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

Abstract

The present invention relates to the therapeutic use of non-classical major histocompatibility complex (MHC), also known as MHC class Ib molecules, in combination with defined peptides. The present invention more specifically relates to the targeted immunomodulatory effects of defined peptide binding proteins or binding molecules, said proteins comprising one or more domains of non-classical MHC class Ib molecules, said molecules interfering with the interaction of MHC class Ib molecules and their receptors. The present invention also relates to methods of producing such proteins, pharmaceutical compositions comprising the proteins, and their use in the treatment of diseases for which antigen-specific immune responses are beneficial, including cancer and infectious diseases, or detrimental, including autoimmune diseases, organ / tissue rejection, immune responses to drug compounds, or reproductive disorders. In addition, since the present invention discloses a new mode of action of MHC class Ib molecules in the process of antigen-specific tolerance induction, the present invention also relates to methods of interfering with the mechanism in cases where induction of antigen-specific immune tolerance is desired, but such induction is physiologically impeded by the mechanism.

Description

Technical Field

[0001] The present invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also known as MHC class Ib molecules) in combination with peptide antigens. The present invention more specifically relates to peptide antigen binding proteins or binding molecules, the proteins comprising one or more domains of non-classical MHC class Ib molecules, the molecules hindering the binding of non-classical MHC class Ib molecules to their receptors. The present invention also relates to methods for producing such proteins, pharmaceutical compositions comprising the proteins, and for the treatment of conditions that benefit from antigen-specific immune responses, including cancer and infectious diseases, or harmful diseases, including autoimmune diseases, organ / tissue rejection, immune responses to drug compounds, or reproductive disorders. Background Art

[0002] Three major classes of major histocompatibility complex (MHC) antigens are known, namely 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 (α1, α2 and α3). The MHC I complex also contains β2-microglobulin and the presented peptide bound to the peptide binding groove, which includes the α1 and α2 domains. Therefore, peptide-loaded conventional classical MHC Ia class molecules can initiate peptide-specific, T cell-mediated immune responses, which may lead to the lysis of presenting cells. This mechanism is crucial for vaccination strategies, which may include shorter or longer peptides (Slingluff, Cancer J. 2011 Sep; 17(5): 343–350), nucleic acids encoding antigens (Restifo et al., Gene Ther. 2000 Jan; 7(2): 89–92), proteins, or attenuated organisms that are often developed or used clinically to induce immune responses to specific antigens. Antigens may include viral, bacterial, or tumor-associated antigens.

[0003] Unlike conventional classical MHC class Ia molecules expressed in most human tissues, non-classical MHC class Ib antigens (such as HLA-G) show only very limited tissue expression. Physiologically, the chorionic trophoblast of normal human placenta expresses high levels of HLA-G, which may act as an immunomodulator to protect the fetus from the maternal immune system (without maternal rejection). According to this hypothesis, previous studies have shown that HLA-G proteins can inhibit allogeneic reactions, such as proliferative T lymphocyte cell reactions, cytotoxic T lymphocyte-mediated cytolysis, and NK cell-mediated cytolysis (Rouas-Freiss N. et al., Proc. Natl. Acad. Sci., 1997, 94, 5249-5254; Semin Cancer Biol 1999, vol 9, p. 3).

[0004] The sequence of the HLA-G gene has been described (e.g., Geraghty et al. Proc. Natl. Acad. Sci. USA, 1987, 84, 9145-9149; Ellis et al., J. Immunol., 1990, 144, 731-735), and contains 4396 base pairs. The gene consists of 8 exons, 7 introns, and 1 3' untranslated end, which correspond to the following domains: exon 1: signal sequence, exon 2: α1 extracellular domain, exon 3: α2 extracellular domain, exon 4: α3 extracellular domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and 3' untranslated region. 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) (see, e.g., Carosella et al., Blood 2008, vol. 111, p 4862). The mature HLA-G1 protein isoform contains three external domains (α1-α3), a transmembrane region, and a cytoplasmic domain, and the mature HLA-G5 protein isoform contains three external domains (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 can also be produced by cleavage of membrane-bound isoforms.

[0005] HLA-G interacts with specific receptors such as Kir2DL4, ILT2 (LILRB1) and ILT4 (LILRB2, Clements et al., Proc Natl Acad Sci US A. 2005 Mar 1; 102(9): 3360-5) in a peptide-independent manner. The most significant immunosuppressive effect of HLA-G on T cells is mediated by ILT2 and ILT4. Since these receptors interact with the α-3 domain contained in HLA-G and other MHC class Ib molecules, such as HLA-F (Lepin et al., Eur. J. Immunol. 2000. 30: 3552–3561), the α3 domain-dependent effect observed for the representative MHC class Ib molecule HLA-G can also be induced by other MHC class Ib molecules.

[0006] It is further known that MHC class Ib molecules present peptides through their α1 and α2 domains. These peptides are usually composed of 8-10 amino acids and contain certain anchor residues (Diehl et al. Curr Biol. 1996 Mar 1; 6 (3): 305-14, Lee et al. Immunity. 1995 Nov; 3 (5): 591-600.). However, to the best of the inventor's knowledge, the peptide-specific interaction of human MHC Ib class molecules with cognate T cell receptors has not yet been studied. Similarly, there are no clear data from animal models. Swanson et al. pointed out that murine MHC Ib molecules may induce peptide-specific immune responses (Swanson et al., An MHC class Ib-restricted CD8 T cell response confers antiviral immunity, JEM 2008), while Wang et al. described the inhibition of murine Qa2 molecules on peptide-specific immune responses. (Wang et al, Sci. Rep. 36064, 31. Oct. 2016). However, human and murine MHC Ib molecules differ greatly (Pratheek et al., Indian J Hum Genet. 2014 Apr-Jun; 20(2): 129–141). Because HLA-G and Qa-2 share only 67% sequence identity based on analysis using proteinblast reference sequences Q5RJ85 (Q5RJ85_HUMAN) and P79568 (P79568_MOUSE) on UniProtKB, conclusions drawn from Qa-2 must be treated with caution, and it is not possible to predict whether those conclusions will also apply to human HLA-G. A recent review article outlined the great difficulties in defining mouse models that are suitable for studying HLA-G function in basic science and preclinical studies (Nguyen-Lefebvre et al., 2016).

[0007] Based on the available data, it has been proposed that HLA-G protein can be used to treat transplant rejection in allogeneic or xenogeneic organ / tissue transplantation. It has also been proposed that HLA-G protein can be used to treat hematological malignancies (EP1 054688), inflammatory diseases (EP1 189 627), and more general immune-related diseases. In addition, HLA-G is often expressed in human tumors (Carosella et al. Trends Immunol. 2008 Mar; 29 (3): 125-32), and it is reported that its function is similar to that of immunosuppressive immune checkpoint molecules, which non-specifically inhibit immune responses in the tumor microenvironment (Carosella ED et al., Adv Immunol. 2015; 127: 33-144). However, none of these studies analyzed the peptides presented on HLA-G. Therefore, the question of whether the presented peptides can indicate (direct) the observed MHC Ib class-mediated effects has not even been proposed.

[0008] Given the inherent limitations of all mouse models for studying human MHC class Ib molecules, it is imperative to explore the effects of human MHC class Ib molecules on human T cells in vitro in order to gain a mechanistic understanding that can 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 critical. Although cytotoxic CD8 + Both effector T cells (cytotoxic T lymphocytes, CTLs) and regulatory T cells (Tregs) are able to detect antigenic peptides presented on MHC molecules, but CTLs are able to destroy cells expressing their cognate antigens, while regulatory T cells, especially when their cognate antigens are presented by their respective tissues, have a tissue protective effect (Wright et al., 2009 PNAS vol.106no.45, 19078–83). Importantly, if antigen-specific regulatory T cells are activated by cognate antigens in target tissues, they can also play a bystander effect and suppress immune responses against other antigens. Therefore, CTLs may be beneficial to cancer patients (Gajewski et al., Nat. Immunol. 14, 1014–1022, 2013), but harmful to autoimmune diseases. Treg cells that suppress immune responses play the opposite role. Insufficient activity or function of Tregs can lead to severe autoimmune diseases in mice and may also be associated with human autoimmune diseases (Bluestone et al., J Clin Invest. 2015; 125(6): 2250–2260). Therefore, strategies for inhibiting (or disinhibiting) cytotoxic T cells and inducing (or inhibiting) Tregs are needed.

[0009] In current clinical practice, regardless of the targeted antigen, diseases caused by pathological immune responses (e.g., autoimmune diseases) are usually treated with immunosuppressive therapies, which may cause serious and often dose-limiting side effects and increase the risk of opportunistic infections. Therefore, there is a need to improve methods and uses for treating such diseases. Therefore, there is a need in the art to improve methods and uses for therapeutically modulating the immune system by more targeted and antigen-specific methods.

[0010] Conversely, there is a need for improved methods and uses for treating diseases (including cancer) that are immune responses to specific antigens. For example, many vaccination methods that have been documented for cancer immunotherapy have proven to be ineffective due to the immunosuppressive mechanisms of cancer. Therefore, there is a need for improved methods and uses for treating such diseases, including cancer. Summary of the invention

[0011] The inventors were surprised to find that human MHC class Ib molecules, such as HLA-G, are capable of inducing antigen-specific tolerance to presented peptide antigens. Therefore, although their structure and sequence are similar to classical human MHC class I molecules that induce antigen-peptide-specific immune responses, 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 inducing antigen-specific regulatory T cells that recognize self-antigens expressed in tissues or another target antigen that is susceptible to autoimmune attack. Based on the above, the inventors have shown that exemplary human MHC class Ib molecules can be used to eliminate cytotoxic effector T cells (such as cytotoxic effector T cells) in an antigen-peptide-specific manner. Figure 1 ) and induction of tolerogenic regulatory T cells (as shown Figure 7 In a non-limiting embodiment, these effects can be achieved by binding specific peptide antigens to membrane-bound or soluble MHC class Ib molecules. Conversely, in cases where an antigen-specific immune response needs to be induced, it is necessary to destroy the immune tolerance associated with MHC class Ib. The inventors have found that this can be achieved by agents that block the binding of human MHC class Ib molecules to their receptors.

