Difunctionally linked polypeptide

Abstract

To provide safer and more effective PD-1 agonist therapy for the treatment of autoimmune diseases. [Solution] A bifunctional conjugated polypeptide comprising a pMHC binding moiety and a PD-1 agonist is provided, based on the fact that a molecule containing a PD-1 agonist fused to a peptide-MHC binding moiety results in efficient inhibition of PD-1 signaling. The pMHC binding moiety may include a TCR variable region and / or an antibody variable region. The pMHC binding moiety may be a T cell receptor (TCR) or a TCR-like antibody. The PD-1 agonist may be a soluble extracellular form of PD-L1 or a functional fragment thereof.

Description

[Technical Field]

[0001] The PD-1 pathway regulates the balance between suppressive and stimulative signals in the immune system. It is known to play an important role in this. Activation of the PD-1 pathway reduces immune activity. It regulates peripheral immune tolerance and prevents autoimmunity (Keir et al., Annu Rev Immunol, 26:677-704, 2008; Okazaki et al., Int Immunol 19:813-824, 2007). PD-1 is a transmembrane receptor protein expressed on the surface of activated immune cells such as T cells, B cells, NK cells, and monocytes (Agata et al., Int Immunol 8:765-772, 1996). The cytoplasmic tail of PD-1 contains the immune receptor tyrosine It contains the base inhibitory motif (ITIM). PD-L1 and PD-L2 are native ligands for PD-1. It is expressed on the surface of antigen-presenting cells (Dong et al., Nat Med., 5:1365-1369, 1999; Freeman et al., J Exp Med 192:1027-1034, 2000; Latchman et al., Nat Immunol 2:261-268, 2001). When a ligand is involved, a phosphatase is recruited to the ITIM region of PD-1, inhibiting TCR-mediated signaling, and subsequently reducing lymphocyte proliferation, cytokine secretion, and cytotoxic activity. PD-1 may also induce apoptosis in T cells through its ability to inhibit co-stimulatory survival signals (Keir et al., Annu Rev Immunol, 26:677-704, 2008).

[0002] The central role of the PD-1 pathway in the regulation of autoimmunity was first demonstrated by the observation that PD-1 knockout mice developed late-onset progressive arthritis, lupus-like glomerulonephritis, and autoimmune cardiomyopathy (Nishimura et al., Immunity 11:141-151, 1999; Nishimura et al., Science 291:319-322, 2001). Furthermore, when PD-1 deficiency was introduced into non-obese diabetic (NOD) mice, the incidence of diabetes increased significantly, with all mice developing diabetes by 10 weeks of age (Wang et al., PNAS 102:11823-11828, 2005). In humans, PD-1 also appears to exhibit a similar regulatory function. Single nucleotide polymorphisms in the PD-1 gene are associated with various autoimmune diseases such as lupus erythematosus, multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and Graves' disease (Prokunina et al., Arthritis Rheum 50:1770, 2004; Neilson et al., Tissue Antigens 62:492, 2003). Kroner et al., Ann Neurol 58:50, 2005; Okazaki et al., Int Immunol 19:813-824, 2007); PD-1 pathway perturbation has also been reported in other autoimmune diseases (Kobayashi et al., J Rheumatol 32:215, 2005; Mataki et al., Am J Gastroenterol 102:302, 2007). Finally, blockade of the PD-1 pathway by antagonistic antibodies is associated with autoimmune side effects in cancer patients. (Michot et al., Eur J Cancer 54:139-148, 2016). [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] Therapeutic strategies that lead to activation of the PD-1 pathway represent a promising approach for treating autoimmune conditions. This provides a solution. For example, artificial dendritic cells overexpressing PD-L1 have been shown to reduce spinal cord inflammation and the clinical severity of experimental autoimmune encephalomyelitis in a mouse model (Hirata et al., J Immunol 174:1888-1897, 2005). Furthermore, PD-L1 with blockade of co-stimulatory molecules Recombinant adenoviruses expressing PD-1 have been shown to prevent lupus nephritis in BXSB mice (Ding et al., Clin Immunol 118:258-267, 2006). Many PD-1 agonist antibodies have been developed for the treatment of various autoimmune diseases in humans (see, for example, WO2013022091, WO2004056875, WO2010029435, WO2011110621, WO2015112800). However, such trials Despite drug development, soluble drugs remain efficient at inducing PD-1 signaling. There is little evidence to suggest this, and to our knowledge, only one such molecule is in clinical trials for the treatment of psoriasis (see NCT03337022). Administration of PD-1 agonists may also cause systemic immune effects away from the disease site, leading to clinical toxicity. Therefore, safer and more effective PD-1 agonists for the treatment of autoimmune diseases are needed. Therapy is necessary. [Means for solving the problem]

[0004] To our surprise, we discovered that molecules containing a PD-1 agonist fused to a peptide-MHC binding site efficiently inhibit PD-1 signaling.

[0005] Without being constrained by theory, the inventors believe that efficient inhibition of T cell activation is achieved through immunotherapy. We assume that localization of PD-1 agonists to the PUSS is necessary. TCR or TCR-like antibody Attaching a PD-1 agonist to the portion of a disease-specific peptide that binds to MHC allows the agonist to be induced in immune synapses, creating a safer and more potent strategy for modulating the PD-1 pathway. It will be provided.

[0006] The T cell receptor (TCR) is CD4 + and CD8 + It is spontaneously expressed by T cells. TCRs are major components. MHC molecules (In humans, MHC molecules are also known as human leukocyte antigens (HLA)) CD8 cells, also known as cytotoxic T cells, are designed to recognize short peptide antigens displayed on the surface of antigen-presenting cells that have formed a complex with them (Davis et al., (1998), Annu Rev Immunol 16:523-544). + T cells specifically recognize peptides bound to MHC class I cells and generally play a role in detecting and mediating the destruction of infected or cancer cells.

[0007] It is desirable that the TCR used in immunotherapy strongly recognizes the target antigen. This is because it elicits a strong response. In order to exert its effect, the TCR has high affinity and / or a long binding half-life for the target antigen. This means that. Naturally occurring TCRs usually have low affinity for their target antigens (my Because the chromole range is low, to improve antigen binding, the following is performed on a given TCR sequence. It is necessary to identify mutations that include, but are not limited to, substitutions, insertions, and / or deletions. For use as a soluble targeting agent, the TCR antigen binding affinity must be nanomolar. It is preferable that the binding half-life is several hours within the picomolar range. The therapeutic TCR is the target antigen. It is also desirable to exhibit a high level of specificity for the target antigen, thereby reducing the risk of toxicity in clinical applications due to off-target binding. Such high specificity is considered to be due to the natural degeneracy of TCR antigen recognition. In some cases, obtaining it can be particularly difficult (Wooldridge et al., (2012), J Biol Chem 287(2):1168-1177; Wilson et al., (2004), Mol Immunol 40(14-15):1047-1055). Subsequently, it is desirable that therapeutic TCRs can be expressed and purified in a very stable manner.

