Combinations of MHC class ib molecules and peptides for targeted therapeutic immunomodulation

Abstract

To provide non-classical major histocompatibility complex (MHC) known as MHC class Ib molecules in combination with specific peptides.SOLUTION: The invention more specifically relates to targeted immunomodulatory effects of defined peptides in combination with proteins comprising one or more domains of a non-classical MHC class lb molecule or in combination with molecules that interfere with the interaction of MHC class lb molecules and their receptors. The invention also relates to methods of producing such proteins, pharmaceutical compositions comprising the same, as well as their uses for treating medical conditions to which antigen-specific immune reactions are beneficial, e.g., cancer and infectious diseases, or to which antigen-specific immune reactions are harmful e.g., autoimmune diseases, organ / tissue rejection, immune reactions towards pharmaceutical compounds or reproductive disorders. Moreover, since the invention reveals a novel mode of action for MHC class lb molecules during antigen-specific tolerance induction, it also relates to methods for interfering with this mechanism in situation where induction of antigen-specific immune tolerance is wanted but physiologically prevented by this mechanism.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also referred to as MHC class Ib molecules) in combination with peptide antigens. More specifically, the invention relates to peptide antigens in combination with proteins comprising one or more domains of non-classical MHC class Ib molecules or in combination with molecules that inhibit the binding of MHC class Ib molecules to their receptors. The invention also relates to methods for the production of said proteins, pharmaceutical compositions comprising said proteins, and their use for the treatment of disease conditions in which an antigen-specific immune response is beneficial, including cancer and infectious diseases, or in which an antigen-specific immune response is detrimental, including autoimmune diseases, organ / tissue rejection, immune responses to pharmaceutical compounds, or reproductive disorders. [Background technology]

[0002] There are three known types of major histocompatibility complex (MHC) antigens: class I antigens (HLA-A, B, C, E, F, G), class II antigens (HLA-DP, HLA-DQ, and HLA-DR), and class III antigens. Class I antigens include conventional / classical MHC Ia antigens, HLA-A, HLA-B, and HLA-C, and non-classical MHC Ib antigens, HLA-E, HLA-F, and HLA-G. Class I antigens contain three globular domains ([α]1, [α]2, and [α]3). The MHC I complex further contains β2-microglobulin and the presented peptide bound to a peptide-binding groove that includes the [α]1 and [α]2 domains. Thus, peptide-loaded conventional MHC Ia molecules can initiate a peptide-specific T cell-mediated immune response that may result in lysis of the presenting cell. This mechanism is essential for vaccination strategies, and shorter or longer peptides (Non-Patent Document 1), nucleic acids encoding antigens (Non-Patent Document 2), proteins, or often attenuated organisms have been developed and used clinically to induce immune responses against specific antigens. Antigens may be viral, bacterial, or tumor-associated antigens.

[0003] Unlike conventional MHC Ia molecules, which are expressed in most human tissues, non-classical MHC Ib antigens such as HLA-G show expression only in significantly restricted tissues. Physiologically, high levels of HLA-G are expressed by the extravillous trophoblast of normal human placenta and likely function as an immunomodulatory agent that protects the fetus from the maternal immune system (lack of maternal rejection). In accordance with this hypothesis, previous studies have shown that HLA-G protein can inhibit allogeneic responses such as proliferative T lymphocyte cell responses, cytotoxic T lymphocyte-mediated cytolysis, and NK cell-mediated cytolysis (Non-Patent Document 3; Non-Patent Document 4).

[0004] The sequence of the HLA-G gene has been described (e.g., Non-Patent Documents 5 and 6) and contains 4396 base pairs. This gene is composed of 8 exons, 7 introns and a 3' untranslated end, which correspond to exon 1: signal sequence, exon 2: [α]1 extracellular domain, exon 3: [α]2 extracellular domain, exon 4: [α]3 extracellular domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and a 3' untranslated region, respectively. Seven isoforms of HLA-G have been identified, of which four are membrane-bound (HLA-G1, HLA-G2, HLA-G3 and HLA-G4) and three are soluble (HLA-G5, HLA-G6 and HLA-G7) (e.g., Non-Patent Document 7). The mature HLA-G1 protein isoform contains three ectodomains (α1-α3), a transmembrane region and a cytoplasmic domain, whereas the mature HLA-G5 protein isoform contains three ectodomains (α1-α3) and a short sequence encoded by intron 4, but lacks the transmembrane and intracellular domains. All soluble HLA-G isoforms lack the transmembrane and cytoplasmic domains and are also produced by cleavage of the membrane-bound isoforms.

[0005] HLA-G interacts in a peptide-independent manner with specific receptors such as Kir2DL4, ILT2 (LILRB1) and ILT4 (LILRB2, Non-Patent Document 8). The most prominent immunosuppressive effect of HLA-G on T cells is mediated by ILT2 and ILT4. The receptor interacts with the [α]-3 domain contained in HLA-G and other MHC class Ib molecules such as HLA-F (Non-Patent Document 9), so the [α]-3 domain-dependent effect observed with the representative MHC class Ib molecule HLA-G can be induced by alternative MHC class Ib molecules.

[0006] Moreover, MHC class Ib molecules are known to present peptides via their [α]1 and [α]2 domains. The peptides usually consist of 8-10 amino acids and contain specific anchor residues (Non-Patent Document 10, Non-Patent Document 11). However, to the inventors' knowledge, peptide-specific interactions of human MHC class Ib molecules with the cognate T cell receptors have not yet been studied. Similarly, there are no clear data from animal models. Swanson et al. suggested that mouse MHC Ib molecules may induce peptide-specific immune responses (Non-Patent Document 12), and Wang et al. reported the suppression of peptide-specific immune responses by mouse Qa2 molecules (Non-Patent Document 13). However, human and mouse MHC Ib molecules are significantly different (Non-Patent Document 14). Because HLA-G and Qa-2 share only 67% sequence identity analyzed using protein blast with the UniProtKB reference sequences Q5RJ85 (Q5RJ85_HUMAN) and P79568 (P79568_MOUSE), conclusions drawn from Qa-2 must be treated with great caution and cannot be predicted to be applicable to human HLA-G. The significant difficulties in identifying suitable mouse models for studying HLA-G function in basic science and preclinical research have been recently outlined in a review article (15).

[0007] Based on already available data, it has been proposed that HLA-G protein can be used to treat graft rejection in allogeneic or xenogeneic organ / tissue transplantation. HLA-G protein has also been proposed for the treatment of hematological malignancies (Patent Document 1), inflammatory diseases (Patent Document 2), and more generally immune-related diseases. Furthermore, HLA-G is often expressed by human tumors (Non-Patent Document 16) and is thought to function like an immunosuppressive immune checkpoint molecule that nonspecifically suppresses immune responses in the tumor microenvironment (Non-Patent Document 17). However, none of these studies have analyzed peptides presented by HLA-G. As a result, the question of whether presented peptides can induce the observed MHC class Ib-mediated effects has not been raised. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] European Patent Application Publication No. 1054688 [Patent Document 2] European Patent Application Publication No. 1189627 [Non-patent literature]

[0009] [Non-Patent Document 1] Slingluff, Cancer J.2011 Sep;17(5):343-350 [Non-Patent Document 2] Restifo et al.,Gene Ther.2000 Jan;7(2):89-92 [Non-Patent Document 3] Rouas-Freiss N.et al.,Proc.Natl.Acad.Sci.,1997,94,5249-5254 [Non-Patent Document 4] Rouas-Freiss N. et al.,Semin Cancer Biol 1999,vol9,3 [Non-Patent Document 5] Geraghty et al.Proc.Natl.Acad.Sci.USA,1987,84,9145-9149

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Non-licensed literature 9

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Non-licensed Document 16

[0010] Given the inherent limitations of all mouse models studying human MHC class Ib molecules, their effects on human T cells need to be explored in vitro to understand mechanisms that may predict MHC class Ib-dependent functions in vivo. In the context of antigen-specific immune responses, the regulation of cytotoxic and tolerogenic T cells is important. Both cytotoxic CD8+ effector T cells (cytotoxic T lymphocytes, CTLs) and regulatory T cells (Tregs) can detect antigen peptides presented on MHC molecules, but CTLs can destroy cells expressing their cognate antigens, whereas regulatory T cells are tissue-protective, especially when their cognate antigens are presented by the respective tissues (Wright et al., 2009 PNAS vol.106 no.45,19078-83). Importantly, when antigen-specific regulatory T cells are activated by cognate antigens in target tissues, they can exert a bystander effect and suppress immune responses against other antigens. Thus, CTLs can be beneficial in cancer patients (Gajewski et al., Nat. Immunol. 14, 1014-1022, 2013), but are harmful in autoimmune diseases. The opposite is true for the function of Treg cells to suppress immune responses. Insufficient activity or function of Tregs can cause severe autoimmune diseases in mice and may also be relevant in human autoimmune diseases (Bluestone et al., J Clin Invest. 2015; 125(6): 2250-2260). Thus, strategies for inhibition (or deactivation) of cytotoxic T cells and induction (or inhibition) of Tregs are needed.

[0011] In current clinical practice, diseases caused by pathological immune responses (e.g., autoimmune diseases) are usually treated with therapeutic agents that suppress immune responses regardless of the target antigen, but this induces severe, often dose-limiting side effects and increases the risk of opportunistic infections.Therefore, there is a need for improved means and uses for the treatment of such diseases.As a result, there is a need in the art for improved means and uses for therapeutic modulation of the immune system by more targeted and antigen-specific means.

