TCR possesses high affinity and specificity for the preproinsulin peptide ALWGPDPAAA bound to HLA-A2*02.

Abstract

The present invention relates to a binding molecule comprising a peptide-major histocompatibility complex (pMHC) binding domain that binds to the ALWGPDPAAA (SEQ ID NO: 1) peptide (derived from human preproinsulin (PPI) protein) presented as a peptide-HLA-A*02 complex. The binding molecule may include a CDR sequence embedded within a framework sequence. The CDR and framework sequence may correspond to a T cell receptor (TCR) variable domain and may further contain non-natural mutations compared to the native TCR variable domain. The binding molecule of the present invention has high affinity and high specificity for the ALWGPDPAAA (SEQ ID NO: 1)-HLA-A*02 complex. The binding molecule may further include an immunosuppressant and / or half-life extension domain. Such a binding molecule is particularly useful in the development of soluble immunotherapeutic reagents for the treatment of autoimmune diseases such as diabetes.

Description

[Technical Field]

[0001] This invention relates to a binding molecule comprising a peptide-major histocompatibility complex (pMHC) binding domain containing a TCR variable domain. The binding molecule may further contain an immunosuppressant and / or half-life extension domain. This invention also relates to the use of the binding molecule in the treatment or diagnosis of autoimmune diseases such as diabetes. [Background technology]

[0002] Autoimmune diseases are often chronic and debilitating, representing an area of ​​clinical need. Given the potential serious adverse events associated with systemic immunosuppression, organ-specific immunosuppression, rather than systemic immunosuppression, may be a more beneficial therapeutic pathway.

[0003] In autoimmunity, there is accumulating evidence that dysfunction of the PD-1 pathway plays a crucial role in disease development. Polymorphisms in the PD-1, PD-L1, and PD-L2 genes are associated with several autoimmune diseases. Abnormally low PD-L1 expression has been observed in samples from patients with type 1 diabetes and Crohn's disease. Therefore, activating PD-1 on autoreactive lymphocytes may act as a mechanism to treat autoimmune diseases. However, few PD-1 agonists have reached the clinical trial stage, and their effectiveness in patients has not yet been demonstrated.

[0004] Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by metabolic dysfunction, particularly dysregulation of glucose metabolism, and is associated with characteristic long-term vascular and neurological complications. T1DM is characterized by absolute insulin deficiency, and patients rely on exogenous insulin for survival. Before the acute clinical onset of T1DM with hyperglycemic symptoms, there is a long asymptomatic preclinical period during which insulin-producing beta cells are gradually destroyed. Autoimmune destruction of beta cells (β cells) is associated with lymphocyte infiltration.

[0005] CD8 +Numerous pieces of evidence support the involvement of T cells in the pathogenesis leading to T1DM. Histological analysis of pancreatic islet tissue from affected individuals reveals CD8 + T cell infiltration is observed. In the animal model of T1DM, CD8 + It is possible to use T cells to transfer the disease process from an infected animal to a healthy animal.

[0006] Systemic immunotherapy based on anti-CD3 blocking antibodies has recently received FDA approval for the treatment of T1DM stage 2 disease. However, because this drug is non-targeted, there are high safety concerns, with lymphopenia being the main side effect in the early stages of treatment. Antigen-specific (or tissue-specific) immunotherapy for type 1 diabetes in the early stages after onset may halt disease progression and preserve residual islet cell function while avoiding systemic immune inactivation. Safe immunotherapy can also be considered for the protection of islet allogeneic grafts and for prevention in cases with a strong genetic predisposition to type 1 diabetes. Islet beta cells express Foxp3 and regulatory CD4 + It has been established that T cells (Tregs) provide natural protection from pathogenic T cells, and that recognition of islet cell antigens is necessary for protection mediated by adoptive T cells.

[0007] Several diabetes-specific human autoreactive CD8 + T cells have been isolated from patients (Skowera et al. 2008 J Clin Invest. 118:3390-402 and Lieberman et al. Proc Natl Acad Sci USA 2003 Jul 8;100(14):8384-8). These T cells primarily possess T cell receptors (TCRs) that recognize peptide epitopes of β-cell antigens such as preproinsulin (PPIs). ALWGPDPAAA 15-24The (SEQ ID NO: 1) peptide is one such peptide derived from the signal sequence of a human PPI (Skowera et al. 2008 J Clin Invest. 118:3390-402 and International Publication No. 2009004315). This peptide is loaded into the HLA-A*02 molecule and presented on the surface of insulin-producing β-cells. Thus, the ALWGPDPAAA-HLA-A*02 complex provides a human β-cell specific marker recognizable by the TCR. High expression of this PPI peptide is detectable on the β-cell surface regardless of disease stage, which means that PPI-targeted therapies may be effective at earlier disease stages compared to existing immunotherapies.

[0008] International Publication No. 2015092362 discloses TCRs and fusion molecules that bind to the ALWGPDPAAA peptide-HLA-A*02 complex. International Publication No. 2019219709 discloses TCRs that bind to the ALWGPDPAAA peptide-HLA-A*02 complex, as well as such TCRs fused with a PD1 agonist (natural ligand PDL1 or anti-PD1scFv). Curnock et al, 2021, JCI Insight. 2021;6(20):e15246 discloses a bispecific molecule consisting of a soluble TCR specific to the ALWGPDPAAA peptide-HLA-A*02 complex and an effector terminus containing a PD1 agonist.

[0009] While TCRs isolated from human donors are generally considered preferable from a safety standpoint, this is not necessarily the case for autoreactive TCRs, as TCRs that recognize self-antigens are likely to be eliminated during the thymic selection process. Furthermore, patient-derived autoreactive TCRs generally have lower affinity compared to TCRs specific to cancer antigens or pathogen antigens (Dolton G. et al., Frontiers Imm. 2018), which may be due to their inability to properly dock with HLA.

[0010] There remains a need for a potent, specific, and effective tissue-targeted immunosuppressive composition optimized for the diagnosis and treatment of autoimmune diseases, such as T1DM, that avoids the risks associated with systemic immunosuppression. Effective therapeutic agents for autoimmune diseases include those with favorable risk profiles (e.g., high levels of target and tissue specificity) and those that allow for reduced administration frequency.

[0011] The creation of TCRs engineered to have high affinity, especially when balanced with other desirable properties, is not easy and is usually accompanied by a high dropout rate. Firstly, those skilled in the art need to identify a suitable initiation sequence or scaffold sequence. Typically, such sequences are obtained from natural sources, e.g., antigen-responsive T cells extracted from donor blood, or from TCR libraries containing alpha and beta chains obtained from the natural repertoire. Since T cells specific to autoantigens such as PPIs are rare in the natural repertoire, it is often necessary to screen many donors, e.g., 20 or more, to find reactive T cells. This screening process can take weeks or months, and even if reactive T cells are found, they may not be suitable for use in immunotherapy. For example, the response may be too weak and / or not specific to the target antigen. Alternatively, it may not be possible to generate a clonal T cell population, or to grow or maintain a specific T cell line to produce enough material to identify the correct TCR chain sequence. Similarly, it may not be possible to identify antigen-specific TCRs from natural libraries. A TCR sequence suitable as a starter or scaffold sequence should possess one or more of the following characteristics: good affinity to the target peptide-HLA complex, e.g., ≥200 μM; high level of target specificity, e.g., relatively weak or no binding to alternative peptide-HLA complexes (which is particularly important in the treatment of autoimmune diseases); suitability for use in display libraries such as phage displays; ability to refold and / or purify in high yield from the associated expression system; and stability in the purified form containing the fusion protein. Given the degenerate nature of TCR recognition, determining whether a particular scaffold TCR sequence has a specificity profile suitable for manipulation for therapeutic applications is extremely difficult, even for experienced researchers (Wooldridge, et al., J Biol Chem. 2012 Jan 6;287(2):1168-77).

[0012] The next challenge is to engineer TCRs to have a higher affinity for a target antigen while maintaining desirable properties such as specificity and yield. This is because naturally occurring TCRs have a weaker affinity (in the low micromolar concentration range) for target antigens compared to antibodies. This weak affinity means that therapeutic TCRs for immunotherapy often require engineering to increase their affinity for the target antigen, thereby generating a strong response. Such affinity improvement is essential for soluble TCR-based reagents. In such cases, an antigen-binding affinity in the nanomolar to picomolar concentration range with a binding half-life of several hours is desirable. The improvement in the effect generated by high-affinity antigen recognition at low epitope numbers is shown in FIGS. 1e and 1f of Liddy et al. (Liddy, et al., Nat Med. 2012 Jun;18(6):980-7). In the affinity maturation process, typically, one skilled in the art needs to engineer specific mutations, including but not limited to substitutions, insertions, and / or deletions, to the starting TCR sequence to enhance the strength of antigen recognition. Affinity maturation techniques are known in the art and include, for example, the use of display libraries (Li et al., Nat Biotechnol. 2005 Mar;23(3):349-54; Holler et al., Proc Natl Acad Sci U S A. 2000 May 9;97(10):5387-92). However, to significantly enhance the affinity of a particular TCR for a specific target, one skilled in the art may sometimes have to engineer mutations from a large number of alternative candidates. The specific mutations that significantly enhance affinity are unpredictable, resulting in a high dropout rate. In many cases, it may be impossible to achieve a significant improvement in affinity with a particular TCR starting sequence.

[0013] In the process of affinity maturation, the need to maintain the antigen specificity of the TCR must also be considered. Increasing the affinity of the TCR for the target antigen brings a substantial risk of cross-reactivity with other unintended targets as a result of the degeneracy inherent in TCR antigen recognition (Wooldridge, et al., J Biol Chem. 2012 Jan 6;287(2):1168-77; Wilson, et al., Mol Immunol 2004,40(14-15):1047-55; Zhao et al., J Immunol 2007,179(9):5845-54). At natural levels of affinity, the recognition of cross-reactive antigens may be too low to elicit a response. If cross-reactive antigens are presented on normal healthy cells, there is a high likelihood of off-target binding in vivo, which can lead to clinical toxicity. Therefore, in addition to increasing antigen binding strength, those skilled in the art must engineer mutations and / or combinations of mutations that allow the TCR to retain high specificity for the target antigen and exhibit a favorable safety profile in preclinical trials. Again, the appropriate mutations and / or combinations of mutations are unpredictable. The dropout rate at this stage is even higher and in many cases may not be achievable at all from a particular TCR starting sequence.

Summary of the Invention

[0014] Binding molecule In a first aspect, the present invention provides a binding molecule comprising a peptide-major histocompatibility complex (pMHC) binding domain having the property of binding to the ALWGPDAAA (SEQ ID NO: 1) HLA-A*02 complex, wherein the pMHC binding domain comprises (i) an alpha chain comprising at least the variable domain of the TCR alpha chain, and (ii) a beta chain comprising at least the variable domain of the TCR beta chain, wherein (a) the variable domain of the TCR alpha chain has the following sequence: CDR1 - DKHSQG (SEQ ID NO: 23), optionally having one, two, or three mutations, CDR2 - IYSQGD (Sequence ID 27) is a sequence having any one, two, or three mutations. A sequence of CDR3-AVRGNEKLT (Sequence ID 7) having any one, two, or three mutations. Includes CDR1, CDR2, and CDR3, and / or (b) The TCR beta chain variable domain has the following sequence: A sequence of CDR1-LQHSY (sequence number 35) having any one, two, or three mutations, CDR2-SVGVGF (Sequence ID 29) is a sequence having one, two, or three mutations, A sequence of CDR3-ASAYMTGELF (Sequence ID 30) having any one, two, or three mutations. Includes CDR1, CDR2, and CDR3.

[0015] The inventors have, surprisingly, identified a binding molecule containing a TCR variable domain that exhibits particularly high affinity (picomolecular concentration range) and high antigen specificity for the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. When prepared as a soluble reagent fused with an immunosuppressive factor, the molecule exhibits potent protection against PPI-positive cells. Therefore, the molecule of the present invention has a profile particularly suitable for therapeutic applications. The specific binding molecule of the present invention is engineered from a suitable scaffold (i.e., "wild-type" or "natural-type") TCR sequence (including the alpha chain sequence of SEQ ID NO: 2 and the beta chain sequence of SEQ ID NO: 12), into which numerous mutations have been introduced to improve affinity, manufacturability, and / or stability while maintaining high specificity.

[0016] The conjugation molecules of the present invention differ from prior molecules shown to bind to the ALWGPDPAAA peptide-HLA-A*02 complex, such as those disclosed in International Publication No. 2015092362, International Publication No. 2019219709, and those described above by Curnock et al. For example, the conjugation molecules of the present invention exhibit improved specificity to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex and are produced in higher yields compared to the TCR disclosed in International Publication No. 2015092362 (see Example 3 hereof). Furthermore, certain conjugation molecules of the present invention have been engineered to have extended half-lives, making them suitable for the treatment of autoimmune diseases while overcoming the challenges of maintaining specificity and potency.

[0017] The therapeutic agent based on the conjugated molecule of the present invention is CD8 + To prevent destruction by T cells, immunosuppressive substances can be delivered to beta cells. Such immunosuppressive substances include antibody fragments or cytokines.

[0018] The binding molecule of the present invention can also be used in a therapeutic process known as adoptive therapy. For example, T regulatory cells (Tregs) transfected with MHC class I restrictive TCRs such as the binding molecule of the present invention may enhance the suppression of T effector cells compared to untransfected Tregs (Plesa et al. 2012 Blood. 119(15):3420-3430), and such cells have great potential in the treatment of autoimmune diseases (Wright et al. 2011 Expert Rev Clin Immunol. 7(2):213-25). Regulatory T cells (Tregs) make up a small fraction (5-10%) of the total number of CD4+ T lymphocytes (Powrie et al., (2003) Science 299(5609): 1030-1) and are characterized by constitutive expression of CD25 and Foxp3 transcription factors.

[0019] The conjugation molecule of the present invention can also be used as a diagnostic reagent to detect cells that present the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. In this case, the molecule can be fused with a detectable label.

[0020] To ensure effective targeting of β-cells presenting ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02, the binding molecule of the present invention may have improved binding affinity and / or binding half-life to the peptide-HLA complex. For use in therapeutic drug delivery or diagnostics, it is desirable that the specific binding molecule of the present invention has high affinity and / or a slow dissociation rate (off-rate) to the peptide-HLA complex. The inventors have also found that the binding molecule exhibits remarkably high specificity for the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex.

[0021] Peptide ALWGPDPAAA (SEQ ID NO: 1) corresponds to amino acids 15-24 of human preproinsulin (Uniprot P01308).

[0022] As used herein, the term “binding molecule” generally refers to a molecule capable of binding to one or more target antigens. A binding molecule may include one or more polypeptide chains. As used herein, the term “polypeptide chain” refers to a polymer (i.e., a chain) of amino acids, typically consisting of 20 or more amino acids linked by peptide bonds, with an N-terminus and a C-terminus. As is known in the art, proteins may comprise multiple polypeptide chains assembled by non-covalent or covalent interactions.

[0023] The binding molecule of the present invention comprises a "pMHC binding domain," which refers to a protein domain capable of binding to a peptide-MHC complex. The pMHC binding domain comprises (a) an alpha chain containing at least a TCR alpha chain variable domain, and (b) a beta chain containing at least a TCR beta chain variable domain. In this context, the term "alpha chain" refers to the region of the binding molecule containing the TCR alpha chain variable domain, and the term "beta chain" refers to the region of the binding molecule containing the TCR beta chain variable domain. The alpha and beta chains may be located on the same or different polypeptide chains within the binding molecule. The pMHC binding domain may be a TCR, for example, a soluble TCR, or may contain one.

[0024] The TCR alpha-chain variable domain and TCR beta-chain variable domain within the pMHC-binding domain each contain three CDRs and four framework regions, arranged as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is the framework region and CDR is the complementarity-determining region.

[0025] The alpha and beta chains may or may not contain the TCR constant domains described herein. The TCR variable domains bind to each other to form a TCR binding site capable of binding to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. Such molecules can take on a number of different forms, as discussed herein. Furthermore, fragments of the binding molecule of the present invention are also envisioned. A fragment refers to a portion of the binding molecule that retains binding ability to the target antigen.