[0008] As a first aspect of this invention, the present invention relates to a bifunctional binding compound comprising a pMHC binding moiety and a PD-1 agonist. The product provides a lipeptide. The pMHC binding region includes a TCR variable region and / or an antibody variable region. It is visible. The pMHC binding site may be a T cell receptor (TCR) or a TCR-like antibody. pMHC binding site The fraction may be a heterodimeric alpha / beta TCR polypeptide pair or a single-stranded alpha / beta TCR polypeptide. PD-1 agonists are the soluble extracellular form of PD-L1 or its mechanism. The active fragment may contain or be derived from the following sequence: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCY. The PD-1 agonist may be a full-length antibody or a fragment thereof, for example, an scFv antibody.

[0009] PD-1 agonists can be fused to the C or N terminus of the pMHC binding site, and via a linker... It can be fused to the pMHC binding site. The linker length is up to 25 amino acids. Preferably, the linker is 2, 3, 4, 5, 6, 7, or 8 amino acids long.

[0010] If the pMHC binding region is a TCR, the TCR may contain a non-natural disulfide bond between the constant region of the alpha chain and the constant region of the beta chain, and may be able to specifically bind to the peptide antigen.

[0011] As a further aspect of the present invention, there is provided a bifunctional binding polypeptide according to the first aspect of the present invention for use in the treatment of autoimmune diseases such as alopecia areata, ankylosing spondylitis, atopic dermatitis, Graves' disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, vitiligo and inflammatory bowel disease.

[0012] The present invention also provides a pharmaceutical composition comprising a bifunctional binding polypeptide according to the first aspect.

[0013] There are provided a nucleic acid encoding a bifunctional binding polypeptide according to the first aspect, and an expression vector containing such a nucleic acid.

[0014] The nucleic acid encoding the bifunctional binding polypeptide may be present as a single open reading frame or as two separate open reading frames encoding the alpha and beta chains of the TCR, respectively. There is further provided a host cell containing such a nucleic acid or such a vector.

[0015] There is also provided a method for producing a bifunctional binding polypeptide according to the first aspect, the method comprising maintaining a host cell of the present invention under any conditions for the expression of the nucleic acid and isolating the bifunctional binding peptide of the first aspect.

[0016] There is also included in the present invention a method for treating an autoimmune disorder comprising administering a bifunctional binding polypeptide according to the first aspect to a patient who needs it.

BRIEF DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention The present invention provides, as a first aspect, a bifunctional binding polypeptide comprising a pMHC binding moiety and a PD-1 agonist. The pMHC binding moiety may comprise a TCR variable region. Alternatively, the pMHC binding moiety may be a peptide or polypeptide having a specific binding affinity for pMHC. The binding region may include an antibody variable region. The pMHC binding region is a T cell receptor (TCR) or TCR-like antibody. It could be a body.

[0018] TCR sequences are described in most cases with reference to the IMGT nomenclature, which is widely known and accessible to those skilled in the art of TCR research. For example, LeFranc and LeFranc, (2001), "T cell Receptor Factsbook," Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 2011(6). See 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10; and Lefranc, (2003), Leukemia 17(1):260-266. Briefly, the αβTCR has two disulfide molecules. It consists of filoid-bonded chains. Each chain (alpha and beta) is generally considered to have two regions: a variable region and a constant region. The short binding region connects the variable and constant regions and is usually considered part of the alpha variable region. In addition, the beta chain usually contains a short variability region adjacent to the binding region. This is usually considered part of the beta variable region.

[0019] The variable region of each chain is located at the N-terminus and consists of three complementary sequences embedded in the framework sequence (FR). It contains a sex-determining region (CDR). The CDR contains the peptide-MHC binding recognition site. Alpha chain is possible. There are several genes encoding variable (Vα) regions and several genes encoding beta-chain variable (Vβ) regions, which are distinguished by frameworks, CDR1 and CDR2 sequences, and a partially defined CDR3 sequence. Vα and Vβ genes are referred to by the prefixes TRAV and TRBV, respectively, in IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Similarly, there are several genes called TRAJ or TRBJ for the alpha and beta chains, respectively. There are several binding genes or J genes, and the beta chain has a diversity called TRBD or D gene (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2):107-114; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2):97-106; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). T cell receptor The enormous diversity of the strands is due to the various V, J, and D gene interactions, including allele variants. TCR ALF is caused by binaural rearrangement and binding diversity (Arstila et al., (1999), Science 286(5441):958-961; Robins et al., (2009), Blood 114(19):4099-4107). The constant or C regions of the α and β chains are called TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10).

[0020] If the pMHC binding site is a TCR, the TCR may not be naturally occurring and / or may be purified and / or genetically engineered. Compared to a natural TCR, One or more mutations may be present in the pha-chain variable region and / or the beta-chain variable region. Mutations preferably occur within the CDR region. Such mutations are typically associated with specific peptide antibodies. It is introduced to improve the binding affinity of the binding site (e.g., TCR) to the proto-HLA complex.

[0021] The pMHC binding site may be a TCR-like antibody. A TCR-like antibody is a peptide presented by MHC. In this technical field, antibody molecules possessing TCR-like specificity to an antigen are referred to as such. These antibodies typically have a higher affinity for the antigen than natural TCRs (Dahan et al., Expert Rev Mol Med 14:e6, 2012). Such antibodies each contain a variable region and a constant region. It may contain heavy and light chains. Functional fragments of such antibodies include scFv, Fab fragments, etc. As is well known in the field, this is included in the present invention.

[0022] The bifunctional conjugated polypeptide of the present invention has the property of binding to a specific peptide antigen-MHC complex. The polypeptide of the present invention exhibits specificity in presenting the peptide antigen-MHC complex. While it has minimal ability to recognize target cells that do not present peptide antigen-MHC complexes, it does not have the ability to recognize target cells that do present peptide antigen-MHC complexes. This relates to their ability to recognize target cells.

[0023] The bifunctional conjugated polypeptide of the present invention may have an ideal safety profile for use as a therapeutic reagent. An ideal safety profile means that, in addition to exhibiting good specificity, the polypeptide of the present invention may pass further preclinical safety tests. Examples of such tests include confirming a low probability of recognition of surrogate HLA types. includes a homologous antigen reactivity test for this purpose.

