical compositions) comprising the multimeric polypeptides of the present disclosure.

[0037] The present disclosure provides a multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory domain; iii) a second MHC polypeptide; and ii) an Ig Fc polypeptide. The present disclosure provides a multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide. In some cases, the first MHC polypeptide is a β2 microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide. In some cases, the β2 microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some cases, the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain. In some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some cases, the first MHC polypeptide is an MHC class II α chain polypeptide; and wherein the second MHC polypeptide is an MHC class II β chain polypeptide. In some cases, the epitope is a T cell epitope. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgAFc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5. Figures 24A-24CIn some cases, the first polypeptide and the second polypeptide are non-covalently associated. In some cases, the first polypeptide and the second polypeptide are covalently linked. In some cases, the covalent linkage is via a disulfide bond. In some cases, the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, and the disulfide linkage is between the first Cys residue and the second Cys residue. In some cases, the multimeric polypeptide comprises a first linker between the epitope and the first MHC polypeptide. In some cases, the immunomodulatory polypeptide is selected from the group consisting of: a 4-1BBL polypeptide, a B7-1 polypeptide; a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2 polypeptide. In some cases, the first polypeptide or the second polypeptide comprises two or more immunomodulatory polypeptides. In some cases, the two or more immunomodulatory polypeptides are in series. In some cases, the multimeric polypeptide comprises a third polypeptide, wherein the third polypeptide comprises an immunomodulatory polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the immunomodulatory polypeptide of the first polypeptide. In some cases, the third polypeptide is covalently linked to the first polypeptide. In some cases, wherein the second polypeptide comprises, in order from N-terminus to C-terminus:

[0038] i) a second MHC polypeptide; ii) an immunoglobulin (Ig) Fc polypeptide; and iii) an affinity tag.

[0039] The present disclosure provides a multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an Ig Fc polypeptide or a non-Ig scaffold, wherein the multimeric polypeptide optionally comprises an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory domains, wherein the one or more immunomodulatory domains: A) are located at the C-terminus of the first polypeptide; B) are located at the N-terminus of the second polypeptide; C) are located at the C-terminus of the second polypeptide; or D) are located at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide. In some cases, the multimeric polypeptide comprises a single immunomodulatory polypeptide. In some cases, the multimeric polypeptide comprises two immunomodulatory polypeptides (e.g., two copies of the same immunomodulatory polypeptide). In some cases, the multimeric polypeptide comprises three immunomodulatory polypeptides (e.g., three copies of the same immunomodulatory polypeptide). In some cases, the multimeric polypeptide comprises four immunomodulatory polypeptides (e.g., four copies of the same immunomodulatory polypeptide). In some cases, the multimeric polypeptide comprises a single immunomodulatory polypeptide. In some cases, the multimeric polypeptide comprises two immunomodulatory polypeptides (e.g., two copies of the same immunomodulatory polypeptide). In some cases, the multimeric polypeptide comprises three immunomodulatory polypeptides (e.g., three copies of the same immunomodulatory polypeptide). In some cases, the multimeric polypeptide comprises four immunomodulatory polypeptides (e.g., four copies of the same immunomodulatory polypeptide). In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory domain; iii) a second MHC polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide. In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory domain. In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an immunomodulatory domain.In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) an immunomodulatory domain; and ii) a second MHC polypeptide. In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) a second MHC polypeptide. In some cases, the non-Ig scaffold is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide. In some cases, the first MHC polypeptide is a β2 microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide. In some cases, the β2 microglobulin polypeptide comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:4. In some cases, the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain. In some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some cases, the first MHC polypeptide is an MHC class II α chain polypeptide; and wherein the second MHC polypeptide is an MHC class II β chain polypeptide. In some cases, the epitope is a T cell epitope. In some cases, the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises and. Figures 24A-24CIn some cases, the first polypeptide and the second polypeptide are non-covalently linked. In some cases, the first polypeptide and the second polypeptide are covalently linked. In some cases, the covalent linkage is via a disulfide bond. In some cases, the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, and the disulfide bond is between the first Cys residue and the second Cys residue. In some cases, the multimeric polypeptide comprises a first linker between the epitope and the first MHC polypeptide. In some cases, the immunomodulatory polypeptide is selected from the group consisting of: a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2 polypeptide. In some cases, the multimeric polypeptide comprises two or more immunomodulatory polypeptides. In some cases, the two or more immunomodulatory polypeptides are linked in series. In some cases, the multimeric polypeptide comprises a third polypeptide, wherein the third polypeptide comprises an immunomodulatory polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the immunomodulatory polypeptide of the first or second polypeptide. In some cases, the third polypeptide is covalently linked to the first polypeptide. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: i) a second MHC polypeptide; ii) an Ig Fc polypeptide; and iii) an affinity tag.

[0040] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a polypeptide chain of a multimeric polypeptide of the present disclosure; in some cases, the nucleic acid is present in a recombinant expression vector. The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a recombinant polypeptide, i) wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope; b) a first MHC polypeptide; c) an immunomodulatory polypeptide; d) a proteolytically cleavable linker or ribosomal skipping signal; e) a second MHC polypeptide; and f) an immunoglobulin (Ig) Fc polypeptide or a non-Ig-based scaffold; or ii) wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope; b) a first MHC polypeptide; c) a proteolytically cleavable linker or ribosomal skipping signal; d) an immunomodulatory polypeptide; e) a second MHC polypeptide; and f) an Ig Fc polypeptide or a non-Ig-based scaffold. In some cases, the first MHC polypeptide is a β2 microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide. In some cases, the β2 microglobulin polypeptide comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:4. In some cases, the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain. In some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some cases, the first MHC polypeptide is an MHC class II α chain polypeptide; and wherein the second MHC polypeptide is an MHC class II β chain polypeptide. In some cases, the epitope is a T cell epitope. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgAFc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises Figures 24A-24CIn some cases, the immunomodulatory polypeptide is selected from the group consisting of: 4-1BBL polypeptide, B7-1 polypeptide, B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide, and PD-L2 polypeptide. In some cases, the immunomodulatory polypeptide is selected from the group consisting of: CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, and HVEM. In some cases, the proteolytically cleavable linker or ribosomal skipping signal comprises an amino acid sequence selected from the group consisting of: a) LEVLFQGP (SEQ ID NO:37); b) ENLYTQS (SEQ ID NO:34); c) a furin cleavage site; d) LVPR (SEQ ID NO:36); e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:64); f) GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:65); g) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:66); and h) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:67). In some cases, the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) a first leader peptide; b) an epitope; c) a first MHC polypeptide; d) an immunomodulatory polypeptide; e) a proteolytically cleavable linker or ribosomal skipping signal; f) a second leader peptide; g) a second MHC polypeptide; and h) an immunoglobulin (Ig) Fc polypeptide. In some cases, the first and second leader peptides are β2-M leader peptides. In some cases, the nucleotide sequence is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the first MHC polypeptide or the linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, and wherein the first and second Cys residues provide a disulfide bond between the first and second MHC polypeptides. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids described above or elsewhere herein. In some cases, the recombinant expression vector is a viral vector. In some cases, the recombinant expression vector is a non-viral vector. The present disclosure provides host cells genetically modified with a recombinant expression vector as described above or elsewhere herein. In some cases, the host cell is in vitro. In some cases, the host cell is genetically modified such that the cell does not produce endogenous MHC β2 microglobulin polypeptide.In some cases, the host cell is a T lymphocyte.

[0041] The present disclosure provides a composition comprising: a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide comprising, from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a first nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising, from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide or a non-Ig-based scaffold. The present disclosure provides a composition comprising: a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide comprising, from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a first nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising, from N-terminus to C-terminus: i) an immunomodulatory domain; ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first and / or second nucleic acid is present in a recombinant expression vector. The present disclosure provides host cells genetically modified with a nucleic acid composition as described above or elsewhere herein.

[0042] The present disclosure provides a method for preparing a multimeric polypeptide as described above or elsewhere herein, the method comprising: a) culturing a host cell as described above or elsewhere herein in vitro in a culture medium under conditions such that the host cell synthesizes the multimeric polypeptide; and b) isolating the multimeric polypeptide from the host cell and / or culture medium. In some cases, the second polypeptide comprises an affinity tag, and wherein the isolating comprises contacting the multimeric polypeptide produced by the cell with a binding partner for the affinity tag, wherein the binding partner is immobilized, thereby immobilizing the multimeric polypeptide. In some cases, the method comprises eluting the immobilized multimeric polypeptide.

[0043] The present disclosure provides a method for selectively regulating the activity of epitope-specific T cells, the method comprising contacting the T cell with a multimeric polypeptide as described above or elsewhere herein, wherein the contact selectively regulates the activity of the epitope-specific T cells. In some cases, the immunomodulatory polypeptide is an activating polypeptide, and wherein the multimeric polypeptide activates the epitope-specific T cells. In some cases, the immunomodulatory polypeptide is an inhibitory polypeptide, and wherein the multimeric polypeptide inhibits the epitope-specific T cells. In some cases, the contact is performed in vitro. In some cases. The contact is performed in vivo.

[0044] The present disclosure provides a method for selectively regulating the activity of epitope-specific T cells in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide as described above or elsewhere herein, to effectively selectively regulate the activity of epitope-specific T cells in the individual. In some cases, the immunomodulatory polypeptide is an activating polypeptide, and wherein the multimeric polypeptide activates the epitope-specific T cells. In some cases, the epitope is an epitope associated with cancer, and wherein the administration selectively increases the activity of T cells specific for the cancer-associated epitope. In some cases, the immunomodulatory polypeptide is an inhibitory polypeptide, and wherein the multimeric polypeptide suppresses the activity of the epitope-specific T cells. In some cases, the epitope is an autologous epitope, and wherein the administration selectively suppresses the activity of T cells specific for the autologous epitope.

[0045] The present disclosure provides a method for treating an infection in an individual, the method comprising administering to the individual an effective amount of a) a multimeric polypeptide as described above or elsewhere herein; or b) one or more recombinant expression vectors comprising a nucleotide sequence encoding the multimeric polypeptide; or c) one or more mRNA comprising a nucleotide sequence encoding the multimeric polypeptide, wherein the epitope is a pathogen-associated epitope, wherein the immunomodulatory polypeptide is an activating polypeptide, and wherein the administration effectively selectively regulates the activity of pathogen-associated epitope-specific T cells in the individual. In some cases, the pathogen is a virus, bacterium, or protozoa. In some cases, the administration is subcutaneous administration (i.e., the administration is performed via subcutaneous administration). In some cases, the administration is intravenous administration (i.e., the administration is performed via intravenous administration). In some cases, the administration is intramuscular administration (i.e., the administration is performed via intramuscular administration). In some cases, the administration is systemic administration. In some cases, the administration is remote from the treatment site (ie, the administration is via subcutaneous administration), the administration is local (ie, the administration is via subcutaneous administration), the administration is at or near the treatment site.

[0046] The present disclosure provides a composition comprising: a) a multimeric polypeptide as described above or elsewhere herein; and b) a pharmaceutically acceptable excipient.

[0047] The present disclosure provides a composition comprising: a) a nucleic acid as described above or elsewhere herein or a recombinant expression vector as described above or elsewhere herein; and b) a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1:SynTac: Artificial immune synapse for T cell activation. The illustration on the left depicts the traditional two-signal hypothesis for T cell activation. That is, targeted T cell engagement by a unique TCR:MHC-epitope interaction between a T cell and an antigen presenting cell (APC), followed by stimulation or inhibition by co-stimulatory molecule engagement. The middle illustration is a schematic diagram of the synTac molecule, followed by the mode of action of synTac (right). Similar to the natural response (left), the synTac fusion protein allows highly specific cell targeting through MHC-epitopes. It is followed by a T cell regulatory domain ("MOD"), which plays a role via co-stimulatory molecule engagement and can provide activation or inhibition. This triggers a clonal rather than comprehensive T cell response. It is worth noting that MOD can be any known or approved antibody, antibody fragment, co-stimulatory molecule or other literature-verified payload (cytokine, toxin, etc.) as well as new entrants.

[0049] Figures 2A-2C SynTac Fc fusion constructs. One strategy utilizes Fc fusion constructs to increase the potency, stability, and therapeutic window of related products. Briefly, the Fc region is a naturally occurring covalent homodimer formed by the interaction of two identical immunoglobulin CH2-CH3 domains (referred to as Fc) and stabilized by two disulfide bonds (shown as two thin lines) between the CH2 domains. Figure 2A A single-chain peptide MHC protein is shown, its carboxyl terminus linked to the IgG Fc region. To introduce alternative protein linkages (such as MOD), the construct is split into the corresponding heavy and light chains and both the peptide and protein are fused to each terminus. One construct ( Figure 2B ) results in the amino terminus of the peptide being associated with a light chain (β2 microglobulin, B2M), followed by extension of the carboxyl terminus of the light chain to the MOD effector molecule. In this case, the heavy chain (HLA-molecule, HC) is fused to the Fc region. The construct is covalently held together by a disulfide bridge (labeled SS). An alternative orientation ( Figure 2C ) The MOD amino terminus of Fc was fused to the heavy chain, while the peptide remained linked to the B2M light chain.

[0050] Figures 3A-3B : Overall design of two basic synTac molecules. This construct utilizes the natural human B2M leader sequence (LEADER) to allow efficient secretion and ER processing, followed by candidate epitopes (labeled as peptides (PEPTIDE)). For light chain bonding (LC, Figure 3A), which is coupled to the native B2M via linker L1, while MOD is coupled via linker L2. The complete cassette is linked to another B2M leader sequence, MHC heavy chain and IgG1 Fc domain via a porcine Teschovirus-1 (P2A) "self-cleaving" peptide to allow stoichiometric expression of each chain. Heavy chain (HC, Figure 3B ) linkage is similar, however, now the viral P2A peptide follows B2M, while MOD follows the second leader peptide. Both constructs terminate with an 8x His tag.

[0051] Figure 4 :CRISPR / CAS mediated knockout of endogenous beta-2 microglobulin. A guide RNA for endogenous B2M was designed, transfected with a plasmid encoding CRISPR / CAS and cultured for three days. The cultured cells were surface stained for B2M and counter-selected by fluorescence-activated cell sorting (FACS) (sorted based on loss of fluorescence). The sorted cells were recovered and two or more rounds of staining, counter-sorting, and recovery (3 rounds in total) were performed to ensure effective knockout. The quality of the final pool was checked by monitoring the surface expression of B2M via FACS (as shown above).

[0052] Figures 5A-5B : SynTac constructs with engineered disulfide bonds were prepared and tested for activity. One construct (H236-L12, labeled synTac 18) demonstrated high expression, while a second construct (H237-L12, synTac 17) demonstrated moderate expression. A dt-SCT disulfide solution was used as a positive control (labeled synTac 2). Non-reducing PAGE showed formation of the expected high molecular weight, disulfide-linked fraction ( Figure 5A All expression constructs were scaled up to 100 ml, purified, and tested for activity by binding to the cognate TCR expressed on the surface of HEK cells (designated A6), as monitored by FACS fluorescence, indicating correct folding and activity ( Figure 5B Cells expressing a non-cognate TCR (called AS01) were used as a negative control.

[0053] Figures 6A-6C : Expression of various synTac protein fusions. The light chain of the synTac fusion was successfully expressed ( Figure 6A ), the synTac fusions have various targeting peptides and HLA isotypes with the PD-L1 MOD domain, specifically 1) HTLV-human HLA-A02, 2) IGRP-murine H2-Kd, and 3) TUM-murine H2-Kd ( Figure 6B) ) which is a syuTac fusion based on IGRP with various MOD domains, 4) Ig variable domain of PD-L1, 5) 4-1BBL, 6) anti-CD28 single chain Fv and 7) B7-1 W88A ( Figure 6C ) ) which is a synTac fusion based on IGRP with various MODs expressed with heavy chain linkage), 8) PD-L1 and 9) anti-CD28 single chain Fv.

[0054] Figures 7A-7B TCR-synTac-PD1 bridge: Verify the integrity of the synTac protein components. HEK cells expressing the cognate TCR (A6) were used as a positive control, and cells expressing a non-cognate TCR (AS01) were generated and used as a negative control along with untransduced parental cells. Cells were challenged with a non-fluorescent purified HTLV-PD-L1 synTac variant and incubated with its cognate receptor, PD1, fused to mouse IgG2a. The PD1-Fc fusion was detected using a FITC-labeled anti-mouse secondary antibody. Figure 7A Show the reaction schematic diagram, Figure 7B FAC results are shown. As expected, colocalized fluorescence was observed only when the homologous (A6) HEK cell line was challenged with HTLV presenting synTac with PD-L1 MOD. Notably, no fluorescence was observed when HEK cells with non-cognate TCRs or parental cells were challenged, when challenged with FITC-PDL-Fc alone, or when MOD was absent.

[0055] Figures 8A-8D SynTac in action: In vitro T cell analysis. CD8+ T cells from 8.3 transgenic NOD mice were cultured in the presence of immobilized anti-CD3 antibody to stimulate polyclonal T cell activation. Figure 8A ) or synTac TUM-PD-L1( Figure 8C ) were used to treat stimulated cultures to examine the antigen specificity of any inhibitory effect. A form of synTac IGRP without PD-L1 ( Figure 8B ) served as an effector control for the MOD domain. Before inoculation, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) to monitor the extent of cell proliferation induced by T cell activation. Cells were harvested on day 5 and flow cytometry was used to examine viability and proliferation. Multiplex flow cytometry microbead analysis was also used to examine the expression of CD8+ T cell effector cytokines IFNγ and TNFα in the supernatant. All CD8+ T cell activation parameters examined were suppressed in an antigen-specific and effector (i.e., MOD) domain-dependent manner ( Figure 8D ).