[0012] On the other hand, the means and use for the treatment of diseases in which immune response against specific antigens is desired, including cancer, also need to be improved.For example, many of the vaccination approaches reported for cancer immunotherapy have been shown to be ineffective due to the immunosuppressive mechanisms exerted by cancer.Therefore, the means and use for the treatment of such diseases, including cancer, also need to be improved. [Means for solving the problem]

[0013] The inventors have surprisingly found that human MHC class Ib molecules, such as HLA-G, are capable of inducing antigen-specific tolerance to presented peptide antigens. Thus, while the structure and sequence of classical human MHC class Ia molecules that induce antigenic peptide-specific immune responses are similar, according to the present invention, MHC class Ib molecules can be advantageously used to suppress immune responses in an antigen-specific manner. Antigen-specific suppression of immune responses to specific antigens can be induced by eliminating antigen-specific cytotoxic T cells or by inducing antigen-specific regulatory T cells that recognize either the respective autoantigens or other target antigens expressed in tissues susceptible to autoimmune attack. In accordance with the above, the inventors have shown that exemplary human MHC class Ib molecules can be used to eliminate cytotoxic effector T cells (as exemplarily shown in FIG. 1) and induce tolerogenic regulatory T cells (as exemplarily shown in FIG. 7) in an antigenic peptide-specific manner. In a non-limiting embodiment, the effect can be achieved by combining a specific peptide antigen with either a membrane-bound or soluble MHC class Ib molecule. On the other hand, in situations where it is desired to induce an antigen-specific immune response, it is necessary to break MHC class Ib-associated immune tolerance, and the inventors have found that this can be achieved via agents that block the binding of human MHC Ib molecules to their receptors.

[0014] Thus, according to the present invention, peptides combined with MHC class Ib molecules can be advantageously used to suppress antigen- or tissue-specific immune responses, which represents a significant advantage compared to many conventional therapeutic agents that suppress immune responses regardless of the target antigen, the lack of specificity of which induces severe, dose-limiting side effects and increases the risk of opportunistic infections.

[0015] Furthermore, the inventors have surprisingly found that for the suppression of an immune response according to the invention, molecules other than naturally occurring MHC class Ib molecules may be used, in particular polypeptides comprising at least one domain of an MHC class Ib molecule, preferably at least the [α]3 domain of an MHC class Ib molecule alone. As illustrated in Figures 7 and 8, the [α]1 and [α]2 domains of a variable class Ia molecule can productively combine with the [α]3 domain of a human MHC class Ib molecule, thereby suppressing the immune response against peptides presented by the antigen.

[0016] Thus, in accordance with the present invention, the use of, for example, the immunosuppressive [α]3 domain of an MHC class Ib molecule in combination with a target antigen presented by, for example, MHC class I [α]1 and 2 domains may be beneficial in many autoimmune diseases.

[0017] On the other hand, the new surprising findings of the present inventors also show that the suppression of antigen-specific immune responses induced by MHC class Ib molecules can be rescued by agents that interfere with the binding of MHC class Ib molecules to their receptors. Thus, according to the present invention, inhibitors such as antibodies against MHC class Ib molecules (illustrated in FIG. 2) or their receptors, including ILT2 and ILT4 (illustrated in FIG. 1), can be advantageously used to treat diseases in which an immune response against a specific antigen is desired. These include cancers such as gastric cancer, gastrointestinal stromal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, breast cancer, uterine cancer, ovarian cancer, cervical cancer, vulvar cancer, vaginal cancer, urothelial cancer, testicular cancer, colorectal cancer, pancreatic cancer, skin cancer and sarcoma (see, e.g., http: / / medicalgenome.kribb.re.kr / GENT / search / view_result.php), as well as diseases such as trypanosomiasis (see, e.g., Gineau et al., Clin Infect Dis. 2016 Nov.1;63(9):1189-1197), cytomegalovirus infection (see, e.g., Cosman et al., Immunity. 1997 Aug;(2):273-82), HTLV-1 infection (see, e.g., Ciliao Alves et al., J Gen Virol. 2016 Oct;97(10):2742-2752), hepatitis C infection (see, e.g., Ding Infectious diseases include, but are not limited to, pulmonary malaria (see, e.g., Garcia et al., Infect Genet Evol. 2013 Jun;16:263-9), or Plasmodium falciparum infection (see, e.g., Garcia et al., Infect Genet Evol. 2013 Jun;16:263-9).

[0018] In situations where it is necessary to first induce a specific immune response against a selected antigen, vaccines containing peptides or proteins, or attenuated pathogens, or DNA or RNA encoding proteins, are commonly used in the art. However, such vaccinations may fail to elicit a response or may even elicit undesirable resistance (Non-Patent Document 1). Presentation of MHC class Ib molecules may be the cause of this obstacle, since tumor cells (Non-Patent Document 16) and virus-infected cells (Rizzo et al., Front Immunol. 2014; 5: 592) express MHC class Ib molecules, such as HLA-G antigen presentation on MHC class Ib molecules. Therefore, according to the present invention, agents that specifically block the binding of MHC class Ib molecules to their receptors can be used to enhance the efficacy of treatments that induce peptide- or protein-specific immune responses using specific antigenic proteins or peptides. This includes therapies based on exogenously administered vaccines, but can also be extended to treatments in which antigenic material released by dying tumor cells can induce antigen-specific T cell responses, such as radiotherapy or chemotherapy (see, for example, Zitvogel et al., Nature Reviews Immunology 8, 59-73, January 2008). On the other hand, the undesirable vaccination effect induced by biologic or gene therapy can be counteracted by adding MHC class Ib-based constructs to prevent the development of anti-drug antibodies.