[0012] According to the present invention, peptides that bind to MHC class Ib molecules can be advantageously used to suppress immune responses in an antigen-specific or tissue-specific manner. This is a significant advantage over many traditional therapeutic approaches, which suppress immune responses regardless of the target antigen, because their lack of specificity produces severe and dose-limiting side effects and increases the risk of opportunistic infections.

[0013] In addition, the inventors surprisingly found that other molecules besides naturally occurring MHC class Ib molecules, in particular polypeptides comprising only at least one domain of an MHC class Ib molecule, preferably at least one α3 domain of an MHC class Ib molecule, can be used to inhibit immune responses as described in the present invention: Figure 7 and Figure 8 As shown, the α1 and α2 domains of various classical class Ia molecules can effectively bind to the α3 domain of human MHC class Ib molecules to inhibit the immune response to peptides presented by these antigens.

[0014] Thus, according to the present invention, the use of, for example, the immunosuppressive α3 domain of an MHC class Ib molecule in combination with a targeting antigen presented, for example, by the α1 and α2 domains of an MHC class I molecule would be beneficial for a number of autoimmune diseases.

[0015] On the contrary, the inventors' new and surprising findings also indicate that the inhibition of antigen-specific immune responses caused by MHC class Ib molecules can be reversed by agents that interfere with the binding of MHC class Ib molecules to their receptors. Therefore, according to the present invention, such blocking agents, such as antibodies to MHC class Ib molecules (such as Figure 2 ) or its receptors including ILT2 and ILT4 (such as Figure 1 ) can be advantageously used to treat diseases for which an immune response to a specific antigen is necessary. These diseases include cancers such as gastric cancer, gastrointestinal stromal tumors, head and neck cancer, kidney cancer, liver cancer, lung cancer, breast cancer, uterine cancer, ovarian cancer, cervical cancer, vaginal cancer, urethral cancer, testicular cancer, colon cancer and intestinal cancer, pancreatic cancer, skin cancer and sarcoma (see, e.g., http: / / medicalgenome.kribb.re.kr / GENT / search / view_result.php), and infectious diseases including, but not limited to, 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; 7(2): 273-82), HTLV-1 infection (see, e.g., Alves et al., J Gen Virol. 2016 Oct; 97(10): 2742-2752.), hepatitis C infection (see, e.g., Ding et al., Med Sci Monit. 2016 Apr 26; 22: 1398-402.), or malaria infection with Plasmodium falciparum (see, e.g., Garcia et al., Infect Genet Evol. 2013 Jun; 16: 263-9.).

[0016] In the case where it is first necessary to induce a specific immune response to a selected antigen, vaccines comprising peptides or proteins or attenuated pathogens or DNA or RNA encoding proteins are generally used in the art. However, such vaccinations may not cause a response, or even cause unwanted tolerance (Slingluff, Cancer J. 2011 Sep; 17 (5): 343–350). Due to tumor cells (Carosella et al. Trends Immunol. 2008 Mar; 29 (3): 125-32) and virus-infected cells (Rizzo et al., Front Immunol. 2014; 5: 592) expressing MHC Ib class molecules, such as antigen presentation of HLA-G on MHC Ib class molecules, may be the cause of such failure. Therefore, according to the present invention, agents that specifically block the binding of MHC Ib class molecules to their receptors can be used to improve the efficacy of the therapy of inducing peptide-specific or protein-specific immune responses using specific antigen proteins or peptides. These therapies include those based on external vaccines, but can also extend to therapies where antigenic material released by dying tumor cells can induce antigen-specific T cell responses, such as radiation therapy or chemotherapy (for such therapies, see, for example, Zitvogel et al., Nature Reviews Immunology 8, 59-73, January 2008). Conversely, the unwanted effects of vaccination caused by treatment with biologics or gene therapy can be counteracted by adding MHC class Ib-based constructs to prevent the emergence of anti-drug antibodies.

[0017] Therefore, the present invention relates to the following preferred embodiments:

[0018] 1. A pharmaceutical composition comprising:

[0019] a) a human non-classical MHC class I molecule, or a polypeptide capable of presenting a peptide antigen to a T cell, wherein the polypeptide comprises an α3 domain of a human non-classical MHC class I molecule or a derivative of the α3 domain of a human non-classical MHC class I molecule, and the derivative is capable of binding to ILT2 or ILT4, and

[0020] b) a peptide antigen, which is presented by the non-classical MHC class I molecule or polypeptide according to a).

[0021] 2. A pharmaceutical composition according to item 1, wherein the composition comprises the polypeptide capable of presenting a peptide antigen according to a), wherein the polypeptide comprises, preferably in N-terminal to C-terminal order, the α1 and α2 domains of the classical MHC class I molecule, and the classical MHC class I molecule is followed by the α3 domain or the derivative.

[0022] 3. The pharmaceutical composition according to item 1 or 2, wherein the α3 domain or derivative comprised by the non-classical MHC class I molecule or polypeptide is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0023] 4. The pharmaceutical composition according to item 3, wherein the α3 domain or derivative comprised by the non-classical MHC class I molecule or polypeptide is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 92% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0024] 5. The pharmaceutical composition according to item 3, wherein the α3 domain or derivative comprised by the non-classical MHC class I molecule or polypeptide is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 94% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0025] 6. The pharmaceutical composition according to item 3, wherein the α3 domain or derivative comprised by the non-classical MHC class I molecule or polypeptide is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 96% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0026] 7. The pharmaceutical composition according to item 3, wherein the α3 domain or derivative comprised by the non-classical MHC class I molecule or polypeptide is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 98% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0027] 8. The pharmaceutical composition according to item 3, wherein the α3 domain or derivative comprised by the non-classical MHC class I molecule or polypeptide is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 99% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0028] 9. The pharmaceutical composition according to item 3, wherein the α3 domain or derivative contained in the non-classical MHC class I molecule or polypeptide has the same amino acid sequence as the α3 domain shown in SEQ ID No:11.

[0029] 10. The pharmaceutical composition according to any one of the above items, wherein the non-classical MHC class I molecule according to a) or the polypeptide capable of presenting a peptide antigen according to a) can bind to ILT2 or ILT4, and the affinity constant K obtained by surface plasmon resonance spectroscopy is d Less than 40μM.

[0030] 11. A pharmaceutical composition according to any one of the above items, wherein the non-classical MHC class I molecule according to a) or the polypeptide capable of presenting a peptide antigen according to a) can bind to ILT2 or ILT4, and the affinity constant K obtained by surface plasmon resonance spectroscopy is d Less than 20μM.

[0031] 12. The pharmaceutical composition according to any one of the above items, wherein the non-classical MHC class I molecule according to a) or the polypeptide capable of presenting a peptide antigen according to a) can bind to ILT2 or ILT4, and the affinity constant K obtained by surface plasmon resonance spectroscopy is d Less than 10μM.

[0032] 13. A pharmaceutical composition according to any of the above items, wherein the pharmaceutical composition further comprises a polypeptide domain, the polypeptide domain comprising the amino acid sequence as shown in SEQ ID No: 6, or a sequence that is at least 90% identical to the amino acid sequence as shown in SEQ ID No: 6, preferably at least 95% identical to the amino acid sequence as shown in SEQ ID No: 6, and more preferably at least 98% identical to the amino acid sequence as shown in SEQ ID No: 6, and the polypeptide domain is preferably comprised by the polypeptide capable of presenting peptide antigens according to a).

[0033] 14. The pharmaceutical composition according to any one of the above items, wherein the non-classical MHC class I molecule according to a) or the polypeptide capable of presenting a peptide antigen according to a) further comprises one or more linker sequences, preferably a (GGGGS)n linker sequence.

[0034] 15. The pharmaceutical composition according to any one of the above items, wherein the non-classical MHC class I molecule according to a) or the polypeptide capable of presenting a peptide antigen according to a) is a dimer or a multimer.

[0035] 16. The pharmaceutical composition according to any one of the above items, wherein the peptide antigen has a length of 7 to 11 amino acids, preferably 8 to 10 amino acids.

[0036] 17. The pharmaceutical composition according to items 1 and 3 to 16, wherein the composition comprises the non-classical MHC class I molecule according to a), and the non-classical MHC class I molecule is HLA-E, HLA-F or HLA-G.

[0037] 18. The pharmaceutical composition according to item 17, wherein the non-classical MHC class I molecule is HLA-G.

[0038] 19. The pharmaceutical composition according to item 17 or 18, wherein the non-classical MHC class I molecule is a human non-classical MHC class I molecule.

[0039] 20. The pharmaceutical composition according to any one of the above items, wherein the peptide antigen according to b) is covalently bound to the non-classical MHC class I molecule or polypeptide according to a).

[0040] 21. The pharmaceutical composition according to item 20, wherein the peptide antigen according to b) is covalently bound to the non-classical MHC class I molecule or polypeptide according to a) via a peptide bond, and the peptide antigen is part of a single polypeptide chain.

[0041] 22. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, from N-terminus to C-terminus,

[0042] i) a peptide antigen, wherein the peptide antigen is presented by the recombinant polypeptide;

[0043] ii) optionally, a first linker sequence;

[0044] iii) optionally, a sequence of a human polypeptide domain, the sequence of the human polypeptide domain comprising the 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 as shown in SEQ ID No: 6;

[0045] iv) optionally, a second linker sequence;

[0046] v) optionally, an α1 domain of an MHC molecule;

[0047] vi) optionally, an α2 domain of an MHC molecule;

[0048] vii) a derivative of the α3 domain of an MHC class Ib molecule or the α3 domain of a non-classical MHC class I molecule, which derivative is capable of binding to ILT2 or ILT4;

[0049] viii) optionally, a protease cleavage site, and

[0050] ix) Optionally, an affinity tag.

[0051] 23. The recombinant polypeptide according to item 22, wherein

[0052] v) the α1 domain and vi) the α2 domain are from a classical MHC class I molecule.

