Recombinant peptide-MHC complex binding proteins and their production and uses

Abstract

The present invention relates to methods for producing recombinant binding proteins with binding specificity for peptide-MHC (pMHC) complexes. The invention also relates to recombinant binding proteins with binding specificity for pMHC complexes, which comprise one, two or more designed repeat domains, preferably designed ankyrin repeat domains, and to such binding proteins which further comprise a binding factor with binding specificity for a protein expressed on the surface of immune cells, preferably T cells. In addition, the invention relates to nucleic acids encoding such binding proteins or repeat domains, pharmaceutical compositions comprising such binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating or diagnosing diseases, including cancer, infectious diseases and autoimmune diseases.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to European Patent Nos. 19215433.4, filed December 11, 2019, 19215434.2, filed December 11, 2019, 19215435.9, filed December 11, 2019, 19215436.7, filed December 11, 2019, 20161059.9, filed March 4, 2020, and 20181234.4, filed June 19, 2020. The disclosures of these six patent applications are incorporated herein by reference in their entirety for all purposes.

[0002] Field of Disclosure The present invention relates to methods for producing recombinant binding proteins with binding specificity for peptide-MHC (pMHC) complexes. The invention also relates to recombinant binding proteins with binding specificity for pMHC complexes, which comprise one, two or more designed repeat domains, preferably designed ankyrin repeat domains, and to such binding proteins which further comprise a binding factor with binding specificity for a protein expressed on the surface of immune cells, preferably T cells. In addition, the invention relates to nucleic acids encoding such binding proteins or repeat domains, pharmaceutical compositions comprising such binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating or diagnosing diseases, including cancer, infectious diseases and autoimmune diseases. [Background technology]

[0003] Major histocompatibility complex (MHC) class I molecules play an important role in intracellular surveillance of abnormal or foreign proteins. Peptides derived from endogenous proteins are loaded into the peptide-binding groove of MHC class I molecules and then presented on the cell surface. Such MHC class I complexes are expressed in all nucleated cells, including malignant cells. Peptide-MHC (pMHC) complexes are recognized by T cell receptors (TCRs) on CD8+ cytotoxic T lymphocytes (CTLs). Presentation of peptide-MHC complexes provides circulating CTLs with a snapshot of the intracellular environment (Reeves and James, Immunology 150:16-24 (2016)). CTLs are activated in response to detecting abnormal or foreign antigens, such as cancer proteins, bacterial proteins, or viral proteins, leading to the destruction of the presenting cell. CTLs are also activated in certain autoimmune diseases when they misrecognize "self" antigens (Bodis et al., Rheumatoi. Ther. 5:5-20 (2018)).

[0004] MHC class I complexes presenting tumor- or infectious agent-specific peptides serve as a unique and promising class of cell surface targets for immunotherapy of cancer and infectious diseases. Different approaches have been developed to exploit this target class, including vaccines, adoptive cell therapy, and TCR-like antibodies. Among tumor-associated antigens, cancer-testis antigens (CTAs) are considered good candidate targets for immunotherapy because they are characterized by restricted expression in normal somatic tissues and re-expression in tumor tissues. One of the most frequently reported CTAs across various cancer types is melanoma-associated antigen A3 (MAGE-A3) (J Exp Clin Cancer Res. 2019 Jul 8;38(1):294. doi:10.1186 / s13046-019-1272-2). Furthermore, several CTAs have been found to induce spontaneous immune responses, with NY-ESO-1 being one of the most immunogenic (Thomas et al., Front Immunol. 2018;9:947). Among infectious agent-specific peptides, EBV nuclear antigen 1 (EBNA-1) is the only viral protein found in all EBV-associated malignancies (J Gen Virol. 2009 Sep;90(Pt 9):2251-2259). Furthermore, sequences 18-27 of the hepatitis B virus (HBV) nucleocapsid antigen are widely recognized by CTLs from HLA-A2-positive patients suffering from acute self-limited HBV infection and serve as a key component of peptide-based therapeutic vaccines aimed at stimulating antiviral CTL responses in patients with chronic hepatitis B (Hepatology. 1997 Oct;26(4):1027-34.c).

[0005] One obstacle to the therapeutic use of virus- or tumor-specific peptide-MHC complexes has been the inherently low affinity of TCRs for the peptide-MHC complexes after thymic selection. This low affinity presents a limitation, especially considering that virus- or tumor-specific peptide-MHC complexes are typically present at low density on the surface of virus-infected or tumor cells. To overcome this obstacle, affinity-enhanced TCRs have been developed, and engineered T cells expressing such affinity-enhanced TCRs have been tested in clinical trials. Affinity-enhanced TCRs can lack specificity, and in some cases, engineered T cells expressing affinity-enhanced TCRs have caused serious, sometimes potentially fatal, medical complications due to unexpected recognition by the TCR of epitopes derived from unrelated proteins (e.g., Linette et al., Blood 122(6):863-871 (2013)). Affinity-enhanced TCRs have also been developed as soluble TCRs, but soluble TCR expression is challenging.

[0006] Several TCR-like antibodies with binding specificity to MHC class I complexes presenting virus-specific or tumor-specific peptides have been reported. Some of these TCR-like antibodies were isolated using hybridoma technology. However, the isolation of pMHC-specific TCR-like antibodies using hybridoma technology is hindered by several factors, including the need to screen hundreds or even thousands of clones, low immunogenicity, some unique clones due to immunodominance, and insufficient control of fine specificity (see, for example, Porgador et al., Immunity 6:715-726 (1997); Bernardeau et al., Eur. J. Immunol. 35(10):2864-2875 (2005); Skora et al., Proc. Natl. Acad. Sci. USA 112(32):9967-9972 (2015)). Many more TCR-like antibodies have been isolated using phage display. However, the affinity of TCR-like antibodies isolated from phage display libraries is generally relatively low and often insufficient for therapeutic purposes (see, e.g., Chames et al., Proc. Natl. Acad. Sci. USA 97:7969-7974 (2000)). To generate TCR-like antibodies with sufficiently high affinity, systems for affinity maturation have been developed. For example, one such system for affinity maturation of TCR-like antibodies combines mutagenesis, library and yeast display, structure determination, and molecular modeling (Zhao et al., Leukemia 29(11):2238-2247 (2015)). Using only this complex and labor-intensive affinity maturation approach, Zhao et al. were able to improve the binding affinity of a TCR-like antibody by approximately 100-fold to obtain a pMHC-specific binding protein with sufficient affinity. In summary, developing molecules that specifically bind disease-associated peptide-MHC complexes with sufficient affinity has so far been difficult, and current approaches generally involve difficult expression systems and / or laborious procedures such as screening large numbers of hybridoma clones or affinity maturation.

[0007] Thus, there remains a need for new methods of producing pMHC-specific binding proteins, a need for new pMHC-specific binding proteins, and a need for therapeutic and diagnostic approaches for the treatment and characterization of diseases that benefit from pMHC-specific binding, including cancer, autoimmune diseases, and infectious diseases. Summary of the Invention

[0008] The present invention provides methods for producing recombinant binding proteins with binding specificity for peptide-MHC (pMHC) complexes. The present invention also provides recombinant binding proteins with binding specificity for pMHC complexes, comprising one, two or more designed repeat domains, preferably designed ankyrin repeat domains, and such binding proteins further comprising a binding factor with binding specificity for a protein expressed on the surface of immune cells, preferably T cells. In addition, the present invention provides nucleic acids encoding such binding proteins or repeat domains, pharmaceutical compositions comprising such binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating or diagnosing diseases such as cancer, autoimmune diseases and infectious diseases in mammals, including humans.

[0009] The methods of the present invention for producing recombinant binding proteins with binding specificity for pMHC complexes are surprisingly efficient and effective in generating binding proteins that bind with high affinity and / or specificity to selected target pMHC complexes. To date, it has been difficult to develop molecules that specifically bind disease-associated pMHC complexes with sufficient affinity, and current approaches generally involve difficult expression systems and / or laborious procedures, such as screening multiple hybridoma clones or affinity maturation. The methods of the present invention disclosed herein do not involve difficult expression systems or require the time-consuming and laborious procedures described above. Furthermore, the binding interaction between the binding proteins of the present invention and the target peptide-MHC complex unexpectedly involves a relatively large number of amino acid residues in the target peptide. The large number of interacting residues in the target peptide is believed to reflect highly selective or specific binding interactions with the target peptide-MHC complex among a large region of different pMHC complexes. The pMHC-specific repeat domains of the binding proteins of the present invention appear to provide a binding surface that sterically fits very well with the complex peptide-MHC surface in specific binding interactions. Furthermore, the pMHC-specific repeat domains of the binding proteins of the invention may comprise specific amino acid sequence motifs within the N-terminal and / or C-terminal capping modules, which may lead to improved pharmacokinetic properties of the designed repeat domains and proteins comprising them. The methods and binding proteins of the invention further offer the advantage that two or more of the same and / or different pMHC-specific repeat domains can be easily combined in one binding protein (e.g., a bivalent, biparatopic or bispecific binding protein), thereby allowing the avidity, binding affinity, binding specificity and / or potency of the binding protein to be tailored and optimized.Moreover, the binding proteins of the invention may even further comprise binding factors that have binding specificity for proteins expressed on the surface of immune cells, such as proteins that are part of the T cell receptor complex expressed on cytotoxic T cells or activating receptors expressed on natural killer (NK) cells. The binding proteins of the invention in such immune cell engager formats (e.g., T cell engager formats or NK cell engager formats) can be advantageously used in methods for activating immune cells (e.g., T cells or NK cells) and / or engaging the immune system in a localized and targeted manner. Furthermore, the length of the linker connecting one or more pMHC-specific repeat domains to the binding factor unexpectedly affects the potency of the binding proteins of the invention for use in immune cell engager formats, such as T cell engager formats. Furthermore, the methods and binding proteins of the invention enable, inter alia, the specific targeting of intracellular proteins, thereby facilitating many new diagnostic and therapeutic opportunities for, e.g., cancer, infectious diseases, and autoimmune diseases.

[0010] In one aspect, the invention provides a method for producing a peptide-MHC (pMHC) specific binding protein, the binding protein comprising engineered repeat domains that have binding specificity for a target peptide-MHC complex, the method comprising: (a) providing a collection of designed repeat domains; (b) providing a recombinant target peptide-MHC complex; (c) screening the collection of designed repeat domains for specific binding to the target peptide-MHC complex to obtain at least one designed repeat domain having binding specificity for the target peptide-MHC complex. In a preferred embodiment, the designed repeat domain is a designed ankyrin repeat domain.

[0011] In another aspect, the present invention provides a recombinant binding protein comprising an engineered repeat domain obtainable by the above method.

[0012] In another aspect, the present invention provides a recombinant binding protein comprising a first designed repeat domain, wherein the first repeat domain has binding specificity for a first target peptide-MHC complex. In a preferred embodiment, the first target peptide is derived from a protein associated with a disease or disorder. By way of example, in one particularly preferred embodiment, the first target peptide is selected from the group consisting of: (i) a peptide derived from a protein expressed in tumor cells, (ii) a peptide derived from a protein of an infectious agent, preferably a viral infectious agent, and (iii) a peptide derived from a protein associated with an autoimmune disorder.

[0013] In a specific embodiment, the first target peptide is derived from an intracellular protein, preferably an intracellular protein expressed in tumor cells, such as NY-ESO-1 or MAGE-A3. In a preferred embodiment, the target peptide derived from NY-ESO-1 comprises or consists of the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34, and the target peptide derived from MAGE-A3 comprises or consists of the amino acid sequence of SEQ ID NO: 155. In another specific embodiment, the first target peptide is derived from a protein of a viral infectious agent, preferably a virus-specific protein such as EBNA-1 or HBV core antigen (HBcAg). In a preferred embodiment, the target peptide derived from EBNA-1 comprises or consists of the amino acid sequence of SEQ ID NO: 92, and the target peptide derived from HBcAg comprises or consists of the amino acid sequence of SEQ ID NO: 255.

[0014] In one preferred aspect, the invention provides such a recombinant binding protein comprising a first designed repeat domain having binding specificity for a first target peptide-MHC complex, wherein said first repeat domain is a designed ankyrin repeat domain.

[0015] In one particular aspect, the present invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein the first target peptide is derived from NY-ESO-1, and the first ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37 to 72 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 37 to 72 are substituted by another amino acid. In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein said first target peptide is derived from NY-ESO-1, and said first ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20 to 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20 to 33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20 to 33 is optionally substituted by N.

[0016] In one particular aspect, the present invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein the first target peptide is derived from MAGE-A3, and the first ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 175 to 217 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 175 to 217 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein said first target peptide is derived from MAGE-A3, and said first ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 156 to 173, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 156 to 173 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 156 to 173 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 156 to 173 is optionally substituted by N.

[0017] In a further particular aspect, the present invention provides such recombinant binding proteins comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein the first target peptide is derived from EBNA-1, and the first ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 111 to 154 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 111 to 154 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein said first target peptide is derived from EBNA-1, and said first ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 93 to 110, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 93 to 110 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 93 to 110 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 93 to 110 is optionally substituted by N.

[0018] In one particular aspect, the present invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein the first target peptide is derived from HBcAg, and the first ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 231 to 254 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 231 to 254 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, wherein said first target peptide is derived from HBcAg, and said first ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 220-230, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 220-230 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 220-230 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 220-230 is optionally substituted by N.

[0019] In a further preferred aspect, the present invention provides such recombinant binding proteins comprising a first designed repeat domain having binding specificity for a first target peptide-MHC complex, said first repeat domain being an N-terminal and / or C-terminal capping module.

[0020] In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for a first target peptide-MHC complex, said first ankyrin repeat domain comprising an N-terminal capping module with an amino acid sequence wherein the amino acid at position 8 is Q and / or the amino acid at position 15 is L, and wherein the position numbers of the positions in the N-terminal capping module are determined by alignment with SEQ ID NO: 276 using the position numbers of SEQ ID NO: 276 against SEQ ID NO: 5. SEQ ID NO: 276 is an N-terminal capping module that is identical to SEQ ID NO: 5, except that it is missing the G at position 1 and the S at position 2 of SEQ ID NO: 5. Thus, position 8 in SEQ ID NO: 276 corresponds to position 10 in SEQ ID NO: 5, and position 15 in SEQ ID NO: 276 corresponds to position 17 in SEQ ID NO: 5. In other words, the first ankyrin repeat domain comprises an N-terminal capping module having an amino acid sequence in which the amino acid at position 10 is Q and / or the amino acid at position 17 is L, and the position numbers of the positions of the N-terminal capping module are determined by alignment with the position numbers of SEQ ID NO: 5 against SEQ ID NO: 5. Preferably, the alignment does not contain any amino acid gaps. Generating sequence alignments is a procedure well known in the art.

[0021] In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for a first target peptide-MHC complex, said first ankyrin repeat domain comprising a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and / or the amino acid at position 18 is Q, wherein the position numbers of the positions in the C-terminal capping module are determined by alignment with SEQ ID NO: 13 using the position numbers of SEQ ID NO: 13. Preferably, said alignment does not contain any amino acid gaps.

[0022] In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, said first ankyrin repeat domain comprising (i) an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and the amino acid at position 15 is L, and / or (ii) a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and the amino acid at position 18 is Q. Preferably, the position numbers of the positions of the N-terminal capping module are determined by alignment with SEQ ID NO: 276 using the position numbers of SEQ ID NO: 276, and the position numbers of the positions of the C-terminal capping module are determined by alignment with SEQ ID NO: 13 using the position numbers of SEQ ID NO: 13. Preferably, said alignment does not include an amino acid gap.

[0023] In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for a first target peptide-MHC complex, said first ankyrin repeat domain comprising an N-terminal capping module with the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 276), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 8 and 15.

[0024] In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for a first target peptide-MHC complex, said first ankyrin repeat domain comprising a C-terminal capping module with the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 13), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 14 and 18 of SEQ ID NO: 13.

[0025] In one particular aspect, the invention provides such a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex, said first ankyrin repeat domain comprising: (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 276), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid is located between position 8 and position 15 or greater; and (ii) an N-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 13), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 14 and 18 of SEQ ID NO: 13.

[0026] In one particular aspect, the present invention provides such recombinant binding proteins, further comprising a second designed repeat domain having binding specificity for a second target peptide-MHC complex. In a preferred embodiment, the second target peptide is derived from a protein associated with a disease or disorder. By way of example, in one particularly preferred embodiment, the second target peptide is selected from the group consisting of: (i) a peptide derived from a protein expressed in tumor cells, (ii) a peptide derived from a protein of an infectious agent, preferably a viral infectious agent, and (iii) a peptide derived from a protein associated with an autoimmune disorder.

[0027] In one particular aspect, the second target peptide is derived from the same protein as the first target peptide. In one embodiment, the second target peptide has the same amino acid sequence as the first target peptide. In one embodiment, the second repeat domain has the same amino acid sequence as the first repeat domain. In one embodiment, the second repeat domain has a different amino acid sequence compared to the first repeat domain. In one embodiment, the second target peptide has a different amino acid sequence compared to the first target peptide.

[0028] In one particular embodiment, the second target peptide is derived from a different protein than the protein from which the first target peptide is derived.

[0029] In one preferred embodiment, the present invention provides such recombinant binding proteins further comprising a second designed repeat domain with binding specificity for a second target peptide-MHC complex, wherein the second repeat domain is a designed ankyrin repeat domain. In one particular embodiment, the present invention provides such recombinant binding proteins further comprising a second ankyrin repeat domain with binding specificity for a second target peptide-MHC complex, wherein the second target peptide is derived from NY-ESO-1, and the second ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37 to 72 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 37 to 72 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein further comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, wherein said second target peptide is derived from NY-ESO-1, and said second ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 20-33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20-33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20-33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20-33 is optionally substituted by N.

[0030] In another particular aspect, the present invention provides such recombinant binding proteins further comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, wherein said second target peptide is derived from MAGE-A3, and said second ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 175 to 217 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 175 to 217 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein further comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, wherein said second target peptide is derived from MAGE-A3, and said second ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 156 to 173, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 156 to 173 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 156 to 173 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 156 to 173 is optionally substituted by N.

[0031] In another particular aspect, the present invention provides such recombinant binding proteins further comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, wherein said second target peptide is derived from EBNA-1, and said second ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 111 to 154 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 111 to 154 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein further comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, wherein said second target peptide is derived from EBNA-1, and said second ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 93 to 110, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 93 to 110 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 93 to 110 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 93 to 110 is optionally substituted by N.

[0032] In another specific aspect, the present invention provides such recombinant binding proteins further comprising a second ankyrin repeat domain with binding specificity for a second target peptide-MHC complex, wherein the second target peptide is derived from HBcAg, and the second ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 231 to 254 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 231 to 254 are substituted by another amino acid. In one particular embodiment, the invention provides such a recombinant binding protein further comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, wherein said second target peptide is derived from HBcAg, and said second ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 220-230, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 220-230 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 220-230 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 220-230 is optionally substituted by N.

[0033] In a further preferred aspect, the present invention provides such recombinant binding proteins comprising a second designed repeat domain with binding specificity for a second target peptide-MHC complex, said second repeat domain being an N-terminal and / or C-terminal capping module.

[0034] In one particular aspect, the invention provides such a recombinant binding protein comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, said second ankyrin repeat domain comprising an N-terminal capping module having an amino acid sequence wherein the amino acid at position 8 is Q and / or the amino acid at position 15 is L, the position number of the N-terminal capping module position being determined by alignment with SEQ ID NO: 276 using the position numbers of SEQ ID NO: 276. In other words, said second ankyrin repeat domain comprises an N-terminal capping module having an amino acid sequence wherein the amino acid at position 10 is Q and / or the amino acid at position 17 is L, the position number of the N-terminal capping module position being determined by alignment with SEQ ID NO: 5 using the position numbers of SEQ ID NO: 5. Preferably, said alignment does not contain any amino acid gaps. Generating sequence alignments is a procedure well known in the art.

[0035] In one particular aspect, the invention provides such a recombinant binding protein comprising a second ankyrin repeat domain with binding specificity for a second target peptide-MHC complex, said second ankyrin repeat domain comprising a C-terminal capping module with an amino acid sequence wherein the amino acid at position 14 is R and / or the amino acid at position 18 is Q, and wherein the position numbers of the positions in the C-terminal capping module are determined by alignment with the position numbers of SEQ ID NO: 13 against SEQ ID NO: 13. Preferably, said alignment does not contain any amino acid gaps.

[0036] In one particular aspect, the invention provides such a recombinant binding protein comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, said second ankyrin repeat domain comprising (i) an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and the amino acid at position 15 is L, and / or (ii) a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and the amino acid at position 18 is Q. Preferably, the position numbers of the positions of the N-terminal capping module are determined by alignment with SEQ ID NO: 276 using the position numbers of SEQ ID NO: 276, and the position numbers of the positions of the C-terminal capping module are determined by alignment with SEQ ID NO: 13 using the position numbers of SEQ ID NO: 13. Preferably, said alignment does not include an amino acid gap.

[0037] In one particular aspect, the invention provides such a recombinant binding protein comprising a second ankyrin repeat domain with binding specificity for a second target peptide-MHC complex, said second ankyrin repeat domain comprising an N-terminal capping module with the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 276), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 8 and 15.

[0038] In one particular aspect, the invention provides such a recombinant binding protein comprising a second ankyrin repeat domain with binding specificity for a second target peptide-MHC complex, said second ankyrin repeat domain comprising a C-terminal capping module with the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 13), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 14 and 18 of SEQ ID NO: 13.