[0019] Thus, the present invention relates to the following preferred embodiments. (1) a) a polypeptide capable of presenting a human MHC class Ib molecule, or a peptide antigen, to a T cell, wherein the polypeptide comprises an [α]3 domain of a human MHC class Ib molecule, or a derivative of the [α]3 domain of a human MHC class Ib molecule capable of binding to ILT2 or ILT4; b) a peptide antigen presented by the MHC class Ib molecule or polypeptide of a); 13. A pharmaceutical composition comprising: (2) The pharmaceutical composition according to (1), comprising a polypeptide capable of presenting the peptide antigen of a), and preferably comprising, from the N-terminus to the C-terminus, the [α]1 domain and the [α]2 domain of an MHC class Ia molecule, followed by the [α]3 domain or a derivative thereof, in that order. (3) A pharmaceutical composition described in (1) or (2), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical to or has at least 80%, preferably at least 90%, identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (4) The pharmaceutical composition described in (3), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical or has at least 92% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (5) The pharmaceutical composition described in (3), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical or has at least 94% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (6) The pharmaceutical composition described in (3), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical or has at least 96% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (7) The pharmaceutical composition described in (3), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical or has at least 98% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (8) The pharmaceutical composition described in (3), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical to or has at least 99% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (9) The pharmaceutical composition described in (3), wherein the amino acid sequence of the [α]3 domain or derivative contained in the MHC class Ib molecule or polypeptide is identical to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (10) The pharmaceutical composition according to any one of the above, wherein the MHC class Ib molecule of a) or the polypeptide capable of presenting the peptide antigen of a) is capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 40 μM as measured by surface plasmon resonance spectroscopy. (11) The pharmaceutical composition according to any one of the above, wherein the MHC class Ib molecule of a) or the polypeptide capable of presenting the peptide antigen of a) is capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 20 μM as measured by surface plasmon resonance spectroscopy. (12) The pharmaceutical composition according to any one of the above, wherein the MHC class Ib molecule of a) or the polypeptide capable of presenting the peptide antigen of a) is capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 10 μM as measured by surface plasmon resonance spectroscopy. (13) The pharmaceutical composition according to any one of the above, further comprising a polypeptide domain comprising the amino acid sequence of SEQ ID NO: 6 or a sequence that is at least 90%, preferably 95%, more preferably 98% identical to the amino acid sequence of SEQ ID NO: 6, and wherein the polypeptide domain is preferably contained in a polypeptide capable of presenting the peptide antigen of a). (14) The pharmaceutical composition according to any one of the above, wherein the MHC class Ib molecule of a) or the polypeptide capable of presenting the peptide antigen of a) further comprises one or more linker sequences, preferably a (GGGS)n linker sequence. (15) The pharmaceutical composition according to any one of the above, wherein the MHC class Ib molecule or the polypeptide capable of presenting the peptide antigen of a) is a dimer or multimer. (16) The pharmaceutical composition according to any one of the above, wherein the peptide antigen is 7 to 11 amino acids long, preferably 8 to 10 amino acids long. (17) The pharmaceutical composition according to any one of (1) and (3) to (16), wherein the pharmaceutical composition comprises the MHC class Ib molecule of a), and the MHC class Ib molecule is HLA-E, HLA-F or HLA-G. (18) The pharmaceutical composition according to (17), wherein the MHC class Ib molecule is HLA-G. (19) The pharmaceutical composition according to (17) or (18), wherein the MHC class Ib molecule is a human MHC class Ib molecule. (20) The pharmaceutical composition according to any one of the above, wherein the peptide antigen of b) is covalently bound to the MHC class Ib molecule or polypeptide of a). (21) The pharmaceutical composition according to (20), wherein the peptide antigen of b) and the MHC class Ib molecule or polypeptide of a) are covalently linked via a peptide bond and are part of a single polypeptide chain. (22) A recombinant polypeptide capable of presenting a peptide antigen, comprising, from the N-terminus to the C-terminus: i) a peptide antigen presented by the recombinant polypeptide; ii) optionally, a first linker sequence; iii) optionally a sequence of a human polypeptide domain comprising a sequence of human β2-microglobulin or an amino acid sequence at least 90% identical to the amino acid sequence of human β2-microglobulin set forth in SEQ ID NO:6; iv) optionally, a second linker sequence; v) optionally the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [α]3 domain of an MHC class Ib molecule capable of binding to ILT2 or ILT4; and viii) optionally, a protease cleavage site; ix) optionally, affinity tags; A recombinant polypeptide comprising, in that order: (23) The recombinant polypeptide according to (22), wherein the [α]1 domain of v) and the [α]2 domain of vi) are derived from an MHC class Ia molecule. (24) A recombinant polypeptide according to (22) or (23), wherein the amino acid sequence of the [α]3 domain or derivative is identical to or has at least 80%, preferably at least 90%, identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (25) The recombinant polypeptide described in (24), wherein the amino acid sequence of the [α]3 domain or derivative is identical to or has at least 92% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (26) The recombinant polypeptide described in (24), wherein the amino acid sequence of the [α]3 domain or derivative is identical to or has at least 94% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (27) The recombinant polypeptide described in (24), wherein the amino acid sequence of the [α]3 domain or derivative is identical to or has at least 96% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (28) The recombinant polypeptide described in (24), wherein the amino acid sequence of the [α]3 domain or derivative is identical to or has at least 98% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11. (29) The recombinant polypeptide described in (24), wherein the amino acid sequence of the [α]3 domain or derivative is identical to or has at least 99% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO:11. (30) The recombinant polypeptide described in (24), wherein the amino acid sequence of the [α]3 domain is identical to the amino acid sequence of the [α]3 domain of SEQ ID NO:11. (31) The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 40 μM as measured by surface plasmon resonance. (32) The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 20 μM as measured by surface plasmon resonance. (33) The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 10 μM as measured by surface plasmon resonance. (34) The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is a dimer or a multimer. (35) The recombinant polypeptide according to any one of the above, wherein the peptide antigen sequence of i) is 7 to 11 amino acids long, preferably 8 to 10 amino acids long. (36) A recombinant polypeptide according to any one of the above, comprising all of i) to vii), but preferably not comprising viii) to ix). (37) A recombinant polypeptide according to any one of (22) to (35), comprising all of i) to ix). (38) The recombinant polypeptide according to any one of the above, further comprising an N-terminal secretory signal peptide sequence. (39) The pharmaceutical composition according to any one of (1) to (21) or the recombinant polypeptide according to any one of (22) to (38), for use in medicine. (40) The pharmaceutical composition according to any one of (1) to (21) or the recombinant polypeptide according to any one of (22) to (38), used in a method for peptide antigen-specific immunomodulation in a subject, wherein the immunomodulation is specific to the peptide antigen contained in the pharmaceutical composition or the recombinant polypeptide. (41) The pharmaceutical composition or recombinant polypeptide described in (40), wherein the immunomodulatory method induces immunological tolerance to the peptide antigen contained in the pharmaceutical composition or recombinant polypeptide. (42) The pharmaceutical composition or recombinant polypeptide according to (40) or (41), wherein the method for immunomodulation is a method for suppressing an autoimmune disease, an allergy, an immune response to a biological preparation, an immune response to an embryonic antigen, or an immune response to a transplanted cell, tissue or organ. (43) The pharmaceutical composition or recombinant polypeptide according to (42), wherein the method for immunomodulation is a method for inducing immune tolerance, wherein the autoimmune disease affects multiple organs, hormone-producing organs, nerves, joints, skin, gastrointestinal system, eyes, blood components or blood vessels. (44) The pharmaceutical composition or recombinant polypeptide according to (41), wherein the method is a method for suppressing an immune response in Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, rheumatoid arthritis, psoriasis, scleroderma, neuromyelitis optica, or type 1 diabetes. (45) A nucleic acid encoding the polypeptide according to any one of (22) to (38) or the polypeptide or MHC class Ib molecule according to any one of (1) to (21). (46) The nucleic acid according to (45), wherein the nucleic acid is a vector. (47) A pharmaceutical composition comprising the nucleic acid according to (45) or (46). (48) A recombinant host cell comprising the nucleic acid molecule or vector according to (45) or (46). (49) A method for producing the polypeptide according to any one of (22) to (38), comprising culturing the recombinant host cell according to (48) under conditions capable of expressing the nucleic acid molecule, and recovering the produced polypeptide. (50)a1) an antigenic protein or peptide antigen, or a nucleic acid encoding said antigenic protein or peptide antigen, or an attenuated organism containing said antigenic protein or peptide antigen; or a2) a cell presenting the peptide antigen of a1); and b) Agents capable of blocking the binding of MHC class Ib molecules to their receptors; A combination of The combination for use in a method for inducing an immune response to said antigenic protein or peptide antigen in a human subject. (51) The combination according to (50), wherein the agent is capable of binding to the human MHC class Ib molecule and / or its receptor. (52) The combination according to any one of the preceding claims, wherein the agent is capable of binding to HLA-G. (53) The combination according to any one of (50) to (52), wherein the agent is an antibody capable of binding to HLA-G, preferably a monoclonal antibody. (54) The combination according to any one of the preceding claims, wherein the agent is capable of binding to ILT2 or ILT4. (55) The combination according to any of the above, wherein the agent is an antibody capable of binding to ILT2 or ILT4, preferably a monoclonal antibody. (56) The combination according to any one of the preceding claims, wherein the agent comprises an Fc domain of an antibody or a fragment thereof. (57) The combination according to any one of the preceding claims, wherein the agent comprises the [α]3 domain of an MHC class Ib molecule. (58) The combination according to any of the above, wherein the agent comprises one or more extracellular domains of the ILT2 or ILT4 receptor, preferably at least two N-terminal extracellular domains of the ILT2 or ILT4 receptor, and wherein the agent more preferably comprises a soluble ILT2 or ILT4 receptor. (59) The combination according to any one of the preceding claims, wherein the agent is administered simultaneously with, prior to, or subsequent to the antigenic protein or peptide antigen, or the nucleic acid encoding the antigenic protein or peptide antigen, or the attenuated organism containing the antigenic protein or peptide antigen. (60) The combination according to any one of the above, wherein the combination is a combination of: a) an antigenic protein or peptide antigen; and b) an agent capable of blocking the binding of the MHC class Ib molecule to its receptor. (61) The combination according to any one of (50) to (59), wherein the combination is a combination of: a) a nucleic acid encoding an antigenic protein or peptide antigen; and b) a drug capable of blocking the binding of the MHC class Ib molecule to its receptor. (62) The combination according to any one of (50) to (59), wherein the combination is a combination of: a) an attenuated organism comprising an antigenic protein or peptide antigen; and b) an agent capable of blocking the binding of the MHC class Ib molecule to its receptor. (63) The combination according to (62), wherein the attenuated organism containing the antigenic protein or peptide antigen is an attenuated virus. (64) The combination according to any one of (50) to (62), wherein the antigenic protein or peptide antigen of a) is a tumor antigen or an antigen that is at least 77% identical to the tumor antigen and can induce cross-protection against the antigen. (65) The combination according to any one of the preceding claims, wherein the method is a method for T cell-based immunotherapy. (66) The combination according to any one of (50) to (63) and (65), wherein the antigenic protein or peptide antigen is detectable in a pathogenic microorganism or virus. (67) The combination according to any one of the preceding claims, wherein the method is a method for the treatment or prevention of an infectious or malignant disease. (68) The combination according to (67), wherein the disease is cancer and the peptide antigen is a tumor antigen. (69) The combination according to (68), wherein the cancer is selected from the group consisting of melanoma, renal cancer, ovarian cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic ductal adenocarcinoma, prostate cancer, B-cell lymphoma, T-cell lymphoma, and lung cancer. (70) The combination according to any one of the preceding claims, wherein the combination is present in one pharmaceutical composition. (71) The combination according to any one of the preceding claims, wherein the immune response against the antigenic protein or peptide antigen is specific for the antigenic protein or peptide antigen. (72) A drug capable of blocking the binding of the MHC class Ib molecule according to any one of (50) to (62) to its receptor, for use in a method for treating cancer in a human subject, the method comprising a treatment that results in the release of a cancer antigen from cells of the cancer. (73) The agent according to (72), wherein the treatment that results in the release of the cancer antigen is chemotherapy or radiation therapy. (74) The pharmaceutical composition or recombinant polypeptide according to (41), wherein the method for inducing immunological tolerance to a peptide antigen further comprises treatment with a peptide drug, and the peptide antigen is: 1) identical to the peptide drug, or 2) a fragment of the peptide drug, or 3) a derivative of the fragment of the peptide drug capable of inducing immunological tolerance to the peptide drug. (75) The pharmaceutical composition or recombinant polypeptide described in (41), wherein the method for inducing immunological tolerance to a peptide antigen further includes treatment with a protein drug, and the peptide antigen is 1) a fragment of the protein drug, or 2) a derivative of the fragment of the protein drug capable of inducing immunological tolerance to the protein drug. (76) The pharmaceutical composition or recombinant polypeptide according to (74), wherein the peptide drug is administered in the form of the peptide drug itself. (77) The pharmaceutical composition or recombinant polypeptide according to (75), wherein the protein drug is administered in the form of the protein drug itself. (78) The pharmaceutical composition or recombinant polypeptide according to (74), wherein the peptide drug is administered by gene therapy using a gene encoding the peptide drug. (79) The pharmaceutical composition or recombinant polypeptide according to (75), wherein the protein drug is administered by gene therapy using a gene encoding the protein drug.