[0024] The bifunctional binding polypeptide of the present invention can be applied to high-yield purification. The yield can be determined based on the amount of material retained during the purification process (i.e., the amount of correctly folded material obtained at the end of the purification process relative to the amount of solubilized material obtained before refolding), and / or the yield can be determined based on the amount of correctly folded material obtained at the end of the purification process relative to the original culture amount. A high yield means more than 1%, more preferably more than 5%, or a higher yield. A high output means more than 1 mg / ml more preferably more than 3 mg / ml, or more than 5 mg / ml, or a higher output.

[0025] The bifunctional binding polypeptide of the present invention has appropriate binding affinity for the peptide antigen and PD-1. The binding affinity (inversely proportional to the equilibrium constant K D ) and the binding half-life (represented as T 1 / 2 ) are known to those skilled in the art. In a preferred embodiment, the binding affinity and the binding half-life are determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), for example, using a BIAcore instrument or an Octet instrument, respectively. It will be understood that when the affinity of the binding polypeptide doubles, K D becomes half. T 1 / 2 is calculated as ln2 divided by the off-rate (k off ). Therefore, when T 1 / 2 doubles, k off becomes half. K <00000!1>and k offThe values ​​are typically measured for soluble polypeptides. To account for variability between independent measurements, particularly in interaction with dissociation times exceeding 20 hours, the binding affinity and / or binding half-life of a given polypeptide can be measured several times, e.g., three or more times, using the same assay protocol, and the average of the obtained results can be taken. Yes, it is possible. To compare binding data between two samples (i.e., two different polypeptides and / or two preparations of the same polypeptide), it is preferable to perform the measurements using the same assay conditions (e.g., temperature).

[0026] In the case of the bifunctional conjugated polypeptide of the present invention, the pMHC binding portion includes a TCR variable region, that region The α and β variable regions may be present. If the pMHC binding region is a TCR, such a TCR may be an αβ heterodimer. In some cases, the pMHC binding region may be a γ and δ TCR variable region. This includes [specific components]. If the pMHC binding portion is a TCR, such a TCR may be a γδ heterodimer.

[0027] The pMHC binding moiety of the present invention may include an extracellular alpha-chain TRAC constant region sequence and / or an extracellular beta-chain TRBC1 or TRBC2 constant region sequence. The constant region may be truncated so that the transmembrane and cytoplasmic regions are absent. One or both of the constant regions. These sequences may contain mutations, substitutions, or deletions compared to the natural TRAC and / or TRBC 1 / 2 sequences. The terms TRAC and TRBC 1 / 2 also encompass natural polymorphic variants, such as the N-to-K substitution at position 4 of TRAC (Bragado et al., International Immunology, 1994 Feb; 6(2):223-30).

[0028] Alternatively, there may be no TCR steady region, either full length or truncated steady region. Therefore, the pMHC binding portion of the present invention is the variable region of the TCR alpha and beta chains. It can be composed of these.

[0029] If the pMHC binding region includes a TCR variable region, such a TCR variable region may be, for example, a single-stranded TCR. It can be any single-stranded form. The single-stranded forms include, but are not limited to, αβTCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ type (where Vα and Vβ are TCRα and β is a variable region, Cα and Cβ are the extracellular constant regions of TCRα and β, respectively, and L is the linker sequence) (Weidanz et al., (1998) J Immunol Methods. Dec 1; 221(1-2):59-76; Epel et al., (2002), Cancer Immunol Immunother. Nov; 51(10):565-73; WO 2004 / 033685; WO9918129). If present, one or both of the extracellular constant regions are of full length. They may be truncated as described above and / or may include mutations. In certain embodiments, the single-stranded TCR variable region and / or single-stranded TCR of the present invention may be WO As described in 2004 / 033685, single-stranded TCRs may have disulfide bonds introduced between residues in each constant region. Single-stranded TCRs can also be found in WO2004 / 033685; WO98 / 39482; WO01 / 62908; Weidanz et al. (1998) J Immunol Methods 221(1-2):59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829. It is described in ).

[0030] In the case of the bifunctional binding polypeptide of the present invention, where the pMHC binding portion is a TCR, the alpha and beta chain constant region sequences of such a TCR are Cys4 and TRBC1 or TRBC2 of exon 2 of the TRAC. To remove the natural disulfide bond between xon 2 and Cys2, truncation or substitution is used. It may be modified by the following. The alpha and / or beta chain constant region sequences may have disulfide bonds introduced between the residues of each constant region, as described, for example, in WO 03 / 020763. In a preferred embodiment, the alpha and beta constant regions may be modified by substitution of cysteine ​​residues at position Thr 48 and position Ser 57 of TRBC1 or TRBC2 in the TRAC, wherein the cysteine ​​is disulfide bonded between the alpha and beta constant regions of the TCR. The αβ heterodimer of the present invention is formed by: TRBC1 or TRBC2 further comprising a cysteine-to-alanine mutation at position 75 of the constant region and an asparagine-to-aspartic acid mutation at position 89 of the constant region. One or both of the extracellular constant regions present in the αβ heterodimer of the present invention are C-terminal For example, up to 15, up to 10, or up to 8 amino acids can be truncated. One or both of the extracellular constant regions present in the αβ heterodimer of the present invention may be, for example, up to 1 Five, up to ten, or up to eight amino acids can be truncated at the C-terminus. The C-terminus of the extracellular constant region of the chain can be truncated by 8 amino acids.

[0031] Non-natural disulfide bonds may be present between extracellular constant regions. These non-natural disulfide bonds are further described in WO03020763 and WO06000830. The non-natural disulfide bond may be between position Thr 48 of the TRAC and position Ser 57 of TRBC1 or TRBC2. One or both of the constant regions may have one or more mutations compared to the native TRAC and / or TRBC1 / 2 sequences. This may include substitutions or deletions.

[0032] In another preferred form of a bifunctional conjugated polypeptide in which the pMHC binding region includes a TCR variable region, the TCR variable region and the PD-1 agonist region are located alternately on separate polypeptide chains. This may result in dimerization. Such a form is described in WO2019012138. Briefly, the first polypeptide chain may contain (from N-terminus to C-terminus) a first antibody variable region, followed by a TCR variable region, and optionally a Fc region. The second chain may contain (from N-terminus to C-terminus) a TCR variable region followed by a second antibody variable region, and optionally a Fc region. Given a linker of appropriate length, the chains may dimerize to form a multispecific molecule, which may optionally contain an Fc region. Molecules in which regions are located on different chains in this manner are sometimes called diabodies, which are also intended herein. Additional chains and regions Additionally, triabodies can be formed, for example.