[0039] In one particular aspect, the invention provides such a recombinant binding protein comprising a second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex, said second ankyrin repeat domain comprising: (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 276), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid is located between positions 8 and 15; and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 13), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 14 and 18 of SEQ ID NO: 13.

[0040] In one particular aspect, the present invention provides such a recombinant binding protein, wherein the binding protein comprises a polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 16-18.

[0041] In a preferred aspect, binding of the repeat domain to its target peptide-MHC complex comprises interaction of the repeat domain with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acid residues of the target peptide. In one embodiment, binding of the repeat domain to its target peptide-MHC complex alternatively or additionally comprises interaction of the repeat domain with at least one amino acid residue of the MHC.

[0042] In one particular aspect, the present invention provides such pMHC-specific recombinant binding proteins, which further comprise a binding factor having binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell or an NK cell, more preferably a CD8+ cytotoxic T cell. In one embodiment, the protein expressed on the surface of a T cell is a protein that is part of the T cell receptor complex. By way of example, in one particular embodiment, the present invention provides such pMHC-specific recombinant binding proteins, which further comprise a binding factor having binding specificity for CD3. In another embodiment, the protein expressed on the surface of an NK cell is an activating receptor for NK cells. Examples of such activating receptors for NK cells include CD16 (also known as FcγRIIA), NKG2D, SLAM family members, and the natural cytotoxic receptors NKp30, NKp44, and NKp46.

[0043] In one preferred embodiment, the present invention provides such a recombinant binding protein, further comprising a binding factor having binding specificity for a protein expressed on the surface of an immune cell, wherein said binding factor is an engineered repeat domain, preferably an engineered ankyrin repeat domain.

[0044] In another aspect, the present invention provides a nucleic acid encoding a designed repeat domain of the invention or a nucleic acid encoding a pMHC-specific recombinant binding protein of the invention, as well as pharmaceutical compositions comprising the pMHC-specific recombinant binding protein or nucleic acid of the invention and a pharmaceutically acceptable carrier and / or diluent.

[0045] In another aspect, the invention provides a method for tumor-localized activation of immune cells, such as T cells or NK cells, in a mammal, preferably a human, comprising the step of administering to the mammal a pMHC-specific recombinant binding protein or nucleic acid of the invention, wherein the binding protein further comprises a binding factor having binding specificity for a protein expressed on the surface of an immune cell, and wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein expressed in a tumor cell.

[0046] In another aspect, the invention provides a pMHC-specific recombinant binding protein or nucleic acid according to the invention for use in a method for tumour-localised activation of immune cells such as T cells or NK cells in a mammal, preferably a human, wherein the binding protein further comprises a binding factor having binding specificity for a protein expressed on the surface of an immune cell, and wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein expressed in a tumour cell.

[0047] In another aspect, the invention provides a method for infectious disease-limited activation of immune cells, such as T cells or NK cells, in a mammal, preferably a human, comprising the step of administering to the mammal a pMHC-specific recombinant binding protein or nucleic acid of the invention, wherein the binding protein further comprises a binding factor having binding specificity for a protein expressed on the surface of an immune cell, and wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein of an infectious agent.

[0048] In another aspect, the invention provides a method for treating a medical condition, the method comprising administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a pMHC-specific recombinant binding protein, nucleic acid, or pharmaceutical composition of the invention.

[0049] In another aspect, the invention provides a pMHC-specific recombinant binding protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of treating a medical condition.

[0050] In another aspect, the present invention provides a method of diagnosing a medical condition in a mammal, preferably a human, comprising: (i) contacting a cell or tissue sample obtained from said mammal with a pMHC-specific recombinant binding protein of the invention; (ii) detecting specific binding of said binding protein to said cell or tissue sample.

[0051] In one particular aspect, the medical condition is cancer, an infectious disease, preferably a viral infection, or an autoimmune disease. In one embodiment, the medical condition is cancer. In one embodiment, the medical condition is an infectious disease, preferably a viral infection. In one embodiment, the medical condition is an autoimmune disease.

[0052] In another aspect, the invention provides a method of targeting tumor cells in a patient suffering from a tumor for their destruction, the method comprising the step of administering to the patient a therapeutically effective amount of a pMHC-specific recombinant binding protein, nucleic acid or pharmaceutical composition of the invention, wherein the first targeting peptide and / or, if the binding protein comprises the second repeat domain, the second targeting peptide is derived from a protein expressed in tumor cells, preferably an intracellular protein expressed in tumor cells. In one embodiment, the binding protein further comprises a virulence factor capable of killing tumor cells.

[0053] In another aspect, the invention provides a pMHC-specific recombinant binding protein, nucleic acid or pharmaceutical composition of the invention for use in a method of targeting tumour cells in a patient suffering from a tumour for their destruction, wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein expressed in tumour cells, preferably an intracellular protein expressed in tumour cells. In one embodiment, the binding protein further comprises a virulence factor capable of killing tumour cells.

[0054] In another aspect, the invention provides a method of targeting infected cells in a patient suffering from a viral infection for their destruction, the method comprising the step of administering to the patient a therapeutically effective amount of a pMHC-specific recombinant binding protein, nucleic acid or pharmaceutical composition of the invention, wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein expressed in infected cells, preferably a virus-specific protein. In one embodiment, the binding protein further comprises a virulence factor capable of killing infected cells.

[0055] In another aspect, the invention provides a pMHC-specific recombinant binding protein, nucleic acid or pharmaceutical composition of the invention for use in a method of targeting infected cells in a patient suffering from a viral infection for their destruction, wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein expressed in infected cells, preferably a virus-specific protein. In one embodiment, the binding protein further comprises a virulence factor capable of killing infected cells. [Brief explanation of the drawings]

[0056] [Figure 1A]Quality control test of biotinylated pMHC complexes (biotin-pMHC). Preparative size exclusion chromatography (SEC) to isolate biotinylated NYESOpMHC complexes, biotinylated NYESOAApMHC complexes, and biotinylated EBNApMHC complexes. Ultraviolet (UV) absorbance at 280 nm was measured in the eluent. [Figure 1B] Quality control test of biotinylated pMHC complexes (biotin-pMHC). SDS-PAGE analysis of concentrated and refolded complexes in the absence or presence of streptavidin. [Figure 1C] Quality control test of biotinylated pMHC complexes (biotin-pMHC). Analytical size exclusion chromatography of ternary pMHC complexes. UV absorbance at 280 nm and 230 nm was measured in the eluent. [Figure 2] Efficient expression and purification of a binding protein containing a repeat domain with binding specificity for a target peptide-MHC complex. A highly soluble binding protein with binding specificity for NYESOpMHC was purified from E. coli culture with a high level of purity. A representative SDS-PAGE gel using elution fractions obtained after a SEC AKTAxpress™ run (post-IMAC) for NYESOpMHC-specific DARPin® protein #21 is shown. The gel was stained with Coomassie blue. [Figure 3A] Specific and high-affinity binding of binding proteins of the invention to target peptide-MHC complexes. A representative SPR trace for DARPin® protein #21 binding to NYESO pMHC is shown. DARPin® protein #21 bound with high affinity to NYESO pMHC, while no binding to the other two pMHC complexes was detected. The usual spikes in the binding trace are artifacts from the instrument and can be ignored. [Figure 3B]Specific and high-affinity binding of binding proteins of the invention to target peptide-MHC complexes. A representative SPR trace for DARPin® protein #21 binding to NYESOAApMHC is shown. DARPin® protein #21 bound with high affinity to NYESOpMHC, while no binding to the other two pMHC complexes was detected. The usual spikes in the binding trace are artifacts from the instrument and can be ignored. [Figure 3C] Specific and high-affinity binding of binding proteins of the invention to target peptide-MHC complexes. A representative SPR trace for DARPin® protein #21 binding to EBNA pMHC is shown. DARPin® protein #21 bound with high affinity to NYESO pMHC, while no binding to the other two pMHC complexes was detected. The usual spikes in the binding trace are artifacts from the instrument and can be ignored. [Figure 4A] Good biophysical properties and specific binding of the binding protein of the invention to target peptide-MHC complexes. Results of a representative homogeneous time-resolved fluorescence (HTRF) assay for DARPin® Protein #21 showed highly specific target binding to NYESOpMHC. No binding of DARPin® Protein #21 to NYESOAApMHC or EBNApMHC was detected. [Figure 4B] Good biophysical properties and specific binding of the binding protein of the invention to the target peptide-MHC complex. Size exclusion chromatography (SEC) of DARPin® Protein #21 showed a single monodisperse peak eluting at a position corresponding to the expected mass of an individual DARPin® Protein #21 molecule. No traces of aggregates or multimers were detected. [Figure 5A]Representative binding proteins to cells. T2 cells were pulsed with NY-ESO-1-9V (157-165) peptide (NY-ESO-pulsed cells) or EBNA-1 (562-570) peptide (EBNA-pulsed cells), or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to NY-ESO-pulsed cells are shown. [Figure 5B] Representative binding proteins to cells. T2 cells were pulsed with NY-ESO-1-9V (157-165) peptide (NY-ESO-pulsed cells) or EBNA-1 (562-570) peptide (EBNA-pulsed cells), or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to unpulsed cells are shown. [Figure 5C] Representative binding proteins to cells. T2 cells were pulsed with NY-ESO-1-9V (157-165) peptide (NY-ESO-pulsed cells) or EBNA-1 (562-570) peptide (EBNA-pulsed cells), or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to NY-ESO-pulsed cells are shown. [Figure 5D] Representative binding proteins to cells. T2 cells were pulsed with NY-ESO-1-9V (157-165) peptide (NY-ESO-pulsed cells) or EBNA-1 (562-570) peptide (EBNA-pulsed cells), or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to NY-ESO-pulsed cells are shown. [Figure 5E]Representative binding proteins to cells. T2 cells were pulsed with NY-ESO-1-9V (157-165) peptide (NY-ESO-pulsed cells) or EBNA-1 (562-570) peptide (EBNA-pulsed cells), or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to unpulsed cells are shown. [Figure 5F] Representative binding protein binding to cells. T2 cells were pulsed with NY-ESO-1-9V(157-165) peptide (NY-ESO-pulsed cells) or EBNA-1(562-570) peptide (EBNA-pulsed cells), or treated with buffer containing no peptide (unpulsed cells). The titration binding curve of TCE DARPin® Protein #21 to EBNA-pulsed cells is also shown. [Figure 6A]T cell activation assay using T2 cells as target cells. T2 cells pulsed with NY-ESO-1-9V(157-165) peptide (black bars) and non-pulsed T2 cells (gray bars) were incubated with effector CD8+ T cells (BK112) in the presence of different TCE DARPin® proteins (1 pM). (1) TCE DARPin® Protein #23, (2) TCE DARPin® Protein #24, (3) TCE DARPin® Protein #25, (4) TCE DARPin® Protein #26, (5) TCE DARPin® Protein #27, (6) TCE DARPin® Protein #28, (7) TCE DARPin® Protein #29, (8) TCE DARPin® Protein #30, (9) TCE DARPin® Protein #31, (10) TCE DARPin® Protein #32, (11) TCE DARPin® Protein #33, (12) TCE DARPin® Protein #21, (13) TCE DARPin® Protein #20, (14) TCE DARPin® Protein #22, and (15) no TCE DARPin® protein. Intracellular interferon-γ (IFN-γ) in T cells was detected by FACS. [Figure 6B] In a similar T cell activation assay, TCE DARPin® Protein #21 was titrated over a wider concentration range (0.01 pM to 1 nM) as indicated. [Figure 6C] In a similar T cell activation assay, TCE DARPin® Protein #32 was titrated over a wider concentration range (0.01 pM to 1 nM) as indicated. [Figure 7]T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) or MCF-7 tumor cells (triangle symbols) were incubated with effector CD8+ T cells (BK112) in the presence of different concentrations of TCE DARPin® Protein #21. As a control, effector CD8+ T cells (BK112) were incubated in the presence of different concentrations of TCE DARPin® Protein #21 but in the absence of tumor cells (square symbols). Intracellular IFN-γ in T cells was detected by FACS. Ag: antigen (here, NY-ESO-1-9V(157-165) peptide). Both tumor cell lines are HLA-A2+. [Figure 8A] T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 8B] T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 8C]T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 8D] T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 8E] T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 9A] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Interferon-γ (IFN-γ) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 9B]T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Interferon-γ (IFN-γ) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 9C] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Interferon-γ (IFN-γ) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 9D] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Interferon-γ (IFN-γ) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 9E] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Interferon-γ (IFN-γ) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 10A] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Tumor necrosis factor alpha (TNF-α) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 10B]T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Tumor necrosis factor alpha (TNF-α) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 10C] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Tumor necrosis factor alpha (TNF-α) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 10D] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Tumor necrosis factor alpha (TNF-α) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 10E] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins, as per Figure 8. Tumor necrosis factor alpha (TNF-α) levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 11A] Target peptide binding analyzed by alanine scanning mutagenesis. Functional binding of the indicated binding proteins to the NY-ESO-1-9V(157-165) peptide and a series of alanine mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. The amino acid residues and positions in the NY-ESO-1-9V(157-165) peptide that were replaced with alanines are indicated. Non-pulsed T2 cells served as a control. Intracellular IFN-γ was detected as a measure of T cell activation. [Figure 11B] Target peptide binding analyzed by alanine scanning mutagenesis. Functional binding of the indicated binding proteins to the NY-ESO-1-9V(157-165) peptide and a series of alanine mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. The amino acid residues and positions in the NY-ESO-1-9V(157-165) peptide that were replaced with alanines are indicated. Non-pulsed T2 cells served as a control. Intracellular IFN-γ was detected as a measure of T cell activation. [Figure 11C] Target peptide binding analyzed by alanine scanning mutagenesis. Functional binding of the indicated binding proteins to the NY-ESO-1-9V(157-165) peptide and a series of alanine mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. The amino acid residues and positions in the NY-ESO-1-9V(157-165) peptide that were replaced with alanines are indicated. Non-pulsed T2 cells served as a control. Intracellular IFN-γ was detected as a measure of T cell activation. [Figure 11D] Target peptide binding analyzed by alanine scanning mutagenesis. Functional binding of the indicated binding proteins to the NY-ESO-1-9V(157-165) peptide and a series of alanine mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. The amino acid residues and positions in the NY-ESO-1-9V(157-165) peptide that were replaced with alanines are indicated. Non-pulsed T2 cells served as a control. Intracellular IFN-γ was detected as a measure of T cell activation. [Figure 11E]Target peptide binding analyzed by alanine scanning mutagenesis. Functional binding of the indicated binding proteins to the NY-ESO-1-9V(157-165) peptide and a series of alanine mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. The amino acid residues and positions in the NY-ESO-1-9V(157-165) peptide that were replaced with alanines are indicated. Non-pulsed T2 cells served as a control. Intracellular IFN-γ was detected as a measure of T cell activation. [Figure 12] Cytotoxicity assay showing target pMHC-dependent target cell killing by effector cells mediated by a representative binding protein of the invention. Effector cells (peripheral blood mononuclear cells (PBMC)) and target cells (either pulsed T2 cells (PT2) or non-pulsed T2 cells (NPT2)) were incubated in the presence of different concentrations (0.01 nM, 0.1 nM, 1 nM) of NYESO pMHC-specific binding protein in the TCE format (TCE DARPin® Protein #21(D)). The level of apoptosis (total green area) at different incubation times is shown. [Figure 13]T cell activation assay using T2 cells as target cells. T2 cells pulsed with the NY-ESO-1-9V(157-165) peptide (black bars) and unpulsed T2 cells (gray bars) were incubated with effector CD8+ T cells (BK112) in the presence of different bivalent or biparatopic binding proteins in TCE format (0.1 pM) or as a control. (1) TCE DARPin® protein with binding specificity for human serum albumin, and (5) TCE BP DARPin® protein #20 / #21 with an ankyrin repeat domain instead of no TCE DARPin® protein; (2) TCE BP DARPin® protein #21 / #22; (3) TCE BV DARPin® protein #21 / #21; (4) an ankyrin repeat domain with binding specificity for NYESOpMHC. Intracellular interferon-γ (IFN-γ) in T cells was detected by FACS. [Figure 14A] Binding of a representative binding protein containing two repeat domains with binding specificity for a target pMHC complex to cells. T2 cells were pulsed with NY-ESO-1-9V(157-165) peptide (pulsed cells) or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to pulsed cells are shown. [Figure 14B] Figure 1 shows the binding of a representative binding protein containing two repeat domains with binding specificity for a target pMHC complex to cells. T2 cells were pulsed with NY-ESO-1-9V(157-165) peptide (pulsed cells) or treated with buffer containing no peptide (unpulsed cells). Titration binding curves of the indicated binding proteins to unpulsed cells (B) are shown. [Figure 15A]T cell activation assay using tumor cells as target cells. IM9 tumor cells (circular symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins, which contain two repeat domains with binding specificity for the target pMHC complex. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 15B] T cell activation assay using tumor cells as target cells. IM9 tumor cells (circular symbols) or MCF-7 tumor cells (triangle symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated binding proteins, which contain two repeat domains with binding specificity for the target pMHC complex. As a control, PBMCs were incubated in the presence of different concentrations of the indicated binding proteins but in the absence of tumor cells (square symbols). CD25 expression on CD8+ T cells was detected by FACS. [Figure 16A] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins containing two repeat domains with binding specificity for the target pMHC complex, as shown in Figure 15. IFN-γ levels were quantified in cell supernatants as an additional measure of T cell activation. [Figure 16B] T cell activation assay using tumor cells as target cells. IM9 or MCF-7 tumor cells were incubated with donor PBMCs as effector cells in the presence of different concentrations of the indicated binding proteins containing two repeat domains with binding specificity for the target pMHC complex, as shown in Figure 15. The level of TNF-α was quantified in the cell supernatant as an additional measure of T cell activation. [Figure 17]Target peptide binding analyzed by alanine scanning mutagenesis. Functional binding of the indicated binding proteins, containing two repeat domains with binding specificity for target pMHC complexes, to the NY-ESO-1-9V(157-165) peptide and a series of alanine mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. The amino acid residues and positions in the NY-ESO-1-9V(157-165) peptide that were replaced with alanines are indicated. Non-pulsed T2 cells were used as a control. Intracellular IFN-γ was detected as a measure of T cell activation, which is dependent on binding of the binding protein to target cells. [Figure 18A] T cell activation assay using tumor cells as target cells. U266B1 tumor cells (circle symbols) or Colo205 tumor cells (square symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated representative binding proteins of the invention (in a TCE format). TCE DARPin® Protein #21 was used. CD69 expression on CD8+ T cells was detected by FACS. Ag:NYESOpMHC. [Figure 18B] T cell activation assay using tumor cells as target cells. U266B1 tumor cells (circle symbols) or Colo205 tumor cells (square symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated representative binding proteins of the invention (in a TCE format). TCE DARPin® Protein #32 was used. CD69 expression on CD8+ T cells was detected by FACS. Ag:NYESOpMHC. [Figure 18C]T cell activation assay using tumor cells as target cells. MCF-7 tumor cells transfected to express NY-ESO-1 (circular symbols) or non-transfected MCF-7 tumor cells (square symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated representative binding proteins of the present invention (in a TCE format). TCE DARPin® Protein #21 was used. CD69 expression on CD8+ T cells was detected by FACS. Ag:NYESOpMHC. [Figure 18D] T cell activation assay using tumor cells as target cells. MCF-7 tumor cells transfected to express NY-ESO-1 (circular symbols) or non-transfected MCF-7 tumor cells (square symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated representative binding proteins of the present invention (in a TCE format). TCE DARPin® Protein #32 was used. CD69 expression on CD8+ T cells was detected by FACS. Ag:NYESOpMHC. [Figure 18E] T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) and MCF-7 tumor cells (square symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated representative binding proteins of the invention (in a TCE format). TCE DARPin® Protein #21 was used. CD69 expression on CD8+ T cells was detected by FACS. Ag:NYESOpMHC. [Figure 18F]T cell activation assay using tumor cells as target cells. IM9 tumor cells (circle symbols) and MCF-7 tumor cells (square symbols) were incubated with donor peripheral blood mononuclear cells (PBMCs) as effector cells in the presence of different concentrations of the indicated representative binding proteins of the present invention (in a TCE format). TCE DARPin® Protein #32 was used. CD69 expression on CD8+ T cells was detected by FACS. Ag:NYESOpMHC. [Figure 19A] Cytotoxicity assay showing target pMHC-dependent target cell killing by effector cells mediated by representative binding proteins of the invention. Pulsed T2 cells (P T2) or non-pulsed T2 cells (NP T2) were incubated with effector CD8+ T cells in the presence or absence of NYESO pMHC-specific binding protein in TCE format (Protein #21 in TCE DARPin®) (1 nM) as target cells. Different effector cell:target cell (E:T) ratios were used (30:1, 10:1, 5:1, and 1:1) as indicated. The percentage of specific lysis of T2 cells obtained by chromium release assay was plotted for different effector:target ratios. Ag:NYESO pMHC. [Figure 19B] Cytotoxicity assay showing target pMHC-dependent target cell killing by effector cells mediated by representative binding proteins of the invention. HLA-A2+ / NY-ESO-1+ tumor cells (IM9, U266B1) or HLA-A2+ / NY-ESO-1- tumor cells (MCF-7) (B) were incubated with effector CD8+ T cells in the presence or absence of NYESO pMHC-specific binding protein in TCE format (Protein #21 in TCE DARPin®) (1 nM) as target cells. Different effector cell:target cell (E:T) ratios were used (30:1, 10:1, 5:1, and 1:1) as indicated. The percentage of specific lysis of tumor cells (B) was plotted for different effector:target ratios. Ag:NYESO pMHC. [Figure 20A]Cytotoxicity assay showing target pMHC-dependent target cell killing by effector cells mediated by a representative binding protein of the invention. These figures show experiments as described in Figures 19A and 19B, but using a different NYESO pMHC-specific binding protein in the TCE format (TCE DARPin® Protein #32). [Figure 20B] Cytotoxicity assay showing target pMHC-dependent target cell killing by effector cells mediated by a representative binding protein of the invention. These figures show experiments as described in Figures 19A and 19B, but using a different NYESO pMHC-specific binding protein in the TCE format (TCE DARPin® Protein #32). [Figure 21A] Target peptide binding analyzed by X-ray scanning mutagenesis. NYESOpMHC-specific binding protein in TCE format (TCE Functional binding of DARPin® Protein #21 to the NY-ESO-1-9V(157-165) peptide and its series of single-mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. Each amino acid in the target peptide sequence was replaced with one of the other 19 standard amino acids. The amino acid residue and position in the NY-ESO-1-9V(157-165) peptide that was replaced with another amino acid are indicated on the left side of the table, and the replaced amino acid is indicated above the table. Intracellular IFN-γ was detected as a measure of T cell activation, which depends on binding of the binding protein to target cells. Each experiment was performed in two independent replicates. Values ​​were averaged and normalized to 100% for the matching wild-type residue (darkly shaded) at each position. All values ​​above 30% indicate no or incomplete loss of T cell activation and are marked in bold font and lightly shaded color. [Figure 21B]Target peptide binding analyzed by X-ray scanning mutagenesis. Functional binding of the NYESO pMHC-specific binding protein in the TCE format (TCE DARPin® Protein #32) to the NY-ESO-1-9V(157-165) peptide and its series of single-mutation variants was tested in a T cell activation assay using pulsed T2 cells as target cells and BK112T cells as effector cells. Each amino acid in the target peptide sequence was replaced with one of the other 19 standard amino acids. The amino acid residue and position in the NY-ESO-1-9V(157-165) peptide that was replaced with another amino acid are indicated on the left side of the table, and the replaced amino acid is indicated above the table. Intracellular IFN-γ was detected as a measure of T cell activation, which depends on binding of the binding protein to target cells. Each experiment was performed in two independent replicates. Values ​​were averaged and normalized to 100% for the matching wild-type residue (dark shaded area) at each position. All values ​​above 30% indicate no or less than complete loss of T cell activation and are labeled in bold font and lightly shaded color. [Figure 22] T cell activation by pMHC-specific binding proteins in the TCE format depending on the length of the linker connecting the pMHC-specific binding domain and the CD3-specific binding factor. HLA-A2+ / NY-ESO-1+ tumor cells (IM9) (solid line) or HLA-A2+ / NY-ESO-1- tumor cells (MCF-7) (dashed line) were incubated with PBMCs for 48 hours in the presence or absence of TCE DARPin® protein #21 with different linker lengths (Figure 22) or in the presence or absence of TCE DARPin® protein #32 with different linker lengths (Figure 23). After 48 hours, CD25 expression was measured in CD8+ T cells. Results obtained in the presence of TCE DARPin® protein are shown. No T cell activation was observed in the absence of TCE DARPin® protein. Ag: antigen / NY-ESO-1. Linker lengths: standard, XXS, XS, S, and L (see Example 11). [Figure 23]T cell activation by pMHC-specific binding proteins in the TCE format depending on the length of the linker connecting the pMHC-specific binding domain and the CD3-specific binding factor. HLA-A2+ / NY-ESO-1+ tumor cells (IM9) (solid line) or HLA-A2+ / NY-ESO-1- tumor cells (MCF-7) (dashed line) were incubated with PBMCs for 48 hours in the presence or absence of TCE DARPin® protein #21 with different linker lengths (Figure 22) or in the presence or absence of TCE DARPin® protein #32 with different linker lengths (Figure 23). After 48 hours, CD25 expression was measured in CD8+ T cells. Results obtained in the presence of TCE DARPin® protein are shown. No T cell activation was observed in the absence of TCE DARPin® protein. Ag: antigen / NY-ESO-1. Linker lengths: standard, XXS, XS, S, and L (see Example 11). [Figure 24] T cell activation by pMHC-specific binding proteins in the TCE format depending on the length of the linker connecting the pMHC-specific binding domain and the CD3-specific binding factor. HLA-A2+ / NY-ESO-1+ tumor cells (U266B1) (solid line) or HLA-A2+ / NY-ESO-1- tumor cells (Colo205) (dashed line) were incubated with PBMCs for 48 hours in the presence or absence of TCE DARPin® protein #21 with different linker lengths (Figure 24) or in the presence or absence of TCE DARPin® protein #32 with different linker lengths (Figure 25). After 48 hours, CD25 expression was measured in CD8+ T cells. Results obtained in the presence of TCE DARPin® protein are shown. No T cell activation was observed in the absence of TCE DARPin® protein. Ag: antigen / NY-ESO-1. Linker lengths: standard, XXS, XS, S, and L (see Example 11). [Figure 25]T cell activation by pMHC-specific binding proteins in the TCE format depending on the length of the linker connecting the pMHC-specific binding domain and the CD3-specific binding factor. HLA-A2+ / NY-ESO-1+ tumor cells (U266B1) (solid line) or HLA-A2+ / NY-ESO-1- tumor cells (Colo205) (dashed line) were incubated with PBMCs for 48 hours in the presence or absence of TCE DARPin® protein #21 with different linker lengths (Figure 24) or in the presence or absence of TCE DARPin® protein #32 with different linker lengths (Figure 25). After 48 hours, CD25 expression was measured in CD8+ T cells. Results obtained in the presence of TCE DARPin® protein are shown. No T cell activation was observed in the absence of TCE DARPin® protein. Ag: antigen / NY-ESO-1. Linker lengths: standard, XXS, XS, S, and L (see Example 11). [Figure 26A] Pharmacokinetic properties in mice of designed ankyrin repeat domain mutants (each genetically linked to the same designed ankyrin repeat domain with binding specificity for serum albumin via the same polypeptide linker). Pharmacokinetic properties in mice of protein #281 and mutant proteins #282 and #283. This experiment was performed as described in Example 17 using Balb / c mice and an intravenous administration of 1 mg / kg. Proteins #281 to #295 (comprising SEQ ID NOs: 281 to 295, respectively, each with a His tag (SEQ ID NO: 326) at the N-terminus; symbols are indicated in the figures) were produced and purified as described in Example 17. C: concentration [nM], t: time [h]. [Figure 26B]Pharmacokinetic properties in mice of designed ankyrin repeat domain mutants (each genetically linked to the same designed ankyrin repeat domain with binding specificity for serum albumin via the same polypeptide linker). Pharmacokinetic properties in mice of protein #284 and mutant proteins #285, #286, and #287. This experiment was performed as described in Example 17 using Balb / c mice and intravenous administration of 1 mg / kg. Proteins #281 to #295 (comprising SEQ ID NOs: 281 to 295, respectively, each with a His tag (SEQ ID NO: 326) at the N-terminus; symbols are indicated in the figures) were produced and purified as described in Example 17. C: concentration [nM], t: time [h]. [Figure 26C] Pharmacokinetic properties of designed ankyrin repeat domain mutants in mice (each genetically linked to the same designed ankyrin repeat domain with binding specificity for serum albumin via the same polypeptide linker). Pharmacokinetic properties of protein #288 in mice, as well as mutant proteins #289, #290, and #291. This experiment was performed as described in Example 17 using Balb / c mice and intravenous administration of 1 mg / kg. Proteins #281 to #295 (comprising SEQ ID NOs: 281 to 295, respectively, each with a His tag (SEQ ID NO: 326) at the N-terminus; symbols are indicated in the figures) were produced and purified as described in Example 17. C: concentration [nM], t: time [h]. [Figure 26D]Pharmacokinetic properties in mice of designed ankyrin repeat domain mutants (each genetically linked to the same designed ankyrin repeat domain with binding specificity for serum albumin via the same polypeptide linker). Pharmacokinetic properties in mice of protein #292 and mutant proteins #293, #294, and #295. This experiment was performed as described in Example 17 using Balb / c mice and intravenous administration of 1 mg / kg. Proteins #281 to #295 (comprising SEQ ID NOs: 281 to 295, respectively, each with a His tag (SEQ ID NO: 326) at the N-terminus; symbols are indicated in the figures) were produced and purified as described in Example 17. C: concentration [nM], t: time [h]. DETAILED DESCRIPTION OF THE INVENTION