[0020] The present invention may be used with any mammalian subject, preferably a human subject.

[0021] Indications for which the above combinations of immunostimulatory T cell directed therapy, preferably with blocking agents against MHC class Ib or ILT2 / 4, should be used include viral infections and tumors where elevated levels of HLA-G or other MHC class Ib molecules are detectable in tumor exudates, blood samples, biopsies, or other means of malignant or non-malignant cells by polymerase chain reaction, ELISA, Western blotting, immunofluorescence, immunohistochemistry, and other methods (as described in Paul et al., Hum Immunol. 2000 Nov;61(11):1177-95). Since HLA-G is not expressed in many tissues, but even small amounts are remarkably potent, detectable levels of expression in otherwise HLA-G deficient tissues, or 50% above physiological levels in tissues that show basal HLA-G expression, are considered preferred elevated levels according to the present invention. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 shows that cells expressing MHC Ib molecules loaded with specific peptides selectively eliminate CTLs specific for the presented peptide. (A) and (B) HLA-A2-restricted CD8+ effector T cells recognizing the model antigen STEAP1 (CD8st) can be selectively eliminated when their cognate peptide is presented on the tumor cell line JEG-3, which shows high expression of the non-classical MHC class 1b molecule HLA-G, but hardly any classical MHC class 1a molecules are detected. Note that STEAP1 is generally referred to herein as "STEAP", "steap", or "st". These terms are used synonymously and interchangeably. Co-cultured CD8+ effector T cells specific for the antigen PRAME (CD8pr) are not affected. (C) When STEAP1-specific T cells are loaded with cognate peptides, HLA-G tumor cells expressing JEG-3 are not eliminated in this process. This indicates that MHC Ib molecules expressed by tumor or infected cells may render these cells resistant to targeting by antigen-specific T cells, which are usually the main effectors of peptide-based vaccination strategies. (D) Induction of apoptosis in STEAP1-specific T cells by HLA-G-presented STEAP1 peptides could be strongly attenuated by a neutralizing antibody against the HLA-G interacting partner ILT-2 expressed on T cells. [Diagram 2]Figure 1: MHC Ib molecules loaded with specific peptides impair the cytotoxic potential of allogeneic CTLs in an antigen- and HLA-G-dependent manner. HLA-A2-restricted T cell clones specific for STEAP1 or PRAME, respectively, were mixed and pretreated with control (+) or STEAP1 peptide-loaded (st) JEG-3 cells. Where indicated, a neutralizing anti-human HLA-G antibody (clone 87G) was added at 10 μg / ml. After 16 h, the cytotoxic potential of STEAP1-specific T cells was tested against luciferase-expressing naive (grey bars) or STEAP1-peptide (black bars) melanoma cells loaded with HLA-A2+ UACC-257 in a 2:1 ratio. After 8 h, D-luciferin was added and the viability of target cells was measured in a luminometer using a biophotonic viability assay (Brown et al., J Immunol Methods. 2005 Feb;297(1-2):39-52). Addition of STEAP1 peptide to HLA-G expressing JEG-3 cells reduced the lytic ability of STEAP1-specific CTL by more than 90%, whereas the inhibition in naive JEG-3 cells was not significant. This effect can be significantly attenuated by the presence of partially neutralizing HLA-G antibodies, and it can be concluded that peptide-added HLA-G can be used to inhibit T cell-mediated immune responses against selected antigens. According to the present invention, this effect can be extended to further MHC class Ib molecules. Meanwhile, induction of antigen-specific T cell-mediated immune responses according to the present invention can be achieved by agents that block MHC Ib. [Diagram 3]Figure 1: MHC Ib molecules combined with specific peptides inhibit allogeneic CTLs, while immune responses against other antigens are largely unaffected. HLA-A2-restricted T cell clones specific for STEAP1, HLA-A2-STEAP1 and HLA-A2-PRAME specific PRAME T cell clones were mixed and pretreated with untreated (ctrl) or control (JEG-3) or STEAP1 peptide-loaded (JEG-3st) JEG-3 cells. After 8 hours, the peptide-specific cytotoxic potential of both T cell clones was tested in a 1:1 ratio against luciferase expressing PRAME-peptide (dark grey bars) or STEAP1-peptide-loaded luciferase expressing HLA-A2+UACC-257 melanoma cells (light grey bars). Pretreatment with STEAP1 peptide-loaded JEG-3 cells inhibited STEAP1 peptide-specific T cell-mediated immune responses by approximately 50%, whereas PRAME-specific immune responses were largely unchanged by naive or STEAP1 peptide-loaded JEG-3 cells. [Figure 4] Figure 1 shows peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutic antigen-specific immune modulation. The presented peptide antigens are shown as dot spheres, the HLA-Gα1-3 domains in light grey and the β2-microglobulin domain in dark grey. Optional linkers connecting the antigenic peptides to the β2-microglobulin molecules are shown as grey sticks and optional disulfide traps are shown as black spheres. The figure was generated using Pymol as disclosed in Hansen et al., Trends Immunol. 2010 Oct;31(10):363-9 and in [Diagram 5]FIG. 1 shows an example of a vector-based construct encoding a single-chain MHC Ib molecule suitable for therapeutic peptide-specific immunomodulation. HLA-G1 and HLA-G5 each consist of three [α] domains (here in black), a non-covalently bound β2-microglobulin subunit (here in dark grey), and an antigenic peptide (short black arrow) presented on HLA-G. HLA-G1 further comprises a transmembrane domain and a short intracellular chain (not shown here). As shown herein, the [α]-3 domain can bind to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci USA. 2003 July 22; 100(15):8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci USA. 2006 October 31; 103(44):16412-7) on immune cells. Physiologically, these sequences form a non-covalently bound MHC class I complex. Two protein tags (myc and His(6x)) were introduced to allow subsequent removal by factor Xa cleavage, simplifying purification of the complex MHC Ib molecule. In addition, the antigen peptide, β2-microglobulin and MHC Ib[α] chain can be linked to increase stability. Vector maps were generated using Snapgene Viewer Software. [Figure 6]Figure 1 shows that soluble peptide HLA-G / peptide-MHC Ib complexes can be combined with dendritic cells (DC-10) to selectively eliminate CD8+ effector T cells that recognize the presented target antigen. Dendritic cells were generated from monocytes in the presence of GM-CSF, IL4, and IL10 (DC-10) and then cell culture supernatants containing soluble peptide MHC Ib constructs were added for 4 hours. Disulfide-trap stabilized single-chain HLA-G5 constructs containing presented Melan-A / MART1 (dtGmelA) or STEAP1 (dtGsteap) peptides were used. Binding of these constructs to DC-10 cells was previously confirmed. Added DC-10 cells were then washed and co-cultured with control CTLs (PRAME-specific, CD8pr) or target CTLs (STEAP1-specific, CD8st) at a 1:1 ratio for 48 hours. These data suggest that dendritic cells loaded with soluble MHC Ib-peptide constructs almost completely deplete allogeneic T cell clones, whereas non-allogeneic CTLs are unaffected. [Figure 7]Figure 1: Peptide-loaded MHC Ib complexes induce human antigen-specific regulatory T cells that recognize presented peptides. A) Peripheral blood mononuclear cells (PBMCs) were obtained from different healthy donors and co-cultured with irradiated JEG-3 cells loaded with Melan-A / MART1 (MART1) peptide or STEAP1 (STEAP) peptide for 14 days in RPMI1640 medium containing 5% hAB serum, 5ng / ml TGFβ1, 20ng / ml IL2 (Treg medium). On day 7, PBMCs were transferred to fresh medium, irradiated and loaded again with peptide-loaded JEG-3 cells. Treg expansion beads from Miltenyi Biotec (anti-CD3, anti-CD28 and anti-CD2) were used as positive control. The resulting cells were stained with antibodies against human CD4 and CD25, HLA-A2 STEAP1 peptide dextramer (STEAP1 dex) and analyzed by flow cytometry. A significant enrichment of STEAP1-specific cells within the CD4+ CD25high Treg population was observed when STEAP1-supplemented JEG-3 cells were present. B) Disulfide-trapped single-chain HLA-G constructs presenting Melan-A (dtGmelA) or STEAP1 (dtGsteap) peptides were added to 4×105 DC-10 cells per well as described in FIG. 6, followed by 4×106 PBLs from the same donor and cells cultured in Treg medium for 7 days. An equal amount of 4×105 DC-10 was then added to each well. After a total of 14 days, Melan-A-specific IL-10-producing Treg cells were quantified by flow cytometry. The number of Melan-A-specific Tregs was significantly increased in conditions where PBLs were co-cultured with DC-10 supplemented with single-chain Melan A HLA-G molecules compared to control molecules (STEAP1) or untreated PBLs. [Figure 8]Figure 1: Single peptide MHC constructs containing human MHC Ibα3 domains in combination with DCs induce mouse Treg cells specific for the presented peptide. Mouse DCs (mDCs) were generated by culturing bone marrow-derived cells for 7 days in complete RPMI-1640 supplemented with 10% GM-CSF-containing supernatant from Ag8653 myeloma cells transfected with the mouse GM-CSF gene. mDCs were supplemented with control CHO supernatants (CHO / ctrl) or with supernatants from CHO cells transfected with a plasmid encoding a single peptide MHC molecule containing the human HLA-Gα3 domain and an ovalbumin peptide presented by mouse H-2Kbα1 and 2 domains (H2Kb). A similar construct containing human HLA-A2α1 and 2 domains (A2G) instead of mouse H-2Kbα1 and 2 domains was included as a control. Expression of the constructs was confirmed by Western blotting of the supernatants. A and B: Splenocytes from OT1 mice expressing only a transgenic T cell receptor that recognizes the H-2Kb-presenting OVA peptide (SIINFEKL) were cultured in Treg-permissive medium (complete RPMI, 5 ng / ml IL-2, 5 ng / ml TGF-β1) with added mDCs at a ratio of 2.5:1 for 14 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Definitions and General Techniques Unless otherwise defined below, terms used herein are to be understood according to their common meanings known to those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0024] All proteins according to the invention, including the polypeptides and MHC molecules according to the invention, may be produced by methods known in the art, including methods for producing recombinant proteins. It will be understood that proteins according to the invention, including the polypeptides and MHC molecules according to the invention, are meant to optionally include a secretion signal peptide sequence. Similarly, proteins according to the invention are also meant to include an affinity tag, for example to facilitate purification, and optionally a protease cleavage site between the tag and the protein, for example to facilitate removal of the tag by protease cleavage. Similarly, it will be understood that the proteins according to the invention, including the polypeptides according to the invention and the MHC molecules, include the respective propeptides. It will also be appreciated that the polypeptides and MHC molecules of the present invention may be in their soluble or membrane-bound form.