[0056] Figures 9A-9F Provided are the amino acid sequence and domain structure of the synTac polypeptide.

[0057] Figures 10A-10C Depicts constructs for 4-1BBL trimer expression. Schematic representation of the monomeric form of the native 4-1BBL extracellular domain (residues 50-254) ( Figure 10A ), showing the membrane proximal (Memb Prox, MP) and TNF homology (TNF-H) domains; ( Figure 10B )4-1BBL dimer synTac; and ( Figure 10C ) A fully active ditrimeric form of 4-1BBL synTac was generated by co-expressing a conventional synTac construct with a "free" 4-1BBL extracellular domain (residues 50-254, Figure 10A All constructs were assembled when expressed together in mammalian cells. Purification via the Fc region (Protein A / G) followed by size exclusion allowed the separation of the 4-1BBL trimer synTac from free BBL.

[0058] Figures 11A-11B Multi-angle light scattering (MALS) analysis of synTac protein with trimeric 4-1BBL. Figure 11A ) Molecular weights of the major species identified by MALS, showing examples of multiple independent measurements. ( Figure 11B ) Representative curve from MALS of synTac40+51, which has relatively high levels of light scattering and low ultraviolet (UV) absorbance, reflecting the presence of small amounts of high molecular weight protein. Low molecular weight buffer components result in large changes in refractive index (positive or negative) without associated changes in UV absorbance.

[0059] Figure 12SynTac 4-1BBL receptor binding. Protein A microbeads were coated to saturation with recombinant human or mouse 4-1BB-Fc fusion proteins and used to bind synTac constructs (as co-regulatory domains) with 4-1BB ligands (dimers and trimers), followed by fluorescent detection antibodies specific for synTac heavy chain isotypes. The degree to which synTac 4-1BBL specifically binds to bead-derived 4-1BB was then measured by high-throughput flow cytometry. Using this system, the cross-reactivity and relative affinity of 4-1BBL to both human and mouse 4-1BB were explored in the context of the synTac scaffold. synTac with 4-1BBL was shown to bind to the cognate receptor, but not to “receptor-free” (referred to as MOD-free) Fc-bound microbeads, indicating that it was a well-folded and active protein reagent. Notably, the trimer bound with an affinity range expected for a dual trimeric engagement, whereas the native dimer displayed a 10-fold decrease in binding affinity, and all constructs cross-reacted between the murine and human receptors.

[0060] Figure 13 CD8 from 8.3 transgenic NOD mice were cultured in the presence of immobilized anti-CD3 antibody. + T cells were used to stimulate polyclonal T cell activation. The stimulated cultures were treated with soluble forms of synTac TUM-41BBL (A) or synTac IGRP-41BBL (B and C) to examine the antigen specificity of any stimulation. Control treatments were culture medium alone (-CNTRL) or immobilized anti-CD3 (+CNTRL) to benchmark response amplitude. Cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) to monitor the degree of cell proliferation induced by T cell activation. After 4 days, cells were harvested and flow cytometry was used to examine viability and proliferation. CD8 + Expression of T cell effector cytokines IFNγ and TNFα. + T cell activation parameters are all activated in an antigen-specific and effector (ie, MOD) domain-dependent manner.

[0061] Figure 14 Single-dose in vivo T cell stimulation analysis. NOD mice were injected intraperitoneally with synTac IGRP-41BBL, synTac TUM-41BBL, or PBS. Six days after injection, mice were sacrificed, and splenocytes were examined for the relative frequency of IGRP-specific CD8 T cells by flow cytometry using the appropriate peptide-MHC pentamer dye. IGRP-41BBL treatment was associated with significantly higher frequencies of IGRP-specific CD8 T cells relative to controls, supporting significant in vivo expansion induced by a single dose.

[0062] Figure 15 Multiple-dose in vivo T cell stimulation analysis. NOD mice were injected intraperitoneally with three doses of synTac IGRP-41BBL, synTac TUM-41BBL, or PBS over a two-week period. Seven days after injection, mice were sacrificed and PBMCs (from blood) were examined for relative frequencies of IGRP-specific CD8 T cells by flow cytometry using appropriate peptide-MHC pentamer dyes. IGRP-41BBL treatment was associated with significantly higher frequencies of IGRP-specific CD8 T cells relative to controls, supporting the significant in vivo expansion induced by multiple doses, including rare tumor-specific T cells (TUM).

[0063] Figures 16A-16B Schematic diagram of the optimized construct for 4-1BBL trimer expression. Disulfide lock ( Figure 16A , DL) and single-chain trimers ( Figure 16B , SCT).

[0064] Figure 17 .SynTac 4-1BBL receptor binding. Protein A microbeads are coated to saturation with recombinant human or mouse 4-1BB-Fc fusion protein and used to bind synTac constructs (as co-regulatory domains) with 4-1BB ligands (disulfide-locked trimers (69, 70, and 71) and single-chain trimers (SCT), followed by specific fluorescent detection antibodies for synTac heavy chain isotypes. The natural trimer shown is a binding control (trimer). The degree of synTac 4-1BBL specific binding to bead-derived 4-1BB is then measured by high-throughput flow cytometry. synTac with 4-1BBL has been shown to bind to cognate receptors, but does not bind to Fc-bound microbeads with "no receptor" (referred to as MOD-free), indicating that it is a well-folded and active protein reagent. All constructs cross-react between mouse and human receptors.

[0065] Figure 18. Expression verification of optimized 4-1BBL construct. SynTac produced by coexpression, it has original 4-1BBL regulator (synTac40 / 51, no disulfide bond locking, marked as "O" (for indicating original));And three containing engineered disulfide bond locking to limit the optimized construct of trimeric configuration. In each construct, two native residues are replaced by cysteine ​​residues (Q94C: P245C (marked as "DL1" in gel), Q94C: P242C "DL2" and Q89C: L115C "DL3", respectively referred to as synTac 69, 70 and 71), which is coexpressed in human cells with a "free" non-labeled form with the same mutation (respectively referred to as 98, 99, 100) to allow covalent locking in cells. The disulfide bonding degree was observed by the amount of released (non-covalently bound) "free" 4-1BBL in non-reducing SDS-PAGE analysis. Free BBL will migrate to ~ 20kDa (BOX), thereby confirming the disulfide bond locking of the engineered construct. The SynTac carrying the 4-1BBL in single-chain trimer form (SCT) also showed the following affinity and gel filtration purification (labeled "SCT"). Accurate mass was confirmed by multi-angle light scattering (MALS).

[0066] Figures 19A-19I . Schematic diagram of an embodiment of the synTac construct of the present disclosure. Figures 19A-19C Describe respectively relative to Figures 2A-2C The construct. Figure 19B and 19C In the figure, the P2A uncleaved polypeptide is depicted (top), and the cleaved polypeptide (by P2A-mediated autocleavage) is depicted (bottom), with the disulfide bond (SS) mediated by the cysteine ​​substitution (*) as shown. Figures 19D-19F Describe the above relative to Figures 8A-8C The construct; Figures 19D-19F In each panel, the P2A uncleaved form is depicted above (top), and the P2A-mediated self-cleavage polypeptide (bottom) has a disulfide bond (SS) mediated by a cysteine ​​substitution (*) as indicated. Figure 19G Describe relative to Figure 9B Generalized format of the synTac40 construct, showing the uncleaved (top) and self-cleaved / disulfide-bonded (bottom) polypeptides. Figure 19H Describe relative to Figures 9C-9E Generalized formats of synTac69, synTac70, and synTac71 constructs are shown, showing uncleaved (top) and self-cleaved / disulfide-bonded (bottom) polypeptides, with additional cysteine ​​substitutions in the 4-1BBL domain indicated (*). Figure 19IDescribe relative to Figure 9F Generalized form of the synTac 4-1BBL single-chain trimer (SCT) shown with uncleaved (top) and self-cleaved / disulfide-bonded (bottom) polypeptides.

[0067] Figure 20 Provided is a multiple amino acid sequence alignment of the beta-2 microglobulin (B2M) precursor (i.e., including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO:78), Pantroglodytes (NP_001009066.1; SEQ ID NO:79), Macaca mulatta (NP001040602.1; SEQ ID NO:80), Bos Taurus (NP 776318.1; SEQ ID NO:81), and Mus musculus (NP 033865.2; SEQ ID NO:82).

[0068] Figure 21 The domain structure of the construct of SEQ ID NO: 6 is provided.

[0069] Figure 22 The domain structure of the construct of SEQ ID NO: 7 is provided.

[0070] Figure 23 Describe the effects of in vivo administration of synTac IGRP-PDL1, synTac TUM-PDL1, or phosphate-buffered saline (PBS) on IGRP-specific CD8 + Effect of T cell frequency.

[0071] Figures 24A-24C The amino acid sequence of an immunoglobulin Fc polypeptide is provided.

[0072] Figures 25A-25C The amino acid sequence of a human leukocyte antigen (HLA) class I heavy chain polypeptide is provided.

[0073] Figures 26A-26B The amino acid sequence of the PD-L1 polypeptide is provided.

[0074] Figure 27 The amino acid sequence of the 4-1BBL polypeptide is provided.

[0075] Figure 28 The amino acid sequence of the ICOS-L polypeptide is provided.

[0076] Figure 29 The amino acid sequence of the OX40L polypeptide is provided.

[0077] Figure 30 The amino acid sequence of the PD-L2 polypeptide is provided.

[0078] Figure 31 The amino acid sequence of the CD80 (B7-1) polypeptide is provided.

[0079] Figure 32 The amino acid sequence of the CD86 (B7-2) polypeptide is provided.

[0080] Figure 33 The amino acid sequence of the Fas ligand (FAS-L) polypeptide is provided.

[0081] Figures 34A-34H Schematic diagrams of embodiments of the disclosed synTac constructs are provided, showing disulfide bonds (SS) mediated by cysteine ​​substitutions (*). In these embodiments, the disulfide bonds are formed between MHC (eg, HLA) polypeptides present in separate polypeptides.

[0082] definition

[0083] As used herein, "leader sequence" includes any signal peptide that can be processed by mammalian cells, including the human B2M leader sequence. Such sequences are well known in the art.

[0084] Unless specifically indicated otherwise, "adjacent" as used herein with respect to, for example, element A and element B, means that element A is adjacent to element B and is bound, preferably via a covalent bond, to element B. For example, with respect to a first amino acid sequence being adjacent to a second amino acid sequence, the C-terminus of the first amino acid sequence may be linked to the N-terminus of the second amino acid sequence via a peptide bond.

[0085] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term also includes polypeptides with co-translational modifications (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like. In addition, "polypeptide," as used herein, refers to proteins including modifications, such as deletions, additions, and substitutions (generally conservative substitutions known to those skilled in the art) of the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional (e.g., by site-directed mutagenesis) or may be accidental (such as by mutations in the host producing the protein or errors due to PCR amplification or other recombinant DNA methods).

[0086] As used herein, the term "recombinant" used to describe nucleic acid molecules refers to polynucleotides of genomic, cDNA, viral, semisynthetic and / or synthetic origin that are not associated with all or a portion of the polynucleotide sequences with which they are naturally associated, due to their origin or manipulation. As used herein, the term "recombinant" with respect to proteins or polypeptides refers to polypeptides prepared by expression of recombinant polynucleotides. As used herein, the term "recombinant" with respect to host cells or viruses refers to host cells or viruses into which recombinant polynucleotides have been introduced. As used herein, the term "recombinant" with respect to substances (e.g., cells, nucleic acids, proteins, or vectors) also refers to substances that have been modified by the introduction of heterologous substances (e.g., cells, nucleic acids, proteins, or vectors).

[0087] The terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to include polymeric forms of nucleotides, whether ribonucleotides or deoxyribonucleotides. The terms refer only to the primary structure of the molecule. Thus, the terms include triple-stranded, double-stranded, and single-stranded DNA, as well as triple-stranded, double-stranded, and single-stranded RNA. The terms also include such molecules with modifications (such as those modified by methylation and / or by capping) and unmodified forms of polynucleotides. More specifically, the terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" include polydeoxynucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as those found in DNA and RNA.

[0088] As used herein, the term "vector" refers to a vehicle capable of transferring a nucleic acid sequence to a target cell. For example, a vector may comprise a coding sequence capable of being expressed in a target cell. As used herein, "vector construct," "expression vector," and "gene transfer vector" generally refer to any nucleic acid construct capable of directing the expression of a gene of interest and capable of being used to transfer a gene of interest to a target cell. Therefore, the term includes cloning and expression vehicles, as well as integrating vectors and non-integrating vectors. Therefore, a vector can transfer a nucleic acid sequence to a target cell, and in some cases, a vector is used to manipulate a nucleic acid sequence, such as a recombinant nucleic acid sequence (i.e., for the preparation of a recombinant nucleic acid sequence), etc. For purposes of this disclosure, examples of vectors include, but are not limited to, plasmids, bacteriophages, transposons, cosmids, viruses, etc.

[0089] As used herein, "expression cassette" encompasses any nucleic acid construct capable of directing the expression of any RNA transcript (including a gene / coding sequence of interest and untranslated RNA). Such cassettes can be constructed into "vectors," "vector constructs," "expression vectors," or "gene transfer vectors" to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. The transcript of the expression cassette can be stably or transiently expressed and can be expressed from a cassette that is integrated into the host genome (in a targeted or non-targeted manner), or can remain unintegrated as desired.

[0090] "Operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. As used herein, the terms "heterologous promoter" and "heterologous control region" refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a "transcription control region that is heterologous to a coding region" is a transcription control region that is not normally associated with the coding region in nature.

[0091] As used herein, the term "immunological synapse" or "immunological synapse" generally refers to the natural interface between two interacting immune cells of an adaptive immune response, including, for example, the interface between an antigen presenting cell (APC) or target cell and an effector cell (e.g., lymphocyte, effector T cell, natural killer cell, etc.). The immunological synapse between an APC and a T cell is typically initiated by an interaction between the T cell antigen receptor and a major histocompatibility complex molecule (e.g., as described in Bromley et al., Annu Rev Immunol. 2001; 19: 375-96; the disclosure of which is incorporated herein by reference in its entirety).

[0092] As used herein, the term "heterologous" used with respect to nucleic acid sequences, proteins or polypeptides means that these molecules are not naturally present in the cell from which the heterologous nucleic acid sequence, protein or polypeptide is derived. For example, a nucleic acid sequence encoding a human polypeptide inserted into a cell of a non-human cell is a heterologous nucleic acid sequence in this particular context. Although heterologous nucleic acids can be derived from different organisms or animal species, such nucleic acids do not need to be derived from a separate biological species that is heterologous. For example, in some cases, a synthetic nucleic acid sequence or a polypeptide encoded therefrom can be heterologous to the cell into which this nucleic acid sequence or the polypeptide encoded therefrom is introduced, because the cell did not previously contain the synthetic nucleic acid. Therefore, a synthetic nucleic acid sequence or a polypeptide encoded therefrom can be considered heterologous to a human cell, for example, even if one or more components in the synthetic nucleic acid sequence or the polypeptide encoded therefrom are initially derived from a human cell.

[0093] As used herein, "host cell" refers to a eukaryotic cell in vivo or in vitro or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which can be or has been used as a recipient of a nucleic acid (e.g., an expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present invention), and includes progeny of the original cell that has been genetically modified by the nucleic acid. It should be understood that the progeny of a unicellular cell are not necessarily identical to the original parent in morphology or in the genome or entire DNA complement sequence due to natural, accidental, or intentional mutations. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid (e.g., an expression vector) has been introduced. For example, a genetically modified eukaryotic host cell is genetically modified by introducing a heterologous nucleic acid (e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in a eukaryotic host cell) into a suitable eukaryotic host cell.

[0094] In some cases, nucleic acid or amino acid sequences (including polypeptides and nucleic acids encoding polypeptides) are referred to based on "sequence similarity" or "sequence identity" (e.g., compared to one or more reference sequences). In other cases, mutant or variant sequences can be referred to based on comparison with one or more reference sequences. For sequence comparison, typically one sequence serves as a reference sequence, to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0095] When necessary or desired, optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), which is incorporated herein by reference, by the homology alignment algorithm of Needleman and Wunsch, J. MoI. Biol. 48:443-53 (1970), which is incorporated herein by reference, by the search similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), which is incorporated herein by reference, by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. (See generally, Ausubel et al. (eds.), Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999)).

[0096] "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), T regulatory cells (Tregs), and NK-T cells.

[0097] As used herein, the term "costimulatory ligand" includes molecules on antigen presenting cells (e.g., APCs, dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on T cells, thereby providing signals that mediate T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided, for example, by the binding of the TCR / CD3 complex to the peptide-loaded MHC molecule. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L and Fas ligand (FasL), inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also encompass antibodies that specifically bind to co-stimulatory molecules presented on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, among others.

[0098] The terms "purification", "isolation" and the like refer to removing a desired substance (e.g., a recombinant protein) from a solution containing an undesirable substance (e.g., a contaminant) or removing an undesirable substance from a solution containing a desired substance, thereby leaving essentially only the desired substance. In some cases, the purified substance may be substantially free of other substances, such as contaminants. Purification as used herein can refer to a range of different purity levels produced, such as where the purified substance accounts for more than 80% of all substances in the solution, including more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, etc. As will be understood by those skilled in the art, in general, when determining the purity of a substance, the solution components themselves, such as water or buffer or salts, are not taken into account.

[0099] As used herein, the term "treat" and the like refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and / or therapeutic in terms of partially or completely curing a disease and / or attributing adverse effects of the disease. As used herein, the term "treat" encompasses the treatment of any disease in a mammal (e.g., in a human) and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing the disease to regress.