[0026] The binding molecules of the present invention include TCR variable domains, which may correspond to those derived from natural TCRs, but more preferably the TCR variable domains may be engineered (i.e., containing mutations compared to the natural sequence). Natural TCR variable domains are also called wild-type, native, parental, unmutated, or scaffold domains. The binding molecules of the present invention may have ideal therapeutic properties such as hyperphysiological affinity to the target, a long binding half-life, high specificity to the target, and good stability. The present invention also includes multispecific (e.g., bispecific), multifunctional (e.g., bifunctional), or fusion molecules incorporating the TCR variable domains described herein and a therapeutic moiety, such as an immunosuppressant. These molecules include CD8 + By suppressing T cells, potent and specific protection against PPI-positive cells can be mediated. Furthermore, the use of binding molecules with hyperphysiological affinity facilitates the recognition of beta cells presenting low levels of target peptide-HLA complexes. Alternatively, the binding molecules may further contain other therapeutic and / or diagnostic agents (e.g., by fusion).

[0027] The binding molecule of the present invention may be in the form of a TCR comprising a TCR alpha-chain variable domain and a TCR beta-chain variable domain. The TCR may be a soluble TCR, i.e., a TCR that does not contain a transmembrane domain and does not contain an intracellular / cytoplasmic domain. The TCR domain sequence can be defined by referring to the widely known and accessible IMGT nomenclature for those involved in the TCR field. See, for example, LeFranc and LeFranc, (2001). “T cell Receptor Factsbook”, Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10; and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly, the αβTCR consists of two chains linked by a disulfide bond. Each chain (alpha and beta) is generally thought to have two domains: a variable domain and a constant domain. A short junction region connects the variable and constant domains, and this is usually considered part of the alpha variable region. In addition, the beta chain usually contains a short diversity region adjacent to the junction region, which is also typically considered part of the beta variable region. The variable domain of each chain is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in a framework sequence (FR). The CDRs contain the peptide-MHC binding recognition site. There are multiple genes encoding the alpha chain variable (Vα) region and multiple genes encoding the beta chain variable (Vβ) region, which are distinguished by the framework, CDR1 and CDR2 sequences, and a partially defined CDR3 sequence.The Vα and Vβ genes are called in the IMGT nomenclature with the prefixes TRAV and TRBV, respectively (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 also multiple joining genes or J genes, which are called TRAJ or TRBJ for the alpha chain and beta chain, respectively, and for the beta chain, the diversity gene or D gene is called TRBD (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). The vast diversity of the T cell receptor chains results from combinatorial rearrangements between various V genes, J genes, and D genes, including allelic variants, and junctional diversity (Arstila, et al., (1999), Science 286(5441): 958-961; Robins et al., (2009), Blood 114(19): 4099-4107). The constant regions or C regions of the TCR alpha and beta chains are called TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10).

[0028] The specific binding molecule of the present invention has a K for the ALWGPDPAAA (SEQ ID NO: 1)-HLA-A*02 complex D which is preferably greater (i.e., stronger) than that of the corresponding natural TCR (also called non-mutated or scaffold TCR). High affinity means K DA lower value indicates a stronger bond. For example, K D The concentration can be in the range of 1 pM to 50 μM. The binding molecule of the present invention is K for the target complex. D However, the K2 concentration may be in the range of approximately (i.e., ±10%) 1 pM to approximately 400 nM, approximately 1 pM to approximately 1000 pM, approximately 1 pM to approximately 500 pM, or approximately 1 pM to approximately 100 pM. The binding molecule may further, or instead, have a binding half-life (T1 / 2) in the range of approximately 0.5 minutes to approximately 50 hours, approximately 20 minutes to approximately 30 hours, or approximately 20 minutes to approximately 25 hours relative to the complex. Preferably, the binding molecule of the present invention has a K2 concentration of approximately 1 pM to approximately 100 pM relative to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. D It has and / or a binding half-life of about 5 to about 25 hours. Such high affinity is preferable for the binding molecule in its soluble form when bound to a therapeutic agent and / or a detectable label. The affinity of the binding molecule can be measured at 25°C, or at 37°C. A method for measuring the affinity of the binding molecule is described herein.

[0029] The conjugation molecule of the present invention, which contains a natural TCR variable domain, has K for the complex D The concentration may be approximately 1 μM to 200 μM, or approximately 1 μM to 100 μM. Such binding molecules may be preferable in adoption therapy applications.

[0030] Certain preferred mutant binding molecules exhibit substantially higher binding affinity and / or binding half-life to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex than the corresponding native TCR. Increasing the binding affinity of the native TCR may reduce its specificity to its peptide-MHC ligand; this has been demonstrated in Zhao et al., (2007) J. Immunol, 179:9, 5845-5854. However, the specific binding molecules of the present invention surprisingly exhibit high levels of specificity to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex while having substantially higher binding affinity than the native TCR.

[0031] The binding molecule of the present invention preferably has the property of specifically binding to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. In this specification, "specific" binding refers to a binding molecule that binds to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex with higher affinity than other peptide-HLA complexes. The highly specific binding molecule of the present invention is particularly suitable for therapeutic applications because it reduces the risk of off-target effects. The specificity of the binding molecule of the present invention can be determined by its ability to recognize antigen-positive target cells while having minimal ability to recognize antigen-negative target cells.

[0032] Specificity can be determined by evaluating the ability of the binding molecule to bind to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex with higher affinity than a panel of alternative peptide-HLA complexes. For example, this can be determined by surface plasmon resonance (SPR) as described herein, e.g., in Example 1. The panel may comprise at least two, at least three, at least five, or at least ten alternative peptide-HLA complexes. The alternative peptides may share low or high levels of sequence identity with SEQ ID NO: 1 and may be naturally presented in vivo. Preferably, the alternative peptides are derived from commonly expressed proteins and / or proteins expressed in healthy human tissues. Suitable alternative peptides having high sequence similarity to ALWGPDPAAA(SEQ ID NO: 1) include the “mimetics” described in Example 1 and provided in SEQ ID NOs: 67 and 88-91. The binding of the binding molecule to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex may be at least twice as strong as the binding to other naturally presented peptide-HLA complexes, more preferably at least 10 times, or at least 50 times, or at least 100 times, and even more preferably at least 1000 times stronger. Naturally occurring variants of the ALWGPDPAAA(SEQ ID NO: 1) peptide may be excluded from the definition of the alternative peptide-HLA complex.

[0033] An alternative or additional approach to determining the specificity of the binding molecule may be to identify the peptide recognition motif of the binding molecule using sequential mutagenesis of the target peptide, e.g., an alanine / serine substitution scan, as described in Example 1. The residues that form part of the binding motif are residues that cannot be substituted. An unacceptable substitution can be defined as a peptide position in which the binding affinity of the binding molecule is reduced by at least 50%, or preferably at least 80%, compared to the binding affinity to the non-mutant peptide. This approach is further described in Cameron et al. (2013), Sci Transl Med. 2013 Aug 7; 5(197): 197ra103 and International Publication No. 2014096803. In this case, the specificity of the binding molecule can be determined by identifying alternative motif-containing peptides, particularly alternative motif-containing peptides in the human proteome, and testing whether these peptides bind to the binding molecule. The binding of one or more alternative peptides by the binding molecule may indicate a lack of specificity. In this case, further validation of the specificity of the binding molecule by cell assays may be necessary. The low tolerance for (alanine / serine) substitution in the central portion of the peptide indicates that the TCR has high specificity and therefore a low risk of cross-reactivity with alternative peptides.

[0034] A binding molecule that has the property of binding to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex may bind to this complex with higher affinity compared to other peptide-HLA-A*02 complexes. The binding molecule may bind to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex with an affinity at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 10 times, at least 100 times, at least 500 times, or at least 1000 times higher than its affinity to the ALLGPDPAAA(SEQ ID NO: 67)-HLA-A*02 complex. The difference in affinity between the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex and the ALLGPDPAAA(SEQ ID NO: 67)-HLA-A*02 complex can be called the "affinity window". Notably, ALLGPDPAAA(SEQ ID NO: 67) differs from ALWGPDPAAA(SEQ ID NO: 1) by only one amino acid position. As shown in Example 4, the inventors identified a binding molecule that has low picomolar affinity for the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex and has an affinity window of at least 500 times (for SEQ ID NO: 67). Such a high-affinity and high-specific binding molecule is suitable for use, for example, as a soluble therapeutic agent.

[0035] The specific binding molecules of the present invention can in vitro bind to antigen-positive cells, particularly cells presenting low levels of antigen (i.e., on the order of 5-100), and generate highly potent anti-inflammatory responses such as CD8+ cell killing and / or CD4+ inflammation inhibition. Such binding molecules are in a soluble form and can be bound to immunosuppressive factors such as PD-1 agonists, or to interleukins or cytokines such as IL-2, IL-4, IL-10, or IL-13. The measured anti-inflammatory response may be CD8+ cell killing and / or CD4+ inflammation inhibition, as well as inhibition of the CD8+ T cell signaling pathway. Suitable methods for evaluating the anti-inflammatory response are known in the art and include the Jurkat NFAT cell reporter assay described in Example 4. Preferably, the IC in the pM range, i.e., ≤1000 pM. 50The response with a value is highly potent. Preferably, the maximum inhibition rate obtained in the reporter assay is greater than 50%, for example, 80% or more.

[0036] The term "mutation" is used synonymously with "modification" and includes designed substitutions, insertions, and deletions (e.g., manipulations or designed substitutions, insertions, and deletions). A "mutation" refers to a difference in the amino acid sequence and does not necessarily require the substitution of one amino acid with another. Mutations to the native (also called parent, natural, non-mutant, wild-type, or scaffold) binding molecule can confer beneficial therapeutic properties such as higher affinity, higher stability, higher specificity, and / or higher potency. For example, mutations can include the binding affinity (k) of the binding molecule to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. D This may include substances that increase the binding half-life (T1 / 2) and / or other factors.

[0037] In the context of this invention, the term “stability” refers to physical and chemical stability, which can be qualitatively and / or quantitatively assessed using various analytical techniques described in the Art and outlined, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Such methods include assessment of aggregate formation (e.g., using size exclusion chromatography (SEC)), measurement of turbidity (e.g., by dynamic light scattering (DLS) or light shielding (LO)), and / or visual inspection (e.g., by determining color and clarity). Stability may be assessed under stress conditions such as high temperatures (e.g., 37°C) or multiple freeze-thaw cycles (e.g., 3, 4, 5 or more).

[0038] In the binding molecule of the present invention, at least one mutation may be present in the TCR alpha chain variable region. Within the alpha chain CDR (i.e., in total across all three CDRs), there may be one, two, three, four, five, six, seven, eight, nine, ten, or more mutations. For example, there may be four mutations in the alpha chain CDR. There may be three mutations in alpha chain CDR1 and / or one mutation in alpha chain CDR2 and / or one mutation in alpha chain CDR3.

[0039] In the binding molecule of the present invention, mutations in the alpha chain CDR may be conserved, semi-conserved, acceptable, or otherwise phenotypically silent mutations, as described herein. Other suitable conserved, semi-conserved, acceptable, or otherwise phenotypically silent mutations will be apparent to those skilled in the art.

[0040] Mutations in the alpha chain CDR can be selected from K28R(CDR1), H29G(CDR1), G32S(CDR1), and Q53N(CDR2), which are numbered by Sequence ID No. 26. Therefore, any or all of these mutations may exist in any combination with other mutations.

[0041] A mutated alpha-chain variable domain can be paired with any beta-chain variable domain as defined herein.

[0042] Mutations in the beta-chain CDR may be conserved, semi-conserved, tolerable, or otherwise phenotypically silent, as described herein. Other suitable conserved, semi-conserved, tolerable, or otherwise phenotypically silent mutations will be apparent to those skilled in the art.

[0043] The TCR beta chain variable region of the binding molecule of the present invention may contain at least one mutation. The beta chain CDR (i.e., the total across all three CDRs) may contain one, two, three, four, five, six, seven, eight, nine, ten, or more mutations. For example, the beta chain CDR may contain six or seven mutations. Beta chain CDR1 may contain one, two, or three mutations, and / or beta chain CDR2 may contain two mutations, and / or beta chain CDR3 may contain one mutation.

[0044] Mutations in the beta-chain CDR can be selected from L27M(CDR1), Q28N(CDR1), S30N(CDR1), V52A(CDR2), F54I(CDR2), and A104S(CDR3), which are numbered by Sequence ID No. 74.

[0045] The mutated beta-chain variable domain can be paired with any alpha-chain variable region as defined herein.

[0046] Mutations in the CDR of the native sequence may improve the binding affinity or stability of the binding molecule of the present invention, but may also result in other benefits, such as improved specificity or efficacy when fused to an immunoeffector, either additionally or alternatively. Mutations may also reduce the risk of destabilization due to post-translational modifications such as deamidation. Mutations at one or more positions may also additionally or alternatively affect the interaction between the adjacent site and the corresponding pMHC complex, for example, by providing a more favorable angle of interaction. Mutations may include those that result in reduced nonspecific binding, i.e., mutations that result in reduced binding to surrogate antigens compared to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex. Mutations may also include those that improve folding and / or stability and / or manufacturability efficiency. Some mutations may contribute to all of these properties; others may contribute, for example, affinity but not specificity, or for example, specificity but not affinity, or stability but not affinity.

[0047] The binding molecule of the present invention may include any of the following combinations of TCR alpha chain variable domain CDRs and TCR beta chain variable domain CDRs: (a) The alpha-chain CDR1, CDR2, and CDR3 amino acid sequences of DRGSQS (SEQ ID NO: 5), IYSNGD (SEQ ID NO: 6), and AVRGNEKLT (SEQ ID NO: 7), respectively, and the beta-chain CDR1, CDR2, and CDR3 amino acid sequences of MNHNY (SEQ ID NO: 15), SVGAGI (SEQ ID NO: 16), and ASSYMTGELF (SEQ ID NO: 17), respectively; (b) The alpha-chain CDR1, CDR2, and CDR3 amino acid sequences of DKHSQG (SEQ ID NO: 23), IYSNGD (SEQ ID NO: 6), and AVRGNEKLT (SEQ ID NO: 7), respectively, and the beta-chain CDR1, CDR2, and CDR3 amino acid sequences of MNHSY (SEQ ID NO: 28), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30), respectively; (c) The alpha-chain CDR1, CDR2, and CDR3 amino acid sequences of DKHSQG (SEQ ID NO: 23), IYSNGD (SEQ ID NO: 6), and AVRGNEKLT (SEQ ID NO: 7), respectively, and the beta-chain CDR1, CDR2, and CDR3 amino acid sequences of MQHSY (SEQ ID NO: 32), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30), respectively; or (d) The alpha-chain CDR1, CDR2, and CDR3 amino acid sequences of DKHSQG (SEQ ID NO: 23), IYSQGD (SEQ ID NO: 27), and AVRGNEKLT (SEQ ID NO: 7), respectively, and the beta-chain CDR1, CDR2, and CDR3 amino acid sequences of LQHSY (SEQ ID NO: 35), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30), respectively.

[0048] The binding molecules preferably include the alpha-chain CDR1, CDR2, and CDR3 amino acid sequences of DKHSQG (SEQ ID NO: 23), IYSQGD (SEQ ID NO: 27), and AVRGNEKLT (SEQ ID NO: 7), respectively, as well as the beta-chain CDR1, CDR2, and CDR3 amino acid sequences of LQHSY (SEQ ID NO: 35), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30), respectively. These are the CDR sequences present in the TCR referred to as "a19b19," "a19b20," "a19b21," and "a19b22" in the examples.

[0049] Mutations may be introduced additionally or alternatively outside the CDR region and within the framework region; such mutations may result in improvements in therapeutic properties, such as increased affinity and / or specificity and / or stability of the binding molecule and / or yield of the purified soluble form. For example, the binding molecule of the present invention may contain one or more mutations, additionally or alternatively, at the N-terminus of FR1 (the first N-terminal framework region) of one or both of the alpha and beta chain variable domains, compared to the standard framework sequence for given TRAV and TRBV chains. Such mutations may improve the efficiency of N-terminal methionine cleavage. Removal of N-terminal initiator methionine is often important for protein function and stability. Insufficient cleavage can be detrimental to therapeutic agents because it can lead to heterogeneous protein products, and / or because the presence of initiator methionine may exhibit immunogenicity in humans. In some cases, initiator methionine may be present in the binding molecule of the present invention.