[0053] 24. A recombinant polypeptide according to item 22 or 23, wherein the α3 domain or derivative is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0054] 25. The recombinant polypeptide according to item 24, wherein the α3 domain or derivative is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 92% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0055] 26. The recombinant polypeptide according to item 24, wherein the α3 domain or derivative is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 94% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0056] 27. The recombinant polypeptide according to item 24, wherein the α3 domain or derivative is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 96% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0057] 28. The recombinant polypeptide according to item 24, wherein the α3 domain or derivative is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 98% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0058] 29. The recombinant polypeptide according to item 24, wherein the α3 domain or derivative is identical to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11, or has at least 99% amino acid sequence identity compared to the amino acid sequence of the α3 domain as shown in SEQ ID No: 11.

[0059] 30. The recombinant polypeptide according to item 24, wherein the α3 domain has the same amino acid sequence as the α3 domain shown in SEQ ID No:11.

[0060] 31. The recombinant polypeptide according to any one of the above items, wherein the polypeptide can bind to ILT2 or ILT4, and the affinity constant K obtained by surface plasmon resonance measurement is d Less than 40μM.

[0061] 32. The recombinant polypeptide according to any one of the above items, wherein the polypeptide can bind to ILT2 or ILT4, and the affinity constant K obtained by surface plasmon resonance measurement is d Less than 20μM.

[0062] 33. The recombinant polypeptide according to any one of the above items, wherein the polypeptide can bind to ILT2 or ILT4, and the affinity constant K obtained by surface plasmon resonance measurement is d Less than 10μM.

[0063] 34. The recombinant polypeptide according to any one of the above items, wherein the polypeptide is a dimer or a multimer.

[0064] 35. The recombinant polypeptide according to any of the above items, wherein the length of the peptide antigen sequence according to i) is 7 to 11 amino acids, preferably 8 to 10 amino acids.

[0065] 36. The recombinant polypeptide according to any one of the above items, wherein the recombinant polypeptide comprises all components from i) to vii), but preferably does not comprise components from viii) to ix).

[0066] 37. The recombinant polypeptide according to any one of items 22 to 35, wherein the recombinant polypeptide comprises all components i) to ix).

[0067] 38. The recombinant polypeptide according to any one of the above items, further comprising an N-terminal secretion signal peptide sequence.

[0068] 39. A pharmaceutical composition according to any one of items 1 to 21, or a recombinant polypeptide according to any one of items 22 to 38, for medical use.

[0069] 40. A pharmaceutical composition according to any one of items 1 to 21, or a recombinant polypeptide according to any one of items 22 to 38, for use 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.

[0070] 41. The pharmaceutical composition or recombinant polypeptide according to item 40 for use according to item 40, wherein the immunomodulatory method is used to induce immune tolerance to the peptide antigen contained in the pharmaceutical composition or recombinant polypeptide.

[0071] 42. A pharmaceutical composition or recombinant polypeptide according to any one of items 40 to 41 for use according to any one of items 40 to 41, wherein the immunomodulatory method is a method for suppressing an autoimmune disease, suppressing an allergic reaction, suppressing an immune response to a biotherapeutic drug, suppressing an immune response to an embryonic antigen, or suppressing an immune response to a transplanted cell, tissue or organ.

[0072] 43. A pharmaceutical composition or recombinant polypeptide according to claim 42 for use according to claim 42, wherein the immunomodulatory method is a method of inducing immune tolerance and the autoimmune disease affects multiple organs, hormone-producing organs, nerves, joints, skin, gastrointestinal system, eyes, blood components or blood vessels.

[0073] 44. A pharmaceutical composition or recombinant polypeptide according to item 41 for use according to item 41, wherein the method is a method of inhibiting 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.

[0074] 45. A nucleic acid encoding the polypeptide according to any one of items 22 to 38 or the polypeptide or non-classical MHC class I molecule according to any one of items 1 to 21.

[0075] 46. ​​The nucleic acid according to item 45, wherein the nucleic acid is a vector.

[0076] 47. A pharmaceutical composition comprising the nucleic acid according to item 45 or 46.

[0077] 48. A recombinant host cell comprising the nucleic acid molecule or vector according to item 45 or 46.

[0078] 49. A method for producing a polypeptide according to any one of items 22 to 38, the method comprising culturing the recombinant host cell of item 48 under conditions where the nucleic acid molecule is expressed, and recovering the produced polypeptide.

[0079] 50. A composition comprising:

[0080] a1) an antigenic protein or peptide antigen, or a nucleic acid encoding the antigenic protein or peptide antigen, or an attenuated organism comprising the antigenic protein or peptide antigen, or a2) a cell presenting the peptide antigen according to a1);

[0081] and b) agents capable of blocking the binding of non-classical MHC class I molecules to their receptors;

[0082] The invention relates to a method for inducing an immune response to the antigenic protein or peptide antigen in a subject.

[0083] 51. A composition for use according to item 50, wherein the agent is capable of binding to the human non-classical MHC class I molecule and / or its receptor.

[0084] 52. A composition for use according to any one of the above claims, wherein the agent is capable of recruiting HLA-G.

[0085] 53. The composition for use according to items 50 to 52, wherein the agent is an antibody, preferably a monoclonal antibody, which is capable of binding to HLA-G.

[0086] 54. A composition for use according to any preceding claim, wherein the agent is capable of binding to ILT2 or ILT4.

[0087] 55. A composition for use according to any one of the above items, wherein the agent is an antibody, preferably a monoclonal antibody, which is capable of binding to ILT2 or ILT4.

[0088] 56. A composition for use according to any preceding claim, wherein the agent comprises the Fc domain of an antibody or fragment thereof.

[0089] 57. A composition for use according to any preceding claim, wherein the agent comprises the α3 domain of a non-classical MHC class I molecule.

[0090] 58. A composition for use according to any one of the above claims, wherein the agent comprises one or more extracellular domains of an ILT2 or ILT4 receptor, preferably at least the two N-terminal extracellular domains of an ILT2 or ILT4 receptor.

[0091] 59. A composition for use according to any one of the above purposes, wherein the agent can be administered simultaneously with, before or after administering the antigenic protein or peptide antigen or the nucleic acid encoding the antigenic protein or peptide antigen or the attenuated organism comprising the antigenic protein or peptide antigen.

[0092] 60. A composition for use according to any one of the above items, wherein the composition is a composition of a) an antigenic protein or peptide antigen and b) an agent capable of blocking the binding of the non-classical MHC class I molecule to its receptor.

[0093] 61. A composition for use according to any one of items 50 to 59, wherein the composition is a composition of a) a nucleic acid encoding an antigenic protein or peptide antigen and b) an agent capable of blocking the binding of the non-classical MHC class I molecule to its receptor.

[0094] 62. A composition for use according to any one of items 50 to 59, wherein the composition is a composition of a) an attenuated organism comprising an antigenic protein or peptide antigen and b) an agent capable of blocking the binding of the non-classical MHC class I molecule to its receptor.

[0095] 63. The composition for use according to item 62, wherein the attenuated organism comprising the antigenic protein or peptide antigen is an attenuated virus.

[0096] 64. A composition for use according to any one of items 50 to 62, wherein the antigenic protein or peptide antigen according to a) is a tumor antigen or an antigen that is at least 77% identical to the tumor antigen and is capable of inducing cross protection to the antigen.

[0097] 65. A composition for use according to any one of the above, wherein the method is a T cell-based immunotherapy method.

[0098] 66. A composition for use according to any one of items 50 to 63 and 65, wherein the antigenic protein or peptide antigen is detectable in a pathogenic microorganism or a virus.

[0099] 67. A composition for use according to any one of the above, wherein the method is a method for treating or preventing an infectious or malignant disease.

[0100] 68. The composition for use according to item 67, wherein the disease is cancer and the peptide antigen is a tumor antigen.

[0101] 69. A composition for use according to item 68, wherein the cancer is selected from melanoma, renal cancer, ovarian cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic ductal adenocarcinoma, prostate cancer, B and T cell lymphoma and lung cancer.

[0102] 70. A composition for use according to any one of the preceding claims, wherein the composition is present in a pharmaceutical composition.

[0103] 71. A composition for use according to any one of the above uses, wherein the immune response to the antigenic protein or peptide antigen is specific to the antigenic protein or peptide antigen.

[0104] 72. An agent capable of blocking the binding of a non-classical MHC class I molecule and its receptor according to any one of items 50 to 62 for use in a method of treating cancer in a human subject, the method comprising a therapy that causes the release of a cancer antigen from cells of the cancer.

[0105] 73. The medicament for use according to item 72, wherein the therapy that causes the release of cancer antigens is chemotherapy or radiotherapy.

[0106] 74. A pharmaceutical composition or recombinant polypeptide according to item 41 for the use according to item 41, wherein the method for inducing immune tolerance to a peptide antigen also includes peptide drug treatment, and the peptide antigen 1) is the same as the peptide drug or 2) is a fragment of the peptide drug or 3) is a derivative of a fragment of the peptide drug capable of inducing immune tolerance to the peptide drug.

[0107] 75. A pharmaceutical composition or recombinant polypeptide according to item 41 for the use according to item 41, wherein the method for inducing immune tolerance to peptide antigens also includes protein drug treatment, and the peptide antigen is 1) a fragment of the protein drug or 2) a derivative of the fragment of the protein drug that can induce immune tolerance to the protein drug.

[0108] 76. The pharmaceutical composition or recombinant polypeptide according to item 74 for use according to item 74, wherein the peptide drug can be administered in the form of the peptide drug itself.

[0109] 77. The pharmaceutical composition or recombinant polypeptide according to item 75 for the use according to item 75, wherein the protein drug can be administered in the form of the protein drug itself.

[0110] 78. The pharmaceutical composition or recombinant polypeptide according to item 74 for the use according to item 74, wherein the peptide drug can be administered by gene therapy, and the gene therapy is a gene therapy comprising a gene encoding the peptide drug.

[0111] 79. The pharmaceutical composition or recombinant polypeptide according to item 75 for the use according to item 75, wherein the protein drug can be administered by gene therapy, and the gene therapy is a gene therapy comprising a gene encoding the protein drug.

[0112] The present invention can be used with any mammalian subject, preferably with a human subject.