[0057] As disclosed and exemplified herein, the present disclosure provides designed repeat proteins, preferably designed ankyrin repeat proteins, that specifically target peptide-MHC complexes. Designed repeat protein libraries, including designed ankyrin repeat protein libraries (WO 2002 / 020565, Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701, 2008), can be used to select target-specific designed repeat domains that bind to targets with high affinity. Such target-specific designed repeat domains can then be used as valuable components of recombinant binding proteins for the treatment of diseases. It has not been shown whether designed repeat protein libraries can be used to identify proteins that specifically and with high affinity bind to complex epitopes, such as proteins presented by peptide-MHC complexes. It has clearly been difficult to generate molecules that specifically bind disease-related peptide-MHC complexes with sufficient affinity.

[0058] Designed ankyrin repeat proteins are a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies and thus enable novel therapeutic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or a combination of two, three, four, five, or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today 13,695-701, 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins containing only a single designed ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These characteristics, along with the possibility of combining two, three, four, five, or more designed ankyrin repeat domains in a single protein, make designed ankyrin repeat proteins ideal candidates for agonistic, antagonistic, and / or inhibitory drugs. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely new drug formats. Collectively, designed ankyrin repeat proteins represent a next-generation example of protein therapeutics with the potential to surpass existing antibody drugs.

[0059] DARPin® is a trademark owned by Molecular Partners AG, Switzerland.

[0060] In one aspect, the invention provides a method for producing a peptide-MHC (pMHC) specific binding protein, the binding protein comprising engineered repeat domains that have binding specificity for a target peptide-MHC complex, the method comprising: (a) providing a collection of designed repeat domains; (b) providing a recombinant target peptide-MHC complex; (c) screening the collection of designed repeat domains for specific binding to the target peptide-MHC complex to obtain at least one designed repeat domain that has binding specificity for the target peptide-MHC complex.

[0061] In one embodiment, the designed repeat domain is a designed ankyrin repeat domain and the collection of designed repeat domains is a collection of designed ankyrin repeat domains.

[0062] In one embodiment, the collection comprises designed repeat domains comprising fixed positions and randomized positions, wherein the designed repeat domains of the collection differ from each other at at least one of the randomized positions.

[0063] In one embodiment, the collection of designed repeat proteins is provided by ribosome display.

[0064] In one embodiment, the method further comprises (i) providing a second recombinant peptide-MHC complex, wherein the peptide of the second peptide-MHC complex comprises an amino acid sequence that differs from the target peptide by at least one amino acid residue; and (ii) removing from the collection by a negative selection designed repeat domain that has binding specificity for the second recombinant peptide-MHC complex.

[0065] In one embodiment, the repeat domain having binding specificity for a target peptide-MHC complex comprises 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10-9 Below M or 5 x 10 -10 Below M or 3 x 10 -10 Less than M or 2 x 10 -10 Below M or 10 -10 Dissociation constant (K D In one embodiment, the repeat domain having binding specificity for a target peptide-MHC complex binds to the target peptide-MHC complex in PBS at 10 -7 Dissociation constant (K D ) to join them.

[0066] In one embodiment, binding of the repeat domain with binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises interaction of the repeat domain with at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid residues of the target peptide. Thus, in one embodiment, binding of the repeat domain with binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises interaction of the repeat domain with at least one amino acid residue of the target peptide. In one embodiment, binding of the repeat domain with binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises interaction of the repeat domain with at least two amino acid residues of the target peptide. In one embodiment, binding of the repeat domain with binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises interaction of the repeat domain with at least three amino acid residues of the target peptide. In one embodiment, the binding of the repeat domain having binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises an interaction between the repeat domain and at least four amino acid residues of the target peptide. In one embodiment, the binding of the repeat domain having binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises an interaction between the repeat domain and at least five amino acid residues of the target peptide. In one embodiment, the binding of the repeat domain having binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises an interaction between the repeat domain and at least six amino acid residues of the target peptide. In one embodiment, the binding of the repeat domain having binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises an interaction between the repeat domain and at least seven amino acid residues of the target peptide.In further or alternative embodiments, binding of the repeat domain having binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises interaction of the repeat domain with at least one amino acid residue of the MHC.

[0067] Methods for determining amino acid residues involved in binding interactions between proteins or between a protein and a peptide, such as, for example, alanine scanning mutagenesis, are well known to those skilled in the art.

[0068] In the context of the present invention, a typical and preferred determination of amino acid residues of a target peptide involved in the binding interaction between a binding protein or a designed repeat domain having binding specificity for a target peptide-MHC complex and the target peptide-MHC complex is performed by alanine scanning mutagenesis as described in Example 7. Accordingly, in one embodiment, the amino acid residues of a target peptide involved in the binding interaction between a binding protein or a designed repeat domain having binding specificity for a target peptide-MHC complex and the target peptide-MHC complex are determined by alanine scanning mutagenesis. In one embodiment, the amino acid residues of a target peptide involved in the binding interaction between a binding protein or a designed repeat domain having binding specificity for a target peptide-MHC complex and the target peptide-MHC complex are determined by alanine scanning mutagenesis as described in Example 7.

[0069] In the context of the present invention, the phrase "binding of the repeat domain having binding specificity for a target peptide-MHC complex to the target peptide-MHC complex comprises interaction of the repeat domain with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 amino acid residues of the target peptide" or similar phrases refers to any amino acid residue of a target peptide that, when mutated to alanine, reduces T cell activation as described in Example 7 by at least 50% compared to the wild-type peptide.

[0070] The present inventors have discovered that a surprisingly large number of peptide residues are important for the specific interaction of a binding protein of the present invention comprising an ankyrin repeat domain with binding specificity for a target peptide-MHC complex with the target peptide. For example, in the accompanying Examples, several peptide residues important for the specific interaction of a binding protein of the present invention comprising an ankyrin repeat domain with binding specificity for NYESOpMHC with the NY-ESO-1 target peptide have been identified (see Figure 11). The inventors theorize that this finding may reflect differences in the structure of the binding surface formed by the designed ankyrin repeat domain of the present invention and the binding surface formed by other binding proteins, such as antibodies, with the T cell receptor (TCR). Without being bound by any theory, it is believed that the greater the number of amino acid residues involved in the binding between the repeat domain and its target peptide, the higher the binding specificity. In one embodiment, the target peptide is selected from the group consisting of (i) peptides derived from proteins expressed in tumor cells, (ii) peptides derived from proteins of infectious agents, such as bacterial or viral infectious agents, preferably viral infectious agents, and (iii) peptides derived from proteins associated with autoimmune disorders. In one embodiment, the target peptide is derived from an intracellular protein, preferably an intracellular protein expressed in tumor cells. In one embodiment, the target peptide is derived from NY-ESO-1. In one embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34. In one embodiment, the target peptide is derived from MAGE-A3. In one embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 155. In one embodiment, the target peptide is derived from a protein of an infectious agent, such as, for example, a viral infectious agent, preferably a virus-specific protein. In one embodiment, the target peptide is derived from EBNA-1. In one embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 92. In one embodiment, the target peptide is derived from HBcAg. In one embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 255.

[0071] In one embodiment, the MHC is MHC class I. In one embodiment, the MHC class I is HLA-A * 02. In one embodiment, the HLA-A * 02 is HLA-A * 0201. In one embodiment, the HLA-A * 0201 has the amino acid sequence of SEQ ID NO: 73. Alternatively, in another embodiment, the MHC is HLA-A * In one embodiment, the MHC class I is not HLA-A. * 01. In one embodiment, the HLA-A * 01 is HLA-A * In one embodiment, the HLA-A * 0101 has the amino acid sequence of SEQ ID NO: 218. Alternatively, in another embodiment, the MHC is HLA-A * Not 01.

[0072] In another aspect, the present invention relates to a recombinant binding protein comprising an engineered repeat domain obtainable by one of the methods of the invention described herein.

[0073] In another aspect, the present invention relates to a recombinant binding protein comprising a first designed repeat domain, said first repeat domain having binding specificity for a first target peptide-MHC complex.

[0074] In a preferred embodiment, the binding protein of the invention comprises a first designed repeat domain having binding specificity for a first target peptide-MHC complex, said first target peptide being selected from the group consisting of: (i) a peptide derived from a protein expressed in a tumor cell; (ii) a peptide derived from a protein of an infectious agent, such as a bacterial or viral infectious agent, preferably a viral infectious agent; and (iii) a peptide derived from a protein associated with an autoimmune disorder.

[0075] In one embodiment, the first target peptide is derived from an intracellular protein, preferably an intracellular protein expressed in a tumor cell. In one embodiment, the first target peptide is derived from a tumor-specific intracellular protein. In one embodiment, the first target peptide is derived from NY-ESO-1. In one embodiment, the first target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34. In one embodiment, the first target peptide is derived from MAGE-A3. In one embodiment, the first target peptide has the amino acid sequence of SEQ ID NO: 155. In one embodiment, the target peptide is derived from a protein of an infectious agent, such as, for example, a viral infectious agent, preferably a virus-specific protein. In one embodiment, the target peptide is derived from EBNA-1. In one embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 92. In one embodiment, the target peptide is derived from HBcAg. In one embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 255. In a preferred embodiment, the first MHC is MHC class I. In one embodiment, the first MHC class I is HLA-A * 02. In one embodiment, the HLA-A * 02 is HLA-A * 0201. In one embodiment, the HLA-A * 0201 has the amino acid sequence of SEQ ID NO: 73. Alternatively, in another embodiment, the first MHC is HLA-A * In one embodiment, the first MHC class I is not HLA-A. * 01, the HLA-A * 01 is HLA-A * In one embodiment, the HLA-A * 0101 has the amino acid sequence of SEQ ID NO: 218. Alternatively, in another embodiment, the first MHC is HLA-A * Not 01.

[0076] In one embodiment, the first repeat domain is delivered to the first target peptide-MHC complex in PBS at 10 -7Dissociation constant (K D ) to join them.

[0077] In one embodiment, the binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid residues of the first target peptide. Thus, in one embodiment, the binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least one amino acid residue of the first target peptide. In one embodiment, the binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least two amino acid residues of the first target peptide. In one embodiment, the binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least three amino acid residues of the first target peptide. In one embodiment, binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least four amino acid residues of the first target peptide. In one embodiment, binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least five amino acid residues of the first target peptide. In one embodiment, binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least six amino acid residues of the first target peptide. In one embodiment, binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least seven amino acid residues of the first target peptide. In further or alternative embodiments, binding of the first repeat domain to the first target peptide-MHC complex comprises interaction of the first repeat domain with at least one amino acid residue of the first MHC.

[0078] In a preferred embodiment, the first designed repeat domain is a designed ankyrin repeat domain. In a particularly preferred embodiment, the first designed ankyrin repeat domain is a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex as more particularly described in any of the aspects or embodiments herein.

[0079] In one embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module.

[0080] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-72 and (2) the sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in any of SEQ ID NOs: 37-72 is substituted by another amino acid. Thus, in one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-72 and (2) the sequences in which up to 9 amino acids in any of SEQ ID NOs: 37-72 are substituted by another amino acid. Thus, in one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-72 and (2) the sequences in which up to 3 amino acids in any of SEQ ID NOs: 37-72 are substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37 to 72 and (2) sequences in which up to two amino acids in any of SEQ ID NOs: 37 to 72 are substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37 to 72 and (2) sequences in which up to one amino acid in any of SEQ ID NOs: 37 to 72 is substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 72. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0081] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42, 54, 55 and 67-72; and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in any of SEQ ID NOs: 37-42, 54, 55 and 67-72 is substituted by another amino acid. Thus, in one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42, 54, 55 and 67-72; and (2) sequences in which up to 3 amino acids in any of SEQ ID NOs: 37-42, 54, 55 and 67-72 are substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42, 54, 55, and 67-72; and (2) sequences in which up to two amino acids in any of SEQ ID NOs: 37-42, 54, 55, and 67-72 are substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42, 54, 55, and 67-72; and (2) sequences in which up to one amino acid in any of SEQ ID NOs: 37-42, 54, 55, and 67-72 is substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, 54, 55, and 67-72. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0082] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42 and 67-69; and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in any of SEQ ID NOs: 37-42, and 67-69 is substituted by another amino acid. Thus, in one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42, and 67-69; and (2) sequences in which up to 3 amino acids in any of SEQ ID NOs: 37-42, and 67-69 are substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37 to 42, and 67 to 69; and (2) sequences in which up to two amino acids in any of SEQ ID NOs: 37 to 42, and 67 to 69 are substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37 to 42, and 67 to 69; and (2) sequences in which up to one amino acid in any of SEQ ID NOs: 37 to 42, and 67 to 69 is substituted by another amino acid. In one embodiment, the ankyrin repeat module comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 42, and 67 to 69. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0083] In another particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 111 to 154 and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 111 to 154 is substituted by another amino acid. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 92.

