Multispecific molecules for modulating T cell activity and uses thereof

Abstract

The present invention provides a multispecific molecule capable of binding to an antigen-specific T cell receptor (TCR) expressed on the surface of a cell, the multispecific molecule comprising a first molecule and a second molecule, the first molecule being a polypeptide comprising (i) a peptide (p) presented in the context of a major histocompatibility complex (MHC) molecule (pMHC complex), and (ii) a first multimerization domain, and the second molecule being a polypeptide comprising (i) a domain that specifically binds to a molecule expressed on the surface of a cell expressing the TCR, and (ii) a second multimerization domain, as well as the use of such a molecule for modulating T cell activity and treating diseases such as cancer, infectious diseases, and autoimmune disorders.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of Provisional U.S. Application No. 63 / 349,770, filed on June 7, 2022, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on June 1, 2023, is named 250298_000494_SL.xml and has a size of 105,334 bytes.

[0003] Multispecific molecules, and the use of such molecules for modulating T cell activity and treating diseases are described herein.

Background Art

[0004] One of the central components of the adaptive immune system is the T cell, which, in response to the recognition of a specific antigen, is activated, proliferates, and differentiates into effector cells (CD8 + cytotoxic T cells or CD4 + helper T cells). The key molecular event leading to T cell activation is the engagement of the T cell receptor (TCR) with an antigen peptide presented in the groove of a major histocompatibility complex (MHC) protein on an antigen - presenting cell (e.g., dendritic cell). The TCR is a membrane - bound heterodimer with an antibody - like binding site that recognizes specific antigens. In addition to antigen recognition by the TCR, T cell activation requires co - stimulatory signals generated by the engagement of cell - surface proteins on the T cell and the antigen - presenting cell. CD28 is a widely recognized cell - surface molecule expressed on T cells that binds to CD80 / CD86 on antigen - presenting cells to provide co - stimulatory signals. Class I MHC proteins engage CD8 + T cells that can be activated to form cytotoxic T cells, and class II MHC proteins engage CD4 + T cells that can be activated to produce helper T cells.

[0005] T cells can not only be activated to proliferate and differentiate, but T cells can also be inhibited from proliferation and made tolerant (anergic) via inhibitory signals produced by engagement of cell surface proteins on T cells and antigen-presenting cells. CTLA-4 is a widely recognized cell surface molecule expressed on T cells that binds to CD80 / CD86 on antigen-presenting cells (with a significantly higher affinity than CD28 in vitro) to provide an inhibitory signal. Inhibition of T cells induces T cell tolerance via cell cycle arrest.

[0006] Despite previous attempts to create molecules for modulating T cell-induced immune responses, there remains a need in the art for molecules that can effectively regulate T cell activity to effect either suppression (in the case of autoimmune disorders) or induction (in the case of infections or cancer) of an immune response. SUMMARY OF THE INVENTION

[0007] Generally, the present invention provides multispecific molecules (e.g., bispecific molecules) and their use, where the multispecific molecule comprises a first molecule for engaging a T cell receptor and a second molecule for engaging a T cell surface molecule for regulating the activity of T cells. Regulation of T cell activity includes activation and / or proliferation (e.g., when the immunomodulatory molecule is a costimulatory molecule such as CD28), or suppression of activity, anergy, and / or T cell death (e.g., when the immunomodulatory molecule is an inhibitory molecule such as CTLA-4). In various embodiments, the first molecule of the multispecific molecule comprises a fusion of a peptide and a major histocompatibility complex (MHC) protein such that the peptide is disposed in the groove of the MHC protein (peptide in the groove, or PiG) for presentation and engagement by a T cell receptor (TCR) of a T cell having specificity for the peptide, and the second molecule of the multispecific molecule comprises a domain for binding to a T cell surface molecule on a T cell having specificity for the peptide. In this way, T cells having specificity for the peptide can be activated to generate an immune response (e.g., in the case of infection or cancer) or inhibited to suppress an immune response (e.g., in the case of an autoimmune disorder).

[0008] In one aspect, the present invention provides a multispecific molecule capable of binding to an antigen-specific T cell receptor (TCR) expressed on the surface of a cell, the multispecific molecule comprising a first molecule and a second molecule, the first molecule being a polypeptide comprising (i) a peptide (p) presented in the context of a major histocompatibility complex (MHC) molecule capable of binding to an antigen-specific T cell receptor (TCR) expressed on the surface of the cell (pMHC complex), and (ii) a first multimerization domain, and the second molecule being a polypeptide comprising (i) a domain that specifically binds to a molecule expressed on the surface of a cell expressing the TCR, and (ii) a second multimerization domain.

[0009] In some embodiments of the multispecific molecule, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is an antigen-binding domain. In some embodiments, the antigen-binding domain is a monovalent antigen-binding domain. In certain embodiments, the antigen-binding domain binds to an immune regulatory molecule (e.g., CD28 or CTLA4) on the surface of a cell expressing a TCR. In some embodiments, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is a small molecule, protein, fusion protein, peptide, aptamer, avimer, or a derivative or fragment thereof. In certain embodiments, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is a T cell co-immunoregulatory molecule that regulates the activity of T cells when it binds to a molecule on the surface of a cell expressing a TCR (e.g., the domain can be a co-stimulatory molecule such as CD80 or CD40, or an inhibitory molecule such as PD-L1 or B7-H3). In certain embodiments, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is a protein, or a derivative or fragment thereof, and the protein is an adhesion molecule (e.g., ICAM).

[0010] In some embodiments of the multispecific molecule, the first and / or second multimerization domain is an immunoglobulin Fc domain. In some embodiments, the first and / or second Fc domain is a human IgG Fc domain, e.g., a human IgG1 Fc domain or a human IgG4 Fc domain, or a human IgM Fc domain. In some embodiments, the first and second Fc domains are the same. In some embodiments, the first Fc domain and / or the second Fc domain contains a CH3 mutation to facilitate purification of the multispecific molecule. In some embodiments, the CH3 mutation is a knob-into-hole mutation or a charge mutation. In some embodiments, the first Fc domain or the second Fc domain contains a CH3 mutation to facilitate purification of the multispecific molecule via affinity chromatography. In some embodiments, the first and / or second Fc domain exhibits enhanced Fcγ receptor binding activity relative to wild-type human IgG1.

[0011] In some embodiments of the multispecific molecule, the antigen-binding domain of the second molecule comprises a Fab or a single-chain Fv (scFv). In some embodiments of the multispecific molecule, the first and / or second multimerization domain comprises a second antigen-binding domain that specifically binds to a cell surface molecule. In some embodiments, such binding is used to immobilize the multispecific molecule on the cell surface (which can be used as a multispecific carrier). In some embodiments, the second antigen-binding domain specifically binds to a B cell surface molecule or a tumor-associated antigen. In some embodiments, the B cell surface molecule is CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD35, CD38, CD180, or CD40. In one embodiment, the B cell surface molecule is CD20. In one embodiment, the B cell surface molecule is CD180. In some embodiments, the second antigen-binding domain comprises an scFv. In some embodiments, the scFv is attached to the C-terminus of the first and / or second multimerization domain. An scFv linked to the C-terminus of a multimerization domain (e.g., an IgG Fc domain) is alternatively referred to herein as a "Stahl body."

[0012] In some embodiments of the multispecific molecule, the pMHC complex displays a peptide in a class I MHC polypeptide, or a fragment thereof (e.g., the peptide-binding groove), variant, or derivative. In some embodiments, the pMHC complex comprises (i) a peptide, (ii) a β2-microglobulin polypeptide, or a fragment, variant, or derivative thereof, (iii) a class I MHC α-chain domain, or a fragment, variant, or derivative thereof, and (iii) an immunoglobulin (Ig) Fc domain. In some embodiments, the pMHC complex comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) a β2-microglobulin polypeptide, or a fragment, variant, or derivative thereof, (iii) a class I α-chain domain, or a fragment, variant, or derivative thereof, and (iv) an Ig Fc domain. In some embodiments, the pMHC complex comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) an optional first linker, (iii) a β2-microglobulin polypeptide, or a fragment, variant, or derivative thereof, (iv) an optional second linker, (v) class I MHC α-chain domains 1, 2, and / or 3, or fragments, variants, or derivatives thereof, (vi) an optional third linker, and (vii) an Ig Fc domain. In some embodiments, the MHC comprises the α1 and α2 domains of a class I MHC polypeptide, or fragments, variants, or derivatives thereof. In some embodiments, the MHC comprises the α1, α2, and / or α3 domains of a class I MHC polypeptide, or fragments, variants, or derivatives thereof. In some embodiments, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC polypeptide is a mouse class I MHC polypeptide selected from the group consisting of H-2K, H-2D, H 2L, H-2Q, H-2M, and H-2T.

[0013] In some embodiments of the multispecific molecule, the pMHC complex displays a peptide in a class II MHC polypeptide or a fragment thereof (e.g., the peptide-binding groove), variant, or derivative. In some embodiments, the pMHC complex comprises (i) a peptide, (ii) a class II MHC α-chain domain, or a fragment, variant, or derivative thereof, (iii) a class II MHC β-chain domain, or a fragment, variant, or derivative thereof, and (iv) an Ig Fc domain. In some embodiments, the MHC class II α-chain consists of the extracellular domain of the class II α-chain. In some embodiments, the MHC class II beta-chain consists of the extracellular domain of the class II beta-chain. In some embodiments, the pMHC complex comprises, from N-terminus to C-terminus, (i) a peptide, (ii) a class II MHC α-chain extracellular domain, or a fragment, variant, or derivative thereof, (iii) a class II MHC β-chain extracellular domain, or a fragment, variant, or derivative thereof, and (iv) an Ig Fc domain. In some embodiments, the pMHC complex comprises, from N-terminus to C-terminus, (i) a peptide, (ii) a class II MHC β-chain extracellular domain, or a fragment, variant, or derivative thereof, (iii) a class II MHC α-chain extracellular domain, or a fragment, variant, or derivative thereof, and (iv) an Ig Fc domain. In some embodiments, the pMHC complex comprises, from N-terminus to C-terminus, (i) a peptide, (ii) an optional first linker, (iii) class II MHC α-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (iv) an optional second linker, (v) class II MHC β-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (vi) an optional third linker, and (vii) an Ig Fc domain. In some embodiments, the pMHC complex comprises, from N-terminus to C-terminus, (i) a peptide, (ii) an optional first linker, (iii) class II MHC β-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (iv) an optional second linker, (v) class II MHC α-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (vi) an optional third linker, and (vii) an Ig Fc domain.In certain embodiments, the MHC comprises the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) of a human class II MHC complex selected from the group consisting of HLA DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO. In another specific embodiment, the MHC comprises the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) of a mouse H-2A or H-2E class II MHC complex.

[0014] In some embodiments of the multispecific molecule, the T cell surface molecule is a T cell co-stimulatory molecule, a T cell immunomodulatory molecule that specifically binds to a domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR. In some embodiments, the T cell co-stimulatory molecule is selected from the group consisting of CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM, 2B4, CD226, TIM1, TIM2, and CD2.

[0015] In some embodiments of the multispecific molecule, the T cell surface molecule is a T cell inhibitory molecule, a T cell immunomodulatory molecule that specifically binds to a domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR. In some embodiments, the inhibitory molecule is selected from the group consisting of BTLA, B7-1, B7-H1, CD160, CTLA4, LAG3, LAIR1, MHC-I, PD1, TIGIT, or TIM3.

[0016] In some embodiments of the multispecific molecule, the peptide consists of about 5 to about 40 amino acid residues, about 6 to about 30 amino acid residues, about 8 to about 20 amino acid residues, or about 9, 10, or 11 amino acid residues.

[0017] In some embodiments of the multispecific molecule, the peptide is derived from a viral antigen. In some embodiments, the viral antigen is derived from a virus selected from the group consisting of adenovirus, astrovirus, chikungunya, cytomegalo, dengue, Ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, Lassa, measles, metapneumovirus, mumps, norovirus, oropauche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, usutu, vaccina, varicella, West Nile, yellow fever, or Zika.

[0018] In some embodiments of the multispecific molecule, the peptide is derived from a bacterial antigen. In some embodiments, the bacterial antigen is an antigen associated with bacteria resistant to conventional antibiotic therapies such as methicillin-resistant Staphylococcus Aureus (MRSA), Clostridium Difficile, carbapenem-resistant Enterobacteriaceae, drug-resistant Neisseria Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, extended-spectrum β-lactamase-producing bacteria, vancomycin-resistant enterococci, multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, vancomycin-resistant Staphylococcus Aureus, erythromycin-resistant group A Streptococcus, or clindamycin-resistant group B Streptococcus.

[0019] In some embodiments of the multispecific molecule, the peptide is derived from a tumor-associated antigen. In some embodiments, the tumor-associated antigen is adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, ALK, BAGE protein (e.g., BAGE-1), BIRC5 (survivin), BIRC7, beta-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CAGE-1-8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen (“ETA”), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE protein (e.g., GAGE-1-8), GD2, GD3, GloboH, glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein,Selected from the group consisting of Lengsin, LMP2, M-CSF, MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, -A9, -A10, -A12, -C1, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE protein (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, seselin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvel antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1. In some embodiments, the peptide is a neoantigen. In some embodiments, the peptide is a tumor-specific antigen.,

[0020] In some embodiments of the multispecific molecule, the peptide is derived from an antigen associated with an autoimmune disorder. In some embodiments, the antigen is gliadin (celiac disease, e.g., (i) an α-gliadin fragment corresponding to amino acids 57-73, or (ii) a γ-gliadin fragment corresponding to amino acids 139-153, or (iii) an ω-gliadin fragment corresponding to amino acids 102-118), GAD 65, IA-2, and insulin B chain (in the case of type 1 diabetes), glatiramer acetate (GA) (in the case of multiple sclerosis), acetylcholine receptor (AChR) (in the case of myasthenia gravis), p205, insulin, thyroid stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)), selected from the group consisting of. In some embodiments, the antigen associated with the autoimmune disorder is selected from the group consisting of IL-4R, IL-6R, and DLL4.

[0021] In some embodiments of the multispecific molecule, the first immunoglobulin Fc domain, the second immunoglobulin Fc domain, or both the first and second immunoglobulin Fc domains can bind to another Fc domain or an Fcγ receptor expressed on a T cell, and can cluster four or more T cells bound to the multispecific molecule.

[0022] In another aspect, the present invention provides a pharmaceutical composition comprising the multispecific molecule described above or discussed herein, and a pharmaceutically acceptable carrier or diluent. In another aspect, the present invention provides a multimer comprising two or more multispecific molecules of the present invention, and a multispecific carrier molecule comprising a plurality of first binding molecules and a plurality of second binding molecules, wherein each first binding molecule is a polypeptide comprising a single-chain peptide-major histocompatibility complex (pMHC) fusion, and each second binding molecule is a polypeptide comprising an antigen-binding domain that specifically binds to a T cell surface molecule.

[0023] In some embodiments, multimerization of the multispecific molecules of the invention can be achieved using, for example, IgG, streptavidin, streptactin, polysaccharides, dextran, micelles, liposomes, cells, polymers, beads, and other types of solid supports, or small organic molecules that carry reactive groups capable of binding to the multispecific molecules of the invention and other molecules, or that carry chemical motifs. Multimerization can involve the use of one or more carriers, and / or one or more scaffolds, and / or one or more linkers that connect the carrier to the scaffold, carrier to carrier, and scaffold to scaffold. In one embodiment, the scaffold can consist of an organic molecule carrying a reactive group that can react with a reactive group on the multimerized multispecific molecule. Non-limiting examples of useful small organic molecules include, for example, molecules having a cyclic structure such as a functionalized cycloalkane or a functionalized aromatic ring structure.

[0024] In some embodiments, multimerization of the multispecific molecule can involve covalent or non-covalent interactions. Non-limiting examples of covalent bonds include, for example, acylation such as amide formation, pyrazolone formation, isoxazolone formation, alkylation, vinylation, disulfide formation, addition to a carbon-hetero multiple bond (e.g., alkene formation by reaction of a phosphonate with an aldehyde or ketone, alkylation of arenes / heteroarenes by reaction with an arylboronate or enol ether), nucleophilic substitution using activation of a nucleophile (e.g., alkylation of an aliphatic halide or tosylate with an enol ether or enamine), and cycloaddition. Non-limiting examples of molecule pairs and molecules capable of forming non-covalent interactions include, for example, streptavidin / biotin, avidin / biotin, antibody / antigen, DNA / DNA, DNA / PNA, DNA / RNA, PNA / PNA, LNA / DNA, leucine zipper, e.g., Fos / Jun, IgG dimer protein, IgM multivalent protein, acid / base coiled-coil helix, chelate / metal ion binding chelate, streptavidin (SA) and avidin and their derivatives, biotin, immunoglobulins, antibodies (monoclonal, polyclonal, and recombinant), antibody fragments and their derivatives, leucine zipper domain of AP-1 (jun and fos), hexa-his (SEQ ID NO: 29) (metal chelate moiety), hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), Strep tag, cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase C epitope, VSV epitope, carbohydrates, lipids, lectins that mediate binding to diverse compounds including proteins, e.g., Con A (jack bean) or WGA (wheat germ agglutinin) and tenascin, or protein A or G (antibody affinity). Combinations of such binding entities can also be used.

[0025] When multimerization involves the use of a solid surface (e.g., beads or plates, including glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids), such a surface can be coated covalently or non-covalently with a multimer or a single multispecific molecule via a non-cleavable or cleavable linker. As an example, the surface can be coated with streptavidin monomers that then associate with biotinylated multispecific molecules of the invention, or the surface can be coated with streptavidin tetramers, each of which associates with 0, 1, 2, 3, or 4 biotinylated multispecific molecules of the invention, or the surface can be coated with a molecule-dextranmer (e.g., a divinylsulfone-activated dextran backbone) where the reactive groups of the molecule-dextranmer react with nucleophilic groups on the surface to form a covalent bond between the dextran of the dextranmer and the surface. The surface can further include a flexible or rigid water-soluble linker that allows the immobilized multispecific molecule to interact efficiently with T cells. In yet another embodiment, the linker is cleavable and allows release of the multispecific molecule from the surface. Non-limiting examples of linker molecules that can be used in the present invention include calmodulin-binding peptide (CBP), 6×HIS (SEQ ID NO: 29), protein A, protein G, biotin, avidin, streptavidin, Strep-tag, cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, GST-tagged protein, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase C epitope, VSV epitope.

[0026] In some embodiments, the multimerized multispecific molecular species are chemically cross-linked multispecific molecules of the invention (e.g., cross-linked to a dendrimer). For example, the multispecific molecules of the invention can be genetically modified by including a sequence encoding an amino acid residue having a chemically reactive side chain such as Cys or His. Such amino acids having chemically reactive side chains can be placed at various positions of the multispecific molecule (e.g., distal to the presentation peptide and the binding domain of the pMHC complex). Suitable side chains can be used to chemically link two or more multispecific molecules of the invention to a suitable dendrimer particle to produce a multimerized molecule. Dendrimers are synthetic chemical polymers that can have any one of several different functional groups on their surface [D. Tomalia, Aldrichimica Acta, 26:91:101 (1993)]. Non-limiting examples of useful dendrimers include, for example, polyamidoamine, polyamidoalcohol, polyalkyleneimine, polyalkylene, polyether, polythioether, polyphosphonium, polysiloxane, polyamide, and polyaryl polymers.

[0027] For further disclosure of means for multimerization, see, for example, U.S. Patent Nos. 8,268,964, 7,074,905, and U.S. Patent Application Publication No. 2005 / 0003431.

[0028] In another aspect, the present invention provides a multispecific carrier comprising a plurality of first molecules for engaging a T cell receptor and a plurality of second molecules for engaging a T cell surface molecule(s) to modulate the activity of T cells, wherein each first molecule is a polypeptide comprising a peptide (p) presented in the context of a major histocompatibility complex (MHC) molecule, or a fragment, variant, or derivative thereof (pMHC complex), each second molecule comprises a domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR, and the first and second molecules can be separate molecules that are not bound together to form a multispecific molecule. In certain embodiments, each first molecule of the plurality of first molecules is the same. In certain embodiments, each first molecule of the plurality of first molecules comprises at least 2, 3, 4, 5, or 6 different types of first molecules. In certain embodiments, each second molecule of the plurality of second molecules is the same. In certain embodiments, each second molecule of the plurality of second molecules comprises at least 2, 3, 4, 5, or 6 different types of first molecules.

[0029] In some embodiments of the multispecific carrier, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is an antigen-binding domain. In some embodiments, the antigen-binding domain is a monovalent antigen-binding domain. In certain embodiments, the antigen-binding domain binds to a T cell co-regulatory molecule (e.g., CD28 or CTLA4) on the surface of a cell expressing a TCR. In some embodiments, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is a small molecule, protein, fusion protein, peptide, aptamer, avimer, or a derivative or fragment thereof. In certain embodiments, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is a T cell co-regulatory molecule that regulates the activity of T cells when it binds to a molecule on the surface of a cell expressing a TCR (e.g., a co-stimulatory molecule such as CD80 or CD86, or an inhibitory molecule such as PD-L1 or B7-H3). In certain embodiments, the domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR is a protein, or a derivative or fragment thereof, and the protein is an adhesion molecule (e.g., ICAM).

[0030] In some embodiments of the multispecific carrier, the MHC comprises (i) a class I MHC polypeptide, or a fragment thereof (e.g., the peptide binding groove), variant, or derivative, and optionally, (ii) a β2-microglobulin polypeptide, or a fragment, variant, or derivative thereof. In some embodiments, the MHC comprises the α1 and α2 domains of a class I MHC polypeptide, or fragments, variants, or derivatives thereof. In some embodiments, the MHC comprises the α1, α2, and / or α3 domains of a class I MHC polypeptide, or fragments, variants, or derivatives thereof. In some embodiments, the MHC comprises a class I MHC polypeptide (or a portion thereof) and β2-microglobulin. In some embodiments, the class I MHC (or a portion thereof) and the β2-microglobulin polypeptide are linked, for example, by a peptide linker. In some embodiments, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC polypeptide is a mouse class I MHC polypeptide selected from the group consisting of H-2K, H-2D, H-2L, H-2Q, H-2M, and H-2T.

[0031] In some embodiments of the multispecific carrier, the MHC comprises a class II MHC polypeptide, or a fragment, variant, or derivative thereof. In some embodiments, the MHC comprises the α and β polypeptides of the class II MHC complex, or fragments, variants, or derivatives thereof, respectively. In some embodiments, the MHC comprises the α1 and β1 domains of the α and β polypeptides of the class II MHC complex, respectively, or fragments, variants, or derivatives thereof. In some embodiments, the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) are linked by a peptide linker. In one particular embodiment, the MHC comprises the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) of a human class II MHC complex selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO. In another particular embodiment, the MHC comprises the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) of a mouse H-2A or H-2E class II MHC complex.

[0032] In some embodiments of the multispecific carrier, the T cell surface molecule to which the domain specifically binding to a molecule expressed on the surface of a cell expressing a TCR specifically binds is a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM.2B4, CD226, TIM1, TIM2, and CD2.

[0033] In some embodiments of the multispecific molecule, the molecule T cell surface molecule to which the domain specifically binding to a molecule expressed on the surface of a cell expressing a TCR specifically binds is a T cell inhibitory molecule. In some embodiments, the inhibitory molecule is selected from the group consisting of CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, MHC-1, B7-1, and B7-H1.

[0034] In various embodiments of the multispecific carrier, the peptide consists of about 5 to about 40 amino acid residues, about 6 to about 30 amino acid residues, about 8 to about 20 amino acid residues, or about 9, 10, or 11 amino acid residues.

[0035] In some embodiments, the peptide is derived from a viral antigen or a bacterial antigen. In some embodiments, the viral antigen is derived from a virus selected from the group consisting of adenovirus, astrovirus, chikungunya, cytomegalo, dengue, Ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, Lassa, measles, metapneumovirus, mumps, norovirus, Oropouche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, Usutu, vaccine, varicella, West Nile, yellow fever, and Zika, or the bacterial antigen is derived from a bacteria selected from the group consisting of methicillin-resistant Staphylococcus Aureus (MRSA), Clostridium Difficile, carbapenem-resistant Enterobacteriaceae, drug-resistant Neisseria Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, extended-spectrum beta-lactamase-producing bacteria, vancomycin-resistant enterococci, multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, vancomycin-resistant Staphylococcus Aureus, erythromycin-resistant group A Streptococcus, and clindamycin-resistant group B Streptococcus.