[0025] According to the present invention, the MHC molecule is preferably a human MHC molecule. The proteins and polypeptides of the invention, including the MHC molecules used according to the invention, the polypeptides of the invention and the antibodies according to the invention, are preferably isolated. The proteins and polypeptides of the invention, including the MHC molecules used according to the invention, the polypeptides of the invention and the antibodies according to the invention, are preferably recombinant.

[0026] Methods for preparing polypeptides capable of binding and presenting peptide antigens of the invention will be understood. For example, peptide antigen binding domains, such as [α]1 and [α]2 domains, are known and such domains may be modified. The binding ability of the polypeptides of the invention and peptide antigens binding to MHC molecules may be determined by techniques known in the art, including, but not limited to, exploratory methods such as MHC peptide elution followed by mass spectrometry and in silico bioinformatic prediction, and confirmatory methods such as MHC peptide multimer binding methods and stimulation assays. With respect to the peptide antigens used in the present invention, it will be understood that any length of these peptide antigens described herein (e.g., "7-11 amino acids long") refers to the length of the peptide antigen itself. Thus, the length of the peptide antigens described herein does not include length contributed by additional amino acids that are not part of the peptide antigen, such as additional amino acids from a possible linker sequence.

[0027] As used herein, the term "autoimmune disease" is not limited to a particular autoimmune disease by its common meaning known to those skilled in the art. In all embodiments of the present invention, the autoimmune disease is preferably an autoimmune disease involving an autoimmune response against a peptide autoantigen.

[0028] In the present invention, the term "comprising" may optionally be replaced with the term "consisted of".

[0029] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or antibody-related methods) are methods known in the art, e.g., as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology." Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0030] Protein-protein binding, such as the binding of an antibody to each target protein, may be assessed by methods known in the art. Protein-protein binding, such as the binding of an antibody to each target protein, is preferably assessed by surface plasmon resonance spectroscopy. For example, the binding of MHC class Ib molecules or polypeptides according to the invention to their receptors, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class Ib molecules or polypeptides according to the invention to their receptors is assessed by surface plasmon resonance spectroscopy at 25° C. Suitable conditions for such surface plasmon resonance spectroscopy measurements are described by Shiroishi et al., Proc Natl Acad Sci USA. 2003 July 22; 100(15): 8856-8861.

[0031] Sequence alignment of the sequences of the present invention is performed using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215. p. 403-410.; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402). Suitable parameters for sequence alignment of short peptides suitable for peptide antigens according to the present invention by the BLAST algorithm are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters of sequence alignment for short input sequences. In one embodiment, the following parameters are used: maximum target sequence 10; word size 3; BLOSUM62 matrix; gap cost: existence 11, extension 1; adjustment of conditional composition score matrix; Thus, the terms "identity" or "identity" and the like when used in reference to sequences preferably refer to the identity value obtained by using the BLAST algorithm.

[0032] Preparation of the Composition of the Present Invention The compositions according to the invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions. For example, the compositions are prepared so that they may be appropriately stored and administered, for example, by using pharma- ceutically acceptable ingredients such as carriers, excipients and / or stabilizers. The pharma- ceutically acceptable ingredients are non-toxic in the amounts used when the pharmaceutical composition is administered to a patient. The pharma- ceutically acceptable ingredients added to a pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the particular use of the pharmaceutical composition, and the route of administration. Generally, pharma- ceutically acceptable ingredients used in connection with the present invention are used in accordance with knowledge available in the art, e.g., from Remington's Pharmaceutical Sciences, Ed. A. R. Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.

[0033] [Peptide antigen of the invention] Peptide antigens that can be used in the present invention, including the peptide antigens identified above, are not particularly limited except for their ability to be presented on MHC molecules. Peptides that can be presented on MHC molecules can be prepared by methods known in the art (e.g., Rammensee, Bachmann, Emmerich, Bachor, Stevanovic. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov; 50(3-4): 213-9; Pearson et al. MHC class I-associated peptides derive from selective regions of the human genome. J Clin Invest. 2016 Dec 1; 126(12): 4690-4701; and Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov; 37(11): 724-737). Peptide antigens are generally known in the art. In general, the peptide antigens of the present invention can bind to MHC class I proteins. Those skilled in the art will understand that for each MHC class Ib molecule or polypeptide that can present the peptide of the present invention, a peptide antigen that can bind to the MHC class Ib molecule or polypeptide is preferably used. These peptide antigens can be selected based on methods known in the art.

[0034] Binding of a peptide antigen to an MHC class Ib molecule or a polypeptide having peptide antigen-binding ability of the present invention can be carried out by a method known in the art, for example, the following method: -Rammensee,Bachmann,Emmerich,Bachor,Stevanovic.SYFPEITHI:database for MHC ligands and peptide motifs.Immunogenetics.1999 Nov;50(3-4):213-9; -Pearson et al.MHC class I-associated peptides derive from selective regions of the human genome.J Clin Invest. 2016 Dec 1;126(12):4690-4701;and -Rock,Reits,Neefjes.Present Yourself!By MHC Class I and MHC Class II Molecules. Trends Immunol.2016 Nov;37(11):724-737; It can be evaluated. Such methods include experimental methods and predictive methods of peptide-antigen binding. Anchor residues that anchor peptide antigens to MHC class I molecules and ensure binding of peptide antigens to MHC class I molecules are known in the art. In a preferred aspect of all embodiments of the invention, the peptide antigens used according to the invention comprise any anchor residues or preferred amino acid residues at predicted positions for MHC class I molecules. Such prediction may preferably be carried out by the methods described in any of the following publications: -Rammensee et al, SYFPEITHI: database for MHC ligands and peptide motifs.Immunogenetics(1999)50:213-219; - Nielsen et al, Protein Sci(2003)12:1007-1017; - Neefjes et al.Nat Rev Immunol.2011 Nov 11;11(12):823-36; - Non-Patent Document 10; - Non-Patent Document 11; - Desai&Kulkarni-Kale,T-cell epitope prediction methods:an overview.Methods Mol Biol.2014;1184:333-64.

[0035] In a preferred aspect of all embodiments of the present invention, the peptide antigen is derived from a human protein. Alternatively, the non-anchor amino acid residues of the peptidic antigens of the invention may be identical to or have at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% sequence identity with the corresponding amino acid residues of the peptidic antigens derived from a human protein. Alternatively, the non-anchor amino acid residues of the peptidic antigens of the invention may comprise conservative substitutions, preferably no more than two conservative substitutions, more preferably one conservative substitution, with respect to the corresponding amino acid residues of the peptidic antigens derived from a human protein. In a preferred embodiment, the human protein is a protein expressed in a tissue or cell affected by a pathological immune response.

[0036] The peptide antigens of the present invention may be naturally occurring or non-naturally occurring peptides. The peptide antigens of the present invention are preferably composed of naturally occurring amino acids. However, non-naturally occurring amino acids, such as modified amino acids, may be used. For example, in one embodiment, the peptide antigens used according to the present invention may be peptidomimetics. Methods for synthesizing peptidic antigens, including those of the present invention, are well known in the art.