[0084] In a further particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 175 to 217 and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 175 to 217 is substituted by another amino acid. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 155.

[0085] In a further particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 231 to 254 and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 231 to 254 is substituted by another amino acid. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 255.

[0086] In one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first ankyrin repeat module and a second ankyrin repeat module, which in one embodiment is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain.

[0087] In one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first ankyrin repeat module, a second ankyrin repeat module and a third ankyrin repeat module, which in one embodiment are located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and which in one embodiment are located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain.

[0088] In one particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first, a second, and optionally a third ankyrin repeat module, wherein each of said first, said second, and, if present, said third ankyrin repeat module independently comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-72 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in any of SEQ ID NOs: 37-72 is substituted by another amino acid. In one embodiment, the first, the second, and, if present, the third ankyrin repeat module each independently comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42, 54, 55 and 67-72; and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in any of SEQ ID NOs: 37-42, 54, 55 and 67-72 is substituted by another amino acid. In one embodiment, the first, the second, and, if present, the third ankyrin repeat modules each independently comprise an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 37-42 and 67-69, and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in any of SEQ ID NOs: 37-42, and 67-69 is substituted by another amino acid. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0089] In one particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first ankyrin repeat module and a second ankyrin repeat module, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 54 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in SEQ ID NO: 54 is substituted by another amino acid; and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 55 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in SEQ ID NO: 55 is substituted by another amino acid. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0090] In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 54 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 54 are substituted by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 55 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 55 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 54 and (2) sequences in which up to 5 amino acids in SEQ ID NO: 54 are substituted by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 55 and (2) sequences in which up to 5 amino acids in SEQ ID NO: 55 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 54 and (2) sequences in which up to four amino acids in SEQ ID NO: 54 are substituted by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 55 and (2) sequences in which up to four amino acids in SEQ ID NO: 55 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 54 and (2) sequences in which up to three amino acids in SEQ ID NO: 54 are substituted by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 55 and (2) sequences in which up to three amino acids in SEQ ID NO: 55 are substituted by other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 54 and (2) sequences in which up to two amino acids in SEQ ID NO: 54 are substituted by another amino acid, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 55 and (2) sequences in which up to two amino acids in SEQ ID NO: 55 are substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 54 and (2) sequences in which one amino acid in SEQ ID NO: 54 is substituted by another amino acid, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 55 and (2) sequences in which one amino acid in SEQ ID NO: 55 is substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 54 and the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 55. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0091] In one particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 37 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in SEQ ID NO: 37 is substituted by another amino acid; and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 38 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 38 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 39 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 39 is substituted by another amino acid. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0092] In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 37 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 38 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 39 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 39 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which up to five amino acids in SEQ ID NO: 37 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which up to five amino acids in SEQ ID NO: 38 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 39 and (2) sequences in which up to five amino acids in SEQ ID NO: 39 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which up to four amino acids in SEQ ID NO: 37 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which up to four amino acids in SEQ ID NO: 38 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 39 and (2) sequences in which up to four amino acids in SEQ ID NO: 39 are substituted by other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which up to three amino acids in SEQ ID NO: 37 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which up to three amino acids in SEQ ID NO: 38 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 39 and (2) sequences in which up to three amino acids in SEQ ID NO: 39 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which up to two amino acids in SEQ ID NO: 37 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which up to two amino acids in SEQ ID NO: 38 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 39 and (2) sequences in which up to two amino acids in SEQ ID NO: 39 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which one amino acid in SEQ ID NO: 37 is substituted by another amino acid, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which one amino acid in SEQ ID NO: 38 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 39 and (2) sequences in which one amino acid in SEQ ID NO: 39 is substituted by another amino acid.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 37, the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 38, and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 39. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0093] In one particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 40 and (2) SEQ ID NO: 40 in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid is substituted by another amino acid; and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 41 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 41 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 42 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 42 is substituted by another amino acid. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0094] In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 40 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 40 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 41 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 41 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 42 and (2) sequences in which up to 6 amino acids in SEQ ID NO: 42 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 40 and (2) sequences in which up to five amino acids in SEQ ID NO: 40 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 41 and (2) sequences in which up to five amino acids in SEQ ID NO: 41 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 42 and (2) sequences in which up to five amino acids in SEQ ID NO: 42 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 40 and (2) sequences in which up to four amino acids in SEQ ID NO: 40 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 41 and (2) sequences in which up to four amino acids in SEQ ID NO: 41 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 42 and (2) sequences in which up to four amino acids in SEQ ID NO: 42 are substituted by other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 40 and (2) sequences in which up to three amino acids in SEQ ID NO: 40 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 41 and (2) sequences in which up to three amino acids in SEQ ID NO: 41 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 42 and (2) sequences in which up to three amino acids in SEQ ID NO: 42 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 40 and (2) sequences in which up to two amino acids in SEQ ID NO: 40 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 41 and (2) sequences in which up to two amino acids in SEQ ID NO: 41 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 42 and (2) sequences in which up to two amino acids in SEQ ID NO: 42 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 40 and (2) sequences in which one amino acid in SEQ ID NO: 40 is substituted by another amino acid, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 41 and (2) sequences in which one amino acid in SEQ ID NO: 41 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 42 and (2) sequences in which one amino acid in SEQ ID NO: 42 is substituted by another amino acid.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 40, the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 41, and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 42. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0095] In one particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the invention comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 67 and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 67 is substituted by another amino acid; and the second ankyrin repeat module comprises: (1) and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 68 and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 68 is substituted by another amino acid and SEQ ID NO: 68, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 69 is substituted by another amino acid. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0096] In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 67 and (2) a sequence in which up to 6 amino acids of SEQ ID NO: 67 are substituted by another amino acid, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 68 and (2) a sequence in which up to 6 amino acids of SEQ ID NO: 68 are substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which up to 6 amino acids of SEQ ID NO: 69 are substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 67 and (2) a sequence in which up to 5 amino acids of SEQ ID NO: 67 are substituted by another amino acid, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 68 and (2) a sequence in which up to 5 amino acids of SEQ ID NO: 68 are substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which up to 5 amino acids of SEQ ID NO: 69 are substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 67 and (2) a sequence in which up to four amino acids of SEQ ID NO: 67 are substituted by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 68 and (2) a sequence in which up to four amino acids of SEQ ID NO: 68 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which up to four amino acids of SEQ ID NO: 69 are substituted by other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 67 and (2) a sequence in which up to three amino acids in SEQ ID NO: 67 are substituted by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 68 and (2) a sequence in which up to three amino acids in SEQ ID NO: 68 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which up to three amino acids in SEQ ID NO: 69 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 67 and (2) a sequence in which up to two amino acids in SEQ ID NO: 67 are replaced by another amino acid, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 68 and (2) a sequence in which up to two amino acids in SEQ ID NO: 68 are replaced by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which up to two amino acids in SEQ ID NO: 69 are replaced by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 67 and (2) a sequence in which one amino acid in SEQ ID NO: 67 is substituted by another amino acid, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 68 and (2) a sequence in which one amino acid in SEQ ID NO: 68 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 69 and (2) a sequence in which one amino acid in SEQ ID NO: 69 is substituted by another amino acid.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 67, the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 68, and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 69. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0097] In one particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 70 and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid in SEQ ID NO: 70 is substituted by another amino acid; and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 71 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 71 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 72 and (2) sequences in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 amino acid of SEQ ID NO: 72 is substituted by another amino acid. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0098] In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 70 and (2) a sequence in which up to 6 amino acids of SEQ ID NO: 70 are substituted by another amino acid, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 71 and (2) a sequence in which up to 6 amino acids of SEQ ID NO: 71 are substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 72 and (2) a sequence in which up to 6 amino acids of SEQ ID NO: 72 are substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 70 and (2) sequences in which up to 5 amino acids in SEQ ID NO: 70 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 71 and (2) sequences in which up to 5 amino acids in SEQ ID NO: 71 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 72 and (2) sequences in which up to 5 amino acids in SEQ ID NO: 72 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 70 and (2) sequences in which up to four amino acids in SEQ ID NO: 70 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 71 and (2) sequences in which up to four amino acids in SEQ ID NO: 71 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 72 and (2) sequences in which up to four amino acids in SEQ ID NO: 72 are substituted by other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 70 and (2) sequences in which up to three amino acids in SEQ ID NO: 70 are substituted by other amino acids, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 71 and (2) sequences in which up to three amino acids in SEQ ID NO: 71 are substituted by other amino acids, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 72 and (2) sequences in which up to three amino acids in SEQ ID NO: 72 are substituted by other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 70 and (2) sequences in which up to two amino acids in SEQ ID NO: 70 are substituted by another amino acid, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 71 and (2) sequences in which up to two amino acids in SEQ ID NO: 71 are substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 72 and (2) sequences in which up to two amino acids in SEQ ID NO: 72 are substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 70 and (2) sequences in which one amino acid in SEQ ID NO: 70 is substituted by another amino acid, the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 71 and (2) sequences in which one amino acid in SEQ ID NO: 71 is substituted by another amino acid, and the third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 72 and (2) sequences in which one amino acid in SEQ ID NO: 72 is substituted by another amino acid.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 70, the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 71, and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 72. In one embodiment, the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located N-terminal to the third ankyrin repeat module within the ankyrin repeat domain. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0099] In another particular embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first, a second, and optionally a third ankyrin repeat module, wherein each of the first, the second, and, if present, the third ankyrin repeat module independently comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 111-154 and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 111-154 is substituted by another amino acid. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 92.

[0100] In a further particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a first, a second, and optionally a third ankyrin repeat module, wherein each of the first, the second, and, if present, the third ankyrin repeat module independently comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 175 to 217 and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 175 to 217 is substituted by another amino acid. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 155.

[0101] In one preferred embodiment, all of the amino acid substitutions of the ankyrin repeat module as described and referred to herein occur in framework positions of the ankyrin repeat module, and typically the overall structure of the module is not affected by the substitutions.

[0102] In one preferred embodiment, all of said amino acid substitutions of said ankyrin repeat modules as described and referred to herein occur at positions other than randomized positions 3, 4, 6, 14 and 15 of said ankyrin repeat modules of SEQ ID NOs: 37-60, 62-66 and 68-72, or other than randomized positions 3, 4, 6, 13 and 14 of said ankyrin repeat modules of SEQ ID NOs: 61 and 67. In another preferred embodiment, all of said amino acid substitutions of said ankyrin repeat modules as described and referred to herein occur at positions other than randomized positions 3, 4, 6, 14 and 15 of said ankyrin repeat modules of SEQ ID NOs: 111-122 and 124-154, or other than randomized positions 3, 4, 6, 15 and 16 of said ankyrin repeat module of SEQ ID NO: 123. In another preferred embodiment, all of said amino acid substitutions of said ankyrin repeat modules as described and referred to herein occur at positions other than randomized positions 3, 4, 6, 14 and 15 of said ankyrin repeat modules of SEQ ID NOs: 175 to 217. In another preferred embodiment, all of said amino acid substitutions of said ankyrin repeat modules as described and referred to herein occur at positions other than randomized positions 3, 4, 6, 14 and 15 of said ankyrin repeat modules of SEQ ID NOs: 231 to 254.

[0103] In a further preferred embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention further comprises an N-terminal and / or C-terminal capping module.

[0104] According to the present invention, the N-terminal capping module of the ankyrin repeat domain of the invention having binding specificity for a target peptide-MHC complex has an amino acid sequence which may or may not comprise the amino acid G at position 1 and / or the amino acid S at position 2. An example of such an N-terminal capping module having an amino acid sequence comprising the amino acid G at position 1 and the amino acid S at position 2 is provided in SEQ ID NO: 5. An example of such an N-terminal capping module having an amino acid sequence which does not comprise the amino acid G at position 1 and the amino acid S at position 2 is provided in SEQ ID NO: 276. Thus, SEQ ID NO: 5 and SEQ ID NO: 276 are identical, with the proviso that the G at position 1 and / or the S at position 2 of SEQ ID NO: 5 is missing in SEQ ID NO: 276. Therefore, those skilled in the art will readily understand that the position numbering provided above for the N-terminal capping module comprising the surface design of the present invention may vary depending on whether these amino acid residues are present or not. Thus, an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and / or the amino acid at position 15 is L, the position numbers being defined by reference to SEQ ID NO: 276, corresponds to an N-terminal capping module having an amino acid sequence in which the amino acid at position 10 is Q and / or the amino acid at position 17 is L, the position numbers being defined by reference to SEQ ID NO: 5, which includes a "GS" sequence at its N-terminus, and SEQ ID NO: 276 is then generally used as the reference sequence.

[0105] In one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and / or the amino acid at position 15 is L, wherein the position numbers of the positions in the N-terminal capping module are determined by alignment with SEQ ID NO: 276 using the position numbers of SEQ ID NO: 276. Preferably, the alignment does not contain any amino acid gaps. Generating sequence alignments is a procedure well known in the art.

[0106] In one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and / or the amino acid at position 18 is Q, wherein the position numbers of the positions in the C-terminal capping module are determined by alignment with SEQ ID NO: 13 using the position numbers of SEQ ID NO: 13. Preferably, the alignment does not contain any amino acid gaps.

[0107] In one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the invention comprises (i) an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and / or the amino acid at position 15 is L, and / or (ii) a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and / or the amino acid at position 18 is Q. In one embodiment, a designed ankyrin repeat domain of the invention comprises (i) an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and / or the amino acid at position 15 is L, and (ii) a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and / or the amino acid at position 18 is Q. In one embodiment, a designed ankyrin repeat domain of the invention comprises (i) an N-terminal capping module having an amino acid sequence in which the amino acid at position 8 is Q and / or the amino acid at position 15 is L, and (ii) a C-terminal capping module having an amino acid sequence in which the amino acid at position 14 is R and / or the amino acid at position 18 is Q. Preferably, the position number of the position of the N-terminal capping module is determined by alignment with SEQ ID NO: 276 using the position number of SEQ ID NO: 276, and the position number of the position of the C-terminal capping module is determined by alignment with SEQ ID NO: 13 using the position number of SEQ ID NO: 13. Preferably, the alignment does not include an amino acid gap.

[0108] In a further embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an N-terminal capping module with the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 276), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid at positions other than position 8 and position 15 are optionally replaced by other amino acids.

[0109] In one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises a C-terminal capping module with the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 13), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid are optionally replaced by other amino acids at positions other than positions 14 and 18 of SEQ ID NO: 13.

[0110] In one embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the invention comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 276), in which up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid is substituted by other amino acids at positions other than positions 8 and 15. and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 13), wherein up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid is optionally replaced by other amino acids at positions other than positions 14 and 18 of SEQ ID NO: 13.

[0111] In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to 9 amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids. In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to 8 amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids.In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to 7 amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids. In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to 6 amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids.In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to five amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids. In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to four amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids.In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to three amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids. In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to two amino acids at positions other than positions 8 and 15 are optionally replaced by other amino acids, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid at positions other than positions 14 and 18 are optionally replaced by other amino acids.In one embodiment, the designed ankyrin repeat domain comprises (i) an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA, in which up to one amino acid at a position other than positions 8 and 15 is optionally replaced by another amino acid, and (ii) a C-terminal capping module having the amino acid sequence QDKSGKTPADLAARAGHQDIAEVLQKAA, in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid at a position other than positions 14 and 18 is optionally replaced by another amino acid.

[0112] The inventors surprisingly found that ankyrin-binding domains comprising one or more of the above-mentioned amino acids at the above-mentioned positions in the N-terminal capping module (i.e., positions 8 and 15) and / or the C-terminal capping module of a designed ankyrin repeat domain (i.e., positions 14 and 18) result in improved pharmacokinetic properties, including a prolonged terminal half-life, of the designed ankyrin repeat domain and of proteins comprising the designed ankyrin repeat domain (Example 17 and Figure 26).

[0113] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 20-33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20-33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20-33 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 20-33 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 20-33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20-33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20-33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20-33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 20-33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 20-33, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 20-33. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, and in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 33.Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs: 20 to 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20 to 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20 to 33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20 to 33 is optionally substituted by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0114] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 20, 21, 27, 32 and 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20, 21, 27, 32 and 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20, 21, 27, 32 and 33 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 20, 21, 27, 32 and 33 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 20, 21, 27, 32 and 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20, 21, 27, 32 and 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20, 21, 27, 32 and 33 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 20, 21, 27, 32 and 33 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 20, 21, 27, 32 and 33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs: 20, 21, 27 and 33, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs: 20, 21, 27, 32 and 33.In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 20, 21, 27, 32 and 33, in one embodiment the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 20, 21, 27, 32 and 33. Thus, in one embodiment the ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs: 20, 21, 27, 32 and 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20, 21, 27, 32 and 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20, 21, 27, 32 and 33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20, 21, 27, 32 and 33 is optionally substituted by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0115] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 20, 21 and 32, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20, 21 and 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20, 21 and 32 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 20, 21 and 32 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 20, 21 and 32, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20, 21 and 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20, 21 and 32 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20, 21 and 32 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 20, 21 and 32. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 20, 21 and 32, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 20, 21 and 32. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 20, 21 and 32; in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 20, 21 and 32.Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs: 20, 21 and 32, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20, 21 and 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20, 21 and 32 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20, 21 and 32 is optionally substituted by N. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0116] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 20, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 20 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 20 is optionally substituted by L and / or A at the last position of SEQ ID NO: 20 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 20, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 20 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 20 is optionally substituted by L and / or A at the last position of SEQ ID NO: 20 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 20. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 20, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 20. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 20, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 20. Thus, in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 20, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 20 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 20 is optionally substituted by L and / or A at the last position of SEQ ID NO: 20 is optionally substituted by N. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0117] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 21, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 21 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 21 is optionally substituted by L and / or A at the last position of SEQ ID NO: 21 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 21, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 21 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 21 is optionally substituted by L and / or A at the last position of SEQ ID NO: 21 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 21. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 21, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 21. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 21, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 21. Thus, in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 21, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 21 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 21 is optionally substituted by L and / or A at the last position of SEQ ID NO: 21 is optionally substituted by N. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0118] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 27, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 27 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 27 is optionally substituted by L and / or A at the last position of SEQ ID NO: 27 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 27, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 27 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 27 is optionally substituted by L and / or A at the last position of SEQ ID NO: 27 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 27. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 27, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 27. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 27, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 27. Thus, in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 27, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 27 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 27 is optionally substituted by L and / or A at the last position of SEQ ID NO: 27 is optionally substituted by N. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0119] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 32, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 32 is optionally substituted by L and / or A at the last position of SEQ ID NO: 32 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 32, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 32 is optionally substituted by L and / or A at the last position of SEQ ID NO: 32 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 32. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 32, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 32. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 32, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 32. Thus, in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 32, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 32 is optionally substituted by L and / or A at the last position of SEQ ID NO: 32 is optionally substituted by N. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0120] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 33, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 33 is optionally substituted by L and / or A at the last position of SEQ ID NO: 33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 33, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 33 is optionally substituted by L and / or A at the last position of SEQ ID NO: 33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 33, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 33. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 33, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 33. Thus, in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 33, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 33 is optionally substituted by L and / or A at the last position of SEQ ID NO: 33 is optionally substituted by N. In a preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0121] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 93-110, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 93-110 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 93-110 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 93-110 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 93 to 110, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 93 to 110 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 93 to 110 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 93 to 110 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 93 to 110. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 93 to 110, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 93 to 110. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 93 to 110, and in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 93 to 110.Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs: 93 to 110, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 93 to 110 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 93 to 110 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 93 to 110 is optionally substituted by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 92.

[0122] In one particular embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 156-173, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 156-173 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 156-173 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 156-173 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 156-173, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 156-173 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 156-173 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 156-173 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 156-173. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 156-173, and in a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 156-173. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 156-173, and in one embodiment, the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 156-173.Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs: 156 to 173, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 156 to 173 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 156 to 173 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 156 to 173 is optionally substituted by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 155.

[0123] In one embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention is -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Below M or 3 x 10 -10 Less than M or 2 x 10 -10 Below M or 10 -10 Dissociation constant (K D ) to bind the target peptide-MHC complex in PBS. Thus, in one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 10 -7 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 5×10 -8 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 3×10 -8 Dissociation constant (K DIn one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 2×10 -8 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 10 -8 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 5×10 -9 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 3×10 -9 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 2×10 -9 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 10 -9 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 5×10 -10 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 3×10 -10 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 2×10 -10 Dissociation constant (K D In one embodiment, the ankyrin repeat domain binds the target peptide-MHC complex in PBS at 10 -10 Dissociation constant (K D ) to join them.