[0100] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0101] "Therapeutically effective amount" or "effective amount" refers to the amount of an agent or the combined amount of two agents that, when administered to a mammal or other subject for treating a disease, is sufficient to achieve such treatment for the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0102] Before further describing the present invention, it should be understood that the present invention is not limited to the particular embodiments described, as such embodiments may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0103] When a range of values ​​is provided, it is understood that unless the context clearly indicates otherwise, each intervening value (to one-tenth of the unit of the lower limit) between the upper and lower limits of the range and any other stated or intermediate values ​​within the stated range are included within the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller range and are also included within the present invention, subject to any specifically excluded limits in the stated range. When the stated range includes one or two limits, ranges excluding one or both of those included limits are also included in the present invention.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials listed in the publications.

[0105] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a multimeric polypeptide" includes a plurality of such polypeptides, and reference to "the immunomodulatory polypeptide" includes reference to one or more immunomodulatory polypeptides and equivalents thereof known to those skilled in the art. It is further noted that claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely" or "only" in connection with the recitation of claim elements or for use of a "negative" limitation.

[0106] It will be appreciated that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for simplicity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the present invention are expressly contemplated herein and disclosed herein, as are each and every combination individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also expressly contemplated herein and disclosed herein, as are each and every subcombination individually and expressly disclosed herein.

[0107] The publications discussed herein are provided solely as of the date of their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed. Detailed Description of the Invention

[0108] Here we describe a new protein-based therapeutic platform that recapitulates the classical immune response, the artificial immune synapse for T cell activation (synTac). A novel fusion protein that links a costimulatory molecule to an MHC epitope allows precise T cell engagement and clonal T cell activation or inhibition depending on the MOD molecule moiety.

[0109] Multimeric polypeptides

[0110] The present disclosure provides multimeric (e.g., heterodimers, heterotrimers) polypeptides. The present disclosure provides multimeric protein precursors of the multimeric polypeptides of the present disclosure. The present disclosure provides precursor gene products, such as multimeric protein precursors of the multimeric polypeptides of the present disclosure, and mRNA gene products encoding two or more polypeptide chains of the multimeric polypeptides of the present disclosure.

[0111] The present invention also provides a recombinant polypeptide construct comprising (i) a candidate epitope peptide bound to a first amino acid linker sequence, the first amino acid linker sequence being adjacent to an amino acid sequence comprising a sequence identical to a natural human B2M peptide sequence, the amino acid sequence comprising a sequence identical to a natural human B2M peptide sequence being adjacent to a second amino acid linker sequence, the second amino acid linker sequence being adjacent to a T cell regulatory domain peptide, wherein (i) is bound by one or more disulfide bonds to (ii) an amino acid sequence having an MHC heavy chain sequence, the amino acid sequence having an MHC heavy chain sequence being adjacent to a third amino acid linker sequence, the third amino acid linker sequence being adjacent to an amino acid sequence identical to an immunoglobulin Fc domain. In one embodiment, the recombinant polypeptide construct comprises

[0112] LLFGYPVYVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTSGSGATNFSLLKQAGDVEENPGPMSRSVALAVLALLSLSGLEAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPAAAGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSHHHHHHHH(SEQ ID NO:6)。

[0113] The present invention also provides a recombinant polypeptide construct comprising (i) a candidate epitope peptide bound to a first amino acid linker sequence, the first amino acid linker sequence being adjacent to an amino acid sequence comprising a sequence identical to a natural human B2M peptide sequence, wherein (i) is bound via one or more disulfide bonds to (ii) a T cell regulatory domain peptide, the T cell regulatory domain peptide being adjacent to a second amino acid linker sequence, the second amino acid linker sequence being adjacent to an amino acid sequence having an MHC heavy chain sequence, the amino acid sequence having an MHC heavy chain sequence being adjacent to a third amino acid linker sequence, the third amino acid linker sequence being adjacent to an amino acid sequence identical to an immunoglobulin Fc domain. In one embodiment, the recombinant polypeptide construct comprises

[0114] LLFGYPVYVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSSGSGATNFSLLKQAGDVEENPGPMSRSVALAVLALLSLSGLEAFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPAAAGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSHHHHHHHH(SEQ ID NO:7)。

[0115] The present invention also provides a protein comprising two recombinant polypeptide constructs described herein, wherein the two recombinant polypeptide constructs are linked by one or more disulfide bonds between the corresponding immunoglobulin Fc domains.

[0116] The present invention also provides a protein comprising two recombinant polypeptide constructs described herein, wherein the two recombinant polypeptide constructs are linked by one or more disulfide bonds between the corresponding immunoglobulin Fc domains.

[0117] The present invention also provides the synTac platform: an artificial immune synapse for targeted T cell activation.

[0118] In one embodiment, the β2 microglobulin has the same sequence as human β2 microglobulin. In one embodiment, the histocompatibility complex heavy chain sequence has the same sequence as a human HLA-A sequence. In one embodiment, the histocompatibility complex heavy chain transmembrane domain has the same sequence as a human major histocompatibility complex I (MHC I) heavy chain transmembrane domain. In one embodiment, the histocompatibility complex heavy chain transmembrane domain has the same sequence as a human major histocompatibility complex II (MHC II) heavy chain transmembrane domain.

[0119] The invention also provides compositions comprising a plurality of constructs.

[0120] In one embodiment, the candidate epitope peptide is a peptide of 8, 9, 10, 11 or 12 amino acids. In one embodiment, the candidate epitope peptide is a peptide of 13, 14, 15, 16 or 17 amino acids. In one embodiment, the candidate epitope peptide is a nonamer (9 amino acids in length).

[0121] The present disclosure provides multimeric polypeptides comprising two or more (e.g., 2, 3, 4 or more) polypeptide chains. In some cases, the multimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first major histocompatibility complex (MHC) polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory domains, wherein the one or more immunomodulatory domains are: A) located at the C-terminus of the first polypeptide; B) located at the N-terminus of the second polypeptide; C) located at the C-terminus of the second polypeptide; or D) located at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide.

[0122] In some cases, the multimeric polypeptides of the present disclosure comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, in order from amino terminus (N-terminus) to carboxyl terminus (C-terminus): a) an epitope (e.g., a T cell epitope); b) a first major histocompatibility complex (MHC) polypeptide, and c) an immunomodulatory polypeptide; and wherein the second polypeptide comprises, in order from N-terminus to C-terminus: a) a second MHC polypeptide; and b) an immunoglobulin (Ig) Fc polypeptide. In other cases, the multimeric polypeptides of the present disclosure comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope (e.g., a T cell epitope); and b) a first MHC polypeptide; and wherein the second polypeptide comprises, in order from N-terminus to C-terminus: a) an immunomodulatory polypeptide; b) a second MHC polypeptide; and c) an Ig Fc polypeptide. In some cases, the first and second MHC polypeptides are MHC class I polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class I polypeptide β2 microglobulin (B2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H chain). In other cases, the first and second MHC polypeptides are MHC class II polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide, and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide, and the second MHC polypeptide is an MHC class II α-chain polypeptide. In some cases, the multimeric polypeptide comprises two or more immunomodulatory polypeptides. When the multimeric polypeptide of the present disclosure comprises two or more immunomodulatory polypeptides, in some cases, the two or more immunomodulatory polypeptides are present in the same polypeptide chain and can be in series. When the multimeric polypeptide of the present disclosure comprises two or more immunomodulatory polypeptides, in some cases, the two or more immunomodulatory polypeptides are present in separate polypeptides. In some cases, the multimeric polypeptides of the present disclosure are heterodimers. In some cases, the multimeric polypeptides of the present disclosure are trimeric polypeptides.

[0123] In some cases, the multimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory domain. In some cases, the multimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an immunomodulatory domain. In some cases, the multimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory domain; and ii) a second MHC polypeptide. In some cases, the multimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and iii) an immunomodulatory domain; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide. In some cases, when the multimeric polypeptides of the present disclosure comprise a non-Ig scaffold, the non-Ig scaffold is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide.

[0124] In some cases, the multimeric polypeptides of the present disclosure are monovalent. In some cases, the multimeric polypeptides of the present disclosure are multivalent. For example, depending on the Fc polypeptide present in the multimeric polypeptide of the present disclosure, the multimeric polypeptide can be a homodimer, wherein two molecules of the multimeric polypeptide are present in the homodimer, wherein the two molecules of the multimeric polypeptide can be connected to each other by a disulfide bond, for example, via the Fc polypeptide present in the two molecules. For another example, the multimeric polypeptide of the present disclosure can comprise three, four, or five molecules of the multimeric polypeptide, wherein the molecules of the multimeric polypeptide can be connected to each other by a disulfide bond, for example, via the Fc polypeptide present in the molecule.

[0125] Linker

[0126] The multimeric polypeptides of the present disclosure may comprise a linker peptide, for example, inserted between an epitope and an MHC polypeptide, between an MHC polypeptide and an immunomodulatory polypeptide, or between an MHC polypeptide and an Ig Fc polypeptide.

[0127] Suitable linkers (also called "spacers") can be readily selected and can be of any of a variety of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0128] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n (SEQ ID NO:8) and (GGGS) n (SEQ ID NO: 9), wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and thus can serve as neutral links between components. Glycine polymers can be used; glycine is even more accessible to the phi-psi space than alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can include, but are not limited to, amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 10), GGSGG (SEQ ID NO: 11), GSGSG (SEQ ID NO: 12), GSGGG (SEQ ID NO: 13), GGGSG (SEQ ID NO: 14), GSSSG (SEQ ID NO: 15), and the like.

[0129] In some cases, the linker polypeptide present in the first polypeptide of the multimeric polypeptide of the present disclosure includes a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in the second polypeptide of the multimeric polypeptide of the present disclosure. For example, in some cases, a suitable linker comprises the amino acid sequence GCGASGGGGSGGGGS (SEQ ID NO: 16).

[0130] Epitope

[0131] The epitope present in the multimeric polypeptide of the present disclosure may have a length of about 4 amino acids to about 25 amino acids, for example, the epitope may have a length of 4 amino acids (aa) to 10aa, 10aa to 15aa, 15aa to 20aa, or 20aa to 25aa. For example, the epitope present in the multimeric polypeptide of the present disclosure may have a length of 4 amino acids (aa), 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa. In some cases, an epitope present in a multimeric polypeptide of the disclosure has a length of 5 amino acids to 10 amino acids, e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

[0132] The epitope present in the multimeric polypeptide of the present disclosure specifically binds to a T cell, that is, the epitope specifically binds to an epitope-specific T cell. The epitope-specific T cell binds to an epitope having a reference amino acid sequence, but does not substantially bind to an epitope different from the reference amino acid sequence. For example, an epitope-specific T cell binds to an epitope having a reference amino acid sequence, but if it binds to an epitope different from the reference amino acid sequence, it does so at a rate of less than 10 -6 M, less than 10 -5 M or less than 10 -4 M with an affinity of at least 10. -7 M, at least 10 -8 M, at least 10 -9 M or at least 10 -10 M binds to its specific epitope with high affinity.

[0133] Non-limiting examples of epitopes include, for example, the human T-lymphotropic virus-1 epitope LLFGYPVYV (SEQ ID NO: 17); the tumor epitope KYQAVTTTL (SEQ ID NO: 18); and the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes VYLKTNVFL (SEQ ID NO: 19) or TYLKTNLFL (SEQ ID NO: 20). Yang et al. (2006) J. Immunol. 176: 2781.

[0134] MHC peptides

[0135] As described above, the multimeric polypeptides of the present disclosure include MHC polypeptides. For the purposes of the present disclosure, the term "major histocompatibility complex (MHC) polypeptide" is intended to include MHC polypeptides of various species, including human MHC (also known as human leukocyte antigen (HLA)) polypeptides, rodent (e.g., mouse, rat, etc.) MHC polypeptides and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, dogs, cats, ungulates (e.g., horses, cattle, sheep, goats, etc.). The term "MHC polypeptide" is intended to include MHC class I polypeptides (e.g., beta-2 microglobulin and MHC class I heavy chain) and MHC class II polypeptides (e.g., MHC class II alpha polypeptide and MHC class II beta polypeptide).

[0136] As described above, in some embodiments of the multimeric polypeptides of the present disclosure, the first and second MHC polypeptides are MHC class I polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class I beta2 microglobulin (B2M) polypeptide, and the second MHC polypeptide is an MHC class I heavy chain (H chain). In other cases, the first and second MHC polypeptides are MHC class II polypeptides; for example, in some cases, the first MHC polypeptide is an MHC class II α chain polypeptide and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β chain polypeptide and the second MHC polypeptide is an MHC class II α chain polypeptide.

[0137] In some cases, the MHC polypeptide of the multimeric polypeptide of the present disclosure is a human MHC polypeptide, wherein human MHC polypeptides are also referred to as "human leukocyte antigen" ("HLA") polypeptides. In some cases, the MHC polypeptide of the multimeric polypeptide of the present disclosure is a class I HLA polypeptide, such as a β2 microglobulin polypeptide or a class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides, and HLA-G heavy chain polypeptides. In some cases, the MHC polypeptide of the multimeric polypeptide of the present disclosure is a class II HLA polypeptide, such as a class II HLA α chain or a class II HLA β chain. MHC class II polypeptides include MCH class II DP α and β polypeptides, DM α and β polypeptides, DOA α and β polypeptides, DOB α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides.

[0138] For example, the MHC class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure may comprise Figure 25A An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-365 of the amino acid sequence of the human HLA-A heavy chain polypeptide depicted in .

[0139] For example, the MHC class I heavy chain polypeptide of the multimeric polypeptides of the present disclosure may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-365 of the following human HLA-A heavy chain amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQ SEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 5).

[0140] For another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure may comprise Figure 25B An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-362 of the amino acid sequence of the human HLA-B heavy chain polypeptide depicted in .

[0141] For another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure may comprise Figure 25C An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-362 of the amino acid sequence of the human HLA-C heavy chain polypeptide depicted in .

[0142] As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptides of the present disclosure may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

[0143] GPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQEGPEYWEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLLRGYQQFAYDGRDYIALNEDLKTWTAADTA ALITRRKWEQAGDAEYYRAYLEGECVEWLRRYLELGNETLLRTDSPKAHVTYHPRSQVDVTLRCWALGFYPADITLTWQLNGEDLTQDMELVETRPAGDGTFQKWAAVVVPLGKEQNYTCHVHHKGLPEPLTLRW(SEQ ID NO:22).

[0144] The β2 microglobulin (B2M) polypeptide of the multimeric polypeptide of the present disclosure can be a human B2M polypeptide, a non-human primate B2M polypeptide, a mouse B2M polypeptide, etc. In some cases, the B2M polypeptide comprises Figure 20 The B2M amino acid sequences depicted in have an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity.

[0145] In some cases, the MHC polypeptide comprises a single amino acid substitution relative to a reference MHC polypeptide (wherein the reference MHC polypeptide can be a wild-type MHC polypeptide), wherein the single amino acid substitution replaces the amino acid with a cysteine ​​(Cys) residue. Such a cysteine ​​residue, when present in the MHC polypeptide of the first polypeptide of the multimeric polypeptide of the present disclosure, can form a disulfide bond with a cysteine ​​residue present in the second polypeptide chain of the multimeric polypeptide of the present disclosure.

[0146] In some cases, the first MHC polypeptide in the first polypeptide of the multimeric polypeptide of the present disclosure and / or the second MHC polypeptide in the second polypeptide of the multimeric polypeptide of the present disclosure comprises an amino acid substitution with a cysteine-substituted amino acid, wherein the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with a cysteine ​​in the second MHC polypeptide, wherein the cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide, or wherein the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide.

[0147] For example, in some cases, one of the following residue pairs in HLA β2-microglobulin and HLA class I heavy chain is substituted with cysteine: 1) B2M residue 12, HLA class I heavy chain residue 236; 2) B2M residue 12, HLA class I heavy chain residue 237; 3) B2M residue 8, HLA class I heavy chain residue 234; 4) B2M residue 10, HLA class I heavy chain residue 235; 5) B2M residue 24, HLA class I heavy chain residue 236; 6) B2M residue 28, HLA class I heavy chain residue 232; 7) B2M residue 98, HLA class I heavy chain residue 192; 8) B2M residue 99, HLA class I heavy chain residue 234; 9) B2M residue 3, HLA class I heavy chain residue 120; 10) B2M residue 31, HLA class I heavy chain residue 96; 11) B2M residue 53, HLA class I heavy chain residue class I heavy chain residue 35; 12) B2M residue 60, HLA class I heavy chain residue 96; 13) B2M residue 60, HLA class I heavy chain residue 122; 14) B2M residue 63, HLA class I heavy chain residue 27; 15) B2M residue Arg3, HLA class I heavy chain residue Gly120; 16) B2M residue His31, HLA class I heavy chain residue Gln96; 17) B2M residue Asp53, HLA class I heavy chain residue Arg35; 18) B2M residue Trp60, HLA class I heavy chain residue Gln96; 19) B2M residue Trp60, HLA class I heavy chain residue Asp122; 20) B2M residue Tyr63, HLA class I heavy chain residue Tyr27; 21) B2M residue Lys6, HLA Class I heavy chain residue Glu232: 22) B2M residue Gln8, HLA class I heavy chain residue Arg234; 23) B2M residue Tyr10, HLA class I heavy chain residue Pro235; 24) B2M residue Ser11, HLA class I heavy chain residue Gln242: 25) B2M residue Asn24, HLA class I heavy chain residue Ala236; 26) B2M residue Ser28, HLA class I heavy chain residue Glu232; 27) B2M residue Asp98, HLA class I heavy chain residue His192; and 28) B2M residue Met99, HLA class I heavy chain residue Arg234. The amino acid numbering of the MHC / HLA class I heavy chain refers to the mature MHC / HLA class I heavy chain without the signal peptide. For example, in Figure 25A In the amino acid sequence including the signal peptide depicted in , Gly120 is Gly144; Gln96 is Gln120; and so on.