[0033] Therefore, this specification also provides a bispecific polypeptide molecule selected from the group of molecules comprising a first polypeptide chain and a second polypeptide chain, where, The first polypeptide chain comprises a first binding region (VD1) of the variable region of the PD-1 agonist antibody, a first binding region (VR1) of the variable region of the TCR that specifically binds to the MHC-binding peptide epitope, and a first linker (LINK1) connecting the said regions. The second polypeptide chain includes a second binding region (VR2) of the variable region of the TCR that specifically binds to the MHC-binding peptide epitope, a second binding region (VD2) of the variable region of the PD-1 agonist antibody, and a second linker (LINK2) connecting the said regions. Here, the first binding region (VD1) and the second binding region (VD2) associate to form the first binding site (VD1)(VD2), The first binding region (VR1) and the second binding region (VR2) combine to form the MHC bond. It forms a second binding site (VR1)(VR2) that binds to the peptide epitope, Here, the two polypeptide chains are the human IgG hinge region and / or the human IgG Fc region It is fused to the region or its dimerized portion, and Here, the two polypeptide chains are connected by covalent and / or non-covalent bonds between the hinge regions and / or between the Fc regions, and Here, the bispecific polypeptide molecule can simultaneously stimulate PD-1 (agonizing) and bind to an MHC-binding peptide epitope, and the bispecific polypeptide molecule In this configuration, the order of the binding regions of the two polypeptide chains is selected from VD1-VR1 and VR2-VD2, or VD1-VR2 and VR1-VD2, or VD2-VR1 and VR2-VD1, or VD2-VR2 and VR1-VD1, and these regions are connected by either LINK1 or LINK2.

[0034] PD-1 agonists may correspond to the soluble extracellular domain or functional fragment of PD-L1 (Uniprot ref: Q9NZQ7) or PD-L2 (Q9BQ51). PD-L1 may contain or consist of sequences such as those described below.

[0035] The full-length PD-L1 has the following sequence. FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSY RQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVV DPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIF YCTFRRLDPEENHTAELVIPELPLAHPPNER

[0036] The truncated form of PD-L1 is assumed to retain its ability to bind to and stimulate PD-1. It can be fused to the pMHC binding site. Such truncated fragments are shown in the following sequence. FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSY RQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY Alternatively, shorter or longer trimmings may also be fused to the pMHC binding site.

[0037] PD-1 agonists may be full-length antibodies or scFv antibodies or fragments thereof such as Fab fragments or nanobodies. Examples of such antibodies are provided in WO2011110621, WO2010029434, and WO2018024237. The antibody molecules of the present invention may include, but are not limited to, complete antibody molecules or fragments thereof having full-length heavy and light chains, but include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-region antibodies (e.g., VH or VL or VHH), scFv, 2, 3 or 4-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies These may be dy, nanobody, or any of the epitope-binding fragments described above.

[0038] PD-1 agonists can be fused to the C or N terminus of the pMHC binding site, 2, 3, 4, 5, 6, 7, Alternatively, it can be fused to the pMHC binding site via an 8-amino acid-length linker. The linker is 10, 12 The linker sequence can be 15, 16, 18, 20, or 25 amino acid lengths. Longer linkers can be formed by repeating the linker sequence. Each linker can be formed by 1, 2, 3, or 4 repeats of a shorter linker sequence. Linker sequences are usually flexible in that they are mainly composed of amino acids such as glycine, alanine, and serine, which do not have bulky side chains that may limit flexibility. Alternatively, linkers with higher rigidity may be desirable. The usable or optimal length of the linker sequence can be easily determined. Linkers are up to 25 amino acid lengths. Often, linker sequences are about 12 or less, for example, 10 or less, or 2 to 8 amino acid lengths. Examples of suitable linkers that can be used in the TCR of the present invention include, but are not limited to, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP, and GGEGGGSEGGGS (as described in WO2010 / 133828).

[0039] The bifunctional conjugated polypeptide of the present invention may further comprise a pK-modified moiety. When an immunoglobulin Fc region is used, it may be any antibody Fc region. This Fc region is the tail region of the antibody that interacts with Fc receptors on the cell surface and several proteins of the complement system. The Fc region typically consists of two or three heavy chain constant regions (called CH2, CH3, and CH4) together. It contains two polypeptide chains having a CH2 and a hinge region, and these two chains are linked by a disulfide bond within the hinge region. The Fc region from immunoglobulin subclasses IgG1, IgG2, and IgG4 binds to FcRn and undergoes recycling via FcRn, resulting in a long circulating half-life (3-4 weeks). The interaction between IgG and FcRn covers the CH2 and CH3 regions, and the Fc region It is localized to a portion of the region. Preferred immunoglobulin Fc for use in the present invention includes, but is not limited to, Fc regions from IgG1 or IgG4. Preferably, the Fc region is derived from a human sequence. The Fc region also preferably contains KiH mutations that promote dimerization, and It may contain mutations that prevent interaction with the activating receptor (i.e., functionally dormant molecules). The immunoglobulin Fc region may contain other regions (i.e., the TCR variable region or immunoeffect region) It can be fused to the C or N terminus of the immunoeffector. Immunoglobulin Fc can be fused to other regions (i.e., TCR variable region or immunoeffector) via a linker. The linker sequence is Flexible is generally characterized by being composed primarily of amino acids such as glycine, alanine, and serine, which typically lack bulky side chains that could limit flexibility. Alternatively, linkers with higher rigidity may be desirable. The usable or optimal length of the linker sequence can be easily determined. Often, the linker sequence is approximately 12 or less, for example, 10 or less, or 2 to 10 amino acids long, with linkers being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 The amino acid lengths may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Suitable linkers that may use the multi-domain binding molecules of the present invention include, but are not limited to, GGGSGGGG, GGGGS, GGGSG, GGSGG, GGGGG, GGGGP, GGEPS, GGEGGGP, and GGEGGGSEGGGS (as described in WO2010 / 133828). When immunoglobulin Fc is fused to the TCR, it may be fused to either the alpha or beta chain, with or without a linker. Furthermore, individual chains of Fc may be fused to individual chains of the TCR.

[0040] Preferably, the Fc region may originate from the IgG1 or IgG4 subclass. The two chains are CH2 and The CH3 steady region and the hinge region may be all or part of the CH3 steady region. The hinge region may substantially or partially correspond to the hinge region from IgG1, IgG2, IgG3, or IgG4. The hinge is The hinge region may include all or part of the core hinge region and all or part of the lower hinge region. Preferably, the hinge region includes at least one disulfide bond connecting the two chains.