[0124] In one embodiment, the designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex according to the present invention bind said target peptide-MHC complex in PBS at 10 -7 Dissociation constant (K D ), and comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 20-33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20-33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20-33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20-33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 20-33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, in a further embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, in one embodiment the ankyrin repeat domain comprises an amino acid sequence of any one of SEQ ID NOs: 20 to 33. Thus, in one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention is capable of binding to the target peptide-MHC complex in PBS for 10 -7 Dissociation constant (K D) and comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20 to 33 are optionally missing, and A at the penultimate position of SEQ ID NOs: 20 to 33 is optionally substituted with L, and / or A at the last position of SEQ ID NOs: 20 to 33 is optionally substituted with N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0125] In one embodiment, the designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex according to the present invention bind said target peptide-MHC complex in PBS at a concentration of 5×10 -8 Dissociation constant (K D), and comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 20-33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20-33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20-33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20-33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 20-33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, in a further embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, in one embodiment the ankyrin repeat domain comprises an amino acid sequence of any one of SEQ ID NOs: 20 to 33. Thus, in one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention binds the target peptide-MHC complex in PBS at a concentration of 5x10 -8 Dissociation constant (K D ) and comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20 to 33 are optionally missing, and A at the penultimate position of SEQ ID NOs: 20 to 33 is optionally substituted with L, and / or A at the last position of SEQ ID NOs: 20 to 33 is optionally substituted with N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0126] In one embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention binds said first target peptide-MHC complex in PBS at a concentration of 2×10 -8 Dissociation constant (K D ), and comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 20-33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20-33 are optionally missing, and wherein A at the penultimate position of SEQ ID NOs: 20-33 is optionally substituted by L and / or A at the last position of SEQ ID NOs: 20-33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 20-33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, in a further embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 20 to 33, in one embodiment the ankyrin repeat domain comprises an amino acid sequence of any one of SEQ ID NOs: 20 to 33. Thus, in one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention binds the target peptide-MHC complex in PBS at a concentration of 2×10 -8 Dissociation constant (K D) and comprises the amino acid sequence of any one of SEQ ID NOs: 20 to 33, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 20 to 33 are optionally missing, and A at the penultimate position of SEQ ID NOs: 20 to 33 is optionally substituted with L, and / or A at the last position of SEQ ID NOs: 20 to 33 is optionally substituted with N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0127] In one embodiment, the designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex according to the present invention bind said target peptide-MHC complex in PBS at 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Dissociation constant (K D), and comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 20, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 20 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 20 is optionally substituted by L and / or A at the last position of SEQ ID NO: 20 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 20. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to SEQ ID NO: 20. In a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 20. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to SEQ ID NO: 20, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 20. Thus, in one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention binds the target peptide-MHC complex in PBS for 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Dissociation constant (K D ), and comprises the amino acid sequence of SEQ ID NO:20, wherein G at position 1 and / or S at position 2 of SEQ ID NO:20 are optionally missing, and wherein A at the penultimate position of SEQ ID NO:20 is optionally replaced by L, and / or A at the last position of SEQ ID NO:20 is optionally replaced by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0128] In one embodiment, the designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex according to the present invention bind said target peptide-MHC complex in PBS at 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Dissociation constant (K D ), and comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 21, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 21 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 21 is optionally substituted by L and / or A at the last position of SEQ ID NO: 21 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 21. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to SEQ ID NO: 21. In a further embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 21. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 21, and in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 21. Thus, in one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention binds the target peptide-MHC complex in PBS for 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10-8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Dissociation constant (K D ), and comprises the amino acid sequence of SEQ ID NO:21, wherein G at position 1 and / or S at position 2 of SEQ ID NO:21 are optionally missing, and wherein A at the penultimate position of SEQ ID NO:21 is optionally replaced by L, and / or A at the last position of SEQ ID NO:21 is optionally replaced by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0129] In one embodiment, the designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention binds said first target peptide-MHC complex in PBS for 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Dissociation constant (K D), and comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 27, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 27 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 27 is optionally replaced by L and / or A at the last position of SEQ ID NO: 27 is optionally replaced by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence with at least 90% amino acid sequence identity to SEQ ID NO: 27. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to SEQ ID NO: 27, in a further embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 27. In one embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to SEQ ID NO: 27, in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 27. Thus, in one embodiment a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention is capable of binding the target peptide-MHC complex in PBS for 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Dissociation constant (K D), and comprises the amino acid sequence of SEQ ID NO:27, wherein G at position 1 and / or S at position 2 of SEQ ID NO:27 are optionally missing, and wherein A at the penultimate position of SEQ ID NO:27 is optionally replaced by L, and / or A at the last position of SEQ ID NO:27 is optionally replaced by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34.

[0130] In one embodiment, the designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex according to the present invention bind said target peptide-MHC complex in PBS at 10 -7 Below M or 5 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Dissociation constant (K D), and comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 32, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 32 is optionally substituted by L and / or A at the last position of SEQ ID NO: 32 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence with at least 90% amino acid sequence identity to SEQ ID NO: 32. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to SEQ ID NO: 32, in a further embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 32. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to SEQ ID NO: 32, in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 32. Thus, in one embodiment, a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention is capable of binding the target peptide-MHC complex in PBS for 10 -7 Below M or 5 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Dissociation constant (K D ), and comprises the amino acid sequence of SEQ ID NO: 32, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 32 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 32 is optionally replaced by L, and / or A at the last position of SEQ ID NO: 32 is optionally replaced by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0131] In one embodiment, the designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex according to the present invention bind said target peptide-MHC complex in PBS at 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Below M or 3 x 10 -10 Less than M or 2 x 10 -10 Dissociation constant (K D), and comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 33, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 33 is optionally substituted by L and / or A at the last position of SEQ ID NO: 33 is optionally substituted by N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence with at least 90% amino acid sequence identity to SEQ ID NO: 33. In another embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to SEQ ID NO: 33, in a further embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 33. In one embodiment the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to SEQ ID NO: 33, in one embodiment the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 33. Thus, in one embodiment a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex according to the present invention is used to bind to the first target peptide-MHC complex in PBS for 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Below M or 3 x 10 -10 Less than M or 2 x 10 -10 Dissociation constant (K D), and comprises the amino acid sequence of SEQ ID NO: 33, wherein G at position 1 and / or S at position 2 of SEQ ID NO: 33 are optionally missing, and wherein A at the penultimate position of SEQ ID NO: 33 is optionally replaced by L, and / or A at the last position of SEQ ID NO: 33 is optionally replaced by N. In one preferred embodiment, the target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0132] In the context of the present invention, the dissociation constant (K) of the recombinant binding protein or engineered repeat domain of the present invention with binding specificity to a target peptide-MHC complex by surface plasmon resonance (SPR) analysis is D An exemplary and preferred determination of the binding specificity of a recombinant binding protein or designed repeat domain of the invention to a target peptide-MHC complex is determined in PBS by surface plasmon resonance (SPR), as described in Example 2. Thus, in one embodiment, the binding specificity of a recombinant binding protein or designed repeat domain of the invention to a target peptide-MHC complex is determined in PBS by surface plasmon resonance (SPR), as described in Example 2.

[0133] In one aspect of the invention, the recombinant binding protein of the invention further comprises a second engineered repeat domain that has binding specificity for a second target peptide-MHC complex.

[0134] In a preferred embodiment, the second target peptide is selected from the group consisting of: (i) a peptide derived from a protein expressed in a tumor cell; (ii) a peptide derived from a protein of an infectious agent, such as a bacterial infectious agent or a viral infectious agent, preferably a viral infectious agent; and (iii) a peptide derived from a protein associated with an autoimmune disorder.

[0135] In one embodiment, the second target peptide is derived from an intracellular protein, preferably an intracellular protein expressed in a tumor cell. In one embodiment, the second target peptide is derived from an intracellular protein expressed in a tumor cell.

[0136] In one embodiment, the second target peptide is derived from a protein of an infectious agent, preferably a viral infectious agent, hi one embodiment, the second target peptide is derived from a virus-specific protein.

[0137] In one embodiment, the second repeat domain having binding specificity for a second target peptide-MHC complex is 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10 -8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Below M or 3 x 10 -10 Less than M or 2 x 10 -10 Below M or 10 -10 Dissociation constant (K D In one embodiment, the second repeat domain having binding specificity for a second target peptide-MHC complex binds to the second target peptide-MHC complex in PBS at 10 -7 Dissociation constant (K D ) to join them.

[0138] In one embodiment, said binding of said second repeat domain to said second target peptide-MHC complex comprises interaction of said second repeat domain with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acid residues of said second target peptide. Thus, in one embodiment, said binding of said second repeat domain to said second target peptide-MHC complex comprises interaction of said second repeat domain with at least one amino acid residue of said second target peptide. In one embodiment, said binding of said second repeat domain to said second target peptide-MHC complex comprises interaction of said second repeat domain with at least two amino acid residues of said second target peptide. In one embodiment, said binding of said second repeat domain to said second target peptide-MHC complex comprises interaction of said second repeat domain with at least three amino acid residues of said second target peptide. In one embodiment, the binding of the second repeat domain to the second target peptide-MHC complex comprises interaction of the second repeat domain with at least four amino acid residues of the second target peptide. In one embodiment, the binding of the second repeat domain to the second target peptide-MHC complex comprises interaction of the second repeat domain with at least five amino acid residues of the second target peptide. In one embodiment, the binding of the second repeat domain to the second target peptide-MHC complex comprises interaction of the second repeat domain with at least six amino acid residues of the second target peptide. In one embodiment, the binding of the second repeat domain to the second target peptide-MHC complex comprises interaction of the second repeat domain with at least seven amino acid residues of the second target peptide. In further or alternative embodiments, the binding of the second repeat domain to the second target peptide-MHC complex comprises interaction of the second repeat domain with at least one amino acid residue of the second MHC.

[0139] In one embodiment, the second MHC is MHC class I. In one embodiment, the second MHC is HLA-A* 02. In one embodiment, the HLA-A * 02 is HLA-A * 0201. In one embodiment, the HLA-A * 0201 has the amino acid sequence of SEQ ID NO: 73. Alternatively, in another embodiment, the second MHC is HLA-A * Not 02.

[0140] In one embodiment, the second MHC is MHC class I. In one embodiment, the second MHC is HLA-A * 01. In one embodiment, the HLA-A * 01 is HLA-A * In one embodiment, the HLA-A * 0101 has the amino acid sequence of SEQ ID NO: 218. Alternatively, in another embodiment, the second MHC is HLA-A * Not 01.

[0141] In one embodiment, the second target peptide is derived from the same protein as the first target peptide. In one embodiment, the second target peptide has the same amino acid sequence as the first target peptide. In a further embodiment, the second repeat domain has the same amino acid sequence as the first repeat domain. Alternatively, in another further embodiment, the second repeat domain has a different amino acid sequence compared to the first repeat domain. In one embodiment, the second target peptide has a different amino acid sequence compared to the first target peptide.

[0142] In another embodiment, the second target peptide is derived from a different protein than the protein from which the first target peptide is derived.

[0143] In a preferred embodiment, the second designed repeat domain is a designed ankyrin repeat domain. In a particularly preferred embodiment, the second designed ankyrin repeat domain is a designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex as more particularly described in any of the aspects and embodiments herein.

[0144] In one aspect of the invention, the recombinant binding protein of the invention further comprises a third designed repeat domain having binding specificity for a third target peptide-MHC complex. In a preferred embodiment, the third designed repeat domain is a designed ankyrin repeat domain. In a particularly preferred embodiment, the third designed ankyrin repeat domain is a designed ankyrin repeat domain having binding specificity for a target peptide-MHC complex as more particularly described in any of the aspects and embodiments herein.

[0145] In the context of the present invention, a recombinant binding protein of the present invention is bivalent when it comprises first and second designed repeat domains, and the first and second repeat domains have binding specificity for the same target peptide-MHC complex, i.e., when the second target peptide has the same amino acid sequence as the first target peptide, and the first and second repeat domains are identical in sequence. In the context of the present invention, a recombinant binding protein of the present invention is biparatopic when it comprises first and second designed repeat domains, and the first and second repeat domains have binding specificity for the same target peptide-MHC complex, i.e., when the second target peptide has the same amino acid sequence as the first target peptide, and the first and second repeat domains are different in sequence. In the context of the present invention, a recombinant binding protein of the present invention is bispecific when it comprises first and second designed repeat domains, and the first and second repeat domains have binding specificity for different target peptide-MHC complexes, i.e., when the second target peptide has a different amino acid sequence compared to the first target peptide. The different target peptides may be derived from the same protein or from different proteins. In the context of the present invention, the same definition applies as for a recombinant binding protein of the present invention comprising first, second and third designed repeat domains, each of which has binding specificity for a target peptide-MHC complex.

[0146] In one embodiment, a recombinant binding protein of the invention is monovalent, bivalent, trivalent, multivalent, monoparatopic, biparatopic, triparatopic, multiparatopic, monospecific, bispecific, trispecific, or multispecific. In one particular embodiment, a recombinant binding protein of the invention is monovalent. In one particular embodiment, a recombinant binding protein of the invention is bivalent. In one particular embodiment, a recombinant binding protein of the invention is trivalent. In one particular embodiment, a recombinant binding protein of the invention is multivalent. In one particular embodiment, a recombinant binding protein of the invention is monoparatopic. In one particular embodiment, a recombinant binding protein of the invention is biparatopic, in one particular embodiment, a recombinant binding protein of the invention is triparatopic, and in one particular embodiment, a recombinant binding protein of the invention is multiparatopic. In one particular embodiment, a recombinant binding protein of the invention is monospecific. In one particular embodiment, the recombinant binding protein of the invention is bispecific. In one particular embodiment, the recombinant binding protein of the invention is trispecific. In one particular embodiment, the recombinant binding protein of the invention is multispecific. It should be understood that the preceding embodiments can be combined with each other. As an example, a recombinant binding protein of the invention comprising two identical repeat domains with specificity for a first pMHC complex and further comprising a third repeat domain with specificity for a second, different pMHC complex would be simultaneously bivalent and bispecific.Thus, in one particular embodiment, the recombinant binding proteins of the invention are monovalent, bivalent, trivalent, or multivalent, and are monoparatopic, biparatopic, triparatopic, or multiparatopic, and / or monospecific, bispecific, trispecific, or multispecific.

[0147] As a non-limiting example, a bivalent binding protein according to the invention can comprise at least two occurrences of a repeat domain having the amino acid sequence of SEQ ID NO: 21. An example of such a bivalent protein is provided by SEQ ID NO: 16. As a further non-limiting example, a biparatopic binding protein according to the invention can comprise at least one repeat domain having the amino acid sequence of SEQ ID NO: 20 and at least one repeat domain having the amino acid sequence of SEQ ID NO: 21, or the binding protein can comprise at least one repeat domain having the amino acid sequence of SEQ ID NO: 21 and at least one repeat domain having the amino acid sequence of SEQ ID NO: 22. Examples of such biparatopic proteins are provided by SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0148] In one embodiment, when a recombinant binding protein of the invention comprises two or more ankyrin repeat domains, for example when a recombinant binding protein of the invention comprises two or three ankyrin repeat domains, the two or more ankyrin repeat domains, for example the two or three ankyrin repeat domains, can be linked with a peptide linker. In one embodiment, the peptide linker is a proline-threonine-rich peptide linker. In one embodiment, the peptide linker is a proline-threonine-rich peptide linker of SEQ ID NO: 1 or 2. In one embodiment, the two or more ankyrin repeat domains, for example the two or three ankyrin repeat domains, are proline-threonine-rich peptide linkers of SEQ ID NO: 1 or 2. In another embodiment, the peptide linker is a glycine-serine-rich peptide linker. In one embodiment, the peptide linker is a glycine-serine-rich peptide linker of SEQ ID NO: 3. In one embodiment, the two or more ankyrin repeat domains, for example the two or three ankyrin repeat domains, are linked with a glycine-serine-rich peptide linker of SEQ ID NO: 3. In one embodiment, when a recombinant binding protein of the invention comprises three or more ankyrin repeat domains, the three or more ankyrin repeat domains can be linked with different peptide linkers, for example a proline-threonine-rich peptide linker and a serine-glycine-rich peptide linker, such as the peptide linkers of SEQ ID NOs: 1 to 3.

[0149] In one embodiment, the recombinant binding protein of the invention comprises two or three ankyrin repeat domains, each of said two or three ankyrin repeat domains independently comprising an ankyrin repeat module as more particularly described in any of the aspects and embodiments herein.

[0150] In one embodiment, the recombinant binding protein comprises two ankyrin repeat domains with binding specificity for a target peptide-MHC complex, each of the two ankyrin repeat domains independently comprising an ankyrin repeat module as more particularly described in any of the aspects and embodiments herein.

[0151] In one embodiment, a recombinant binding protein of the invention comprises two ankyrin repeat domains with binding specificity for a target peptide-MHC complex, wherein each of the two ankyrin repeat domains comprises a first ankyrin repeat module and a second ankyrin repeat module as more particularly described in any of the aspects and embodiments herein.

[0152] In one embodiment, a recombinant binding protein of the invention comprises two ankyrin repeat domains with binding specificity for a target peptide-MHC complex, each of said two ankyrin repeat domains comprising a first ankyrin repeat module, a second ankyrin repeat module and a third ankyrin repeat module as more particularly described in any of the aspects and embodiments herein.

[0153] In one embodiment, a recombinant binding protein of the invention comprises two or three ankyrin repeat domains with binding specificity for a target peptide-MHC complex, each of the two or three ankyrin repeat domains independently comprising an amino acid sequence as more particularly described in any of the aspects and embodiments herein.

[0154] In one embodiment, a recombinant binding protein of the invention comprises exactly two ankyrin repeat domains with binding specificity for a target peptide-MHC complex, each of said two ankyrin repeat domains independently comprising an amino acid sequence as more particularly described in any of the aspects and embodiments herein.

[0155] In one embodiment, a recombinant binding protein of the invention comprises three ankyrin repeat domains with binding specificity for a target peptide-MHC complex, each of the three ankyrin repeat domains independently comprising an amino acid sequence as more particularly described in any of the aspects and embodiments herein.

[0156] In one embodiment, a recombinant binding protein of the invention comprises exactly three ankyrin repeat domains with binding specificity for a target peptide-MHC complex, each of the three ankyrin repeat domains independently comprising an amino acid sequence as more particularly described in any of the aspects and embodiments herein.

[0157] In one embodiment, the recombinant binding protein comprises a polypeptide consisting of first and second ankyrin repeat domains having binding specificity for first and second target peptide-MHC complexes, respectively, linked by a peptide linker, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 16-18, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 16-18 are optionally missing, and wherein A at the penultimate position of each of the ankyrin repeat domains of SEQ ID NOs: 16-18 is optionally substituted by L and / or A at the last position of each of the ankyrin repeat domains of SEQ ID NOs: 16-18 is optionally substituted by N. Thus, in one embodiment, the polypeptide comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 16-18. In one embodiment, the polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 16-18. In one embodiment, the polypeptide comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs: 16-18. In one embodiment, the polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs: 16-18. In one embodiment, the polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs: 16-18. In one embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 16-18. In one embodiment, the polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 16-18. In one embodiment, the recombinant binding protein comprising a polypeptide consisting of two pMHC-specific ankyrin repeat domains is 10 -7 Below M or 5 x 10 -8 Below M or 3 x 10 -8 Less than M or 2 x 10-8 Below M or 10 -8 Below M or 5 x 10 -9 Below M or 3 x 10 -9 Less than M or 2 x 10 -9 Below M or 10 -9 Below M or 5 x 10 -10 Below M or 3 x 10 -10 Less than M or 2 x 10 -10 Below M or 10 -10 Dissociation constant (K D ) to bind target peptide-MHC complexes in PBS. In one preferred embodiment, the first and second target peptides independently have the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34.

[0158] In one embodiment, the recombinant binding protein of the invention comprises a single designed ankyrin repeat domain with binding specificity for a target peptide-MHC complex as more particularly described in any of the aspects and embodiments herein, or comprises a combination of two, three, four, five or more designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex as more particularly described in any of the aspects and embodiments herein.

[0159] In one aspect of the invention, the recombinant binding protein of the invention further comprises a binding factor.

[0160] In one particular embodiment, the binding agent has binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell. In a further embodiment, the protein expressed on the surface of a T cell is a protein that is part of the T cell receptor complex. By way of example, in one particular embodiment, the recombinant binding protein of the invention further comprises a binding agent that has binding specificity for cluster of differentiation 3 (CD3).

[0161] In the context of the present invention, a binding agent can be an antibody, an antibody mimetic comprising a scaffold protein or a repeat protein, an engineered repeat domain, preferably an engineered ankyrin repeat domain, or any other suitable binding molecule known in the art. In one embodiment, the binding agent is an antibody. In another embodiment, the binding agent is an engineered repeat domain, preferably an engineered ankyrin repeat domain.

[0162] In one particular embodiment, the binding agent comprises or consists of an antibody having binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell. In a further embodiment, the protein expressed on the surface of a T cell is a protein that is part of the T cell receptor complex. By way of example, in one particular embodiment, the recombinant binding protein of the invention further comprises an antibody having binding specificity for CD3.

[0163] In one particular embodiment, the binding agent comprises or consists of an engineered repeat domain, preferably an engineered ankyrin repeat domain, with binding specificity for a protein expressed on the surface of immune cells, preferably T cells, even more preferably CD8+ cytotoxic T cells. In a further embodiment, the protein expressed on the surface of T cells is a protein that is part of the T cell receptor complex. By way of example, in one particular embodiment, the recombinant binding protein of the invention further comprises an engineered ankyrin repeat domain with binding specificity for CD3.

[0164] In one embodiment, the binding agent is linked, conjugated, fused or otherwise physically associated with the pMHC-specific ankyrin repeat domain or the two, three or more pMHC-specific ankyrin repeat domains. In one embodiment, the binding agent is covalently linked to the pMHC-specific ankyrin repeat domain or the two, three or more pMHC-specific ankyrin repeat domains. In one embodiment, the binding agent is covalently linked to the pMHC-specific ankyrin repeat domain or the two, three or more pMHC-specific ankyrin repeat domains using a peptide linker. In one embodiment, the peptide linker is a proline-threonine-rich peptide linker. In one embodiment, the peptide linker is a proline-threonine-rich peptide linker of SEQ ID NO: 1 or 2. In one embodiment, the binding agent is covalently linked to the pMHC-specific ankyrin repeat domain or to the two, three or more pMHC-specific ankyrin repeat domains using a proline-threonine-rich peptide linker of SEQ ID NO: 1 or 2. In another embodiment, the peptide linker is a glycine-serine-rich peptide linker. In one embodiment, the peptide linker is a glycine-serine-rich peptide linker of SEQ ID NO: 3. In one embodiment, the binding agent is covalently linked to the pMHC-specific ankyrin repeat domain or to the two, three or more pMHC-specific ankyrin repeat domains using a glycine-serine-rich peptide linker of SEQ ID NO: 3.