[0050] In the binding molecule of the present invention, the alpha-chain variable domain framework region may include the following sequence: FR1 - AKEVEQNSGPLSVPEGAIASLQCTYS (Sequence ID 25) is a sequence having one, two, or three mutations, FR2 - FFWYRQYSGKSPELIMS (Sequence ID 9) is a sequence having any one, two, or three mutations. FR3 - KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLC (Sequence ID 10) is a sequence having one, two, or three mutations, FR4 - FGTGTRLTIIP (Sequence ID 11) is a sequence having any one, two, or three mutations. and / or the beta-chain variable domain framework region contains the following sequence: A sequence of the form FR1 - NAGVTQTPKFRILKIGQSMTLQCAQD (Sequence ID 18) having any one, two, or three mutations, FR2 - MYWYRQDPGMGLKPIYY (Sequence ID 19) is a sequence having any one, two, or three mutations. FR3 - TDKGEVPQGYQVSRSTTEDFPLRLESAAPSQTSVYFC (Sequence ID 75) is a sequence having one, two, or three mutations, A sequence of the form FR4 - FGEGSRLTVL (sequence number 21) that optionally has one, two, or three mutations.

[0051] The alpha chain framework regions FR1, FR2, and FR3 may contain amino acid sequences corresponding to the TRAV12-2*02 chain, and / or the beta chain framework regions FR1, FR2, and FR3 may contain amino acid sequences corresponding to the TRBV6-6*02 chain.

[0052] The FR4 region may contain the junction regions for the alpha and beta variable chains (TRAJ and TRBJ, respectively). The TRAJ region may contain the amino acid sequence corresponding to TRAJ48*01. The TRBJ region may contain the amino acid sequence corresponding to TRBJ2-2*01.

[0053] The alpha chain variable domain framework region may have a total of 1, 2, 3, 4, 5 or more mutations compared to the above sequence. The alpha chain variable domain framework region may have 2 mutations compared to the above sequence. Mutations(s) in the TCR alpha chain variable domain framework region may be selected from A1Q and Q22N, numbered according to Sequence ID No. 26. The alpha chain variable domain framework region may not contain any mutations (other than those listed above).

[0054] The beta-chain variable domain framework region may have a total of 1, 2, 3, 4, 5 or more mutations compared to the above sequence. The beta-chain variable domain framework region may have one mutation compared to the above sequence. The mutation(s) in the TCR beta-chain variable domain framework region may be selected from Q62N, Q62E, Q62D, and Q65N, numbered according to Sequence ID No. 74. The beta-chain variable domain framework region may not contain any other mutations compared to the above sequence.

[0055] The TCR alpha chain variable domain of the binding molecule of the present invention may contain each framework amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with respect to SEQ ID NOs. 25, 9, 10, and 11. The TCR beta chain variable domain of the binding molecule of the present invention may contain each framework amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with respect to SEQ ID NOs. 18, 19, 75, and 21. Alternatively, the stated percentage identity may refer to the entire framework sequence as a whole.

[0056] The TCR alpha-chain variable domain may contain one of the amino acid sequences of SEQ ID NOs: 3, 22, 24, or 26, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with one of SEQ ID NOs: 3, 22, 24, or 26. The TCR beta-chain variable domain may contain one of the amino acid sequences of SEQ ID NOs: 13, 68, 31, 34, 74, 76, or 78, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with one of SEQ ID NOs: 13, 68, 31, 34, 74, 76, or 78. Since all alpha-chain variable domains and beta-chain variable domains originate from the same scaffolding TCR sequence (i.e., SEQ ID NOs: 2 and SEQ ID NOs: 12, respectively), all alpha-chain variable domain sequences are expected to be compatible with all beta-chain variable domain sequences. Therefore, the alpha chain variable domain may contain an amino acid sequence described in any one of SEQ ID NOs: 3, 22, 24, or 26, or an amino acid sequence having at least 90% identity with these, and the beta chain variable domain may contain an amino acid sequence described in any one of SEQ ID NOs: 13, 68, 31, 34, 74, 76, or 78, or an amino acid sequence having at least 90% identity with these.

[0057] The binding molecule may contain any of the following combinations of alpha-chain and beta-chain variable domains: (a) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 22, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 68; (b) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 24, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 31; (c) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 34; (d) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 74; (e) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 76; or (f) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 78.

[0058] Preferably, the alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 26[a19], and the beta chain variable domain comprises the amino acid sequence of SEQ ID NO: 74[b20]. In this regard, the present invention provides a binding molecule comprising a peptide-major histocompatibility complex (pMHC) binding domain having the property of binding to ALWGPDPAAA (SEQ ID NO: 1) complexed with HLA-A*02, wherein the pMHC binding domain comprises (i) an alpha chain comprising at least a TCR alpha chain variable domain, and (ii) a beta chain comprising at least a TCR beta chain variable domain, wherein the TCR alpha chain variable domain comprises the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 26, and the TCR beta chain variable domain comprises the amino acid sequence described in SEQ ID NO: 74, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 74.

[0059] In the binding molecule of the present invention, the variable domain, and if present, the constant domain and / or other domains, may be configured in any suitable form / sequence that enables antigen binding. Hereinafter, “form” of the binding molecule refers in particular to the defined spatial arrangement within the variable domain and any constant domain. Characteristics of such protein forms include the number of polypeptide chains (single polypeptide chain, double polypeptide chain, or multiple polypeptide chains), the type and length of linkers connecting different domains, the number of antigen-binding moieties (and therefore the number of binding valencies), the number of different antigen-binding moieties (and therefore the number of specificities for different antigens, e.g., bispecificity, polyspecificity), and the order and orientation of the variable domains (e.g., crossover, parallel). For example, the alpha and beta chains of the pMHC-binding domain may be arranged in a monoclonal TCR form, where the two chains are linked by disulfide bonds either within the constant domain or within the variable domain, or the variable domain is fused to one or more dimerizing domains. Alternatively, the variable domain may be arranged in a single polypeptide chain form with or without one or more constant domains, or the variable domain may be arranged in a diabody form. Other appropriate formats are also available.

[0060] The alpha and / or beta chains of the pMHC-binding domain may contain the TCR constant domain or a fragment thereof, e.g., the alpha chain TRAC constant domain and / or the beta chain TRBC1 or TRBC2 constant domain. Thus, the alpha chain may contain the TCR alpha chain constant domain, and / or the beta chain may contain the TCR beta chain constant domain. As those skilled in the art will understand, the terms TRAC and TRBC1 / 2 also encompass natural polymorphic variants, e.g., N to K at position 4 of TRAC (Bragado et al International immunology. 1994 Feb;6(2):223-30).

[0061] If present, one or both constant domains may contain mutations, substitutions, or deletions compared to the native constant domain sequence. The constant domain may be cleaved, i.e., lacking a transmembrane or cytoplasmic domain. Therefore, the terms “TCR alpha chain constant domain” and “TCR beta chain constant domain” include such cleaved amino acid sequences, as long as they retain a length sufficient to facilitate the binding of the alpha and beta chains from the native TCR constant region. For example, the binding molecule of the present invention may include the extracellular region of the TCR alpha chain constant domain and / or the extracellular region of the TCR beta chain constant domain. Alternatively, the constant domain may be full-length, meaning that the extracellular domain, transmembrane domain, and cytoplasmic domain are all present. The domain sequences of TRAC and TRBC may be modified by cleavage or substitution, and the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2 may be removed. The constant domain sequences of the alpha and / or beta chains may have disulfide bonds introduced between residues in each constant domain, as described, for example, in International Publication No. 03 / 020763. Therefore, the binding molecule may include unnatural covalent disulfide bonds linking residues in the TCR alpha chain constant domain to residues in the TCR beta chain constant domain. Preferably, the alpha and beta constant domains may be modified by substitution of cysteine ​​residues at the Thr48 position of TRAC and the Ser57 position of TRBC1 or TRBC2, where the cysteine ​​forms unnatural disulfide bonds between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may further include a cysteine-to-alanine mutation at position 75 of the constant domain and an asparagine-to-aspartic acid mutation at position 89 of the constant domain. For example, one or both of the extracellular constant domains present in an αβ heterodimer may be further cleaved at the C-terminus (or multiple C-terminus) by, for example, up to 15, up to 10, or up to 8 amino acids.For example, one or both of the extracellular constant domains present in an αβ heterodimer may be cleaved at the C-terminus (or multiple C-terminus) by, for example, up to 15, 10, or 8 amino acids. The C-terminus of the extracellular constant domain of the alpha chain may be cleaved by 8 amino acids.

[0062] The binding molecule of the present invention may include the extracellular region of the TCR alpha chain constant domain, which may be cleaved at the C-terminus by up to 15 amino acids, and / or the extracellular region of the TCR beta chain constant domain, which may be cleaved at the C-terminus by up to 15 amino acids.

[0063] The TCR alpha chain constant domain may contain the amino acid sequence described in SEQ ID NO: 37, or an amino acid sequence having at least 90% identity with the sequence described in SEQ ID NO: 37, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, and / or the TCR beta chain constant domain may contain the amino acid sequence described in SEQ ID NO: 39, or an amino acid sequence having at least 90% identity with the sequence described in SEQ ID NO: 39, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0064] The pMHC-binding domain may include an alpha chain containing the TCR alpha chain constant domain with the amino acid sequence described in SEQ ID NO: 37, and a beta chain containing the TCR beta chain constant domain with the amino acid sequence described in SEQ ID NO: 39. The binding molecule may not contain the transmembrane domain or cytoplasmic domain of the TCR.

[0065] Alternatively, the TCR constant domain may be absent, either in the case of a full-length or cleaved constant domain. Therefore, the pMHC-binding domain may consist of TCR alpha and beta variable domains and may have additional domains as described herein. These additional domains may include, but are not limited to, immunosuppressive domains (e.g., antibody domains), Fc domains or albumin-binding domains, therapeutic agents, or detectable labels.

[0066] The binding molecule can contain both an alpha and a beta chain in the form of a single polypeptide chain (i.e., the alpha and beta chains can reside on the same polypeptide chain). The single polypeptide chain form includes, but is 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 the TCRα and β variable regions, respectively, Cα and Cβ are the TCRα and β constant regions, 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; International Publication No. 2004 / 033685; International Publication No. 9918129).

[0067] As used herein, the term “linker” refers to one or more amino acid residues inserted between domains or between a domain and an agent, which provide sufficient mobility for a domain or element, such as the domain of the binding molecule of the present invention, to fold correctly to form an antigen-binding site. Each linker may be inserted at the amino acid sequence level between variable domains or between a variable domain and a constant domain (or other domain) in a transition region. The transition regions between domains can be identified from the fact that the approximate sizes of antibody domains and TCR domains are well known to those skilled in the art. The precise location of the domain transition region can be determined by locating peptide elongation regions that do not form secondary structural elements such as beta sheets or alpha helices, as demonstrated by experimental data or estimated by modeling or secondary structure prediction techniques.

[0068] Linker sequences are typically mobile, mainly because they consist of amino acids that do not have bulky side chains that could restrict mobility, such as glycine, alanine, and serine. Alternatively, a more rigid linker may be desirable. The usable or optimal length of a linker sequence can be easily determined. Linker sequences are often less than about 12, e.g., less than 10, or between 2 and 10 amino acids. Linkers can 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 acids long. Suitable linkers usable in the binding molecules of the present invention include, but are not limited to, the following: GGGGS (SEQ ID NO: 73), GGGSG (SEQ ID NO: 55), GGSGG (SEQ ID NO: 56), GSGGG (SEQ ID NO: 57), GSGGGP (SEQ ID NO: 58), GGEPS (SEQ ID NO: 59), GGEGGGP (SEQ ID NO: 60), GGEGGGSEGGGS (SEQ ID NO: 61), GGGSGGGG (SEQ ID NO: 62), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), EAAAK (SEQ ID NO: 65), and EAAAKEAAAKEAAAK (SEQ ID NO: 66).

[0069] A single polypeptide chain TCR may be soluble, i.e., it does not contain a transmembrane domain or a cytoplasmic domain. A single polypeptide chain TCR may have disulfide bonds introduced between residues of each constant domain, as described in International Publication No. 2004 / 033685. A single polypeptide chain TCR is further described in International Publication No. 2004 / 033685, International Publication No. 98 / 39482, International Publication No. 01 / 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.

[0070] Alternatively, the binding molecule may contain two or more polypeptide chains, with the alpha and beta chains being contained within separate polypeptide chains.

[0071] The TCR variable domain may be arranged in a diabody configuration. In the diabody configuration, two single polypeptide chain fragments form a dimer in a head-to-tail orientation, resulting in a compact molecule with a molecular weight similar to that of a tandem scFv (approximately 50 kDa).

[0072] Particularly suitable alpha chain sequences include, but are not limited to, one of sequence numbers 2, 70, 36, and 40. Particularly suitable beta chain sequences include, but are not limited to, one of sequence numbers 12, 69, 38, 41, 80, 81, and 82. These sequences do not contain transmembrane or cytoplasmic domains. All alpha chain sequences (i.e., sequence numbers 2, 70, 36, and 40) are expected to be compatible with all beta chain sequences (i.e., sequence numbers 12, 69, 38, 41, 80, 81, and 82) because they all originate from the same native (scaffolding) TCR sequence (sequence numbers 2 and 12, respectively). Therefore, the alpha chain may contain an amino acid sequence described in any one of SEQ ID NOs: 2, 70, 36, and 40, or an amino acid sequence having at least 90% identity with an amino acid sequence described in any one of SEQ ID NOs: 2, 70, 36, and 40, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, and The beta chain may contain an amino acid sequence described in any one of SEQ ID NOs: 12, 69, 38, 41, 80, 81, and 82, or an amino acid sequence having at least 90% identity with an amino acid sequence described in any one of SEQ ID NOs: 12, 69, 38, 41, 80, 81, and 82, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0073] More specifically, the pMHC binding domain is (a) an alpha chain containing the amino acid sequence of SEQ ID NO: 2 and a beta chain containing the amino acid sequence of SEQ ID NO: 12; (b) an alpha chain containing the amino acid sequence of SEQ ID NO: 70 and a beta chain containing the amino acid sequence of SEQ ID NO: 69; (c) Alpha chain containing the amino acid sequence of SEQ ID NO: 36 and beta chain containing the amino acid sequence of SEQ ID NO: 38; (d) an alpha chain containing the amino acid sequence of SEQ ID NO: 40 and a beta chain containing the amino acid sequence of SEQ ID NO: 41; (e) an alpha chain containing the amino acid sequence of SEQ ID NO: 40 and a beta chain containing the amino acid sequence of SEQ ID NO: 80; (f) an alpha chain containing the amino acid sequence of SEQ ID NO: 40 and a beta chain containing the amino acid sequence of SEQ ID NO: 81; or (g) May include an alpha chain containing the amino acid sequence of SEQ ID NO: 40 and a beta chain containing the amino acid sequence of SEQ ID NO: 82.

[0074] Preferably, the pMHC-binding domain includes an alpha chain containing the amino acid sequence of SEQ ID NO: 40 and a beta chain containing the amino acid sequence of SEQ ID NO: 80. In this regard, the present invention provides a binding molecule comprising a pMHC binding domain having the property of binding to ALWGPDPAAA (SEQ ID NO: 1) that has formed a complex with HLA-A*02, wherein the pMHC binding domain comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 40, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and a beta chain comprising the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 80, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0075] The conjugation molecules of the present invention are useful for delivering detectable labels or therapeutic agents to antigen-presenting cells and tissues containing antigen-presenting cells. Therefore, they may contain, or be conjugated (covalently or otherwise) a detectable label (for diagnostic purposes, used to detect the presence of cells that present the corresponding antigen in which the conjugation molecule is used), and / or a therapeutic agent including an immunoeffector, and / or a pharmacokinetic (PK) modifier.

[0076] Examples of PK modification include, but are not limited to, PEG (Dozier et al., (2015) Int J Mol Sci. Oct 28;16(10):25831-64 and Jevsevar et al., (2010) Biotechnol J. Jan; 5(1):113-28), PAS modification (Schlapschy et al., (2013) Protein Eng Des Sel. Aug;26(8):489-501), albumin and albumin-binding domains (Dennis et al., (2002) J Biol Chem. Sep 20;277(38):35035-43), and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec;27(12):1186-90). Furthermore, the PK modification portion includes an immunoglobulin Fc domain. The PK modification portion may play a role in extending the in vivo half-life of the binding molecule of the present invention. Therefore, the PK modification portion can function as a half-life extension domain.