[0148] Immunomodulatory peptides

[0149] The immunomodulatory polypeptide of the multimeric polypeptide of the present disclosure can be an activating immunomodulatory polypeptide or an inhibitory immunomodulatory polypeptide. In some cases, the multimeric polypeptide of the present disclosure comprises a single immunomodulatory polypeptide. In some cases, the multimeric polypeptide of the present disclosure comprises two immunomodulatory polypeptides. In some cases, the two immunomodulatory polypeptides are connected in series in a single polypeptide chain. In some cases, the two immunomodulatory polypeptides are in separate polypeptide chains. In some cases, the two immunomodulatory polypeptides are in separate polypeptide chains and are connected to each other by a disulfide bond.

[0150] In some cases, the immunomodulatory polypeptide of the multimeric polypeptide of the present disclosure is a T cell modulatory polypeptide. In some cases, the T cell modulatory polypeptide is a stimulatory (activating) T cell modulatory polypeptide. In some cases, the T cell modulatory polypeptide is an inhibitory T cell modulatory polypeptide. The T cell modulatory polypeptide can be an antibody, a peptide ligand, a T cell costimulatory polypeptide, a cytokine or a toxin.

[0151] In some cases, the immunomodulatory polypeptide of the multimeric polypeptide of the present disclosure is an antibody-based or non-antibody-based recognition portion that specifically binds to a co-stimulatory polypeptide expressed on the surface of an epitope-specific T cell. Antibody-based recognition portions include, for example, antibodies; antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, single-chain Fv (scFv), and Fd fragments; chimeric antibodies; humanized antibodies; single-chain antibodies (scAb), single-domain antibodies (dAb); single-domain heavy chain antibodies; single-domain light chain antibodies; and the like. Suitable non-antibody-based recognition portions include, for example, affibodies; engineered Kunitz domains; monomers (adnectin); anticalins; aptamers; designed ankyrin repeat domains (DARPins); binding sites for cysteine-rich polypeptides (e.g., cysteine-rich knottin peptides); high-affinity polymers (avimers); afflins; and the like. Antibody-based or non-antibody-based recognition moieties specifically bind to costimulatory polypeptides expressed on the surface of epitope-specific T cells, wherein such costimulatory polypeptides include but are not limited to CTLA4, PD1, ICOS, OX40, CD20 and 4-1BB. Costimulatory polypeptides expressed on the surface of epitope-specific T cells are known in the art.

[0152] In some cases, the immunomodulatory polypeptide of the multimeric polypeptide of the present disclosure is a T cell co-stimulatory polypeptide. In some cases, the immunomodulatory polypeptide of the multimeric polypeptide of the present disclosure is a T cell co-stimulatory polypeptide and a member of the tumor necrosis factor (TNF) superfamily; such as FasL polypeptide, 41BBL polypeptide, CD40 polypeptide, OX40L polypeptide, CD30L polypeptide, CD70 polypeptide, etc. In some cases, the immunomodulatory polypeptide of the multimeric polypeptide of the present disclosure is a T cell co-stimulatory polypeptide and a member of the immunoglobulin (Ig) superfamily; such as CD7 polypeptide, CD86 polypeptide, ICAM polypeptide, etc.

[0153] Suitable immunomodulatory polypeptides of the multimeric polypeptides of the present invention include, but are not limited to, CD80 (B7-1), CD86 (B7-2), 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1, FasL, and PD-L2. Suitable immunomodulatory polypeptides of the multimeric polypeptides of the present invention include, for example, CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, and HVEM.

[0154] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 26A or Figure 26B An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 19-290 of the PD-L1 amino acid sequence depicted in .

[0155] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is a 4-1BBL polypeptide. In some cases, the 4-1BBL polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 27 An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 50-254 of the 4-1BBL amino acid sequence depicted in .

[0156] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is an ICOS-L polypeptide. In some cases, the ICOS-L polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 28An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 19-302 of the ICOS-L amino acid sequence depicted in .

[0157] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is an OX40L polypeptide. In some cases, the OX40L polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 29 An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 1-183 of the OX40L amino acid sequence depicted in .

[0158] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is a PD-L2 polypeptide. In some cases, the PD-L2 polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 30 An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 20-273 of the PD-L2 amino acid sequence depicted in .

[0159] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is a CD80 (B7-1) polypeptide. In some cases, the CD80 polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 31 An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 35-288 of the CD80 amino acid sequence depicted in .

[0160] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is a CD86 polypeptide. In some cases, the CD86 polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 32 An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 31-329 of the CD86 amino acid sequence depicted in .

[0161] In some cases, the T cell modulating polypeptide of the multimeric polypeptide of the present disclosure is a FasL polypeptide. In some cases, the FasL polypeptide of the multimeric polypeptide of the present disclosure comprises Figure 33An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 1-281 of the FasL amino acid sequence depicted in .

[0162] Additional T cell regulatory domains (MODs) that can be used in the present invention include naturally occurring or synthetic human gene products (proteins), affinity reagents targeting human gene products (e.g., antibodies, antibody fragments, single chain Fvs, aptamers, nanobodies), including but not limited to all secreted proteins from canonical and atypical (e.g., FGF2, IL1, S100A4) secretion mechanisms, and the extracellular domains of all cell surface proteins anchored by naturally occurring gene-encoded protein fragments (single or multiple transmembrane) or post-translational modifications (such as GPI linkages). Any naturally occurring or synthetic affinity reagents (e.g., antibodies, antibody fragments, single chain Fvs, aptamers, nanobodies, lectins, etc.) targeting cell surface glycans or other post-translational modifications (e.g., sulfation). Examples include, but are not limited to, members of the TNF / TNFR family (OX40L, ICOSL, FASL, LTA, LTBTRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70) or affinity reagents to members of the TNF / TNFR family; members of the immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chains, ICOS, ICOS ligand, HHLA2, butyrophilin, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILR), killer cell immunoglobulin-like receptors (KIR)), lectin superfamily members, selectins, cytokines / chemokines and cytokine / chemokine receptors, growth factors and growth factor receptors), adhesion molecules (integrins, fibronectin, cadherins) or extracellular domains of multi-span integral membrane proteins or affinity reagents directed against the immunoglobulin superfamily and listed gene products.In addition, active homologs / orthologs of these gene products include, but are not limited to, viral sequences (e.g., CMV, EBV), bacterial sequences, fungal sequences, eukaryotic pathogens (e.g., Schistosoma, Plasmodium, Babesia, Eimeria, Theileria, Toxoplasma, Entamoeba, Leishmania, and Trypanosoma), and coding regions of mammalian origin. In addition, MODs may comprise small molecule drugs targeting human gene products.

[0163] Fc polypeptide

[0164] The multimeric polypeptides of the present disclosure comprise an Fc polypeptide or other suitable scaffold polypeptide.

[0165] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, for example, Hassouneh et al. (2012) Methods Enzymol. 502: 215; for example, polypeptides comprising a pentapeptide repeat unit of (Val-Pro-Gly-X-Gly), wherein X is any amino acid other than proline), albumin-binding polypeptides, silk-like polypeptides (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357: 165), silk-elastin-like polypeptides (SELP; see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54: 1075), etc. Suitable XTEN polypeptides include, for example, those disclosed in WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, US 2010 / 0189682, and US 2009 / 0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27: 1186). Suitable albumin polypeptides include, for example, human serum albumin.

[0166] Suitable scaffold polypeptides will be polypeptides with extended half-life in some cases. Thus, in some cases, suitable scaffold polypeptides extend the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide compared to a control multimeric polypeptide lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide extends the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, or more than 100 times, compared to a control multimeric polypeptide lacking the scaffold polypeptide. For example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to a control multimeric polypeptide lacking the Fc polypeptide.

[0167] The Fc polypeptide of the multimeric polypeptide of the present disclosure can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises Figure 24A -C has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity. In some cases, the Fc region comprises an amino acid sequence that is identical to Figure 24A In some cases, the Fc polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the human IgG1 Fc polypeptide depicted in Figure 24A , at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG2 Fc polypeptide depicted in Figure 24A In some cases, the Fc polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to amino acids 99-325 of the human IgG2 Fc polypeptide depicted in . Figure 24AThe human IgG3 Fc polypeptide depicted in the foregoing has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity; for example, the Fc polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the foregoing; Figure 24A In some cases, the Fc polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to amino acids 19-246 of the human IgG3 Fc polypeptide depicted in . Figure 24B , at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide depicted in; for example, the Fc polypeptide comprises an amino acid sequence that is identical to Figure 24B In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide depicted in . Figure 24C The human IgAFc polypeptides depicted in the foregoing have an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the human IgAFc polypeptides depicted in the foregoing; for example, the Fc polypeptides comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the human IgAFc polypeptides depicted in the foregoing; Figure 24C amino acids 1-234 of the human IgAFc polypeptide depicted in ATCC 5011 having an amino acid sequence of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity.

[0168] Other peptides

[0169] The polypeptide chains of the multimeric polypeptides of the present invention may comprise one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides may be included at the N-terminus of the polypeptide chain of the multimeric polypeptide of the present invention, at the C-terminus of the polypeptide chain of the multimeric polypeptide of the present invention, or within the polypeptide chain of the multimeric polypeptide of the present invention.

[0170] Epitope tag

[0171] Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 23); FLAG (e.g., DYKDDDDK (SEQ ID NO: 24); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 25); and the like.

[0172] Affinity domain

[0173] Affinity domains include peptide sequences that can interact with binding partners (e.g., such as those immobilized on a solid support for identification or purification). DNA sequences encoding multiple consecutive single amino acids (such as histidine) when fused to the expressed protein can be used for single-step purification of the recombinant protein by high-affinity binding to a resin column (such as nickel agarose). Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 26), HisX6 (HHHHHH) (SEQ ID NO: 27), C-myc (EQKLISEEDL) (SEQ ID NO: 25), Flag (DYKDDDDK) (SEQ ID NO: 24), StrepTag (WSHPQFEK) (SEQ ID NO: 28), hemagglutinin, e.g., HA tag (YPYDVPDYA) (SEQ ID NO: 23), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 30), Phe-His-His-Thr (SEQ ID NO: 31), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 32), SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 NO:32), a metal binding domain, e.g., a zinc binding domain or a calcium binding domain, such as those from calcium binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, D28K) and calretinin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequence, and maltose binding protein.

[0174] Modification

[0175] The multimeric polypeptides of the present disclosure may include one or more non-polypeptide moieties covalently linked to the multimeric polypeptide. Suitable non-polypeptide moieties include, for example, biocompatible fatty acids and derivatives thereof; hydroxyalkyl starch (HAS), such as hydroxyethyl starch (HES); poly(ethylene glycol); hyaluronic acid (HA); heparin precursor (heparosan) polymer (HEP); phosphorylcholine-based polymers; dextran; polysialic acid (PSA); and the like. In some cases, the non-polypeptide moiety prolongs the in vivo half-life of the multimeric polypeptide compared to a control multimeric polypeptide not comprising the non-polypeptide moiety.

[0176] In some cases, the multimeric polypeptides of the present disclosure comprise a detectable label. Suitable detectable labels include radioisotopes, such as 1231 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), 67 Ga (gallium), radioactive Gd isotope ( 153 Gd); contrast agents such as gadolinium (Gd), dysprosium and iron; enzymes that produce a detectable product (e.g., luciferase, β-galactosidase, horseradish peroxidase, alkaline phosphatase, etc.); fluorescent proteins; chromogenic proteins, dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, etc.); fluorescent metals, such as 152 Eu or other lanthanide elements; chemiluminescent compounds, such as luminol, isoluminol, acridinium salts, etc.; bioluminescent compounds; etc.

[0177] active

[0178] Depending on the nature of the immunomodulatory ("MOD") polypeptide present in the multimeric polypeptide of the present disclosure, the multimeric polypeptide can activate or inhibit target T cells. The multimeric polypeptide of the present disclosure selectively activates or inhibits target T cells that are specific for the epitope present in the multimeric polypeptide. "Target T cells" include epitope-specific CD4 + T cells, antigen-specific CD8 + In some cases, the target CD4 + The T cells are helper T cells (e.g., Th1, Th2, or Th17 cells). In some cases, the target CD4 + T cells are CD4 + / CD25 + / FoxP3 + Regulatory T (Treg) cells. In some cases, the target T cells are CD8 +In some cases, the target T cell is a memory T cell, which can be a CD4 + T cells or CD8 + T cells, of which memory T cells are usually CD45RO + In some cases, the target T cell is a NK-T cell.

[0179] In some cases, the multimeric polypeptides of the present invention enhance T cell homing and transport. For example, in some cases, the multimeric polypeptides of the present invention increase the extravasation of the target T cells to the treatment site when in contact with target T cells. In some cases, compared to the extravasation level of target T cells not in contact with the multimeric polypeptides, when the multimeric polypeptides of the present invention are in contact with target T cells, the target T cells that extravasate into the treatment site can be increased by at least 10%, at least 15%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 100 times or more than 100 times. Increased extravasation can increase the number of T cells at the treatment site. In some cases, the multimeric polypeptides of the present invention can increase the number of T cells at the treatment site when in contact with target T cells.

[0180] In some cases, the multimeric polypeptides of the present invention increase the expression of one or more proteins by target T cells that mediate or regulate the transport of target T cells to lymphocytes. For example, in some cases, the multimeric polypeptides of the present invention increase the level of one or more adhesion molecules and / or chemokine receptor molecules in target T cells when in contact with target T cells. For example, in some cases, when compared to the level of adhesion molecules and / or chemokine receptor molecules produced by target T cells that are not exposed to the multimeric polypeptides, the multimeric polypeptides of the present invention increase the expression of one or more adhesion molecules and / or chemokine receptors by at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 100 times or more than 100 times when in contact with target T cells. Examples of adhesion molecules include adhesion molecules produced by CD8 T cells, wherein examples of such adhesion molecules include but are not limited to CD44, LFA-1 and VLA-4. Examples of chemokine receptors include chemokine receptors produced by CD8 T cells, where examples of such chemokine receptors include, but are not limited to, CCR5, CCR7, and CXCR3.

[0181] In some cases, the multimeric polypeptides of the present invention result in the generation of memory T cells that can produce a rapid cytotoxic response to previously experienced epitopes. For example, in some cases, the multimeric polypeptides of the present invention, when in contact with target T cells, result in the generation of memory T cells comprising 0.5% or more of a pool of antigen-specific T cells. For example, in some cases, the multimeric polypeptides of the present invention, when in contact with target T cells, result in the generation of memory T cells comprising 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, or 20% or more of a pool of antigen-specific T cells. An example of a cell surface marker for memory T cells is CD45RO.

[0182] In some cases, the multimeric polypeptides of the present disclosure increase target T cell proliferation. For example, in some cases, the multimeric polypeptides of the present disclosure, when in contact with target T cells, can increase the proliferation of target T cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more than 100-fold, compared to the proliferation of target T cells that have not been contacted with the multimeric polypeptide.

[0183] In some cases, the multimeric polypeptides of the present disclosure increase the cytotoxic activity of T cells against target cells. For example, in some cases, the multimeric polypeptides of the present disclosure, when in contact with target T cells, can increase the cytotoxic activity of T cells against target cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more than 100-fold, compared to the cytotoxic activity of T cells against target cells that have not been contacted with the multimeric polypeptide. Targets of T cells include virus-infected cells, cancer cells, and the like.

[0184] In some cases, the multimeric polypeptides of the present invention increase the production of cytokines by target T cells. For example, in some cases, the multimeric polypeptides of the present invention, when in contact with target T cells, increase the production of cytokines by T cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more than 100-fold, compared to the level of cytokines produced by target T cells that have not been contacted with the multimeric polypeptide. Examples of cytokines include cytokines produced by Th1 cells, such as IL-2, IFN-γ, and TNF-α; cytokines produced by Th17 cells, such as IL-17, IL-21, and IL-22; and cytokines produced by Treg cells, such as TGF-β, IL-35, and IL-10.

[0185] In some cases, the multimeric polypeptides of the present invention inhibit the production of cytokines by target T cells. For example, in some cases, the multimeric polypeptides of the present invention inhibit the production of cytokines by target T cells when in contact with target T cells, compared to the level of cytokines produced by target T cells that have not been contacted with the multimeric polypeptide. By at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more than 90%. Examples of cytokines include cytokines produced by Th2 cells, such as IL-4, IL-5, IL-6, IL-10, and IL-13.