[0165] The present inventors have surprisingly and unexpectedly discovered that the shorter the linker between the binding agent and the pMHC-specific ankyrin repeat domain (or the two, three or more pMHC-specific ankyrin repeat domains), the higher the potency of the construct in a T cell engager format (see Example 11). Thus, in a further preferred embodiment, the length of the amino acid sequence of the peptide linker described herein is less than 38 amino acids, preferably at most 37, more preferably at most 24, even more preferably at most 18, even more preferably at most 11 and most preferably at most 6 amino acids. In another preferred embodiment, the length of the amino acid sequence of the peptide linker is 1-37, 1-24, 1-23, 1-18, 1-17, 1-11, 1-10, 1-6, 6-37, 6-24, 6-23, 6-18, 6-17 or 6-11 amino acid residues. Preferably, the peptide linker is a proline-threonine-rich peptide linker. In a more preferred embodiment, the length of the amino acid sequence of the peptide linker is less than 18 amino acids and at least 1, i.e., the length of the peptide linker is 1 to 17 amino acids. In one embodiment, the amino acid sequence of the linker is as provided in any one of SEQ ID NOs: 1 and 277-280. In a preferred embodiment, the amino acid sequence of the peptide linker is as provided in SEQ ID NO: 278. In a more preferred embodiment, the amino acid sequence of the peptide linker is as provided in SEQ ID NO: 279. In a most preferred embodiment, the amino acid sequence of the peptide linker is as provided in SEQ ID NO: 280.

[0166] In one aspect of the invention, the recombinant binding protein of the invention further comprises a CD3-specific binding agent comprising one, two, three or more pMHC-specific ankyrin repeat domains as more particularly described in any of the aspects and embodiments herein, wherein the linker between the binding agent and the pMHC-specific ankyrin repeat domain (or the two, three or more pMHC-specific ankyrin repeat domains) is a peptide linker as described in any of the previous embodiments. Preferably, the peptide linker is a proline-threonine-rich peptide linker 1 to 17 amino acids in length (such as a peptide linker having an amino acid sequence as provided in SEQ ID NO: 279 or SEQ ID NO: 280).

[0167] In one aspect of the invention, a recombinant binding protein of the invention, which comprises one, two, three or more pMHC-specific ankyrin repeat domains as more particularly described in any of the aspects and embodiments herein, and which further comprises a CD3-specific binding factor, is capable of promoting infection-localized or tumor-localized activation of T cells. In such cases, the recombinant binding protein of the invention may be used in a T cell engager format to locally activate T cells against tumor cells and / or infected cells presenting target peptide-MHC complexes.

[0168] In one aspect of the invention, the recombinant binding protein of the invention is capable of inhibiting recognition of the first target peptide-MHC complex and / or, if the binding protein comprises the second repeat domain, inhibiting recognition of the second target peptide-MHC complex by a T cell receptor. In other words, the recombinant binding protein of the invention is capable of inhibiting the immune system's ability to recognize a target peptide-MHC complex. In such cases, the recombinant binding protein of the invention may be used to treat autoimmune diseases. Measuring recognition of a target peptide-MHC complex by a T cell receptor can be carried out by any suitable method known to those skilled in the art or by a suitable T cell activation assay, such as the assays described in the Examples or variations thereof of the present application.

[0169] In one embodiment, the recombinant binding protein of the present invention further comprises a polypeptide tag. In the context of the present invention, a polypeptide tag is an amino acid sequence attached to a polypeptide / protein that is useful for purifying, detecting, or targeting the polypeptide / protein, or that improves the physicochemical behavior of the polypeptide / protein, or that has an effector function. Individual polypeptide tags of a binding protein can be linked to other parts of the binding protein directly or via a peptide linker. Polypeptide tags are well known in the art and are readily available to those skilled in the art. Examples of polypeptide tags are small polypeptide sequences, such as His-tags, HA-tags, myc-tags, FLAG-tags, or Strep-tags, or polypeptides such as enzymes (e.g., alkaline phosphatase), that allow detection of the polypeptide / protein, or polypeptides that can be used for targeting (e.g., immunoglobulins or fragments thereof) and / or as effector molecules.

[0170] In one embodiment, the recombinant binding protein of the present invention further comprises a peptide linker. In the context of the present invention, a peptide linker is an amino acid sequence capable of linking, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-protein compound or a polymer such as polyethylene glycol, a protein domain and a biologically active molecule, a protein domain and a T cell-specific ankyrin repeat domain, or two sequence tags. Peptide linkers are known to those skilled in the art. A list of examples is provided in patent application WO 2002 / 020565. Specific examples of such linkers are glycine-serine linkers and variable-length proline-threonine linkers. Examples of glycine-serine linkers are the amino acid sequence GS and the amino acid sequence of SEQ ID NO:3, and examples of proline-threonine linkers are the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:277-280.

[0171] In another aspect, the present invention relates to a nucleic acid encoding the amino acid sequence of a designed repeat domain of the present invention or the amino acid sequence of a pMHC-specific recombinant binding protein of the present invention. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of a pMHC-specific recombinant binding protein of the present invention. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of a designed repeat domain of the present invention. In one embodiment, the present invention relates to a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 33. In one embodiment, the present invention relates to a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 25, 32, and 33. In one embodiment, the present invention relates to a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, and 33. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 20. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 21. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 27. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 32. In one embodiment, the present invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 33. In one embodiment, the present invention relates to a nucleic acid sequence of SEQ ID NO: 78, which encodes the amino acid sequence of SEQ ID NO: 20. In one embodiment, the present invention relates to the nucleic acid sequence of SEQ ID NO: 79, which encodes the amino acid sequence of SEQ ID NO: 21. In one embodiment, the present invention relates to the nucleic acid sequence of SEQ ID NO: 80, which encodes the amino acid sequence of SEQ ID NO: 32. Furthermore, the present invention relates to a vector comprising any of the nucleic acids of the present invention. Nucleic acids are well known to those skilled in the art. In the examples, the nucleic acids were used to produce the designed ankyrin repeat domains or recombinant binding proteins of the present invention in E. coli.

[0172] In another aspect, the present invention relates to a pharmaceutical composition comprising a pMHC-specific recombinant binding protein of the invention and / or a nucleic acid of the invention, optionally together with a pharmaceutically acceptable carrier and / or diluent.

[0173] Pharmaceutically acceptable carriers and / or diluents are known to those skilled in the art and are described in more detail below. Still further, diagnostic compositions are provided that comprise one or more of the above-described recombinant binding proteins and / or nucleic acids, particularly the recombinant binding proteins of the invention.

[0174] The pharmaceutical composition may comprise a recombinant binding protein and / or nucleic acid, preferably a recombinant binding protein and / or nucleic acid as more particularly described in any of the aspects or embodiments herein, and a recombinant binding protein and / or nucleic acid, e.g., as described in Remington's Pharmaceutical Sciences 16 th and a pharmaceutically acceptable carrier, excipient, or stabilizer, as described in Osol, A. Ed., 1980.

[0175] Suitable carriers, excipients, or stabilizers known to those skilled in the art include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Pharmaceutical compositions may also be combination preparations containing additional active ingredients, such as anti-cancer or anti-angiogenic agents, or additional biologically active compounds.

[0176] Formulations to be used for in vivo administration must be sterile, or must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0177] In one embodiment, a pharmaceutical composition comprises at least one recombinant binding protein as described herein, a detergent, e.g., a non-ionic detergent, a buffer, e.g., a phosphate buffer, and a sugar, e.g., sucrose. In one embodiment, such a composition comprises a recombinant binding protein as described above and PBS.

[0178] In another aspect, the invention provides a method for tumor-localized activation of immune cells, preferably T cells, in a mammal, preferably a human, comprising the step of administering to the mammal a pMHC-specific recombinant binding protein of the invention, comprising a first repeat domain having binding specificity for a first target peptide-MHC complex, optionally comprising a second repeat domain having binding specificity for a second target peptide-MHC complex, and further comprising a binding factor having binding specificity for a protein expressed on the surface of immune cells, preferably T cells, wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein expressed in a tumor cell. In one embodiment, the first and / or, if present, the second target peptide is derived from an intracellular protein expressed in a tumor cell. In one embodiment, the first and / or, if present, the second target peptide is derived from a tumor-specific protein. In one embodiment, the first and / or, if present, the second target peptide is derived from an intracellular tumor-specific protein. In one embodiment, the first and / or, if present, the second target peptide is derived from NY-ESO-1. In one embodiment, the first and / or, if present, the second target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34. In one embodiment, the first and / or, if present, the second target peptide is derived from MAGE-A3. In one embodiment, the first and / or, if present, the second target peptide has the amino acid sequence of SEQ ID NO: 155. In one embodiment, the binding agent has binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell. In one embodiment, the binding agent is an antibody having binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell. In one embodiment, the binding agent is an engineered repeat domain having binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell.In one embodiment, the binding agent is an engineered ankyrin repeat domain with binding specificity for a protein expressed on the surface of immune cells, preferably T cells, even more preferably CD8+ cytotoxic T cells. In a preferred embodiment, the protein expressed on the surface of immune cells is a protein that is part of the T cell receptor complex, such as CD3. In one embodiment, the first ankyrin repeat domain and / or, if present, the second ankyrin repeat, are independently designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex, as more particularly described in any of the aspects and embodiments herein.

[0179] Further provided is a pMHC-specific recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention for use in a method of tumour-localised activation of immune cells as described herein.

[0180] In another aspect, the invention provides a method for infectious disease-limited activation of immune cells, preferably T cells, in a mammal, preferably a human, comprising the step of administering to the mammal a pMHC-specific recombinant binding protein of the invention, comprising a first repeat domain having binding specificity for a first target peptide-MHC complex, optionally comprising a second repeat domain having binding specificity for a second target peptide-MHC complex, and further comprising a binding factor having binding specificity for a protein expressed on the surface of immune cells, preferably T cells, wherein the first target peptide and / or, if the binding protein comprises the second repeat domain, the second target peptide is derived from a protein of an infectious agent. In one embodiment, the infection is a viral infection. In one embodiment, the first and / or, if present, the second target peptide is derived from a protein of a viral infectious agent. In one embodiment, the first and / or, if present, the second target peptide is derived from a virus-specific protein. In one embodiment, the first and / or, if present, the second target peptide is derived from EBNA-1. In one embodiment, the first and / or, if present, the second target peptide has the amino acid sequence of SEQ ID NO: 92. In one embodiment, the first and / or, if present, the second target peptide is derived from HBcAg. In one embodiment, the first and / or, if present, the second target peptide has the amino acid sequence of SEQ ID NO: 255. In one embodiment, the binding agent has binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell. In one embodiment, the binding agent is an antibody having binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell. In one embodiment, the binding agent is an engineered repeat domain having binding specificity for a protein expressed on the surface of an immune cell, preferably a T cell, even more preferably a CD8+ cytotoxic T cell.In one embodiment, the binding agent is an engineered ankyrin repeat domain with binding specificity for a protein expressed on the surface of immune cells, preferably T cells, even more preferably CD8+ cytotoxic T cells. In a preferred embodiment, the protein expressed on the surface of immune cells is a protein that is part of the T cell receptor complex, such as CD3. In one embodiment, the first ankyrin repeat domain and / or, if present, the second ankyrin repeat, are independently designed ankyrin repeat domains with binding specificity for a target peptide-MHC complex, as more particularly described in any of the aspects and embodiments herein.

[0181] Further provided is a pMHC-specific recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention for use in a method for infection-limited activation of immune cells as described herein.

[0182] In another aspect, the present invention provides a method for treating a medical condition, the method comprising administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a pMHC-specific recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention.

[0183] Further provided is a pMHC-specific recombinant binding protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of treating a medical condition as described herein.

[0184] In another aspect, the present invention provides a method of diagnosing a medical condition in a mammal, preferably a human, comprising: (i) contacting a cell or tissue sample obtained from said mammal with a recombinant binding protein of the invention; (ii) detecting specific binding of said binding protein to said cell or tissue sample. In one embodiment, said tissue is tumor tissue.

[0185] In the context of the present invention, the terms "medical condition," "disease," and "disorder" are used interchangeably and include, but are not limited to, cancer, infectious diseases, and autoimmune diseases. In a preferred embodiment, the medical condition is cancer, an infectious disease, preferably a viral infection, or an autoimmune disease. In a preferred embodiment, the medical condition is cancer. In one embodiment, such cancer is selected from the group consisting of epithelial malignancies (primary and metastatic), including, but not limited to, lung cancer, colon cancer, gastric cancer, bladder cancer, ovarian cancer, and breast cancer; hematologic malignancies, including, but not limited to, leukemia, lymphoma, and myeloma; sarcomas, including, but not limited to, osteosarcoma and soft tissue sarcoma; and melanoma. In a preferred embodiment, such cancer is selected from the group consisting of liposarcoma, neuroblastoma, synovial sarcoma, melanoma, and ovarian cancer. In another preferred embodiment, the cancer is selected from the group consisting of melanoma, lung cancer, liver cancer, gastric cancer, skin cancer, neuroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, and ovarian cancer. In one preferred embodiment, the medical condition is an infectious disease, preferably a viral infection. In one embodiment, the infectious disease is a viral infection caused by hepatitis B virus (HBV). In another embodiment, the infectious disease is a viral infection caused by Epstein-Barr virus (EBV). In one preferred embodiment, the medical condition is an autoimmune disease. In one embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes.

[0186] In one embodiment, the present invention relates to the use of a pharmaceutical composition, nucleic acid, or recombinant binding protein according to the present invention for the treatment of a disease. For this purpose, the pharmaceutical composition, nucleic acid, or recombinant binding protein according to the present invention is administered to a patient in need thereof in a therapeutically effective amount. Administration can include topical administration, oral administration, and parenteral administration. A typical route of administration is parenteral administration. For parenteral administration, the pharmaceutical composition of the present invention is formulated in a unit dose injection form such as a solution, suspension, or emulsion together with a pharmaceutically acceptable excipient as defined above. The dose and method of administration will vary depending on the individual and the particular disease being treated.

[0187] Furthermore, any of the pharmaceutical compositions or recombinant binding proteins described above are considered for the treatment of disorders.

[0188] In one embodiment, the recombinant binding protein or pharmaceutical composition as described herein is administered intravenously. For parenteral administration, the recombinant binding protein or pharmaceutical composition can be injected in a therapeutically effective amount as a bolus injection or by slow infusion.

[0189] In one embodiment, the present invention relates to a method for treating a medical condition, comprising administering to a patient in need of such treatment a therapeutically effective amount of a recombinant binding protein, nucleic acid, or pharmaceutical composition of the present invention. In one embodiment, the present invention relates to the use of a recombinant binding protein, nucleic acid, or pharmaceutical composition of the present invention for the treatment of a medical condition. In one embodiment, the present invention relates to a recombinant binding protein, nucleic acid, or pharmaceutical composition of the present invention for use in the treatment of a medical condition. In one embodiment, the present invention relates to the use of a pharmaceutical composition, recombinant binding protein, or nucleic acid of the present invention as a medicament for the treatment of a medical condition. In one embodiment, the present invention relates to the use of a pharmaceutical composition, recombinant binding protein, or nucleic acid of the present invention for the manufacture of a medicament. In one embodiment, the present invention relates to the use of a pharmaceutical composition, recombinant binding protein, or nucleic acid of the present invention for the manufacture of a medicament for the treatment of a medical condition. In one embodiment, the present invention relates to a process for the manufacture of a medicament for the treatment of a medical condition, wherein the pharmaceutical composition, recombinant binding protein, or nucleic acid molecule of the present invention is the active ingredient of the medicament. In one embodiment, the present invention relates to a process for treating a medical condition using a pharmaceutical composition, recombinant binding protein, or nucleic acid molecule of the present invention.

[0190] In a further embodiment, the present invention relates to the use of a recombinant binding protein, nucleic acid or pharmaceutical composition of the invention for the manufacture of a medicament used for the treatment of a medical condition, preferably a neoplastic disease, more preferably cancer.

[0191] In one embodiment, the present invention relates to a recombinant binding protein comprising any of the above-mentioned designed ankyrin repeat domains for therapeutic purposes.

[0192] In one aspect, the invention relates to a kit comprising a recombinant binding protein of the invention. In one embodiment, the invention relates to a kit comprising a nucleic acid encoding a recombinant binding protein of the invention. In one embodiment, the invention relates to a kit comprising a pharmaceutical composition of the invention. In one embodiment, the invention relates to a kit comprising a recombinant binding protein of the invention, and / or a nucleic acid encoding a recombinant binding protein of the invention, and / or a pharmaceutical composition of the invention. In one embodiment, the invention relates to a kit comprising a recombinant binding protein comprising one or more peptide-MHC-specific ankyrin repeat domains of the invention, such as one or more peptide-MHC-specific ankyrin repeat domains independently having the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 32 or SEQ ID NO: 33, and / or a nucleic acid comprising a recombinant binding protein comprising one or more peptide-MHC-specific ankyrin repeat domains of the invention, such as one or more peptide-MHC-specific ankyrin repeat domains independently having the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 32 or SEQ ID NO: 33, and / or a pharmaceutical composition comprising a recombinant binding protein comprising one or more peptide-MHC-specific ankyrin repeat domains of the invention, such as one or more peptide-MHC-specific ankyrin repeat domains independently having the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 32 or SEQ ID NO: 33.

[0193] In another aspect, the invention provides a method of targeting tumor cells in a patient suffering from a tumor for their destruction, comprising administering to the patient a therapeutically effective amount of a binding protein, nucleic acid, or pharmaceutical composition of the invention, wherein the first targeting peptide and / or, if the binding protein comprises the second repeat domain, the second targeting peptide is derived from a protein expressed in tumor cells, preferably an intracellular protein expressed in tumor cells. In one embodiment, the binding protein further comprises a virulence factor capable of killing tumor cells.

[0194] Further provided is a recombinant binding protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of targeting tumor cells in a patient suffering from a tumor for their destruction, wherein the first targeting peptide and / or, if the binding protein comprises the second repeat domain, the second targeting peptide is derived from a protein expressed in tumor cells, preferably an intracellular protein expressed in tumor cells. In one embodiment, the binding protein further comprises a virulence factor capable of killing tumor cells.

[0195] In another aspect, the invention provides a method of targeting infected cells in a patient suffering from a viral infection for their destruction, comprising administering to the patient a therapeutically effective amount of a binding protein, nucleic acid, or pharmaceutical composition of the invention, wherein the first targeting peptide and / or, if the binding protein comprises the second repeat domain, the second targeting peptide is derived from a protein expressed in infected cells, preferably a virus-specific protein. In one embodiment, the binding protein further comprises a virulence factor capable of killing infected cells.

[0196] Further provided is a recombinant binding protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of targeting infected cells in a patient suffering from a viral infection for their destruction, wherein the first targeting peptide and / or, if the binding protein comprises the second repeat domain, the second targeting peptide is derived from a protein expressed in infected cells, preferably a virus-specific protein. In one embodiment, the binding protein further comprises a virulence factor capable of killing infected cells.

[0197] In a preferred embodiment, the medical condition treated by the method of treatment or by the binding protein, nucleic acid or pharmaceutical composition of the invention is a cancer or tumor selected from the group consisting of liposarcoma, neuroblastoma, synovial sarcoma, melanoma and ovarian cancer. In another preferred embodiment, the recombinant binding protein of the invention has binding specificity for a peptide-MHC (pMHC) complex, and the peptide is derived from NY-ESO-1.

[0198] The present invention is not limited to the specific embodiments described in the examples.

[0199] This specification references a number of amino acid sequences, nucleic acid sequences, and SEQ ID NOs, which are disclosed in the accompanying Sequence Listing, which is incorporated herein by reference in its entirety.

[0200] definition Unless otherwise defined herein, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0201] In the context of the present invention, the term "collection" refers to a group that includes at least two different entities or members. Preferably, such a collection includes at least 10 5 pcs, more preferably 10 7 More than 10, and most preferably 10 9 A "collection" may also be referred to as a "library" or a "plurality."

[0202] The term "nucleic acid molecule" refers to a polynucleotide molecule that can be either a single-stranded or double-stranded ribonucleic acid (RNA) molecule or a deoxyribonucleic acid (DNA) molecule, including modified and artificial forms of DNA or RNA. The nucleic acid molecule can be present in either isolated form or contained in a recombinant nucleic acid molecule or vector.

[0203] In the context of the present invention, the term "protein" refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or can assume a defined three-dimensional configuration by forming secondary, tertiary, and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. When a protein comprises two or more polypeptide chains, the individual polypeptide chains can be linked by non-covalent or covalent bonds, for example, by disulfide bonds between two polypeptides. Portions of a protein that have or can assume a defined three-dimensional configuration by individually forming secondary and / or tertiary structures are referred to as "protein domains." Such protein domains are well known to those skilled in the art.