[0077] The binding molecule may contain a half-life extension domain. As used herein, “half-life extension domain” refers to a protein domain that extends the half-life of the binding molecule of the present invention compared to a binding molecule lacking the corresponding half-life extension domain. For example, the half-life extension domain may include an immunoglobulin Fc domain. The immunoglobulin Fc domain can be any antibody Fc region. Thus, the binding molecule of the present invention may include an Fc region, for example, an IgG Fc region. The Fc region is the tail region of an antibody that interacts with cell surface Fc receptors and several proteins of the complement system. The Fc region typically consists of two polypeptide chains, i.e., two “parts” of FC1 and FC2, both of which have two or three heavy chain constant domains (called CH2, CH3, and CH4) and a hinge region. The two parts may be linked by one or more disulfide bonds within the hinge region. Fc regions derived from immunoglobulin subclasses IgG1, IgG2, and IgG4 bind to FcRn and extend the half-life of the binding molecule through FcRn-mediated recycling. The interaction between IgG and FcRn is localized within the Fc region that covers a portion of the CH2 and CH3 domains. Immunoglobulin Fc particularly suitable for use in the present invention includes, but is not limited to, Fc domains derived from IgG1 or IgG4. For example, the Fc region may be an IgG1 Fc region, i.e., the FC1 and FC2 regions may be IgG1 Fc sequences. The Fc region may be derived from a human sequence, for example, the wild-type human IgG1 Fc region (SEQ ID NO: 92). In this regard, the FC1 and FC2 regions may each contain, or consist of, an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 92.

[0078] The two portions of the Fc region may contain mutations compared to the wild-type or unmodified Fc sequence. Mutations include substitutions, insertions, and deletions. Such mutations may be introduced to introduce desired therapeutic properties. For example, a knob-in-hole (KiH) mutation may be manipulated in the CH3 domain to promote heterodimerization. Thus, the half-life extension domain may contain one or more amino acid substitutions that promote dimerization of the FC1 and FC2 regions. Such substitutions include "knob-in-hole" substitutions. In this case, one strand (i.e., one of the FC1 or FC2 regions) is manipulated to contain a bulky protruding residue (i.e., a knob) such as Y, and the other strand (i.e., the other of the FC1 and FC2 regions) is manipulated to contain a complementary pocket (i.e., a hole). For example, a knob can be constructed by substituting a small amino acid side chain for a large side chain. A hole can be constructed by substituting a large amino acid side chain for a small side chain. While not intended to be constrained by theory, this is thought to stabilize the heterodimer of the FC1 and FC2 regions by prioritizing the formation of heterodimers over other species, such as homomultimers of FC1 and FC2, thereby improving the stability and manufacturability of the binding molecule of the present invention.

[0079] Suitable locations and substitutions for KiH mutations, as well as other mutations that promote dimerization of the Fc region, are known in the art and include those described in Merchant et al., Nat Biotechnol 16:677 (1998), Ridgway et al., Prot Engineering 9:617 (1996), and Atwell et al. J Mol Biol 270,1 (1997): 26-35. For example, substitutions that form knobs and holes corresponding to two Fc regions may correspond to one or more paired combinations shown in the table below:

[0080] [Table 1]

[0081] In the table above, substitutions are listed in the following order: original residue, position using the EU numbering system, and then the introduced residue (all residues are indicated by a single-letter amino acid code). Multiple substitutions are separated by colons.

[0082] The FC1 and FC2 regions may contain one or more substitutions as shown in the table above. For example: (i) Either the FC1 region or the FC2 region may contain one or more amino acid substitutions selected from the group consisting of T366S, L368A, T394S, F405A, Y407A, Y407T, and Y407V according to the EU numbering system; and (ii) The other of the FC1 region and the FC2 region may contain one or more amino acid substitutions selected from the group consisting of T366W, T366Y, T366W, T394W, and F405W according to the EU numbering system. The substitutions in (i) and (ii) are hole-forming substitutions and knob-forming substitutions, respectively. The FC1 region may contain one or more of the substitutions in (i), and the FC2 region may contain one or more of the substitutions in (ii).

[0083] for example: (i) Either the FC1 region or the FC2 region may contain one or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V according to the EU numbering system; and (ii) The other of the FC1 and FC2 regions may contain the amino acid substitution T366W according to the EU numbering system. The FC1 region may contain one or more of the substitutions in (i), and the FC2 region may contain the substitution in (ii).

[0084] Preferably, (i) one of the FC1 region and the FC2 region contains the amino acid substitutions T366S, L368A, and Y407V according to the EU numbering system; and (ii) the other of the FC1 region and the FC2 region contains the amino acid substitution T366W according to the EU numbering system. For example, the FC1 region may contain the amino acid substitutions T366S, L368A, and Y407V according to the EU numbering system; and the FC2 region may contain the amino acid substitution T366W according to the EU numbering system.

[0085] The Fc region may contain one or more mutations that attenuate the effector function of the Fc region. Examples of effector functions include, but are not limited to, complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC). Modifications that attenuate effector function may be modifications that alter the glycosylation pattern of the Fc region, for example, modifications that result in a non-glycosylated Fc region. Alternatively, modifications that attenuate effector function may be modifications that do not alter the glycosylation pattern of the Fc region. Modifications that attenuate effector function may reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. FC1 and / or FC2 may contain one or more amino acid substitutions that prevent or reduce binding to FcγR. For example, FC1 and / or FC2 may contain one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A, D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A, and P331S according to the EU numbering system. Specific modifications include the N297G or N297A substitution (EU numbering) in the Fc region of human IgG1. Other suitable modifications include L234A, L235A, and P329G substitutions in the Fc region of human IgG1 (EU numbering), which result in attenuation of effector function. The FC1 and / or FC2 regions may include substitutions at the N297 residue, numbered by the EU index. For example, the substitution may be an N297G or N297A substitution. Other suitable mutations (e.g., mutations at the N297 residue) are known to those skilled in the art.

[0086] A reduced-effector Fc region refers to a mutant that reduces effector function (e.g., activity such as binding to CDC, ADCC, and / or FcR) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region that may have other mutations but does not have mutations that reduce effector function). A reduced-effector Fc mutant may also be an Fc mutant that has completely lost detectable effector function compared to a wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

[0087] In vitro and / or in vivo cytotoxic assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can confirm that an Fc region or fusion protein lacks FcγR binding (and is therefore likely to lack ADCC activity) but retains FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is outlined in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0088] The FC1 and FC2 regions may be subjected to substitutions that inhibit or reduce binding to the Fcγ receptor and / or increase binding to FcRn and / or prevent Fab arm replacement and / or remove the protease site. In this regard, the half-life extension domain may contain one or more amino acid substitutions that prevent or reduce binding to the activating receptor. The half-life extension domain may contain one or more amino acid substitutions that prevent or reduce binding to FcγR. For example, the FC1 and / or FC2 regions may contain the N297G amino acid substitution according to the EU numbering system. Both the FC1 and FC2 regions may also contain the N297G amino acid substitution.

[0089] The serum half-life of binding molecules containing an Fc region can be further extended by increasing the binding affinity of the Fc region to FcRn; therefore, half-life extension domains may include one or more modifications (e.g., amino acid substitutions, insertions, or deletions) that promote binding to FcRn. One or more modifications are compared to the corresponding wild-type Fc region (e.g., human IgG1 or IgG4 Fc region). Methods for measuring binding to FcRn are known. In vivo binding and serum half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered with polypeptides having mutant Fc regions. International Publication No. 2004 / 42072 (Presta) describes antibody substitutions that improve or decrease FcR binding. In particular, Mackness et al., MAbs. 11:1276-1288 (2019) describes appropriate amino acid substitutions in the antibody Fc region to enhance binding to FcRn.

[0090] As used herein, the term “half-life” refers to a pharmacokinetic property of a binding molecule, which is a measure of the mean survival time of the binding molecule after administration to a subject. The half-life of a binding molecule can be expressed as the time required for a known amount of the binding molecule to be removed by 50% from the subject’s body (or other mammal) or a specific site, for example, in the serum, i.e., by its circulating half-life, or by measurement in other tissues.

[0091] An increased half-life allows for a reduction in the amount of drug administered to the patient and a decrease in the frequency of administration. An increased half-life may also be beneficial in the treatment of autoimmune diseases or conditions, for example. It is also possible to generate binding proteins with increased half-lives by modifying amino acid residues identified as being involved in the interaction between Fc and the FcRn receptor. Binding proteins having an Fc region containing one or more modifications that promote binding to FcRn may have an increased half-life of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, or more compared to binding proteins containing the native Fc region. Binding proteins having an Fc region containing one or more modifications that promote binding to FcRn may have a half-life that is approximately 2, 3, 4, 5, 10, 20, 50 or more times longer than binding proteins containing a native Fc region, or they may have a half-life that is 2 to 10, 5 to 25, or 15 to 50 times longer.

[0092] Modifications (multiple) that promote binding to FcRn in the Fc region (e.g., amino acid substitutions, insertions, or deletions) are listed in Kabat and numbered according to the EU index: 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265 The Fc region may be one or more positions selected from the group consisting of 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. The Fc region may optionally contain non-natural amino acid residues at additional and / or alternative positions known in the art.

[0093] More specifically, the Fc region is numbered according to the EU index listed in Kabat: 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E , 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 2 52Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M , 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 2 65Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L , 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 2 96D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I , 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 3 16D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N , 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 3 28A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F , 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 33 1V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F,The substitution may include at least one selected from the group consisting of 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 434W. The Fc region is optional and may contain additional and / or alternative non-natural amino acid residues known in the art.

[0094] Modifications (multiple) that promote binding to FcRn in the Fc region (e.g., amino acid substitutions, insertions, or deletions) may be located at one or more positions selected from the group consisting of 234, 235, and 331, numbered according to the EU index described in Kabat. For example, the Fc region may contain at least one substitution selected from the group consisting of 234F, 235F, 235Y, and 331S, numbered according to the EU index described in Kabat.

[0095] Modifications (multiple) that promote binding to FcRn in the Fc region (e.g., amino acid substitutions, insertions, or deletions) may be located at one or more positions selected from the group consisting of 239, 330, and 332, numbered according to the EU index described in Kabat. For example, the Fc region may contain at least one substitution selected from the group consisting of 239D, 330L, and 332E, numbered according to the EU index described in Kabat.

[0096] Modifications that facilitate binding to FcRn in the Fc region (e.g., amino acid substitutions, insertions, or deletions) may be located at one or more positions selected from the group consisting of 252, 254, and 256, numbered according to the EU index described in Kabat. For example, the Fc region may contain at least one substitution selected from the group consisting of 252Y, 254T, and 256E, numbered according to the EU index described in Kabat, as described in U.S. Patent No. 7,083,784, the entirety of which is incorporated herein by reference. The Fc region may contain the following substitutions, numbered according to the EU index described in Kabat: 252Y, 254T, and 256E.

[0097] The substitutions listed above that promote binding to FcRn are compared to the corresponding wild-type Fc region (e.g., the Fc region of human IgG1 or IgG4) and may be present in one or preferably both of the FC1 and FC2 portions of the Fc region. In other words, these substitutions refer to amino acids that are not normally present in the corresponding wild-type Fc region, e.g., the human IgG1 or IgG4 Fc region. In this regard, as used herein, “substitution” refers to the presence of one of the listed amino acids in the polypeptide and does not necessarily require the substitution of one amino acid with another.

[0098] Additionally, or alternatively, mutations may be introduced for manufacturing reasons, for example, by removing or substituting amino acids that may be subject to post-translational modifications such as glycosylation, as described herein.

[0099] The two Fc regions, FC1 and FC2, may each contain all or part of the CH2 and CH3 constant domains and hinge sequences. The hinge sequences may substantially or partially correspond to the hinge regions of IgG1, IgG2, IgG3, or IgG4. The hinge sequence may be an IgG1 hinge sequence, such as the amino acid sequence described in SEQ ID NO: 50 or 95. Alternatively, the hinge may be an IgG4 hinge sequence, such as the amino acid sequence described in SEQ ID NO: 96. The preferred IgG hinge sequence is SEQ ID NO: 50. The hinge may contain all or part of the core hinge domain and all or part of the lower hinge region.

[0100] As described above, the binding molecule of the present invention may include a half-life extension domain comprising a first portion (FC1) of the IgG Fc region and a second portion (FC2) of the IgG1 Fc region. FC1 and FC2 dimerize to form an Fc region. The FC1 region may contain, or consist of, an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 49 or 93, and the FC2 region may contain, or consist of, an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 52 or 94. The FC1 region may contain, or consist of, an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 49 or 93, and the FC2 region may contain, or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 52 or 94. The FC1 region may contain or consist of the amino acid sequence described in SEQ ID NO: 49, and the FC2 region may contain or consist of the amino acid sequence described in SEQ ID NO: 52. Alternatively, the FC1 region may contain or consist of the amino acid sequence described in SEQ ID NO: 93, and the FC2 region may contain or consist of the amino acid sequence described in SEQ ID NO: 94.

[0101] As those skilled in the art will understand, the sequences described above for FC1 and FC2 may be interchangeable. For example, (a) either FC1 or FC2 may contain the amino acid sequence described in SEQ ID NO: 49, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence described in SEQ ID NO: 49, and (b) the other of FC1 and FC2 may contain the amino acid sequence described in SEQ ID NO: 52, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence described in SEQ ID NO: 52. Similarly, (a) either FC1 or FC2 may contain the amino acid sequence described in SEQ ID NO: 93, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence described in SEQ ID NO: 93, and (b) the other of FC1 and FC2 may contain the amino acid sequence described in SEQ ID NO: 94, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence described in SEQ ID NO: 94.

[0102] The Fc region may be fused to other domains of the binding molecule of the present invention (e.g., alpha or beta chains) via linker and / or hinge sequences in any suitable orientation. Alternatively, a linker may not be used. Preferred forms of binding molecules containing the Fc region are described below.

[0103] Alternatively, the half-life extension domain may be albumin or an albumin-binding domain. As is well known in the art, albumin has a long circulating half-life of 19 days, partly due to its size exceeding the renal threshold and specific interactions and recycling via FcRn. Binding to albumin is a well-known strategy to improve the in vivo circulating half-life of therapeutic molecules. Albumin can be bound non-covalently using a specific albumin-binding domain, or covalently by conjugation or direct gene fusion. Examples of therapeutic molecules that utilize albumin binding for half-life extension are shown in Sleep et al., Biochim Biophys Acta. 2013 Dec;1830(12):5526-34.

[0104] The albumin-binding domain is any portion capable of binding to albumin and includes any known albumin-binding portion. The albumin-binding domain may be selected from endogenous or exogenous ligands, small organic compounds, fatty acids, peptides, and proteins that specifically bind to albumin. Examples of preferred albumin-binding domains include short peptides such as those described in Dennis et al., J Biol Chem. 2002 Sep 20;277(38):35035-43 (e.g., peptide QRLMEDICLPRWGCLWEDDF), proteins engineered to bind albumin, such as antibodies, antibody fragments, and antibody-like scaffolds, such as Albudab® (commercially available from GSK, O'Connor-Semmes et al., Clin Pharmacol Ther. 2014 Dec;96(6):704-12) and Nanobody® (commercially available from Ablynx, Van Roy et al., Arthritis Res Ther. 2015 May 20;17:135); and proteins based on naturally occurring albumin-binding domains, such as Streptococcal G protein (Stork et al., Eng Des Sel. 2007). (Nov;20(11):569-76), for example, Albumod®, commercially available from Affibody. Preferably, the albumin is human serum albumin (HSA). The affinity of the albumin-binding domain for human albumin can range from picomolar to micromolar concentrations. Considering that the albumin concentration in human serum is extremely high (35-50 mg / ml, about 0.6 mM), it can be calculated that substantially all of the albumin-binding domains bind to albumin in vivo.

[0105] The albumin-binding moiety can be fused to the C-terminus or N-terminus of other domains (i.e., the TCR variable domain and / or the TCR constant domain and / or the immunoeffector domain) in any suitable order or configuration. The albumin-binding moiety can be fused to one or more of the other domains (i.e., the TCR variable domain and / or the TCR constant domain and / or the immunoeffector domain) via a linker. Suitable linkers are known in the art and include those described herein. When the albumin-binding moiety is fused to the TCR, it can be fused to either the alpha or beta chain, with or without a linker.