[0041] The Fc region may contain mutations in the WT sequence. Mutations include substitutions, insertions, and deletions. Such mutations may be made to introduce desired therapeutic properties. For example, a knobs into holes (KiH) mutation can be incorporated into the CH3 region to promote heterodimerization. In this case, one strand contains a bulky, protruding residue (i.e., a knob) such as Y. One strand is designed to contain a complementary pocket (i.e., a hole). The appropriate locations for KiH mutations are known in the art. Additionally or alternatively, they inactivate or reduce binding to the Fcy receptor and / or increase binding to FcRn, and / Alternatively, mutations can be introduced to prevent Fab arm replacement or to remove the protease site. .

[0042] The PK modification moiety may also be an albumin-binding domain that can act to extend the half-life. As is known in the art, albumin has a long circulating half-life of 19 days, partly due to its size, exceeding the renal threshold, its specific interactions, and recycling via FcRn. Attachment to albumin improves the circulating half-life of therapeutic molecules in vivo. This is a well-known strategy for achieving this. Albumin can be covalently bound through the use of specific albumin-binding domains, or covalently by conjugation or direct gene fusion. An example of a therapeutic molecule that utilizes attachment to albumin to improve half-life is Sleep et al., Biochim This is described in BiophysActa. December 2013; 1830(12):5526-34.

[0043] The albumin-binding region can be any region capable of binding to albumin, including any known albumin-binding portion. The albumin-binding region can be selected from endogenous or exogenous ligands, small organic molecules, fatty acids, peptides, and proteins that specifically bind to albumin. Examples of preferred albumin-binding regions include short peptides (e.g., peptides) as described by Dennis et al., J Biol Chem. 2002 / 9 / 20; 277(38):35035-43. Proteins designed to bind to albumin, such as antibodies, antibody fragments, and antibody-like scaffolds, including Albudab® (commercially available from GSK, O'Connor-Semmes et al., Clin Pharmacol Ther. December 2014; 96(6):704-12) and Nanobody® (commercially available from Ablynx, Van Roy et al., Arthritis Res Ther. May 20, 2015; 17:135) and Streptococcal G proteins. (Stork et al., Eng Des Sel. November 2007; 20(11):569-76) such as those found in nature This includes albumin-binding domain-based proteins, such as Albumod®, commercially available from Affibody.

[0044] Preferably, the albumin is human serum albumin (HSA). The affinity of the albumin-binding region can range from picomolar to micromolar. When the concentration of albumin in human serum is very high (35-50 mg / ml, approximately 0.6 mM), virtually all of it is affected. It is calculated that the albumin-binding region of this molecule binds to albumin in vivo.

[0045] The albumin-binding region can be linked to the C or N terminus of other regions (i.e., the TCR variable region or immunoeffector). It can be linked to the TCR variable region or immunoeffector. The linker sequence is usually responsive. It is flexible in that it is mainly composed of amino acids such as glycine, alanine, and serine, which do not have bulky side chains that may limit flexibility. Alternatively, a linker with higher rigidity may be desirable. The usable or optimal length of the linker sequence can be easily determined. Often, the linker sequence is about 12 or less, for example 10 or less, or 2 to 10 amino acid lengths, and the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid lengths. Examples of suitable linkers that can use the multi-region binding molecules of the present invention include GGGSGGGG, GGGGS This includes, but is not limited to, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP, and GGEGGGSEGGGS (as described in WO2010 / 133828). The albumin-binding moiety, when bound to the TCR, is of the alpha or beta chain, with or without a linker. It can be connected to either of them.

[0046] Further aspects of the present invention include alopecia areata, ankylosing spondylitis, atopic dermatitis, Graves' disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, vitiligo, inflammatory bowel disease, Crohn's disease, ulcerative colitis, childhood steatorrhea (celiac disease), eye diseases (e.g., uveitis), cutaneous lupus erythematosus and lupus nephritis, and PD-1 / PD-L1 angioplasty. The present invention provides a bifunctional conjugated polypeptide according to a first aspect of the present invention for use in the treatment of autoimmune diseases, such as autoimmune diseases induced in cancer patients by tagagonists.

[0047] The present invention also provides a bifunctional conjugated polypeptide according to a first aspect of the present invention for use in the treatment or prevention of pain, particularly pain associated with inflammation.

[0048] Optionally, the bifunctional polypeptide of the present invention is intended for use in the treatment of type 1 diabetes, inflammatory bowel disease, and rheumatoid arthritis.

[0049] The present invention also provides a pharmaceutical composition comprising a bifunctional conjugated polypeptide according to the first aspect.

[0050] In a further aspect, the present invention provides nucleic acids encoding the bifunctional binding polypeptide of the present invention. In some embodiments, the nucleic acid is cDNA. In some embodiments, the nucleic acid may be mRNA. In some embodiments, the present invention provides a variable α-chain of the TCR of the present invention. The present invention provides nucleic acids comprising sequences that encode a region. In some embodiments, the present invention provides nucleic acids comprising sequences that encode a β-chain variable region of the TCR of the present invention. In this context, the present invention provides nucleic acids comprising sequences encoding the light chain of a TCR-like antibody. In several embodiments, the present invention provides nucleic acids comprising sequences encoding the heavy chain of a TCR-like antibody. Provided. In some embodiments, the present invention provides all or part of a PD-1 agonist. For example, the present invention provides a nucleic acid containing a sequence that encodes all or part of an agonist PD-1 antibody, such as PD-L1 or a truncated form thereof, or the light and / or heavy chain of the antibody. These may not exist in nature and / or may be purified and / or genetically engineered. Nucleic acid sequences may be codon-optimized depending on the expression system used. As is known to those skilled in the art, the expression system may include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or they may be cell-free expression systems.

[0051] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention. Preferably, the vector This vector is a suitable expression vector.

[0052] The present invention also provides cells that accommodate the vectors of the present invention. Suitable cells include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells. The vectors may comprise the nucleic acids of the present invention encoding the alpha and beta chains of a TCR, or the light and heavy chains of a TCR-like antibody, on a single open reading frame, or on two separate open reading frames, respectively.

[0053] Another aspect provides cells having a first expression vector comprising nucleic acids encoding the alpha / light chain of the polypeptide TCR / TCR-like antibody of the present invention, and a second expression vector comprising nucleic acids encoding the beta / heavy chain of the TCR / TCR-like antibody of the present invention. The cells of the present invention may be isolated and / or recombinant and / or non-naturally occurring and / or genetically engineered.