[0204] The term "recombinant," when used in reference to recombinant protein, recombinant polypeptide, etc., means that the protein or polypeptide is produced by use of recombinant DNA techniques well known to those of skill in the art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or into DNA that allows for in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into appropriate bacteria (e.g., E. coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The polypeptide or protein produced accordingly is referred to as a recombinant polypeptide or recombinant protein.

[0205] In the context of the present invention, the term "binding protein" refers to a protein comprising a binding domain. A binding protein may also comprise two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. More preferably, the binding protein of the present invention comprises an ankyrin repeat domain that has binding specificity for a pMHC complex.

[0206] Additionally, any such binding protein may comprise additional polypeptides (e.g., polypeptide tags, peptide linkers, fusions to other proteinaceous domains with binding specificity, cytokines, hormones, or antagonists, etc.) or chemical modifications (e.g., coupling to polyethylene glycol, toxins, small molecules, antibiotics, etc.) known to those of skill in the art. In some embodiments, the binding protein further comprises a binding factor that has binding specificity for a protein expressed on the surface of an immune cell. In some embodiments, the binding protein further comprises a virulence factor that can kill tumor cells and / or infected cells. Examples of virulence factors include, but are not limited to, vinblastine, doxorubicin, topoisomerase I inhibitors, calicheamicin, duocarmycin-hydroxybenzamide-azaindole (DUBA), pyrrolobenzodiazepine dimers (PBD), and derivatives of the microtubule inhibitor family, such as auristatin and maytansine, e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), the drug maytansinoid 1 (DM1), and the drug maytansinoid 4 (DM4).

[0207] The term "binding domain" refers to a protein domain that exhibits binding specificity for a target. Preferably, the binding domain is a recombinant binding domain.

[0208] The term "target" refers to an individual molecule, such as a nucleic acid molecule, peptide, polypeptide, or protein, carbohydrate, or any other naturally occurring molecule containing any portion of such an individual molecule, or a complex consisting of two or more such molecules, or a whole cell or tissue sample, or any non-natural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein containing a chemical modification, e.g., modified by naturally occurring or non-natural phosphorylation, acetylation, or methylation. In the context of the present invention, peptide-MHC complexes and peptide-MHC-presenting cells and tissues are targets of pMHC-specific binding proteins. Additionally, CD3 and CD3-expressing cells, e.g., T cells, are targets of pMHC-specific binding proteins of the present invention, which further comprise a binding factor having binding specificity for a protein expressed on the surface of immune cells, such as CD3.

[0209] In the context of the present invention, the term "polypeptide" relates to a molecule consisting of a chain of multiple, i.e., two or more, amino acids linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked by cysteine ​​S-S bridges. Polypeptides are well known to those skilled in the art.

[0210] Patent application 2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Pluckthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of the characteristics of repeat proteins and repeat domains, their characteristics, techniques, and applications. The term "repeat protein" refers to a protein containing one or more repeat domains. Preferably, a repeat protein contains one, two, three, four, five, or six repeat domains. Furthermore, the repeat protein may contain further non-repeat protein domains, polypeptide tags, and / or peptide linkers. The repeat domain may be a binding domain.

[0211] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, which repeat modules have structural and sequence homology. Preferably, the repeat domain also comprises an N-terminal and / or a C-terminal capping module. For clarity, the capping module may be a repeat module. Such repeat domains, repeat modules and capping modules, sequence motifs and structural and sequence homologies are well known to those skilled in the art from examples such as ankyrin repeat domains (Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al., 2004, loc.cit.; WO 2002 / 020565; WO 2012 / 069655), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, E.R., Xiong, Y., Cocco, M.J., D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003) and armadillo repeat domains (WO 2009 / 040338). It is further known to those skilled in the art that such repeat domains are distinct from proteins containing repeated amino acid sequences, all of which can form individual domains (e.g., the FN3 domain of fibronectin).

[0212] The term "ankyrin repeat domain" refers to a repeat domain that contains two or more consecutive ankyrin repeat modules as a structural unit, wherein the ankyrin repeat modules have structural and sequence homology.

[0213] The term "designed" as used in designed repeat proteins, designed repeat domains, etc. refers to the property that such repeat proteins and repeat domains, respectively, are artificial and do not occur in nature. The binding proteins of the present invention are designed repeat proteins, which comprise at least one designed repeat domain. Preferably, the designed repeat domain is a designed ankyrin repeat domain.

[0214] The term "target interaction residues" refers to amino acid residues of a repeat module that contribute to direct interaction with the target.

[0215] The term "framework residue" or "framework position" refers to an amino acid residue of a repeat module that contributes to the folding topology, i.e., contributes to the folding of said repeat module or contributes to interactions with neighboring modules. Such contributions may be interactions with other residues within the repeat module, or influence on the polypeptide backbone structure found in α-helices or β-sheets, or participation in amino acid stretches that form linear polypeptides or loops.

[0216] Such framework and target interaction residues can be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known relevant structural information well known to those skilled in the art in structural biology and / or bioinformatics.

[0217] The term "repeat module" refers to the repeated amino acid sequence and structural unit of a designed repeat domain, originally derived from the repeat units of naturally occurring repeat proteins. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, preferably the ankyrin repeat protein family. Furthermore, each repeat module comprised in a repeat domain may comprise a "repeat sequence motif" obtained from a repeat domain selected on a target, e.g., as described in Example 1, and deduced from homologous repeat modules with the same target specificity.

[0218] Thus, the term "ankyrin repeat module" refers to a repeat module that is originally derived from the repeat units of a naturally occurring ankyrin repeat protein. Ankyrin repeat proteins are well known to those skilled in the art.

[0219] A repeat module may comprise positions with amino acid residues that are not randomized in the library for the purpose of selecting target-specific repeat domains ("non-randomized positions" or "fixed positions", used interchangeably herein) and positions with amino acid residues that are randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). Non-randomized positions include framework residues. Randomized positions include target-interaction residues. "Randomized" means, for example, that more than one amino acid is allowed at an amino acid position of a repeat module, and that any of the 20 common naturally occurring amino acids is allowed, or that amino acids other than cysteine, or most of the 20 naturally occurring amino acids are allowed, such as amino acids other than glycine, cysteine, and proline. For the purposes of this patent application, amino acid residues 3, 4, 6, 14, and 15 of SEQ ID NOs: 37-60, 62-66, 68-72, 111-122, 124-154 175-217, and 231-254, as well as amino acid residues 3, 4, 6, 13, and 14 of SEQ ID NOs: 61 and 67, and amino acid residues 3, 4, 6, 15, and 16 of SEQ ID NO: 123, are randomized positions in ankyrin repeat modules of the invention.

[0220] The term "repeat sequence motif" refers to an amino acid sequence deduced from one or more repeat modules. Preferably, the repeat modules are derived from repeat domains with binding specificity for the same target. Such a repeat sequence motif comprises framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of a repeat module. Similarly, the target interaction residue positions correspond to the target interaction residue positions of a repeat module. A repeat sequence motif comprises non-randomized and randomized positions.

[0221] The term "repeat unit" refers to an amino acid sequence that contains one or more naturally occurring protein sequence motifs, which are found in multiple copies and exhibit a defined folding topology common to all such motifs that determines protein folding. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0222] The terms "binding specificity," "having binding specificity for a target," "specifically binds to a target," "binds with high specificity to a target," "specific for a target," or "target specificity," etc., mean that a binding protein or binding domain binds to a target in PBS with a lower dissociation constant (i.e., binds with higher affinity) than it binds to an unrelated protein, such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant in PBS for the target ("K D ") is at least 10 times larger than the corresponding dissociation constant for MBP. 2 times, more preferably at least 10 3 times, more preferably at least 10 4 times, or more preferably at least 10 5 Methods for measuring the dissociation constant of a protein-protein interaction, such as surface plasmon resonance (SPR)-based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The K of a particular protein-protein interaction D Measurements of K may vary when measured under different conditions (e.g., salt concentration, pH). D The measurement of the dissociation constant (K) of the recombinant binding protein of the present invention having binding specificity for pMHC is preferably carried out using a standardized solution of the protein and a standardized buffer solution such as PBS. D An exemplary and preferred determination of ) by surface plasmon resonance (SPR) analysis is described in Example 2.

[0223] The term "about" means + / - 20% of the stated value, for example, "about 50" shall mean 40-60.

[0224] The term "PBS" refers to an aqueous phosphate buffer solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4.

[0225] The major histocompatibility complex (MHC) is a group of genes that encode proteins found on the surface of cells that help the immune system recognize foreign substances. Proteins encoded by the major histocompatibility complex or complexes of such proteins are called "MHC proteins" or "MHC molecules." MHC proteins are found in all higher vertebrates. Most notably, the MHC class I and class II glycoproteins present peptides to immune cell receptors, such as T cell receptors. In humans, the major histocompatibility complex is also called the human leukocyte antigen (HLA) system.

[0226] As used herein, the term "MHC" refers to the major histocompatibility complex, preferably a mammalian major histocompatibility complex, and even more preferably a human major histocompatibility complex. As used herein, the term "HLA" refers to the human leukocyte antigen system. MHC molecules present peptides to the immune system of vertebrates. The terms "peptide antigen" and "MHC peptide antigen" are used interchangeably herein and refer to MHC ligands that can bind in the peptide-binding groove of MHC molecules. Peptide antigens typically have 8 to 25 amino acids linked via peptide bonds. MHC peptide antigens can be either self-peptides or non-self-peptides. Peptide antigens can typically be presented to the immune system by MHC molecules. MHC class I molecules typically present peptide antigens to CD8-positive T cells, whereas MHC class II molecules typically present peptide antigens to CD4-positive T cells. In the context of the present invention, peptide antigens that are specifically bound by the binding proteins of the present invention when the peptide antigens bind to MHC molecules are also referred to as "target peptides." Thus, the terms "peptide-MHC complex," "pMHC complex," "peptide-MHC," and "pMHC" are used interchangeably in this application and refer to a complex formed by binding of a peptide antigen to an MHC molecule. Preferably, the MHC molecule is an MHC class I molecule. Also preferably, the peptide is a peptide derived from a protein expressed in tumor cells, a protein of an infectious agent, e.g., a viral infectious agent, or a protein associated with an autoimmune disorder. In one preferred embodiment, the protein expressed in tumor cells is a tumor-specific protein.

[0227] The term "tumor-specific protein" refers to a protein that is expressed in tumor cells and not expressed or expressed at a lower level in many or all non-tumorigenic tissues, or expressed only in a limited number of non-tumorigenic tissues in addition to tumor tissues. Preferably, the tumor-specific protein has the ability to induce a cancer-specific immune response. Examples of tumor-specific proteins are known to those skilled in the art and include, but are not limited to, MAGE-A1, MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, CT83, SSX2, etc.

[0228] The term "virus-specific protein" refers to a protein that is expressed from the viral genome in virus-infected cells and is not expressed in uninfected host cells. Examples of virus-specific proteins are known to those skilled in the art and include, but are not limited to, EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2, NSP1, NSP2, NSP4, NSP5, NSP6, E1, E2, HBx, HBsAg, HBcAg, etc.

[0229] Examples of proteins associated with autoimmune disorders are known to those of skill in the art and include, but are not limited to, carboxypeptidase H, chromogranin A, insulin, beta-arrestin, aquaporin-4, citrullinated proteins, and the like.

[0230] "Target peptide-MHC complex" refers to a peptide-MHC complex in which the peptide antigen is a target peptide.

[0231] More preferably, the peptide antigen is derived from an intracellular protein, even more preferably an intracellular protein expressed in tumor cells or a tumor-specific protein.

[0232] Most preferably, the peptide antigen is derived from NY-ESO-1. NY-ESO-1, or New York esophageal squamous cell carcinoma 1, is a well-known cancer-testis antigen (CTA) that is re-expressed in numerous cancer types (WO 98 / 14464; Chen YT et al., Proc Natl Acad Sci USA 1997, 94:1914-18; Scanlan et al., 2004, Cancer Immunity 4, 1). Its ability to induce spontaneous humoral and cellular immune responses, along with its restricted expression pattern, has made it a good candidate target for cancer immunotherapy (Thomas R et al., Front Immunol. 2018;9:947). In particular, NY-ESO-1 is expressed at very high frequencies in, for example, liposarcoma, neuroblastoma, synovial sarcoma, melanoma, and ovarian cancer. The protein and polynucleotide sequences for NY-ESO-1 are provided in Genbank Accession Number U87459, Edition U87459.1. Preferably, the peptide antigen derived from NY-ESO-1 has the amino acid sequence SLLMWITQV (SEQ ID NO: 19) or SLLMWITQC (SEQ ID NO: 34).

[0233] Alternatively, the peptide antigen can be derived from MAGE-A3. MAGE-A3, or melanoma-associated antigen 3, is a protein encoded by the MAGEA3 gene in humans. The cancer / testis antigen MAGE-A3 is a member of the melanoma antigen gene (MAGE) family, whose expression is restricted to the testis and aberrantly expressed in cancer cells. MAGE-A3 has been shown to be widely expressed in various malignancies, including melanoma, breast cancer, head and neck cancer, lung cancer, gastric cancer, cutaneous squamous cell carcinoma, and colorectal cancer. The relatively restricted expression of MAGE-A3 and its immunogenicity make it an ideal target for immunotherapy (Int J Med Sci. 2018;15(14):1702-1712). The protein and polynucleotide sequences for MAGE-A3 are provided in GenPept Accession No. NP_005353, rev. NP_005353.1. Preferably, the peptide antigen derived from MAGE-A3 has the amino acid sequence EVDPIGHLY (SEQ ID NO: 155).

[0234] The peptide antigen can also be derived from a protein of an infectious agent, for example, a protein of a viral infectious agent, preferably a virus-specific protein. The viral infectious agent can be Epstein-Barr virus (EBV), and the EBV protein is preferably a virus-specific protein. The viral infectious agent can be hepatitis B virus (HBV), and the HBV protein is preferably a virus-specific protein.

[0235] The peptide antigen can be derived from EBNA-1. EBNA-1, or Epstein-Barr nuclear antigen 1, was the first Epstein-Barr virus (EBV) protein detected and is the most widely studied. EBNA1 is expressed in both latent and lytic modes of EBV infection, but has been primarily studied during latency, where it plays multiple important roles. The importance of EBNA1 in EBV latency is reflected by the fact that EBNA1 is the only viral protein expressed in proliferating cells in all latency states and in all EBV-associated tumors (Scientifica (Cairo). 2012; 2012:438204). Preferably, the peptide antigen derived from EBNA-1 has the amino acid sequence FMVFLQTHI (SEQ ID NO: 92).

[0236] Alternatively, the peptide antigen can be derived from the Hepatitis B virus core antigen (HBcAg). Preferably, the peptide antigen derived from HBcAg is sequences 18-27 of the HBV core antigen, abbreviated herein as "HBVc18" or "c18 peptide," and has the amino acid sequence FLPSDFFPSV (SEQ ID NO: 255).

[0237] The term "peptide derived from," as used in "peptides derived from tumor-specific proteins," "peptides derived from intracellular proteins," "peptides derived from proteins of infectious agents," "peptides derived from proteins associated with autoimmune disorders," and the like, refers to molecular fractions or peptide fragments of individual proteins. Such peptide fragments can result from the degradation of normal or pathogenic proteins to be presented, i.e., displayed, in the context of MHC molecules on the surface of cells for recognition by certain lymphocytes, such as T cells. The presented peptides can be either self or non-self, which an organism's immune system can normally distinguish between, thus preventing the organism's immune system from targeting its own cells. Alternatively, such peptide fragments can be produced by methods known in the art. Preferably, the peptides are derived from intracellular proteins.

[0238] The term "binding agent" refers to any molecule capable of specifically binding a target molecule, including, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., J Biol Chem 266:5182-5190 (1991)), antibody mimetics, repeat proteins, e.g., designed aniculin repeat proteins, receptor proteins, and any other naturally occurring interaction partners of the target molecule, including naturally occurring proteins and proteins that have been modified or engineered, e.g., to include non-naturally occurring residues and / or to lack natural residues.

[0239] The term "negative selection" or "deselection" of binding domains refers to the selective removal of binding domains that are not suitable or less suitable for the purposes of the present invention from a collection of binding domains. In the context of the present invention, negative selection or deselection preferably refers to the selective removal from a collection of designed repeat domains of undesired designed repeat domains that have binding specificity for peptide-MHC complexes other than the target peptide-MHC complex or that have binding specificity for a common HLA-A scaffold of a different pMHC complex. Methods for negatively selecting or deselecting undesired members of a collection, such as undesired binding domains of a collection of binding domains, are known to those skilled in the art, such as the method used in Example 1 of the present application.

[0240] As used herein, the term "peptide-MHC-presenting cell" refers to any cell capable of expressing or displaying peptide-MHC on the cell surface, including, but not limited to, tumor cells, infected cells, cells associated with autoimmune disorders, conventional antigen-presenting cells such as dendritic cells, macrophages, and B cells, and cells engineered to display MHC molecules bound to exogenously administered peptides, such as peptide-pulsed T2 cells.

[0241] As used herein, the term "CD3-expressing cell" refers to any cell that expresses CD3 (cluster of differentiation 3) on the cell surface, including, but not limited to, T cells such as cytotoxic T cells (CD8+ T cells) and T helper cells (CD4+ T cells).

[0242] The term "tumor-localized activation of T cells" means that T cells are preferentially activated in tumor tissue compared to non-tumor tissue.

[0243] The term "infection-limited activation of T cells" means that T cells are preferentially activated in infected tissue compared to non-infected tissue.

[0244] The term "medical condition" (or "disorder" or "disease") includes, but is not limited to, autoimmune disorders, inflammatory disorders, retinopathies (especially proliferative retinopathy), neurodegenerative disorders, infectious diseases, metabolic diseases, and neoplastic diseases. Any of the recombinant binding proteins described herein can be used in the preparation of a medicament for the treatment of such disorders, particularly disorders selected from the group including autoimmune disorders, inflammatory disorders, infectious diseases (e.g., viral or bacterial infections), and neoplastic diseases. A "medical condition" may be one characterized by inappropriate cell proliferation. A medical condition may be a hyperproliferative condition. The present invention particularly relates to methods of treating a medical condition, comprising administering to a patient in need of such treatment a therapeutically effective amount of a recombinant binding protein or pharmaceutical composition of the present invention. In a preferred embodiment, the medical condition is a neoplastic disease. The term "neoplastic disease," as used herein, refers to an abnormal state or condition of cells or tissues characterized by rapidly proliferating cell proliferation or tumors. In one embodiment, the medical condition is a malignant neoplastic disease. In one embodiment, the medical condition is cancer. In another preferred embodiment, the medical condition is an infectious disease. In another preferred embodiment, the medical condition is an autoimmune disease. The term "therapeutically effective amount" means an amount sufficient to produce a desired effect in a patient.

[0245] The term "antibody" refers not only to intact antibody molecules, but also to any fragments and variants of antibody molecules that retain immunogen-binding ability. Such fragments and variants are also well known in the art and are routinely used both in vitro and in vivo. Thus, the term "antibody" encompasses intact immunoglobulin molecules, antibody fragments such as Fab, Fab', F(ab')2, and single-chain V-region fragments (scFv), bispecific antibodies, chimeric antibodies, humanized antibodies, antibody fusion polypeptides, and non-conventional antibodies.

[0246] The terms "cancer" and "cancerous" are used herein to refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer encompasses solid and liquid tumors, as well as primary and metastatic tumors. A "tumor" contains one or more cancerous cells. Solid tumors typically also contain tumor stroma. Examples of cancer include, but are not limited to, primary and metastatic cancers, lymphoma, blastoma, sarcoma, myeloma, melanoma, and leukemia, as well as any other epithelial and hematologic malignancies. More specific examples of such cancers include brain cancer, bladder cancer, breast cancer, ovarian cancer, renal cancer, colon cancer, gastric cancer, head and neck cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, osteosarcoma, soft tissue sarcoma, carcinoma, squamous cell carcinoma, clear cell renal carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, small cell lung cancer (SCLC), triple negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CL), and malignant melanoma. L), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), squamous cell carcinoma of the head and neck (SCCHN), chronic myeloid leukemia (CML), small lymphocytic lymphoma (SLL), malignant mesothelioma, liposarcoma, neuroblastoma, or synovial sarcoma.

[0247] The term "therapeutically effective amount" refers to an amount sufficient to induce a desired biological, pharmacological, or therapeutic result in a subject. A therapeutically effective amount, in the context of this invention, means a sufficient amount of the binding protein to treat or prevent a disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment.

[0248] The terms "treatment" or "treating" refer to both therapeutic and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0249] The term "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sport, or pet animals such as dogs, horses, cats, cows, etc. The terms "autoimmune disease" and "autoimmune disorder" are used herein to refer to or describe disorders in which a mammal's immune system mounts a humoral or cellular immune response against the mammal's own tissues or against antigens that are not inherently harmful to the mammal, thereby resulting in tissue damage in such mammal. Examples of autoimmune disorders are many and include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, and type I diabetes. Autoimmune diseases also include acute glomerulonephritis, Addison's disease, adult-onset idiopathic parathyroid deficiency (AOIH), alopecia totalis, amyotrophic lateral sclerosis, ankylosing spondylitis, autoimmune aplastic anemia, autoimmune hemolytic anemia, Behçet's disease, celiac disease, chronic active hepatitis, CREST syndrome, Crohn's disease, dermatomyositis, dilated cardiomyopathy, eosinophilia-myalgia syndrome, epidermolysis bullosa acquisita (EBA), giant cell arteritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, hemochromatosis, and Henoch-Schonlein syndrome. These include purpura, idiopathic IgA nephropathy, insulin-dependent diabetes mellitus (IDDM), juvenile rheumatoid arthritis, Lambert-Eaton syndrome, linear IgA dermatosis, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocarditis, narcolepsy, necrotizing vasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome, pemphigoid, pemphigus, polymyositis, primary sclerosing cholangitis, psoriasis, rapidly progressive glomerulonephritis (RPGN), Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, stabilizing systemic syndrome, thyroiditis, and ulcerative colitis.