[0037] 〔array〕 Preferred amino acid sequences described in this application may be independently selected from the following sequences, which are presented in N-terminal to C-terminal order and represented in the one-letter amino acid code: Leader peptides: Examples

[0038] [ka] (SEQ ID NO:1) Peptide antigen: any MHC class I peptide corresponding to the MHC class I[α]1 and 2 domains, e.g.

[0039] [ka] (SEQ ID NO:2) or

[0040] [ka] (Ova) (SEQ ID NO: 3) Linker 1 (disulfide trap stabilization): Example

[0041] [ka] (SEQ ID NO: 4) or

[0042] [ka] (SEQ ID NO:5) For example, β2-microglobulin from humans or other species:

[0043] [ka] (SEQ ID NO:6, human β3 microglobulin) For example, Linker 2

[0044] [ka] (SEQ ID NO:7) [α]1 and 2 domains from either human HLA-G or other MHC class I [α]1 and 2 domains suitable for presenting a selected antigenic peptide, where Y84 can be C in the DT mutant.

[0045] [ka] (SEQ ID NO:8) e.g., mouse H2Kb[α]1 and 2 domains (Y84C)

[0046] [ka] (SEQ ID NO:9) or: human HLA-A2[α] 1 and 2 domains

[0047] [ka] (SEQ ID NO:10) Human HLA-G [α]3 domain (or any MHC Ib[α]3 domain, such as HLA-F, that interacts with the ILT2 and ILT4 receptors), e.g.

[0048] [ka] (SEQ ID NO: 11; sequence of HLA-G[α]3). Note that the amino acids in the above sequence underlined below are relevant for ILT2 or ILT4 receptor interaction.

[0049] [ka] Factor Xa restriction site:

[0050] [ka] (SEQ ID NO:12) Myc Tag:

[0051] [ka] (SEQ ID NO:13) Additional sequence: NSAVD His tag: HHHHHH* (sequence number 14).

[0052] Examples of mature full-length proteins of the invention: disulfide trap_Ova_Linker1_humanbeta2microglobulin_Linker2_H2Kbalpha1&2_HLA-Galpha3_XaSite_myc&hisTAG (dtH2Gova)

[0053] [ka] (SEQ ID NO:15) It should be noted that the sequence of the peptide antigen of the full-length protein (herein, "SIINFEKL") may be replaced by any peptide antigen sequence according to the present invention.

[0054] [ka] (SEQ ID NO:16) It should be noted that the sequence of the peptide antigen of the full-length protein (here, MLAVFLPIV) above may be replaced by any peptide antigen sequence according to the present invention.

[0055] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of said receptors according to the invention are as follows:

[0056] [ka] (SEQ ID NO:17)

[0057] [ka] (SEQ ID NO:18) The present invention is further illustrated by the following non-limiting examples.

[0058] [General notes] All steps were performed under sterile conditions and protective containers were only opened under a laminar flow hood. Cells were always centrifuged at 350 × g for 5 min unless otherwise stated. All live cells were incubated at 37 °C and 5% CO 2 The cells were kept in an incubator with 5% CO and >95% humidity. A water bath set at 37°C was used to pre-warm the medium, PBS, or other solutions added to the cells. A Neubauer chamber was used for cell counting. Statistical analysis was performed using Student's T test, with a p value of less than 0.05 considered significant. EXAMPLES

[0059] Cells expressing MHC Ib molecules loaded with a particular peptide selectively eliminate CTLs specific for the presented peptide. Materials and Methods JEG-3 is a human choriocarcinoma cell line that expresses high levels of HLA-G but little classical MHC class I molecules (Rinke de Wit et.al., J Immunol. 1990 Feb 1;144(3):1080-7). JEG-3 cells were cultured in complete RPMI 1640 medium ("complete RPMI") containing 10% fetal bovine serum, 0.5% sodium pyruvate solution (100 mM), and 1% penicillin (10 kU / ml) and streptomycin (10 mg / ml) solutions. 3 × 10 5 JEG-3 cells were seeded in 12-well plates in 1 ml of complete RPMI.

[0060] Where indicated, 1 μl of stock solution containing STEAP1 (292.2L-9mer, MLAVFLPIV) peptide (5 μg / μl) or PRAME (435-9mer, NLTHVLYPV) peptide (5 μg / μl) was added. The next day, JEG-3 cells were washed three times with PBS and 300 μl of CellGro DC supplemented medium (5% human serotype AB serum, 25–50 U / ml IL-2, 5 ng / ml IL-15) was added to each well.

[0061] Clonal HLA-A*02 (HLA-A2)-restricted STEAP1 (st) or PRAME (pr) peptide-specific CD8+ T cells (STEAP1- / PRAME "specific") were generated according to Woelfl et al, Nat Protoc. 2014 Apr;9(4):950-66. STEAP1-specific CD8+ T cells were stained with the cell proliferation dye eFluor® 670 according to the manufacturer's instructions and resuspended in complete RPMI 1640 medium as described above. 1.5×10 5 cells are added to each well along with peptide-loaded JEG-3. Similarly, unstained PRAME-specific CD8+ T cells were pelleted, resuspended, and added to each well. In the experiment shown in D, anti-ILT-2 antibody (clone HP-F1) or isotype control antibody was added to a final concentration of 10 μg / ml.

[0062] After 16 hours, cells were harvested and stained with 5 μM CellEvent Caspase-3 / 7 Green (Life Technologies) according to the manufacturer's instructions. Non-adherent cells were then harvested and stained with 1:100 dilutions of anti-human CD8 (PE / Cy7, clone RPA-T8) and anti-human CD4 (PE / Dye647, clone MEM-241) antibodies for 30 minutes on ice and analyzed by flow cytometry. Because CTLs are CD8+CD4-, CD4 staining could exclude potential CD4+ / CD8+ double positive cells and autofluorescent cells. Total cell numbers were determined based on the number of cells per μl. Viability of adherent JEG-3 cells was quantified by crystal violet assay.

[0063] Results: A) In control conditions without JEG-3 cells or with HLA-G+DMSO-treated control JEG-3 cells, apoptosis was detected in less than 5% of caspase 3 / 7+eFluor670 PRAME-specific or eFluor670+STEAP1-specific CD8+ T cells. In contrast, after coculture with STEAP1-supplemented JEG-3 cells, more than 90% of STEAP1-specific CD8+ T cells were lost or showed apoptosis, but no significant effect on PRAME-specific T cells was observed. STEAP1-specific CD8+ T cells were easily distinguished from PRAME-specific T cells by bright eFluor670 staining. The dot plot is a representative result of one of three experiments. B) Statistical analysis of three independent experiments showed that these effects were highly significant, indicating that STEAP1-specific T cells could be selectively eliminated by coculture with HLA-G+JEG-3 cells supplemented with the cognate peptide. C) The survival of JEG-3 cells is not reduced by the addition of a peptide recognized by the co-cultured T cells. D) Under the same conditions, the addition of the antibody ILT2, which blocks the HLA-G receptor, partially inhibited the loss of STEAP1-specific T cells from the target cells.

[0064] Conclusion: This experiment shows that peptide-specific CD8+ T cells can be selectively deleted when they come into contact with human MHC Ib+ cells, such as JEG-3 cells, that present their cognate antigen. This is surprising because MHC Ia+ target cells that present cognate peptides to activated CD8+ T cells are usually deleted during the survival of the T cells. In contrast to MHC Ia+ targets, the addition of peptide to JEG-3 cells did not reduce viability, suggesting that the function of MHC Ib molecules may be antagonistic to that of MHC Ia molecules. Furthermore, MHC Ib molecules and their receptor ILT2 cooperate to achieve this effect, as shown by the inhibition of this effect achieved by agents that block the interaction, such as ILT2 blocking antibodies. Thus, according to the present invention, such blocking agents may be used to promote the induction of peptide-specific immune responses in the presence of MHC Ib molecules. EXAMPLES

[0065] Using specific peptide-loaded MHC Ib molecules, it is possible to inhibit the potential cytotoxicity of allogeneic CTLs in an antigen-specific manner. [Materials and Methods] 1×10 6 JEG-3 cells were either untreated or added with STEAP1 peptide ("st", see Example 1) in 1 ml of complete RPMI 1640 in a 6-well plate. 5 5 x 10 STEAP1-specific CD8+ T cells 5 PRAME-specific CD8+ T cells (effectors) were mixed and these were left untreated or co-cultured with JEG-3. 10 μg / ml of neutralizing anti-human HLA-G antibody (clone 87G, BioLegend Germany) was added where indicated. The next day, firefly luciferase expressing HLA-A2+ UACC-257 melanoma cells (targets) were detached using Accutase solution (PAA Germany) and treated with STEAP1 peptide (5 μg / ml, "st loaded") or the same amount of DMSO ("unloaded") for 4 h on a shaker at 37°C, then washed and incubated at 1 × 10 per well. 4 UACC cells were seeded in white round-bottom 96-well plates. Non-adherent mixed T cells were then harvested and 4 × 10 4 Primary T cells (2 × 10 4 Equivalents of 1000 μg / ml of 1000 μg ...