[0186] Exemplary embodiments

[0187] Non-limiting examples of multimeric polypeptides of the present disclosure include:

[0188] 1) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a 4-BBL polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0189] 2) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a PD-L1 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0190] 3) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) an ICOS-L polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0191] 4) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) an OX40L polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0192] 5) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a CD80 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0193] 6) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a CD86 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0194] 7) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a PD-L2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0195] 8) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a 4-BBL polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0196] 9) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a PD-L1 polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0197] 10) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) an ICOS-L polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0198] 11) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) an OX40L polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0199] 12) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a CD80 polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0200] 13) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a CD86 polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0201] 14) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a PD-L2 polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0202] 15) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; and ii) an MHC class I β2 microglobulin polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) a FasL polypeptide; ii) an MHC class I heavy chain polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0203] 16) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem 4-BBL polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0204] 17) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem PD-L1 polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0205] 18) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem ICOS-L polypeptides; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0206] 19) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem OX40L polypeptides; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0207] 20) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem CD80 polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0208] 21) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem CD86 polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0209] 22) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem PD-L2 polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0210] 23) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) two tandem FasL polypeptides; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0211] 24) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first 4-1BBL polypeptide; b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second 4-1BBL polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond; and the first polypeptide and the third polypeptide are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are connected to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; In some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide a cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are connected to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second 4-1BBL polypeptides. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0212] 25) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first PD-L1 polypeptide; b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second PD-L1 polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second PD-L1 polypeptides. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0213] 26) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first ICOS-L polypeptide; b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second ICOS-L polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second ICOS-L polypeptides. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0214] 27) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first OX40L polypeptide; b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second OX40L polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second OX40L polypeptides. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0215] 28) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first CD80 polypeptide; b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second CD80 polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second CD80 polypeptides. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0216] 29) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first CD86 polypeptide; b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second CD86 polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second CD86 polypeptides. In some cases, the IgFc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0217] 30) A multimeric polypeptide comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a CD80 polypeptide; b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a CD86 polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the CD80 polypeptide and the CD86 polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0218] 31) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first PD-L2 polypeptide; b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second PD-L2 polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second PD-L2 polypeptides. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgAFc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide;

[0219] 32) A multimeric polypeptide comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T cell epitope; ii) an MHC class I β2 microglobulin polypeptide; and iii) a first FasL polypeptide; b) a second polypeptide comprising, from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and c) a third polypeptide comprising a second FasL polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the first and second polypeptides are linked to each other by a disulfide bond; and the first and third polypeptides are linked to each other by a disulfide bond. In some cases, the first polypeptide comprises a linker polypeptide between the epitope and the β2 microglobulin polypeptide. In some cases, the first and second polypeptides are linked to each other by a disulfide bond via a cysteine ​​residue present in the linker polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide. In some cases, the first polypeptide and the second polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in the MHC class I β2 microglobulin polypeptide and a cysteine ​​residue present in the MHC class I heavy chain polypeptide; in some of these embodiments, the MHC class I β2 microglobulin polypeptide and / or the MHC class I heavy chain polypeptide include amino acid substitutions to provide cysteine ​​that participates in the disulfide bond. In some cases, the first polypeptide and the third polypeptide are linked to each other by a disulfide bond via a cysteine ​​residue present in (or substituted into) the first and second FasL polypeptides. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG2 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG3 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgA Fc polypeptide or an IgM Fc polypeptide. In some cases, an MHC class II polypeptide is used instead of an MHC class I polypeptide. In some cases, the multimeric polypeptide comprises an epitope tag and / or affinity domain at the C-terminus of the Fc polypeptide.

[0220] Polyprotein precursor

[0221] The present invention provides a recombinant polypeptide as described below, which comprises an amino acid sequence identical to a first B2M leader sequence, the amino acid sequence identical to the first B2M leader sequence is adjacent to a candidate epitope peptide, the candidate epitope peptide is adjacent to a first amino acid linker sequence, the first amino acid linker sequence is adjacent to an amino acid sequence identical to a natural human B2M peptide sequence, the amino acid sequence identical to a natural human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a T cell regulatory domain peptide sequence, the T cell regulatory domain peptide sequence is adjacent to a third amino acid linker, the third amino acid linker is adjacent to a second B2M leader sequence, the second B2M leader sequence is adjacent to an amino acid sequence identical to an MHC heavy chain, the amino acid sequence identical to an MHC heavy chain is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain.

[0222] In one embodiment, the first amino acid can be any amino acid sequence having 50 amino acids or less to at least 5 amino acids. In one embodiment, the second amino acid linker can be any amino acid sequence having 70 amino acids or less to at least 5 amino acids. In one embodiment, the third amino acid linker can be a viral 2A peptide or a peptide with known protease cleavage ability (e.g., in non-limiting embodiments, a furin cleavage site, a tobacco etch virus [TEV] sequence, a precision protease site, or a thrombin protease). In one embodiment, the first amino acid comprises GGGGSGGGGSGGGGS (SEQ ID NO: 1). In one embodiment, the second amino acid linker comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 2). In one embodiment, the third amino acid linker comprises SGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 3).

[0223] The present invention also provides a recombinant polypeptide as described below, which comprises an amino acid sequence identical to a first B2M leader sequence, the amino acid sequence identical to the first B2M leader sequence is adjacent to a candidate epitope peptide, the candidate epitope peptide is adjacent to a first amino acid linker sequence, the first amino acid linker sequence is adjacent to an amino acid sequence identical to a natural human B2M peptide sequence, the amino acid sequence identical to a natural human B2M peptide sequence is adjacent to a second amino acid linker sequence, the second amino acid linker sequence is adjacent to a second B2M leader sequence, the second B2M leader sequence is adjacent to a T cell regulatory domain peptide sequence, the T cell regulatory domain peptide sequence is adjacent to a third amino acid linker sequence, the third amino acid linker sequence is adjacent to an amino acid sequence identical to an MHC heavy chain, and the amino acid sequence identical to an MHC heavy chain is adjacent to an amino acid sequence identical to an immunoglobulin Fc domain.

[0224] Linker

[0225] In one embodiment, the first amino acid can be any amino acid sequence having 50 amino acids or less to at least 5 amino acids. In one embodiment, the second amino acid linker can be any amino acid sequence having 70 amino acids or less to at least 5 amino acids. In one embodiment, the third amino acid linker can be a viral 2A peptide or a peptide with known protease cleavage ability (e.g., in non-limiting embodiments, a furin cleavage site, a tobacco etch virus [TEV] sequence, a precision protease site, or a thrombin protease). In one embodiment, the first amino acid comprises GGGGSGGGGSGGGGS (SEQ ID NO: 1). In one embodiment, the second amino acid linker comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 2). In one embodiment, the third amino acid linker comprises SGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 3).

[0226] In one embodiment of the recombinant polypeptide, the third amino acid linker is self-cleaving. In one embodiment of the recombinant polypeptide, the second amino acid linker is self-cleaving. In one embodiment of the recombinant polypeptide, the self-cleaving peptide is a viral 2A peptide or has its sequence. In one embodiment, the viral 2A peptide is porcine teschovirus-1 (P2A), foot-and-mouth disease virus (F2A), Thosea asigna virus (T2A), equine rhinitis virus type A (E2A) or viral porcine teschovirus-1 (P2A) peptide or has the sequence of one of them. Alternatively, this can also be delivered as two separate plasmids (or viruses) with the 2A sequence completely removed.

[0227] The proteolytically cleavable linker may comprise a protease recognition sequence that is recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C. C), IgA-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methylthioyl aminopeptidase, myxobacterium, nardilysin, pancreatic endopeptidase E, picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, yeast pepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, snake venom serine protease A A), snake venom serine protease AB and Xaa-Pro aminopeptidase. In some cases, the proteolytically cleavable linker can include a protease recognition sequence that can be recognized by a host enzyme, such as an enzyme naturally produced by the host cell.

[0228] For example, the proteolytically cleavable linker can comprise a matrix metalloproteinase cleavage site, such as a cleavage site for an MMP selected from the group consisting of collagenase-1, collagenase-2, and collagenase-3 (MMP-1, MMP-8, and MMP-13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, MMP-10, and MMP-11), matrix lytic factor (matrilysin) (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-XX-Hy (wherein X represents any residue; Hy is a hydrophobic residue), such as Pro-XX-Hy-(Ser / Thr), such as Pro-Leu / Gln-Gly-Met-Thr-Ser (SEQ ID NO: 33) or Pro-Leu / Gln-Gly-Met-Thr (SEQ ID NO: 21). Another example of a protease cleavage site is a plasminogen activator cleavage site, such as a uPA or tissue plasminogen activator (tPA) cleavage site. Specific examples of cleavage sequences for uPA and tPA include sequences containing Val-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, such as ENLYTQS (SEQ ID NO: 34), wherein the protease cleaves between glutamine and serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, such as DDDDK (SEQ ID NO: 35), wherein cleavage occurs after a lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, such as LVPR (SEQ ID NO: 36). Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a furin cleavage site, such as Arg-X-(Arg / Lys)-Arg, wherein X is any amino acid. Additional suitable linkers comprising a protease cleavage site include linkers comprising one or more of the following amino acid sequences: LEVLFQGP (SEQ ID NO: 37), which is cleaved by PreScission protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase; Walker et al. (1994) Biotechnol. 12: 601); a thrombin cleavage site, such as CGLVPAGSGP (SEQ ID NO: 38); SLLKSRMVPNFN (SEQ ID NO: 39) or SLLIARRMPNFN (SEQ ID NO: 40), which is cleaved by cathepsin B;SKLVQASASGVN (SEQ ID NO: 41) or SSYLKASDAPDN (SEQ ID NO: 42), cleaved by Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO: 43), cleaved by MMP-3 (matrilysin); SLRPLALWRSFN (SEQ ID NO: 44), cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO: 45), cleaved by MMP-9; DVDERDVRGFASFL (SEQ ID NO: 46), cleaved by thermolysin-like MMP; SLPLGLWAPNFN (SEQ ID NO: 47), cleaved by matrix metalloproteinase 2 (MMP-2); SLLIFRSWANFN (SEQ ID NO: 48), cleaved by cathepsin L; SGVVIATVIVIT (SEQ ID NO: 49), cleaved by cathepsin D; SLGPQGIWGQFN (SEQ ID NO: 50), cleaved by cathepsin D; ID NO:50), cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV (SEQ ID NO:51), cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG (SEQ ID NO:52), cleaved by type 1 membrane matrix metalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO:53), cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase, and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO:54), cleaved by matrix metalloproteinase 13 (collagenase-3); GGSGQRGRXALE (SEQ ID NO:55), cleaved by tissue plasminogen activator (tPA); SLSALLSSDIFN (SEQ ID NO:56), cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO:57). NO: 57), cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO: 58), cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO: 59), cleaved by calpain (calcium-activated neutral protease). Additional examples of suitable proteolytically cleavable linkers include: 1) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 60); 2) EGRGSLLTCGDVEENPGP (SEQ ID NO: 61); 3) QCTNYALLKLAGDVESNPGP (SEQ ID NO: 62);and 4) VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 63). Additional examples of suitable proteolytically cleavable linkers include: 1) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 64); 2) GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 65); 3) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 66); and 4) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 67). ;

[0229] Examples of suitable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker includes a picornavirus 2A-like linker, a porcine teschovirus (P2A), a CHYSEL sequence of a beta-tetrasomal virus of the genus Thunbergia (T2A), and combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence may comprise Asp-Val / Ile-Glu-X-Asn-Pro-Gly 2A -Pro 2BMotif, which results in cleavage between 2A glycine and 2B proline. For the purposes of this disclosure, P2A (GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 64)), T2A (GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 65)), E2A (GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 66)), and F2A (GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 67)) can be considered "proteolytic cleavage sites" or "ribosomal skipping signals" (CHYSELs). See, e.g., Kim et al. (2011) PLoS ONE 6: e18556. The mechanism by which the encoded polypeptide is produced as two polypeptide chains may be self-cleavage of the linker, ribosomal skipping, or translational shunting. Regardless of the mechanism, at least two polypeptide chains in the multimeric polypeptides of the present disclosure can be generated using P2A, T2A, E2A or F2A sequences. Suitable linkers include polypeptides comprising amino acid sequences such as GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 64), GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 65), GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 66), GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 67), or amino acid sequences having 1 to 5 amino acid substitutions relative to the amino acid sequences set forth in SEQ ID NOs: 64-67 (e.g., amino acid sequences having 1 to 5 conservative amino acid substitutions relative to the amino acid sequences set forth in SEQ ID NOs: 64-67). Suitable linkers include polypeptides comprising an amino acid sequence such as GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 64), GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 65), GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 66), GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 67), or an amino acid sequence having 1 to 10 amino acid substitutions relative to the amino acid sequences set forth in SEQ ID NOs: 64-67 (e.g., an amino acid sequence having 1 to 10 conservative amino acid substitutions relative to the amino acid sequences set forth in SEQ ID NOs: 64-67).

[0230] Epitope

[0231] In one embodiment of the recombinant polypeptide, the candidate epitope comprises 7-20 amino acids. In one embodiment of the recombinant polypeptide, the epitope peptide is 5-20 amino acids for MHC class I. In one embodiment, the epitope peptide is 8-11 amino acids for MHC class I. In one embodiment, the epitope peptide is 5-40 amino acids for MHC class II. In one embodiment, the epitope peptide is 13-17 amino acids for MHC class II. In one embodiment, the epitope peptide is any naturally occurring or mutated human sequence or any sequence derived from a pathogen.

[0232] MHC peptides

[0233] In one embodiment of the recombinant polypeptide, the first and / or second B2M leader sequence has the sequence of a human B2M leader sequence.

[0234] In some cases, the leader peptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human B2M leader sequence: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 68).

[0235] In some cases, the B2M leader sequence described herein can be a mammalian B2M leader sequence, including but not limited to, for example, a human B2M leader sequence, a primate B2M leader sequence, a rodent B2M leader sequence, etc. In some cases, the B2M leader sequence described herein comprises Figure 20 An amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to one of the B2M leader sequences depicted in .

[0236] In one embodiment, the B2M comprises the following sequence:

[0237] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 4).

[0238] In some cases, the B2M polypeptide comprises Figure 20 The B2M amino acid sequences depicted in are amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity.

[0239] In one embodiment of the recombinant polypeptide, the MHC heavy chain is a human MHC heavy chain. In one embodiment of the recombinant polypeptide, the MHC heavy chain is an MHC I molecule. Exemplary MHC I heavy chains include the α chains of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and HLA-L. In one embodiment of the recombinant polypeptide, the MHC heavy chain is HLA-A02:01. In one embodiment, the HLA is HLA-A02. In one embodiment, the HLA-A02 comprises the following sequence:

[0240] GSHSMRYFFTSVSRPGRGEPFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAA QTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP(SEQ ID NO:5).

[0241] In one embodiment of the recombinant polypeptide, the MHC heavy chain is an MHC II molecule. Exemplary MHC II heavy chains include those of HLA-D.

[0242] In one embodiment of the recombinant polypeptide, the recombinant polypeptide further comprises mutations in its human native B2M peptide sequence and its heavy chain sequence so as to realize a disulfide bond between the B2M peptide sequence and the heavy chain sequence.

[0243] In one embodiment of the recombinant polypeptide, the heavy chain sequence of the recombinant polypeptide is HLA and the disulfide bond connects one of the following residue pairs:

[0244] B2M residue 12, HLA residue 236;

[0245] B2M residue 12, HLA residue 237;

[0246] B2M residue 8, HLA residue 234;

[0247] B2M residue 10, HLA residue 235;

[0248] B2M residue 24, HLA residue 236;

[0249] B2M residue 28, HLA residue 232;

[0250] B2M residue 98, HLA residue 192;

[0251] B2M residue 99, HLA residue 234;

[0252] B2M residue 3, HLA residue 120;

[0253] B2M residue 31, HLA residue 96;

[0254] B2M residue 53, HLA residue 35;

[0255] B2M residue 60, HLA residue 96;

[0256] B2M residue 60, HLA residue 122;

[0257] B2M residue 63, HLA residue 27;

[0258] B2M residue Arg3, HLA residue Gly120;

[0259] B2M residue His31, HLA residue Gln96;

[0260] B2M residue Asp53, HLA residue Arg35;

[0261] B2M residue Trp60, HLA residue Gln96;

[0262] B2M residue Trp60, HLA residue Asp122;

[0263] B2M residue Tyr63, HLA residue Tyr27;

[0264] B2M residue Lys6, HLA residue Glu232;

[0265] B2M residue Gln8, HLA residue Arg234;

[0266] B2M residue Tyr10, HLA residue Pro235;

[0267] B2M residue Ser11, HLA residue Gln242;

[0268] B2M residue Asn24, HLA residue Ala236;

[0269] B2M residue Ser28, HLA residue Glu232;

[0270] B2M residue Asp98, HLA residue His192; and

[0271] B2M residue Met99, HLA residue Arg234

[0272] (See SEQ ID NOs: 4 and 5 for B2M and HLA sequences).

[0273] In one embodiment of the recombinant polypeptide, the heavy chain sequence is HLA and wherein the disulfide bond connects one of the following residue pairs:

[0274] first linker position Gly2, heavy chain (HLA) position Tyr84;

[0275] Light chain (B2M) position Arg12, HLA Ala236; and / or

[0276] B2M residue Arg12, HLA residue Gly237.

[0277] Fc polypeptide

[0278] In one embodiment of the recombinant polypeptide, the immunoglobulin Fc domain is an IgG Fc domain. In one embodiment of the recombinant polypeptide, the immunoglobulin Fc domain is an IgA Fc domain. In one embodiment of the recombinant polypeptide, the immunoglobulin Fc domain is an IgM Fc domain. In one embodiment of the recombinant polypeptide, the immunoglobulin Fc domain is a human immunoglobulin Fc domain. In one embodiment of the recombinant polypeptide, the immunoglobulin Fc domain is an IgG1 Fc domain.

[0279] Immunomodulatory peptides

[0280] In one embodiment of the recombinant polypeptide, the T cell regulatory domain is an inhibitory domain.

[0281] In one embodiment of the recombinant polypeptide, the T cell regulatory domain is a stimulatory domain.

[0282] In one embodiment of the recombinant polypeptide, the T cell regulatory domain is an antibody, an antibody fragment, a peptide ligand, a T cell co-stimulatory peptide, a cytokine, or a toxin.

[0283] In one embodiment of the recombinant polypeptide, the T cell regulatory domain comprises a PD-L1 peptide, an Ig variable domain of a PD-L1 peptide, the T cell regulatory domain comprises 4-1BBL, the T cell regulatory domain comprises B7-1W88A, or the T cell regulatory domain comprises an anti-CD28 single-chain Fv.