[0036] In some embodiments, the peptide is derived from a tumor-associated antigen. In some embodiments, the tumor-associated antigen is adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, ALK, BAGE protein (e.g., BAGE-1), BIRC5 (survivin), BIRC7, beta-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CAGE-1-8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen (“ETA”), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE protein (e.g., GAGE-1-8), GD2, GD3, GloboH, glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, LMP2,Selected from the group consisting of M-CSF, MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, -A9, -A10, -A12, -C1, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE protein (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, seselinin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvel antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1. In some embodiments, the peptide is a neoantigen. In some embodiments, the peptide is a tumor-specific antigen.,

[0037] In some embodiments, the peptide is derived from an antigen associated with an autoimmune disorder. In some embodiments, the antigen is selected from the group consisting of gliadin (celiac disease, e.g., (i) an α-gliadin fragment corresponding to amino acids 57-73, or (ii) a γ-gliadin fragment corresponding to amino acids 139-153, or (iii) an ω-gliadin fragment corresponding to amino acids 102-118), GAD 65, IA-2 and insulin B chain (in the case of type 1 diabetes), glatiramer acetate (GA) (in the case of multiple sclerosis), acetylcholine receptor (AChR) (in the case of myasthenia gravis), p205, insulin, thyroid stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)).

[0038] In certain embodiments of the multispecific carrier, the carrier is a cell. In certain embodiments, the cell is a cell line such as, but not limited to, CHO, HEK293 (embryonic kidney), HMEC (epithelial), HIVE-55 (endothelial), HIVS-125 (smooth muscle), and tumor cells (e.g., HN5). In certain embodiments, the cell is an antigen-presenting cell. In certain embodiments, the antigen-presenting cell is a macrophage or a dendritic cell. In certain embodiments, the cell is a B cell.

[0039] In certain embodiments of the multispecific carrier, the multispecific carrier is a virus-like particle. In another aspect, the present invention provides a pharmaceutical composition comprising the multispecific carrier described above or discussed herein and a pharmaceutically acceptable carrier.

[0040] In another aspect, the present invention provides a set or series of nucleic acid molecules encoding (i) a first transmembrane polypeptide and (ii) a second transmembrane polypeptide, wherein the first transmembrane polypeptide comprises a first extracellular domain comprising a single-chain peptide-major histocompatibility complex (pMHC) fusion and a first transmembrane domain, and the second transmembrane polypeptide comprises a second extracellular domain comprising an antigen-binding domain that specifically binds to a T cell surface molecule and a second transmembrane domain. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), and the scFv is connected to the second extracellular domain either directly or via a linker.

[0041] In some embodiments of the nucleic acid molecule, the pMHC complex comprises a class I MHC polypeptide. In some embodiments, the pMHC complex comprises a peptide, a β2-microglobulin peptide, and a class I alpha chain domain or a fragment thereof. In some embodiments, the pMHC complex comprises, from N-terminus to C-terminus, a peptide, a β2-microglobulin peptide, and a class I alpha chain domain or a fragment thereof. In some embodiments, the pMHC complex comprises, from N-terminus to C-terminus, a peptide, an optional first linker, a β2-microglobulin peptide, an optional second linker, and class I MHC alpha chain domains 1, 2, and / or 3.

[0042] In some embodiments of the nucleic acid molecule, the pMHC complex comprises a class II MHC polypeptide. In some embodiments, the pMHC complex comprises a peptide, a class II MHC alpha chain domain or a fragment thereof, and a class II MHC beta chain domain or a fragment thereof. In some embodiments, the MHC class II alpha chain consists of the extracellular domain of the class II alpha chain. In some embodiments, the MHC class II beta chain consists of the extracellular domain of the class II beta chain. In some embodiments, the pMHC complex comprises, from the N-terminus to the C-terminus, a peptide, the extracellular domain of the class II MHC alpha chain, and the extracellular domain of the class II MHC beta chain. In some embodiments, the pMHC complex comprises, from the N-terminus to the C-terminus, a peptide, an optional first linker, class II MHC alpha chain domains 1 and 2, an optional second linker, and class II MHC beta chain domains 1 and 2.

[0043] In some embodiments of the nucleic acid molecule, the T cell surface molecule is a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM.2B4, CD226, TIM1, TIM2, and CD2.

[0044] In some embodiments of the nucleic acid molecule, the T cell surface molecule is a T cell inhibitory molecule. In some embodiments, the inhibitory molecule is selected from the group consisting of CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, MHC-1, B7-1, and B7-H1.

[0045] In various embodiments of the nucleic acid molecule, the peptide consists of about 5 to about 40 amino acid residues, about 6 to about 30 amino acid residues, about 8 to about 20 amino acid residues, or about 9, 10, or 11 amino acid residues.

[0046] In some embodiments of the nucleic acid molecule, the peptide is derived from a viral antigen or a bacterial antigen. In some embodiments, the viral antigen is derived from a virus selected from the group consisting of adenovirus, astrovirus, chikungunya, cytomegalo, dengue, Ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, Lassa, measles, metapneumovirus, mumps, norovirus, Oropouche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, Usutu, vaccine, varicella, West Nile, yellow fever, and Zika, or the bacterial antigen is derived from a bacteria selected from the group consisting of methicillin-resistant Staphylococcus Aureus (MRSA), Clostridium Difficile, carbapenem-resistant Enterobacteriaceae, drug-resistant Neisseria Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, extended-spectrum beta-lactamase-producing bacteria, vancomycin-resistant enterococci, multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, vancomycin-resistant Staphylococcus Aureus, erythromycin-resistant group A Streptococcus, and clindamycin-resistant group B Streptococcus.

[0047] In some embodiments of the nucleic acid molecule, the peptide is derived from a tumor-associated antigen. In some embodiments, the tumor-associated antigen is adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, ALK, BAGE protein (e.g., BAGE-1), BIRC5 (survivin), BIRC7, beta-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CAGE-1-8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen (“ETA”), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE protein (e.g., GAGE-1-8), GD2, GD3, GloboH, glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin,Selected from the group consisting of LMP2, M-CSF, MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, -A9, -A10, -A12, -C1, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE protein (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, cessernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-β, TMPRSS2, Thompson-Newell antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1. In some embodiments, the peptide is a neoantigen. In some embodiments, the peptide is a tumor-specific antigen.,

[0048] In some embodiments of the nucleic acid molecule, the peptide is derived from an antigen associated with an autoimmune disorder. In some embodiments, the antigen is gliadin (celiac disease, e.g., (i) an α-gliadin fragment corresponding to amino acids 57-73, or (ii) a γ-gliadin fragment corresponding to amino acids 139-153, or (iii) an ω-gliadin fragment corresponding to amino acids 102-118), GAD 65, IA-2, and insulin B chain (in the case of type 1 diabetes), glatiramer acetate (GA) (in the case of multiple sclerosis), acetylcholine receptor (AChR) (in the case of myasthenia gravis), p205, insulin, thyroid stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)), and is selected from the group consisting of.

[0049] In another aspect, the present invention provides a vector(s) comprising any of the nucleic acid molecules described above or herein. In some embodiments, the vector(s) is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the vector(s) is a lentiviral vector.

[0050] In another aspect, the present invention provides a method of modulating T cell activity in a subject, comprising administering to the subject a multispecific molecule or a multispecific carrier described above or herein, whereby the administration of the multispecific molecule or the multispecific carrier modulates the activation, proliferation, and / or survival of T cells.

[0051] In some embodiments of the method, the pMHC complex comprises a class I MHC polypeptide, and the multispecific molecule or the multispecific carrier molecule is CD8 + Modulates the activation, proliferation, and / or survival of T cells.

[0052] In some embodiments of the method, the T cell surface molecule is a T cell costimulatory molecule, whereby the administration of the multispecific molecule or the multispecific carrier molecule is CD8+ Stimulates the activation, proliferation, and / or survival of T cells. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, and CD226.

[0053] In some embodiments of the method, the T cell surface molecule is a T cell inhibitory molecule, whereby administration of the multispecific molecule or multispecific carrier molecule is CD8 + Inhibits the activation, proliferation, and / or survival of T cells. In some embodiments, the inhibitory molecule is selected from the group consisting of CTLA4, PD1, BTLA, TIM3, CD160, LAG3, LAIR1, B7-1, and B7-H1. In some embodiments, CD8 + Inhibition of T cell activation, proliferation, and / or survival results in induction of T cell anergy or T cell death.

[0054] In some embodiments of the method, the pMHC complex comprises a peptide and a class II MHC polypeptide, and the multispecific molecule or multispecific carrier molecule is CD4 + Regulates the activation, proliferation, and / or survival of T cells.

[0055] In some embodiments of the method, the T cell surface molecule is a T cell costimulatory molecule, whereby administration of the multispecific molecule or multispecific carrier molecule is CD4 + Stimulates the activation, proliferation, and / or survival of T cells. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, and TIM2.

[0056] In some embodiments of the method, the T cell surface molecule is a T cell inhibitory molecule, whereby administration of the multispecific molecule or multispecific carrier molecule is CD4 +Inhibits the activation, proliferation, and / or survival of T cells. In some embodiments, the inhibitory molecule is selected from the group consisting of CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, and B7-H1. In some embodiments, CD4 + Inhibition of the activation, proliferation, and / or survival of T cells results in the induction of T cell anergy or T cell death.

[0057] In another aspect, the present invention provides a method for treating an infectious disease in a subject, comprising administering to the subject a multispecific molecule or a multispecific carrier molecule comprising a class I MHC protein as described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM.2B4, CD226, TIM1, TIM2, and CD2.

[0058] In another aspect, the present invention provides a method for treating an infectious disease in a subject, comprising administering to the subject a multispecific molecule or a multispecific carrier molecule comprising a class II MHC protein as described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM.2B4, CD226, TIM1, TIM2, and CD2.

[0059] In various embodiments of the method for treating an infectious disease, the pathogen causing the infectious disease is a virus and the peptide is a fragment of a viral protein. In some embodiments, the virus can be any one of the viruses described above or discussed herein.

[0060] In another aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject a multispecific molecule or a multispecific carrier molecule comprising a class I MHC protein as described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM.2B4, CD226, TIM1, TIM2, and CD2.

[0061] In another aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject a multispecific molecule or a multispecific carrier molecule comprising a class II MHC protein as described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM.2B4, CD226, TIM1, TIM2, and CD2.

[0062] In another aspect, the invention provides a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject a multispecific molecule or a multispecific carrier molecule comprising a class I MHC protein as described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CTLA4, PD1, BTLA, TIM3, CD160, LAG3, LAIR1, B7-1, and B7-H1.

[0063] In another aspect, the present invention provides a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject a multispecific molecule or a multispecific carrier molecule comprising the class II MHC protein described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, and B7-H1.

[0064] In another aspect, the present invention provides a method of treating an infectious disease in a subject, the method comprising administering to the subject the engineered cell described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2. In some embodiments, the pathogen causing the infectious disease is a virus and the peptide is a fragment of a viral protein.

[0065] In another aspect, the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject the engineered cell described above or discussed herein, wherein the antigen-binding domain specifically binds to a T cell costimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2.

[0066] In another aspect, the invention provides a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject the engineered cells described above or herein, wherein the antigen-binding domain specifically binds to a T cell co-stimulatory molecule. In some embodiments, the antigen-binding domain specifically binds to CTLA4, PD1, BTLA, TIM3, CD160, LAG3, LAIR1, B7-1, or B7-H1.

[0067] In another aspect, the invention provides a method of modulating the activity of T cells ex vivo, the method comprising obtaining CD8 + T cells from a subject and culturing the CD8 + T cells with a plurality of multispecific molecules or multispecific carrier molecules under conditions and for a period sufficient to modulate the activity of the CD8 + T cells, wherein each of the plurality of multispecific molecules or multispecific carrier molecules comprises a class I MHC protein described above or herein, whereby the activity of CD8 + T cells having a T cell receptor (TCR) specific for a peptide is modulated.

[0068] In some embodiments of the method, the T cell surface molecule is a T cell co-stimulatory molecule, and the CD8 + T cells having a TCR specific for a peptide are activated and / or proliferate. In some embodiments, the T cell co-stimulatory molecule is selected from the group consisting of CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, and CD226.

[0069] In some embodiments of the method, the T cell surface molecule is a T cell inhibitory molecule, and the CD8 + T cells having a TCR specific for a peptide are inactivated. In some embodiments, the inhibitory molecule is selected from the group consisting of CTLA4, PD1, BTLA, TIM3, CD160, LAG3, LAIR1, B7-1, and B7-H1.

[0070] In some embodiments of the method, CD8 + T cells are tumor-infiltrating lymphocytes. In another aspect, the present invention is a method of modulating the activity of T cells ex vivo, comprising obtaining CD4 + T cells from a subject and culturing the CD4 + T cells with a plurality of multispecific molecules or multispecific carrier molecules under conditions and for a period sufficient to modulate the activity of the CD4 + T cells, wherein each of the plurality of multispecific molecules or multispecific carrier molecules comprises a class II MHC protein as described above or discussed herein, whereby a CD4 + T cell having a T cell receptor (TCR) specific for a peptide has its activity modulated. A method is provided.

[0071] In some embodiments of the method, the T cell surface molecule is a T cell costimulatory molecule, and the CD4 + T cell having a TCR specific for a peptide is activated and / or proliferates. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, and TIM2.

[0072] In some embodiments of the method, the T cell surface molecule is a T cell inhibitory molecule, and the CD4 + T cell having a TCR specific for a peptide is inactivated. In some embodiments, the inhibitory molecule is selected from the group consisting of CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, and B7-H1.

[0073] In various embodiments of the ex vivo methods discussed above, multiple multispecific molecules are bound to a scaffold in a clustered arrangement. In some embodiments, the multiple multispecific molecules are clustered via one or more linkers. In some embodiments, the one or more linkers are multivalent antibodies that bind to the first and / or second Fc domains of the multispecific molecule. In some embodiments, the ratio of multivalent antibody to multispecific molecule is about 1:1 in culture.

[0074] In another aspect, the invention provides a method of treating or ameliorating a disease or disorder in which T cells modulated by the ex vivo methods discussed above are reintroduced into a subject. In some embodiments, the disease or disorder is an infectious disease, cancer, or autoimmune disorder.

[0075] In another aspect, the invention provides a method of treating or ameliorating an infectious disease (e.g., viral or bacterial infection) or cancer in a subject, comprising: (a) obtaining CD8 + T cells from the subject; (b) culturing the CD8 + T cells with a plurality of multispecific molecules or multispecific carrier molecules under conditions and for a period sufficient to activate and / or expand the CD8 + T cells, wherein each of the plurality of multispecific molecules or multispecific carrier molecules comprises a class I MHC protein as discussed above or herein; and (c) reintroducing the activated and / or expanded CD8 + T cells having a TCR specific for a peptide into the subject, thereby treating or ameliorating the viral infection or cancer.

[0076] In another aspect, the invention provides a method of treating or ameliorating an autoimmune disorder in a subject, comprising: (a) obtaining CD4 + T cells from the subject; (b) culturing the CD4 + T cells with a plurality of multispecific molecules or multispecific carrier molecules under conditions and for a period sufficient to inactivate the CD4 +Culturing T cells, wherein each of a plurality of multispecific molecules or multispecific carrier molecules comprises a class II MHC protein as described above or discussed herein; and (c) inactivated CD4 having a TCR specific for a peptide + Reintroducing the T cells into a subject, thereby treating or ameliorating an autoimmune disorder. A method is provided that includes these steps.

[0077] In another aspect, the invention provides the use of a multispecific molecule as described above or discussed herein for the treatment and / or prevention of an infectious disease (e.g., a viral infectious disease), cancer, and / or an autoimmune disorder. In other aspects, the invention provides the use of a multispecific molecule, a multispecific carrier molecule, or an engineered cell as described above or discussed herein in the manufacture of a medicament for the treatment and / or prevention of an infectious disease, cancer, and / or an autoimmune disorder.

[0078] Other embodiments will be apparent from a review of the detailed description and the accompanying drawings. Any reference to a multispecific molecule above or herein also refers to a bispecific molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0079]

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Modes for Carrying Out the Invention

[0080] Before the present invention is described, it is to be understood that the present invention is not limited to such methods and conditions since the specific methods and experimental conditions described may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention is limited only by the appended claims and the full scope of equivalents to which such claims are entitled.

[0081] 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. As used herein, the terms "about" or "approximately" include within the statistically significant range of a value. Such a range can be within the digit of a given value or range, preferably within 50% of a given value or range, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The allowable variations encompassed by the term "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art. For example, as used herein, the expression "about 100" includes 99 and 101, and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0082] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and this description includes both the case where the event or circumstance occurs and the case where it does not.

[0083] The term "and / or" refers to any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0084] The term "or" means any one member of a particular list and also includes any combination of members of that list. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading the present disclosure.

[0085] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those of ordinary skill in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims along with the full scope of equivalents to which the appended claims are entitled.

[0086] All patents, applications, and non-patent publications mentioned herein are hereby incorporated by reference in their entirety. Definitions All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human forms of each protein, polypeptide, or protein fragment unless specifically identified as being from non-human species. For example, the expression "CD28" means human CD28 unless specified as being derived from non-human species, such as "mouse CD28," "monkey CD28," etc.

[0087] The term "T cell," as used herein, refers to all types of immune cells that express CD3, including CD4+ cells (helper T cells), CD8+ cells (cytotoxic T cells), regulatory T cells (Tregs), and tumor-infiltrating lymphocytes.

[0088] The term "antigen" refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) that, when introduced into a host, animal, or human (either directly or, for example, upon expression such as in a DNA vaccine), has the ability to be recognized by the host's immune system and to induce an immune response.

[0089] As described herein, a T cell receptor (TCR) recognizes a peptide presented in the context of a major histocompatibility complex (MHC) molecule. The peptide MHC (pMHC) complex is recognized by the TCR, and the peptide (epitope) and TCR idiotype provide the specificity of the interaction. Thus, the term "antigen" encompasses a peptide presented in the context of an MHC molecule. Peptides displayed on MHC molecules may also be referred to as "epitopes" or "antigenic determinants". As used herein, the terms "peptide", "antigenic determinant", and "epitope" refer not only to those naturally presented by antigen-presenting cells (APCs), but also to any desired peptide, for example, if appropriately presented to cells of the immune system and recognized by immune cells. For example, peptides having an artificially prepared amino acid sequence may also be used as epitopes.

[0090] The terms "major histocompatibility complex", "MHC", and "MHC molecule" include naturally occurring MHC molecules, as well as the individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2-microglobulin, MHC class II α chain, MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1 and / or α2 subunits of the MHC class II α chain, β1 and / or β2 subunits of the MHC class II β chain), and their fragments, variants, and various derivatives (including fusion proteins), and such fragments, variants, and derivatives retain the ability to display antigenic peptides for recognition by a TCR, e.g., an antigen-specific TCR. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy α chain that can accommodate peptides of about 8-10 amino acids in length. Despite the fact that both classes of MHC bind to the core of about 9 amino acids within the peptide, the MHC class II peptide-binding groove (the α1 domain of the class II MHC polypeptide that associates with the β1 domain of the class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II usually vary in length between 13 and 17 amino acids, although shorter or longer lengths are not uncommon. As a result, the peptide may shift within the MHC class II peptide-binding groove, altering which 9mer is directly positioned within the groove at any given time. Conventional designations of specific MHC variants are used herein. For example, HLA-B17 refers to the human leukocyte antigen from gene locus (known as the locus) number 17 of the B gene group (and thus class I MHC), and the gene HLA-DR11 refers to the human leukocyte antigen encoded by the gene from gene locus number 11 of the DR region (and thus class II MHC).

[0091] The antigenic determinants contained in the multispecific molecules described herein can include any peptide that can bind to an MHC protein, preferably specifically, in such a way that the pMHC complex can bind to a TCR. In certain embodiments, such binding induces a T cell response. Examples include peptides produced by hydrolysis, and most typically, randomly generated peptides, specifically designed peptides, and synthetically produced peptides including peptides in which at least some of the amino acid positions are conserved between several peptides and the remaining positions are random.

[0092] In nature, peptides produced by hydrolysis are hydrolyzed before the antigen binds to the MHC protein. Class I MHC typically presents peptides derived from proteins actively synthesized in the cytoplasm of the cell. In contrast, class II MHC typically presents peptides derived from either exogenous proteins that enter the endocytic pathway of the cell or proteins synthesized in the ER. Intracellular trafficking enables the peptide to associate with the MHC protein.

[0093] Binding of the peptide to the MHC peptide-binding groove can control the spatial arrangement of the MHC and / or peptide amino acid residues recognized by the TCR. Such spatial control is partly due to hydrogen bonds formed between the peptide and the MHC protein. Based on knowledge of how peptides bind to various MHCs, the major MHC anchor amino acids and surface-exposed amino acids that differ between different peptides can be determined.

[0094] Preferably, the length of the MHC-binding peptide is from about 5 to about 40 amino acid residues, more preferably from about 6 to about 30 amino acid residues, even more preferably from about 8 to about 20 amino acid residues, even more preferably from about 9 to about 11 amino acid residues, and includes peptides of any size from 5 to 40 amino acids in integer units (i.e., 5, 6, 7, 8, 9...40). Of course, MHC class II-binding peptides vary from about 9 to 40 amino acids, and in almost all cases, the peptide can be shortened to a 9 to 11 amino acid core without loss of MHC-binding activity or T cell recognition.

[0095] Peptides include peptides containing at least a portion of a protein selected from the group consisting of self-proteins associated with autoimmune disorders, proteins of infectious disease agents, and tumor-associated proteins, e.g., an antigenic determinant.

[0096] Non-limiting examples of self-proteins associated with autoimmune disorders include, for example, gliadin (celiac disease, e.g., (i) an α-gliadin fragment corresponding to amino acids 57 - 73, or (ii) a γ-gliadin fragment corresponding to amino acids 139 - 153, or (iii) an ω-gliadin fragment corresponding to amino acids 102 - 118), GAD 65, IA-2 and insulin B chain (in the case of type 1 diabetes), glatiramer acetate (GA) (in the case of multiple sclerosis), acetylcholine receptor (AChR) (in the case of myasthenia gravis), p205, insulin, thyroid stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)).

[0097] In certain embodiments, the antigen comprises a peptide (e.g., an antigenic determinant of a protein) that is a target of autoreactive T cells involved in celiac disease. For example, the peptide may be derived from and include a portion of a gluten peptide such as α-gliadin, γ-gliadin, and / or glutenin. In certain embodiments, the epitope is derived from α-gliadin (33mer (57-89) and its truncated forms, 25mer (64-89), 18mer (71-89), 17mer (57-73), 13mer (57-68), and glia-20), γ-gliadin (DQ2-γ-I, DQ2-γ-II, DQ2-γ-III, DQ2-γ-IV, and DQ2-γ-V, 14mer-1 (105-118) and 14mer-2 (173-186)), glutenin (Glt-19-39 and glt-156 (42-56)), and / or glu-5. In certain embodiments, the antigen-derived peptide may include α-gliadin (57-73), γ-gliadin (139-153), and / or ω-gliadin (102-118). See, for example, Camarca et al., Endocrine, Metabolic & Immune Disorders - Drug Targets, 12:207-219 (2012), Camarca et al., J. Immunol., 182(7):4158-4166 (2009).

[0098] In certain embodiments, the peptide comprises an antigenic determinant of a protein that is a target of autoreactive T cells involved in psoriasis, such as BV3 and / or BV13S1. In certain embodiments, the peptide comprises an antigenic determinant of a protein that is a target of autoreactive T cells involved in multiple sclerosis, such as BV5S2, BV6S5, and / or BV13SI.

[0099] In certain embodiments, the peptide comprises an antigenic determinant of a protein that is a target of autoreactive T cells involved in rheumatoid arthritis, such as BV3, BV14, and / or BV17.

[0100] Non-limiting examples of viral proteins from which the peptides used in the multispecific molecules described herein may be derived include LCMV gp33, CMV pp65, HIV gag, EBV BMLF1, and antigens derived from influenza virus (e.g., surface glycoprotein hemagglutinin (HA) and neuraminidase (NA)), immunodeficiency virus (e.g., human immunodeficiency virus antigens (HIV) such as gp120, gp160, p18 antigen Gag p17 / p24, Tat, Pol, Nef, and Env), herpes virus (e.g., glycoproteins derived from herpes simplex virus (HSV), Marek's disease virus, cytomegalovirus (CMV), or Epstein-Barr virus), hepatitis virus (e.g., hepatitis B surface antigen (HBsAg)), papillomavirus, Rous-related virus (e.g., RAV-1env), infectious bronchitis virus (e.g., matrix and / or prepro), flavivirus (e.g., Japanese encephalitis virus (JEV) antigen, yellow fever antigen, or dengue virus antigen), morbillivirus (e.g., canine distemper virus antigen, measles antigen, or rinderpest antigens such as HA or F), rabies (e.g., rabies glycoprotein G), parvovirus (e.g., canine parvovirus antigen), poxvirus (e.g., ectromelia antigen, canarypox virus antigen, or fowlpox virus antigen), varicella virus (varicella zoster antigen), infectious bursal disease virus (e.g., VP2, VP3, or VP4), Hantaan virus, and mumps virus.