[0113] Preferably, indications for combining immunostimulatory T cell directed therapy with MHC class Ib or ILT2 / 4 blockers as described above include viral infections and tumors, in which elevated levels of HLA-G or other MHC class Ib molecules can be detected in tumor effusions, blood samples, biopsies by methods such as polymerase chain reaction, ELISA, immunoblotting, fluorescent immunoassay, immunohistochemistry, and other methods (as described in Paul et al., Hum Immunol. 2000 Nov; 61(11): 1177-95) or by other means on malignant or non-malignant tumor cells. Since HLA-G is not expressed in many tissues, even small amounts of HLA-G are effective, therefore, according to the present invention, expression at detectable levels in tissues that are otherwise deficient in HLA-G or an increase of more than 50% of physiological levels in tissues that show basal HLA-G expression is considered a preferred elevated level. SEQUENCE LISTING <110> Julius Maximilian University of Würzburg Valentin Brutal <120> Combinations of MHC class I molecules and peptides for targeted therapeutic immunomodulation <130> P19114536WP <150> EP 17172444.6 <151> 2017-05-23 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Leader peptide <400> 1 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala 20 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide antigen: STEAP1 <400> 2 Met Leu Ala Val Phe Leu Pro Ile Val 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide antigen: Ova <400> 3 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 (disulfide trap fixed) <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 (disulfide trap fixed) <400> 5 Gly Cys Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 6 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> Human beta 3 - microglobulin <400> 6 Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu 1 5 10 15 Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro 20 25 30 Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys 35 40 45 Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60 Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys 65 70 75 80 Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp 85 90 95 Arg Asp Met <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Linker 2 <400> 7 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 8 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> [Alpha] 1 & 2 domains <400> 8 Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr 50 55 60 Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr 65 70 75 80 Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln 85 90 95 Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys 130 135 140 Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Met Leu Gln Arg Ala 180 <210> 9 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Mouse H2Kb [alpha]1 & 2 domains (Y84C) <400> 9 Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr Val Asp Asp Thr Glu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro Arg 35 40 45 Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu Thr 50 55 60 Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg Thr 65 70 75 80 Leu Leu Gly Cys Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Ile Gln 85 90 95 Val Ile Ser Gly Cys Glu Val Gly Ser Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Gln Gln Tyr Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Lys Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr Lys 130 135 140 His Lys Trp Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly Asn 165 170 175 Ala Thr Leu Leu Arg Thr 180 <210> 10 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Human HLA-A2 [alpha]1 & 2 domains <400> 10 Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr 50 55 60 Arg Lys Val Lys Ala His Ser Gln Thr His Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Val Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp Arg Phe Leu Arg Gly 100 105 110 Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys 130 135 140 His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr 180 <210> 11 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> HLA-G [alpha]3 sequence <400> 11 Asp Pro Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu 1 5 10 15 Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile 20 25 30 Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu 35 40 45 Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala 50 55 60 Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln 65 70 75 80 His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Ser Lys Glu Gly 85 90 95 Asp Gly Gly Ile Met Ser Val Arg Glu Ser Arg Ser Leu Ser Glu Asp 100 105 110 Leu <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Factor Xa restriction site <400> 12 Ile Glu Gly Arg Thr Gly Thr Lys Leu Gly Pro 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Myc tag <400> 13 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> His tag <400> 14 His His His His His His 1 5 <210> 15 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> dtH2Gova <400> 15 Ser Ile Ile Asn Phe Glu Lys Leu Gly Cys Gly Ala Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile Gln Val 20 25 30 Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys 35 40 45 Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys 50 55 60 Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser 65 70 75 80 Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr 85 90 95 Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln 100 105 110 Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro 130 135 140 His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly Leu Gly 145 150 155 160 Glu Pro Arg Tyr Met Glu Val Gly Tyr Val Asp Asp Thr Glu Phe Val 165 170 175 Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro Arg Ala Arg 180 185 190 Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu Thr Gln Lys 195 200 205 Ala Lys Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg Thr Leu Leu 210 215 220 Gly Cys Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Ile Gln Val Ile 225 230 235 240 Ser Gly Cys Glu Val Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Gln 245 250 255 Gln Tyr Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn Glu Asp Leu 260 265 270 Lys Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr Lys His Lys 275 280 285 Trp Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr Leu Glu Gly 290 295 300 Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly Asn Ala Thr 305 310 315 320 Leu Leu Arg Thr Asp Pro Pro Lys Thr His Val Thr His His Pro Val 325 330 335 Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro 340 345 350 Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln 355 360 365 Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln 370 375 380 Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr 385 390 395 400 Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp 405 410 415 Ser Lys Glu Gly Asp Gly Gly Ile Met Ser Val Arg Glu Ser Arg Ser 420 425 430 Leu Ser Glu Asp Leu Ile Glu Gly Arg Thr Gly Thr Lys Leu Gly Pro 435 440 445 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His 450 455 460 His His His His His 465 <210> 16 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> dtGsteap <400> 16 Met Leu Ala Val Phe Leu Pro Ile Val Gly Cys Gly Ala Ser Gly Gly 1 5 10 15 Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile Gln 20 25 30 Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn 35 40 45 Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu 50 55 60 Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe 65 70 75 80 Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro 85 90 95 Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser 100 105 110 Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg 145 150 155 160 Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe 165 170 175 Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg Ala 180 185 190 Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg 195 200 205 Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu 210 215 220 Arg Gly Cys Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp 225 230 235 240 Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr 245 250 255 Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp 260 265 270 Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg 275 280 285 Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu 290 295 300 Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu 305 310 315 320 Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His Pro 325 330 335 Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr 340 345 350 Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr 355 360 365 Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe 370 375 380 Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr 385 390 395 400 Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg 405 410 415 Trp Ser Lys Glu Gly Asp Gly Gly Ile Met Ser Val Arg Glu Ser Arg 420 425 430 Ser Leu Ser Glu Asp Leu Ile Glu Gly Arg Thr Gly Thr Lys Leu Gly 435 440 445 Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp 450 455 460 His His His His His His 465 470 <210> 17 <211> 650 <212> PRT <213> Artificial Sequence <220> <223> ILT2 <400> 17 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540 Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val 545 550 555 560 Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565 570 575 Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590 Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605 Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620 Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val 625 630 635 640 Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 645 650 <210> 18 <211> 598 <212> PRT <213> Artificial Sequence <220> <223> ILT4 <400> 18 Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Asp Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser 35 40 45 Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn 65 70 75 80 Gly Gln Phe His Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr 85 90 95 Gly Cys Gln Tyr Tyr Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp Pro 100 105 110 Leu Val Leu Val Met Thr Gly Ala Tyr Pro Lys Pro Thr Leu Ser Ala 115 120 125 Gln Pro Ser Pro Val Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys 130 135 140 Glu Ser Gln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu 145 150 155 160 Glu Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser 165 170 175 Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn Arg Arg Trp 180 185 190 Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro Tyr Val Trp Ser 195 200 205 Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys 210 215 220 Pro Ser Leu Ser Val Gln Pro Gly Pro Val Val Ala Pro Gly Glu Ser 225 230 235 240 Leu Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu 245 250 255 Tyr Lys Glu Gly Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln Pro 260 265 270 Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg 275 280 285 Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Ser 290 295 300 Glu Cys Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln 305 310 315 320 Ile Arg Gly Thr Pro Phe Ile Ser Val Gln Pro Gly Pro Thr Val Ala 325 330 335 Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe His 340 345 350 Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg Leu 355 360 365 Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser 370 375 380 Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu 385 390 395 400 Asn Ser Asp Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu 405 410 415 Val Val Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly Pro 420 425 430 Ile Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Val Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys Asp Thr Gln Pro Glu Asp Gly 530 535 540 Val Glu Met Asp Thr Arg Ala Ala Ala Ser Glu Ala Pro Gln Asp Val 545 550 555 560 Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr Glu 565 570 575 Pro Pro Pro Ser Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile Tyr 580 585 590 Ala Thr Leu Ala Ile His 595 BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 Cells expressing nonclassical MHC class I molecules loaded with defined peptides selectively exclude CTLs specific for the presented peptides

[0115] A, B) HLA-A2 restricted CD8 recognizing the pattern antigen STEAP1 (CD8st) when their cognate peptides were presented on the tumor cell line JEG-3. + Effector T cells can be selectively excluded. The tumor cell line JEG-3 shows high expression of the MHC Ib molecule HLA-G while the classical MHC class I molecules are almost undetectable. Note that in the present invention, STEAP1 is also often referred to as "STEAP", "steap" or simply "st". These terms are synonymous and can be interchanged. Co-cultured CD8 with specificity for the antigen PRAME (CD8pr) + Effector T cells were not affected. (C) Despite loading with cognate peptides for STEAP1-specific T cells, JEG-3 tumor cells expressing HLA-G were not excluded in the process. This suggests that MHC Ib molecules expressed by tumor or infected cells can render these cells resistant to targeting by antigen-specific T cells, which are often the main effectors of peptide-based vaccination strategies. (D) Apoptosis of STEAP1-specific T cells induced by STEAP1 peptide presented on HLA-G was greatly attenuated by neutralizing antibodies against ILT-2, an HLA-G interacting partner expressed on T cells.

[0116] Figure 2 : MHC Ib molecules loaded with defined peptides impair the cytotoxic potential of cognate CTLs in an antigen- and HLA-G-dependent manner.

[0117] 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. Neutralizing anti-human HLA-G antibody (clone 87G) was added at 10 μg / ml as indicated. After 16 h, STEAP1-specific T cells were activated against naive (grey bars) or STEAP1-peptide loaded (black bars) HLA-A2 expressing luciferase. + The cytotoxic potential of UACC-257 melanoma cells was tested at a ratio of 2:1. After 8 hours, D-luciferin was added and the survival of the target cells was determined in a cold light instrument using a biophoton viability assay (Brown et al., J Immunol Methods. 2005 Feb; 297(1-2): 39-52). When loaded with STEAP1 peptide, JEG-3 cells expressing HLA-G reduced the lytic potential of STEAP1-specific CTL by more than 90%, while naive JEG-3 cells did not cause significant inhibition. Since this effect can be significantly attenuated by the presence of partially neutralizing HLA-G antibodies, it can be concluded that peptide-loaded 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 other MHC class Ib molecules. In contrast, the induction of antigen-specific T cell-mediated immune responses according to the present invention can be achieved by agents that block MHC Ib.