[0066] Results: Presentation of peptide antigen by HLA-G+ JEG-3 cells inhibited the cytotoxic potential of a CD8+ T cell clone that recognized this specific peptide antigen in an MHC-Ib-dependent manner. In the described setting, STEAP1-specific control CTLs or CTLs pretreated with HLA-G+ JEG-3 cells lysed approximately 90% of all target cells loaded with cognate peptide, whereas naive target cells were not eliminated. In contrast, pretreatment with JEG-3 cells and cognate peptide resulted in almost complete protection of antigen-presenting target cells. Antibodies capable of partially blocking the HLA-G-dependent effect (87G) partially reversed this peptide-specific immunosuppressive effect. This means that peptide-loaded MHC class Ib molecules may also suppress unwanted cytotoxic (auto)immune responses against presented antigens in a clinical setting.

[0067] Furthermore, MHC Ib-positive tumor cells contacted with peptides (e.g., via radiation, chemotherapy, or peptide vaccination regimens) can specifically suppress CD8+ T cell-mediated antitumor immune responses, but this effect can be blocked with agents that block the interaction of MHC Ib molecules with their receptors. EXAMPLES

[0068] Combining MHC Ib molecules with specific peptides inhibits allogeneic CTLs but has little effect on immune responses to other antigens. Materials and Methods In the experiment shown in Figure 3, HLA-A2 STEAP1-specific CD8+ T cells (CD8st) and PRAME-specific CD8+ T cells (CD8pr) were mixed and co-cultured for 8 hours with JEG-3 cells that were left untreated or supplemented with or without STEAP1 peptide (see Figure 2 for methods). T cells in suspension were then collected and combined with luciferase-expressing PRAME-peptide (dark grey bars) or STEAP1-peptide (light grey bars) supplemented with HLA-A2+UACC-257 melanoma cells (T cells were not counted after pretreatment, initial effector:target ratio 1:1).

[0069] 〔result〕 In the context of MHC Ib positive cell lines, pre-exposure of mixed CD8 T cell clones to one allogeneic peptide reduced the cytotoxic potential of allogeneic T cells to about 50%, while the cytotoxic activity of the other T cell clones remained at about 90%, which was comparable to the peptide-independent immunosuppressive effect of HLA-G+JEG-3 cells alone. As a result, this approach shows that tolerance can be induced against specific (auto)immune-associated target antigens without simultaneously compromising the desired immune response against different (e.g. viral) antigens. Based on the MHC pattern displayed by JEG-3 cells and previous experiments with neutralizing antibodies, it will be understood that these peptide-specific effects are mediated through HLA-G. This experiment suggests that presentation of antigenic peptides on MHC Ib molecules may inhibit the cytolytic capacity of allogeneic CD8+ T cells. EXAMPLES

[0070] Therapeutic drug construction strategy: Soluble single-chain constructs containing antigenic peptides, MHC class I-based [α]1 and [α]2 domains, [α]3 domains derived from HLA-G (or other MHC class Ib molecules) and [β]2-microglobulin [Design of MHC Ib peptide complex] MHC class Ib molecules, such as HLA-G, naturally consist of three polypeptide molecules in a complex, which can be linked with a linker to improve stability, as shown in Figure 4. Alternatively, all components may be displayed linearly, as shown in FIG. The sequences used in certain embodiments are listed below. The coding sequence consists of: Leader peptide: e.g.

[0071] [ka] Secretion-inducing leader peptides such as (SEQ ID NO:1) Presented peptide antigens: MHC class I [α] 1 and 2 domains, e.g.

[0072] [ka] (STEAP1) (SEQ ID NO:2) or

[0073] [ka] Any peptide with 8 to 12 amino acid anchor residues that allows presentation by SIINFEKL(Ova) (SEQ ID NO:3). (Disulfide trap stabilized (underlined)) Linker 1:

[0074] [ka] (SEQ ID NO:4) or

[0075] [ka] (SEQ ID NO:5) β2-microglobulin of human or other species origin

[0076] [ka] SEQ ID NO:6 Linker 2

[0077] [ka] (SEQ ID NO:7) [α]1 and 2 domains from human HLA-G or any other MHC class I [α]1 and 2 domains suitable for presenting a selected antigenic peptide, Y84 may be C in the DT variant

[0078] [ka] (SEQ ID NO:8) For example, mouse H2Kb[α]1 and 2 domains ( Y84C )

[0079] [ka] (SEQ ID NO:9) or: human HLA-A2[α] 1 and 2 domains

[0080] [ka] (SEQ ID NO:10) Human HLA-G[α]3 domain (or any MHC Ib[α]3 domain such as HLA-F, which also interacts with the ILT2 and ILT4 receptors; the underlined amino acids are relevant for interaction with ILT-2 or ILT-4)

[0081] [ka] (SEQ ID NO:11) Factor Xa restriction site: (SEQ ID NO: 12) Myc tag: (SEQ ID NO: 13) Additional sequence: NSAVD His tag: HHHHHH* (SEQ ID NO: 14) Examples of mature full-length proteins:

[0082] [ka] EXAMPLES

[0083] Soluble peptide-MHC Ib complexes in combination with dendritic cells (DC-10) can selectively eliminate CD8+ effector T cells that recognize the presented target antigen. Materials and Methods To test whether the soluble peptide-MHC Ib constructs could eliminate effector T cells in an antigen-dependent manner, the constructs were added to dendritic cells grown in the presence of IL-4, GM-CSF, and IL-10 (DC-10). 5x10 purified MHC Ib cells from healthy donors were cultured in DC-10-Medium (complete RPMI 1640 medium, 10ng / ml IL-4, 10ng / ml IL-10, 100ng / ml GM-CSF). 6 MACS-purified (CD14 beads, Miltenyi, Germany) CD14+ cells / ml were cultured for 7 days to generate DC-10. Fresh medium was added on days 3 and 5. The resulting DC-10 cells did not adhere to the cell culture dish. Then, 4x10 5 DC-10 cells were transfected with CHO cells (1x10) transiently transfected by Lipofection with pCDNA3.1 expression vectors for single chain disulfide-trapped peptide HLA-G constructs containing STEAP1 peptide (dtGsteap, see Example 4 for sequence), Melan A / MART-1 peptide (ELAGIGILTV, dtGmelA), or control supernatant. 6 The DC-10 cells were then mixed with the same amount of supernatant from day 5 of cell culture (10 μg / ml) for 4 h. The DC-10 cells were then washed three times with PBS and incubated for 5 h with AB serum + IL-2 (10 μg / ml). 6 The cells were then resuspended in 50 μl of RPMI 1640 medium containing 5 × 10 4 Peptide-MHC Ib-loaded DC-10 cells were combined with HLA-A2-restricted antigen-specific CD8+ T cells recognizing either STEAP1 (CD8st) or PRAME (CD8pr) at a 1:1 ratio for 16 hours. Cells were then stained with CellEvent Caspase-3 / 7 Green (5 μM, Life Technologies) and antibodies specific for human CD4 (clone EDU-2) and CD8 (clone RPA-T8) according to the manufacturer's instructions (see Example 2). CD8+CD4 Caspase-3 / 7 cells were quantified by flow cytometry. 〔result〕 As shown in Figure 6, in two independent experiments, STEAP1-specific T cells were almost completely eliminated within 16 hours when combined with DC-10 cells loaded with a single-chain MHC Ib construct presenting the cognate peptide. The same conditions did not adversely affect the survival of T cells specific for a control peptide (CD8pr). A control containing a control peptide (dtGmelA) slightly reduced the survival of STEAP1-specific CD8+ T cells. This indicates that soluble MHC Ib molecules, when bound to a specific peptide, can selectively eliminate effector T cells specific for the presented peptide, i.e., selectively modulate the immune response against a particular antigen. EXAMPLES

[0084] Peptide-loaded MHC Ib complexes induce human antigen-specific regulatory T cells that recognize presented peptides In the experiment shown in Figure 7A, 5 × 10 6 Peripheral blood mononuclear cells (PBMCs) were obtained from two independent healthy donors and were cultured at 1 × 10 6 The irradiated JEG-3 cells were co-cultured for 14 days in 2 ml of RPMI 1640 medium containing 5% human serotype AB serum, 5 ng / ml TGF-β1, 20 ng / ml IL-2 (Treg medium) (supplemented as above). Fresh medium was added on day 3. On day 7, medium was changed and PBMCs were freshly irradiated and supplemented with peptide at 1 × 10 61000 cells were transferred to JEG-3 cells. Treg expansion beads (Miltenyi Biotec, anti-CD3 / CD28) were used as a positive control according to the manufacturer's instructions. The resulting cells were stained with antibodies against human CD4 (clone EDU-2, Immunotools) and CD25 (Miltenyi 120-001-311) and HLA-A2 STEAP1 dextramer (STEAP1 dex, Immudex Denmark, all dilutions 1:100) for 30 min on ice. The frequency of STEAP1-specific T cells among CD4+CD25high Treg cells was quantified by flow cytometry (Shevach et al., 2002, Nat. Rev. Immunol. 2:389). STEAP1-specific CD4+ CD25high Treg cells were not detected when PBMCs were cultured alone (ctrl) or in the presence of control peptide (melA), but a significant population was repeatedly observed when PBMCs were cocultured with JEG-3 cells presenting the cognate antigen, with a lower range in the positive control setting (aCD3 / 28).