[0101] Bacterial peptides that can be used in the multispecific molecules described herein, for example, non-limiting examples of bacterial proteins that can be sources of antigenic determinants include lipopolysaccharides isolated from Gram-negative bacterial cell walls, and staphylococcus-specific, streptococcus-specific, pneumococcus-specific (see, for example, PspA, PCT Publication No. 92 / 14488), Neisseria gonorrhea-specific, Borrelia-specific (e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdorferi, Borrelia afzelli, and Borrelia garinii (see, for example, U.S. Patent No. 5,523,089, PCT Publication Nos. 90 / 04411, 91 / 09870, 93 / 04175, 96 / 06165, 93 / 08306, PCT / US92 / 08697, Bergstrom et al., Mol. Microbiol., 1999; 3:479486, Johnson et al., Infect. and Immun. 1992; 60:1845-1853, Johnson et al., Vaccine 1995; 13:1086-1094, The Sixth International Conference on Lyme Borreliosis: Progress on the Development of Lyme Disease Vaccine, Vaccine 1995; 13:133-135]), and pseudomonas-specific proteins or peptides.Non-limiting additional examples of bacterial antigens include, for example, Neisseria gonorrhea, Mycobacterium tuberculosis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, and Actinobaccilus seminis.

[0102] Non-limiting examples of malaria-specific proteins from which antigenic determinants can be isolated include circumsporozoite (CS) protein, thrombospondin-related adhesive (anonymous) protein (TRAP), also known as sporozoite surface protein 2 (SSP2), LSA I, hsp70, SALSA, STARP, Hep17, MSA, RAP-1, RAP-2.

[0103] Non-limiting examples of fungal proteins from which antigenic determinants can be isolated include those isolated from Candida (e.g., MP65 from Candida albicans), trichophyton, and ptyrosporum.

[0104] Non-limiting examples of tumor-associated proteins from which antigenic determinants can be isolated include, for example, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, ALK, BAGE proteins (e.g., BAGE-1), BIRC5 (survivin), BIRC7, beta-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CAGE-1-8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen (“ETA”), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE proteins (e.g., GAGE-1-8), GD2, GD3, GloboH, glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, LMP2, M-CSF,MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, -A9, -A10, -A12, -C1, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE protein (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, seselin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1. In some embodiments, the peptide is a neoantigen. In some embodiments, the peptide is a tumor-specific antigen.,

[0105] The term "neoantigen" or "neoantigenic" refers to a class of tumor antigens that result from tumor-specific mutation(s) that alter one or more amino acids compared to the parental (i.e., genomically encoded) protein. For example, a neoantigen may be a tumor-associated neoantigen, and the term "tumor-associated neoantigen" includes peptides or proteins that contain amino acid modifications resulting from tumor-specific mutations.

[0106] In some embodiments, the multispecific molecules described herein are exposed to a library of synthetically produced peptides in order to identify the epitopes recognized by specific T cells. Such peptide libraries include, for example, peptide libraries produced by PCR (including those by introducing random mutations at various positions of the template peptide). The peptide library can contain up to 209 or 2×1011 members, or only a few hundred to a few thousand members, depending on the knowledge of the peptide-binding properties of a given MHC. Generally, since 4-5 amino acids are involved in MHC binding and cannot directly contact the TCR, prior knowledge of the nature of these amino acids means that only about 5-7 amino acids in the peptide are required, and thus a library of typically 106-109 members is sufficient. In addition, in some embodiments, T cell recognition is dominated by only a few amino acids in the core of the peptide, and in these cases, a library with only a few hundred to a few thousand members may be sufficient to identify functional peptide-MHC complexes.

[0107] Extensive knowledge regarding the binding of peptides to MHC complexes is available to those skilled in the art, so those skilled in the art can design MHC-groove binding peptides that vary in a range less than all available positions for a given MHC complex. For example, MHCBN is a comprehensive database of major histocompatibility complex (MHC) binding and non-binding peptides compiled from published literature and existing databases. The latest version of the database has 19,777 entries, including 17,129 MHC binding factors and 2,648 MHC non-binding factors for over 400 MHCs. The database has (a) the source protein of the peptide and (b) the sequence and structural data of the MHC. MHCBN has many web tools such as: (i) mapping of peptides on a query sequence, (ii) searching on any field, (iii) creation of a dataset, and (iv) online data submission (Bioinformatics 2003 Mar.22;19(5):665-6).

[0108] In one particular embodiment, a library of candidate peptides is created by genetically engineering the library using polymerase chain reaction (PCR) or any other suitable technique for constructing DNA fragments encoding the peptides. In the PCR technique, by using oligonucleotides randomly mutated within a particular triplet codon, the resulting pool of fragments encodes all possible combinations of codons at these positions. Preferably, certain amino acid positions are kept constant, which are the conserved amino acids required for binding to the MHC peptide binding groove and do not contact the T cell receptor. See, for example, U.S. Patent Application Publication No. 2004 / 0110253).

[0109] In this screening method, the target TCR is a TCR for which it is desired to identify the peptide epitope recognized by the receptor. In one embodiment, the target TCR is derived from a patient having a T cell-mediated disease such as an autoimmune disease, an infectious disease, or cancer. See, for example, Rossjohn and Koning, Mucosal Immunology, 9(3):583-586(2016), Qiao et al., J Immunol., 187:3064-3071(2011), Broughton et al., Immunity, 37:611-621(2012), Qiao et al., International Immunology, 26(1):13-19.

[0110] Binding a peptide to an MHC class I or MHC class II molecule via a flexible linker has the advantage that the peptide is guaranteed to occupy the MHC and remain associated during biosynthesis, transport, and presentation. However, there may be situations where this linker interferes with peptide binding to the MHC or TCR recognition of the complex. As an alternative approach, in some embodiments, the MHC and peptide are expressed separately. Thus, in certain embodiments, separately expressed peptides are loaded onto MHC molecules.

[0111] A "single-chain peptide-major histocompatibility complex (pMHC) fusion" is a single-chain polypeptide comprising a peptide fused to one or more domains of an MHC protein, and optionally, the peptide and one or more domains of the MHC protein are joined together by one or more linkers.

[0112] As used herein, the term "fusion" means a polypeptide formed by the expression of a chimeric gene typically created by combining two or more sequences such that two genes encode a single continuous polypeptide, typically by cloning one gene into an expression vector in-frame with a second gene (but not limited thereto). For example, recombinant cloning techniques such as polymerase chain reaction (PCR) and restriction endonuclease cloning are well known in the art. In addition to being made by recombinant techniques, portions of polypeptides can be fused to each other to form "fusions" by chemical reactions, or by other means known in the art for making custom polypeptides.

[0113] T cell surface molecules (e.g., immunomodulatory molecules) targeted by the antigen-binding domain of a second molecule contained within a multispecific molecule described herein can anchor the multispecific molecule and / or can mediate T cell responses including, but not limited to, proliferation, activation, differentiation, etc. Immunomodulatory molecules, particularly T cell immunomodulatory molecules, include, for example, CD28, CD80, CD86, CD3, CD4, CD7, CD8, CD27, CD47, CD70, CD83, BTLA, 4-1BB, 4-1BBL, OX40, OX40L, CD30, CD40, CD40L, CD70, CD160, CTLA4, PD1, PD-L1, PDL2, ICOS, ICOS-L, galectin 9, GITR, GITRL, ILT3, ILT4, lymphocyte function-associated antigen-1 (LFA-1), LFA-3, LIGHT, MHC-1, NKG2C, TIM1, TIM3, TIM4, Toll ligand receptor, B7-H3, B7-H4, HVEM, 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, cadherin), or ectodomains of multi-span integral membrane proteins. In certain embodiments, the T cell surface molecule is targeted by a domain that specifically binds to a molecule expressed on the surface of a cell expressing the TCR of the second molecule described herein (e.g., CD28, CD27, BTLA, 4-1BB, OX40, CD40L, CD160, CTLA4, PD1, ICOS, galectin 9, GITR, lymphocyte function-associated antigen-1 (LFA-1), MHC-1, TIM1, HVEM, CD2, etc.).In certain embodiments, the T cell surface molecule is a domain that specifically binds to a molecule expressed on the surface of a cell expressing the TCR of the second molecule described herein (e.g., CD80, CD86, CD40, ICOS-L, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, PDL-1, PD-L2, B7-H3, B7-H4, HVEM, ILT3, ILT4, 2B4, CD226, etc.).

[0114] As used herein, the expression "T cell immunomodulatory molecule" encompasses "T cell costimulatory molecule" and "T cell inhibitory molecule". As used herein, a "T cell costimulatory molecule" refers to a protein expressed by a T cell that binds to a cognate ligand or receptor (e.g., on an antigen-presenting cell) to provide a stimulatory signal, which, in combination with the primary signal provided by the engagement of the T cell's TCR with peptide / MHC, stimulates the activity of the T cell. The result of stimulation can only be achieved when combined with the primary TCR signal. Stimulation of T cells can include activation, proliferation, and / or survival of T cells. Non-limiting examples of T cell costimulatory molecules include CD28, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, galectin 9, LFA-1, DR3, CD30, SLAM, 2B4, CD226, TIM1, TIM2, and CD2.

[0115] As used herein, a "T cell inhibitory molecule" refers to a protein expressed by a T cell that binds to a cognate ligand or receptor (e.g., on an antigen-presenting cell) to provide an inhibitory signal, which, in combination with the primary signal provided by the engagement of the T cell's TCR with peptide / MHC, inhibits the activity of the T cell. Inhibition of T cells can include anergy, suppression of activity or proliferation, and / or death of T cells. Non-limiting examples of T cell inhibitory molecules include CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, MHC-1, B7-1, and B7-H1.

[0116] As used herein, the expressions "cell surface expression" or "cell surface molecule" mean one or more proteins expressed on the surface of a cell in vitro, ex vivo, or in vivo, at least a portion of which is exposed on the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody or the antigen-binding domain of a multispecific molecule discussed herein.

[0117] As used in the present invention, the term "antigen-binding domain" means any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD28 or CTLA-4). The terms "monovalent antigen-binding domain" or "single-arm antibody" include an immunoglobulin molecule that contains two polypeptide chains (one heavy (H) chain and one light (L) chain) interconnected by disulfide bonds. The heavy chain includes a heavy-chain variable region (abbreviated herein as HCVR or V H and a heavy-chain constant region. The heavy-chain constant region includes three domains, C H 1, C H 2, and C H 3. The light chain includes a light-chain variable region (abbreviated herein as LCVR or V L and a light-chain constant region. The light-chain constant region includes one domain (C L 1). The V H region and the V L region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the antigen-binding domain can be identical to the human germline sequences or can be modified naturally or artificially. Amino acid consensus sequences can be defined based on the parallel analysis of two or more CDRs.

[0118] The terms "antigen-binding domain" and "monovalent antigen-binding domain" also include, as used herein, antigen-binding fragments of the molecules discussed above. Antigen-binding fragments include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments can be obtained from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion techniques or recombinant genetic engineering techniques involved in the manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains. Such DNA is known and / or can be readily obtained, for example, from commercial suppliers, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA is sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, and modify, add, or delete amino acids. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) Fab' fragments, (iii) Fd fragments, (iv) Fv fragments, and (v) single-chain Fv (scFv) molecules.

[0119] An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V domain bound to H In an antibody-binding fragment having a H V domain and a L V domain, the V domains can be arranged relative to each other in any suitable configuration.

[0120] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the invention include the following: (i) VH -C H 1. (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1 - C H 2. (v) V H -C H 1 - C H 2 - C H 3. (vi) V H -C H 2 - C H 3. (vii) V H -C L . (viii) V L -C H 1. (ix) V L -C H 2. (x) V L -C H 3. (xi) V L -C H 1 - C H 2. (xii) V L -C H 1 - C H 2 - C H 3. (xiii) V L -C H 2 - C H 3. and (xiv) V L -C L . In any arrangement of the variable domain and the constant domain, including any of the exemplary arrangements listed above, the variable domain and the constant domain can be either directly linked to each other or linked by a full or partial hinge region or linker region. The hinge region can consist of at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the present invention comprises one or more monomeric V H domains or V LIn the context of non-covalent association with a domain, it may include a homodimer or heterodimer (or other multimer) of either of the variable domain conformations and constant domain conformations listed above.

[0121] In certain embodiments of the invention, the antigen-binding domain and / or the MHC moiety of the pMHC fusion are human. As used herein, the term "human antigen-binding domain" is intended to include antigen-binding domains having variable and constant regions derived from human germline immunoglobulin sequences. The human antigen-binding domains of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutations in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antigen-binding domain" is not intended to include antigen-binding domains in which CDR sequences from another mammalian species (e.g., mouse) have been transplanted into a human framework sequence. Similarly, as used herein, the term "human MHC" is intended to refer to MHC molecules in which the various domains are encoded by human MHC genes.

[0122] In certain embodiments of the invention, the antigen-binding domain of the second molecule contained within the multispecific molecule of the invention is a monoclonal antibody, synthetic antibody, recombinantly produced antibody, multispecific antibody, human antibody, chimeric antibody, camelized antibody, single-chain Fv (scFv), single-chain antibody, Fab fragment, F(ab’) fragment, disulfide-linked Fv (sdFv), intrabody, or an epitope-binding fragment of any of the foregoing. In certain embodiments, the antigen-binding domain of the second molecule contained within the multispecific molecule of the invention or an antigen-binding fragment thereof is a covalently linked diabody such as Ig-DARTS or those disclosed in U.S. Patent Application Publication No. 2007:0004909.

[0123] The antigen-binding domain of the second molecule contained within the multispecific molecule of the present invention can be humanized by any method known in the art for modifying proteins for therapeutic use in humans. In addition to methods generally known in the art for combining heterologous CDR sequences with human frameworks and / or constant domains, the term "humanized" also includes methods of protein and / or antibody resurfacing, such as those disclosed in, for example, U.S. Patent Nos. 5,770,196, 5,776,866, 5,821,123, and 5,896,619.

[0124] The antigen-binding domain of the second molecule contained within the multispecific molecule of the present invention can be derived from any species (e.g., rabbit, mouse, rat, donkey, cow, camel, llama, sheep, goat, horse, primate), but is preferably derived from a human immunoglobulin molecule that can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The antigen-binding domain of the second molecule contained within the multispecific molecule of the present invention can be produced by any method known in the art, such as chemical synthesis or recombinant techniques.

[0125] The present invention encompasses an antigen-binding domain that includes, for example, one or more amino acid modifications that alter antibody binding or effector function. See, for example, U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514, U.S. Patents Nos. 5,624,821 and 5,648,260, European Patent No. 0307434, International Patent Application Publication Nos. 04 / 029207, 04 / 029092, 04 / 028564, 99 / 58572, 99 / 51642, 98 / 23289, 89 / 07142, 88 / 07089, U.S. Patents Nos. 5,843,597 and 5,642,821). In some embodiments, the amino acid mutations of the protein result in the generation of equivalent or even improved second-generation molecules. For example, certain amino acids can be replaced by other amino acids in the protein structure without a detectable loss of affinity or binding.

[0126] The present invention encompasses an antigen-binding domain that is fused to a heterologous polypeptide or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous polypeptide. In some embodiments, such fusion proteins include a linker sequence.

[0127] The modified antibody or fragment thereof can be produced, for example, by error-prone PCR-induced random mutagenesis, random nucleotide insertion, or recombination, or other previous methods such as DNA shuffling.

[0128] In some uses, it may be preferable to use human or chimeric antibodies or fragments thereof. Fully human antibodies are particularly desirable for the therapeutic treatment of human subjects. Human antibodies can be produced by various methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111, and International Patent Application Publications Nos. 98 / 46645, 98 / 50433, 98 / 24893, 98 / 16654, 96 / 34096, 96 / 33735, and 91 / 10741.

[0129] Single domain antibodies, e.g., antibodies lacking a light chain, can be produced by methods well known in the art. See, for example, Riechmann et al., 1999, J. Immunol. 231:25-38, Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1(3):253-263, Muylderman, 2001, J. Biotechnol. 74(4):277302, U.S. Pat. No. 6,005,079, and International Patent Application Publications Nos. 94 / 04678, 94 / 25591, and 01 / 44301.

[0130] Terms such as "specifically binds", "binds in a specific manner", and "antigen-specific" indicate that the molecules involved in specific binding can form relatively stable complexes with each other under physiological conditions and cannot form non-specifically stable complexes with other molecules outside the designated binding pair. In certain embodiments, the antigen is a TCR and the pMHC complex functions as a TCR-binding molecule. Thus, a pMHC complex that specifically binds to an antigen-specific TCR indicates not only that the pMHC complex forms a stable complex with the antigen-specific TCR, but also that the antigen-specific TCR does not form a stable complex with a different antigen. Thus, a pMHC complex that specifically binds to an antigen-specific TCR may be considered an antigen to which the TCR is specific. For example, the pMHC complex may (i) not target at all the constant domain(s) of the TCR or other components of the TCR complex (e.g., CD3), (ii) target the constant domain(s) of the TCR or other components of the TCR complex (e.g., CD3) in addition to targeting the variable domain(s) and / or idiotype of the TCR, or (iii) target only the variable domain(s) and / or idiotype of the TCR. Specific binding may be characterized by an equilibrium dissociation constant (KD) of about 3000 nM or less (i.e., a smaller KD represents stronger binding), about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 1 nM or less, or about 0.5 nM or less. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0131] As used herein, a "multimerization domain" is any macromolecule that has the ability to (cooperatively or non-cooperatively) associate with a second macromolecule of the same or similar structure or composition. For example, a multimerization domain is an immunoglobulin C HIt may be a polypeptide containing 3 domains. Non-limiting examples of multimerization domains are the Fc portions of immunoglobulins of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, such as the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, and allotypes within each isotype group. In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence having a length of 1 to about 200 amino acids and containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, helix-loop motif, or coiled-coil motif.

[0132] As used herein, the terms “nucleic acid” or “polynucleotide” refer to nucleotides and / or polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications can include, for example, replacement of one or more hydroxyl groups with halogen, alkyl, amine, and azide groups, or the sugar can be functionalized as an ether or ester. Further, the entire sugar moiety can be replaced with sterically and electronically similar structures such as azasugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single-stranded or double-stranded.

[0133] Nucleic acids are said to have a "5' end" and a "3' end" because the mononucleotides react in such a way that the 5' phosphate of one mononucleotide pentose ring binds in one direction via a phosphodiester bond to the adjacent 3' oxygen. The end of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of the mononucleotide pentose ring. The end of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence may also be said to have 5' and 3' ends even if it is present within a larger oligonucleotide. In either a linear or circular DNA molecule, distinct elements are referred to as "upstream" or "downstream" 5' or 3' elements.

[0134] As used herein, the term "recombinant" is intended to include all molecules prepared, expressed, made, or isolated by recombinant means, such as multispecific molecules (e.g., bispecific molecules) expressed using a recombinant expression vector transfected into a host cell (described further below), multispecific molecules (e.g., bispecific molecules) isolated from an animal (e.g., a mouse) transgenic for a human immunoglobulin gene (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or molecules prepared, expressed, made, or isolated by any other means including splicing to other DNA sequences of human immunoglobulin and / or MHC gene sequences. Such recombinant multispecific molecules can include antigen-binding domains having variable and constant regions derived from human germline immunoglobulin sequences.

[0135] As used interchangeably herein, the terms "subject", "individual", "animal", or "patient" include all members of the animal kingdom, including non-human primates and humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and laboratory animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human. In one embodiment, the patient is a human having a disease or disorder, such as an infectious disease, cancer, or autoimmune disorder.

[0136] The term "treatment" or "treating" of a state, disorder, or condition includes (1) preventing, delaying, or reducing the likelihood of the occurrence and / or appearance of at least one clinical or subclinical symptom of a state, disorder, or condition in a subject who may be or is predisposed to having the state, disorder, or condition but has not yet experienced or exhibited the clinical or subclinical symptoms of the state, disorder, or condition, or (2) inhibiting the state, disorder, or condition, i.e., preventing, reducing, or delaying the onset or recurrence of the disease or at least one of its clinical or subclinical symptoms, or (3) alleviating the disease, i.e., causing regression of at least one of the state, disorder, or condition, or its clinical or subclinical symptoms. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0137] The term "effective" as applied to amount or quantity refers to that amount of a compound or pharmaceutical composition sufficient to provide the desired activity when administered to a subject in need of that activity. It should be noted that when combinations of active ingredients are administered, the effective amount of the combination may or may not include the amounts of the individual ingredients that were effective when administered separately. The exact amount required will vary from subject to subject, depending on the species, age, and general health of the subject, the severity of the condition being treated, the particular drug or drugs used, the mode of administration, and the like.

[0138] The phrase "pharmaceutically acceptable" when used in reference to the compositions described herein refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and typically produce no adverse reactions when administered to a mammalian (e.g., human) subject. Preferably, the term "pharmaceutically acceptable" means that the entity or ingredient has been approved by a regulatory agency of the Federal or a State government or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0139] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, indicate a nucleotide sequence identity of at least about 95%, more preferably about 96%, 97%, about 98%, or 99% nucleotide base, as measured by any well-known algorithm of sequence identity such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0140] As used interchangeably herein, the terms “protein,” “polypeptide,” and “peptide” include polymeric forms of amino acids of any length, including coded and non-coded amino acids, and chemically or biochemically modified or derivatized amino acids. The term also includes modified polymers, such as polypeptides having modified peptide backbones. The terms “protein,” “polypeptide,” and “peptide” include all types of naturally occurring and synthetic proteins of all lengths, including protein fragments, fusion proteins, and modified proteins, including glycoproteins, and all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP ribosylation, pegylation, biotinylation, etc.), but are not limited thereto.

[0141] Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N-terminus” refers to the beginning of a protein or polypeptide terminated by an amino acid having a free amine group (-NH2). The term “C-terminus” refers to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH).

[0142] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT, using a defined gap weight. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference). Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445 (incorporated herein by reference). A "moderately conservative" replacement is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0143] Sequence similarity to a polypeptide, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similar measurements assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used with the default parameters for determining sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein, including programs such as Gap and Bestfit. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, which is a program of GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the best overlapping regions and percent sequence identity between a query sequence and a search sequence (Pearson (2000) supra). Another preferred algorithm when comparing the sequences of the present invention to a database containing a number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each incorporated herein by reference.

[0144] As used herein, the terms "vector" and "expression vector" include, but are not limited to, linear or circular DNA or RNA molecules that can consist of viral vectors, plasmids, RNA vectors, or chromosomal, episomal, semisynthetic, or synthetic nucleic acids. In some embodiments, the vector is capable of autonomous replication (episomal vector) and / or expression of the nucleic acids to which they are ligated (expression vector). A number of suitable vectors are known to those skilled in the art and are commercially available. Viral vectors include negative-strand RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), picornaviruses, and alphaviruses, as well as double-stranded DNA viruses such as adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, canarypox). Other viruses include, for example, norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian type C, type B virus, type D virus, HTLV-BLV group, and lentivirus.

[0145] According to the disclosure herein, conventional molecular biology, microbiology, and recombinant DNA techniques within the art can be used. Such techniques are well described in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (hereinafter "Sambrook et al., 1989"), DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985), Oligonucleotide Synthesis (M.J. Gait ed. 1984), Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)], Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)], Animal Cell Culture [R.I. Freshney, ed. (1986)], Immobilized Cells And Enzymes [IRL Press, (1986)], B. Perbal, A Practical Guide To Molecular Cloning (1984), Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.These techniques include site-directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Patent No. 5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263:357-360 (1999), Kim and Maas, BioTech. 28:196-198 (2000), Parikh and Guengerich, BioTech. 24:428-431 (1998), Ray and Nickoloff, BioTech. 13:342-346 (1992), Wang et al., BioTech. 19:556-559 (1995), Wang and Malcolm, BioTech. 26:680-682 (1999), Xu and Gong, BioTech. 26:639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3:74-75 (2001), U.S. Patent Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30:486-488 (2001), Wang and Wilkinson, Biotech. 29:976-978 (2000), Kang et al., Biotech. 20:44-46 (1996), Ogel and McPherson, Protein Engineer. 5:467-468 (1992), Kirsch and Joly, Nucl. Acids Res. 26:1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178:3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids Res. 22:541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57:229-237, and Pons et al., Meth. Molec. Biol. 67:209-218.

[0146] First and second molecules of a multispecific molecule and a multispecific carrier The multispecific molecule of the present invention is composed of two heterologous binding molecules for engaging T cells and either suppressing or inducing an immune response. In certain embodiments, the multispecific molecule comprises (i) a peptide (p) presented in the context of a major histocompatibility complex (MHC) molecule (pMHC complex), and (ii) a first multimerization domain, and a second molecule that comprises (i) a domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR, and (ii) a second multimerization domain. The multispecific molecule of the present invention does not contain an MHC protein or MHC domain on both binding molecules.