[0284] Additional T cell regulatory domains (MODs) that can be used in the present invention include naturally occurring or synthetic human gene products (proteins), affinity reagents targeting human gene products (e.g., antibodies, antibody fragments, single chain Fvs, aptamers, nanobodies), including but not limited to all secreted proteins from canonical and atypical (e.g., FGF2, IL1, S100A4) secretion mechanisms, and the extracellular domains of all cell surface proteins anchored by naturally occurring gene-encoded protein fragments (single or multiple transmembrane) or post-translational modifications (such as GPI linkages). Any naturally occurring or synthetic affinity reagents (e.g., antibodies, antibody fragments, single chain Fvs, aptamers, nanobodies, lectins, etc.) targeting cell surface glycans or other post-translational modifications (e.g., sulfation). Examples include, but are not limited to, members of the TNF / TNFR family (OX40L, ICOSL, FASL, LTA, LTBTRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70) or affinity reagents to members of the TNF / TNFR family; members of the immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chain, ICOS, ICOS ligand, HHLA2, butyrophilin, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILR), killer cell immunoglobulin-like receptors (KIR)), lectin superfamily members, selectins, cytokines / chemokines and cytokine / chemokine receptors, growth factors and growth factor receptors), adhesion molecules (integrins, fibronectin, cadherins) or extracellular domains of multi-spanning membrane proteins or affinity reagents for the immunoglobulin superfamily and listed gene products. In addition, active homologs / orthologs of these gene products include but are not limited to viral sequences (e.g., CMV, EBV), bacterial sequences, fungal sequences, eukaryotic pathogens (e.g., Schistosoma, Plasmodium, Babesia, Eimeria, Theileria, Toxoplasma, Entamoeba, Leishmania and Trypanosoma) and coding regions of mammalian origin. In addition, MODs may comprise small molecule drugs targeting human gene products.

[0285] Other peptides

[0286] In one embodiment of the recombinant polypeptides, they further comprise a His-8 tag adjacent to the C-terminus of the recombinant polypeptide.

[0287] Nucleic Acids

[0288] The present invention provides nucleic acids encoding any of the recombinant polypeptides described herein. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is cDNA. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA.

[0289] In one embodiment, the recombinant nucleic acid is a vector. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a lentiviral vector.

[0290] The present disclosure provides nucleic acids comprising nucleotide sequences encoding multimeric polypeptides of the present disclosure. In some cases, each polypeptide chain of a multimeric polypeptide of the present disclosure is encoded in a separate nucleic acid. In some cases, all polypeptide chains of a multimeric polypeptide of the present disclosure are encoded in a single nucleic acid. In some cases, a first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the present disclosure; and a second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a multimeric polypeptide of the present disclosure. In some cases, a single nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the present disclosure and a second polypeptide of a multimeric polypeptide of the present disclosure. In some cases, a nucleic acid comprises a nucleotide sequence encoding a multimeric protein precursor as described above.

[0291] Separate nucleic acids encoding each polypeptide chain of a multimeric polypeptide

[0292] The present disclosure provides nucleic acids comprising nucleotide sequences encoding multimeric polypeptides of the present disclosure. As described above, in some cases, each polypeptide chain of a multimeric polypeptide of the present disclosure is encoded in a separate nucleic acid. In some cases, the nucleotide sequences encoding the individual polypeptide chains of a multimeric polypeptide of the present disclosure are operably linked to a transcriptional control element, e.g., a promoter, such as a promoter that functions in eukaryotic cells, wherein the promoter can be a constitutive promoter or an inducible promoter.

[0293] The present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the present disclosure, wherein the first polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope (e.g., a T cell epitope); b) a first MHC polypeptide; and c) an immunomodulatory polypeptide; and wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a multimeric polypeptide of the present disclosure, wherein the second polypeptide comprises, in order from N-terminus to C-terminus: a) a second MHC polypeptide; and b) an Ig Fc polypeptide. Suitable T cell epitopes, MHC polypeptides, immunomodulatory polypeptides, and Ig Fc polypeptides are described above. In some cases, the nucleotide sequence encoding the first and second polypeptides is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the nucleic acids are present in separate expression vectors.

[0294] The present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the present disclosure, wherein the first polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope (e.g., a T cell epitope); b) a first MHC polypeptide; and wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a multimeric polypeptide of the present disclosure, wherein the second polypeptide comprises, in order from N-terminus to C-terminus: a) an immunomodulatory polypeptide; b) a second MHC polypeptide; and c) an Ig Fc polypeptide. Suitable T cell epitopes, MHC polypeptides, immunomodulatory polypeptides, and Ig Fc polypeptides are described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the nucleic acids are present in separate expression vectors.

[0295] Nucleic acids encoding two or more polypeptides present in a multimeric polypeptide

[0296] The present disclosure provides nucleic acids comprising a nucleotide sequence of a first polypeptide and a second polypeptide encoding at least a multimeric polypeptide of the present disclosure. In some cases where the multimeric polypeptide of the present disclosure comprises the first, second, and third polypeptides, the nucleic acid comprises a nucleotide sequence encoding the first, second, and third polypeptides. In some cases, the nucleotide sequence of the first polypeptide and the second polypeptide encoding the multimeric polypeptide of the present disclosure comprises a proteolytically cleavable linker between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequence of the first polypeptide and the second polypeptide encoding the multimeric polypeptide of the present disclosure comprises an internal ribosome entry site (IRES) between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequence of the first polypeptide and the second polypeptide encoding the multimeric polypeptide of the present disclosure comprises a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. Examples of nucleic acids are described below in which a proteolytically cleavable linker is provided between the nucleotide sequences encoding the first and second polypeptides of the multimeric polypeptides of the present disclosure; in any of these embodiments, an IRES or ribosomal skipping signal can be used in place of the nucleotide sequence encoding the proteolytically cleavable linker.

[0297] In some cases, a first nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a first polypeptide chain of a multimeric polypeptide of the present disclosure; and a second nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a second polypeptide chain of a multimeric polypeptide of the present disclosure. In some cases, the nucleotide sequence encoding the first polypeptide and the second nucleotide sequence encoding the second polypeptide are each operably linked to a transcriptional control element, e.g., a promoter, such as a promoter that functions in eukaryotic cells, wherein the promoter can be a constitutive promoter or an inducible promoter.

[0298] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope (e.g., a T cell epitope); b) a first MHC polypeptide; c) an immunomodulatory polypeptide; d) a proteolytically cleavable linker; e) a second MHC polypeptide; and f) an immunoglobulin (Ig) Fc polypeptide. The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) a first leader peptide; b) an epitope; c) a first MHC polypeptide; d) an immunomodulatory polypeptide; e) a proteolytically cleavable linker; f) a second leader peptide; g) a second MHC polypeptide; and h) an Ig Fc polypeptide. The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope; b) a first MHC polypeptide; c) a proteolytically cleavable linker; d) an immunomodulatory polypeptide; e) a second MHC polypeptide; and f) an Ig Fc polypeptide. In some cases, the first leader peptide and the second leader peptide are β2-M leader peptides. In some cases, the nucleotide sequence is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that can function in eukaryotic cells.

[0299] Suitable MHC polypeptides are described above. In some cases, the first MHC polypeptide is a β2 microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide. In some cases, the β2 microglobulin polypeptide comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:4. In some cases, the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L heavy chain. In some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some cases, the first MHC polypeptide is an MHC class II α chain polypeptide; and wherein the second MHC polypeptide is an MHC class II β chain polypeptide.

[0300] Suitable Fc polypeptides are described above. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises Figures 24A-24C The amino acid sequences depicted in are amino acid sequences having at least 85% amino acid sequence identity.

[0301] Suitable immunomodulatory polypeptides are described above. In some cases, the immunomodulatory polypeptide is selected from the group consisting of: 4-1BBL polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide, and PD-L2 polypeptide. In some cases, the immunomodulatory polypeptide is selected from the group consisting of: CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, and HVEM.

[0302] Suitable proteolytically cleavable linkers are described above. In some cases, the proteolytically cleavable linker comprises an amino acid sequence selected from the group consisting of: a) LEVLFQGP (SEQ ID NO: 37); b) ENLYTQS (SEQ ID NO: 34); c) DDDDK (SEQ ID NO: 35); d) LVPR (SEQ ID NO: 36); and e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 64).

[0303] In some cases, the linker between the epitope and the first MHC polypeptide comprises a first Cys residue and the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, such that the first and second Cys residues provide a disulfide bond between the linker and the second MHC polypeptide. In some cases, the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue and the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, such that the first Cys residue and the second Cys residue provide a disulfide bond between the first MHC polypeptide and the second MHC polypeptide.

[0304] Recombinant expression vector

[0305] The present disclosure provides recombinant expression vectors comprising the nucleic acids of the present disclosure. In some cases, the recombinant expression vector is a non-viral vector. In some embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a non-integrating viral vector, etc.

[0306] Suitable expression vectors include, but are not limited to, viral vectors (e.g., based on vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543-2549, 1994; Borras et al., Gene Ther 6:515-524, 1999; Li and Davidson, PNAS 92:7700-7704, 1995; Sakamoto et al., H Gene Ther 5:1088-1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81-86, 1998; Flannery et al., PNAS 94:691-696); 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997; Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239; Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165 and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:103-19 23, 1997; Takahashi et al., J Virol 73:7812-7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses (such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0307] Many suitable expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided by way of illustration; for use with eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used as long as it is compatible with the host cell.

[0308] Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements may be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0309] In some embodiments, the nucleotide sequence encoding the DNA-targeting RNA and / or the site-directed modifying polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element can function in eukaryotic cells, e.g., mammalian cells; or prokaryotic cells (e.g., bacterial or archaeal cells). In some embodiments, the nucleotide sequence encoding the DNA-targeting RNA and / or the site-directed modifying polypeptide is operably linked to a plurality of control elements that allow the nucleotide sequence encoding the DNA-targeting RNA and / or the site-directed modifying polypeptide to be expressed in prokaryotic and eukaryotic cells.

[0310] Non-limiting examples of suitable eukaryotic promoters (promoters that function in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, and mouse metallothionein-1. The selection of appropriate vectors and promoters is well within the skill of those skilled in the art. The expression vector may also contain ribosome binding sites for translation initiation and transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.

[0311] Genetically modified host cells

[0312] The present invention provides cells transformed with nucleic acids encoding any of the recombinant polypeptides described herein. Examples of cells that can be transformed with nucleic acids encoding any of the recombinant polypeptides include isolated mammalian cells, including but not limited to human embryonic kidney (HEK), Chinese hamster ovary (CHO), NSO (murine myeloma) cells, human amniotic fluid cells (CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia pastoris), plant cells (including but not limited to tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g., High5)), or bacterial cells (including but not limited to Escherichia coli (E. coli)).

[0313] The present disclosure provides genetically modified host cells, wherein the host cell is genetically modified with a nucleic acid of the present disclosure.

[0314] Suitable host cells include eukaryotic cells, such as yeast cells, insect cells, and mammalian cells. In some cases, the host cell is a cell of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0315] In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC β2-M.

[0316] Method for preparing multimeric polypeptides

[0317] The present disclosure provides a method for preparing the multimeric polypeptides of the present disclosure. Generally speaking, the method involves: culturing a host cell genetically modified by a recombinant expression vector comprising a nucleotide sequence encoding a multimeric polypeptide in a culture medium; and isolating the multimeric polypeptide from the genetically modified host cell and / or culture medium. A host cell genetically modified by a recombinant expression vector comprising a nucleotide sequence encoding a multimeric polypeptide is also referred to as an "expression host." As described above, in some cases, each polypeptide chain of the multimeric polypeptides of the present disclosure is encoded in a separate recombinant expression vector. In some cases, all polypeptide chains of the multimeric polypeptides of the present disclosure are encoded in a single recombinant expression vector.

[0318] Multimeric polypeptides can be isolated from the expression host cells (eg, from a lysate of the expression host cells) and / or the culture medium in which the host cells are cultured using standard protein purification methods.

[0319] For example, a lysate from an expression host can be prepared and purified using high performance liquid chromatography (HPLC), size exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. Alternatively, when the multimeric polypeptide is secreted into a culture medium from the expression host cell, HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques can be used to purify the multimeric polypeptide from the culture medium. In some cases, the composition used will comprise at least 80 % by weight, at least about 85 % by weight, at least about 95 % by weight or at least about 99.5 % by weight of the desired product relative to the pollutants relevant to the product preparation and its purification method. This percentage can be calculated by total protein.

[0320] In some cases, for example, where the multimeric polypeptide comprises an affinity tag, the multimeric polypeptide can be purified using an immobilized binding partner for the affinity tag.

[0321] Composition

[0322] The present disclosure provides compositions, including pharmaceutical compositions comprising the multimeric polypeptides of the present disclosure. The present disclosure provides compositions, including pharmaceutical compositions comprising the nucleic acids or recombinant expression vectors of the present disclosure.

[0323] Compositions comprising multimeric polypeptides

[0324] In addition to the multimeric polypeptides of the present disclosure, the compositions of the present disclosure may further comprise one or more of the following: salts, such as NaCl, MgCl, KCl, MgSO4, etc.; buffers, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizing agents; detergents, such as non-ionic detergents, such as Tween-20, etc.; protease inhibitors; glycerol; and the like.

[0325] The composition may comprise a pharmaceutically acceptable excipient, many of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are fully described in a variety of publications, including, for example, "Remington: The Science and Practice of Pharmacy," 19th edition (1995) or the latest edition, Mack Publishing Co; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th edition, Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AK Hibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0326] Pharmaceutical compositions may comprise a multimeric polypeptide of the present disclosure and a pharmaceutically acceptable excipient. In some cases, the subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile. For example, in some embodiments, the subject pharmaceutical composition will be suitable for administration to a human subject, e.g., wherein the composition is sterile and does not contain detectable pyrogens and / or other toxins.

[0327] The protein composition may contain other components such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonate, etc. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate, solvate (e.g., mixed ionic salts, water, organic matter), hydrate (e.g., water), etc.

[0328] For example, the composition may include an aqueous solution, a powder form, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc. The composition can be formulated according to various administration routes as described below.

[0329] When the multimeric polypeptides of the present disclosure are administered directly into the tissue in the form of an injection (e.g., subcutaneously, intraperitoneally, and / or intravenously), the preparation can be provided in a ready-to-use dosage form or in a non-aqueous form (e.g., a resolvable storage-stable powder) or in an aqueous form (e.g., a liquid consisting of a pharmaceutically acceptable carrier and excipient). Protein-containing preparations can also be provided to extend the serum half-life of the subject protein after administration. For example, the protein can be provided in a liposome formulation, prepared as a colloid or other conventional technology for extending the serum half-life. A variety of methods can be used to prepare liposomes, such as described in, for example, Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9: 467, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. The preparation can also be provided in a controlled-release or sustained-release form.

[0330] Other examples of preparations suitable for parenteral administration include isotonic sterile injection solutions, antioxidants, antibacterial agents, and solutes, suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives that make the preparation isotonic with the blood of the intended recipient. For example, the subject pharmaceutical composition can be present in a container, for example, a sterile container, such as a syringe. The preparation can be present in unit dose or multi-dose sealed containers (such as ampoules and vials) and can be stored in freeze-dried (lyophilized) conditions that only require adding sterile liquid excipients (such as water for injection) shortly before use. Real-time injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0331] The concentration of the multimeric polypeptides of the present disclosure in the formulation can vary widely (e.g., from less than about 0.1% by weight, typically at or at least about 2% by weight to as much as 20% to 50% by weight or more), and will generally be selected primarily based on fluid volume, viscosity, and patient-based factors according to the particular mode of administration selected and the patient's needs.

[0332] The present disclosure provides a container comprising a composition (e.g., a liquid composition) of the present disclosure. The container can be, for example, a syringe, an ampoule, etc. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

[0333] Compositions comprising nucleic acids or recombinant expression vectors

[0334] The present disclosure provides compositions comprising the nucleic acid or recombinant expression vector of the present disclosure, such as pharmaceutical compositions. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been described in detail in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th edition, Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0335] Compositions of the present disclosure may include: a) a subject nucleic acid or recombinant expression vector; and b) one or more of the following: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizing agent, a thickening agent, a stabilizer, a bacteriostat, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), and the like. Suitable salts include, for example, NaCl, MgCl2, KCl, MgSO4, and the like.

[0336] Pharmaceutical preparations of the present disclosure may comprise a nucleic acid or recombinant expression vector of the present disclosure in an amount of about 0.001% to about 90% (w / w). In the description of the preparation below, it will be understood that "the subject nucleic acid or recombinant expression vector" includes a nucleic acid or recombinant expression vector of the present disclosure. For example, in some embodiments, the subject preparation comprises a nucleic acid or recombinant expression vector of the present disclosure.

[0337] The subject nucleic acid or recombinant expression vector can be compounded, encapsulated, coupled or otherwise associated with other compounds or mixtures of compounds; such compounds may include, for example, liposomes or receptor targeting molecules. The subject nucleic acid or recombinant expression vector can be combined in a formulation with one or more components that facilitate uptake, distribution and / or absorption.

[0338] The subject nucleic acid or recombinant expression vector composition can be formulated into any of many possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The subject nucleic acid or recombinant expression vector composition can also be formulated into a suspension in an aqueous, non-aqueous, or mixed medium. The aqueous suspension can further contain substances that can increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension can also contain a stabilizer.

[0339] The preparation comprising the subject nucleic acid or recombinant expression vector can be a liposomal preparation. As used herein, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical double layer or double layer. Liposomes are unilamellar or multilamellar vesicles having a membrane formed by a lipophilic material and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form a stable complex. pH-sensitive or negatively charged liposomes are believed to entrap DNA rather than complex with it. Both cationic and non-cationic liposomes can be used to deliver the subject nucleic acid or recombinant expression vector.