[0054] As is well known in the art, polypeptides can undergo post-translational modifications. Glycosylation is one such modification, involving the covalent bonding of an oligosaccharide moiety to a defined amino acid of a TCR / TCR-like antibody / PD-L1 or PD-1 antibody or other PD-1 agonist. For example, asparagine residues or serine / threonine residues are well known sites for oligosaccharide bonding. The glycosylation state of a particular protein depends on several factors, including the protein sequence, the protein's three-dimensional structure, and the availability of specific enzymes. Furthermore, the glycosylation state (i.e., the type of oligosaccharide, covalent bonds, and total number of bonds) can affect protein function. Therefore, controlling glycosylation is often desirable when producing recombinant proteins. Controlled glycosylation has been used to improve antibody-based therapies. (Jefferis et al., (2009) Nat Rev Drug Discov Mar; 8(3):226-34.) In the case of the soluble TCR of the present invention, glycosylation can be controlled, for example, by using specific cell lines (including, but not limited to, mammalian cell lines such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells) or by chemical modification. Such modifications may be desirable because glycosylation improves pharmacokinetics, reduces immunogenicity, and allows for a more rigorous mimicry of native human proteins (Sinclair and Elliott, (2005) Pharm Sci. Aug; 94(8):1626-35).

[0055] For administration to a patient, the bifunctional conjugated polypeptide of the present invention may be provided as part of a sterile pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. This pharmaceutical composition may be in any suitable form (depending on the desired method of administering it to the patient). It may be provided in the form of unit doses, generally in sealed containers, and may be provided as part of a kit. Such a kit usually (but not necessarily) includes instructions for use. It may comprise multiple of the aforementioned unit dose forms.

[0056] Pharmaceutical compositions can be adapted for administration by any suitable route, including parenteral (including subcutaneous, intramuscular, intrathecal, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes. Such compositions can be prepared by any method known in the art of pharmaceuticals, for example, by mixing an active ingredient with a carrier or excipient under sterile conditions.

[0057] The dosage of the substance of the present invention may vary within a wide range depending on the disease or disorder being treated, the age and condition of the individual being treated, etc. The appropriate dosage range for the bifunctional conjugated polypeptide is 25 ng / kg to 50 μg / kg or 1 μg to 1 g. Determine the dosage.

[0058] The bifunctional conjugated polypeptides, pharmaceutical compositions, vectors, nucleic acids, and cells of the present invention are substantially pure forms, for example, at least 80%, at least 85%, at least 90%, and at least Provided in 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure form. It is possible.

[0059] Nucleic acids encoding a bifunctional binding polypeptide bind the alpha and beta chains of the TCR. A host cell containing a nucleic acid or vector is further provided, which may exist as a single open reading frame or as two separate open reading frames, each encoding a different signal.

[0060] A method for producing a bifunctional conjugated polypeptide of the first aspect is also provided, which comprises maintaining a host cell of the present invention under any conditions for the expression of the nucleic acid of the present invention and isolating the bifunctional conjugated peptide of the first aspect.

[0061] Preferred features of each aspect of the present invention are also applicable to each of the other aspects (with necessary modifications). Prior art documents described herein are incorporated to the maximum extent permitted by law.

[0062] Herein, the present invention will be described with reference to the following non-limiting examples and figures. [Brief explanation of the drawing]

[0063] [Figure 1] Figure 1 shows dose-dependent inhibition of NFAT reporter activity by the bifunctional polypeptide of the present invention, which contains soluble TCR and truncated PD-L1, in the presence of peptide pulsed target cells. [Figure 2] Figure 2 shows the inhibition of NFAT reporter activity by the bifunctional polypeptide of the present invention, which contains a soluble TCR and a PD-1 agonist scFv antibody fragment, in the presence of peptide pulsed target cells. [Figure 3] Figure 3 shows the inhibition of primary human T cell activation by the bifunctional polypeptide of the present invention, which contains a soluble TCR and a PD-1 agonist scFv antibody fragment, in the presence of peptide pulsed target cells. [Figure 4] Figure 4 shows the inhibition of NFAT reporter activity by the bifunctional polypeptide of the present invention, which comprises one of two soluble TCRs with different specificities and a PD-1 agonist scFv antibody fragment, in the presence of peptide pulsed target cells. [Examples]

[0064] Example 1 The following example demonstrates that a PD-1 agonist fused to a soluble TCR can effectively inhibit T cell activation when targeting immune synapses.

[0065] The soluble TCR used in this bifunctional conjugating polypeptide is human prepro insulin. It is an affinity-enhanced version of the native TCR that specifically recognizes the derived HLA-A*02 restriction peptide (such molecules are described in WO2015092362). PD-1 agonists are truncated versions of the extracellular region of PD-L1, including the PD-1 interaction site (Zak et al., Structure 23:2341-2348, 2015). PD-L1 is fused to the N-terminus of the TCR alpha chain via a standard 5-amino acid linker.

[0066] Using the Jurkat NFAT luciferase PD-1 reporter assay, HEK293T antigen presentation target cells were identified. We measured the inhibition of T cell NFAT activity via TCR-PD1 agonist fusion molecules in the presence of cells.

[0067] method Expression, refolding, and purification of TCR-PD1 agonist fusion molecules Expression of the TCR-PD1 agonist fusion molecule was performed using a high-yield transient expression system (ExpiCHO Expression system, Thermo Fisher) based on Chinese hamster ovary (CHO) cells adapted for suspension culture. A mammalian expression plasmid containing the TCR chain fused to the PD-1 agonist was used. Cells were cotransfected using the product according to the manufacturer's instructions. After harvesting, the cell culture supernatant was clarified by centrifugation at 4000-5000 xg for 30 minutes in a refrigerated centrifuge. The supernatant was filtered through a 0.22 μm filter and collected for further purification.

[0068] Alternatively, expression of the TCR-PD1 agonist fusion molecule was performed using *E. coli* as the host organism. Expression plasmids containing the alpha and beta chains were expressed separately in *E. coli* strain BL21pLysS. The transformed cells were seeded on LB agar plates containing 100 μg / mL ampicillin. A platinum loopful of colonies was collected and incubated in LB medium (containing 100 μg / mL ampicillin and 1% glucose) at 37°C. 600It was allowed to multiply until it reached approximately 0.5 to 1.0. Next, the LB starter Add the luture to the automated induction medium (Foremedium), and incubate the cells at 37°C for approximately 3 hours, followed by incubating at 30°C. Cells were grown in the evening. Cells were collected by centrifugation and lysed with Bugbuster (Novagen). Inclusion bodies (IBs) were extracted by removing cell fragments and membranes by performing two Triton washes (50 mM Tris pH 8.1, 100 mM NaCl, 10 mM EDTA, 0.5% Triton). After each wash, the IBs were collected by centrifugation at 10000 g for 5 minutes. To remove surfactants, the IBs were washed with 50 mM Tris pH 8.1, 100 mM NaCl, and 10 mM EDTA. Finally, the IBs were resuspended in 50 mM Tris pH 8.1, 100 mM NaCl, and 10 mM EDTA buffer. Protein yield was measured. Therefore, IB was dissolved in 8M urea buffer, and its concentration was measured by absorbance at 280 nM.