[0147] The multispecific carrier molecule of the present invention expresses on its surface two heterologous transmembrane molecules for engaging T cells and either suppressing or inducing an immune response. In certain embodiments, the multispecific carrier comprises a plurality of first molecules and a plurality of second molecules, each first molecule being a peptide (p) presented in the context of a major histocompatibility complex (MHC) molecule (pMHC complex), and each second molecule being a polypeptide comprising a domain that specifically binds to a molecule expressed on the surface of a cell expressing a TCR.

[0148] First molecule of a multispecific molecule or a multispecific carrier The first molecule of a multispecific molecule or a multispecific carrier can comprise one or more domains of a class I or class II MHC protein, or a fragment, variant, or derivative thereof, fused to a peptide that can be recognized by the TCR of a T cell. In some embodiments, for example, where the target T cell is a CD8+ T cell, the multispecific molecule or multispecific carrier described herein comprises a class I MHC polypeptide, or a fragment, variant, or derivative thereof. In some embodiments, for example, where the target T cell is a CD4+ T cell, the multispecific molecule or multispecific carrier described herein comprises a class II MHC polypeptide, or a fragment, variant, or derivative thereof.

[0149] In one embodiment, the first molecule comprises one or more domains of a class I MHC protein, or a fragment, variant, or derivative thereof. For example, the first molecule may comprise one or more of the alpha1, alpha2, and alpha3 domains of a class I MHC protein, and the beta2 microglobulin domain. In one embodiment, the first molecule comprises all of the alpha1, 2, and 3 chain domains, as well as the beta2 microglobulin domain.

[0150] In one embodiment of the multispecific molecule, the first molecule comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) a beta2 microglobulin peptide, or a fragment, variant, or derivative thereof, (iii) a class I alpha domain, or a fragment, variant, or derivative thereof, and (iv) a multimerization domain (e.g., an immunoglobulin Fc domain). In one embodiment of the multispecific molecule, the first molecule comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) a beta2 microglobulin peptide, or a fragment, variant, or derivative thereof, (iii) an alpha1 class I domain peptide, or a fragment, variant, or derivative thereof, (iv) an alpha2 class I domain peptide, or a fragment, variant, or derivative thereof, (v) an alpha3 domain peptide, or a fragment, variant, or derivative thereof, and (vi) a multimerization domain (e.g., an immunoglobulin Fc domain). In one embodiment of the multispecific molecule, the first molecule comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) an alpha3 domain peptide, or a fragment, variant, or derivative thereof, (iii) an alpha2 class I domain peptide, or a fragment, variant, or derivative thereof, (iv) an alpha1 class I domain peptide, or a fragment, variant, or derivative thereof, (v) a beta2 microglobulin peptide, or a fragment, variant, or derivative thereof, and (vi) a multimerization domain (e.g., an immunoglobulin Fc domain). In some embodiments, the peptide, class I MHC domain, and / or multimerization domain are linked by one or more peptide linkers.

[0151] In one embodiment of the multispecific carrier, the first molecule, from the N-terminus to the C-terminus, comprises (i) a peptide, (ii) a beta-2 microglobulin peptide, or a fragment, variant, or derivative thereof, (iii) a class I alpha domain, or a fragment, variant, or derivative thereof, and (iv) a transmembrane domain. In one embodiment of the multispecific carrier, the first molecule of the multispecific carrier, from the N-terminus to the C-terminus, comprises (i) a peptide, (ii) a beta-2 microglobulin peptide, or a fragment, variant, or derivative thereof, (iii) an alpha-1 class I domain peptide, or a fragment, variant, or derivative thereof, (iv) an alpha-2 class I domain peptide, or a fragment, variant, or derivative thereof, (v) an alpha-3 domain peptide, or a fragment, variant, or derivative thereof, and (vi) a transmembrane domain. In one embodiment of the multispecific carrier, the first molecule, from the N-terminus to the C-terminus, comprises (i) a peptide, (ii) an alpha-3 domain peptide, or a fragment, variant, or derivative thereof, (iii) an alpha-2 class I domain peptide, or a fragment, variant, or derivative thereof, (iv) an alpha-1 class I domain peptide, or a fragment, variant, or derivative thereof, (v) a beta-2 microglobulin peptide, or a fragment, variant, or derivative thereof, and (vi) a transmembrane domain. In some embodiments, the peptide, class I MHC domain, and / or transmembrane domain are linked by one or more peptide linkers.

[0152] In some embodiments, the class I MHC polypeptide is a human class I MHC polypeptide such as, but not limited to, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another embodiment, the class I MHC polypeptide is a mouse class I MHC polypeptide such as, but not limited to, H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0153] In some embodiments, the MHC class I alpha heavy chain is fully human. In some embodiments, the MHC class I alpha heavy chain is humanized. Humanized MHC class I alpha heavy chains are described, for example, in U.S. Patent Publications Nos. 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467. In some embodiments, the MHC class I alpha heavy chain comprises a human extracellular domain (human alpha 1, alpha 2, and / or alpha 3 domain) and a cytoplasmic domain of another species. In some embodiments, the class I alpha heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence can be the HLA-A*0201 sequence. In various aspects, the peptide-MHC can include all domains of the MHC class I heavy chain.

[0154] In some embodiments, β2-microglobulin is fully human. In some embodiments, β2-microglobulin is humanized. Humanized β2-microglobulin polypeptides are described, for example, in U.S. Patent Publications Nos. 2013 / 0111617 and 2013 / 0185819.

[0155] In some embodiments, the MHC class I molecule comprises mutations in the β2-microglobulin (β2m or B2M) polypeptide and the heavy chain sequence so as to affect the disulfide bond between B2M and the heavy chain. In some embodiments, the heavy chain is HLA, and the disulfide bond links to one of the following residue pairs: Β2M residue 12, HLA residue 236; Β2M residue 12, HLA residue 237; Β2M residue 8, HLA residue 234; Β2M residue 10, HLA residue 235; Β2M residue 24, HLA residue 236; Β2M residue 28, HLA residue 232; Β2M residue 98, HLA residue 192; Β2M residue 99, HLA residue 234; Β2M residue 3, HLA residue 120; Β2M residue 31, HLA residue 96; Β2M residue 53, HLA residue 35; Β2M residue 60, HLA residue 96; Β2M residue 60, HLA residue 122; Β2M residue 63, HLA residue 27; Β2M residue Arg3, HLA residue Glyl20; Β2M residue His31, HLA residue Gln96; Β2M residue Asp53, HLA residue Arg35; Β2M residue Trp60, HLA residue Gln96; Β2M residue Trp60, HLA residue Aspl22; Β2M residue Tyr63, HLA residue Tyr27; Β2M residue Lys6, HLA residue Glu232; Β2M residue Gln8, HLA residue Arg234; Β2M residue TyrlO, HLA residue Pro235; Β2M residue Serl l, HLA residue Gln242; Β2M residue Asn24, HLA residue Ala236; Β2M residue Ser28, HLA residue Glu232; Β2M residue Asp98, HLA residue His192; and Β2M residue Met99, HLA residue Arg234, first linker position Gly2, heavy chain (HLA) position Tyr84; light chain (Β2M) position Arg12, HLA Ala236; and / or Β2M residue Argl2, HLA residue Gly237. See, for example, International Patent Application Publication No. 2015 / 195531, which is incorporated herein by reference for all intended purposes.

[0156] In certain embodiments, the pMHC complex can include a peptide covalently bound to MHC class I α (heavy) chain via a disulfide bridge (i.e., a disulfide bond between two cysteines). In certain embodiments, the disulfide bond includes a first cysteine constituted by a linker extending from the carboxy terminus of the antigen peptide, and a second cysteine constituted by the MHC class I heavy chain (e.g., the MHC class I α (heavy) chain having a non-covalent binding site for the antigen peptide). In certain embodiments, the second cysteine can be a mutation (addition or substitution) in the MHC class I α (heavy) chain. In certain embodiments, the pMHC complex can include one continuous polypeptide chain and a disulfide bridge. In certain embodiments, the pMHC complex can include two continuous polypeptide chains that are bound via a disulfide bridge as the only covalent bond. In some embodiments, the linking sequence can include at least one amino acid in addition to cysteine, including one or more glycines, one or more alanines, and / or one or more serines.

[0157] In certain embodiments, when the pMHC complex includes a first cysteine in a Gly-Ser linker extending between the C-terminus of the peptide and β2-microglobulin and a second cysteine at an adjacent heavy chain position, the disulfide bridge can link the antigen peptide within the class I groove of the pMHC complex.

[0158] In some embodiments, the β2-microglobulin sequence can include the full-length β2-microglobulin sequence. In certain embodiments, the β2-microglobulin sequence lacks a leader peptide sequence. Thus, in some configurations, the β2-microglobulin sequence can include about 99 amino acids and can be a mouse β2-microglobulin sequence (e.g., Genebank X01838). In some other configurations, the β2-microglobulin sequence can include about 99 amino acids and can be a human β2-microglobulin sequence (e.g., Genebank AF072097.1).

[0159] In some embodiments, the pMHC complex sequence may be as described in U.S. Patent Nos. 4,478,82, 6,011,146, 8,518,697, 8,895,020, 8,992,937, WO96 / 04314, Mottez et al. J. Exp. Med. 181:493-502, 1995, Madden et al. Cell 70:1035-1048, 1992, Matsumura et al., Science 257:927-934, 1992, Mage et al., Proc. Natl. Acad. Sci. USA 89:10658-10662, 1992, Toshitani et al, Proc. Nat’l Acad. Sci. 93:236-240, 1996, Chung et al, J. Immunol. 163:3699-3708, 1999, Uger and Barber, J. Immunol. 160:1598-1605, 1998, Uger et al., J. Immunol. 162, pp. 6024-6028, 1999, White et al., J. Immunol. 162:2671-2676, 1999, Yu et al., J. Immunol. 168:3145-3149, 2002, Truscott et al., J. Immunol. 178:6280-6289, 2007.

[0160] In certain embodiments, the first binding molecule comprises one or more domains of a class II MHC protein. For example, the first binding molecule may comprise one or more of the alpha1, alpha2, beta1, and beta2 domains of a class II MHC protein. In one embodiment, the first binding molecule comprises all of the alpha1, alpha2, beta1, and beta2 domains.

[0161] In certain embodiments of the multispecific molecule, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) a class II alpha domain, or a fragment, variant, or derivative thereof, (iii) a class II beta domain, or a fragment, variant, or derivative thereof, and (iv) a multimerization domain (e.g., an immunoglobulin Fc domain). In one embodiment of the multispecific molecule, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) a class II beta domain, or a fragment, variant, or derivative thereof, (iii) a class II alpha domain, or a fragment, variant, or derivative thereof, and (iv) a multimerization domain (e.g., an immunoglobulin Fc domain). In one embodiment of the multispecific molecule, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) an alpha2-class II domain peptide, or a fragment, variant, or derivative thereof, (iii) an alpha1-class II domain peptide, or a fragment, variant, or derivative thereof, (iv) a beta1 domain peptide, or a fragment, variant, or derivative thereof, (v) a beta2 domain peptide, or a fragment, variant, or derivative thereof, and (vi) a multimerization domain (e.g., an immunoglobulin Fc domain). In one embodiment of the multispecific molecule, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) a beta2 domain, or a fragment, variant, or derivative thereof, (iii) a beta1 domain peptide, or a fragment, variant, or derivative thereof, (iv) an alpha1-class II domain peptide, or a fragment, variant, or derivative thereof, (v) an alpha2-class II domain peptide, or a fragment, variant, or derivative thereof, and (vi) a multimerization domain (e.g., an immunoglobulin Fc domain). In some embodiments, the peptide, class II MHC domain, and / or multimerization domain are linked by one or more peptide linkers.

[0162] In certain embodiments of the multispecific carrier, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) a class II alpha domain, or a fragment, variant, or derivative thereof, (iii) a class II beta domain, or a fragment, variant, or derivative thereof, and (iv) a transmembrane domain. In one embodiment of the multispecific carrier, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) a class II beta domain, or a fragment, variant, or derivative thereof, (iii) a class II alpha domain, or a fragment, variant, or derivative thereof, and (iv) a transmembrane domain. In one embodiment of the multispecific carrier, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) an alpha2-class II domain peptide, or a fragment, variant, or derivative thereof, (iii) an alpha1-class II domain peptide, or a fragment, variant, or derivative thereof, (iv) a beta1 domain peptide, or a fragment, variant, or derivative thereof, (v) a beta2 domain peptide, or a fragment, variant, or derivative thereof, and (vi) a transmembrane domain. In one embodiment of the multispecific carrier, the first binding molecule, from N-terminus to C-terminus, comprises (i) a peptide, (ii) a beta2 domain peptide, or a fragment, variant, or derivative thereof, (iii) a beta1 domain peptide, or a fragment, variant, or derivative thereof, (iv) an alpha1-class II domain peptide, or a fragment, variant, or derivative thereof, (v) an alpha2-class II domain peptide, or a fragment, variant, or derivative thereof, and (vi) a transmembrane domain. In some embodiments, the peptide, class II MHC domain, and / or transmembrane domain are linked by one or more peptide linkers.

[0163] In some embodiments, the MHC comprises the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) of a human class II MHC complex selected from the group consisting of HLA DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO. In another specific embodiment, the MHC comprises the α and β polypeptides (or fragments thereof, such as the α1 and β1 domains) of a mouse H-2A or H-2E class II MHC complex.

[0164] Naturally occurring MHC class II molecules consist of two polypeptide chains, α and β. The chains can be derived from the DP, DQ, or DR gene clusters. There are approximately 40 known different human MHC class II molecules. Although their basic structures are the same, their molecular structures are slightly different. MHC class II molecules bind to peptides that are 13 - 18 amino acids in length.

[0165] In some embodiments, the multispecific molecule comprises one or more MHC class II α chains. In some embodiments, the MHC class II α chain is fully human. In some embodiments, the MHC class II α chain is humanized. Humanized MHC class II α chains are described, for example, in U.S. Pat. Nos. 8,847,005 and 9,043,996, and U.S. Patent Publication No. 2014 / 0245467. In some embodiments, the humanized MHC class II α chain polypeptide comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, the class II α chain polypeptide is a humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA.

[0166] In some embodiments, the multispecific molecule comprises one or more MHC class II β chains. In some embodiments, the MHC class II β chain is fully human. In some embodiments, the MHC class II β chain polypeptide is humanized. Humanized MHC class II β chain polypeptides are described, for example, in U.S. Patent Nos. 8,847,005 and 9,043,996, and U.S. Patent Publication No. 2014 / 0245467. In some embodiments, the humanized MHC class II β chain comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some embodiments, the class II β chain is a humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB.

[0167] The first binding molecule of the multispecific molecule of the present invention is structured such that a peptide can be positioned within a groove formed, for example, by a class I alpha 1 and alpha 2 domain peptide or a class II alpha 1 and beta 1 domain peptide for presentation to the TCR of a T cell. In various embodiments, the peptide can consist of about 5 to about 40 amino acid residues, about 6 to about 30 amino acid residues, about 7 to about 25 amino acid residues, or about 8 to about 20 amino acid residues. In some embodiments, the peptide can consist of about 5 to about 15 amino acid residues, about 8 to about 12 amino acid residues, or about 8, about 9, about 10, about 11, or about 12 amino acid residues.

[0168] In some embodiments, the peptide is derived from a viral antigen. In some embodiments, the viral antigen is a viral protein or a fragment of a viral protein associated with adenovirus, astrovirus, chikungunya, cytomegalo, dengue, Ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, Lassa, measles, metapneumovirus, mumps, norovirus, Oropouche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, Usutu, vaccine, varicella, West Nile, yellow fever, or Zika.

[0169] In some embodiments, the peptide is derived from a bacterial antigen. In some embodiments, the bacterial antigen is an antigen associated with bacteria resistant to conventional antibiotic therapies such as methicillin-resistant Staphylococcus Aureus (MRSA), Clostridium Difficile, carbapenem-resistant Enterobacteriaceae, drug-resistant Neisseria Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, extended-spectrum beta-lactamase-producing bacteria, vancomycin-resistant enterococci, multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, vancomycin-resistant Staphylococcus Aureus, erythromycin-resistant group A Streptococcus, or clindamycin-resistant group B Streptococcus.

[0170] In some embodiments, the peptide is derived from a tumor-associated antigen. As used herein, the term "tumor-associated antigen" refers to a protein that is expressed by tumor cells or is overexpressed (compared to non-tumor cells). In some embodiments, the tumor-associated antigen is adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, ALK, BAGE protein (e.g., BAGE-1), BIRC5 (survivin), BIRC7, beta-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CAGE-1-8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen ("ETA"), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE protein (e.g., GAGE-1-8), GD2, GD3, GloboH, glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1,Selected from the group consisting of KM-HN-1, also known as CCDC110, KMHN1, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, LMP2, M-CSF, MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, -A9, -A10, -A12, -C1, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE proteins (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, selenoprotein 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1. In some embodiments, the peptide is a neoantigen. In some embodiments, the peptide is a tumor-specific antigen.,

[0171] In some embodiments, the peptide is derived from an antigen associated with an autoimmune disorder. In some embodiments, the antigen associated with an autoimmune disorder is gliadin (celiac disease, e.g., (i) an α-gliadin fragment corresponding to amino acids 57-73, or (ii) a γ-gliadin fragment corresponding to amino acids 139-153, or (iii) an ω-gliadin fragment corresponding to amino acids 102-118), GAD 65, IA-2, and insulin B chain (in the case of type 1 diabetes), glatiramer acetate (GA) (in the case of multiple sclerosis), acetylcholine receptor (AChR) (in the case of myasthenia gravis), p205, insulin, thyroid stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)), selected from the group consisting of. In some embodiments, the antigen associated with an autoimmune disorder is selected from the group consisting of IL-4R (interleukin-4 receptor), IL-6R (interleukin-6 receptor), and DLL4 (delta-like ligand 4).

[0172] In some embodiments, the components of the first binding molecule or the peptide are separated by a linker (or "spacer") peptide. Such peptide linkers are well known in the art (e.g., polyglycine) and typically allow for proper folding of one or both of the components of the fusion polypeptide. The linker provides a flexible junction region for the components of the fusion polypeptide and allows the components of the molecule to move independently. Thus, the junction region, in some embodiments, acts as a linker that combines two parts together and as a spacer that allows each of the connected parts to form its own biological structure and not interfere with the other part. Further, the junction region needs to create an epitope that is not recognized as foreign by the immune system of the subject, i.e., is not considered immunogenic. In one embodiment, each of the respective class I or class II MHC domains and the peptide are connected to each other via a peptide linker.

[0173] Suitable linkers for use in MHCs can be any of several suitable lengths, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, etc., and can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid residues or more, but are typically 5 to 25 residues. Examples of linkers include polyglycine linkers such as Gly-Gly (2Gly), Gly-Gly-Gly (3Gly), 4Gly (SEQ ID NO: 30), 5Gly (SEQ ID NO: 31), 6Gly (SEQ ID NO: 32), 7Gly (SEQ ID NO: 33), 8Gly (SEQ ID NO: 34), and 9Gly (SEQ ID NO: 35). Examples of linkers also include Gly-Ser peptides such as Ser-Gly (SG), Gly-Ser (GS), Gly-Gly-Ser (G2S), Ser-Gly-Gly (SG2), G3S (SEQ ID NO: 36), SG3 (SEQ ID NO: 37), G4S (SEQ ID NO: 38), SG4 (SEQ ID NO: 39), G5S (SEQ ID NO: 40), SG5 (SEQ ID NO: 41), G6S (SEQ ID NO: 42), SG6 (SEQ ID NO: 43), (G4S)n (SEQ ID NO: 44), (S4G)n (SEQ ID NO: 45) (n = 1 to 10). Any one of the linkers described herein can be repeated and the linker extended as needed. Other flexible linkers known in the art are disclosed, for example, in Chichili et al, Protein Science, 22:153-167 (2013), which is hereby incorporated by reference in its entirety for all purposes. Glycine and glycine-serine polymers can be used. Since both Gly and Ser are relatively unstructured, they can function as neutral tethers between components. Glycine polymers can be used. Glycine has access to far more phi-psi space than alanine and far fewer restrictions than residues with longer side chains (see Scheraga, Rev. Computational Chem. 1 1173-142 (1992)).Exemplary linkers can include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO: 46), GGSGG (SEQ ID NO: 47), GSGSG (SEQ ID NO: 48), GSGGG (SEQ ID NO: 49), GGGSG (SEQ ID NO: 50), GSSSG (SEQ ID NO: 51), GCGASGGGGSGGGGS (SEQ ID NO: 52), GCGASGGGGSGGGGS (SEQ ID NO: 52), GGGGSGGGGS (SEQ ID NO: 53), GGGASGGGGSGGGGS (SEQ ID NO: 54), GGGGSGGGGSGGGGS (SEQ ID NO: 55), GGGASGGGGS (SEQ ID NO: 56), GGGGSGGGGSGGGGS (SEQ ID NO: 55), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 57) (Table 1). In some embodiments, the linker polypeptide includes a cysteine residue capable of forming a disulfide bond with a cysteine residue present in the second polypeptide.

[0174] [Table 1]

[0175] The second molecule of the multispecific molecule or multispecific carrier The second binding molecule of the multispecific molecule or multispecific carrier can include an antigen-binding domain (e.g., an antibody, a one-armed antibody, or an antigen-binding fragment thereof) that anchors the multispecific molecule and / or specifically binds to a T cell surface molecule to provide a stimulatory or inhibitory signal to the T cell. In various embodiments, the antigen-binding domain comprises at least one complementarity-determining region (CDR) alone or in combination with one or more additional CDRs and / or framework regions (FRs) that specifically bind to a particular antigen, or consists of a protein, polypeptide, or molecular complex. In certain embodiments, the antigen-binding domain is part of a one-armed antibody or a fragment thereof as those terms are defined elsewhere herein.

[0176] In certain exemplary embodiments of the present invention, the antigen-binding domain comprises at least one heavy chain and at least one light chain. The heavy chain may comprise a heavy chain variable region (HCVR) and a heavy chain constant region. The heavy chain constant region may comprise one or more of the CH1, CH2, and / or CH3 domains. The light chain may comprise a light chain variable region (LCVR) and a light chain constant region. The variable regions of the heavy and light chains may each comprise CDRs designated HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively.

[0177] The antigen-binding domain of the second molecule of the multispecific molecule or multispecific carrier specifically binds to an immunomodulatory molecule expressed by a T cell. Binding to the immunomodulatory molecule induces or suppresses T cell activation in conjunction with the primary signal provided by the binding of the TCR of the T cell to the peptide / MHC complex, where the TCR of the T cell specifically binds to the peptide. In some embodiments, the immunomodulatory molecule is a costimulatory molecule that induces T cell activation, proliferation, and / or survival in conjunction with the signal provided by the binding of the TCR of the T cell to the peptide / MHC complex. For example, the antigen-binding domain can be a one-armed or two-armed antibody that specifically binds to CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2. In one embodiment, the T cell is a CD8+ T cell. In one embodiment, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell and the immunomodulatory molecule bound by the antigen-binding domain is a costimulatory molecule such as, but not limited to, CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, and CD226. In some embodiments, the T cell is a CD4+ T cell and the immunomodulatory molecule bound by the antigen-binding domain is a costimulatory molecule such as, but not limited to, CD28, ICOS, HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, TIM1, and TIM2. In other cases, the immunomodulatory molecule is an inhibitory molecule that suppresses T cell activation or induces anergy or T cell death in conjunction with the signal provided by the binding of the TCR of the T cell to the peptide / MHC complex. For example, the antigen-binding domain can be a one-armed or two-armed antibody that specifically binds to CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, or B7-H1. In one embodiment, the T cell is a CD8+ T cell. In one embodiment, the T cell is a CD4+ T cell.In some embodiments, the T cell is a CD8+ T cell, and the immune regulatory molecule bound by the antigen-binding domain is an inhibitory molecule such as, but not limited to, CTLA4, PD1, BTLA, TIM3, CD160, LAG3, LAIR1, B7-1, and B7-H1. In some embodiments, the T cell is a CD4+ T cell, and the immune regulatory molecule bound by the antigen-binding domain is an inhibitory molecule such as, but not limited to, CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, and B7-H1.