[0118] Figure 3 : MHC Ib molecules that bind to defined peptides inhibit cognate CTLs, while immune responses to other antigens are largely unaffected

[0119] HLA-A2 restricted T cell clones specific for STEAP1 or PRAME were mixed with T cell clones specific for HLA-A2-STEAP1 and HLA-A2-PRAME and were left untreated (ctrl) or pretreated with control (JEG-3) or STEAP1 peptide-loaded (JEG-3st) JEG-3 cells. After 8 h, both T cell clones were specific for HLA-A2-STEAP1 (dark grey bars) or STEAP1 peptide-loaded (light grey bars) expressing luciferase. + The cytotoxic potential of UACC-257 melanoma cells was tested at a ratio of 1: 1. Pretreatment of JEG-3 cells with STEAP1-peptide-loaded cells inhibited the STEAP1-peptide-specific T cell-mediated immune response by about 50%, whereas the PRAME-specific immune response was not substantially altered by naive or STEAP1-peptide-loaded JEG-3 cells.

[0120] Figure 4 : Description of peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutic antigen-specific immunomodulation.

[0121] Presented peptide antigen depicted in dashed spheres, HLA-G α1-3 domains drawn in light grey, β2 microglobulin domains shown in dark grey. Optional linkers connecting the antigenic peptide and β2 microglobulin molecules are shown in grey rod-like format, and optional disulfide capture is shown in black spheres. This figure was generated using Pymol and modified from Clements et al., Proc Natl Acad Sci US A. 2005 Mar 1; 102(9): 3360-5 and Hansen et al., Trends Immunol. 2010 Oct; 31(10): 363-9.

[0122] Figure 5 : Examples of vector-based constructs encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation

[0123] HLA-G1 and HLA-G5 are composed of three α domains (shown in black here), one non-covalently associated β2 microglobulin subunit (shown in dark gray here), and an antigenic peptide presented on HLA-G (short black arrow). HLA-G1 also contains a transmembrane domain and a short intracellular chain (not shown here). As shown in the figure, the α-3 domain can bind to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22; 100 (15): 8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci US A. 2006 Oct 31; 103 (44): 16412-7) on immune cells. Physiologically, these sequences form non-covalently linked MHC class 1 complexes. To simplify the purification of the complex MHC Ib molecules, two protein tags (myc and His(6×)) were introduced to allow their subsequent removal by factor Xa cleavage. In addition, the antigenic peptide, β2-microglobulin and MHC Ib α chain can be linked to improve stability. Vector maps were generated using Snapgene Viewer software.

[0124] Figure 6 :Soluble peptide HLA-G / peptide-MHC Ib complexes bound to dendritic cells (DC-10) can selectively eliminate CD8 + Effector T cells.

[0125] Dendritic cells were generated from monocytes in the presence of GM-CSF, IL4 and IL10 (DC-10) for 4 hours before the addition of cell culture supernatant containing soluble peptide MHC Ib constructs. Single-chain HLA-G5 constructs were captured and fixed using disulfide bonds containing presented Melan-A / MART1 (dtGmelA) or STEAP1 (dtGsteap) peptides. The binding of these constructs to DC-10 cells has been previously confirmed. Then, the added DC-10 cells were washed and co-cultured with control CTL (PRAME specific, CD8pr) or target CTL (STEAP1 specific, CD8st) at a 1:1 ratio for 48 hours. These data show that dendritic cells loaded with soluble MHC Ib-peptide constructs can almost completely deplete homologous T cell clones, while non-homologous CTLs are not affected.

[0126] Figure 7 :Peptide-loaded MHC Ib complexes induce human antigen-specific regulatory T cells to recognize presented peptides

[0127] A) Peripheral blood mononuclear cells (PBMCs) were obtained from different healthy donors and co-cultured for 14 days in RPMI1640 medium containing 5% hAB serum, 5ng / ml TGFβ1, 20ng / ml IL2 (Treg medium) with irradiated JEG-3 cells loaded with Melan-A / MART1 (MART1) or STEAP1 (STEAP) peptides. On day 7, PBMCs were transferred to fresh medium and irradiated peptide-loaded JEG-3 cells were added again. Treg expansion beads from MiltenyiBiotec (anti-CD3, anti-CD28 and anti-CD2) were used as positive controls. The resulting cells were stained with antibodies against human CD4 and CD25, HLA-A2STEAP1 peptide multimers (STEAP1dex) and analyzed by flow cytometry. In the presence of JEG-3 cells loaded with STEAP1, STEAP1-specific cells were observed in CD4 + CD25 high Significantly enriched in the Treg population.

[0128] B) 4×10 per well 5 DC-10 cells were added with Figure 6 Then, 4×10 6 PBL cells were cultured in Treg medium for 7 days. Then, 4×10 5The same loaded DC-10 was added to each well. After a total of 14 days, the production of Melan A-specific IL-10 Treg cells was quantified by flow cytometry. Compared with control molecules (STEAP1) or untreated PBL, the number of Melan A-specific Tregs was greatly increased under the conditions where PBL was co-cultured with DC-10 loaded with single-chain Melan A HLA-G molecules.

[0129] Figure 8 : Single-chain peptide MHC constructs containing the human MHC Ib α3 domain that bind DC induce murine Treg cells specific for the presented peptide.

[0130] Murine DCs (mDCs) were generated by culturing bone marrow-derived cells for 7 days in complete RPMI-1640 supplemented with 10% GM-CSF containing supernatant of Ag8653 myeloma cells transfected with the murine GM-CSF gene. mDCs were supplemented with control CHO supernatant (CHO / ctrl) or supernatant of CHO cells transfected with a plasmid encoding a single-chain peptide MHC molecule containing the human HLA-Gα3 domain and the murine H-2Kbα1 and 2 domains presenting an ovalbumin peptide. Controls included a similar construct containing the human HLA-A2α1 and 2 domains (A2G) instead of the murine H-2Kbα1 and 2 domains. A and B: Splenocytes from OT1 mice expressing only a transgenic T cell receptor recognizing an H-2Kb-presented OVA peptide (SIINFEKL) were added to mDCs at a ratio of 2.5:1 and cultured for 14 days in Treg-permissive medium (complete RPMI, 5 ng / ml IL-2, 5 ng / ml TGF-β1). DETAILED DESCRIPTION OF THE INVENTION

[0132] Definition and general techniques

[0133] Unless otherwise specified below, the terms used in the present invention should be understood according to their common meanings known to those skilled in the art.All publications, patents and patent applications cited herein are fully incorporated herein by reference.

[0134] All proteins according to the invention, including polypeptides and MHC molecules according to the invention, can be produced by methods known in the art. These methods include methods for producing recombinant proteins. It is understood that proteins according to the invention, including polypeptides and MHC molecules according to the invention, should optionally contain a secretion signal peptide sequence. Similarly, proteins according to the invention should also optionally contain an affinity tag, for example to facilitate purification, and an optional protease cleavage site between the tag and the protein, for example to facilitate removal of the tag by protease cleavage.

[0135] Likewise, it is to be understood that proteins according to the invention, including polypeptides according to the invention and MHC molecules shall comprise the respective propeptides.

[0136] It will also be appreciated that the polypeptides and MHC molecules according to the present invention may be present in their soluble form or in their membrane bound form.

[0137] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0138] 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.

[0139] 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.

[0140] It is understood how to prepare polypeptides that can bind and present peptide antigens according to the present invention. For example, peptide antigen binding domains, such as α1 and α2 domains, are well known, and people can modify these domains. According to the present invention, the ability of peptide antigens to bind to polypeptides and MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods, such as MHC peptide elution, followed by mass spectrometry analysis and bioinformatics prediction in silico, and confirmatory methods, such as MHC peptide multimer binding and stimulation tests.

[0141] It is understood that, in relation to the peptide antigens used in the present invention, any length of these peptide antigens referred to herein (e.g., "length of 7 to 11 amino acids") refers to the length of the peptide antigen itself. Therefore, the length of the peptide antigen referred to herein does not include the length conferred by additional amino acids that are not part of the peptide antigen (such as additional amino acids in a possible linker sequence, etc.).

[0142] The term "autoimmune disease" is used herein according to the common meaning known to those skilled in the art and is not limited to a specific autoimmune disease. According to all embodiments of the present invention, the autoimmune disease is preferably an autoimmune disease involving an autoimmune response to a peptide autoantigen.

[0143] According to the present invention, each occurrence of the word "comprising" may optionally be replaced by "consisting of".

[0144] Methods and techniques

[0145] Generally, unless otherwise indicated herein, the methods used in the present invention (e.g., cloning methods or methods related to antibodies) are performed according to procedures known in the art, such as those 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.

[0146] Protein-protein binding, e.g. binding of an antibody to its respective target protein, can be assessed by methods known in the art. Protein-protein binding, e.g. binding of an antibody to its respective target protein, is preferably assessed by surface plasmon resonance spectroscopy measurements.

[0147] For example, the binding of the MHC class Ib molecule or polypeptide according to the present invention to its receptor, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of the MHC class Ib molecule or polypeptide according to the present invention to its receptor is assessed by surface plasmon resonance measurement at 25°C. Shiroishi et al. describe suitable conditions for such surface plasmon resonance measurement (Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22; 100(15): 8856-8861.).

[0148] Sequence alignment according to 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 using the BLAST algorithm are known in the art and are suitable for peptide antigens according to the present invention. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment of short input sequences. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment. Therefore, when terms such as "identity" or "identical" are used in connection with sequences, they preferably refer to identity values ​​derived using the BLAST algorithm.

[0149] Preparation of the composition of the present invention

[0150] The compositions according to the present invention are prepared according to known standards for the preparation of pharmaceutical compositions.

[0151] For example, the composition is prepared in such a way that it can be properly stored and administered, such as using pharmaceutically acceptable components such as carriers, excipients and / or stabilizers.

[0152] These pharmaceutically acceptable components are non-toxic in the amounts used when the pharmaceutical composition is administered to a patient. The pharmaceutically acceptable components added to the pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the specific use of the pharmaceutical composition and the route of administration.

[0153] Generally, pharmaceutically acceptable components used in connection with the present invention are used according to knowledge known in the art, for example, see Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.

[0154] Peptide antigens according to the present invention

[0155] Peptide antigens that can be used according to the present invention, including peptide antigens as defined above, are not particularly limited except that they can be presented on MHC molecules. As is known in the art, peptides that can be presented on MHC molecules can be produced (see, for example, Rammensee, Bachmann, Emmerich, Bachor, SYFPEITHI:database for MHC ligands and peptide motifs.Immunogenetics.1999Nov;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).