[0106] Diagnostically detectable labels include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

[0107] For several purposes, the binding molecules of the present invention can aggregate into complexes containing several binding molecules to form polyvalent binding molecule complexes. Numerous human proteins contain multimerizing domains that can be used to produce polyvalent binding molecule complexes. For example, the tetramerizing domain of p53 has been used to tetramerize scFv antibody fragments, resulting in increased serum persistence and a significantly reduced dissociation rate (off-rate) compared to monomeric scFv fragments (Willuda et al. (2001) J. Biol. Chem. 276(17) 14385-14392). Hemoglobin also possesses a tetramerizing domain and is capable of this type of application. The polyvalent binding molecule complexes of the present invention may exhibit improved binding ability compared to the non-multimeric natural-type (also called parent, natural, non-mutant wild-type, or scaffold) T cell receptor heterodimer of the present invention. Therefore, polyvalent complexes of the binding molecules of the present invention are also included in the present invention. Such polyvalent molecular complexes according to the present invention are particularly useful for tracking or targeting cells presenting specific antigens in vitro or in vivo, and are also useful as intermediates for the production of further polyvalent molecular complexes having such applications.

[0108] Examples of therapeutic agents associated with or constituting the binding molecules of the present invention include immunosuppressive factors, such as interleukins, cytokines, or immune checkpoint agonists. To ensure that the therapeutic effect is exerted at the desired site, the agent can be encapsulated in liposomes or other nanoparticle structures linked to the binding molecules, allowing the compound to be released slowly. This prevents adverse effects during transport in the body and ensures that the agent exerts its maximum effect after the binding molecules bind to the relevant antigen-presenting cells.

[0109] Examples of suitable therapeutic agents include, but are not limited to, antibodies or fragments thereof, such as immune checkpoint agonist antibodies (e.g., anti-PD-1 antibodies) or alternative protein scaffolds with antibody-like binding properties (e.g., DARPins). Other suitable therapeutic agents include ligands for immune checkpoint receptors, interleukins, or cytokines. IL-2, IL-4, IL-10, and IL-13 are examples of cytokines suitable for binding with the binding molecules of the present invention.

[0110] The binding molecules of the present invention may be polyspecific. As used herein, the term “polyspecific” refers to a binding molecule comprising two or more binding sites, each containing a pMHC binding domain. Such a binding molecule is capable of binding to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex and one or more other different targets. For example, the binding molecule may be bispecific. Such a binding molecule comprises a pMHC binding domain that binds to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex and another binding site (e.g., an antibody-antigen binding site) that binds to another target.

[0111] The binding molecules of the present invention may include immunosuppressive factors. As used herein, the term “immunosuppressive factor” refers to any molecule, such as a protein, that can inhibit an immune response, including inhibiting T cell activation. Immunosuppressive factors may bind to a target (e.g., an antigen). For example, an immunosuppressive factor may be an immune checkpoint agonist, i.e., a molecule that induces immune checkpoint signaling. Such immunosuppressive factors will be discussed later.

[0112] Immunosuppressive factors may contain antigen-binding moieties capable of binding to antigens. Antigens of immunosuppressive factors may localize on immune cells such as T cells. Binding molecules may include antibodies or their antigen-binding fragments. As used herein, the term “antibody” is intended to include conventional / natural antibodies and engineered antibodies, particularly functional antibody fragments, single-chain antibodies, single-domain antibodies, and bispecific or polyspecific antibodies. “Natural” or “conventional” refers to antibodies that have the same types of domains and domain sequences as naturally occurring antibodies and contain antibody-derived CDR and FR sequences. In natural / conventional antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Variable domains in both the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The binding site of conventional antibodies consists primarily of residues derived from the “antibody complementarity-determining region” (CDR) or hypervariable region. Occasionally, residues from the non-hypervariable region or framework region (FR) may affect the overall domain structure and, consequently, the binding site. CDRs refer to amino acid sequences that determine both the binding affinity and specificity of the natural Fv region of the native antibody binding site. Conventional antibodies have three CDRs each in their light and heavy chains, denoted as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, the antigen-binding site of a conventional antibody contains six CDRs, including the CDR sets derived from both the VH and VL regions.

[0113] "Engineered" antibody forms include functional antibody fragments, single-chain antibodies, single-domain antibodies, and chimeric antibodies, humanized antibodies, bispecific antibodies, or polyspecific antibodies. Engineered antibody forms also include TCR-derived CDRs, which may contain an additional 3, 2, or 1 N-terminal and / or C-terminal framework residues, or constructs in which an entire TCR-derived variable domain is grafted onto the heavy or light chain of an antibody. "Functional antibody fragment" refers to a portion of a full-length antibody that retains its ability to bind to a target antigen, or a protein similar to a portion of a full-length antibody, particularly the antigen-binding or variable region of a full-length antibody. Examples of functional antibody "fragments" include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies. For example, the binding molecule of the present invention may include scFv. Functional antibody fragments may also be single-domain antibodies, such as heavy-chain antibodies. In this regard, the term "single-domain antibody" refers to an antibody composed of a single antibody variable domain (e.g., a heavy-chain variable domain). Therefore, immunosuppressive factors can include, for example, VHH (i.e., the variable domain of a heavy chain antibody). As is known in the art, the antigen-binding site of a single-domain antibody such as VHH may contain three CDRs (in contrast to the six of a conventional four-chain antibody). As used herein, the term “antigen-binding portion of an antibody” includes such a binding site. Alternatively or additionally, the binding molecule may include a Fab or Fv fragment. The term “Fab” (“antigen-binding fragment”) refers to the antigen-binding fragment of an antibody, which includes the antibody light chain (VL-CL) and the variable domain and CH1 domain (VH-CH1) of the antibody heavy chain. The Fab fragment typically has a molecular weight of about 50,000 daltons. The Fv fragment is the N-terminal portion of the Fab fragment of an antibody and consists of one light chain (VL) and one heavy chain (VH) variable portion.

[0114] Immunosuppressive factors may contain a variable region (VH) and a variable region (VL) of the antibody heavy chain, which bind to form an antigen-binding moiety capable of binding to an antigen. Therefore, the antigen-binding moiety may contain VH and VL. For example, an immunosuppressive factor may contain an scFv that includes VH and VL.

[0115] Other suitable antigen-binding moieties include heavy chain antibodies (hcAb), single-domain antibodies (sdAb), minibodies (Tramontano et al (1994) J. Mol. Recognition 7, 9-24), variable domains of camelid heavy chain antibodies (VHH), variable domains of novel antigen receptors (VNAR), afibodies (Nygren PA (2008) FEBS J. 275, 2668-2676), alphabodies (see International Publication No. 2010066740), engineered ankyrin repeat domains (DARPins) (Stumpp et al (2008) Drug Discovery Today 13, 695-701), antikalin (Skerra et al (2008) FEBS J. 275, 2677-2683), and nottin (Kolmar et al (2008) FEBS J. 275, These are the 2684-2690) and the manipulated CH2 domain (nanoantibody, see Dimitrov DS (2009) mAbs 1, 26-28).

[0116] The antigen-binding portion is a heavy chain variable domain comprising, or essentially comprising, four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); or any suitable fragment (holding the antigen-binding site) of such a heavy chain variable domain, or may include such fragments. The antigen-binding portion may be a heavy chain antibody. The antigen-binding portion may be a heavy chain variable domain sequence of a conventional four-chain antibody, e.g., an antibody derived from a VH sequence of a human antibody, but not limited to human antibodies. Preferably, the antigen-binding portion is or includes a variable domain of a heavy chain antibody (e.g., a camelid antibody), e.g., VHH (also referred to herein as the "VHH domain"). Preferably, the antigen-binding portion is VHH.

[0117] As described herein, immunosuppressive factors may include antigen-binding moieties (e.g., antibody-antigen-binding moieties) that bind to antigens localized on immune cells. In the context of the present invention, “immune cells” may refer to, for example, T cells or B cells. In particular, the antigen of the antigen-binding moiety may be a T cell surface antigen.

[0118] The targets (i.e., antigens) of immunosuppressive factors can be immunomodulatory factors. For example, targets are immune checkpoint molecules such as PD-1 (Programmed Death 1 receptor), A2AR (adenosine A2A receptor), A2BR (adenosine A2B receptor), B7-H3 (B7 homolog 3, also known as CD276), B7-H4 (B7 homolog 4, also known as VTCN1), BTLA (B and T lymphocyte attenuation factor, also known as CD272), CTLA-4 (cytotoxic T lymphocyte-associated protein 4, also known as CD152), IDO (indoleamine 2,3-dioxygenase), CD200 receptor, KIR (killer cell immunoglobulin-like receptor), TIGIT (Ig and ITIM receptor) These may include the T cell immune receptor (containing the main), LAG3 (lymphocyte activation gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), TIM-3 (T cell immunoglobulin domain and mucin domain 3), VISTA (V domain Ig suppressor of T cell activation), SIGLEC7 (sialic acid-binding immunoglobulin lectin 7, also known as CD328), and SIGLEC9 (sialic acid-binding immunoglobulin lectin 9, also known as CD329). In this regard, immunosuppressive factors may be agonists of one or more of the above immune checkpoint molecules. Therefore, immunosuppressive factors may be immune checkpoint agonists (i.e., those that suppress immune activation). Suitable immune checkpoint agonists, including natural ligands and antibodies, are outlined, for example, in Paluch et al. Front Immunol, 2018, 9:2306.

[0119] Immunosuppressive factors may contain one of the receptor-ligand pairs instead of the antigen-binding portion of an antibody, thereby enabling the immunosuppressive factor to bind to the other of the receptor-ligand pairs. The target ligand or receptor may be localized on immune cells. For example, an immunosuppressive factor may contain the ligand of the immune checkpoint molecule described above. In particular, an immunosuppressive factor may contain the extracellular domain of PD-L1 (Uniprot ref: Q9NZQ7) or PD-L2 (Q9BQ51), or a functional fragment thereof (i.e., a portion capable of binding to PD-1). For example, an immunosuppressive factor may contain the amino acid sequence described in SEQ ID NO: 102, or an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO: 102. Such an immunosuppressive factor may associate with immune cells by binding to PD-1 and stimulate PD-1 signaling.

[0120] Alternatively, the immunosuppressive factor may include an agonist antibody that binds to an immune checkpoint molecule and preferably stimulates its signaling. For example, the immunosuppressive factor may be or contain a PD-1 agonist antibody (e.g., a single-domain antibody). Preferably, such a PD-1 agonist does not compete with PD-L1 for binding to PD-1. Preferably, such a PD-1 agonist has a high degree of specificity for PD-1 and elicits a potent inhibitory response when tested with the reporter assay described in Example 4. The PD-1 agonist may be a full-length antibody or a fragment thereof, e.g., an scFv antibody or Fab fragment, or a single-domain antibody. Examples of such antibodies are described in International Publication Nos. 2011110621, 2010029434, and 2018024237. Thus, the antigen of the immunosuppressive factor may be PD-1, and the antigen-binding portion of the immunosuppressive factor may be a PD-1 agonist. The antigen-binding portion of the immunosuppressive factor may include a single-domain antibody, and optionally a VHH. Preferably, the immunosuppressive factor is a PD-1 agonist VHH.

[0121] As described above, the immunosuppressive factor is preferably a PD-1 agonist. As used herein, the term “PD-1 agonist” refers to any molecule that can bind to PD-1 and activate PD-1 signaling, including, for example, PD-1 ligands, PD-L1, and PD-1 agonist antibodies. Activation of the PD-1 pathway downregulates immune activity, promotes 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 an immune receptor tyrosine-based repressive motif (ITIM). PD-L1 and PD-L2 are native ligands for PD-1 and are 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). Ligand association recruits phosphatases 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 T cell apoptosis through its ability to inhibit co-stimulatory survival signals (Keir et al., Annu Rev Immunol, 26:677-704, 2008). Therefore, activation targeting the PD-1 pathway offers a therapeutic approach for autoimmune conditions such as T1DM.

[0122] The antigen-binding portion of immunosuppressive factors has the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. In this regard, the antigen-binding portion may be a single-domain antibody that binds to PD-1, and includes CDR1, CDR2, and CDR3, which have the following amino acid sequences: CDR1 - GFTFSSYA (Sequence ID 43) is a sequence having any one, two, or three mutations. CDR2 - IASDGAST (Sequence ID 44) is a sequence having one, two, or three mutations, and CDR3 - CARGGYLTYDRY (SEQ ID NO: 45) is a sequence having one, two, or three mutations. Preferably, CDR1 includes the amino acid sequence described in SEQ ID NO: 43, CDR2 includes the amino acid sequence described in SEQ ID NO: 44, and CDR3 includes the amino acid sequence described in SEQ ID NO: 45. Preferably, the antigen-binding moiety is VHH.

[0123] A single-domain antibody may be a VHH containing the amino acid sequence of SEQ ID NO: 42, or a humanized version thereof, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 42. Alternatively, a single-domain antibody may be a VHH containing the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 71.

[0124] Preferably, the single-domain antibody is a VHH containing the amino acid sequence described in SEQ ID NO: 71.

[0125] Immunosuppressive factors may be covalently linked to the pMHC-binding domain, optionally via a linker sequence, via the C-terminus or N-terminus of the alpha or beta chain, if present. For example, the C-terminus of an immunosuppressive factor may be covalently linked to the N-terminus of the beta chain, optionally via a linker sequence. Suitable linker sequences are known in the art. Linker sequences are usually mobile, as they consist mainly of amino acids that do not have bulky side chains that may restrict mobility, such as glycine, alanine, and serine. Alternatively, a more rigid linker may be desirable. The length of a usable or optimal linker sequence can be easily determined. Linker sequence lengths are often less than about 12, e.g., less than 10, or 2 to 10 amino acids. For example, the immunosuppressant may be covalently linked to the pMHC-binding domain via a linker sequence selected from GGGGS (SEQ ID NO: 73), GGGSG (SEQ ID NO: 55), GGSGG (SEQ ID NO: 56), GSGGG (SEQ ID NO: 57), GSGGGP (SEQ ID NO: 58), GGEPS (SEQ ID NO: 59), GGEGGGP (SEQ ID NO: 60), GGEGGGSEGGGS (SEQ ID NO: 61), GGGSGGGG (SEQ ID NO: 62), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), EAAAK (SEQ ID NO: 65), and EAAAKEAAAKEAAAK (SEQ ID NO: 66) via the C-terminus or N-terminus of the alpha or beta chain.

[0126] Immunosuppressive factors can be covalently linked to the C-terminus or N-terminus of the alpha or beta chain via the GGGGS (SEQ ID NO: 73) linker sequence. For example, the C-terminus of an immunosuppressive factor can be covalently linked to the N-terminus of the beta chain via the GGGGS (SEQ ID NO: 73) linker sequence.

[0127] The binding molecule containing the immunosuppressive factor may contain at least a first polypeptide chain and a second polypeptide chain. For example, the binding molecule may: (a) A first polypeptide chain containing an immunosuppressant and a beta chain of a pMHC-binding domain; and (b) It may contain a second polypeptide chain including an alpha chain of the pMHC binding domain. The C-terminus of the immunosuppressor may optionally be covalently linked to the N-terminus of the beta chain via the linker sequence of SEQ ID NO: 73.

[0128] The first polypeptide may contain the structure N-IS-Beta-C, and the second polypeptide may contain the structure N-Alpha-C, where "IS" refers to an immunosuppressant, "Beta" to the beta chain of the pMHC-binding domain, and "Alpha" to the alpha chain of the pMHC-binding domain. The immunosuppressant may be as described above, and may include, for example, scFv or VHH. The first and / or second polypeptide chain(s) may or may not contain other polypeptide sequences at the N-terminus or C-terminus.

[0129] As described above, representative binding molecules of the present invention that contain two polypeptides include a2b3VHH (consisting of SEQ ID NOs. 70 and 72), a18b16VHH (consisting of SEQ ID NOs. 36 and 46), a19b19VHH (consisting of SEQ ID NOs. 40 and 47), a19b20VHH (consisting of SEQ ID NOs. 40 and 83), a19b21VHH (consisting of SEQ ID NOs. 40 and 84), and a19b22VHH (consisting of SEQ ID NOs. 40 and 85).