[0178] In certain embodiments, the second binding molecule of the multispecific molecule or multispecific carrier can include a small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof that has an affinity for a molecule expressed on the surface of a cell expressing a TCR. In certain embodiments, the small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof specifically binds to an immunomodulatory molecule expressed by T cells. Binding of the immunomodulatory molecule induces or suppresses T cell activation in conjunction with the primary signal provided by binding of the TCR of the T cell to a peptide / MHC complex, where the TCR specifically binds to the peptide. In some embodiments, the immunomodulatory molecule is a costimulatory molecule that induces T cell activation, proliferation, and / or survival in conjunction with the signal provided by binding of the TCR of the T cell to the peptide / MHC complex. For example, the small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof can specifically bind to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2. In one embodiment, the T cell is a CD8+ T cell. In one embodiment, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell and the immunomodulatory molecule bound by the small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof is a costimulatory molecule such as, but not limited to, CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, and CD226. In some embodiments, the T cell is a CD4+ T cell and the immunomodulatory molecule bound by the small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof is a costimulatory molecule such as, but not limited to, CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, and TIM2.In other cases, the immunomodulatory molecule is an inhibitory molecule that suppresses T cell activation or induces anergy or T cell death in combination with the signal provided by the binding of the TCR of the T cell to the peptide / MHC complex. For example, small molecules, proteins, fusion proteins, peptides, aptamers, avimers, or derivatives or fragments thereof can specifically bind to CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, or B7-H1. In one embodiment, the T cell is a CD8+ T cell. In one embodiment, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell, and the immunomodulatory molecule bound by a small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof is an inhibitory molecule such as, but not limited to, CTLA4, PD1, BTLA, TIM3, CD160, LAG3, LAIR1, B7-1, and B7-H1. In some embodiments, the T cell is a CD4+ T cell, and the immunomodulatory molecule bound by a small molecule, protein, fusion protein, peptide, aptamer, avimer, or derivative or fragment thereof is an inhibitory molecule such as, but not limited to, CTLA4, PD1, BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, and B7-H1.

[0179] Multimerization of the first and second molecules The first binding molecule and the second binding molecule can be directly or indirectly connected to each other to form the multispecific molecule of the present invention. In one embodiment, the first binding molecule and the second binding molecule can each be connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two binding molecules, thereby forming the multispecific molecule according to the present invention. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, the multimerization domain is an immunoglobulin C HIt may be a polypeptide containing 3 domains. Non-limiting examples of multimerization domains include the Fc portion of immunoglobulins (C H 2-C H containing 3 domains), for example, the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype within each isotype group. In one embodiment, the multimerization domain is human IgG1. In one embodiment, the multimerization domain is human IgG4.

[0180] The multispecific antigen-binding molecules of the present invention typically contain two multimerization domains, for example, two Fc domains that are individual portions of separate antibody heavy chains. The first and second multimerization domains can be of the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains can be of different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0181] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0182] The multivalent molecules of the present invention may include a multimerization domain, such as an Fc domain, that contains one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring Fc domain. For example, the present invention includes multivalent molecules that include one or more modifications in the Fc domain that result in a modified binding interaction (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the multivalent molecule includes a modification in the CH2 or CH3 region, which modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., within an endosome in the pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include the modifications 428L (e.g., M428L) and 434S (e.g., N434S), the modifications 428L, 259I (e.g., V259I), and 308F (e.g., V308F), the modifications 433K (e.g., H433K) and 434 (e.g., 434Y), the modifications 252, 254, and 256 (e.g., 252Y, 254T, and 256E), the modifications 250Q and 428L (e.g., T250Q and M428L), the modifications 307 and / or 308 (e.g., 308F or 308P).

[0183] The present invention also includes multivalent molecules that include a first multimerization domain and a second multimerization domain (e.g., an Ig Fc domain), wherein the first and / or second multimerization domain includes an amino acid sequence that facilitates purification of the multivalent molecule.

[0184] In one embodiment, the amino acid sequence that facilitates purification of the multispecific molecule is an amino acid substitution that results in a weak binding or no detectable binding to an Fc-binding affinity matrix. In one particular embodiment, one of the two multimerization domains comprises a CH3 domain ( "Fc") that can bind to Protein A, and the other of the two multimerization domains comprises a CH3 domain ( "Fc*") that cannot bind to Protein A. In some embodiments, the second multimerization domain comprises an H435R / Y436F (H95R / Y96F according to the IMGT exon numbering system, according to the EU numbering system) substitution in its CH3 domain ( "Fc*" or "star substitution"), showing weak binding or no detectable binding to an Fc-binding ligand such as Protein A, Protein G, Protein L, or derivatives thereof. The three-component mixture of FcFc* heterodimers and FcFc and Fc*Fc* homodimers can be separated using differential binding affinity chromatography. See, for example, U.S. Patent No. 8,586,713. Further modifications that can be found within the second CH3 include, for example, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT, D356E, L358M, N384S, K392N, V397M, and V422I according to EU) for the IgG1 heavy chain, N44S, K52N, and V82I (IMGT, N384S, K392N, and V422I according to EU) for the IgG2 heavy chain, and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU) for the IgG4 heavy chain. A further example of an additional mutation that abrogates binding to the affinity column is a mutation to the heavy chain variable region (VH) on the same chain as the Fc* mutation, as provided, for example, in U.S. Patent No. 9,493,563 (mutations of VH3 and Fc described as IMGT3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 90).

[0185] In certain embodiments, the multispecific molecules of the invention comprise a protein-protein interface substitution between the CH3 domain of an antibody Fc region and a protein-protein interface found in the constant region of a T cell receptor (TCR). Misfolding of the Lc is avoided by replacing one of the Fab arms of the bispecific IgG with an scFv. For purification purposes, the molecule is designed to lack a protein A binding site on the Hc of the molecule. Thus, homodimeric molecules having 2Hc do not bind to a protein A column, while heterodimeric molecules and homodimeric Fc-scFv molecules exhibit different affinities for protein A because these molecules have one and two binding sites for protein A, respectively. See, e.g., U.S. Pat. Nos. 9,683,052 and 9,683,053, and U.S. Patent Application Publication No. 2015 / 0239991.

[0186] In certain embodiments, the multispecific molecules of the invention comprise a knob-into-hole pair created by amino acid changes of T22Y in strand B of the first CH3 domain and Y86T in strand E of the partner CH3 domain. The change of the T22Y amino acid creates a knob, while Y86T creates a hole in the partner CH3 domain. See, e.g., Ridgway, J.B. et al. Protein Eng. 9(7):617-2 (1996).

[0187] In certain embodiments, the Fc domain can be a chimeric that combines Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain can include some or all of the CH2 sequence derived from the CH2 region of human IgG1, human IgG2, or human IgG4, and some or all of the CH3 sequence derived from human IgG1, human IgG2, or human IgG4. The chimeric Fc domain can also include a chimeric hinge region. For example, the chimeric hinge can include an "upper hinge" sequence derived from the human IgG1 hinge region, the human IgG2 hinge region, or the human IgG4 hinge region, combined with a "lower hinge" sequence derived from the human IgG1 hinge region, the human IgG2 hinge region, or the human IgG4 hinge region. Specific examples of chimeric Fc domains that can be included in any of the antigen-binding molecules described herein include, from N-terminus to C-terminus, [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any of the multispecific molecules described herein includes, from N-terminus to C-terminus, [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the multispecific molecules of the invention are described in U.S. Patent Publication No. 2014 / 0243504, published Aug. 28, 2014, which is incorporated herein by reference in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can alter Fc receptor binding.

[0188] In certain embodiments, the invention provides a multimerization domain that is an antibody heavy chain, and the heavy chain constant region (CH) region comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any wild-type allele of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or IgY.

[0189] Binding characteristics of the multispecific molecules of the invention As used herein, the term "binding," in the context of the binding of a domain, antigen-binding domain, or antibody-binding fragment that specifically binds to a molecule expressed on the surface of a cell (e.g., a T cell or a B cell) to a given antigen, such as a cell surface protein, or a fragment thereof, typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0190] For example, when the binding affinity is determined by surface plasmon resonance (SPR) technology using an antigen as a ligand and an antigen-binding domain as an analyte (or anti-ligand), e.g., in a BIAcore 3000 instrument, it typically corresponds to a KD value of about 10-7 M or less, e.g., about 10-8 M or less, e.g., about 10-9 M or less. Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31, Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0191] Thus, the domain or antigen-binding domain that specifically binds to a molecule expressed on the cell surface of the present invention binds to a given antigen or cell surface molecule (receptor) having an affinity corresponding to a KD value that is at least 10-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, the affinity of a domain that specifically binds to a molecule expressed on the surface of a cell, or an antigen-binding domain corresponding to a KD value 10-fold or less lower than that of a non-specific antigen, may be considered non-detectable binding.

[0192] The term "KD" (M) refers to the dissociation equilibrium constant of a specific domain that specifically binds to a molecule expressed on the surface of a cell or an antigen-binding domain-antigen interaction, or the dissociation equilibrium constant of a domain that specifically binds to a molecule expressed on the surface of an antigen-binding domain that binds to a cell or an antigen. There is an inverse relationship between KD and binding affinity. Thus, the smaller the KD value, the higher the affinity, i.e., the stronger. Therefore, the terms "higher affinity" or "stronger affinity" relate to a higher ability to form an interaction, i.e., a smaller KD value, and conversely, the terms "lower affinity" or "weaker affinity" relate to a lower ability to form an interaction, i.e., a larger KD value. In some situations, compared to the binding affinity of a molecule (e.g., a one-armed antibody) for another interacting partner molecule (e.g., antigen Y), a higher binding affinity (or KD) of a specific molecule (e.g., a one-armed antibody) for that interacting partner molecule (e.g., antigen X) can be expressed as a binding ratio determined by dividing a larger KD value (lower, or weaker, affinity) by a smaller KD (higher, or stronger, affinity), e.g., expressed as 5-fold or 10-fold higher binding affinity in some cases.

[0193] The term "kd" (sec-1 or 1 / sec) refers to the dissociation rate constant of a specific one-armed antibody-antigen interaction, or the dissociation rate constant of a one-armed antibody or an antibody-binding fragment. This value is also referred to as the koff value.

[0194] The term "ka" (M-1×sec-1 or 1 / M) refers to the association rate constant of a specific one-armed antibody-antigen interaction, or the association rate constant of a one-armed antibody or an antibody-binding fragment.

[0195] The term "KA" (M-1 or 1 / M) refers to the association equilibrium constant of a specific one-armed antibody-antigen interaction, or the association equilibrium constant of a one-armed antibody or an antibody-binding fragment. The association equilibrium constant is obtained by dividing ka by kd.

[0196] The term "EC50" or "EC50" refers to the half-maximal effective concentration and includes the concentration of a molecule that binds specifically to a multi-specific molecule, pMHC complex, and / or molecule expressed on the surface of a cell (e.g., a T cell) that induces a response midway between the baseline and the maximum after a specific exposure time. The EC50 essentially represents the concentration of a molecule that binds specifically to a multi-specific molecule, pMHC complex, and / or molecule expressed on the surface of a cell (e.g., a T cell) at which 50% of its maximal effect is observed.

[0197] In one embodiment, the EC50 value represents the concentration of a molecule that binds specifically to a multi-specific molecule, pMHC complex, and / or molecule expressed on the surface of a cell (e.g., a T cell) of the present invention that induces depletion of the maximum half amount of target cells by the cytotoxic activity of the T cell. Thus, an increase in cytotoxic activity (e.g., T cell-mediated tumor cell death) is observed along with a decrease in the EC50, or a decrease in the half-maximal effective concentration value.

[0198] T cell regulatory properties of multi-specific molecules The multispecific molecule of the present invention is useful for modulating the activity of T cells having specificity for the peptide component of the first binding molecule. T cells having specificity for the peptide presented in the groove of the MHC domain component of the first binding molecule bind to the peptide / MHC complex via the T cell receptor (each T cell has approximately 30,000 TCRs, and each of the TCRs contains a variable domain similar to the antigen-binding domain of an antibody). Activation or suppression of T cells is achieved based on the specificity of the antigen-binding domain of the second binding molecule. In some embodiments, the second binding molecule comprises an antigen-binding domain that specifically binds to a costimulatory molecule (e.g., CD28) on the T cell and provides a signal that induces activation, proliferation, and / or survival of the T cell. In other embodiments, the second binding molecule comprises an antigen-binding domain that specifically binds to an inhibitory molecule (e.g., LAG3) on the T cell and provides a signal that suppresses activation or induces anergy or T cell death. In some embodiments, modulation of T cell activity is achieved in vivo by administering the multispecific molecule of the present invention to a subject in need thereof. The subject in need thereof may have a disease or disorder that can be prevented, treated, or ameliorated by modulating T cell activity, or may be at high risk thereof. For example, the subject may have or be at high risk of infection, cancer, or an autoimmune disorder. In some embodiments, modulation of T cell activity is achieved ex vivo. In various embodiments, modulation of T cell activity ex vivo can be performed by obtaining T cells (CD4+ or CD8+) from a subject and culturing the T cells with a plurality of the multispecific molecules of the present invention under conditions and for a period sufficient to modulate the activity of the T cells.

[0199] In some embodiments, the multispecific molecules (e.g., bispecific molecules) of the present disclosure can be used ex vivo to modulate autologous T cells (e.g., to induce activation or anergy) for use in the treatment of diseases or disorders susceptible to T cell modulation (e.g., cancer, infectious diseases, or autoimmune disorders). For example, CD8+ and / or CD4+ T cells are obtained from a subject via apheresis and cultured under conditions that promote the activation and proliferation of T cells (e.g., CD8+ T cells) or induce anergy in T cells (e.g., CD4+ T cells) together with the multispecific molecules discussed herein, and subsequently the T cells can be reintroduced into the subject. As part of the culture process, the T cells can be selected to enrich the proportion of cells having specificity for the peptide (PiG) of the MHC component of the multispecific molecule. In some embodiments, the subject is a cancer patient and the PiG contains a fragment of a tumor-associated antigen. Autologous T cells (e.g., CD8+ T cells) are removed from the patient via apheresis and cultured under conditions that induce the activation and proliferation of T cells having specificity for the peptide together with the multispecific molecules discussed herein (e.g., a pMHC complex displaying a peptide on a class I MHC polypeptide and a bispecific molecule comprising an anti-CD28 binding domain), and subsequently the activated / proliferated T cells are reintroduced into the patient. In some embodiments, the patient is an individual suffering from an infectious disease, and autologous T cells are removed, cultured, and reintroduced in a similar manner except that the PiG contains a fragment of an infectious disease antigen associated with the patient's infection.

[0200] Clustering In some embodiments, the regulatory activity of the multispecific molecule or multispecific carrier molecule of the present invention can be enhanced by clustering T cells in proximity to each other. In some embodiments, clustering comprises bringing four or more T cells into proximity to each other such that they can bind to cytokines secreted by adjacent T cells. In some embodiments, clustering comprises bringing about 1200 or more T cells into proximity to each other such that they can bind to cytokines secreted by adjacent T cells. In various embodiments, the number of "clustered" T cells discussed herein can be about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 500, about 1000, about 5000, about 6000, about 7000, or about 8000 or more. In some embodiments, clustering comprises bringing about 1000 or more T cells together in the presence of an antigen recognized by the TCR and a co-stimulatory signal. In some embodiments, clustering comprises bringing about 1200 or more T cells together in the presence of an antigen recognized by the TCR and a co-stimulatory signal. In some embodiments, clustering comprises bringing 1500 or more T cells together in the presence of an antigen recognized by the TCR and a co-stimulatory signal.

[0201] In an in vivo environment, clustering can be achieved, for example, by binding a first molecule or a multispecific molecule to a carrier (e.g., a cell such as a B cell, a virus-like particle, etc.), thereby bringing TCR and / or co-stimulatory molecules into proximity with each other when they bind to the first molecule and / or multispecific molecule that have been aggregated on the carrier. As an example, the Fc domain (e.g., IgG1 or IgG4) of the first molecule or multispecific molecule can be for an Fcγ receptor (FcγI, FcγIIA, FcγIIB, FcγIIIA, or FcγIIIB) on a cell (e.g., a B cell). In one embodiment, the first molecule or multispecific molecule of the present invention can include an antigen-binding domain that specifically binds to a cell surface molecule or a domain (e.g., an Fc domain) fused thereto. In certain embodiments, the antigen-binding domain is linked to the C-terminus of the first molecule, the second molecule, and / or the multimerization domain. Non-limiting examples of this second antigen-binding domain can have specificity for a surface molecule (e.g., CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD35, CD38, CD180, or CD40) or a tumor-associated antigen (e.g., as defined herein). The binding of this second antigen-binding domain to its antigen on the cell surface brings together the multispecificity and / or the cluster of the first molecule and the T cell bound thereto. In various embodiments, the second antigen-binding domain can be a Fab or scFv (which, when bound to the C-terminus of the antigen-binding domain, is alternatively referred to herein as a "Stahl body").

[0202] In one embodiment, the second antigen-binding domain specifically binds to CD20. CD20 (also known as Bp35, MS4A1, LEU-16, or CVID6) (HGNC(7315), Entrez Gene(931), Ensembl(ENSG00000156738), OMIM(112210), UniProtKB(P11836), each of which is incorporated herein by reference in its entirety) is expressed on the surface of all B cells, starting from the pro-B phase (CD45R+, CD117+) and gradually increasing in concentration until maturation. CD20 is the target of monoclonal antibodies rituximab, ofatumumab, obinutuzumab, ocrelizumab, ibritumomab tiuxetan, tositumomab, and ublituximab.

[0203] In one embodiment, the second antigen-binding domain specifically binds to CD180. CD180 (also known as Bgp-95, LY64, Ly78, or RP105) (HGNC(6726), Entrez Gene(4064), Ensembl(ENSG00000134061), OMIM(602226), UniProtKB(Q99467), NP_005573.1, each of which is incorporated herein by reference in its entirety) belongs to the family of pathogen receptors, Toll-like receptors (TLRs), and is a cell surface molecule consisting of an extracellular leucine-rich repeat (LRR) and a short cytoplasmic tail. See also Miura et al., Genomics 38:299-304, 1996 and Miura et al., Blood 92:2815-2822, 1998, both of which are incorporated herein by reference in their entirety for all intended purposes. CD180 is expressed on antigen-presenting cells (e.g., B cells and dendritic cells). Anti-CD180 antibodies include RP / 14, MHR73, MHR73-11, and G28-8.

[0204] In other cases, in vivo clustering can be achieved, for example, by delivering the components of the multispecific molecules discussed herein (e.g., scGP-33-MHC and anti-CD28 binding domains) aligned on the surface of a carrier molecule. For example, the carrier molecule can include molecules containing surface alignment of pMHC complexes and anti-T cell surface molecule binding domains. The pMHC complex can be any of such molecules discussed herein (e.g., the peptide can be derived from a tumor-associated antigen), and the anti-T cell surface molecule binding domain can be any of such molecules (e.g., anti-CD28 or anti-PD1) discussed herein. In some embodiments, the carrier can be a virus-like particle (VLP) generated by overexpressing the surface proteins of interest (e.g., anti-CD28 and scMHC peptide) in producer cells and harvesting the VLP. Exemplary VLPs containing the alignment of scMHC-gp33 and anti-CD28 binding domains are discussed in Example 10.

[0205] In other embodiments, in vivo clustering can be achieved by expressing the molecules of the multispecific molecule on the surface of engineered cells and introducing the engineered cells into a subject. For example, the subject's cells (e.g., B cells) can be harvested and engineered via transfection with RNA and / or DNA or transduction via a vector (e.g., a lentiviral vector) to express a first transmembrane polypeptide containing an extracellular pMHC complex and a second transmembrane polypeptide containing an extracellular antigen-binding domain specific for a T cell surface molecule (e.g., CD28 or LAG3). In some embodiments, the second transmembrane polypeptide can be a single-chain variable fragment (scFv) containing the light-chain variable region (LCVR) and heavy-chain variable region (HCVR) of an antibody, e.g., an antibody (e.g., anti-CD28 antibody) linked to a transmembrane domain to anchor the polypeptide to the cell surface. In practice, multiple first and second transmembrane polypeptides are expressed on the surface of the engineered cells (e.g., B cells) such that reintroduction of a population of the cells into a subject enables binding and clustering of multiple peptide-specific T cells, promoting modulation of T cell activity (e.g., activation of T cells).

[0206] In an ex vivo environment, clustering can be achieved by artificially aligning the multispecific molecules in a manner that brings together groups of T cells bound to the multispecific molecules. In one embodiment, a plurality of multispecific molecules can be bound to a scaffold in a clustered arrangement in culture. As used herein, "clustered arrangement" refers to an arrangement of multispecific molecules that are close enough to each other such that bound T cells can bind to cytokines (e.g., IL-2) secreted by adjacent T cells. In another embodiment, a plurality of multispecific molecules can be clustered via one or more linkers. In some embodiments, the linker used to cluster the multispecific molecules is a multivalent antibody that is specific for a portion of the Fc domain of the multispecific molecule. In one embodiment, the linker (e.g., multivalent antibody) is provided in a 1:1 ratio with the multispecific molecule. In other embodiments, the ratio of linker (e.g., multivalent antibody) to multispecific molecule is 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, or 1:5. In another embodiment, as discussed above, a second antigen-binding domain (e.g., Fab or scFv) contained within or fused to the Fc domain of the multispecific molecule can be used in an ex vivo environment that includes cells expressing an antigen specific for the second antigen-binding domain or a scaffold containing the antigen.

[0207] Preparation / Activation of B Cells The invention also provides methods for creating cells (e.g., B cells) that express the first and second transmembrane polypeptides described herein. In one embodiment, the method includes transfecting or transducing cells isolated from a subject. In certain embodiments, the cells are isolated from an individual and genetically modified without further manipulation in vitro. In certain embodiments, the B cells are mature B cells. Thus, in certain embodiments, the cells can be directly re-administered to an individual once they are manipulated.

[0208] In a further embodiment, the cells are first stimulated / activated to proliferate in vitro before being genetically modified to express the transmembrane polypeptide. In this regard, the cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express the transmembrane polypeptide described herein). In certain examples, the increased activity can be at a level 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more, than that of non-contact cells or cells contacted with a negative control.

[0209] In certain embodiments, the B cell activating factor can bind to the C-terminus of the first and / or second molecule. In certain embodiments, B cells are activated via a peptide and / or antigen-binding domain that specifically binds to a B cell surface molecule. In some embodiments, the B cell surface molecule is CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD35, CD38, CD180, CD40, Toll-like receptor (TLR) (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13), C-type lectin receptor (CLR), or interleukin-1 receptor. In certain embodiments, the peptide and / or antigen-binding domain that specifically binds to the B cell surface molecule can bind to the C-terminus of the first and / or second molecule. In various embodiments, the antigen-binding domain that specifically binds to the B cell surface molecule can be a Fab or scFv (which, alternatively, is referred to herein as a "Stahl body").

[0210] In certain embodiments, the activation of B cells is enhanced by inducible adaptors including, but not limited to, inducible PRR adaptors (e.g., MyD88, including truncated forms such as those lacking the TIR domain, and TRIF), inducible pattern recognition receptors (e.g., NOD-like receptors such as NOD1 or NOD2), RIG-like helicases (e.g., RIG-I or Mda-5), and the CD40 cytoplasmic domain.

[0211] Prior to the in vitro manipulation or genetic modification of the cells (e.g., B cells) described herein, the cell source can be obtained from a subject. In one embodiment, cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains lymphocytes and includes T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and then placed in an appropriate buffer or medium for subsequent processing. In one embodiment of the invention, the cells are washed with PBS. In alternative embodiments, the wash solution may lack calcium, lack magnesium, or lack many, but not all, divalent cations. As will be recognized by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions, with or without buffers. In certain embodiments, the unwanted components of the apheresis sample can be removed in the cells resuspended directly in the medium.

[0212] Engineer the cells to express the first and / or second molecule The transmembrane polypeptides of the present invention are introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid can be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid can be maintained transiently or represent stable introduction. Transfection can be achieved by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun, but is not limited thereto. Transduction refers to the delivery of gene(s) using a viral vector or retroviral vector, rather by means of viral infection rather than transfection. In certain embodiments, the retroviral vector is transduced by packaging the vector into virions prior to contact with the cell. For example, the nucleic acid encoding the transmembrane polypeptide carried by the retroviral vector can be transduced into the cell via infection and proviral integration.

[0213] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material within a cell. The terms "genetically modified cell", "modified cell", and "redirected cell" are used interchangeably.

[0214] In certain embodiments, the nucleic acid or viral vector is introduced via ex vivo transformation. Methods for transfecting vascular cells and tissues removed from an organism in an ex vivo environment are known to those of skill in the art. Thus, it is contemplated that the polynucleotides presented herein can be used to remove and transfect cells or tissues ex vivo. In certain aspects, the transplanted cells or tissues can be placed within an organism. Thus, it is well within the knowledge of those of skill in the art to isolate antigen presenting cells (e.g., B cells) from an animal (e.g., a human), transfect the cells with an expression vector, and then administer the transfected or transformed cells back to the animal.

[0215] In certain embodiments, the nucleic acid or viral vector is introduced via injection. In certain embodiments, the polynucleotide can be delivered to an organelle, cell, tissue, or organism via one or more injections (i.e., needle injections) such as, for example, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, etc. Methods of injecting vaccines are well known to those of skill in the art (e.g., injection of a composition containing saline). Further embodiments include the introduction of the polynucleotide by direct microinjection. The amount of expression vector used can vary depending on the antigen and the nature of the organelle, cell, tissue, or organism being used.

[0216] In certain embodiments, the polynucleotide is introduced into an organelle, cell, tissue, or organism via electroporation. Electroporation involves exposing cells and a suspension of DNA and / or RNA to a high voltage discharge. In some variations of this method, certain cell wall degrading enzymes such as pectinase are used to make the target recipient cells more susceptible to transformation by electroporation than untreated cells (see U.S. Patent No. 5,384,253, which is incorporated herein by reference).