[0156] Peptide antigens are well known in the art. Generally, the peptide antigens according to the present invention are capable of binding to MHC class I proteins. It will be appreciated by those skilled in the art that for each MHC class Ib molecule or polypeptide capable of presenting a peptide according to the present invention, a peptide antigen capable of binding to said MHC class Ib molecule or polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.

[0157] The binding of the peptide antigen to the MHC class Ib molecule according to the present invention or the polypeptide capable of binding to the peptide antigen can be assessed by methods known in the art, for example:

[0158] Rammensee,Bachmann,Emmerich,Bachor, SYFPEITHI:database forMHC ligands and peptide motifs.Immunogenetics.1999 Nov;50(3-4):213-9;

[0159] 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

[0160] Rock,Reits,Neefjes.Present Yourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016 Nov;37(11):724-737 method.

[0161] These methods include experimental approaches and methods for predicting peptide antigen binding.

[0162] Anchor residues for anchoring peptide antigens to MHC class I molecules and ensuring binding of peptide antigens to MHC class I molecules are known in the art.

[0163] In a preferred embodiment according to all embodiments of the present invention, the peptide antigen used according to the present invention comprises any amino acid residues anchored or preferred at positions predicted for MHC class I molecules.

[0164] Preferably, such predictions are performed as described in any of the following publications:

[0165] -Rammensee et al, SYFPEITHI: database for MHC ligands and peptidemotifs. Immunogenetics (1999) 50:213-219

[0166] -Nielsen et al,Protein Sci(2003)12:1007-1017

[0167] -Neefjes et al. Nat Rev Immunol.2011 Nov 11;11(12):823-36

[0168] -Diehl et al. Curr Biol.1996 Mar 1;6(3):305-14,

[0169] -Lee et al.Immunity.1995 Nov;3(5):591-600.

[0170] -Desai&Kulkarni-Kale, T-cell epitope prediction methods:anoverview. Methods Mol Biol. 2014; 1184:333-64.

[0171] In a preferred embodiment according to all embodiments of the present invention, the peptide antigen is derived from a human protein.

[0172] Alternatively, the non-anchor amino acid residues of the peptide antigen of the present invention may be identical to the corresponding amino acid residues of the peptide antigen derived from human protein, or have at least 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity, further more preferably at least 80% sequence identity, further more preferably at least 90% sequence identity with the corresponding amino acid residues of the peptide antigen derived from human protein. Alternatively, the non-anchor amino acid residues of the peptide antigen of the present invention may contain conservative substitutions, preferably no more than two conservative substitutions, more preferably one conservative substitution, compared to the corresponding amino acid residues of the peptide antigen derived from human protein. In a preferred embodiment, the human protein is a protein expressed in a tissue or cell affected by a pathological immune response.

[0173] The peptide antigens according to the present invention may be naturally occurring peptides or non-naturally occurring peptides. The peptide antigens according to the present invention are preferably composed of naturally occurring amino acids. However, non-naturally occurring amino acids, such as modified amino acids, may also be used. For example, in one embodiment, the peptide antigen used according to the present invention may be a peptidomimetic.

[0174] Methods for synthesizing peptide antigens, including methods for synthesizing peptide antigens according to the present invention, are well known in the art.

[0175] sequence

[0176] The preferred amino acid sequences mentioned in the present application can be independently selected from the following sequences. The sequences are represented in the order from N-terminus to C-terminus; and they are represented by one-letter amino acid codes.

[0177] Leader peptide: for example, MSRSVALAVLALLSLSGLEA (SEQ ID No: 1)

[0178] Peptide antigen: Any MHC class I peptide corresponding to the MHC class I α1 & 2 domains, for example, MLAVFLPIV (STEAP1) (SEQ ID No: 2) or SIINFEKL (Ova) (SEQ ID No: 3)

[0179] Linker 1 (disulfide bond capture fixed) disulfide trap stabilized): For example GGGGSGGGGSGGGGS (SEQ ID No: 4) or G C GASGGGGSGGGGS (SEQ ID No: 5)

[0180] β2-microglobulin from humans or other species, such as:

[0181] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID No: 6, human β3 microglobulin)

[0182] Connector 2, for example

[0183] GGGGSGGGGSGGGGSGGGGS(SEQ ID No:7)

[0184] The α1&2 domains are derived from human HLA-G or other MHC class I α1&2 domains suitable for presenting the selected antigenic peptide. In the DT variant, Y84 can be C

[0185] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRG Y YNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA(SEQ ID No:8)

[0186] For example: mouse H2Kbα1&2 domain ( Y84C )

[0187] GPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLG C YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRT(SEQ ID No:9)

[0188] Or: Human HLA-A2 α1&2 domains

[0189] GSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT(SEQ ID No:10)

[0190] Human HLA-G α3 domain (or any MHC class Ib α3 domain, such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for example:

[0191] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID No: 11; sequence of HLA-Gα3). Note: The following The underlined amino acids are involved in the interaction with ILT2 or ILT4 receptors :

[0192] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL

[0193] Factor Xa restriction site: IEGRTGTKLGP (SEQ ID No: 12)

[0194] Myc tag: EQKLISEEDL (SEQ ID No: 13)

[0195] Additional sequence: NSAVD

[0196] His tag: HHHHHH* (SEQ ID No: 14)

[0197] Examples of mature full-length proteins of the present invention:

[0198] Disulfide bond capture_Ova_Linker 1_Human β2 microglobulin_Linker 2_H2Kbα1&2_HLA-Gα3_Xa site_myc&his tag

[0199] (disulfide trap_Ova_Linker1_humanbeta2microglobulin_Linker2_H2Kbalpha1&2_HLA-Galpha3_XaSite_myc&hisTAG)

[0200] (dtH2Gova)

[0201] SIINFEKLG C GASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVK WDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLG C YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLIEGRTGTKLGPEQKLISEEDLNSAVDHHHHHH*(SEQ ID No:15)

[0202] Note that the sequence of the peptide antigen of the above-mentioned full-length protein (here: SIINFEKL) may be replaced by any peptide antigen sequence according to the present invention.

[0203] Disulfide bond capture_STEAP1_Linker 1_Human β2 microglobulin_Linker 2_HLA-A2α1&2_HLA-Gα3_Xa site_myc&his tag

[0204] (disulfidetrap_STEAP1_Linker1_humanbeta2microglobulin_Linker2_HLA-A2alpha1&2_HLA-Galpha3_XaSite_myc&hisTAG)

[0205] (dtGsteap)

[0206] MLAVFLPIVG C GASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVK WDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRG C YNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLIEGRTGTKLGPEQKLISEEDLNSAVDHHHHHH*(SEQ ID No:16)

[0207] Note that according to the present invention, the sequence of the peptide antigen of the above-mentioned full-length protein (here: MLAVFLPIV) can be replaced by any peptide antigen sequence according to the present invention.

[0208] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of these receptors according to the invention are as follows:

[0209] ILT2:

[0210] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID No:17)

[0211] ILT4:

[0212] (SEQ ID No:18)

[0213] The present invention is further illustrated by the following non-limiting examples. Example

[0214] General Considerations

[0215] All steps were performed under sterile conditions; protective containers were opened only under laminar flow hoods. Unless otherwise stated, cells were always centrifuged at 350×g for 5 minutes. All living cells were kept in an incubator at 37°C, 5% CO2, and >95% humidity. Culture medium, PBS, or other solutions added to cells were preheated using a water bath set to 37°C. A Neubauer chamber was used for cell counting. Statistical analysis was performed using the Student's T test, and p values ​​below 0.05 were considered significant.

[0216] Example 1: Cells expressing MHC Ib molecules loaded with defined peptides selectively eliminate CTLs specific for the presented peptides

[0217] Materials and methods JEG-3 is a human choriocarcinoma cell line that expresses HLA-G at a high level and hardly expresses any 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 RPMI1640 medium containing 10% fetal bovine serum, 0.5% sodium pyruvate solution (100 mM) and 1% penicillin (10 kU / ml) and streptomycin (10 mg / ml) solution ("complete RPMI"). 3×10 5 JEG-3 cells were seeded in 1 ml complete RPMI1640 in 12-well plates.

[0218] As indicated, 1 μl of a stock solution containing STEAP1 (292.2L-9mer, MLAVFLPIV) or PRAME (435-9mer, NLTHVLYPV) peptide (5 μg / μl) was added. The next day, JEG-3 cells were washed three times with PBS, and then 300 μl of supplemented CellGro DC medium (5% human serotype AB serum, 25-50 U / ml IL-2, 5 ng / ml IL-15) was added to each well.

[0219] according to et al, Nat Protoc. 2014 Apr; 9(4): 950-66, produced clones of HLA-A*02 (HLA-A2)-restricted STEAP1 (st) or PRAME (pr) peptide-specific CD8 + T cells (STEAP1- / PRAME "specific"). STEAP1-specific CD8 + Cell Proliferation Dye for T cells 670 were stained according to the manufacturer's instructions and then resuspended in complete RPMI1640 medium as described above. 1.5 × 10 5 Cells were added to each well along with peptide-loaded JEG-3. In the same manner, unstained PRAME-specific CD8 + T cells were pelleted, resuspended and added to each well. In experiments as shown in D, anti-ILT-2 antibody (clone HP-F1) or isotype control antibody was added to a final concentration of 10 μg / ml.

[0220] After 16 hours, cells were collected and stained with 5 μM CellEvent Caspase-3 / 7 Green (Life Technologies) according to the manufacturer's instructions. Non-adherent cells were then collected and stained in a 1:100 dilution of anti-human CD8 (PE / Cy7, clone RPA-T8) and anti-human CD4 (PE / Dye647, clone MEM-241) antibodies on ice for 30 minutes and then analyzed by flow cytometry. + CD4 - Therefore, CD4 staining can exclude possible CD4 + / CD8 + Double positive cells and autofluorescent cells. Total cell number was determined based on the number of cells per microliter. Survival of adherent JEG-3 cells was quantified by crystal violet assay.