[0340] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposomes, result in an extended circulation life relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposomes contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Patent No. 6,287,860, which is incorporated herein by reference in its entirety.

[0341] The formulations and compositions of the present disclosure may also include a surfactant. The use of surfactants in pharmaceuticals, formulations, and emulsions is well known in the art. Surfactants and their uses are further described in U.S. Patent No. 6,287,860.

[0342] In one embodiment, various penetration enhancers are included to achieve effective delivery of nucleic acid. In addition to helping non-lipophilic drugs diffuse through cell membranes, penetration enhancers also enhance the permeability of the lipophilic drug. Penetration enhancers can be divided into one of five major classes (i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants). Penetration enhancers and their use are further described in U.S. Patent No. 6,287,860 (which is incorporated herein by reference in its entirety).

[0343] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, caplets, tablets or mini-tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or adhesives may be required. Suitable oral formulations include those in which the subject antisense nucleic acid is administered together with one or more penetration enhancers, surfactants and chelating agents. Suitable surfactants include, but are not limited to, fatty acids and / or their esters or salts, bile acids and / or their salts. Suitable bile acids / salts and fatty acids and their uses are further described in U.S. Patent No. 6,287,860. Combinations of penetration enhancers are also suitable, such as combinations of fatty acids / salts with bile acids / salts. Exemplary suitable combinations are the sodium salts of lauric acid, capric acid and UDCA. Additional penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofuran methanol and AZONE TM .

[0344] Methods for regulating T cell activity

[0345] The present invention also provides a method for inhibiting a T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cell with an amount of a recombinant peptide as described herein that is effective in inhibiting the T cell clone, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is an inhibitory domain.

[0346] The present invention also provides a method for stimulating a T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cell with an amount of a recombinant peptide as described herein that is effective to stimulate the T cell clone, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is a stimulatory domain.

[0347] The present disclosure provides a method for selectively modulating the activity of epitope-specific T cells, the method comprising contacting a T cell with a multimeric polypeptide of the present disclosure, wherein contacting the T cell with the multimeric polypeptide of the present disclosure selectively modulates the activity of the epitope-specific T cell. In some cases, the contacting occurs in vitro. In some cases, the contacting occurs in vivo. In some cases, the contacting occurs ex vivo.

[0348] In an example where the target T cell is CD8 + In some cases where the target T cell is a CD4 T cell, the multimeric polypeptide comprises a class I MHC polypeptide (e.g., β2 microglobulin and a class I MHC heavy chain). + In some cases of T cells, the multimeric polypeptide comprises a class II MHC polypeptide (e.g., a class II MHC α chain; a class II MHC β chain).

[0349] When the multimeric polypeptides of the present disclosure include immunomodulatory polypeptides that are activating polypeptides, contacting T cells with the multimeric polypeptide activates the epitope-specific T cells. In some cases, the epitope-specific T cells are T cells specific for an epitope present on a cancer cell, and contacting the epitope-specific T cells with the multimeric polypeptide increases the cytotoxic activity of the T cells against the cancer cell. In some cases, the epitope-specific T cells are T cells specific for an epitope present on a cancer cell, and contacting the epitope-specific T cells with the multimeric polypeptide increases the number of the epitope-specific T cells.

[0350] In some cases, the epitope-specific T cells are T cells specific for an epitope present on a cell infected by a virus, and contacting the epitope-specific T cells with the multimeric polypeptide increases the cytotoxic activity of the T cells against the cell infected by the virus. In some cases, the epitope-specific T cells are T cells specific for an epitope present on a cell infected by a virus, and contacting the epitope-specific T cells with the multimeric polypeptide increases the number of the epitope-specific T cells.

[0351] When the multimeric polypeptides of the present disclosure include immunomodulatory polypeptides that are inhibitory polypeptides, contacting T cells with the multimeric polypeptides inhibits the epitope-specific T cells. In some cases, the epitope-specific T cells are autoreactive T cells specific for an epitope present in an autoantigen and the contact reduces the number of autoreactive T cells.

[0352] Treatment

[0353] The present invention also provides a method for treating an autoimmune disease by inhibiting an autoreactive T cell clone that recognizes an epitope peptide, comprising contacting the cloned T cells with a recombinant peptide as described herein that is effective for treating the autoimmune disease, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is an inhibitory domain.

[0354] The present invention also provides a method for treating cancer by stimulating a T cell clone that recognizes an epitope peptide on cancer, comprising contacting the cloned T cells with an amount of a recombinant peptide as described herein effective to treat the cancer, wherein the recombinant peptide comprises the epitope peptide and a T cell regulatory domain that is a stimulatory domain.

[0355] In one embodiment, the cells transformed to express the recombinant polypeptide of the present invention are isolated suspension-adapted cells. In one embodiment of the plurality of isolated suspension-adapted cells or recombinant nucleic acids, the nucleic acid comprises DNA.

[0356] In one embodiment, the T cells comprise peripheral T cells obtained from a subject. In one embodiment, the T cells comprise T cells in a subject. In one embodiment, the T cells comprise peripheral T cells in a subject. In one embodiment of the methods herein, the subject is a human.

[0357] The present invention provides a method for selectively regulating the activity of epitope-specific T cells in an individual, the method comprising administering to the individual an amount of a multimeric polypeptide of the present invention or one or more nucleic acids encoding the multimeric polypeptide that is effective in selectively regulating the activity of epitope-specific T cells in the individual. In some cases, the treatment methods of the present invention comprise administering to an individual in need thereof one or more recombinant expression vectors comprising a nucleotide sequence encoding a multimeric polypeptide of the present invention. In some cases, the treatment methods of the present invention comprise administering to an individual in need thereof one or more mRNA molecules comprising a nucleotide sequence encoding a multimeric polypeptide of the present invention. In some cases, the treatment methods of the present invention comprise administering to an individual in need thereof a multimeric polypeptide of the present invention.

[0358] The present disclosure provides a method for selectively regulating the activity of epitope-specific T cells in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the present disclosure or one or more nucleic acids (e.g., expression vectors; mRNA, etc.) comprising a nucleotide sequence encoding the multimeric polypeptide, wherein the multimeric polypeptide selectively regulates the activity of epitope-specific T cells in the individual. Selectively regulating the activity of epitope-specific T cells can treat an individual's disease or disorder. Therefore, the present disclosure provides a method of treatment comprising administering to an individual in need thereof an effective amount of a multimeric polypeptide of the present disclosure.

[0359] In some cases, the immunomodulatory polypeptide is an activating polypeptide and the multimeric polypeptide activates the epitope-specific T cells. In some cases, the epitope is an epitope associated with cancer, and the multimeric polypeptide increases the activity of T cells specific for the epitope associated with cancer.

[0360] The present disclosure provides a method for treating cancer in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the present disclosure or one or more nucleic acids comprising a nucleotide sequence encoding the multimeric polypeptide (e.g., expression vector; mRNA, etc.), wherein the multimeric polypeptide comprises a T cell epitope that is a cancer epitope, and wherein the multimeric polypeptide comprises a stimulatory immunomodulatory polypeptide. In some cases, an "effective amount" of a multimeric polypeptide is an amount that reduces the number of cancer cells in an individual when administered to an individual in need thereof in one or more doses. For example, in some cases, an "effective amount" of a multimeric polypeptide of the present disclosure is an amount that reduces the number of cancer cells in an individual when administered to an individual in need thereof in one or more doses, compared to the number of cancer cells in an individual before or in a patient in need thereof, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some cases, an "effective amount" of a multimeric polypeptide of the present invention is an amount that, when administered to an individual in need thereof at one or more doses, reduces the number of cancer cells in the individual to an undetectable level. In some cases, an "effective amount" of a multimeric polypeptide of the present invention is an amount that, when administered to an individual in need thereof at one or more doses, reduces the mass of a tumor in the individual. For example, in some cases, an "effective amount" of a multimeric polypeptide of the present invention is an amount that, when administered to an individual in need thereof at one or more doses, reduces the mass of a tumor in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared to the mass of a tumor in an individual before or without the administration of the multimeric polypeptide. In some cases, an "effective amount" of a multimeric polypeptide of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, prolongs the survival of the individual. For example, in some cases, an "effective amount" of a multimeric polypeptide of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, prolongs the survival of the individual by at least 1 month, at least 2 months, at least 3 months, 3 months to 6 months, 6 months to 1 year, 1 year to 2 years, 2 years to 5 years, 5 years to 10 years, or more than 10 years, compared to the expected survival of the individual without the administration of the multimeric polypeptide.

[0361] In some cases, the epitope-specific T cells are T cells specific for an epitope present on a cell infected by a virus, and contacting the epitope-specific T cells with the multimeric polypeptide increases the cytotoxic activity of the T cells against the cell infected by the virus. In some cases, the epitope-specific T cells are T cells specific for an epitope present on a cell infected by a virus, and contacting the epitope-specific T cells with the multimeric polypeptide increases the number of the epitope-specific T cells.

[0362] Thus, the present disclosure provides a method for treating a viral infection in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the present disclosure or one or more nucleic acids comprising a nucleotide sequence encoding the multimeric polypeptide, wherein the multimeric polypeptide comprises a T cell epitope that is a viral epitope, and wherein the multimeric polypeptide comprises a stimulatory immunomodulatory polypeptide. In some cases, an "effective amount" of a multimeric polypeptide is an amount that, when administered to an individual in need thereof at one or more doses, reduces the number of virally infected cells in the individual. For example, in some cases, an "effective amount" of a multimeric polypeptide of the present disclosure is an amount that, when administered to an individual in need thereof at one or more doses, reduces the number of virally infected cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared to the number of virally infected cells in an individual before or without administration of the multimeric polypeptide. In some cases, an "effective amount" of a multimeric polypeptide of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of virus-infected cells in the individual to undetectable levels.

[0363] Thus, the present disclosure provides a method for treating an infection in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the present disclosure or one or more nucleic acids comprising a nucleotide sequence encoding the multimeric polypeptide, wherein the multimeric polypeptide comprises a T cell epitope of an epitope associated with a pathogen, and wherein the multimeric polypeptide comprises a stimulatory immunomodulatory polypeptide. In some cases, an "effective amount" of the multimeric polypeptide is an amount that reduces the number of pathogens in the individual when administered to an individual in need thereof at one or more doses. For example, in some cases, an "effective amount" of the multimeric polypeptide of the present disclosure is an amount that reduces the number of pathogens in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when administered to an individual in need thereof at one or more doses, compared to the number of pathogens in an individual before or without the administration of the multimeric polypeptide. In some cases, an "effective amount" of a multimeric polypeptide of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of pathogens in the individual to undetectable levels. Pathogens include viruses, bacteria, protozoa, and the like.

[0364] In some cases, the immunomodulatory polypeptide is an inhibitory polypeptide and the multimeric polypeptide inhibits the activity of the antigen-specific T cells. In some cases, the epitope is a self-epitope and the multimeric polypeptide selectively inhibits the activity of T cells specific for the self-epitope.

[0365] The present disclosure provides a method for treating an autoimmune disease in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the present disclosure or one or more nucleic acids comprising a nucleotide sequence encoding the multimeric polypeptide, wherein the multimeric polypeptide comprises a T cell epitope that is a self-epitope, and wherein the multimeric polypeptide comprises an inhibitory immunomodulatory polypeptide. In some cases, an "effective amount" of a multimeric polypeptide is an amount that, when administered to an individual in need thereof at one or more doses, reduces the number of autoreactive T cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the number of autoreactive T cells in an individual before or without the administration of the multimeric polypeptide. In some cases, an "effective amount" of a multimeric polypeptide of the present disclosure is an amount that, when administered to an individual in need thereof at one or more doses, reduces the production of Th2 cytokines in the individual. In some cases, an "effective amount" of a multimeric polypeptide of the invention is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with an autoimmune disease in the subject.

[0366] As noted above, in some cases, when performing the subject treatment methods, the multimeric polypeptides of the present invention are administered to a subject in need thereof in the form of the polypeptide itself. In other cases, when performing the subject treatment methods, one or more nucleic acids comprising a nucleotide sequence encoding a multimeric polypeptide of the present invention are administered to a subject in need thereof. Thus, in other cases, one or more nucleic acids of the present invention (e.g., one or more recombinant expression vectors of the present invention) are administered to a subject in need thereof.

[0367] preparation

[0368] Suitable formulations are described above, wherein suitable formulations comprise a pharmaceutically acceptable excipient. In some cases, suitable formulations comprise: a) a multimeric polypeptide of the present disclosure; and b) a pharmaceutically acceptable excipient. In some cases, suitable formulations comprise: a) a nucleic acid comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure; and b) a pharmaceutically acceptable excipient; in some cases, the nucleic acid is mRNA. In some cases, suitable formulations comprise: a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide of the multimeric polypeptide of the present disclosure; b) a second nucleic acid comprising a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present disclosure; and c) a pharmaceutically acceptable excipient. In some cases, suitable formulations comprise: a) a recombinant expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure; and b) a pharmaceutically acceptable excipient. In some cases, a suitable formulation comprises: a) a first recombinant expression vector comprising a nucleotide sequence encoding a first polypeptide of the multimeric polypeptide of the present disclosure; b) a second recombinant expression vector comprising a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present disclosure; and c) a pharmaceutically acceptable excipient.

[0369] Suitable pharmaceutically acceptable excipients are described above.

[0370] dose

[0371] The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's body size, body surface area, age, specific polypeptide or nucleic acid to be administered, the patient's gender, time and route of administration, general health status and other drugs administered simultaneously. The amount of the multimeric polypeptides of the present invention may be between 1 ng / kg body weight and 20 mg / kg body weight per dose, for example, between 0.1 mg / kg body weight and 10 mg / kg body weight, for example, between 0.5 mg / kg body weight and 5 mg / kg body weight; however, dosages lower or higher than this exemplary range are conceivable, especially in view of the aforementioned factors. If the regimen is a continuous infusion, it may also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[0372] In some cases, a suitable dose of the multimeric polypeptides of the present invention is 0.01 μg to 100 g per kilogram of body weight, 0.1 μg to 10 g per kilogram of body weight, 1 μg to 1 g per kilogram of body weight, 10 μg to 100 mg per kilogram of body weight, 100 μg to 10 mg per kilogram of body weight, or 100 μg to 1 mg per kilogram of body weight. One of ordinary skill in the art can easily estimate the repetition rate of administration based on the residence time and concentration of the administered agent measured in body fluids or tissues. After successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent recurrence of the disease state, wherein the multimeric polypeptides of the present invention are administered at a maintenance dose within the following range: 0.01 μg to 100 g per kilogram of body weight, 0.1 μg to 10 g per kilogram of body weight, 1 μg to 1 g per kilogram of body weight, 10 μg to 100 mg per kilogram of body weight, 100 μg to 10 mg per kilogram of body weight, or 100 μg to 1 mg per kilogram of body weight.

[0373] The skilled artisan will readily recognize that dosage levels may vary with the specific multimeric polypeptide, the severity of the symptoms, and the subject's sensitivity to side effects. The preferred dosage of a given compound can be readily determined by those skilled in the art in a variety of ways.

[0374] In some embodiments, multiple doses of the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered. The frequency of administration of the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention may vary according to any of a variety of factors, such as the severity of the symptoms, etc. For example, in some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0375] The duration of administration of the multimeric polypeptides of the present disclosure, the nucleic acids of the present disclosure, or the recombinant expression vectors of the present disclosure (e.g., the administration cycle of the multimeric polypeptides of the present disclosure, the nucleic acids of the present disclosure, or the recombinant expression vectors of the present disclosure) can vary depending on any of a variety of factors (e.g., the patient's response, etc.). For example, the administration cycle of the multimeric polypeptides of the present disclosure, the nucleic acids of the present disclosure, or the recombinant expression vectors of the present disclosure can be from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about one year, from about one year to about two years, or from about two years to about four years, or longer.

[0376] Route of administration

[0377] The active agent (multimeric polypeptide of the disclosure, nucleic acid of the disclosure, or recombinant expression vector of the disclosure) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, as well as systemic and local administration routes.

[0378] Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. If desired, a variety of routes of administration may be combined or adjusted based on the multimeric polypeptide and / or desired effect. The multimeric polypeptides of the present disclosure, nucleic acids of the present disclosure, or recombinant expression vectors may be administered in a single dose or multiple doses.

[0379] In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered intravenously. In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered intramuscularly. In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered topically. In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered intratumorally. In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered peritumorally. In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered intracranially. In some embodiments, the multimeric polypeptides of the present invention, the nucleic acids of the present invention, or the recombinant expression vectors of the present invention are administered subcutaneously.

[0380] In some embodiments, the multimeric polypeptides of the present disclosure are administered intravenously. In some embodiments, the multimeric polypeptides of the present disclosure are administered intramuscularly. In some embodiments, the multimeric polypeptides of the present disclosure are administered topically. In some embodiments, the multimeric polypeptides of the present disclosure are administered intratumorally. In some embodiments, the multimeric polypeptides of the present disclosure are administered peritumorally. In some embodiments, the multimeric polypeptides of the present disclosure are administered intracranially. In some embodiments, the multimeric polypeptides of the present disclosure are administered subcutaneously.

[0381] The multimeric polypeptides of the present invention, the nucleic acids of the present invention or the recombinant expression vectors of the present invention can be administered to the host using any available conventional methods and routes (including systemic or local routes) suitable for delivering conventional drugs. In general, the routes of administration contemplated by the present invention include but are not necessarily limited to enteral, parenteral or inhalation routes.