[0217] In certain embodiments, the polynucleotide is delivered to the cell using DEAE-dextran, followed by polyethylene glycol (see, e.g., Gopal, T.V., Mol Cell Biol. 1985 May;5(5):1188-90), sonication loading (see, e.g., Fechheimer et al., (1987) Proc. Nat’l Acad. Sci. USA, 84, 8463-8467), liposome-mediated transfection, receptor-mediated delivery vehicle transfection (see, e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432, Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990, Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994, Myers, EPO0273085), and / or microprojectile bombardment (see, e.g., U.S. Patent Nos. 5,550,318, 5,538,880, 5,610,042, and PCT Application No. 94 / 09699). Each reference listed in this paragraph is hereby incorporated by reference in its entirety for all intended purposes.

[0218] In certain embodiments, the polynucleotides encoding the first and second transmembrane polypeptides described herein are inserted into a vector(s). A vector refers to a vehicle into which a polynucleotide encoding a protein can be covalently inserted such that the polynucleotide results in expression of the protein and / or cloning of the polynucleotide. Such a vector may also be referred to as an “expression vector.” The isolated polynucleotide can be inserted into the vector using any suitable method known in the art. For example, without limitation, the vector can be digested using appropriate restriction enzymes and then ligated to the isolated polynucleotide having matching restriction ends. An expression vector has the ability to incorporate and express a heterologous or modified nucleic acid sequence encoding at least a portion of a gene product that can be transcribed intracellularly. In most cases, the RNA molecule is then translated into a protein. An expression vector can contain various control sequences, which refer to nucleic acid sequences necessary for transcription or translation of an operably linked coding sequence in a particular host organism. In addition to the control sequences that regulate transcription and translation, vectors and expression vectors can further contain nucleic acid sequences that serve other functions. An expression vector can contain additional elements. For example, an expression vector can have two replication systems and thus be maintained in two organisms, such as human cells for expression and a prokaryotic host for cloning and amplification.

[0219] Expression vectors can have promoter sequences such as CMV, PGK, and EF1 alpha promoters, ribosome recognition and binding TATA boxes, and necessary 5' upstream and 3' downstream regulatory elements such as the 3'UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in their respective host cells. Other suitable promoters include constitutive promoters such as the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters can also be used, including but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, inducible promoters are also contemplated as part of vectors that express transmembrane polypeptides. This provides a molecular switch that can turn on or turn off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include but are not limited to the metallothionein promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0220] Expression vectors can have additional sequences such as 6× histidine (SEQ ID NO: 29), c-Myc, and FLAG tags incorporated into the expressed polypeptide. Thus, expression vectors can be engineered to include 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences that can promote or enhance the efficient transcription of the nucleic acid(s) of interest carried by the expression vector. Expression vectors can also be engineered for replication and / or expression functionality (e.g., transcription and translation) in specific cell types, cell locations, or tissue types. Expression vectors can include selectable markers for maintenance of the vector in the host or recipient cell.

[0221] In various embodiments, the vector is a plasmid, an autonomous replication sequence, and a transposable element. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs) such as artificial chromosomes, bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40), but are not limited thereto. Examples of expression vectors are the Lenti-XTM bicistronic expression system (Neo) vector (Clontech), pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST.TM., pLenti6 / V5-DEST.TM., and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the transmembrane polypeptides disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0222] In certain embodiments, the nucleic acid encoding the transmembrane polypeptide of the invention is provided in a viral vector. The viral vector can be derived from a retrovirus, a lentivirus, or a foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. The viral vector can contain the coding sequences of the various chimeric proteins described herein in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of introducing DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0223] In certain embodiments, the viral vector comprising the coding sequence of the transmembrane polypeptide described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector that contains structural and functional genetic elements mainly derived from retroviruses. The term "lentiviral vector" refers to a vector that contains structural and functional genetic elements outside the LTR mainly derived from lentiviruses.

[0224] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). The "retroviruses" of the present invention also include lentiviruses of the family Retroviridae such as human T cell leukemia virus, HTLV-1 and HTLV-2, and human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0225] The lentiviral vector for use in this specification refers to a vector derived from lentiviruses, which are a group (or genus) of retroviruses that cause slowly developing diseases. Viruses included in this group are HIV (human immunodeficiency virus including HIV type 1 and HIV type 2), Visna / maedi, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Preparation of recombinant lentiviruses can be achieved using the methods by Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72:8463-8471 and Zufferey et al., J. Virol. 1998; 72:9873-9880).

[0226] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present invention can be generated using standard cloning techniques by combining the desired DNA sequences in accordance with the instructions and adaptations described herein (Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals, Eglitis, et al. (1985) Science 230:1395-1398, Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464, Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018, Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145, Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043, Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381, Chowdhury et al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644, Kay et al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895, Hwu et al. (1993) J. Immunol 150:4104-4115, U.S. Patent No. 4,868,116, U.S. Patent No. 4,980,286, PCT Application No. 89 / 07136, PCT Application No. 89 / 02468, PCT Application No. 89 / 05345, and PCT Application No. 92 / 07573).

[0227] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) arrays for use in generating vectors include, for example, genomic RNA and cDNA obtainable from commercial sources including the Type Culture Collection (ATCC), Rockville, Md. The arrays can also be chemically synthesized.

[0228] For expression of the first and second transmembrane polypeptides, the vector(s) can be introduced into a host cell to enable expression of the polypeptide(s) within the host cell. The expression vector can include various elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selectable marker, and a signal sequence. These elements can be appropriately selected by one of ordinary skill in the art as described above. For example, the promoter sequence can be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the β-actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. The enhancer sequence can be selected to enhance transcription of the polynucleotide. The selectable marker can enable selection of host cells into which the vector has been inserted from those that have not, and, for example, the selectable marker can be a gene conferring antibiotic resistance. The signal sequence can be selected to enable the expressed polypeptide to be transported outside of the host cell.

[0229] In cloning of a polynucleotide, the vector is introduced into a host cell (an isolated host cell) to enable replication of the vector itself and thereby amplify copies of the polynucleotide contained therein. Cloning vectors generally can include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by one of ordinary skill in the art. For example, the origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0230] In certain embodiments, the disclosure provides an isolated host cell (e.g., a B cell) comprising the vectors provided herein. A host cell comprising a vector can be useful in the expression or cloning of the polynucleotide contained in the vector.

[0231] Sequence variant The antigen-binding domains of the multispecific molecules of this specification may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding domains may be derived from any of the exemplary amino acid sequences disclosed herein, and one or more amino acids within one or more framework and / or CDR regions may be mutated to the corresponding residue(s) of the germline sequence from which the antibody is derived, or to the corresponding residue(s) of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a number of antigen-binding domains and antigen-binding fragments containing one or more individual germline mutations or combinations thereof starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, the mutated residues are found within the first 8 amino acids of FR1, or the mutated residues are found within the last 8 amino acids of FR4, or the mutated residues are found only within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain was originally derived). Further, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence.Once obtained, an antigen-binding domain comprising one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, etc. Multispecific molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed within the scope of the present invention.

[0232] The invention also includes an antigen-binding domain that includes a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that has one or more conservative substitutions. For example, the invention includes an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. "Conservative amino acid substitution" means that an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445 (incorporated herein by reference). A "moderately conservative" replacement is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0233] The invention also includes an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. When referring to amino acid sequences, the terms "substantial identity" or "substantially identical" mean that two amino acid sequences share at least 95% sequence identity, more preferably at least 98%, or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using a defined gap weight. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference).

[0234] Sequence similarity to a polypeptide, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similar measurements assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used with default parameters for determining sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein, including programs such as Gap and Bestfit. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, which uses default or recommended parameters of the GCG version 6.1 program FASTA. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the best overlapping regions between the query sequence and the search sequence and percent sequence identity (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each incorporated herein by reference.

[0235] Preparation of antigen-binding domains and construction of multispecific molecules Antigen-binding domains specific for a particular antigen (e.g., CD28 or CTLA-4) can be prepared by any antibody generation technique known in the art. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the antigen-binding domains of the present invention are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the antigen-binding domains of the multispecific molecules of the present invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technique), high-affinity chimeric antibodies against a particular antigen (e.g., CD28 or CTLA-4) having human variable regions and murine constant regions are first isolated. Antibodies are characterized and selected for desirable properties, including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the multispecific molecules of the present invention.

[0236] Genetically engineered animals can be used to generate human antigen-binding domains. For example, genetically modified mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene of the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human antigen-binding domains. By fully human is meant an antibody, or an antigen-binding domain or fragment thereof, that contains an amino acid sequence encoded by DNA derived from human sequences over the entire length of each polypeptide of the antibody, or its antigen-binding domain or fragment. In some cases, the fully human-like sequences are derived from proteins that are endogenous to humans. In other examples, the fully human-like proteins or protein sequences contain chimeric sequences in which each component sequence is derived from human sequences. Without being bound by any theory, chimeric proteins or chimeric sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, for example as compared to any wild-type human immunoglobulin region or domain.

[0237] Once obtained, the antigen-binding domain can be appropriately positioned relative to the peptide-MHC polypeptide component and the multimerization domain, and the multispecific molecules of the invention can be produced using conventional methods, for example as discussed in Example 1.

[0238] Biological equivalents The present invention encompasses multispecific molecules having an amino acid sequence that differs from those of the exemplary molecules disclosed herein but retains the ability to bind to a specific antigen (CD28 or CTLA-4) and a specific T cell receptor. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described multispecific molecules.

[0239] The present invention includes multispecific molecules that are biologically equivalent to any of the exemplary multispecific molecules (including antigen-binding domains and peptide-MHC fusion polypeptides) described herein. Two such multispecific molecules are considered to be biological equivalents if, for example, they show no significant difference in absorption rate and extent of absorption when administered at the same molar dose, either as a single dose or multiple doses, under similar experimental conditions, and are pharmaceutical equivalents or pharmaceutical alternatives. Some multispecific molecules may be considered equivalents or pharmaceutical alternatives if the extent of their absorption is equivalent but the absorption rate is not, and such differences in absorption rate are intentional and reflected in the labeling, and are considered to be biologically equivalent, for example, if they are not essential for achieving an effective body drug concentration for long-term use and are considered not medically significant for the particular formulation being studied.

[0240] In one embodiment, two multispecific molecules are biologically equivalent if there are no clinically significant differences in their safety, purity, and efficacy. In one embodiment, two multispecific molecules are biologically equivalent if they can be switched in a patient one or more times without switching between a reference product and a biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or a decrease in efficacy, and compared to a therapy that is sustained.

[0241] In one embodiment, two multispecific molecules are biologically equivalent if they both act by a common mechanism or mechanism of action for the conditions of use (s), to the extent that such a mechanism is known.

[0242] Biological equivalence can be demonstrated by in vivo and / or in vitro methods. Biological equivalence assays include, for example, (a) in vivo tests in humans or other mammals in which the concentration of a multispecific molecule or its metabolite is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro tests that reasonably predict this data, correlated with human bioavailability data, (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of the multispecific molecule (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or biological equivalence of the multispecific molecule.

[0243] Biologically equivalent variants of the exemplary multispecific molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges during regeneration. In other contexts, biologically equivalent multispecific molecules can include variants of the exemplary multispecific molecules described herein (including antigen-binding domains, peptide-MHC polypeptides, and multimerization domains) that contain amino acid changes that modify the glycosylation properties of the molecule, such as mutations that eliminate or remove glycosylation.

[0244] Therapeutic Formulations and Administration The present invention provides a pharmaceutical composition comprising the multispecific molecule of the present invention. The pharmaceutical composition of the present invention is formulated with suitable carriers, excipients, and other agents that provide improved movement, delivery, resistance, etc. Many suitable formulations can be found in the formulary known to all pharmacists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) - containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA, etc.), DNA conjugates, anhydrous absorbent pastes, oil - in - water emulsions and water - in - oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi - solid gels, and semi - solid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238 - 311.

[0245] The dosage of the multispecific molecule administered to a patient can vary depending on the patient's age and physical build, target disease, condition, route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. When the multispecific molecule of the present invention is used for therapeutic purposes in adult patients, it may be advantageous to administer the multispecific molecule of the present invention intravenously in a single dose of usually about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule for administering the multispecific molecule are determined empirically. For example, the patient's progress can be monitored by regular evaluation and the dosage adjusted accordingly. Further, interspecies scaling of dosages can be performed using methods well - known in the art (e.g., Mordenti et al., 1991, Pharmaceut.Res. 8:1351).

[0246] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Routes of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by absorption through the epithelium or mucosal lining (such as oral mucosa, rectal and intestinal mucosa, etc.) by injection or bolus injection, and can be administered together with other biologically active agents. Administration can be systemic or local.

[0247] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen delivery devices are readily applicable for delivering the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0248] Numerous reusable pen delivery devices and auto-injector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Some examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having applications in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ pen (Abbott Labs, Abbott Park IL).

[0249] In certain situations, the pharmaceutical composition can be delivered by a sustained release system. In one embodiment, a pump can be used (see Langer, supra, Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used, see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the sustained release system can be placed in the vicinity of the target of the composition, whereby only a small fraction of the systemic dose is required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other sustained release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0250] Injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection and intramuscular injection, infusion, etc. These injectable preparations can be prepared by publicly known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying the multispecific molecule or a salt thereof described above in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of the aqueous medium for an injection solution include physiological saline, isotonic solutions containing glucose, and other adjuvants, which can be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The injection solution thus prepared is preferably filled into appropriate ampoules.

[0251] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms of suitable unit dosages to match the dosages of the active ingredients. Such dosage forms of unit dosages include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the aforementioned multispecific molecule contained is generally about 5 to about 500 mg per dosage form in unit dosage, and particularly in the form of injection, the aforementioned multispecific molecule is about 5 to about 100 mg, and for other dosage forms, it is preferably contained in an amount of about 10 to about 250 mg.

[0252] Therapeutic use of multispecific molecules The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising a multispecific molecule discussed herein. The therapeutic composition may comprise any of the multispecific molecules disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "a subject in need thereof" means a subject suffering from an infectious disease (e.g., a subject suffering from a bacterial or viral infection, including any of those mentioned herein), cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned herein), an autoimmune disorder (e.g., a subject suffering from any of the autoimmune diseases or disorders mentioned herein), an inflammatory disease, or a human or non-human animal presenting one or more symptoms or signs that would otherwise benefit from the enhancement or suppression of T cell activity.

[0253] In another aspect, a method of treating a disorder in a subject in need of treatment of the disorder is described herein, and comprises administering to the subject an effective amount of a multispecific molecule described herein, wherein the multispecific molecule binds to an antigen-specific TCR, and the antigen recognized by the TCR is associated with the disorder.

[0254] The multispecific molecules of the present invention (and therapeutic compositions containing the same) are useful, inter alia, for treating any disease or disorder in which stimulation or suppression of an immune response targeted to a specific antigen (via T cell modulation) is beneficial. In particular, the multispecific molecules of the present invention can be used for the treatment and prevention of infectious diseases, cancer, or autoimmune disorders.

[0255] When the multispecific molecule described herein comprises a second molecule comprising a domain that specifically binds to a T cell surface molecule that is an activating polypeptide, transduction of T cells with the multispecific molecule activates 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 multispecific molecule increases the cytotoxic activity of the T cells against the cancer cells. In some embodiments, 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 multispecific molecule increases the number of epitope-specific T cells.

[0256] In some embodiments, the epitope-specific T cells are T cells specific for an epitope present on a virus-infected cell, and contacting the epitope-specific T cells with the multispecific molecule increases the cytotoxic activity of the T cells against the virus-infected cells. In some cases, the epitope-specific T cells are T cells specific for an epitope present on a virus-infected cell, and contacting the epitope-specific T cells with the multispecific molecule increases the number of epitope-specific T cells.

[0257] When the multispecific molecule described herein comprises a second molecule comprising a domain that specifically binds to a T cell surface molecule that is an inhibitory polypeptide, contacting the T cells with the multispecific molecule inhibits epitope-specific T cells. In some cases, the epitope-specific T cells are autoreactive T cells specific for an epitope present in a self-antigen, and contacting reduces the number of autoreactive T cells.

[0258] The interaction between a T cell and a multispecific molecule described herein can result in, for example, activation, induction of anergy, or death of the T cell when the TCR of the T cell is bound by a TCR-binding pMHC complex. "Activation of a T cell" refers to the induction of a signal transduction pathway within the T cell that results in the production of cell products (e.g., interleukin-2) by that T cell. "Anergy" refers to a decrease in the responsiveness of a T cell to an antigen. Activation and anergy can be measured, for example, by measuring the amount of IL-2 produced by the T cell after the pMHC complex has bound to the TcR. Anergic cells will have a reduced production of IL-2 when compared to stimulated T cells. Another method for measuring the reduced activity of anergic T cells involves measuring intracellular and / or extracellular calcium mobilization by the T cell upon engagement of the TCR. "T cell death" refers to the permanent cessation of substantially all functions of the T cell.

[0259] T cell phenotypes can be evaluated using well-known methods. For example, T cell activation can be determined by measuring, for example, changes in the expression levels of cytokines and / or T cell activation markers, and / or the induction of antigen-specific proliferating cells. Expression cytokines and T cell activation markers can be measured using techniques known to those skilled in the art, including but not limited to immunoprecipitation followed by Western blot analysis, ELISA, flow cytometry, Northern blot analysis, and RT-PCR. Cytokine release can be measured by measuring the secretion of cytokines including but not limited to interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-16 (IL-16), PDGF, TGF-α, TGF-β, TNF-α, TNF-β, GCSF, GM-CSF, MCSF, IFN-α, IFN-β, IFN-γ, TFN-γ, IGF-I, and IGF-II (see, for example, Isaacs et al., 2001, Rheumatology, 40:724-738, Soubrane et al., 1993, Blood, 81(1):15-19).

[0260] T cell regulation can also be evaluated, for example, by measuring proliferation, for example, by 3H-thymidine incorporation, trypan blue cell counting, and fluorescence-activated cell sorting (FACS).

[0261] The anti-tumor response of T cells after exposure to the multispecific molecules described herein can be determined in a xenograft tumor model. The tumor can be established using any human cancer cell line that expresses a tumor-associated antigen presented by the multispecific molecule. To establish a xenograft tumor model, about 5×106 viable cells can be injected, for example, subcutaneously (s.c.) into nude athymic mice using, for example, Matrigel (Becton Dickinson). The endpoints of the xenograft tumor model can be determined based on tumor size, animal body weight, survival time, and histochemical and histopathological examination of the cancer using methods known to those skilled in the art.

[0262] The anergy or death of T cells after exposure to the multispecific molecules described herein, which may be useful for the treatment of inflammatory and autoimmune disorders, can be tested in vitro or in vivo, for example, by a 51Cr release assay. The ability to mediate depletion of peripheral blood T cells can be evaluated, for example, by measuring T cell counts using flow cytometry analysis.

[0263] Non-limiting examples of useful animal models for analyzing the effect of exposure of T cells to the multispecific molecules described herein on inflammatory diseases include adjuvant-induced arthritis rat models, collagen-induced arthritis rats and mouse models, and antigen-induced arthritis rats, rabbits, and hamster models (see, for example, Crofford L.J. and Wilder R.L., “Arthritis and Autoimmunity in Animals”, in Arthritis and Allied Conditions, A Textbook of Rheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993), Trenthorn et al., 1977, J. Exp. Med. 146:857, Courtenay et al., 1980, Nature 283:665, Cathcart et at, 1986, Lab. Invest. 54:26, Holmdahl, R., 1999, Curr. Biol. 15:R528-530). Other useful animal models of inflammatory diseases include, for example, animal models of inflammatory bowel disease, ulcerative colitis, and Crohn's disease induced by sulfated polysaccharides (e.g., amylopectin, collagen, amylopectin sulfate, dextran sulfate) or chemical stimulants (e.g., trinitrobenzenesulfonic acid (TNBS) or acetic acid). See, for example, Kim et al., 1992, Scand. J. Gastroenterol. 27:529-537, Strober, 1985, Dig. Dis. Sci. 30(12 Suppl):3S-10S).

[0264] Additional useful models include, for example, animal models for asthma such as adoptive transfer models in which airborne allergen challenge of Th1 or Th2 recipient mice results in the migration of TH effector cells to the airways and is associated with a strong neutrophil (TH1) and eosinophil (TH2) pulmonary mucosal inflammatory response (see, e.g., Cohn et al., 1997, J. Exp. Med. 186:1737-1747). A useful animal model for studying the effect of the multispecific molecules of the present invention on multiple sclerosis (MS) is the experimental allergic encephalomyelitis (EAE) model (see, e.g., Zamvil et al, 1990, Ann. Rev, Immunol. 8:579). Animal models that can be used to analyze the effect of the multispecific molecules of the present invention on autoimmune disorders such as type 1 diabetes, thyroid autoimmunity, systemic lupus erythematosus, and glomerulonephritis have also been developed (see, e.g., Bluestone et al., 2004, PNAS 101:14622-14626, Flanders et al., 1999, Autoimmunity 29:235-246, Krogh et al., 1999, Biochimie 81:511-515, Foster, 1999, Semin. Nephrol. 19:12-24).

[0265] The effectiveness of the multispecific molecules disclosed herein for downregulating the immune response in the treatment of autoimmune disorders can be evaluated, for example, by their ability to reduce one or more symptoms of an autoimmune disorder, reduce the mean absolute lymphocyte count, reduce T cell activation, reduce T cell proliferation, reduce cytokine production, or regulate one or more specific cytokine profiles (e.g., interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-16 (IL-16), PDGF, TGF-α, TGF-β, TNF-α, TNF-β, GCSF, GM-CSF, MCSF, IFN-α, IFN-β, IFN-γ, TFN-γ, IGF-I, and IGF-II)) (see, e.g., Isaacs et al., 2001, Rheumatology, 40:724-738; Soubrane et al., 1993, Blood, 81(1):15-19).

[0266] The effectiveness of a multispecific molecule for use in the treatment of diabetes can be evaluated, for example, by its ability to reduce one or more symptoms of diabetes, maintain the C-peptide response to MMTT, reduce the level of HA1 or HA1c, reduce the daily insulin requirement, or reduce T cell activation in pancreatic tissue. The effectiveness in the treatment of arthritis can be evaluated via determination of the joint count of tenderness and swelling, determination of the overall score of pain and disease activity, ESR CRP, determination of the progression of structural joint damage (e.g., quantitative scoring (Sharp method) of hand, wrist, and foot x-rays), determination of changes in functional status (e.g., evaluated using the Health Assessment Questionnaire (HAQ)), or determination of changes in quality of life (e.g., evaluated using the SF-36).

[0267] In related aspects, a method of treating a disorder in a subject in need thereof is disclosed herein, comprising administering to the subject an effective amount of a multispecific molecule described herein, the multispecific molecule binds to an antigen-specific TCR, and the antigen is associated with the disorder. In some embodiments, the disorder is an inflammatory or autoimmune disorder, and the administration results in downregulation of the inflammatory or autoimmune response. In one particular embodiment, the disorder is celiac disease or gluten sensitivity. In one particular embodiment, the antigen comprises gliadin or a fragment thereof (e.g., (i) an α-gliadin fragment corresponding to amino acids 57-73, or (ii) a γ-gliadin fragment corresponding to amino acids 139-153, or (iii) an ω-gliadin fragment corresponding to amino acids 102-118). In one particular embodiment, the multispecific molecule presents a peptide derived from the antigen in the context of class II MHC. In some embodiments, the disorder is a tumor, and the administration results in upregulation of the anti-tumor immune response. In another embodiment, the disorder is an infection caused by an infectious agent, and the administration results in upregulation of the immune response against the infectious agent. In one particular embodiment, the infectious agent is selected from the group consisting of viruses, bacteria, fungi, protozoa, parasites, helminths, and ectoparasites. In one particular embodiment, the infectious agent is lymphocytic choriomeningitis virus (LCMV), and the antigen is the gp33 protein. In one particular embodiment, the multispecific molecule presents a peptide derived from the antigen in the context of class I MHC. In some embodiments, the subject is a mammal (e.g., human).