[0221] result :A) In the absence of JEG-3 cells or with HLA-G + Caspase 3 / 7 was detected in the control JEG-3 cells with apoptosis rate less than 5% under the two control conditions treated with DMSO. + eFluor670 - PRAME specific or eFluor670 + STEAP1-specific CD8 + In contrast, after co-culture with STEAP1-loaded JEG-3 cells, more than 90% of STEAP1-specific CD8 + T cells were eliminated or apoptotic, however, no significant effect on PRAME-specific T cells was observed. Due to bright eFluor 670 staining, STEAP1-specific CD8 + T cells are easily distinguished from PRAME-specific T cells. This dot plot is a representative result of one of three experiments. B) Statistical analysis of three independent experiments shows that these effects are highly significant and selective for the elimination of HLA-G cells loaded with the cognate peptide. + JEG-3 cells co-cultured with STEAP1-specific T cells. C) The survival rate of JEG-3 cells does not decrease due to the peptide recognized by the co-cultured T cells. D) Under the same conditions, the addition of antibodies that block the HLA-G receptor ILT2 partially inhibited the targeted elimination of STEAP1-specific T cells.

[0222] in conclusion :This experiment shows that if the peptide is specific for CD8 + T cells and human MHC Ib +Cells, such as JEG-3 cells presenting their cognate antigen, can be selectively eliminated. This is surprising because activated CD8 + T cells present cognate peptides on MHC Ia + Target cells are usually eliminated while T cells survive. + Compared with the target, peptide loading of JEG-3 cells did not cause a decrease in survival rate, which indicates that MHC class Ib molecules may have an opposite effect to MHC class Ia molecules. In addition, MHC class Ib molecules synergize with their receptor ILT2 to achieve an inhibitory effect achieved by an agent that inhibits their interaction, such as an ILT2 blocking antibody. Therefore, according to the present invention, in the presence of MHC class Ib molecules, such a blocker can be used to promote the induction of a peptide-specific immune response.

[0223] Example 2 : MHC class Ib molecules loaded with defined peptides can be used to inhibit cytotoxic potential in an antigen-specific manner.

[0224] Materials and methods :1×10 6 JEG-3 cells were either untreated or treated with STEAP1 peptide ("st", see Example 1) in 1 ml complete RPMI1640 in 6-well plates. 5 STEAP1-specific CD8 + T cells and 5×10 5 PRAME-specific CD8 + T cells (effectors) were mixed and either untreated or co-cultured with these JEG-3 for 16 h. Where indicated, 10 μg / ml neutralizing anti-human HLA-G antibody (clone 87G, BioLegend, Germany) was added. The next day, HLA-A2 expressing firefly luciferase was induced using Accutase solution (PAA, Germany). + UACC-257 melanoma cells (target) were detached, washed and loaded with STEAP1 peptide (5 μg / ml, “st loaded”) or an equal amount of DMSO (“unloaded”) for 4 h on a shaker at 37°C. Then, 1 × 10 cells were seeded per well in a white round-bottom 96-well plate. 4 Then, non-adherent mixed T cells were collected and an equal amount of 4×10 4 Initial T cells (2×10 4) and firefly D-luciferin (PJK Germany, final concentration 140 μg / ml). After 8 hours, the survival rate of target cells was determined in a luminometer (specific method Brown et al., J Immunol Methods. 2005 Feb; 297(1-2): 39-52.).

[0225] result : In HLA-G + Presentation of peptide antigens on JEG-3 cells impairs CD8 T cells that recognize specific peptide antigens in an MHC class Ib-dependent manner + Cytotoxicity of T cell clones. In the described environment, STEAP1-specific control CTL or HLA-G + CTLs pretreated with JEG-3 cells lysed about 90% of all target cells loaded with the homologous peptide, without eliminating the original target cells. In contrast, pretreatment with JEG-3 cells and the homologous peptide almost completely protected the antigen-presenting target cells. Antibodies (87G) that partially block HLA-G-dependent effects partially restored this peptide-specific immunosuppressive effect. This means that in a clinical setting, MHC class Ib molecules loaded with peptides can also inhibit harmful cytotoxic (auto)immune responses to the presented antigens.

[0226] In addition, MHC class Ib-positive tumor cells that are exposed to peptides (e.g., by radiation, chemotherapy, or peptide vaccination regimens) can specifically inhibit CD8 + T cell-mediated antitumor immune response. However, this effect can be abolished by agents that block the interaction of MHC class Ib molecules with their receptors.

[0227] Example 3 : MHC class Ib molecules bound to defined peptides inhibit cognate CTLs, while immune responses against other antigens are largely unaffected.

[0228] Materials and methods :In Figure 3 In the experiments shown, HLA-A2 STEAP1-specific (CD8st) and PRAME-specific CD8 + T cells (CD8pr) were mixed and left untreated or co-cultured with JEG-3 cells loaded or not loaded with STEAP1 peptide for 8 h (methods see Figure 2 ). T cells in suspension were then collected and incubated with HLA-A2 cells loaded with PRAME peptide expressing luciferase (dark grey bars) or loaded with STEAP1 peptide (light grey bars). + UACC-257 melanoma cell binding (T cells were not counted after pretreatment, initial effector:target ratio 1:1).

[0229] result:

[0230] In the context of an MHC class Ib-positive cell line, pre-exposure of a mixed CD8 T cell clone to one of the cognate peptides reduced the cytotoxic potential of the cognate T cell by about 50%, while the cytotoxic activity of the other T cell clone remained at about 90%, which was consistent with the effect of HLA-G alone. + The peptide-independent immunosuppressive effects of JEG-3 cells are comparable. Therefore, this method shows that tolerance can be induced against target antigens related to specific (auto)immunity without simultaneously destroying beneficial immune responses against different antigens (e.g., viruses). Based on the MHC profile presented by JEG-3 cells and the experiments on neutralizing antibodies shown above, it can be understood that these peptide-specific effects are mediated by HLA-G. This experiment shows that presentation of antigenic peptides on MHC class Ib molecules impairs cognate CD8 + Cytolytic capacity of T cells.

[0231] Example 4 :Construction plan of therapeutic agent: Soluble single-chain construct containing antigenic peptide, α1 and α2 domains based on MHC class I, α3 domain derived from HLA-G (or other MHC class Ib molecules) and β2-microglobulin

[0232] Design of MHC class Ib peptide complexes

[0233] MHC class Ib molecules, such as HLA-G, are naturally composed of three polypeptide molecules in a complex. Figure 4 As shown, such molecules can be linked via linkers to increase stability.

[0234] Or, if Figure 5 As shown, all components can be presented in a linear fashion.

[0235] The sequences used in the specific embodiments are listed below.

[0236] Components of the coding sequence:

[0237] Leader peptide: for example, a leader peptide that induces secretion, such as MSRSVALAVLALLSLSGLEA (SEQ ID No: 1)

[0238] Presented peptide antigen: Any peptide of 8-12 amino acids in length with anchor residues that allow presentation through the MHC class I α1 and α2 domains, such as MLAVFLPIV (STEAP1) (SEQ ID No: 2) or SIINFEKL (Ova) (SEQ ID No: 3)

[0239] Linker 1 (disulfide bond capture fixed): GGGGSGGGGSGGGGS (SEQ ID No: 4) or GCGASGGGGSGGGGS (SEQ ID No: 5)

[0240] β2-microglobulin from human or other species

[0241] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID No:6)

[0242] Connector 2

[0243] GGGGSGGGGSGGGGSGGGGS(SEQ ID No:7)

[0244] The α1&2 domains are derived from human HLA-G or any other MHC class I α1&2 domains suitable for presenting the selected antigenic peptide. In the DT variant, Y84 can be C

[0245] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA(SEQ ID No:8)

[0246] Example: Mouse H2Kbα1&2 domains (Y84C)

[0247] GPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRT(SEQ ID No:9)

[0248] Or: Human HLA-A2 α1&2 domains

[0249] GSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT(SEQ ID No:10)

[0250] The human HLA-G α3 domain (or any MHC class Ib α3 domain, such as HLA-F, which also interacts with ILT2 and ILT4 receptors, The underlined amino acids are involved in the interaction with ILT2 or ILT4). For example:

[0251] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL(SEQ ID No:11)

[0252] Restriction site for factor Xa: IEGRTGTKLGP (SEQ ID No: 12)

[0253] Myc tag: EQKLISEEDL (SEQ ID No: 13)

[0254] Additional sequence: NSAVD

[0255] His tag: HHHHHH* (SEQ ID No: 14)

[0256] Example of mature full-length protein:

[0257] Disulfide trap_Ova_Linker1_human beta2microglobulin_Linker2_H2Kbalpha1&2_HLA-Galpha3_XaSite_myc&hisTAG)

[0258] (dtH2KbGova)

[0259] SIINFEKLG CGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLG C YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLIEGRTGTKLGPEQKLISEEDLNSAVDHHHHHH*(SEQ ID No:15)

[0260] Disulfide trap_STEAP1_Linker 1_Human beta2 microglobulin_Linker 2_HLA-A2 alpha1&2_HLA-G alpha3_Xa site_myc&his tag

[0261] (disulfide trap_STEAP1_Linker 1_Human beta2 microglobulin_Linker 2_HLA-A2 alpha1&2_HLA-G alpha3_Xa Site_myc&his TAG)

[0262] (dtGsteap)

[0263] MLAVFLPIVG CGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVK WDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRG C YNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLIEGRTGTKLGPEQKLISEEDLNSAVDHHHHHH*(SEQ ID No:16)

[0264] Example 5 Soluble peptide-MHC class Ib complexes bound to dendritic cells (DC-10) selectively eliminate CD8 + Effector T cells

[0265] Materials and methods To investigate whether soluble peptide MHC class Ib constructs can eliminate effector T cells in an antigen-dependent manner, these constructs were loaded onto dendritic cells expanded in the presence of IL-4, GM-CSF and IL-10 (DC-10). 5×10 dendritic cells per ml from healthy donors were cultured in DC-10 medium (complete RPMI1640 medium, 10 ng / ml IL-4, 10 ng / ml IL-10, 100 ng / ml GM-CSF). 6 Purification of CD14 by MACS (CD14beans, Miltenyi, Germany) + The cells were cultured for 7 days to generate DC-10. New culture medium was added on days 3 and 5. The DC-10 cells obtained did not adhere to the cell culture dish. Then 4×10 5 DC-10 cells were mixed with an equal amount of CHO cells (1×10 6The CHO cells were transiently transfected with the pCDNA3.1 expression vector for 4 hours by lipofection method. The pCDNA3.1 expression vector was used to contain the STEAP1 peptide (dtGsteap, sequence see Example 4) or Melan A / MART-1 peptide (ELAGIGILTV, dtGmelA) or a control single chain disulfide trapped HLA-G construct. The DC-10 were then washed 3 times with PBS and resuspended in 50 μl RPMI 1640 medium containing 5 hAB serum + IL-2 (106 DC-10 / ml). Then, 5×10 4 Peptide-MHC class Ib DC-10 cells and HLA-A2-restricted antigen-specific CD8 recognizing STEAP1 (CD8st) or PRAME (CD8pr) + T cells were combined 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) (see Example 2) according to the manufacturer's instructions. CD8 + CD4 - Caspase3 / 7 - Quantitative analysis of cells.