[0130] As described above, the binding molecule containing the two polypeptides includes a first polypeptide chain containing an amino acid sequence having at least 90% identity with the amino acid sequence described in any one of SEQ ID NOs: 72, 46, 47, 83, 84, and 85, or an amino acid sequence having at least 90% identity, for example, at least 90%, at least 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% identity with the amino acid sequence described in any one of SEQ ID NOs: 72, 46, 47, 83, 84, and 85, and The second polypeptide chain may include an amino acid sequence described in any one of SEQ ID NOs. 70, 36, and 40, or an amino acid sequence having at least 90% identity with the amino acid sequence described in any one of SEQ ID NOs. 70, 36, and 40, for example, at least 90%, at least 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% identity.

[0131] More specifically, the above type of bonded molecule is, (a) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 72, and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 70; (b) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 46, and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 36; (c) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 47, and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 40; (d) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83, and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 40; (e) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84, and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 40; or (f) The polypeptide chain may include a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 85 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 40.

[0132] Preferably, the binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence described in SEQ ID NO: 83, and the second polypeptide chain comprises the amino acid sequence described in SEQ ID NO: 40. Thus, the present invention provides a binding molecule having the property of binding to ALWGPDPAAA (SEQ ID NO: 1) that forms a complex with HLA-A*02, wherein the binding molecule comprises a first polypeptide chain comprising a TCR beta chain covalently linked to a PD-1 agonist VHH, and a second polypeptide chain comprising a TCR alpha chain, wherein the first polypeptide chain comprises the amino acid sequence described in SEQ ID NO: 83, and the second polypeptide chain comprises the amino acid sequence described in SEQ ID NO: 40.

[0133] As described above, a binding molecule containing two polypeptide chains (i.e., the first and second polypeptide chains) may further contain one or more other polypeptide chains (e.g., a third polypeptide chain). Therefore, the binding molecule of the present invention may contain three polypeptide chains, i.e., the first, second, and third polypeptide chains. Such a binding molecule may include one containing an immunoglobulin Fc region comprising a first portion FC1 and a second portion FC2, as described herein. For example, a binding molecule may be: (a) an immunosuppressive factor and a first polypeptide chain comprising either (i) an alpha chain or (ii) a beta chain of a pMHC-binding domain; (b) FC1 and a second polypeptide chain comprising (i) the alpha chain and (ii) the other of the beta chain; and (c) May include a third polypeptide chain containing FC2.

[0134] The FC1 and FC2 regions may have one or more of the characteristics described above with respect to the half-life extension domain.

[0135] The C-terminus of the immunosuppressant may optionally be covalently linked to the N-terminus of either the (i) alpha chain or the (ii) beta chain of the pMHC-binding domain via a linker sequence. The linker sequence may be, for example, SEQ ID NO: 73. The other C-terminus of the (i) alpha chain and the other C-terminus of the (ii) beta chain may be covalently linked to the N-terminus of FC1. For example, the other C-terminus of the (i) alpha chain and the other C-terminus of the (ii) beta chain may be covalently linked to the N-terminus of FC1 via an IgG hinge sequence. A third polypeptide may contain an IgG hinge sequence at the N-terminus of FC2. This IgG hinge may contain the amino acid sequence of SEQ ID NO: 50.

[0136] The first polypeptide chain may contain a beta chain of the pMHC-binding domain, and the second polypeptide chain may contain an alpha chain of the pMHC-binding domain. Therefore, the C-terminus of the immunosuppressant may optionally be covalently linked to the N-terminus of the beta chain of the pMHC-binding domain via a linker sequence such as SEQ ID NO: 73. The C-terminus of the alpha chain may be covalently linked to the N-terminus of FC1. For example, the C-terminus of the alpha chain may be covalently linked to the N-terminus of FC1 via an IgG hinge sequence such as SEQ ID NO: 50.

[0137] The inventors have confirmed that the “3-chain” form of the molecule described above exhibits the highest activity (i.e., potency and selectivity) among more than 20 different forms tested. In this context, “3-chain” is used to describe a binding molecule that is expressed as three distinct polypeptide chains and binds to each other to form a single three-dimensional folded structure containing i) a pMHC-binding domain (formed by dimerization of the alpha and beta chains), ii) an immunosuppressant (e.g., VHH), and iii) a half-life extension domain including an Fc region. For the binding molecule of the form described immediately above, it is preferable that the first polypeptide chain contains the beta chain of the pMHC-binding domain and the second polypeptide chain contains the alpha chain of the pMHC-binding domain. Such a molecule has the following general structure: - First polypeptide chain: N-IS-Beta-C; - Second polypeptide chain: N-Alpha-FC1-C; and - Having a third polypeptide chain: N-FC2-C, Or, more specifically, the following general structure: - First polypeptide chain: N-IS-linker-Beta-C; - Second polypeptide chain: N-Alpha-Hinge-FC1-C; and - It has a third polypeptide chain: N-hinge-FC2-C.

[0138] Here, "N" and "C" represent the N-terminus and C-terminus of each polypeptide chain, respectively; "IS" is an immunosuppressant; "Alpha" is the alpha chain of the pMHC-binding domain; "Beta" is the beta chain of the pMHC-binding domain; "Linker" is the linker sequence described herein (preferably SEQ ID NO: 73); and "Hinge" is the IgG hinge sequence described herein (SEQ ID NO: 50). In the context of the above three-chain linking molecule, the alpha and beta chains preferably do not include a transmembrane region or a cytoplasmic region.

[0139] Appropriate linker sequences are described above. For example, if present, the linker (i.e., the linker of the first and / or second polypeptide chain) may have an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO: 73), GGGSG (SEQ ID NO: 55), GGSGG (SEQ ID NO: 56), GSGGG (SEQ ID NO: 57), GSGGGP (SEQ ID NO: 58), GGEPS (SEQ ID NO: 59), GGEGGGP (SEQ ID NO: 60), GGEGGGSEGGGS (SEQ ID NO: 61), GGGSGGGG (SEQ ID NO: 62), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), EAAAK (SEQ ID NO: 65), and EAAAKEAAAKEAAAK (SEQ ID NO: 66). Preferably, the C-terminus of the immunosuppressor is linked to the N-terminus of the beta chain of the first polypeptide chain via a linker having the amino acid sequence GGGGS (SEQ ID NO: 73).

[0140] Appropriate IgG hinge sequences are described above. For example, if present, the hinge (e.g., the hinge of the first and / or second and / or third polypeptide chain) may have the amino acid sequence described in SEQ ID NO: 50, or an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 50. Preferably, the C-terminus of the alpha chain is ligated to the N-terminus of FC1 of the second polypeptide chain via an IgG hinge having the amino acid sequence of SEQ ID NO: 50. Alternatively, the IgG hinge having the amino acid sequence of SEQ ID NO: 50 may be located at the N-terminus of the third polypeptide chain (i.e., the N-terminus of FC2).

[0141] The binding molecule of the present invention may include a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, where, a) The first polypeptide comprises, from the N-terminus to the C-terminus, a VHH (preferably a PD1 agonist VHH), a linker sequence (preferably SEQ ID NO: 73), and a beta chain of a pMHC binding domain. b) The second polypeptide comprises, from the N-terminus to the C-terminus, an alpha chain of the pMHC-binding domain, an IgG hinge (preferably SEQ ID NO: 50), and a first portion (FC1) of the Fc region, c) The third polypeptide comprises an IgG hinge (preferably SEQ ID NO: 50) and a second portion (FC2) of the Fc region from the N-terminus to the C-terminus.

[0142] The first polypeptide chain may contain an amino acid sequence described in any one of SEQ ID NOs. 72, 46, 47, 83, 84, and 85, or an amino acid sequence having at least 90% identity with an amino acid sequence described in any one of SEQ ID NOs. 72, 46, 47, 83, 84, and 85, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity; and / or, The second polypeptide chain may contain an amino acid sequence described in any one of SEQ ID NOs: 48, 53, 54, 97, 99, or 100, or an amino acid sequence having at least 90% identity with the amino acid sequence described in any one of SEQ ID NOs: 48, 53, 54, 97, 99, or 100, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity; and / or The third polypeptide chain may include the amino acid sequence of SEQ ID NO: 51 or 98, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 51 or 98, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0143] More specifically, the binding molecules are: (a) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 72, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 48, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 51; (b) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 46, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 53, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 51; (c) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 47, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 54, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 51; (d) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 54, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 51; (e) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 54, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 51; (f) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 85, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 54, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 51; (g) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 72, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 97, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 98; (h) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 46, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 99, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 98; (i) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 47, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 100, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 98; (j) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 100, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 98; (k) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 100, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 98; or (l) It may include a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 85, a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 100, and a third polypeptide chain containing the amino acid sequence of SEQ ID NO: 98.

[0144] For example, the first, second, and third polypeptide chains have the following amino acid sequences: i) Sequence IDs 72, 48, and 51; ii) Sequence IDs 46, 53, and 51; iii) Sequence ID 47; Sequence ID 54 and Sequence ID 51; iv) Sequence ID 83; Sequence ID 54 and Sequence ID 51; v) Sequence ID 84; Sequence ID 54 and Sequence ID 51; vi) Sequence ID 85; Sequence ID 54 and Sequence ID 51; vii) Sequence ID 72; Sequence IDs 97 and 98; viii) Sequence ID 46; Sequence ID 99 and Sequence ID 98; ix) Sequence ID 47; Sequence ID 100 and Sequence ID 98; x) Sequence ID 83; Sequence ID 100 and Sequence ID 98; xi) Sequence ID 84; Sequence ID 100 and Sequence ID 98; or xii) Sequence ID 85; Sequence ID 100 and Sequence ID 98, Alternatively, they may include sequences that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with these.

[0145] The binding molecule is, a) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 83; b) A second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 54; and c) The molecule may include a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 51.

[0146] The binding molecule is, a) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 83; b) A second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 100; and c) The molecule may include a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 98, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 98.

[0147] The binding molecule is, a) A first polypeptide chain having the following amino acid sequence: AVQLVESGGG LVQPGGSLRL SCAASGFTFS SYAMTWVRQA PGKGPEWVSA IASDGASTSY ADSVKGRFTI SRDNSKNTLY LQMNSLRPED TAVYYCARGG YLTYDRYGQG TLVTVSSGGG GSNAGVTQTP KFRILKIGQS MTLQCAQDLQ HSYMYWYRQD PGMGLKPIYY SVGVGFTDKG EVPQGYQVSR STTEDFPLRL ESAAPSQTSV YFCASAYMTG ELFFGEGSRL TVLEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV NGKEVHSGVC TDPQPLKEQP ALQDSRYALS SRLRVSATFW QDPRNHFRCQ VQFYGLSEND EWTQDRAKPV TQIVSAEAWG RAD(sequence number 83); b) A second polypeptide chain having the following amino acid sequence: AKEVEQNSGP LSVPEGAIAS LQCTYSDKHS QGFFWYRQYS GKSPELIMSI YSQGDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRGNEKLTFG TGTRLTIIPN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TQVSQSKDSD VYITDKCVLD MRSMDFKSNS AVAWSQKSDF ACANAFQNSI IPEDTDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YGSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK(SEQ ID NO: 54); and c) A third polypeptide chain having the following amino acid sequence: DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (Sequence ID 51) It may include.

[0148] Furthermore, the present invention also provides a binding molecule having the property of binding to ALWGPDAAA (SEQ ID NO: 1) that has formed a complex with HLA-A*02, wherein the binding molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, each containing the amino acid sequences described in SEQ ID NO: 47, SEQ ID NO: 54, and SEQ ID NO: 51, respectively.

[0149] The binding molecule is, a) A first polypeptide chain having the following amino acid sequence: AVQLVESGGG LVQPGGSLRL SCAASGFTFS SYAMTWVRQA PGKGPEWVSA IASDGASTSY ADSVKGRFTI SRDNSKNTLY LQMNSLRPED TAVYYCARGG YLTYDRYGQG TLVTVSSGGG GSNAGVTQTP KFRILKIGQS MTLQCAQDLQ HSYMYWYRQD PGMGLKPIYY SVGVGFTDKG EVPQGYQVSR STTEDFPLRL ESAAPSQTSV YFCASAYMTG ELFFGEGSRL TVLEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV NGKEVHSGVC TDPQPLKEQP ALQDSRYALS SRLRVSATFW QDPRNHFRCQ VQFYGLSEND EWTQDRAKPV TQIVSAEAWG RAD(sequence number 83); b) A second polypeptide chain having the following amino acid sequence: AKEVEQNSGP LSVPEGAIAS LQCTYSDKHS QGFFWYRQYS GKSPELIMSI YSQGDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRGNEKLTFG TGTRLTIIPN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TQVSQSKDSD VYITDKCVLD MRSMDFKSNS AVAWSQKSDF ACANAFQNSI IPEDTDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLYITR EPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YGSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK(sequence ID 100); and c) A third polypeptide chain having the following amino acid sequence: DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (Sequence ID 98) It may include.

[0150] Furthermore, the present invention provides a binding molecule having the property of binding to ALWGPDAAA (SEQ ID NO: 1), which forms a complex with HLA-A*02, wherein the binding molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, each containing the amino acid sequences described in SEQ ID NO: 83, SEQ ID NO: 100, and SEQ ID NO: 98, respectively.

[0151] The binding molecule of the present invention is preferably composed of a protein. The binding molecule may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinks, etc., or converted to an acid addition salt, and / or optionally dimerized or polymerized, or conjugated. All of these forms are encompassed in the present invention.

[0152] The binding molecule may contain one or more substitutions that remove one or more glycosylation sites, such as N-linked glycosylation sites, in the TCR alpha-chain variable domain or constant domain and / or TCR beta-chain variable domain or constant domain. The substitutions in this context are compared to natural (e.g., wild-type) TCR (e.g., including the alpha chain of SEQ ID NO: 2 and the beta chain of SEQ ID NO: 12). For example, the binding molecule may contain a completely deglycosylated (i.e., non-glycosylated) TCR chain, i.e., the alpha and beta chain sequences may lack N-linked glycosylation sites. In this regard, we have surprisingly found that a completely deglycosylated TCR sequence increases potency compared to a completely glycosylated equivalent sequence.

[0153] The binding molecule may be synthesized, recombinant, isolated, manipulated, and / or purified. “Purified” means that the molecule exists substantially free of other biomacromolecules of the same kind with respect to the polypeptide or nucleotide sequence. In this specification, “purified” means that at least 75%, 85%, 95%, or 98% (by weight) of the biomacromolecule of the same kind is the molecule. A purified nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide in question; however, the molecule may contain additional bases or portions that do not adversely affect the basic properties of the composition.

[0154] The binding molecule may be isolated. The term “isolated” means a state that has been altered or removed from its natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide that has been partially or completely separated from coexisting material in its natural state is “isolated.” Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as in a host cell. An isolated binding molecule substantially does not contain other binding molecules with different antigen specificities. Furthermore, an isolated binding molecule may substantially not contain other cellular material and / or chemicals.

[0155] The conjugated molecule may be a recombinant. A "recombinant" molecule refers to a molecule prepared, expressed, produced, or isolated by recombinant means. In this respect, recombinant molecules do not exist naturally.

[0156] Single-domain antibody Furthermore, the present invention provides single-domain antibodies comprising CDR1, CDR2, and CDR3, which bind to PD-1 and have the following amino acid sequences: CDR1 - GFTFSSYA (Sequence ID 43) is a sequence having any one, two, or three mutations; CDR2 - IASDGAST (Sequence ID 44) is a sequence having any one, two, or three mutations; and A sequence of CDR3 - CARGG YLTYDRY (sequence number 45) which optionally has one, two, or three mutations.

[0157] The single-domain antibody of the present invention may be a PD-1 agonist and / or may contain any one or more of the features described above with respect to the immunosuppressive factor of the binding molecule of the present invention.

[0158] For example, the single-domain antibody of the present invention may be isolated, and / or recombinant, and / or soluble, and / or humanized. The single-domain antibody of the present invention may be VHH. Furthermore, it is preferable that the single-domain antibody does not compete with PD-L1 for binding to PD-1.

[0159] The single-domain antibody of the present invention may contain the amino acid sequence described in SEQ ID NO: 42, or a humanized version thereof, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 42. Alternatively, the single-domain antibody of the present invention may contain the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 71.