[0069] For refolding, the alpha and beta chains were mixed in a 1:1 molar ratio and denatured at 37°C for 30 minutes in 6M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM EDTA, and 20 mM DTT. Next, the denatured chains were treated with 4M urea, 100 mM Tris pH 8.1, and 0.4M L- The refolding buffer, consisting of arginine, 2 mM EDTA, 1 mM cystamine, and 10 mM cysteamine, was added and incubated for 10 minutes with constant stirring. The refolding buffer containing the denatured chains was then dialyzed through a Spectrapore 1 membrane to 10 times its volume of H2O. In contrast, dialysis was performed for approximately 16 hours, approximately 7 hours with 10 times the volume of 10 mM Tris pH 8.1, and approximately 16 hours with 10 times the volume of 10 mM Tris pH 8.1.

[0070] Soluble proteins obtained from mammalian or E. coli expression systems were purified using AKTA pure (GE Healthcare) with a POROS 50 HQ (Thermo Fisher Scientific) anion exchange column using 20 mM Tris pH 8.1 as the loading buffer and 20 mM Tris pH 8.1 and 1 M NaCl as the binding and elution buffers. The proteins were loaded onto the column and eluted with a 0–50% gradient of the elution buffer. The protein-containing fractions were pooled and subjected to second-stage cation exchange chromatography on a POROS 50 HS (Thermos Fisher Scientific) column using 20 mM MES pH 6.0 and 20 mM MES pH 6.0 and 1 M NaCl as binding and elution buffers, respectively. For this purpose, the solution was diluted 20-fold (volume / volume) with 20 mM MES pH 6.0. The results from the cation exchange column were then obtained. The combined protein was eluted using a 0–100% gradient of elution buffer. The cation exchange fraction containing the protein was pooled and further purified using a Superdex 200 HR (GE Healthcare) gel filtration column with PBS as the running buffer. The positive fraction from gel filtration was pooled, concentrated, and stored at -80°C until needed.

[0071] Jurkat NFAT Luc-PD-1 Reporter Assay In HLA-A*02-positive HEK293T target cells, TCR activator plasmid (BPS Bioscience) Catalog number: 60610) is transiently transfected into a TCR-PD1 agonist fusion molecule. Therefore, the cells were pulsed with the recognized peptide. Next, the target cells were subjected to different concentrations of TCR. -Incubate with a PD1 agonist fusion molecule to form a congeneral peptide-HLA-A2 complex. This enabled the fusion. Jurkat NFAT Luc PD-1 effector cells, which constitutively express PD-1, were added to target cells, and NFAT activity was measured after 18-20 hours. The experiment involved (TCR-PD1 agonist fusion). The experiment was performed with or without rinsing after molecular binding. Further control experiments were conducted using unpulsed target cells. HEK293T A2B2M target cells transfected with TCR activator / PD-L1 were included as a positive control.

[0072] result The data shown in Figure 1 demonstrate that dose-dependent inhibition of NFAT reporter activity is observed with the TCR-PD1 agonist fusion molecule in the presence of peptide-pulsed target cells, with or without rinsing. Importantly, minimal inhibition was observed in unpulsed target cells, indicating that targeting to the immune synapse is crucial for PD-1 agonist activity. It is.

[0073] Example 2 The following example shows that when a PD-1 agonist fused to a soluble TCR targets an immune synapse, T This provides further evidence that it can effectively inhibit cell activation.

[0074] The experimental system and methods used in this example are, in this case, except that the PD-1 agonist portion of the TCR-PD1 agonist fusion molecule was an scFv antibody fragment as described in WO2011110621. And it was the same as that described in Example 1.

[0075] The Jurkat NFAT luciferase PD-1 reporter assay described in Example 1 was used to measure the inhibition of T cell NFAT activity via a TCR-PD1 agonist fusion molecule in the presence of HEK293T antigen-presenting target cells.

[0076] result As shown in Figure 2a, peptide pulsed peptides treated with a 100 nM TCR-PD1 agonist fusion molecule were obtained. In cells (labeled +PPI), substantial inhibition of NFAT activity (>60%) was observed. On the other hand, in unpulsed target cells treated with a TCR-PD1 agonist fusion molecule (indicated as -PPI), Minimal inhibition was observed. Control experiments using soluble TCR alone or PD-1 agonists alone (in both scFv and IgG4 forms) did not show inhibition of reporter activity, indicating that targeting of immune synapses by PD-1 agonists is necessary for PD-1 agonist activity. Figure 2b further shows dose-dependent inhibition of NFAT activity. In this case as well, TCR -Only the PD1 agonist fusion molecule form can inhibit NFAT activity. PD-1 agonist antibodies that are not present cannot inhibit its activity.

[0077] In summary, these results suggest that PD-1 agonists target immune synapses, and that PD This indicates that it is important for -1 agonist activity.

[0078] Example 3 The following example shows that when a PD-1 agonist fused to a soluble TCR targets an immune synapse, the T cell This provides further evidence that cell activation can be effectively inhibited.

[0079] The TCR-PD1 agonist fusion molecule used in this example is a PD-1 agonist that can be cleaved by scFv antibodies. It was the same as that described in Example 2, which was a single piece.

[0080] In this case, an alternative assay is used to fused a TCR-PD1 agonist to primary human T cell function. The effects of the compound molecule were evaluated.