[0266] Results: Figure 6 As shown, in two independent experiments, STEAP1-specific T cells bound to DC-10 cells loaded with single-chain MHC class Ib constructs presenting the cognate peptide were almost completely eliminated within 16 hours. The same conditions did not negatively affect the survival of T cells specific for the control peptide (CD8pr). The construct containing the control peptide (dtGmelA) only slightly reduced the survival of STEAP1-specific CD8 + This also suggests that soluble MHC class Ib molecules that bind specific peptides can be used to selectively eliminate effector T cells specific for the presented peptides, thereby selectively modulating the immune response to defined antigens.

[0267] Example 6 :Peptide-loaded MHC class Ib complexes induce human antigen-specific regulatory T cells that recognize the presented peptides

[0268] In such Figure 7 In the experiment shown in A, 5 × 10 6Peripheral blood mononuclear cells (PBMCs) were cultured in 1×10 6 In the presence of irradiated JEG-3 cells, they were co-cultured in 2 ml RPMI1640 medium containing 5% human serotype AB serum, 5 ng / ml TGF-β1, and 20 ng / ml IL-2 (Treg medium) for 14 days. On day 3, fresh medium was added. On day 7, the medium was replaced and PBMCs were transferred to 1×10 6 On JEG-3 cells that had just been irradiated and loaded with peptides. According to the manufacturer's protocol, Treg expansion beads (Treg Expansion Beads) (Miltenyi Biotec, anti-CD3 / CD28) were used as positive controls. The resulting cells were stained on ice for 30 minutes with antibodies against human CD4 (clone EDU-2, Immunotools) and CD25 (Miltenyi 120-001-311) and HLA-A2 STEAP1 polymers (dextramers) (STEAP1dex, Immudex Denmark, all dilutions 1:100). CD4 was quantitatively analyzed by flow cytometry. + CD25 high The incidence of STEAP1-specific T cells in Treg cells (Shevach et al., 2002, Nat. Rev. Immunol. 2:389). When PBMCs were cultured alone (ctrl) or in the presence of a control peptide (melA), no STEAP1-specific CD4 + CD25 high Treg cells, whereas a large population was repeatedly observed when PBMCs were co-cultured with JEG-3 cells presenting the cognate antigen in the setting of a lower positive control (aCD3 / 28).

[0269] In such Figure 7 In the experiment shown in B, 4 × 10 5 DC-10 cells were added with a disulfidetrap single-chain HLA-G construct comprising Figure 6 Then, 4×10 6 PBL from the same donor were cultured in 2 ml Treg medium in 12-well plates for 7 days, with 1 ml of medium replaced on day 3. On day 7, 4 × 10 5Fresh and identically loaded DC-10, the medium was changed again on day 10. On day 14, cells were collected, washed and stained with fluorescently labeled anti-CD4 (clone MEM-241), CD8 (clone RPA-T8) antibodies and HLA-A2-Melan A peptide multimers (Immudex). Intracellular staining of IL-10 (clone JES3-9D7) was performed using an intracellular staining kit (eBiosciences). Melan A-specific IL-10 was expressed when PBLs were co-cultured with DC-10 loaded with single-chain MelanA HLA-G molecules (dtGmelA) compared to control molecules (dtGsteap) or untreated PBLs. + The number of Tregs increased dramatically.

[0270] Example 7: Single-chain peptide MHC constructs containing human MHC class Ib α3 domains that bind DC induce murine Treg cells specific for the presented peptide ( Figure 8 )

[0271] Murine DC (mDC) was generated by culturing bone marrow cells of wild-type C57BL / 6 mice for 7 days in complete RPMI-1640 supplemented with 10% GM-CSF supernatant of Ag8653 myeloma cell line transfected with the murine GM-CSF gene (detailed protocol: Lutz et al., J Immunol Methods 1999, 223(1):77-92). 4×10 400 μl of 500 μl of complete RPMI were added to the culture medium. 5 mDCs were bound for 4 hours to 500 μl of "day 5 CHO supernatant" derived from mock transfected cells (CHO) or CHO cells transfected with pCDNA3.1 vectors encoding single-chain ovalbumin peptides (SIINFEKL), murine H-2Kbα1 and 2 domains (A2G) and human HLA-Gα3 domains (H2Kb, sequence example 4dtH2KbGova) or human HLA-A2α1 and 2 domains (A2G). The presence of the respective constructs in the supernatant was confirmed by Western blotting. Preliminary results suggest that induction is also possible with purified constructs. Here, peptide-loaded MHC constructs were purified using cOmplete His-Tag purification resin (Sigma Aldrich) that bound the constructs, followed by washing with PBS (3 times) and digestion with factor Xa protease (1 U / 100 μl, 6 h at 20°C, Qiagen) to release the constructs. Factor Xa removal resin can then be used to remove Factor Xa (Qiagen, all according to manufacturer's protocol). The sequence is listed in Example 4. The mDCs are then washed with PBS.

[0272] C57BL / 6RAG - / - OT1 mice express T cell receptors that interact almost exclusively with the ova peptide presented by H-2Kb. 6 Splenocytes were cultured in the presence (mDC A2G / CHO / H2Kb OT1) or absence (OT1ctrl) of 4×10 5 The mDCs were cultured in Treg induction medium (complete RPMI, 5ng / ml IL-2, 5ng / ml TGF-β1) added as described above for 14 days. The cells were then stained with fluorescently labeled antibodies specific to murine CD3 (clone KT3, Serotec), Foxp3 (3G3, Miltenyi Biotec) and IL10 (JES5-16E3), and quantitatively analyzed by flow cytometry (see Hünig et al., Brain. 2008 Sep; 131 (Pt 9): 2353-65 for experimental protocols suitable for mice). Under conditions where T cells were combined with cognate peptides / MHC α1 and 2 domains of MHC class Ib molecules and immunosuppressive α3 domains, a significant increase in antigen-specific Tregs was observed. The moderate induction with purified constructs can be explained by the loss of protein during the purification process.

[0273] These experiments show that peptide presentation on MHC class Ib molecules promotes the expansion of cognate Tregs. Such Tregs will be activated preferentially through their T cell receptors in tissues where the antigen is present, and therefore, given the appropriate tissue-specific antigen, it should be possible to target tissue-specific inhibition of autoimmune responses. It should be noted that due to the bystander suppression capacity of antigen-specific Tregs, the tissue-specific "Treg activation antigen" chosen does not have to be the same as the self-antigen driving the pathological immune response.

[0274] Industrial Applicability

[0275] The compositions, polypeptides, nucleic acids, cells, combinations and methods of the invention can be used industrially. For example, they can be used to manufacture or be used as medicines.

Claims

1. An isolated recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus, i) a peptide antigen, wherein the peptide antigen is presented by the recombinant polypeptide; ii) optionally, a first linker sequence; iii) a sequence of a human polypeptide domain, wherein the sequence of the human polypeptide domain comprises the sequence of human β2 microglobulin as shown in SEQ ID NO: 6; iv) optionally, a second linker sequence; v) α1 domain of the MHC molecule; vi) the α2 domain of the MHC molecule; vii) an α3 domain of an MHC class Ib molecule, wherein the α3 domain has the same amino acid sequence as the α3 domain shown in SEQ ID NO: 11; viii) optionally, a protease cleavage site; and ix) optionally, an affinity tag; in, v) the α1 domain and vi) the α2 domain are from a classical MHC class I molecule, and According to i), the length of the peptide antigen sequence is 8 to 11 amino acids.

2. The recombinant polypeptide according to claim 1, wherein the polypeptide can bind to d The affinity constant K for binding to ILT2 or ILT4 is d Measured by surface plasmon resonance.

3. The recombinant polypeptide according to claim 1, wherein the polypeptide can bind to d The affinity constant K for binding to ILT2 or ILT4 is d Measured by surface plasmon resonance.

4. The recombinant polypeptide according to claim 1, wherein the polypeptide can bind to d The affinity constant K for binding to ILT2 or ILT4 is d Measured by surface plasmon resonance. The recombinant polypeptide according to claim 1 , wherein the polypeptide is a dimer or a multimer.

6. The recombinant polypeptide according to claim 1, wherein the length of the peptide antigen sequence according to i) is 8 to 10 amino acids.

7. The recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises all components i) to vii).

8. The recombinant polypeptide according to claim 7, wherein the recombinant polypeptide does not contain components viii) to ix).

9. The recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises all components i) to ix).

10. The recombinant polypeptide according to any one of the preceding claims, further comprising an N-terminal secretion signal peptide sequence.

11. The recombinant polypeptide according to any one of claims 1 to 10 for medical use.

12. A nucleic acid encoding the polypeptide according to any one of claims 1 to 10.

13. The nucleic acid according to claim 12, wherein the nucleic acid is a vector.

14. A pharmaceutical composition comprising the nucleic acid according to claim 12 or 13.

15. A recombinant host cell comprising the nucleic acid molecule or vector according to claim 12 or 13.

16. A method for producing the polypeptide according to any one of claims 1 to 10, the method comprising culturing the recombinant host cell according to claim 15 under conditions allowing expression of the nucleic acid molecule, and recovering the produced polypeptide.

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