[0085] In the experiment shown in Figure 7B, 4x10 5 DC-10 cells / well were loaded with disulfide-trapped single-chain HLA-G constructs containing presented MELAN-A (dtGmelA) or STEAP1 (dtGsteap) peptides as described in Figure 6. Then, 4x10 DC-10 cells / well from the same donor were loaded with disulfide-trapped single-chain HLA-G constructs containing presented MELAN-A (dtGmelA) or STEAP1 (dtGsteap) peptides as described in Figure 6. 6 PBLs were added and the cells were cultured in 2 ml of Treg medium in 12-well plates for 7 days, with 1 ml of medium replaced on day 3. On day 7, 4 × 10 5Fresh, identical DC-10 was added to each well, and the medium was changed again on day 10. On day 14, cells were harvested, washed, and stained with fluorescently labeled antibodies against CD4 (clone MEM-241), CD8 (clone RPA-T8), and HLA-A2-Melan A peptide dextramer (Immudex). Intracellular staining for IL-10 (clone JES3-9D7) was performed using an intracellular staining kit (eBiosciences). The number of MELAN-A-specific IL-10+ Tregs was significantly increased under conditions in which PBL were co-cultured with DC-10 supplemented with single-chain Melan A HLA-G molecule (dtGmelA) compared with control molecule (dtGsteap) or untreated PBL. EXAMPLES

[0086] A single peptide-MHC construct containing the human MHC Ibα3 domain in combination with DC induces mouse Treg cells specific for the presented peptide (FIG. 8). Murine DCs (mDCs) were generated by culturing bone marrow cells from wild-type C57BL / 6 mice for 7 days in complete RPMI-1640 supplemented with 10% GM-CSF supernatant from the Ag8653 myeloma cell line transfected with the mouse GM-CSF gene (detailed protocol: Lutz et al., J Immunol Methods 1999, 223(1):77-92). 4 × 10 5mDCs were mixed for 4 hours with 500 μl of "day 5 CHO supernatant" from mock transfected cells (CHO) or from CHO cells transfected with a pCDNA3.1 vector encoding a single chain ovalbumin peptide (SIINFEKL), mouse H-2Kb α1 domain and mouse H-2Kb α2 domain, and human HLA-G α3 domain (H2Kb, sequence dtH2KbGova in Example 4) or human HLA-A2 α1 and human HLA-A2 α2 domain (A2G). The presence of each construct in the supernatant was confirmed by Western blotting. Preliminary results suggest that induction is possible even with purified constructs. Here, peptide-loaded MHC constructs were purified using cOmplete His-Tag purification resin (Sigma Aldrich) to bind the constructs, followed by washing with PBS (3 times) and factor Xa protease digestion (1U / 100 μl, 20° C., 6 hours, Qiagen) to release the constructs. Factor Xa can then be removed using factor Xa removal resin (Qiagen, all according to manufacturer's instructions). Sequences are described in Example 4. mDCs were then washed with PBS.

[0087] C57BL / 6 RAG- / -OT1 mice express almost exclusively T cell receptors that interact with ova peptides presented on H-2Kb. 6 Splenocytes were cultured at 4×10 in Treg induction medium (complete RPMI medium, 5 ng / ml IL-2, 5 ng / ml TGF-β1) with or without (mDC A2G / CHO / H2Kb OT1) as indicated. 5mDCs were cultured for 14 days. Cells were then stained with fluorochrome-conjugated antibodies specific for mouse CD3 (clone KT3, Serotec), Foxp3 (3G3, Miltenyi Biotec) and IL10 (JES5-16E3) and quantified by flow cytometry (see Huenig et al., Brain. 2008 Sep;131(Pt9):2353-65 for mice and protocol). It was observed that antigen-specific Tregs were significantly increased under all conditions in which T cells were combined with the cognate peptide / MHC α1 and α2 domains of the MHC Ib molecule and the immunosuppressive α3 domain. The moderate induction of purified constructs could be explained by the loss of protein during the purification process.

[0088] These examples imply that peptide presentation on MHC class Ib molecules promotes the expansion of allogeneic Tregs. Such Tregs are preferentially activated via T cell receptors in tissues where antigens are present, i.e., target tissue-specific autoimmune reactions should be suppressed if appropriate tissue-specific antigens are available. Note that due to the bystander inhibitory potential of antigen-specific Tregs, the selected tissue-specific "Treg-activating antigen" does not have to be identical to the autoantigen that drives the pathological immune response. [Industrial Applicability]

[0089] The compositions, polypeptides, nucleic acids, cells, combinations and methods of the present invention have industrial applicability, for example, for use in the manufacture of pharmaceuticals or as pharmaceuticals.

Claims

1. 1. An isolated recombinant polypeptide capable of presenting a peptide antigen, comprising from N-terminus to C-terminus the following components: i) a peptide antigen presented by said isolated recombinant polypeptide; ii) a first linker sequence; iii) a sequence of a human polypeptide domain comprising a sequence of human β2-microglobulin or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2-microglobulin represented by SEQ ID NO:6; iv) a second linker sequence; v) the [α]1 domain of an MHC molecule; vi) the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of said [α]3 domain of said MHC class Ib molecule, which is capable of binding to ILT2 or ILT4, The [α]3 domain is identical to the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of the derivative of the [α]3 domain of the MHC class Ib molecule has at least 99% identity to the amino acid sequence of the [α]3 domain of SEQ ID NO: 11, and is represented by SEQ ID NO: 11: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL The amino acid sequence represented by the formula (I) includes the underlined sequence; viii) a protease cleavage site; and ix) affinity tags; 1. An isolated recombinant polypeptide comprising at least components i), iii), v), vi) and vii), in that order.

2. Ingredients below: ii) a first linker sequence; and / or iv) a second linker sequence; and / or viii) a protease cleavage site; and / or ix) Affinity Tags 2. The isolated recombinant polypeptide of claim 1, comprising:

3. 3. The isolated recombinant polypeptide of claim 1 or 2, wherein the [α]1 domain of component v) and the [α]2 domain of component vi) are derived from an MHC class Ia molecule.

4. binds to ILT2 or ILT4 with an affinity constant Kd of less than 40 μM as measured by surface plasmon resonance; or binds to ILT2 or ILT4 with an affinity constant Kd of less than 20 μM as measured by surface plasmon resonance; or 4. The isolated recombinant polypeptide of any one of claims 1 to 3, capable of binding to ILT2 or ILT4 with an affinity constant Kd of less than 10 μM as measured by surface plasmon resonance.

5. is a dimer or multimer; and / or the peptide antigen sequence of component i) is 7 to 11 amino acids in length, or 8 to 10 amino acids in length; and / or containing all of components i) to vii), or not containing components viii) to ix), or containing all of components i) to ix); and / or The isolated recombinant polypeptide of any one of claims 1 to 4, further comprising an N-terminal secretory signal peptide sequence.

6. 6. An isolated recombinant polypeptide according to any one of claims 1 to 5 for use in medicine.

7. 6. An isolated recombinant polypeptide according to any one of claims 1 to 5 for use in a method for peptide antigen-specific immune modulation in a subject, said peptide antigen-specific immune modulation being specific for said peptide antigen comprised in said isolated recombinant polypeptide.

8. 8. The isolated recombinant polypeptide of claim 7, wherein the method for peptide antigen-specific immune modulation is used to induce immunological tolerance to the peptide antigen contained in the isolated recombinant polypeptide.

9. 9. The isolated recombinant polypeptide of any one of claims 6 to 8, wherein the method for peptide antigen-specific immune modulation is a method for suppressing autoimmune diseases, a method for suppressing allergies, a method for suppressing immune responses to biological agents, a method for suppressing immune responses to embryonic antigens, or a method for suppressing immune responses to transplanted cells, tissues or organs.

10. 10. The isolated recombinant polypeptide of claim 9, wherein the method for peptide antigen-specific immune modulation is a method for inducing immune tolerance and the autoimmune disease affects multiple organs, hormone-producing organs, nerves, joints, skin, gastrointestinal system, eyes, blood components or blood vessels.

11. 8. The isolated recombinant polypeptide of claim 7, wherein the method for peptide antigen-specific immune modulation is a method for suppressing immune responses in Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, rheumatoid arthritis, psoriasis, scleroderma, neuromyelitis optica or type 1 diabetes.

12. 6. A method for producing an isolated recombinant polypeptide according to any one of claims 1 to 5, comprising culturing a recombinant host cell under conditions allowing expression of a nucleic acid molecule and recovering the isolated recombinant polypeptide produced, wherein said recombinant host cell comprises said nucleic acid molecule encoding the isolated recombinant polypeptide according to any one of claims 1 to 5.

13. The method of claim 12, wherein the nucleic acid molecule is a vector.

14. A) the method for inducing immunological tolerance to the peptide antigen further comprises the treatment of a peptide drug, the peptide antigen being: 1) identical to the peptide drug, or 2) a fragment of the peptide drug, or 3) a derivative of the fragment of the peptide drug capable of inducing immunological tolerance to the peptide drug, wherein the peptide drug is administered in the form of the peptide drug itself or by gene therapy using a gene encoding the peptide drug; or B) the method for inducing immunological tolerance to the peptide antigen further includes treatment with a protein drug, the peptide antigen being 1) a fragment of the protein drug, or 2) a derivative of the fragment of the protein drug capable of inducing immunological tolerance to the protein drug; wherein the peptide drug is administered in the form of the peptide drug itself, or by gene therapy using a gene encoding the peptide drug; The isolated recombinant polypeptide of claim 7.

15. A pharmaceutical composition comprising a nucleic acid encoding the isolated recombinant polypeptide of any one of claims 1 to 5.

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