[0268] According to certain aspects, the multispecific molecules of the present invention target tumor cells expressing tumor-associated antigens, such as adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, ALK, BAGE proteins (e.g., BAGE-1), BIRC5 (survivin), BIRC7, beta-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CAGE-1-8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen ("ETA"), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE proteins (e.g., GAGE-1-8), GD2, GD3, GloboH, glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, also known as CCDC110 and KMHN1, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, LMP2, M-CSF,Cancer expressing tumor-associated antigens selected from the group consisting of MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, -A9, -A10, -A12, -C1, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE proteins (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, cerulinin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-beta, TMPRSS2, Thompson-Nouvel antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1 can be used to treat. In some embodiments, the peptide is a neoantigen. In some embodiments, the peptide is a tumor-specific antigen. Specific cancers / tumors treatable by the methods and multispecific molecules of the present invention include various solid malignancies, carcinomas, lymphomas, sarcomas, blastomas, and leukemias, butNot limited. As non-limiting specific examples, for example, breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, kidney cancer, bladder cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain tumors of all histopathological types, angiosarcoma, hemangiosarcoma, osteosarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, Ewing tumor, leiomyosarcoma, Ewing sarcoma, rhabdomyosarcoma, cancer of unknown primary origin (CUP), squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenström macroglobulinemia, papillary thyroid carcinoma, cystadenocarcinoma, bronchiogenic carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, lung cancer, epithelial cancer, cervical cancer, testicular tumor, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, anaplastic glioma, meningioma, retinoblastoma, leukemia, neuroblastoma, small cell lung cancer, bladder cancer, lymphoma, multiple myeloma, myeloid cancer, B-cell lymphoma, T-cell lymphoma, NK-cell lymphoma, large granular lymphocyte lymphoma or leukemia, gamma-delta T-cell lymphoma or gamma-delta T-cell leukemia, mantle cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, hematopoietic tumor, thymoma, sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Epstein-Barr virus (EBV)-induced malignant tumors of all types including EBV-related Hodgkin lymphoma and non-Hodgkin lymphoma, all forms of post-transplant lymphoma including post-transplant lymphoproliferative disorder (PTLD), uterine cancer, renal cell cancer, hepatocellular cancer, hepatoblastoma are included. Cancers that can be treated by the methods and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, cancer includes, but is not limited to,Specifically, it can be of the following histological types: malignant tumor, cancer tumor, undifferentiated cancer tumor, giant cell and spindle cell cancer, small cell cancer, papillary cancer, squamous cell cancer, lymphoepithelial cancer, basal cell cancer, pilomatrix carcinoma, transitional cell cancer, papillary transitional cell cancer, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, cord adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyp, familial polyposis colorectal adenocarcinoma, solid cancer, malignant carcinoid tumor, bronchiolo-alveolar adenocarcinoma, papillary adenocarcinoma, chromophobic cancer, eosinophilic cancer, eosinophilic adenocarcinoma, basophilic cancer, clear cell adenocarcinoma, granular cell cancer, follicular adenocarcinoma, papillary and follicular adenocarcinoma, unencapsulated sclerosing cancer, adrenocortical carcinoma, endometroid cancer, skin appendage cancer, apocrine adenocarcinoma, sebaceous gland carcinoma, ceruminous gland carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, acinar cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant cystic tumor, malignant granulosa cell tumor, and malignant roblastoma, Sertoli cell carcinoma, malignant Leydig cell tumor, malignant lipoid cell tumor, malignant paraganglioma, malignant extra-mammary paraganglioma, pheochromocytoma, glomangiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant melanoma of giant pigmented nevus, epitheloid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, fetal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, Müllerian duct mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, undifferentiated embryonal cell tumor, embryonal carcinoma, malignant teratoma, malignant struma ovarii, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant angioendothelioma, Kaposi sarcoma, malignant pericytic tumor, lymphangiosarcoma, osteosarcoma, juxtacortical osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing sarcoma, malignant odontogenic tumor, ameloblastic odontogenic sarcoma, malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, epithelioma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma,Astrocytoma, astrocytoma, oligodendroglioma, oligoastrocytoma, primitive neuroectodermal, cerebellar sarcoma, ganglioblastoma, neuroblastoma, retinoblastoma, olfactory nerve tumor, malignant meningioma, neurofibrosarcoma, malignant schwannoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, side granuloma, small lymphocyte malignant lymphoma, large cell diffuse malignant lymphoma, follicular malignant lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myelogenous leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblast leukemia, myelosarcoma, and hairy cell leukemia.,

[0269] The present invention also includes a method for treating residual cancer in a subject. As used herein, the term "residual cancer" means the presence or persistence of one or more cancerous cells in a subject after treatment with anti-cancer therapy.

[0270] Non-limiting examples of inflammatory and autoimmune diseases include, for example, inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease, diabetes (e.g., type 1 diabetes), multiple sclerosis, arthritis (e.g., rheumatoid arthritis), Graves' disease, lupus erythematosus, ankylosing spondylitis, psoriasis, Behcet's disease, autistic enteritis, Guillain-Barré syndrome, myasthenia gravis, pemphigus vulgaris, acute disseminated encephalomyelitis (ADEM), transverse myelitis, autoimmune myocarditis, celiac disease, dermatomyositis, Wegener's granulomatosis, allergies, asthma, contact dermatitis, arteriosclerosis (or any other inflammatory condition affecting the heart or vascular system), autoimmune uveitis, and other autoimmune skin pathologies, autoimmune kidney, lung, or liver pathologies, autoimmune neuropathy, asthma, allergies, celiac disease, systemic lupus erythematosus (SLE), scleroderma, sarcoidosis, thyroiditis, multiple sclerosis, spondylitis, periarteritis, eczema, atopic dermatitis, myasthenia gravis, insulin-dependent diabetes, Crohn's disease, Guillain-Barré syndrome, Graves' disease, glomerulonephritis, ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, psoriatic arthritis, reactive arthritis, spondyloarthritis, psoriasis, acute or chronic immune diseases, inflammatory diseases associated with organ transplantation, rejection of skin or organ transplantation, graft-versus-host disease (GVHD), or autoimmune diseases, and administering to a subject a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising a multispecific molecule described herein).Examples of autoimmune diseases include, for example, glomerulonephritis, arthritis, dilated cardiomyopathy-like diseases, ulcerative colitis, Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, rheumatic fever, vitiligo, insulin-dependent diabetes, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture's syndrome, infertility, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, antiphospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, autoimmune achlorhydria, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus erythematosus, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis, some embodiments of lymphopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, lens-induced uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, limited scleroderma (or CREST syndrome), sympathetic ophthalmia, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis, hyperthyroidism, type B insulin resistance, ulcerative colitis, and Wegener's granulomatosis.

[0271] In another embodiment, the methods described herein are used to treat or prevent transplant-related conditions. In another embodiment, the methods described herein are used to treat or prevent graft-versus-host disease. In another embodiment, the methods described herein are used to treat or prevent post-transplant lymphoproliferative disorders.

[0272] According to certain aspects, the multispecific molecules of the invention can be used to treat infectious diseases such as bacterial infections (e.g., bacterial infections resistant to conventional antibiotics) or viral infections. In certain embodiments, the multispecific molecules are designed to present peptides derived from viral or bacterial antigens. In some embodiments, the viral antigen is derived from a virus selected from the group consisting of adenovirus, astrovirus, chikungunya, cytomegalo, dengue, Ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, Lassa, measles, metapneumovirus, mumps, norovirus, Oropouche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, Usutu, vaccine, varicella, West Nile, yellow fever, or Zika. In some embodiments, the bacterial antigen is derived from a bacteria selected from the group consisting of methicillin-resistant Staphylococcus Aureus (MRSA), Clostridium Difficile, carbapenem-resistant Enterobacteriaceae, drug-resistant Neisseria Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, extended-spectrum β-lactamase-producing bacteria, vancomycin-resistant enterococci, multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, vancomycin-resistant Staphylococcus Aureus, erythromycin-resistant group A Streptococcus, or clindamycin-resistant group B Streptococcus.

[0273] The multispecific molecules of the present invention designed to treat cancer or an infectious disease may include an antigen-binding domain (e.g., a one-armed antibody) on a second binding molecule that specifically binds to a T cell co-stimulatory molecule (e.g., CD28) to induce the activation, proliferation (e.g., clonal expansion), and / or survival of T cells (e.g., CD8+ T cells) specific for a peptide presented on a first binding molecule. In some embodiments, T cell activation is restored. In some embodiments, naive T cells are activated or proliferation is induced. Such T cells can enhance or stimulate an immune response against cells (e.g., tumor cells or infected cells) that express a protein containing the peptide presented on the first binding molecule of the multispecific molecule. In various embodiments, the multispecific molecule does not induce the proliferation of non-specific T cells (i.e., T cells that are not specific for the peptide presented on the first binding molecule).

[0274] According to certain aspects, the multispecific molecules of the present invention can be used to treat, prevent, or ameliorate an autoimmune disease or disorder by targeting the activity of T cells specific for a peptide corresponding to an antigen associated with the autoimmune disease or disorder. For example, the antigen can be selected from the group consisting of gliadin (celiac disease, e.g., (i) an α-gliadin fragment corresponding to amino acids 57-73, or (ii) a γ-gliadin fragment corresponding to amino acids 139-153, or (iii) an ω-gliadin fragment corresponding to amino acids 102-118), GAD 65, IA-2, and insulin B chain (in the case of type 1 diabetes), glatiramer acetate (GA) (in the case of multiple sclerosis), acetylcholine receptor (AChR) (in the case of myasthenia gravis), p205, insulin, thyroid stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)). In some embodiments, the antigen can be IL-4R, IL-6R, or DLL4.

[0275] The multispecific molecules of the present invention designed to treat autoimmune disorders include an antigen-binding domain (e.g., a one-armed antibody) on a second binding molecule that specifically binds to a T cell inhibitory molecule (e.g., CTLA-4, LAG3, PD1, etc.) to inhibit the activity of T cells (e.g., CD4+ T cells) specific for a peptide presented on a first binding molecule. Such inhibition or suppression of T cell activity can treat, alleviate, or prevent the recurrence of an autoimmune disease or disorder in which the cells targeted by the individual's immune system express a protein containing the peptide presented on the first binding molecule of the multispecific molecule. In some embodiments, administration of the multispecific molecules of the present invention can be used to render the individual's T cells tolerant to the self-antigen to which the T cells are specific.

[0276] The present invention also includes the use of the multispecific molecules discussed herein in the manufacture of a medicament for preventing, treating, and / or ameliorating an infectious disease, cancer, or autoimmune disorder (e.g., as discussed herein).

[0277] Combination Therapies and Formulations When used to treat various diseases, it is contemplated that the compositions and methods can be combined with other therapeutic agents suitable for the same or similar diseases. Also, two or more embodiments described herein can be co-administered to produce additive or synergistic effects. When co-administered with a second therapeutic agent, the embodiments described herein and the second therapeutic agent can be simultaneous or sequential (in any order). Due to additive or synergistic effects, the preferred therapeutically effective dosage of each agent may be reduced.

[0278] As a non-limiting example, the methods described herein can be combined with other therapies that block inflammation (e.g., via blockade of IL1, INFα / β, IL6, TNF, IL13, IL23, etc.).

[0279] In some embodiments, the compositions and methods disclosed herein are useful for enhancing the effectiveness of vaccines directed against tumors or infectious diseases. Accordingly, the compositions and methods described herein can be administered to a subject prior to (e.g., 1 to 30 days before) the administration of a reagent (including but not limited to small molecules, antibodies, or cellular reagents) that acts to induce an immune response (e.g., for treating cancer or an infectious disease) to the subject.

[0280] The compositions and methods described herein can also be administered in combination with an anti-tumor antibody or antibody directed against a pathogenic antigen or allergen. The compositions or methods of the present disclosure can be used in combination with other immunomodulatory therapies such as, for example, therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to agents that enhance 41BB, OX40, etc.). The inhibitory therapies described herein can also be combined, in any of the foregoing forms or formulations, alone, with each other, or in combination with other agents, with other therapies that have the ability to modulate the NKT function or stability, including but not limited to any other of the antigens, CD1d chimeric antigen receptors (CD1d-CAR), or any of the five known CD1 isomers present in humans (CD1a, CD1b, CD1c, CD1e), whether unloaded or loaded with any of CD1d, CD1d fusion proteins, CD1d dendrimers, or large polymers of CD1d.

[0281] The treatment methods described herein can be combined with additional immunotherapies and therapies. For example, when used to treat cancer, the NKT cells described herein can be used in combination with conventional cancer therapies such as surgery, radiation therapy, chemotherapy, or combinations thereof, depending on the type of tumor, the patient's condition, other health problems, and various factors. In certain embodiments, other therapeutic agents useful in combination cancer therapy with the inhibitors described herein include anti-angiogenic agents. For example, many anti-angiogenic agents have been identified and are known in the art, including TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR, and FLT-1 receptor, placental proliferin-related protein, and those listed by Carmeliet and Jain (2000). In some embodiments, the inhibitors described herein can be used in combination with VEGF antagonists or VEGFR antagonists such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0282] The present invention provides a method comprising administering a pharmaceutical composition comprising any of the exemplary multispecific molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that can be combined with or co-administered with the multispecific molecules of the present invention include, for example, EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), antagonists of other EGFR family members such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibodies, or small molecule inhibitors of ErbB2, ErbB3, or ErbB4 activity), antagonists of EGFRvIII (e.g., antibodies that specifically bind to EGFRvIII), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies), VEGF antagonists (e.g., see VEGF trap, e.g., US7,087,411, also referred to herein as the "VEGF inhibitory fusion protein"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US2009 / 0142354, e.g., REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US2011 / 0027286, e.g., H1H685P), FOLH1 (PSMA) antagonists, PRLR antagonists (e.g., anti-PRLR antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin antibodies), and the like.Other agents that can be beneficially administered in combination with the multispecific molecules of the present invention include cytokine inhibitors, including cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or small molecule cytokine inhibitors and antibodies that bind to their respective receptors. The pharmaceutical compositions of the present invention also include "ICE": ifosfamide (e.g., Ifex (trademark)), carboplatin (e.g., Paraplatin (registered trademark)), etoposide (e.g., Etopophos (registered trademark), Toposar (registered trademark), VePesid (registered trademark), VP-16), "DHAP": dexamethasone (e.g., Decadron (registered trademark)), cytarabine (e.g., Cytosar-U (trademark), cytosine arabinoside, ara-C), cisplatin (e.g., Platinol (registered trademark)-AQ), and "ESHAP": etoposide (e.g., Etopophos (registered trademark), Toposar (trademark), VePesid (registered trademark), VP-16), methylprednisolone (e.g., Medrol (registered trademark)), high-dose cytarabine, cisplatin (e.g., Platinol (registered trademark)-AQ), and can be administered as part of a treatment regimen comprising a combination of one or more therapeutic agents selected therefrom.

[0283] The present invention also encompasses a therapeutic combination comprising any of the antigen-binding molecules referred to herein, and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment, F(ab’)2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or diabody, triabody, tetrabody, minibody, and minimal recognition unit). The multispecific molecules of the present invention can also be administered in combination with and / or co-formulated with an antiviral agent, an antibiotic, an analgesic, a corticosteroid and / or an NSAID. The antigen-binding molecules of the present invention may also be administered as part of a treatment regimen that also includes radiotherapy and / or conventional chemotherapy.

[0284] Non-limiting examples of chemotherapeutic compounds that can be used in combination therapy include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, irinotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0285] These chemotherapeutic compounds can be classified, for example, according to their mechanism of action, into the following groups: metabolic inhibitors / anticancer agents such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)), anti-proliferative / mitotic inhibitors (natural products such as vinca alkaloids (vinblastine, vincristine, vinorelbine)), microtubule-disrupting agents (e.g., taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilone, and navelbine, epipodophyllotoxins (etoposide, teniposide)), DNA-damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16)), antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin), enzymes (L-asparaginase which metabolizes L-asparagine systemically, depleting cells and not having the ability to synthesize its own asparagine), antiplatelet agents, anti-proliferative / anti-mitotic alkylating agents (e.g., nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes - dacarbazine (DTIC), anti-proliferative / anti-mitotic metabolic antagonists (e.g., folic acid analogs (methotrexate)),Platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide, hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole), anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors), fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab, anti-migratory agents, anti-secretory agents (brefeldin), immunosuppressive agents (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil), anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab), and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors), angiotensin receptor blockers, nitric oxide donors, antisense oligonucleotides, antibodies (trastuzumab), cell cycle inhibitors and differentiation inducers (tretinoin), mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, etoposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone), growth factor signaling kinase inhibitors, mitochondrial dysfunction inducers, and caspase activators, and chromatin disruptors.,

[0286] For the treatment of infectious diseases, the combination therapies described herein can include co-administering the compositions and methods described herein with antibiotics, antifungal agents, antiviral agents, anti-parasitic agents, anti-protozoal agents, or combinations thereof.

[0287] Non-limiting examples of useful antibiotics include lincosamides (clindamycin), chloramphenicol, tetracyclines (tetracycline, chlortetracycline, demeclocycline, methacycline, doxycycline, minocycline, etc.), aminoglycosides (gentamicin, tobramycin, netilmicin, amikacin, kanamycin, streptomycin, neomycin, etc.), beta-lactams (penicillin, cephalosporin, imipenem, aztreonam, etc.), vancomycin, bacitracin, macrolides (erythromycin), amphotericin, sulfonamides (sulfanilamide, sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole, sulfacytine, sulfadoxine, mafenide, p-aminobenzoic acid, trimethoprim-sulfamethoxazole, etc.), methenamine, nitrofurantoin, phenazopyridine, trimethoprim, rifampicin, metronidazole, cefazolin, lincomycin, spectinomycin, mupirocin, quinolones (nalidixic acid, cinoxacin, norfloxacin, ciprofloxacin, perfloxacin, ofloxacin, enoxacin, fleroxacin, levofloxacin, etc.), novobiocin, polymyxin, gramicidin, and anti-Pseudomonas aeruginosa drugs (carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, piperacillin, etc.), or any salts or variants thereof.See also Physician’s Desk Reference, 59th edition, (2005), Thomson PDR, Montvale N.J., Gennaro et al., Eds.; Remington’s The Science and Practice of Pharmacy, 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md., Braunwald et al., Eds.; Harrison’s Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY, Berkow et al., Eds.; The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J. Such antibiotics can be purchased, for example, from Daiichi Sankyo, Inc. (Parsipanny, N.J.), Merck (Whitehouse Station, N.J.), Pfizer (New York, N.Y.), Glaxo Smith Kline (Research Triangle Park, N.C.), Johnson & Johnson (New Brunswick, N.J.), AstraZeneca (Wilmington, Del.), Novartis (East Hanover, N.J.), and Sanofi-Aventis (Bridgewater, N.J.). The antibiotics used vary depending on the type of bacterial infection.

[0288] Non-limiting examples of useful antifungal agents include imidazoles (such as griseofulvin, miconazole, terbinafine, fluconazole, ketoconazole, voriconazole, and itraconazole), polyenes (such as amphotericin B and nystatin), flucytosine, and candicidin, or any salts or variants thereof. See also Physician’s Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J., Gennaro et al., Eds. Remington’s The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md., Braunwald et al., Eds. Harrison’s Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY, Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0289] Non-limiting examples of useful antiviral agents include interferon alpha, beta, or gamma, didanosine, lamivudine, zanamivir, lopinavir, nelfinavir, efavirenz, indinavir, valacyclovir, zidovudine, amantadine, rimantadine, ribavirin, ganciclovir, foscarnet, and acyclovir, or any salts or variants thereof. See also Physician’s Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J., Gennaro et al., Eds. Remington’s The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md., Braunwald et al., Eds. Harrison’s Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY, Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0290] Non-limiting examples of useful anti-parasitic agents include chloroquine, mefloquine, quinine, primaquine, atovaquone, sulfasoxine, and pyrimethamine, or any salts or variants thereof. See also Physician’s Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J., Gennaro et al., Eds. Remington’s The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md., Braunwald et al., Eds. Harrison’s Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY, Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0291] Non-limiting examples of useful antiprotozoal drugs include metronidazole, diloxanide, iodoquinol, trimethoprim, sulfamethoxazole, pentamidine, clindamycin, primaquine, pyrimethamine, and sulfadiazine, or any salts or variants thereof. See also Physician’s Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J., Gennaro et al., Eds. Remington’s The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md., Braunwald et al., Eds. Harrison’s Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY, Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0292] Additional therapeutic active component(s) may be administered immediately before, simultaneously with, or immediately after administration of the multispecific molecule of the invention. (For the purposes of this disclosure, such an administration regimen is considered an administration of the multispecific molecule “in combination with” the additional therapeutic active component(s)).

[0293] The present invention includes pharmaceutical compositions in which the multispecific molecule of the invention is co-formulated with one or more of the additional therapeutic active component(s) described elsewhere herein. Administration regimen According to certain embodiments of the invention, multiple doses of the multispecific molecule can be administered to a subject over a defined period of time. A method according to this aspect of the invention comprises continuously administering multiple doses of the multispecific molecule of the invention to a subject. As used herein, "continuously administering" means that each dose of the multispecific molecule is administered to the subject on different days separated by different time points, e.g., at predetermined intervals (e.g., several hours, several days, several weeks, or several months). The invention includes methods comprising continuously administering to a patient a single initial dose of the multispecific molecule, followed by one or more secondary doses of the multispecific molecule, and then optionally one or more tertiary doses of the multispecific molecule.

[0294] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the chronological order of administration of the multispecific molecule of the invention. Thus, the "initial dose" is the dose administered at the start of the treatment regimen (also referred to as the "baseline dose"), the "secondary dose" is the dose administered after the initial dose, and the "tertiary dose" is the dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose can all contain the same amount of the multispecific molecule, but generally can differ from each other with respect to the frequency of administration. However, in certain embodiments, the amount of the multispecific molecule contained in the initial dose, secondary dose, and / or tertiary dose can differ from each other (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency.

[0295] In certain exemplary embodiments of the present invention, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, 13, 13 and 1 / 2, 14, 14 and 1 / 2, 15, 15 and 1 / 2, 16, 16 and 1 / 2, 17, 17 and 1 / 2, 18, 18 and 1 / 2, 19, 19 and 1 / 2, 20, 20 and 1 / 2, 21, 21 and 1 / 2, 22, 22 and 1 / 2, 23, 23 and 1 / 2, 24, 24 and 1 / 2, 25, 25 and 1 / 2, 26, 26 and 1 / 2, or more) weeks after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means, in a series of multiple administrations, the dose of the multispecific molecule administered to a patient prior to the administration of the immediately succeeding dose without an intervening dose.

[0296] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses of the multispecific molecule to a patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0297] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The dosing frequency may also be adjusted by a physician during the course of treatment according to the needs of an individual patient following clinical examination.

Example

[0298] The following examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the methods and compositions of the present invention and are not intended to limit the scope that the inventors regard as the present invention. Similarly, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is limited only by the terms of the appended claims and the full scope of equivalents to which such claims are entitled.

[0299] Efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0300] Example 1: Generation of Multispecific T Cell Activators Multispecific molecules were prepared by expression of two plasmids exemplified in Figure 1 in a CHO cell line. The resulting structure of the multispecific molecules is also shown in Figure 1. The multispecific molecules comprise a first molecule comprising an anti-CD28 specific binding domain (heavy and light chains), and a second molecule comprising, from 5' to 3', a lymphocytic choriomeningitis virus (LCMV) glycoprotein 33 (GP33) peptide, a beta-2 microglobulin protein, an H2-Db MHC protein, and an hIgG4 Fc as a single-chain peptide-MHC-Fc fusion protein. In this example, the Fc regions of both molecules are of the IgG4 isotype and contain a chimeric hinge. The peptide-MHC molecule further comprises an Fc region containing a modified CH3 domain. The modified CH3 domain was prepared with the dipeptide modification H435R / Y436F (H95R / Y96F according to IMGT exon numbering) by EU numbering (also known as FcΔAdp described in US20100331527, which is hereby incorporated by reference in its entirety for all purposes). The presence of the dipeptide modification in one heavy chain of the multispecific monomer facilitates differential purification of the heterodimer via differential affinity for protein A in a chromatography platform, while the other does not facilitate differential purification of the heterodimer via differential affinity for protein A in a chromatography platform.

[0301] The hamster anti-CD28 binding domain was recombinantly produced (PV-1 clone, Abe R, et al. J. Immunol. 154:985-997, 1995). The nucleic acid and amino acid sequence identifiers are listed in Table 2.

[0302]

Table 2

[0303] The anti-CD28 light-chain nucleic acid was cloned upstream of the IgG4 constant light chain (IgG4 CL) and downstream of the CMV promoter in one plasmid (monocistronic plasmid, Figure 1). As shown in Figure 1, both 1) the peptide-MHC-Fc fusion protein and 2) the two Fc-containing polynucleotides encoding the anti-CD28 heavy chain were cloned into a second plasmid (bicistronic plasmid). The GP33 nucleic acid (9-amino acid sequence KAVYNFATM, SEQ ID NO: 6, PDB: 2F74_C) encoding the peptide (PiG) within the groove of the MHC complex was cloned downstream of the signal sequence. The peptide linker binds PiG to β2-microglobulin, β2-microglobulin to the H2-Db MHC protein, and the H2-Db MHC protein to IgG4Fc. Transcription of each Fc-containing polynucleotide is driven by its own upstream CMV promoter / intron.