[0160] Preferably, the single-domain antibody of the present invention contains the amino acid sequence of SEQ ID NO: 71. In this regard, the present invention also provides a protein containing the amino acid sequence of SEQ ID NO: 71.

[0161] A single-domain antibody has an affinity (i.e., K) of approximately 1 nM to approximately 500 nM, or preferably approximately 50 nM to approximately 70 nM, for PD-1. D) may have. A single-domain antibody may have a binding half-life of about 1 second to about 40 seconds, or preferably about 15 seconds to about 20 seconds. The affinity of a single-domain antibody can be measured using the method described above for the binding molecule of the present invention. The affinity of a single-domain antibody can be measured using, for example, the method provided in Example 1 or Example 4.

[0162] Single-domain antibodies exhibit high specificity for PD-1 and may elicit a potent inhibitory response when tested with the reporter assay described in Example 4.

[0163] A single-domain antibody may bind to a PD-1 epitope containing one or more, or all, of the following amino acids: E38, F59, P60, E61, T75, Q76, L77, P78, N79, and G80, numbered according to SEQ ID NO: 101. For example, a single-domain antibody may bind to a PD-1 epitope containing at least 5, at least 6, at least 7, at least 8, or at least 9 of the following amino acids: E38, F59, P60, E61, T75, Q76, L77, P78, N79, and G80, numbered according to SEQ ID NO: 101. A single-domain antibody may bind to a PD-1 epitope containing the following amino acids: E38, F59, P60, E61, T75, Q76, L77, P78, N79, and G80, numbered according to SEQ ID NO: 101.

[0164] The present invention also, (a) comprising the single-domain antibody of the present invention; (b) comprising a pMHC binding domain which may include (i) an alpha chain containing at least a TCR alpha chain variable domain, and (ii) a beta chain containing at least a TCR beta chain variable domain; and (c) Provide a binding molecule which may contain a half-life extension domain.

[0165] The pMHC-binding domain and / or half-life extension domain may include one or more of the features described herein.

[0166] amino acid sequence The scope of the present invention also includes phenotypic silent variants of any molecule disclosed herein. As used herein, the term “phenotypic silent variant” refers to a variant that incorporates one or more further amino acid changes, including substitutions, insertions, and deletions, in addition to those listed above, and such variant is understood to have a phenotype similar to the corresponding molecule without such changes. For the purposes of the present invention, the phenotype is defined as binding affinity (K D This includes the binding half-life and / or specificity. The phenotype of the binding molecule may include immunoactivating potency and purification yield, in addition to binding affinity and specificity. Phenotypic silent variants are those that do not exhibit the aforementioned changes (multiple changes) when measured under identical conditions (e.g., 25°C and / or on the same SPR chip), and the measured K of the corresponding binding molecule. D and / or K for the ALWGPDPAAA(SEQ ID NO: 1)HLA-A*02 complex with a binding half-life of 50% or less, more preferably 30%, 25%, or 20%. D and / or bond half-life may be present. Suitable conditions are further described in the examples.

[0167] Furthermore, phenotypic silent variants may maintain the same or substantially the same therapeutic window between binding to the ALWGPDPAAA(SEQ ID NO: 1)HLA-A*02 complex and binding to one or more alternative peptide-HLA complexes. Phenotypic silent variants may maintain the same or substantially the same therapeutic window between the efficacy of immune cell inhibition in response to cells presenting the ALWGPDPAAA(SEQ ID NO: 1)HLA-A*02 complex and the efficacy of immune cell inhibition against cells presenting one or more alternative off-target peptide-HLA complexes. The therapeutic window can be calculated based on the lowest effective concentration ("LOEL") observed for normal cells and cell lines associated with the indication. The therapeutic window may differ by at least 10-fold, at least 100-fold, at least 1000-fold, or more. Phenotypic variants may share the same or substantially the same recognition motif as determined by the serial mutagenesis techniques described later.

[0168] As is known to those skilled in the art, it may be possible to produce binding molecules that incorporate changes in the variable domain compared to those detailed above, without significantly altering the affinity for interaction with the ALWGPDPAAA(SEQ ID NO: 1)HLA-A*02 complex or other functional properties. In particular, such silent mutations may be incorporated into parts of sequences known not to be directly involved in antigen binding (e.g., framework regions and / or parts of CDRs that do not come into contact with the antigen). Such variants are within the scope of the present invention.

[0169] Phenotypically silent variants may include one or more conserved substitutions and / or one or more permissible substitutions. Permissible substitutions are those that do not meet the definition of conserved, as described below, but are still phenotypically silent. Those skilled in the art recognize that various amino acids have similar properties and are therefore "conserved." It is often possible to substitute one or more such amino acids in a protein, polypeptide, or peptide with one or more other such amino acids without compromising the desired activity of that protein, polypeptide, or peptide.

[0170] Therefore, amino acids such as glycine, alanine, valine, leucine, and isoleucine are often substituted for each other (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used for substitution with each other (because they have relatively short side chains), and valine, leucine, and isoleucine are preferably used for substitution with each other (because they have highly hydrophobic aliphatic side chains). Other amino acids that are often substituted for each other include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains). It will be understood that amino acid substitutions within the scope of the present invention can be carried out using natural or non-natural amino acids. For example, it is assumed herein that the methyl group of alanine may be substituted with an ethyl group, and / or minor modifications may be made to the peptide skeleton. Regardless of whether natural or synthetic amino acids are used, it is preferable that only L-amino acids are present.

[0171] Substitutions of this nature are often referred to as “conservative” or “semi-conservative” amino acid substitutions. Accordingly, the present invention extends to the use of molecules that include any of the amino acid sequences described above but have one or more conservative substitutions and / or one or more acceptable substitutions in the sequence, thereby so that the amino acid sequence of the molecule, or any domain or region thereof, has at least 90% identity with the sequences disclosed herein, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0172] "Identity," as is well known in the art, refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by a comparison between these sequences. In the art, identity is sometimes the degree of sequence relevance between polypeptide sequences or polynucleotide sequences, determined by the degree of fit between the strings of these sequences. There are several methods for measuring identity between two polypeptide sequences or two polynucleotide sequences, but methods commonly used to determine identity are coded into computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0173] Programs such as CLUSTAL can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces into each sequence as needed. It is possible to calculate amino acid identity or similarity (in addition to identity, conservation of amino acid types) for the optimal alignment. Programs such as BLASTx align the longest extensions of similar sequences and quantify the degree of fit. Therefore, if multiple similar regions are found, it is possible to obtain comparisons with different scores for each. Both types of identity analysis are envisioned in this invention.

[0174] The percentage identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison (for example, by introducing a gap in the first sequence to optimally align it with the other sequence) and comparing the amino acid residues or nucleotides at corresponding positions. "Optimal alignment" is the alignment of the two sequences that yields the highest percentage identity. Percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions × 100).

[0175] The determination of percentage identity between two sequences can be achieved using mathematical algorithms known to those skilled in the art. An example of such a mathematical algorithm for comparing two sequences is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The BLASTn and BLASTp programs in Altschul et al. (1990) J. Mol. Biol. 215:403-410 incorporate such algorithms. The determination of percentage identity between two nucleotide sequences can be performed using the BLASTn program. The determination of percentage identity between two protein sequences can be performed using the BLASTp program. To obtain gapped alignments for comparison purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, can be used. Alternatively, PSI-Blast can be used to perform iterative searches to detect distant relative relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters for each program (e.g., BLASTp and BLASTp) can be used. See http: / / www.ncbi.nlm.nih.gov. Examples of default general parameters include Word Size=3 and Expect Threshold=10. It is also possible to select parameters that are automatically adjusted when the input sequence is short. Another example of a mathematical algorithm used for sequence comparison is the algorithm by Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), part of the CGC sequence alignment software package, incorporates this algorithm.Other sequence analysis algorithms known in the art include ADVANCE and ADAM, described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA, described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. For the purpose of evaluating percent identity in this disclosure, BLASTp with default parameters is used as a comparison method. Furthermore, the described percent identity provides non-integer values ​​for amino acids (i.e., if a 25-amino acid sequence with 90% sequence identity provides a value of "22.5", the obtained value is rounded down to the next integer, i.e., "22". Thus, in the presented example, a sequence in which 22 out of 25 amino acids match falls within 90% sequence identity).

[0176] When used herein, if a sequence is described as having sequence identity with another sequence, this sequence retains the function of the other sequence, for example, the general binding properties in the case of a peptide.

[0177] As will be apparent to those skilled in the art, it may be possible to cleave or extend the sequence shown at its C-terminus and / or N-terminus by 1, 2, 3, 4, 5 or more residues without substantially affecting the functional properties of the molecule, such as the TCR portion. The sequence shown at its C-terminus and / or N-terminus can be cleaved or extended by 1, 2, 3, 4, or 5 residues. All such variants are included in the present invention.

[0178] Mutations, insertions, and deletions, including conserved and tolerable substitutions, can be introduced into the provided sequences using any suitable method, including but not limited to polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation-independent cloning (LIC) procedures. These methods are detailed in many standard molecular biology textbooks. For further details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd Ed.) CSHL Press. Further information on ligation-independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The protein sequences provided herein can be obtained by recombinant expression, solid-phase synthesis, or other suitable methods known in the art.

[0179] Evaluation of binding properties and activity of binding molecules Binding affinity (equilibrium constant K D Methods for determining binding affinity (inversely proportional to) and binding half-life (expressed as T1 / 2) are known in the art. Binding affinity and binding half-life can be determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), for example, using a BIAcore or Octet instrument, respectively. For example, the binding affinity of a binding molecule to a peptide-MHC complex can be determined using SPR at 25°C and / or 37°C, where the peptide-MHC complex is immobilized on a solid support (e.g., a sensor chip) and brought into contact with a solution containing the binding molecule. Suitable experimental conditions and methods for determining binding parameters are described in the examples (e.g., Examples 1 and 2).

[0180] Those skilled in the art will know that a higher affinity is K D A lower value indicates a stronger bond. In other words, a doubling of affinity is K DThis refers to the value being halved. T1 / 2 is ln2 = dissociation rate (off-rate) (k off It is calculated by dividing by ). Therefore, when T1 / 2 is doubled, k off It will be halved. TCR's K D Value and k off Values ​​are typically measured in the soluble form of the TCR, i.e., in a cleaved form that removes cytoplasmic and transmembrane domain residues. To account for variability between independent measurements, and especially interactions with dissociation times exceeding 20 hours, the binding affinity and / or binding half-life of a particular protein can be measured multiple times, e.g., three or more times, using the same assay protocol, and the average of the results can be taken. To compare binding data between two samples (i.e., two different proteins, and / or two preparations of the same protein), it is desirable to perform the measurements under identical assay conditions (e.g., temperature). Measurement methods described in relation to the TCR may also be applicable to the binding molecules described herein.

[0181] The specific binding molecules of the present invention, namely immune checkpoint agonists, can potently inhibit the T cell response to antigen-positive cells in vitro. The measured T cell response may be the release of T cell activation markers such as interferon-γ or granzyme B, or target cell killing, or other indicators of T cell activation such as T cell proliferation. Another method for evaluating the inhibition of T cell activation is the Jurkat NFAT cell reporter assay described in Example 4.

[0182] Molecules included in the present invention may have improved half-lives. Methods for determining whether a protein's half-life is improved will be apparent to those skilled in the art. For example, the ability of the protein to bind to the neonatal Fc receptor (FcRn) is evaluated. In this regard, increased binding affinity to FcRn increases the serum half-life of the protein (see, e.g., Kim et al. Eur J Immunol., 24:2429, 1994).

[0183] The half-lives of the proteins disclosed herein can also be measured by pharmacokinetic studies, for example, according to the method described in Kim et al. Eur J of Immunol 24: 542, 1994. According to this method, the radiolabeled protein is administered intravenously to mice, and plasma concentrations are measured periodically as a function of time, for example, from 3 minutes to 72 hours after injection. Alternatively, the unlabeled conjugated molecule of the present invention can be injected, and its plasma concentration can be measured periodically using ELISA. The clearance curve thus obtained should be biphasic, i.e., alpha-phase and beta-phase. To determine the in vivo half-life of the protein, the clearance rate in the beta-phase is calculated and compared to that of the wild-type or unmodified protein.

[0184] Nucleic acids, vectors, and host cells The present invention provides nucleic acids encoding the binding molecule or single-domain antibody of the present invention. The alpha and beta chains of the binding molecule may be encoded in a single open reading frame or in two different open reading frames. Similarly, for a binding molecule containing three polypeptide chains, the polypeptide chains may be encoded in a single open reading frame or in three different open reading frames. Alternatively, the alpha and beta chains of the binding molecule, or two or three polypeptide chains, may be encoded on separate nucleic acids. The term “nucleic acid” includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, which may be single-stranded or double-stranded. Nucleic acids may be present in whole cells, in cell lysates, or in isolated, partially purified, or substantially pure forms. Nucleic acids are considered “substantially pure” if they have been purified by standard techniques from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins. Nucleic acids may be recombinant and / or non-natural and / or engineered. Nucleic acid sequences may be codon-optimized depending on the expression system used. As is known to those skilled in the art, expression systems may include bacterial cells such as Escherichia coli (E. coli), or yeast cells, or mammalian cells, or insect cells, or they may be cell-free expression systems.

[0185] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as described above. In particular, the present invention provides an expression vector comprising the nucleic acid of the present invention. The terms “vector,” “cloning vector,” and “expression vector” refer to a medium for introducing a DNA or RNA sequence (e.g., an exogenous gene) into a host cell, transforming the host, and optionally promoting the expression (e.g., transcription and translation) of the introduced sequence. This vector may be capable of expressing both Foxp3 and the binding molecule of the present invention in T cells.

[0186] The alpha and beta chains of the binding molecule of the present invention can be expressed together with Foxp3 (optionally a GFP / Foxp3 fusion protein). For example, the binding molecule of the present invention and Foxp3 can be expressed from a multi-cistronic retroviral vector using, for example, viral ribosome skipping (2A) and an internal ribosome entry site (IRES). This type of vector can efficiently convert conventional CD4+ T cells into antigen-specific regulatory phenotype T cells (Treg). While not intended to be theoretically constrained, it is expected that by simultaneously delivering the islet antigen-specific binding molecule of the present invention and Foxp3, islet specificity and regulatory activity will not be separated, and as a result, transfected T cells will be able to optimally control the pro-inflammatory environment that would otherwise promote the destruction of islet cells.

[0187] The present invention also provides recombinant host cells comprising one or more of the above constructs. As described above, nucleic acids encoding the binding molecule or single-domain antibody of the present invention constitute aspects of the present invention, and similarly, methods for producing the binding molecule or single-domain antibody, including expression from nucleic acids encoding the binding molecule or single-domain antibody of the present invention, also constitute aspects of the present invention. Expression can be readily achieved by culturing recombinant host cells containing nucleic acids under appropriate conditions. After production by expression, the binding molecule or single-domain antibody can be isolated and / or purified using any appropriate technique and then optionally used.

[0188] Systems for cloning and expressing proteins such as the conjugation molecule or single-domain antibody of the present invention in various different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, and NSO mouse melanoma cells. A generally preferred bacterial host is Escherichia coli. The expression of TCRs, antibodies, and antibody fragments in prokaryotic cells such as Escherichia coli is well established in the art. For a review, see, for example, Plueckthun, Bio / Technology 9:545-551 (1991). Expression in cultured eukaryotic cells is also available to those skilled in the art as a means of producing the binding molecule. For recent reviews, see, for example, Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

[0189] A suitable vector can be selected or constructed containing a suitable regulatory sequence that optionally includes a promoter sequence, terminator sequence, polyadenylation sequence, enhancer sequence, marker gene, and other sequences. The vector may optionally contain a plasmid or viral vector (e.g., a phage or phagemid), and may be any suitable vector known in the art. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual: 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for nucleic acid manipulation, such as preparation of nucleic acid constructs, mutagenesis, sequencing, DNA introduction and gene expression into cells, and protein analysis, are described in detail in Ausubel et al. eds., Short Protocols in Molecular Biology, 2nd Edition, John Wiley & Sons (1992).