[0081] method Primary Human T Cell Assay Primary human T cells were isolated using the pan-T cell isolation kit (Miltenyi, catalog number: 130-096-535). Using this method, newly prepared PBMCs were isolated. HLA-A*02-positive Raji B cells (Raji A2B2M) were pre-loaded with Staphylococcus enterotoxin B (SEB, 100 ng / ml, Sigma S4881) for 1 hour. Subsequently, irradiation was performed with 33 Gy. For pre-activation, primary human T cells were cultured in a 24-well cell culture. In a 1:1 ratio, each cell type was incubated with SEB-loaded Raji A2B2M target cells using 1 x 10E6 cells / ml. Primary human T cells were incubated with SEB-loaded RajiA2B2M cells. Both cells were incubated for 10 days, and IL-2 (50 U / ml) was added on days 3 and 7. On day 10, the pre-activated T cells were washed and resuspended in fresh medium. Fresh RajiA2B2M cells were given 20 μM of the corresponding peptide recognized by the TCR-PD1 agonist fusion molecule. Intermittent pulses were applied, or pulses were left unpulsed. During the last hour of peptide pulsed irradiation, Raji A2B2M cells were loaded with SEB (10 ng / ml) and then irradiated with 33 Gy. Raji A2B2M cells were seeded at 1 x 10⁵ cells / well in 96-well cell culture plates and then pre-incubated for 1 hour with a TCR-PD1 agonist fusion molecule titration. Pre-activated T cells were then seeded at 1 x 10⁵ cells / well in 96-well cell culture plates. The solution was added to RajiA2B2M target cells in a cell / well and incubated for 48 hours. The supernatant was collected. IL-2 levels were determined using MSD ELISA.

[0082] result The data shown in Figure 3 demonstrates that TCR-PD1 agonist fusion molecules dose-dependently inhibit primary human T cell IL-2 production in the presence of peptide pulsed target cells, whereas untargeted TCR-PD1 agonist fusion molecules (i.e., unpulsed target cells) or PD-1 agonists This indicates that scFv alone does not inhibit PD-1. These data suggest that PD-1 agonists inhibit immunity. We showed that targeting epidemiological synapses leads to PD-1 agonist activity in primary cells. Yes, they are.

[0083] Example 4: The following example demonstrates that the same technical effect can be observed using a TCR that recognizes a surrogate antigen. Yes, they are.

[0084] The experimental system and methods used in this example were the same as those described in Example 2. In this case, the PD-1 agonist antibody was fused to two different soluble TCRs.

[0085] The Jurkat NFAT luciferase PD-1 reporter assay described in Example 1 was used to measure the inhibition of T cell NFAT activity via a TCR-PD1 agonist fusion molecule in the presence of HEK293T antigen-presenting target cells.

[0086] result As shown in Figure 4, when two TCR-PD1 agonist fusion molecules (containing PD-1 agonist scFv antibody fragments fused to either TCR1 or TCR2) were administered in the presence of target cells pulsed with their respective peptides (peptide 1 or 2), potent, dose-dependent inhibition was observed. When both TCR-PD1 agonist fusion molecules were studied without the presence of the target peptide, minimal activity was observed.

[0087] These results demonstrate that TCR-PD1 agonist fusion molecules can promote targeted inhibition of T cell activity by directing soluble TCRs, which have specificity for different pMHCs, to various tissues.

Claims

1. A bifunctional conjugated polypeptide containing a pMHC binding moiety and a PD-1 agonist.

2. The pMHC binding portion includes a TCR variable region and / or an antibody variable region, according to claim 1. A bifunctional conjugated polypeptide.

3. The two-device according to claim 1, wherein the pMHC binding portion is a T cell receptor (TCR) or a TCR-like antibody. Potentially binding polypeptide.

4. Claim 1, wherein the pMHC binding portion is a heterodimeric alpha / beta TCR polypeptide pair. A bifunctional conjugated polypeptide as described in any one of items 3 to 3.

5. The pMHC binding portion is a single-chain alpha / beta TCR polypeptide, according to claims 1 to 3. A bifunctional conjugated polypeptide as described in item 1.

6. The TCR contains unnatural disulfides between the constant region of the alpha chain and the constant region of the beta chain. A bifunctional conjugated polypeptide according to any one of claims 3 to 5, comprising a bond.

7. The twin apparatus according to any one of claims 3 to 6, wherein the TCR specifically binds to the peptide antigen. Potentially binding polypeptide.

8. The PD-1 agonist is PD-L1 or a functional fragment thereof, according to any one of claims 1 to 7. The bifunctional conjugated polypeptide described above.

9. The PD-L1 contains or consists of the following sequence, according to claim 8, a bifunctional conjugate Lipeptide. FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY

10. The PD-1 agonist is a full-length antibody or a fragment thereof, any one of claims 1 to 7. The bifunctional conjugated polypeptide described above.

11. The bifunctional conjugated polypeptide according to claim 10, wherein the PD-1 agonist is an scFv antibody. 。

12. Claim 1, wherein the PD-1 agonist is fused to the C or N terminus of the pMHC binding portion. A bifunctional conjugated polypeptide as described in any one of items 11 to 11.

13. Claim 1, wherein the PD-1 agonist is fused to the pMHC binding portion via a linker. A bifunctional conjugated polypeptide as described in any one of items 12.

14. The bifunctional conjugated polypeptide according to claim 13, wherein the linker has a length of 2, 3, 4, 5, 6, 7, or 8 amino acids.

15. A pharmaceutical composition comprising a bifunctional conjugated polypeptide according to any one of claims 1 to 14.

16. A nucleic acid encoding a bifunctional conjugated polypeptide according to any one of claims 1 to 14.

17. An expression vector comprising the nucleic acid described in claim 16.

18. A host cell comprising the nucleic acid according to claim 16 or the vector according to claim 17, wherein the nucleic acid encoding the bifunctional binding polypeptide optionally exists as a single open reading frame encoding an alpha chain and a beta chain, or as two separate open reading frames encoding each of them.

19. A method for producing a bifunctional conjugated polypeptide according to any one of claims 1 to 14, comprising maintaining the host cell according to claim 18 under any conditions for nucleic acid expression and isolating the bifunctional conjugated peptide.

20. A bifunctional conjugated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15, a nucleic acid according to claim 16, and / or a vector according to claim 17, for use in medicine, particularly for treating autoimmune diseases, or for use in treating or preventing pain, particularly pain associated with inflammation.

21. The autoimmune diseases mentioned above include alopecia areata, ankylosing spondylitis, atopic dermatitis, Graves' disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes and vitiligo, inflammatory bowel disease, Crohn's disease, ulcerative colitis, childhood steatorrhea (celiac disease), eye diseases (e.g., uveitis), cutaneous lupus erythematosus and lupus nephritis, and PD-1 / PD-L1 antagonists. A bifunctional conjugated polypeptide, pharmaceutical composition, nucleic acid and / or vector for use according to claim 20, which is one of the autoimmune diseases in cancer patients caused by a gonist.

22. A method for treating an autoimmune disorder, comprising administering to a patient in need a bifunctional conjugated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15, a nucleic acid according to claim 16, and / or a vector according to claim 17.

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