[0304] Example 2: Isolation of IgG4 Antigen (GP33)-Specific Cytotoxic T Cells Spleen cells were isolated from mice previously infected with LCMV Armstrong (2×105 ffui.p., more than 21 days post-infection). CD8+ T cells were enriched from spleen cells using negative selection with the EasySep Mouse CD8+ T Cell Isolation Kit (StemCell Technologies), removing all other (non-CD8+) T cell phenotypes. Confirmation of CD8+ T cell enrichment was performed by flow cytometric gating on singlet, live, lymphocyte cells, staining with live / dead stain (Life Technologies), anti-mCD8a (Biolegend catalog number 100724), and anti-mCD4 (Biolegend catalog number 100428). The gating strategy for the enrichment of CD8+ T cells from C57BL / 6 spleen cells via flow cytometry is shown in Figure 2. For verification of CD8 T cell enrichment, samples were acquired on a BD FACSCanto II and analyzed using FlowJo software (TreeStar). Small resting lymphocytes (gated on SSC-A × FSC-A parameters) were further gated for singlets (FSC-H × FSC-A) and live cells (live / dead stain negative). Next, live singlet cells were plotted for the mCD4 × mCD8a parameters. Spleen cells enriched for CD8+ T cells showed more than 90% of cells staining positive for the CD8 marker, compared to approximately 6% CD8+ cells in non-enriched spleen cells.

[0305] The isolated CD8+ T cells were maintained in T cell growth medium (RPMI 1640, 10% FBS, 1% 100×PSG, 2-mercaptoethanol (5 μM), sodium pyruvate (1 mM), HEPES (20 mM), 8 ng / μl IL-2, delivered IL-7 at 10 ng / ml) for the proliferation experiments discussed in the following examples.

[0306] Example 3: Proliferation of Cytotoxic T Cells Experienced with Antigen (GP33) Cell culture conditions: CD8+ T cells were labeled with the CellTrace Violet Cell Proliferation Kit (Invitrogen) and cultured in a 24-well plate at a final concentration of 2×106 cells / ml in 0.5 ml of medium. As shown in Example 4 below, the cytokines IL-7 (10 μg / ml) and IL-2 (8 ng / μl) were added to the culture on days 1 and 4, respectively.

[0307] A gating strategy for evaluating the proliferation of GP33-specific CD8+ T cells (isolated in Example 2) in the culture is shown in Figure 3. T cells were contacted with the plate-bound version of the multispecific molecule of Example 1, and the corresponding proliferation was compared to an unstimulated control (T cells cultured in the absence of the multispecific molecule). Note that the CellTrace dye intensity decreases by 50% with each cell division, and the degree of stimulation / activation is measured by the degree of loss of the proliferation dye. Cells were stained with CellTrace, placed in the culture under various stimulation conditions, and grown for 7 days. On day 7, the cells were removed and stained with live / dead stain, anti-mCD8a (Biolegend catalog number 100724), and anti-gp33 tetramer. Samples were acquired on a BD FACSCANTO II and analyzed using FlowJo software. Cells were gated using the SSC-A × FSC-A parameters, live / dead stain-negative cells, and mCD8a-positive cells, and then analyzed for the GP33 tetramer × proliferation dye (CellTrace) parameters. Unstimulated cells maintained bright CellTrace staining after culture (right panel), while cells stimulated with scMHC / GP33 × anti-mCD28 multispecificity showed a significant decrease in CellTrace brightness in the tetramer-positive stained cells (approximately 39% tetramer+ proliferation dye dim), indicating a higher degree of cell division.

[0308] Example 4: Effect of plate-bound multispecific GP33-MHC × anti-CD28 on the proliferation of antigen (GP33)-specific CD8+ T cells stimulated with or without cytokines In this example, the effect of cytokines (IL-2 and IL-7) in the culture on the proliferation of in vitro-stimulated GP33-specific CD8+ T cells was examined with plate-bound multivalent GP33-MHC×anti-CD28 (see Example 1). The multivalent molecule of Example 1 was prepared at 30 nM (about 5 μg / ml) in PBS solution, and 300 μl of the solution was added to each well of a 24-well culture plate. The plate was sealed and incubated at 37 °C for 2 hours or overnight at 4 °C. The solution was removed immediately before adding 5×10 5 cells in 0.5 ml of medium. As described above, the CellTrace dye intensity decreased by 50% with each cell division, and the degree of stimulation / activation was measured by the degree of loss of the proliferation dye.

[0309] As shown in Figure 4, CD8+ T cells cultured with the multivalent molecule showed maximal proliferation of GP33-specific T cells that did not occur in unstimulated cultures. Note in Figure 4 that the number of cells specific for the antigen (GP33) increased by nearly 39% in the population of cells stimulated with the plate-bound multivalent molecule and maintained in culture with cytokines (see Quadrant 1 (Q1)). Despite the presence of cytokines, antigen-specific T cells were rarely observed in the population without the plate-bound multivalent molecule. Cytokines in the cell culture supported T cell survival (comparing both stimulation panels), but the proliferation of GP33-specific T cells was dependent on multivalent stimulation and not due to cytokines alone.

[0310] Example 5: Effect of titration of a polyclonal antibody crosslinker against multivalent GP33-MHC×anti-CD28 on the proliferation of antigen (GP33)-specific CD8+ T cells in vitro In this example, the effect of titration of a polyclonal antibody crosslinker (Thermo Scientific Pierce, goat anti-human IgG(H+L), cross-adsorbed secondary antibody, Prod number 31119, 1.8 mg / ml (MW ~144 kDa)) against the multispecific GP33-MHC×anti-CD28 (see Example 1) was examined for the proliferation of GP33-specific CD8+ T cells in vitro. The crosslinking polyclonal Ab was pre-complexed with the multispecific molecule at the ratios shown in Figure 5 before addition to the CD8+ T cells. Briefly, goat polyclonal anti-human IgG(H+L) and the multispecific molecule were combined in a tube at a molar ratio in the range of 5:1 to 1:5 (150 nM:30 nM to 30 nM:150 nM) of pAb / multispecific antibody in 0.25 ml of medium. The mixture was incubated on ice for 15 minutes and then added to 0.25 ml of cells, incubated on ice for a further 15 minutes, and then placed in a 24-well plate at 37 °C. Cytokines (IL-2 and IL-7) were used for cell culture. As described above, the CellTrace dye intensity decreased by 50% with each cell division, and the degree of stimulation / activation was measured by the degree of loss of the proliferation dye.

[0311] Cells were cultured for 7 days with the complex of pAb and multispecific molecule formed by varying the ratio of crosslinking pAb to multispecificity and then analyzed for proliferation as described in Example 3. Cells cultured with complexes derived from crosslinker-to-multispecific ratios greater than 1 generally showed better stimulation of GP33-specific cells than ratios with less crosslinker than multispecificity. A 1:1 ratio (30 nM:30 nM) of polyclonal Ab to multispecific molecule provided efficient crosslinking and thus appeared to provide efficient proliferation of antigen-specific T cells (Q1 = 17.9%). All test ratios of crosslinker to multispecific molecule (5:1 to 1:5) showed significant Ag-specific T cell proliferation compared to control wells containing the multispecific molecule without any crosslinker, as shown in Table 3 below (lower right panel).

[0312]

Table 3

[0313] Example 6: Stimulation of antigen-specific CD8+ T cells with multispecific GP33-MHC×anti-CD28 having IgG1 Fc aligned on human embryonic kidney (HEK293-hFcR1) cells In this example, the stimulation of antigen-specific CD8+ T cells was measured by adding multispecific GP33-MHC×anti-CD28 having IgG1 Fc to the co-culture of CD8+ T cells derived from LCMV-immunized mice with HEK293 cells expressing hFcgR1 (or alternatively, parental HEK293 control cells not expressing hFcgR1). Briefly, HEK293 cells were pretreated with 50 μg / ml mitomycin C for 1 hour at 37°C and washed twice with PBS before co-culturing with T cells. Both cell types were resuspended in T cell culture medium and combined in a 96-well round-bottom culture plate at a 1:1 cell ratio in 0.2 ml of medium. The multispecific GP33-MHC×anti-CD28 IgG1 reagent was added to the co-culture at the concentrations shown in Figure 6 and incubated overnight at 37°C. IL-2 (8 ng / ml) and IL-7 (10 ng / ml) were added, and on day +1, the cultures were expanded in 0.5 ml of medium in 24-well plates. The cultures were grown for 4 days and evaluated for growth as described above. As shown in Figure 6, robust gp33-specific T cell proliferation was observed only when the IgG1 Fc multispecific molecule was co-cultured with 293 cells expressing hFcgR1 and not with parental 293 cells. Furthermore, this effect was titratable with higher concentrations of the multispecific molecule that mediated more robust T cell proliferation, but no T cell proliferation was observed in the absence of the multispecific molecule reagent.

[0314] Nucleic acid and amino acid sequence identifiers are listed in Table 4.

[0315] [Table 4]

[0316] Example 7: Stimulation of antigen-specific CD8+ T cells with multispecific GP33-MHC × anti-CD28 having IgG4 / 2 stealth Fc arrayed on human embryonic kidney (HEK293-anti-hFc scFv) cells In this example, the method described in Example 6 was used to measure the stimulation of antigen-specific CD8+ T cells, and multispecific GP33-MHC × anti-CD28 having IgG4 Fc (Example 1) was bound to HEK293 cells via the binding of IgG4 Fc to anti-human Fc scFv expressed on HEK293 cells. As shown in Figure 7, robust gp33-specific T cell proliferation was observed only when the IgG4 Fc multispecific molecule was co-cultured with 293 cells expressing anti-hFc scFv, and not with parental 293 cells. This effect was titratable with higher concentrations of the multispecific molecule that mediated more robust T cell proliferation, but no T cell proliferation was observed in the absence of the multispecific molecule reagent.

[0317] Nucleic acid and amino acid sequence identifiers are listed in Table 5.

[0318] [Table 5]

[0319] Example 8: Stimulation of antigen-specific CD8+ T cells with multispecific GP33-MHC × anti-CD28 having IgG4 / 2-stealth Fc and C-terminal anti-CD20 scFv arrayed on cells In this example, a multispecific GP33-MHC×anti-CD28 having IgG4 Fc (see Example 1) further includes a C-terminal anti-CD20 scFv (also known as the mCD20 Stahl body), and the stimulation of antigen-specific CD8+ T cells was measured that aligns the multispecific molecule on primary B cells via CD20 binding. Primary mouse B cells were enriched from mouse splenocytes using an immunomagnetic negative selection protocol (EasySep™ Mouse B Cell Isolation Kit, Stem Cell Technologies). Similar to the method of the previous example, the mCD20 Stahl body was added at various concentrations to wells containing CD8+ T cells from LCMV-immunized mice and B cells enriched from naive mouse spleens. As shown in FIG. 8, robust gp33-specific T cell proliferation was observed when the mCD20 Stahl body was co-cultured with primary B cells. This effect was titratable with higher concentrations of the multispecific molecule that mediate more robust T cell proliferation, but no T cell proliferation was observed in the absence of the multispecific molecule reagent. Human CD20 and human CD19 scFv Stahl body variants were also tested and showed similar results.

[0320] Nucleic acid and amino acid sequence identifiers are set forth in Table 6.

[0321]

Table 6

[0322] Example 9: Induction of T Cell Division by Plate-Bound or Cell-Bound Multispecific GP33-MHC×anti-CD28 Having a C-Terminal scFv for Binding to Cell Surface Molecules In this example, a multispecific GP33-MHC × anti-CD28 having IgG4 Fc (Example 1) and further comprising a C-terminal anti-CD20 scFv (referred to herein as Stahl body) was added to T cell cultures under the following four conditions to measure the induction of antigen-specific CD8+ T cell proliferation. 1) Plate-bound reagent, 2) soluble reagent, 3) soluble reagent containing co-cultured primary B cells, or 4) soluble reagent containing co-cultured Jurkat cells. The flow cytometry plots and histogram analysis of Figure 9 show that presenting the mCD20 Stahl body to T cells using B cells results in greater maximum proliferation of Ag-specific T cells compared to cultures of the Stahl body with only T cells and cultures of the Stahl body with T cells and CD20-negative Jurkat cells. The maximum proliferation of T cells from Stahl body / B cell co-cultures was similar to the proliferation profile observed with the plate-bound reagent positive control.

[0323] Example 10: Stimulation of antigen-specific CD8+ T cells with virus-like particles (VLPs) in which scGP33-MHC and anti-CD28 are aligned In this example, the stimulation of antigen-specific CD8+ T cells was measured using single-stranded GP33-MHC and virus-like particles (VLPs) aligned with anti-CD28. The VLPs were produced on the surface with scMHCgp33 or scMHCova257 in combination with a membrane version of the anti-mCD28 antibody (clone PV-1). Briefly, 293T cells were transfected with packaging plasmid psPAX2 and expression constructs for transmembrane scMHCp, transmembrane anti-mCD28 HC, and anti-CD28 LC. The VLPs were harvested from the supernatant and concentrated using ultracentrifugation with a 20% sucrose cushion. The VLP pellet was rehydrated overnight at 4 °C in 40 μl of PBS, aliquoted, and stored at -80 °C. The VLP concentration was evaluated using the Lenti-XTM qRT-PCR Titration Kit (Takara, catalog number 631235). As shown in Figure 10, CD8+ T cells from either LCMV-immunized mice or OT1 mice (specific for the ova257 epitope) were cultured for 4 days with the indicated titers of VLPs and evaluated for proliferation. scMHCgp33 VLPs specifically stimulated and proliferated gp33 T cells from LCMV-immunized mice, while scMHCova257 VLPs specifically stimulated and proliferated OT1 CD8 T cells.

[0324] Example 11: Activation and Proliferation of Antigen-Specific T Cells Using B Cells Presenting Single-Stranded GP33-MHC or Single-Stranded OVA-MHC In this example, the stimulation of antigen-specific CD8+ T cells was measured, in which single-stranded GP33-MHC or single-stranded ovalbumin (OVA) peptide-MHC containing a C-terminal anti-CD20 scFv (alias mCD20 Stahl body) aligned multispecific molecules on primary B cells via CD20 binding. Primary mouse B cells were enriched from mouse spleen cells using an immunomagnetic negative selection protocol (EasySep(™) Mouse B Cell Isolation Kit, Stem Cell Technologies). The B cells were cultured with 50 μg / ml of LPS for 48 hours and then, by the spinoculation method, 5×10 4RV genome / cells were used to transduce with retrovirus (RV) and incubated for 24 hours before the co-culture experiment. As shown in Figure 11A (the first panel), scMHCgp33 B cells co-cultured with CD8 + T cells from LCMV-immunized mice induced specific proliferation of gp33 tetramer-positive cells compared to scMHCova B cells, non-transduced activated B cells, and a control of T cells only (Figure 11A (the third panel and the fourth panel respectively)) (Figure 11A (the second panel)). The numerous tetramer-negative dividing T cells observed with specific scMHCova B cell stimulation may be due to downregulation of the Ag-specific TCR by continuous stimulation during culture. Figure 11B shows OTI CD8 + T cells (T cells obtained from transgenic homozygous mice contain inserts of the murine Tcra-V2 and Tcrb-V5 genes, and the transgenic T cell receptor is designed to recognize ovalbumin residues 257-264 in the context of MHC-Kb) provided further evidence of the in vitro antigen specificity of the engineered B cells observed. scMHCova or irrelevant control scMHC-P15E RV-transduced B cells (5x10 4 RV genome / cells) were co-cultured with a defined mixture of OTI Thy1.2+CD8 T cells and naive Thy1.1+ innate B cells (1:3 ratio). Staining for Thy1.2 confirmed that nearly 100% of the dividing cells responding to scMHCova B cells were OTI cells. T cells co-cultured with scMHC-P15e B cells or non-transduced B cells were similar to the T cell-only control.

[0325] Nucleic acid and amino acid sequence identifiers are listed in Table 7.

[0326]

Table 7

[0327] Example 12: In vivo delivery of scMHCova B cells and non-irrelevant scMHCgp33 B cells is OTI CD8 +Specifically stimulate the proliferation of T cells In this example, primary mouse B cells were enriched from naive mouse splenocytes as described in the previous example and then cultured with 50 μg / ml of LPS for 48 hours. B cells were treated ex vivo under the following conditions to generate cells presenting specific antigen peptides: O...

Claims

1. A multispecific molecule that can bind to antigen-specific T cell receptors (TCRs) expressed on the surface of cells, wherein the multispecific molecule is (i) a peptide (p) (pMHC complex) presented in the context of a major histocompatibility complex (MHC) molecule, and (ii) a first polypeptide comprising a first human IgG Fc domain, and (i) a domain that specifically binds to CD28, and (ii) a second polypeptide comprising a second human IgG Fc domain. A multispecific molecule containing [this component].

2. The multispecific molecule according to claim 1, wherein the first human IgGFc domain and / or the second human IgGFc domain is a human IgG1 Fc domain or a human IgG4 Fc domain.

3. The multispecific molecule according to claim 1 or 2, wherein the first and second human IgGfc domains are the same.

4. The multispecific molecule according to claim 1 or 2, wherein the first human IgGfc domain and / or the second human IgGfc domain includes an amino acid sequence that facilitates the purification of the multispecific molecule.

5. The multispecific molecule according to claim 4, wherein the amino acid sequence that facilitates the purification of the multispecific molecule is a knob-into-hole mutation, or an amino acid substitution that results in weak binding to the Fc-binding affinity matrix or does not result in detectable binding.

6. The multispecific molecule according to claim 1 or 2, wherein the first human IgGfc domain and / or the second human IgGfc domain exhibit enhanced Fcγ-receptor binding activity for wild-type human IgG1.

7. The multispecific molecule according to claim 1 or 2, wherein the domain that specifically binds to CD28 includes Fab or single-stranded Fv (scFv).

8. The multispecific molecule according to claim 1 or 2, wherein the pMHC complex comprises (i) a class I MHC polypeptide, or a fragment, variant, or derivative thereof, and (ii) a β2 microglobulin polypeptide, or a fragment, variant, or derivative thereof.

9. The multispecific molecule according to claim 8, wherein the pMHC complex comprises a class I MHC polypeptide linked to a β2-microglobulin polypeptide via a peptide linker.

10. The multispecific molecule according to claim 1 or 2, wherein the pMHC complex comprises a human class I MHC polypeptide which is at least one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G, or a mouse class I MHC polypeptide which is at least one of H-2K, H-2D, H-2L, H-2Q, H-2M, or H-2T.

11. The first polypeptide is (i) a peptide, (ii) β ２ - A multispecific molecule according to claim 1 or 2, comprising (iii) a microglobulin polypeptide, or a class I MHC alpha chain polypeptide, or a fragment, variant, or derivative thereof, and (iv) the first human IgGfc domain.

12. The multispecific molecule according to claim 11, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) a β2-microglobulin polypeptide, (iii) a class I MHC alpha chain, or a fragment, variant, or derivative thereof, and (iv) the first human IgG Fc domain.

13. The first polypeptide is formed from the N-terminus to the C-terminus by (i) a peptide, (ii) an optional first linker, and (iii) β ２ - A multispecific molecule according to claim 12, comprising (iv) a microglobulin polypeptide, or a fragment, variant, or derivative thereof; (v) an optional second linker; (v) a class I MHC alpha chain domain 1, 2 and / or 3, or a fragment, variant, or derivative thereof; (v) an optional third linker; and (vi) the first human IgG Fc domain.

14. The multispecific molecule according to claim 1 or 2, wherein the pMHC complex comprises a class II MHC polypeptide, or a fragment, variant, or derivative thereof.

15. The multispecific molecule according to claim 14, wherein the pMHC complex comprises class II MHC alpha and beta chain polypeptides, or fragments, variants, or derivatives thereof.

16. The multispecific molecule according to claim 15, wherein the class II MHC alpha and beta chain polypeptides are linked by a peptide linker.

17. The multispecific molecule according to claim 16, wherein the class II MHC alpha and beta chain polypeptides are derived from (i) a human class II MHC which is at least one of HLA DP, HLA-DR, and HLA-DQ, or (ii) a mouse H-2A or H-2E class II MHC complex.

18. The multispecific molecule according to claim 14, wherein the first polypeptide comprises (i) a peptide, (ii) a class II MHC alpha chain polypeptide, or a fragment, variant, or derivative thereof, (iii) a class II MHC beta chain polypeptide, or a fragment, variant, or derivative thereof, and (iv) the first human IgG Fc domain.

19. The multispecific molecule according to claim 1 or 2, wherein the pMHC complex comprises a class II MHC alpha chain extracellular domain or a class II MHC beta chain extracellular domain.

20. The multispecific molecule according to claim 19, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, (i) a peptide, (iii) a class II MHC alpha chain extracellular domain, or a fragment, variant, or derivative thereof, (iii) a class II MHC beta chain extracellular domain, or a fragment, variant, or derivative thereof, and (iv) the first human IgG Fc domain.

21. The multispecific molecule according to claim 19, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, (i) a peptide, (iii) a class II MHC beta chain extracellular domain, or a fragment, variant, or derivative thereof, (iii) a class II MHC alpha chain extracellular domain, or a fragment, variant, or derivative thereof, and (iv) the first human IgG Fc domain.

22. The multispecific molecule according to claim 20, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) an optional first linker, (iii) class II MHC alpha-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (iv) an optional second linker, (v) class II MHC beta-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (vi) an optional third linker, and (vii) the first human IgG Fc domain.

23. The multispecific molecule according to claim 21, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, (i) a peptide, (ii) an optional first linker, (iii) class II MHC beta-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (iv) an optional second linker, (v) class II MHC alpha-chain domains 1 and 2, or fragments, variants, or derivatives thereof, (vi) an optional third linker, and (vii) the first human IgG Fc domain.

24. The multispecific molecule according to claim 1 or 2, wherein the peptide is derived from a viral antigen or a bacterial antigen.

25. The multispecific molecule according to claim 24, wherein the viral antigen is derived from a virus, and the virus is adenovirus, astrovirus, chikungunya, cytomegalovirus, dengue, Ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, lassa, measles, metapneumovirus, mumps, norovirus, oropouche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, usutu, vaccinia, varicella, West Nile, yellow fever, or Zika.

26. The bacterial antigen is derived from bacteria, and the bacteria include methicillin-resistant Staphylococcus aureus (MRSA), Clostridium Difficile, carbapenam-resistant Enterobacteriaceae, and drug-resistant Neisseria. Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, fluconazole-resistant Candida, extended-spectrum β-lactamase-producing bacteria, vancomycin-resistant Enterococcus, multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoid Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, vancomycin-resistant Staphylococcus Aureus, erythomycin-resistant group A Streptococcus, or clindamycin-resistant group B The multispecific molecule according to claim 24, which is Streptococcus.

27. The multispecific molecule according to claim 1 or 2, wherein the peptide is derived from a tumor-associated antigen.

28. The tumor-associated antigens include adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, ALK, BAGE protein (e.g., BAGE-1), BIRC5 (Survivin), BIRC7, β-catenin, BRCA1, BORIS, B-RAF, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING- 4. CA-125, CALCA, carcinoembryonic antigen ("CEA"), CAGE-1 to 8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, Cyclin-B1, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), EphA3, Epithelial tumor antigen ("ETA"), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE ​​protein (e.g., GAGE-1-8), GD2, GD3, GloboH, Glypican-3, GM3, gp100, GAS7, GnTV, gp100 / Pme117, GPNMB, GnTV, HAUS3, Hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13R alpha 2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, LMP2, M-CSF, MAGE protein (e.g., MAGE-A1, -A2, -A3, -A4,-A6, -A9, -A10, -A12, -C1, and -C2), malate enzyme, mammoglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, Mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, Myosin Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP 1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE protein (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, Cesernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, Survivin, SYT-S The multispecific molecule according to claim 27, which is at least one of the following: SX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, telomerase, TPBG, TRAG-3, triose phosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, or WT-1.

29. The multispecific molecule according to claim 1 or 2, wherein the peptide is derived from an antigen associated with autoimmune disorders.

30. The multispecific molecule according to claim 29, wherein the antigen associated with the autoimmune disorder is gliadin, GAD65, IA-2, insulin B chain, glatiramer acetate (GA), acetylcholine receptor (AChR), p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP1, myelin antigen, IL-4R, IL-6R, or DLL4.

31. The multispecific molecule according to claim 1 or 2, wherein the first human IgGfc domain, the second human IgGfc domain, or both of the first and second human IgGfc domains can bind to an Fcγ receptor expressed on a cell, and can cluster approximately 1200 or more peptide-specific T cells.

32. A bispecific molecule, as described in claim 1 or 2.

33. A pharmaceutical composition comprising a multispecific molecule according to claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.

34. A nucleic acid molecule encoding the multispecific molecule described in claim 1 or 2.

35. An in vitro method for regulating the activity, proliferation, or survival of cells expressing a T cell receptor (TCR), comprising contacting the cells with an effective amount of the multispecific molecule described in claim 1 or 2.

36. A pharmaceutical composition for treating a disorder, comprising an effective amount of the multispecific molecule described in claim 1 or 2.

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