[0250] The terms "infectious disease" and "infection" are used herein to refer to or describe the invasion and proliferation of microorganisms in body tissues, particularly causing pathological symptoms. Examples of infectious diseases include, but are not limited to, viral and bacterial diseases such as HIV infection, West Nile virus infection, hepatitis A, B, and C, smallpox, tuberculosis, vesicular stomatitis virus (VSV) infection, respiratory syncytial virus (RSV) infection, human papillomavirus (HPV) infection, SARS, influenza, Ebola virus disease, viral meningitis, herpes, anthrax, Lyme disease, and E. coli infection, among others. [Example]

[0251] The starting materials and reagents disclosed below are known to those of ordinary skill in the art, are commercially available, and / or can be prepared using well-known techniques.

[0252] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). Unless otherwise stated, DNA polymerases, restriction enzymes, and buffers were purchased from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA). Inducible E. coli expression strains were used for cloning and protein production, such as E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA).

[0253] molecular biology Unless otherwise stated, methods are performed according to known protocols (see, for example, Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

[0254] Designed ankyrin repeat protein library Methods for generating designed ankyrin repeat protein libraries are described, for example, in U.S. Pat. No. 7,417,130; Binz et al. 2003, loc.cit.; Binz et al. 2004, loc.cit. Such methods allow the construction of designed ankyrin repeat protein libraries with randomized ankyrin repeat modules and / or randomized capping modules. For example, such libraries could be assembled based on a fixed N-terminal capping module (e.g., an N-terminal capping module of SEQ ID NO: 5, 6, or 7), or a randomized N-terminal capping module according to SEQ ID NO: 8, one or more randomized repeat modules according to the sequence motif of SEQ ID NO: 9, 10, or 11, and a fixed C-terminal capping module (e.g., a C-terminal capping module of SEQ ID NO: 12, 13, or 14), or a randomized C-terminal capping module according to SEQ ID NO: 15. Preferably, such libraries are assembled so as to have none of the amino acids C, G, M, N (before a G residue), and P at the randomized positions of the repeat or capping module. In addition, randomized repeat modules with sequence motifs of SEQ ID NO: 9, 10, or 11 could be further randomized at position 10 and / or position 17; randomized N-terminal capping modules with sequence motifs of SEQ ID NO: 8 could be further randomized at position 7 and / or position 9; and randomized C-terminal capping modules with sequence motifs of SEQ ID NO: 15 could be further randomized at positions 10, 11, and / or 17.

[0255] Such randomized modules in such libraries may also contain additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are antibody complement-determining region (CDR) loop libraries or newly generated peptide libraries. For example, such loop insertions can be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., Nakanishi, M., Kusakabe, Y., Goto, Y., Kitade, Y., Nakamura, K.T., EMBO J. 23(30), 3929-3938, 2004) as a guide. Similar to this ankyrin repeat domain, in which 10 amino acids are inserted into a beta turn located near the boundary between two ankyrin repeats, ankyrin repeat protein libraries can contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted into one or more beta turns of the ankyrin repeat domain.

[0256] Any such N-terminal capping module of an ankyrin repeat protein library preferably has a RILLAA, RILLKA, or RELLKA motif (e.g., present at positions 21 to 26 of SEQ ID NO: 20), and any such C-terminal capping module of an ankyrin repeat protein library preferably has a KLN, KLA, or KAA motif (e.g., present at the last three amino acids of SEQ ID NO: 20).

[0257] The design of such ankyrin repeat protein libraries can be guided by known structures of ankyrin repeat domains that interact with targets. Examples of such structures, identified by unique Protein Data Bank (PDB) accession or identification codes (PDB-IDs), are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70, and 2ZGD.

[0258] Examples of designed ankyrin repeat protein libraries have been described, such as N2C and N3C designed ankyrin repeat protein libraries (U.S. Patent No. 7,417,130, Binz et al. 2003, loc.cit.; Binz et al. 2004, loc.cit.). The N2C and N3C numbers describe the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

[0259] The nomenclature used to define the internal positions of repeat units and modules is based on Binz et al. 2004, loc.cit., with modifications in which the boundaries of ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of an ankyrin repeat module in Binz et al. 2004 (loc.cit.) corresponds to position 2 of an ankyrin repeat module of the present disclosure; consequently, position 33 of an ankyrin repeat module in Binz et al. 2004, loc.cit. corresponds to position 1 of the following ankyrin repeat module of the present disclosure.

[0260] All DNA sequences were confirmed by Sanger sequencing.

[0261] Example 1: Selection of binding proteins containing ankyrin repeat domains with binding specificity for the NY-ESO-1 peptide-MHC complex (NYESOpMHC) overview Using ribosome display (Hanes, J. and Pluckthun, A., PNAS 94, 4937-42, 1997), multiple ankyrin repeat domains with binding specificity for the NY-ESO-1 peptide-MHC complex (NYESOpMHC) were selected from a DARPin® library using a method similar to that described by Binz et al. 2004 (loc.cit.), with specific conditions and additional deselection steps as described below. The binding and specificity of selected clones to recombinant NYESOpMHC and NYESOAApMHC targets were assessed by homogeneous time-resolved fluorescence (HTRF) of crude extracts of E. coli, demonstrating that multiple NYESOpMHC-specific binding proteins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOs: 20-33 constitute the amino acid sequences of selected binding proteins containing ankyrin repeat domains with binding specificity for NYESOpMHC. Individual ankyrin repeat modules from such ankyrin repeat domains with binding specificity for NYESOpMHC are provided, for example, in SEQ ID NOs: 37 to 72. Examples of nucleic acids encoding such ankyrin repeat domains with binding specificity for NYESOpMHC are provided in SEQ ID NOs: 78 to 80.

[0262] Production of biotinylated pMHC complexes as targets and selection materials. HLA-A *The ternary complex of O201 (SEQ ID NO: 73), human beta2-microglobulin (hβ2m, SEQ ID NO: 74), and one of the peptides NY-ESO-1-9V (amino acids 157-165, SLLMWITQV, SEQ ID NO: 19), NY-ESO-1-9VAA (amino acids 157-165, SLLAAITQV, SEQ ID NO: 35), and EBNA-1 (amino acids 562-570, FMVFLQTHI, SEQ ID NO: 36) was produced by established protocols (Garboczi et al., 1992; Celie et al., 2009). The codon-optimized HLA-A complex containing a linker (GSGGSGGSAGG, SEQ ID NO: 75) and an Avi-tag (GLNDIFEAQKIEWHE, SEQ ID NO: 76; Fairhead & Howarth, 2015) for biotinylation was used. * 0201(HLA-A * HLA-A (0201avi, SEQ ID NO: 77) and wild-type human beta2-microglobulin (hβ2m) were expressed in Escherichia coli BL21(DE3) at 37°C as inclusion bodies (IBs), and after IB purification, were dissolved in 50 mM MES, 5 mM EDTA, 5 mM DTT, 8 M urea, pH 6.5. * O201avi and hβ2m molecules were refolded in the presence of each peptide at final concentrations of 25, 30, and 15 mg per 500 mL volume in 50 mM Tris pH 8.3, 230 mM L-arginine, 3 mM EDTA, 255 μM GSSG, 2.5 mM GSH, and 250 μM PMSF. The resulting three pMHC complexes were: (1) NYESOpMHC containing the NY-ESO-1-9V(157-165) peptide, (2) NYESOAApMHC containing the NY-ESO-1-9VAA(157-165) peptide, and (3) EBNApMHC containing the EBNA-1(562-570) peptide (see Table 1).

[0263] For biotinylation, the sample was concentrated to a volume of 7.5 mL and buffer exchanged into 100 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl, pH 7.5 using a PD10 column. *O201avi was biotinylated by adding 5 mM ATP, 400 μM biotin, 200 μM PMSF, and 20 μg of BirA enzyme. BirA was produced in-house according to the procedure described in Shen et al., 2009. The refolded ternary biotinylated complex was isolated using size-exclusion chromatography (Superdex 200 HiLoad 16 / 600) in PBS supplemented with 150 mM NaCl, 1 mM EDTA, and 10% glycerol. Samples were concentrated to approximately 1 mg / mL and flash-frozen in liquid nitrogen in 25 and 50 μL aliquots.

[0264] [Table 1]

[0265] For quality control purposes, 25 μg of biotinylated pMHC complexes (biotin-pMHC) were incubated with 50 μg of streptavidin (IBA Lifesciences) with or without the addition of 100 mM DTT and incubation at 95°C for 5 min. A 50 μg sample was subjected to analytical size-exclusion chromatography (GE Superdex 200 10 / 300 GL). The biotinylated NYESOpMHC, NYESOAApMHC, and EBNApMHC complexes all eluted from the Superdex 200 HiLoad 16 / 600 column with a peak maximum of approximately 82 mL (Figure 1A). The final amounts of NYESOpMHC, NYESOAApMHC, and EBNApMHC complexes obtained after size exclusion were approximately 3.5, 3.5, and 5 mg, respectively. HLA-A * Concentration and thawing of the flash-frozen refolded complex followed by SDS-PAGE analysis demonstrated efficient biotinylation, as O201avi bound almost completely to streptavidin (Figure 1B). Analytical size-exclusion chromatography revealed a single peak at a retention volume corresponding to an apparent molecular weight of 45 kDa, close to the theoretical molecular weight of the ternary pMHC complex of approximately 48 kDa (Figure 1C).

[0266] Selection of NYESOpMHC-specific ankyrin repeat proteins by ribosome display Selection of pMHC-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the NYESO pMHC complex as the target, a library of ankyrin repeat proteins as previously described, and established protocols (see, e.g., Zahnd, C., Amstutz, P., and Pluckthun, A., Nat. Methods 4, 69-79, 2007). The number of reverse transcription (RT)-PCR cycles after each selection round was consistently reduced, adjusting for yield by enrichment of binders. The first four selection rounds employed standard ribosome display selection, increasing the selection pressure from the first to the fourth round by decreasing the target concentration and increasing the washing stringency (Binz et al. 2004, loc. cit.), but incorporating an unusual deselection step.

[0267] During the ribosome display round, a deselection (or negative selection) step was incorporated, in which the ternary complex was preincubated with a corresponding isotype HLA molecule containing a different peptide and then transferred to the target NYESOpMHC complex to identify ankyrin repeat proteins binding to the peptide-embedded epitope, away from the common HLA-A scaffold. In other words, deselection (or negative selection) was performed to deselect ankyrin repeat proteins that primarily bind to the common HLA-A scaffold of the pMHC complex, rather than to the specific epitope provided by the embedded peptide. Furthermore, ankyrin repeat proteins that cross-react with the epitope provided by the embedded peptide used for deselection were also deselected. Here, an EBNApMHC complex containing the EBNA-1(562-570) peptide was used for deselection.

[0268] Specifically, for the deselection step, Nunc MaxiSorp plates were coated with 100 μl of 66 nM neutravidin in PBS and incubated overnight at 4°C. The following day, MaxiSorp 96-well microplates were washed three times with 300 μl of PBST per well, blocked with 300 μl of PBST-BSA for 1 h at 4°C, and rotated at 700 rpm before the deselection step. After emptying the wells, 100 μl of a 50 nM biotinylated pMHC deselection target solution in PBST-BSA was added to each well and rotated at 700 rpm for 1 h at 4°C. During this incubation step, in vitro translation of mRNA using the ribosome display protocol was performed separately. Immediately after in vitro translation and generation of the ternary complex (i.e., mRNA, ribosome, and translated ankyrin repeat protein), the pMHC-PBST-BSA solution was discarded, and Nunc MaxiSorp microplate wells were washed three times with 300 μl of PBST and finally incubated with Tris wash buffer containing BSA (WBT-BSA). Just before the actual deselection step, the WBT-BSA solution was discarded, and 150 μl (for the first selection round) or 100 μl (for selection rounds 2–4) aliquots of the in vitro translated ternary complex were transferred three times consecutively to the prepared Nunc MaxiSorp wells containing the immobilized deselected pMHC complexes and incubated for 20 min at 4°C in each of the three wells. At the end of the deselection process, all 100 μl ternary complex aliquots from each selection pool were combined, and the matching volumes were carried forward for selection on the actual target pMHC complex as described above.

[0269] Selected clones specifically bind to the NYESOpMHC complex as demonstrated by crude extract HTRF. Individually selected ankyrin repeat proteins that specifically bind NYESOpMHC complexes in solution were identified by homogeneous time-resolved fluorescence (HTRF) assay using crude extracts of ankyrin repeat protein-expressing Escherichia coli cells using standard protocols. Ankyrin repeat protein clones selected by ribosome display were cloned into a derivative of the pQE30 (Qiagen) expression vector, transformed into E. coli XL1-Blue (Stratagene), plated on LB-agar (containing 1% glucose and 50 μg / ml ampicillin), and then incubated overnight at 37°C. Single colonies (each clone in a single well) were plated into 96-well plates containing 165 μl of growth medium (LB containing 1% glucose and 50 μg / ml ampicillin) and incubated overnight at 37°C with shaking at 800 rpm. 8.5 μL of overnight culture was inoculated into 150 μL of fresh LB medium containing 50 μg / mL ampicillin in a new 96-deep-well plate. After 120 minutes of incubation at 37°C and 850 rpm, expression was induced with IPTG (final concentration 0.5 mM) and continued for 6 hours. Cells were harvested by centrifugation of the plate, the supernatant was discarded, and the pellet was frozen overnight at -20°C before being resuspended in 8.5 μL of B-PERII (Thermo Scientific) and incubated for 1 hour at room temperature with shaking (600 rpm). 160 μL of PBS was then added, and cell debris was removed by centrifugation (3220 g for 15 minutes).

[0270] The lysed extracts of each clone were diluted 1:200 (final concentration) in PBSTB (PBS supplemented with 0.1% Tween 20® and 0.2% (w / v) BSA, pH 7.4) with 20 nM (final concentration) biotinylated pMHC complex, 1:400 (final concentration) anti-6-His-D2 HTRF antibody-FRET acceptor complex (Cisbio), and 1:400 (final concentration) anti-strep-Tb antibody-FRET donor complex (Cisbio, France) into wells of a 384-well plate and incubated for 120 min at 4°C. HTRF was read on a Tecan M1000 using a 340 nm excitation wavelength and a 620 ± 10 nm emission filter for background fluorescence detection and a 665 ± 10 nm emission filter for detecting the fluorescent signal for specific binding.

[0271] The lysed extracts of each clone were tested for binding to each of three biotinylated pMHC complexes, NYESOpMHC, NYESOAApMHC, and EBNApMHC, to assess binding and specificity to the target NYESOpMHC complex. NYESOAApMHC and EBNApMHC function as pMHC complexes distinct from NYESOpMHC, allowing the selection of ankyrin repeat proteins with high binding specificity for NYESOpMHC.

[0272] To calculate the specificity of each ankyrin repeat protein for NYESOpMHC, the ratio of the HTRF signal for the target NYESOpMHC to the HTRF signal for a distinct NYESOAApMHC was determined. All binders that generated HTRF signals at least 25-fold higher for the target NYESOpMHC than for NYESOAApMHC were considered specific hits and were advanced for sequencing. Surprisingly, screening hundreds of clones by such crude cell extract HTRF analysis revealed many different ankyrin repeat domains with specificity for NYESOpMHC. The specific binding of ankyrin repeat proteins to a composite epitope containing an HLA scaffold and a short peptide, which is distinct from other composite epitopes to which the peptide does not specifically bind, has never been demonstrated before, making it challenging to develop binders specific for such composite epitopes using antibody or TCR technology.

[0273] A total of 95 hits were sequenced using the sequencing service at Microsynth (Balgach, Switzerland). Examples of amino acid sequences of selected ankyrin repeat domains that specifically bind to NYESOpMHC are provided in SEQ ID NOs: 20-33.

[0274] These ankyrin repeat domains with binding specificity for NYESOpMHC were cloned into pQE (QIAgen, Germany)-based expression vectors that provide an N-terminal His tag (SEQ ID NO: 4) to facilitate simple protein purification as described below. For example, expression vectors encoding the following ankyrin repeat proteins were constructed: DARPin® protein #20 (SEQ ID NO: 20, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #21 (SEQ ID NO: 21, with a His tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #22 (SEQ ID NO: 22, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #23 (SEQ ID NO: 23, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #24 (SEQ ID NO: 24, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® Protein #25 (SEQ ID NO: 25, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #26 (SEQ ID NO: 26, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #27 (SEQ ID NO: 27, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #28 (SEQ ID NO: 28, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® Protein #29 (SEQ ID NO: 29, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #30 (SEQ ID NO: 30, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #31 (SEQ ID NO: 31, with a His-tag (SEQ ID NO: 4) fused to its N-terminus). DARPin® protein #32 (SEQ ID NO: 32, with a His tag (SEQ ID NO: 4) fused to its N-terminus), and DARPin® protein #33 (SEQ ID NO: 33, with a His-tag (SEQ ID NO: 4) fused to its N-terminus).

[0275] High-level and soluble expression of NYESOpMHC-specific ankyrin repeat proteins For further analysis, selected clones showing specific NYESOpMHC binding in crude cell extract HTRF as previously described were expressed in E. coli cells and purified using their His tag by standard protocols. A 25 ml static overnight culture (TB, 1% glucose, 50 mg / L ampicillin; 37°C) was used ...

Claims

1. 1. A method for producing a peptide-MHC (pMHC) specific binding protein, said binding protein comprising an engineered ankyrin repeat domain with binding specificity for a target peptide-MHC complex, said method comprising: (a) providing a collection of designed ankyrin repeat domains; (b) providing a recombinant target peptide-MHC complex; (c) screening the collection of designed ankyrin repeat domains for specific binding to the target peptide-MHC complex to obtain at least one designed ankyrin repeat domain having binding specificity for the target peptide-MHC complex.

2. 2. The method of claim 1, wherein the collection comprises designed ankyrin repeat domains comprising fixed and randomized positions, and the designed ankyrin repeat domains of the collection differ from each other in at least one of the randomized positions.

3. The method of claim 1 or 2, wherein the collection of designed ankyrin repeat domains is provided by ribosome display.

4. 4. The method of claim 1, further comprising the steps of: (i) providing a second recombinant peptide-MHC complex, wherein the peptide of the second peptide-MHC complex comprises an amino acid sequence that differs from the target peptide by at least one amino acid residue; and (ii) removing from the collection by negative selection designed ankyrin repeat domains that have binding specificity for the second recombinant peptide-MHC complex.

5. the target peptide is derived from an intracellular protein, and the target peptide is derived from NY-ESO-1 comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34; or the target peptide is derived from EBNA-1 comprising or consisting of the amino acid sequence of SEQ ID NO: 92; or the target peptide is derived from MAGE-A3 comprising or consisting of the amino acid sequence of SEQ ID NO: 155; or the target peptide is derived from HBcAg comprising or consisting of the amino acid sequence of SEQ ID NO: 255; and / or The MHC is MHC class I. The method according to any one of claims 1 to 4.

6. a recombinant binding protein comprising a first designed ankyrin repeat domain, said first ankyrin repeat domain having binding specificity for a first target peptide-MHC complex; The first ankyrin repeat domain is attached to the first target peptide-MHC complex in PBS for 10 －７ A dissociation constant (K Ｄ ) and the first target peptide has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34; A binding protein, wherein the first ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 20-33.

7. the binding protein further comprises a second engineered ankyrin repeat domain, the second ankyrin repeat domain having binding specificity for a second target peptide-MHC complex; 7. The binding protein of claim 6, wherein the second ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 20-33.

8. 8. The binding protein of claim 6 or 7, wherein the binding protein comprises a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs: 16-18.

9. 9. The binding protein of any one of claims 6 to 8, wherein the binding protein further comprises a binding factor having binding specificity for a protein expressed on the surface of an immune cell, wherein the immune cell is a T cell, wherein the T cell is a CD8+ cytotoxic T cell, and wherein the protein expressed on the surface of the immune cell is a protein that is part of a T cell receptor complex, and wherein the protein that is part of the T cell receptor complex is cluster of differentiation 3 (CD3).

10. 10. A nucleic acid encoding said designed ankyrin repeat domain having binding specificity for a first or second target peptide-MHC complex as defined in any one of claims 6 to 9, or encoding a binding protein according to any one of claims 6 to 9.

11. A pharmaceutical composition comprising the binding protein according to any one of claims 6 to 9 or the nucleic acid according to claim 10, and a pharmaceutically acceptable carrier and / or diluent.

12. 11. A medicament for use in a method for tumor-localized activation of T cells in a mammal, the medicament comprising a binding protein according to any one of claims 6 to 9 or a nucleic acid according to claim 10, the method comprising the step of administering to the mammal a binding protein according to any one of claims 6 to 9 or a nucleic acid according to claim 10, wherein the first target peptide is derived from a protein expressed in tumor cells, and / or, if the binding protein comprises a second ankyrin repeat domain, the second target peptide is derived from a protein expressed in tumor cells.

13. 12. A binding protein according to any one of claims 6 to 9, a nucleic acid according to claim 10, or a pharmaceutical composition according to claim 11 for use in a method for treating a medical condition.

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