[0382] Parenteral administration routes other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous routes, i.e., any other route of administration other than through the digestive tract. Parenteral administration can be performed to achieve systemic or local delivery of the multimeric polypeptides of the present disclosure, the nucleic acids of the present disclosure, or the recombinant expression vectors of the present disclosure. When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of the pharmaceutical formulation.

[0383] Subjects suitable for treatment

[0384] Suitable subjects for treatment with the disclosed methods include individuals with cancer, including individuals diagnosed as having cancer, individuals who have received cancer treatment but have not responded to the treatment, and individuals who have received cancer treatment and initially responded but subsequently became refractory to the treatment. Suitable subjects for treatment with the disclosed methods include individuals with infection (e.g., infected by pathogens, such as bacteria, viruses, protozoa, etc.), including individuals diagnosed as having infection, individuals who have received infection treatment but have not responded to the treatment. Suitable subjects for treatment with the disclosed methods include individuals with bacterial infection, including individuals diagnosed as having bacterial infection and individuals who have received bacterial infection treatment but have not responded to the treatment. Suitable subjects for treatment with the disclosed methods include individuals with viral infection, including individuals diagnosed as having viral infection and individuals who have received viral infection treatment but have not responded to the treatment. Suitable subjects for treatment with the disclosed methods include individuals with autoimmune disease, including individuals diagnosed as having autoimmune disease, individuals who have received autoimmune disease treatment but have not responded to the treatment.

[0385] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or clearly contradicted by context.

[0386] The present invention will be better understood from the experimental details below. However, those skilled in the art will readily recognize that the specific methods and results discussed are only for illustrative purposes and the following claims will more fully describe the present invention. Example

[0387] The following examples are put forward so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of the invention as viewed by the inventors nor to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; kb, kilobase; bp, base pairs; nt, nucleotide; im, intramuscular (intramuscular); ip, intraperitoneal (intraperitoneal); sc, subcutaneous (subcutaneous), etc.

[0388] Example 1 :S YN T AC Heterodimer formation

[0389] Aspects of the present disclosure relate to a novel protein-based therapeutic platform, "synTac," that mimics the interaction specificity and regulatory signaling of the immune synapse. SynTac is a fusion protein that links a co-stimulatory molecule to an MHC-epitope to allow precise T cell engagement and clonal T cell activation or inhibition ( Figure 1 ), a soluble form to which the body naturally responds. In this way, synTac combines optimal epitopes, bispecific antibodies, soluble co-stimulatory molecules and ADCs. SynTac allows for highly specific cell targeting via MHC-epitopes, while the "single-chain fusion" design does not allow for cross-presentation of free epitopes ( Figures 2A-2C ). T cell regulatory domain (herein alternatively referred to as "MOD") is also covalently linked, which causes activation or inhibition according to the nature of costimulatory engagement. This causes antigen-specific rather than comprehensive T cell responses. It is worth noting that the MOD may include any known antibody, antibody fragment, costimulatory molecule or other literature-verified payload (cytokine, toxin, etc.) and does not need to be internalized to affect T cells. In addition, the two targets are present on the surface of the same cell, thereby eliminating the "spacing problem" of traditional bispecific antibodies.

[0390] In one embodiment, this strategy utilizes an Fc-fusion construct ( Figures 2A-2C Non-limiting examples are shown) to increase the potency, stability and therapeutic window of related products. Briefly, the Fc region is a natural covalent homodimer and is synthesized by Figures 2A-2C The two disulfide bonds shown in Figure 2 are stabilized by two thin lines. The presence of the Fc domain is known to prolong therapeutic activity by extending plasma half-life, which is attributed to its interaction with the neonatal Fc receptor and the slower renal clearance of the larger bivalent molecule [23,24]. From a biophysical point of view, the Fc domain folds independently and can improve the solubility and stability of the partner molecule in vitro and in vivo

[25] , and the Fc region allows for simple and low-cost purification during production by protein-A / G affinity chromatography

[26] . Figure 2A Single-chain peptide MHC proteins (single-chain trimers

[27] ) are shown with their carboxyl termini linked to the IgG Fc region. As depicted, these single-chain constructs are limited in their ability to extend the system through alternative protein linkages such as MOD. Specifically, linkage is preferably restricted to the C-terminal region of MHC, e.g. Figure 2A The method of expressing the construct using direct bonding is highly dependent on the MOD used. The solution disclosed herein is to separate the construct into the corresponding heavy and light chains and fuse both the peptide and protein to different ends ( Figure 2B and Figure 2COne configuration results in the amino terminus of the peptide being associated with a light chain (β2 microglobulin), which in turn extends from the carboxyl terminus of the light chain to the MOD effector molecule ( Figure 2B In this case, the heavy chain (HLA-molecule) is fused to the Fc region. During the production process, all components associate and self-assemble in eukaryotic cells (e.g. HEK, CHO). The construct is covalently held together by disulfide bridges. An alternative orientation ( Figure 2C ) fuses the MOD amino terminus of the Fc to the heavy chain, while the peptide remains attached to the B2M light chain. Again, all components self-assemble and form stable covalent interactions via disulfide bonds. Traditional bispecific antibodies often attempt to bridge two cells by dimerizing an amino-terminal Fc payload with a carboxyl-terminal Fc payload. In contrast, the configuration disclosed herein directs two different protein payloads (MHC epitope targeting machinery and MOD effector) toward the surface of the same cell, similar to the CH1-light chain interactions found in traditional antibodies. In addition, the use of Fc fusions allows for custom engagement of relevant effector functions (such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or phagocytosis) by modulating binding affinity to Fc receptors through mutations

[28] .

[0391] Figures 3A-3B The design of two basic synTac molecules is presented in Figure 2. Briefly, the construct utilizes the native human B2M leader sequence to allow efficient secretion and ER processing, followed by the candidate epitope (labeled as a peptide). Once in the ER, the leader sequence is completely removed and allows the peptide to be presented in the MHC binding pocket. For the "light chain" linkage (LC, Figure 3A ), which is coupled to the native B2M via linker L1 and to the MOD via linker L2. The complete cassette is linked to another B2M leader sequence, an MHC heavy chain (e.g., human HLA-A02:01 or murine H-2Kd in the examples) and an Fc domain (human IgG1 or murine IgG2a) via a porcine Teschovirus-1 (P2A) "self-cleaving" peptide to allow stoichiometric expression of each chain. The P2A peptide was chosen because it is reported to have the highest "cleavage" efficiency of all viral 2A peptides expressed in mammalian cells

[29] . The "heavy chain" (HC, Figure 3B ) bonding is similar, however, now the viral P2A peptide follows B2M, and MOD follows the second leader peptide, resulting in Figure 2C Both constructs can be terminated with an 8x His tag for ease of purification.

[0392] Specialized expression cells: Although both chains are expressed and co-localized to the ER due to P2A linkage, there is some concern that endogenous B2M from the expression host (suspension-adapted HEK293 cells) may outnumber the recombinant form, as HEK293 cells naturally express both HLA and B2M molecules. This would result in reduced stability (e.g., manifested in reduced overall yield) or a very unsatisfactory heterologous protein sample. To circumvent this complication, the CRISPR / CAS system was utilized to knock out native B2M from a pool of HEK cells

[30] . Briefly, a guide RNA targeting endogenous B2M was designed, transfected with a plasmid encoding CRISPR / CAS, and cultured for three days. The cultured cells were surface stained for anti-B2M and counter-selected by fluorescence activated cell sorting (FACS) (sorted based on loss of fluorescence). The sorted cells were recovered and subjected to two or more rounds of staining, counter-selection, and recovery (three rounds in total) to ensure efficient (~100%) knockout. Figure 4 As shown in, the quality of the final pool was checked via FACS monitoring of B2M surface expression, which showed that endogenous B2M had been completely removed. Then, an experiment using next generation sequencing was performed to quantify the knockout percentage at the genome level. All subsequent experiments were performed using the resulting HEK-293-B2M-KO system (referred to as HEK-KO).

[0393] Engineered disulfide bonds: To increase protein stability and avoid complications associated with possible peptide transfer to cellular MHC molecules (cross-presentation) and B2M release, single-chain constructs are often used [27,31]. However, these single-chain constructs ( Figure 2A (shown in Figure 2) is limited in its ability to extend the system by alternative protein linkages such as MOD. One solution is to split the construct into the corresponding heavy and light chains similar to previous efforts

[32] , but now fuse both the peptide and the protein to different ends as described ( Figure 2B and 2C). However, in the final construct, in order to retain the stability provided by traditional single chain systems, the option of engineering a disulfide bridge between the heavy and light chains (SS as shown in Figure 2), as seen in the disulfide trapped single chain trimer [dt-SCT]

[33] , was investigated. It is noteworthy that because attempts to utilize the dt-SCT disulfide bond scheme when synTac was initially prepared resulted in low expression levels, which further depended on the peptide presented, the dt-SCT disulfide bond configuration was considered unsuitable for split protein systems. Therefore, attempts were made to identify alternative positions to engineer disulfide bridges that would be more suitable for split protein systems such as synTac. Two positions (2, 12) were selected from the light chain that each had a disulfide bond potential for use at two positions in the heavy chain (119, 120 and 236, 237, respectively, from analysis of PDB 2X4R). Notably, these positions are highly conserved residues that are not known to interact with the peptide binding groove

[34] , the TCR complex

[35] , or the CD8 coreceptor

[36] . One construct (H236-L12, where H refers to the heavy chain position and L refers to the light chain, Figures 5A-5B 18) was shown to have high levels of expression, and the second construct (H237-L12, Figures 5A-5B The dt-SCT disulfide bond scheme was used as a positive control (labeled as synTac 2). As seen by non-reducing PAGE gel, a high molecular weight fraction was formed, indicating the formation of stable disulfide bonds ( Figure 5A All expression constructs were scaled up to 100 ml, purified and tested for activity by binding to the cognate TCR expressed on the surface of HEK cells (designated HEK-A6), as monitored by FACS fluorescence, which showed correct folding and activity ( Figure 5B As a negative control, cells expressing a non-cognate TCR (referred to as HEK-AS01) were used. An additional construct was generated with only a C-terminal 8X His tag (monovalent).

[0394] SynTac control: Previous work has focused on autoimmune diabetes

[37] and disease-related model systems, specifically, autoreactive CD8+ 8.3 T cells isolated from pancreatic islets of non-obese diabetic (NOD) mice have been used. Based on this work, a synTac construct was generated with a peptide presented by the mouse class I H-2Kd allele (called IGRP) that is known to interact with 8.3 T cells. The peptide consists of residues 206 to 214 of the islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP206-214). A control synTac presenting a tumor-derived peptide (KYQAVTTTL, SEQ ID NO: 18) that is not recognized by 8.3 T cells was prepared in the same manner (e.g., mouse H-2Kd presentation) and was named TUM. To determine the extent to which the system could tolerate multiple HLA alleles (e.g., mouse H-2Kd, human HLA-A02, etc.), a third synTac variant was constructed with a previously validated human HLA-A02 restricted epitope (human T-cell lymphotropic virus, Tax 11-19) and termed HTLV. To achieve targeted T cell depletion, the initial synTac construct used a light chain-bound form and carried the PD-L1 MOD domain (in Figure 2B Each synTac variant (IGRP, TUM and HTLV) showed positive expression profiles in HEK-KO cells, and the non-reducing SDS-page results are shown in Figure 6A To examine the general utility of this expression system, synTac constructs based on IGRP with variant MOD domains were explored, including two MODs for T cell stimulation (i.e., humanized anti-CD28 single-chain Fv and the extracellular domain of the TNF ligand 4-1BBL), and two additional MODs that allow T cell inhibition (a single-point mutant [W88A] of B7-1, known to bind only to CTLA4

[38] ; and a truncated variant of PD-L1 [only the Ig variable domain]). All constructs expressed well in HEK-KO cells. Figure 6B The expression capacity of the synTac protein in the heavy chain bonded form was further explored ( Figure 2C To this end, using the IGRP epitope as a targeting peptide and PD-L1 or humanized anti-CD28 scFv as MOD, the results again showed a positive expression profile in HEK-KO cells ( Figure 6C These proteins were then prepared at a 1 L or larger scale and purified to homogeneity by Ni2+ IMAC and size exclusion in an endotoxin-free environment. All IGRP and TUM constructs were used in T cell proliferation assays, and HTLV constructs were used in the following TCR-synTac-PD1 bridging experiments.

[0395] TCR-synTac-PD1 bridging: Although the solution profile after size exclusion indicated good protein folding, it was desirable to verify the integrity of each synTac component (both the MHC-epitope targeting mechanism and the MOD) before using these reagents in activity assays. Using the previously described HEK-A6 cells as a positive control, cells expressing a non-cognate TCR (AS01, reactive to an HLA-A0201-restricted Epstein-Barr virus epitope) were generated, along with untransduced ...

Claims

1. A multimeric polypeptide comprising: A heterodimeric polypeptide comprising a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; and ii) first major histocompatibility complex (MHC) class II polypeptide; b) a second polypeptide comprising a second MHC class II polypeptide; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises the at least one immunomodulatory polypeptide, wherein the first MHC class II polypeptide is a human MHC β-chain polypeptide and the second MHC class II polypeptide is a human MHC α-chain polypeptide, wherein the peptide epitope is an epitope of a self-antigen, Wherein the multimeric polypeptide comprises one or more linker peptides.

2. The multimeric polypeptide of claim 1, wherein the multimeric polypeptide comprises an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold.

3. The multimeric polypeptide according to claim 1, wherein the multimeric polypeptide comprises: a1) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; and ii) a first MHC class II polypeptide; and b1) a second polypeptide comprising, in order from N-terminus to C-terminus: i) at least one immunomodulatory polypeptide; ii) a second MHC class II polypeptide; and iii) immunoglobulin (Ig) Fc polypeptide; or a2) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; ii) a first MHC class II polypeptide; and iii) at least one immunomodulatory polypeptide; and b2) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide; and ii) Ig Fc polypeptide; or a3) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; and ii) a first MHC class II polypeptide; and b3) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide; and ii) Ig Fc polypeptide; and iii) at least one immunomodulatory polypeptide; or a4) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; and ii) a first MHC class II polypeptide; and b4) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide; and ii) at least one immunomodulatory polypeptide; or a5) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; and ii) a first MHC class II polypeptide; and b5) a second polypeptide comprising, in order from N-terminus to C-terminus: i) at least one immunomodulatory polypeptide; and ii) a second MHC class II polypeptide; or a6) a first polypeptide comprising, in order from N-terminus to C-terminus: i) peptide epitopes; ii) a first MHC class II polypeptide; and iii) at least one immunomodulatory polypeptide; and b6) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide, Wherein the multimeric polypeptide comprises one or more linker peptides.

4. The multimeric polypeptide of claim 2, wherein the non-Ig scaffold is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide. 5 . The multimeric polypeptide according to claim 1 , wherein the at least one immunomodulatory polypeptide is selected from the group consisting of: a 4-1BBL polypeptide and a PD-L1 polypeptide.

6. The multimeric polypeptide according to any one of claims 1 to 4, comprising 2 or more immunomodulatory polypeptides.

7. The multimeric polypeptide according to claim 6, wherein the two or more immunomodulatory polypeptides are connected in series.

8. A protein comprising two multimeric polypeptides according to any one of claims 1 to 7, wherein each of the two multimeric polypeptides comprises an immunoglobulin (Ig) Fc polypeptide, and wherein the two multimeric polypeptides are linked by one or more disulfide bonds between the respective IgFc polypeptides. 9 . A nucleic acid comprising a nucleotide sequence encoding the first polypeptide and the second polypeptide according to any one of claims 1 to 7 .

10. An expression vector comprising the nucleic acid of claim 9. A host cell genetically modified by the expression vector according to claim 10.

12. A method for selectively inhibiting the activity of autoreactive T cells or reducing the number of autoreactive T cells, the method comprising contacting the T cells with the multimeric polypeptide described in any one of claims 1 to 7 or the protein described in claim 8 in vitro, wherein the contact selectively inhibits the activity of the autoreactive T cells or reduces the number of the autoreactive T cells.

13. Use of the multimeric polypeptide according to any one of claims 1 to 7 or the protein according to claim 8 in the preparation of a medicament, wherein the medicament is used in a method for selectively inhibiting the activity of autoreactive T cells or reducing the number of autoreactive T cells in an individual, wherein the multimeric polypeptide or protein is administered to selectively inhibit the activity of autoreactive T cells or reduce the number of autoreactive T cells in the individual.

14. Use of the multimeric polypeptide according to any one of claims 1 to 7, or the protein according to claim 8, or the nucleic acid according to claim 9, or the expression vector according to claim 10, or one or more mRNAs comprising a nucleotide sequence encoding the multimeric polypeptide according to any one of claims 1 to 7 or the protein according to claim 8 in the preparation of a medicament for use in a method for treating an autoimmune disease in an individual.

15. A composition comprising: a) the multimeric polypeptide of any one of claims 1 to 7, or the protein of claim 8; and b) pharmaceutically acceptable excipients.

16. Use of the multimeric polypeptide according to any one of claims 1 to 7 or the protein according to claim 8 in the preparation of a medicament, wherein the medicament is used in a method for selectively activating target T cells specific for a peptide epitope present in the multimeric polypeptide, wherein the target T cells are epitope-specific CD4 + T cells.

17. Use of the multimeric polypeptide according to any one of claims 1 to 7 or the protein according to claim 8 in the preparation of a medicament, wherein the medicament is used in a method for selectively activating target T cells specific for a peptide epitope present in the multimeric polypeptide, wherein the target T cells are epitope-specific CD8 + T cells.

18. The method according to claim 16, wherein the epitope-specific CD4 + T cells are CD4 + / CD25 + / FoxP3 + Regulatory T cells.

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