[0190] The present invention also provides host cells containing nucleic acids disclosed herein. The present invention also: (a) The expression vector of the present invention; (b) A first expression vector comprising a nucleic acid encoding a first polypeptide including the alpha chain of the binding molecule of the present invention, and a second expression vector comprising a nucleic acid encoding a second polypeptide including the beta chain of the binding molecule of the present invention; or (c) A first expression vector comprising a nucleic acid encoding the first polypeptide of the triple-chain molecule described herein, a second expression vector comprising a nucleic acid encoding the second polypeptide of the triple-chain molecule described herein, and a third expression vector comprising a nucleic acid encoding the third polypeptide of the triple-chain molecule described herein. Provides cells that are easily identifiable.

[0191] Also provided are non-natural, and / or purified, and / or engineered cells, preferably T cells, that present the binding molecule of the present invention. Numerous suitable methods exist for transfecting T cells with nucleic acids (e.g., DNA or RNA) encoding the TCR of the present invention (see, for example, Robbins et al., 2008 J Immunol. 180: 6116-6131 and Plesa et al. 2012 Blood. 119(15):3420-3430). The cells may be CD4+ and / or Foxp3+ T cells. For example, the cells may be Treg cells. Such cells presenting the binding molecule of the present invention may be used in adoptive therapy for the treatment of diabetes.

[0192] Furthermore, the present invention provides a method comprising introducing such nucleic acids into host cells. Any available technique can be used for the introduction. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus, or baculovirus in insect cells. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduction can then induce or allow expression from the nucleic acid, for example, by culturing the host cells under gene expression conditions.

[0193] Host cells suitable for cloning or expressing proteins from the nucleic acids and / or vectors of the present invention are known in the art. Host cells suitable for the expression of (glycosylated) proteins also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in combination for transfection into insect cells, particularly Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the antibody production technology in transgenic plants, "PLANTIBODIES®"). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension culture may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (e.g., 293 or 293T cells described in GRAHAM, FL et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., Mather, JP, Biol. Reprod. 23(1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer tumor cells (MMT 060562); and TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. As described in 383(1982)44-68); MRC5 cells; and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, e.g., DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77(1980)4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for protein production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC(ed.), Humana Press, Totowa, NJ(2004), pp. 255-268. The host cell may be a eukaryotic cell, e.g., Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells). Alternatively, the host cell may be a prokaryotic cell, e.g., E. coli cells.

[0194] The nucleic acids of the present invention may be incorporated into the genome (e.g., chromosomes) of a host cell. This integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0195] Method for creating bond molecules Furthermore, a method for producing the binding molecule or single-domain antibody of the present invention is provided herein. In one embodiment, the method comprises a) maintaining cells of the present invention under conditions suitable for the expression of the binding molecule or single-domain antibody, and b) isolating the binding molecule or single-domain antibody.

[0196] Methods for producing recombinant proteins such as the binding molecule or single-domain antibody of the present invention are well known in the art. The nucleic acid encoding the protein can be cloned into an expression construct or vector, which is then transfected into host cells such as Escherichia coli cells, yeast cells, insect cells, or mammalian cells, e.g., monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not intrinsically produce the protein. Typical mammalian cells used for protein expression are CHO cells, myeloma cells, or HEK cells. Preferred cells for producing the binding molecule of the present invention are Escherichia coli cells. Molecular cloning techniques for achieving these objectives are well known in the art and are described, for example, in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updated versions to date) or Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide range of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art; see, for example, U.S. Patent No. 4,816,567 or No. 5,530,101.

[0197] Nucleic acids may be operationally ligated to a promoter in an expression construct or expression vector for further cloning (DNA amplification) or expression in a cell-free system or intracellularly. As used herein, the term “promoter” is used in its broadest sense and includes transcriptional regulatory sequences of genomic genes, including, for example, TATA boxes or initiation factor elements necessary for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that alter nucleic acid expression in development and / or in response to external stimuli or in a tissue-specific manner. In this context, the term “promoter” is also used to describe recombinant, synthetic, or fusion nucleic acids or derivatives that confer, activate, or enhance the expression of operationally ligated nucleic acids. An exemplary promoter may include additional copies of one or more specific regulatory elements to further enhance the expression of said nucleic acid and / or alter spatial and / or temporal expression. As used herein, the term “operationally ligated” means that the promoter is positioned relative to the nucleic acid so that the expression of the nucleic acid is controlled by the promoter.

[0198] Many vectors for intracellular expression are commercially available. Vector components generally include, but are not limited to, one or more of a signal sequence, a protein-coding sequence (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will recognize sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretory signals (e.g., pe1B, alkaline phosphatase, penicillin-degrading enzyme, Ipp, or thermostable enterotoxin II), yeast secretory signals (e.g., invertase reader, factor α reader, or acid phosphatase reader), or mammalian secretory signals (e.g., herpes simplex gD signal).

[0199] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-α promoter (EF1), micronuclear RNA promoters (Ula and Ulb), α-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Roussarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; CMV enhancer / β-actin promoter; or immunoglobulin promoters, or hybrid regulatory elements containing their active fragments. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cells subcloned for suspension culture growth); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0200] Typical promoters suitable for expression in yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe include, but are not limited to, the ADH1 promoter, GAL1 promoter, GALA promoter, CUP1 promoter, PH05 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0201] Host cells used for protein production can be cultured in various media depending on the type of cell used. Commercial media such as Ham F10 medium (Sigma), Minimum Essential Medium (MEM) (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0202] Methods for isolating proteins are known in the art. If the protein is secreted into a culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, can be included in either of the above steps to inhibit proteolysis, or antibiotics can be included to prevent the growth of accidental contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, by serial centrifugation.

[0203] Proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination thereof.

[0204] These methods are known in the art and are described, for example, in International Publication No. 99 / 57134 or in Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Those skilled in the art also recognize that proteins can be modified to include tags, such as polyhistidine tags, hexahistidine tags, influenza virus hemagglutinin (HA) tags, Simian virus 5 (V5) tags, LLAG tags, and glutathione S-transferase (GST) tags, to facilitate purification or detection. The resulting proteins are purified using methods known in the art, such as affinity purification. For example, proteins containing hexa-his tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexa-his tags immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively or additionally, a ligand or antibody that binds to the tag may be used in affinity purification.

[0205] The molecules of the present invention can be purified in high yield. The yield may be determined based on the amount of substance retained in the purification process (i.e., the amount of properly folded substance obtained at the end of the purification process relative to the amount of solubilized substance obtained before refolding), and / or the yield may be based on the amount of properly folded substance obtained at the end of the purification process, based on the original culture volume. High yield means a yield greater than 1%, greater than 5%, or higher. High yield means a yield greater than 1 mg / ml, greater than 3 mg / ml, greater than 5 mg / ml, or higher.

[0206] Pharmaceutical compositions and medical methods The conjugating molecules, single-domain antibodies, nucleic acids, expression vectors, and / or cells of the present invention can be used in methods for treating or diagnosing autoimmune diseases such as type 1 diabetes. For administration to a patient, the conjugating molecules, single-domain antibodies, nucleic acids, expression vectors, and / or cells of the present invention may be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients (e.g., buffers, also known as "buffers"). This pharmaceutical composition may be provided in any suitable form (e.g., depending on the desired method of administration to a patient). It may also be provided in unit dosage forms, generally in sealed containers, and may be provided as part of a kit. Such kits usually (but not necessarily) include instructions for use. Multiple such unit dosage forms may be included.

[0207] Pharmaceutical compositions can be adapted for administration via any suitable route, such as parenteral (including subcutaneous, intramuscular, intrathecal, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes. For example, pharmaceutical compositions can be adapted for subcutaneous administration (e.g., formulation). Such compositions can be prepared by any method known in the art, for example, by mixing the active ingredient with a carrier(s) or excipient(s) under sterile conditions. Methods for preparing proteins into a form suitable for administration to a target (e.g., pharmaceutical compositions) are known in the art, including, for example, the methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0208] Pharmaceutical compositions generally contain a solution of the binding molecule (or nucleic acid, cell, or vector) of the present invention dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, can be used. The composition may contain pH adjusters and pharmaceutically acceptable auxiliary substances such as buffers and toxicity modifiers, as needed to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of the binding molecule and single-domain antibody of the present invention in these formulations may vary considerably and are mainly selected based on fluid volume, viscosity, body weight, etc., depending on the selected specific mode of administration and patient needs. Typical carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous media such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The media may contain trace amounts of additives to enhance isotonicity and chemical stability, such as buffers and preservatives.

[0209] The conjugation molecules, single-domain antibodies, pharmaceutical compositions, vectors, nucleic acids, and cells of the present invention may be supplied in substantially pure form. For example, they may be supplied with a purity of at least 80%, at least 85%, at least 90%, at least 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%.

[0210] The conjugation molecules of the present invention may have an ideal safety profile for use as therapeutic reagents. As used herein, “safety profile” refers to the ability to distinguish antigen-positive cells, particularly cells presenting the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex, from antigen-negative cells. This ability is often expressed by the “safety window” or “therapeutic window” indicators. In this case, the conjugation molecule may be in a soluble form and may preferably be fused with an immunosuppressant. Suitable immunosuppressants are described herein and include, but are not limited to, immune checkpoint agonists, interleukins, cytokines, antibodies, and antibody-like scaffolds, including fragments, derivatives, and variants thereof that bind to antigens on immune cells such as T cells, B cells, and NK cells (e.g., anti-PD-1 agonist antibodies). An ideal safety profile means that, in addition to exhibiting good specificity, the conjugation molecules of the present invention may have passed further preclinical safety studies. Examples of such tests include whole blood assays that confirm minimal cytokine release in the presence of whole blood and therefore a low risk of inducing potential cytokine release syndrome in vivo, and alloreactivity tests that confirm low recognizability of surrogate HLA types.

[0211] The appropriate dosage of the conjugated molecule or single-domain antibody of the present invention can vary widely depending on the disease or disorder being treated, the age and condition of the subject, etc. Preferably, the subject is human. The physician will determine the appropriate dosage to be used. The administration of the conjugated molecule or single-domain antibody may be a "therapeutically effective dose," which is an amount sufficient to produce a beneficial effect on the patient.

[0212] In other embodiments, the present invention provides conjugating molecules, single-domain antibodies, nucleic acids, vectors, pharmaceutical compositions, or cells for use in pharmaceuticals. In particular, the conjugating molecules, single-domain antibodies, nucleic acids, vectors, pharmaceutical compositions, and cells of the present invention can be used to treat autoimmune diseases such as diabetes. Thus, the present invention also provides conjugating molecules, single-domain antibodies, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention for use in methods of treating diabetes. The diabetes to be treated is preferably type 1 diabetes mellitus (T1DM). For example, a method of treating diabetes may include administering a soluble conjugating molecule, a single-domain antibody, or a pharmaceutical composition of the present invention, or it may be a method of adoptive therapy including administering cells of the present invention (e.g., Treg cells presenting the conjugating molecule of the present invention). As is known to those skilled in the art, there are many suitable methods for carrying out adoptive therapy (see, for example, Rosenberg et al., 2008 Nat Rev Cancer 8(4): 299-308).

[0213] The present invention further provides: • Conjugated molecules, single-domain antibodies, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention for use in pharmaceuticals, preferably in human subjects and / or preferably in methods for treating diabetes; • Conjugated molecules, single-domain antibodies, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention for use in diagnostic methods for diabetes in subjects; • Use of the conjugated molecule, single-domain antibody, nucleic acid, vector, pharmaceutical composition, or cell of the present invention in the manufacture of pharmaceuticals for the treatment of diabetes; A method for treating diabetes in a subject, comprising administering to the subject the conjugated molecule of the present invention, a single-domain antibody, nucleic acid, vector, pharmaceutical composition, or cells; • An injectable formulation for administration to human subjects, comprising the conjugating molecule of the present invention, a single-domain antibody, a nucleic acid, a vector, a pharmaceutical composition, or cells.

[0214] Kits and manufactured products In another embodiment, a kit or product comprising materials useful for the treatment, diagnosis and / or prevention of the above-mentioned disease is provided.

[0215] The kit may include (a) a container containing the conjugation molecule, single-domain antibody, nucleic acid, vector, or cell of the present invention together with an optionally pharmaceutically acceptable carrier or diluent, and (b) a package insert containing instructions for the treatment or diagnosis of a disease (e.g., diabetes) in a subject. The kit may further include (c) at least one additional therapeutic compound or agent.

[0216] The accompanying information may be on the container or associated with the container. Suitable containers include, for example, bottles, vials, and syringes. Containers may be formed from various materials such as glass or plastic. Containers may hold or contain a composition comprising the conjugating molecule, single-domain antibody, nucleic acid, vector, or cell of the present invention and may have a sterile access port (for example, the container may be an intravenous infusion bag or a vial with a stopper puncturable with a subcutaneous needle). At least one activator in the composition is the conjugating molecule, single-domain antibody, nucleic acid, vector, or cell of the present invention. The label or accompanying information may indicate that the composition is for use in the treatment of a target subject, e.g., a person having or prone to developing one of the diseases described herein, along with specific guidelines regarding the dosage and interval of the provided composition and any other pharmacopoeia. The kit may further include additional containers containing pharmaceutically acceptable dilution buffers, e.g., bacteriostatic water for intravenous injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other substances desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes.

[0217] The invention also includes particles presenting the binding molecules or single domain antibodies of the invention, and including said particles in a particle library. Such particles include, but are not limited to, phages, yeast cells, ribosomes, or mammalian cells. Methods for making such particles and libraries are known in the art (see, for example, WO 2004 / 044004; WO 01 / 48145, Chervin et al. (2008) J. Immuno. Methods 339.2: 175-184).

[0218] In addition to what is shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and are included in the appended claims. The preferred features of each aspect of the invention are applicable to other aspects as well. The documents referred to herein are incorporated by reference to the maximum extent legally permitted.

Brief Description of the Drawings

[0219] [Figure 1] Graph showing the recognition of PPI peptides and mimetic peptides by soluble native TCR. [Figure 2] Graph showing the recognition of serine-substituted PPI peptides by native soluble TCR. [Figure 3] Schematic diagram of an exemplary TCR-PD-1 agonist binding molecule incorporating an Fc domain. [Figure 4A] a) Graph showing the inhibition of T cell signaling in a Jurkat NFAT reporter assay by a TCR PD-1 agonist binding molecule, b) Graph comparing the inhibition of T cell signaling in a Jurkat NFAT reporter assay by a TCR PD-1 agonist with or without Fc. [Figure 4B] Same as above. [Figure 5] Graph showing the in vivo concentration of a TCR PD-1 agonist binding molecule in SCID mice over 3 weeks after intravenous (IV) or subcutaneous (SC) administration. [Figure 6]Graph showing inhibition of IL2 release by primary CD4+ T cells in the presence of a TCR PD-1 agonist. [Figure 7] Graphs showing inhibition of B cell killing (a) and IFNγ cytokine release (b) by two autoreactive T cell clones in the presence of a TCR PD-1 agonist binding molecule. [Figure 8] Graph showing inhibition of stimulation in PD1 +ve and PD-1 -ve NK cells, indicated by the % of CD107a and IFNγ positive cells, in the presence of a TCR PD-1 agonist binding molecule (*p≦0.05, **p≦ 0.01; ns = no significant difference). [Figure 9] Image showing the experimental results of analyzing the specificity of the selected PD-1 agonist VHH described in Example 4 using Retrogenix Cell Microarray Technology. The VHH bound to PD-1 on the microarray but did not bind to other control proteins. [Figure 10] The TCR-PD-1 agonist inhibits NK-92-PD-1 cells stimulated in vitro by HLA-A*02+ve K562 target cells pulsed with PPI peptide. (a) Schematic diagram of the experimental model. (b) Representative flow cytometry profile and graph showing specific and concentration-dependent binding of the TCR-PD-1 agonist molecule to target cells. (c) Graph showing the percentage of inhibition of CD107a expression and intracellular IFNγ production by NK92-PD-1 cells stimulated with target cells and incubated with different concentrations of control (grey dots) or TCR-PD-1 agonist (white dots). Each point represents the mean of 3 independent experiments, n = 6, two-way analysis of variance, **p≦0.01, ****p≦0.0001). (d) Representative flow cytometry profile and graph showing CD107a expression and intracellular IFNγ production by NK92-PD-1 cells stimulated with target cells in the presence of 10 nM control (grey) or 10 nM TCR-PD-1 agonist (white). Each point represents 1 sample, n = 6, 3 independent experiments, paired t-test, ***p≦0.001, ****p≦0.0001). [Figure 11] The interaction between TCR-PD-1 agonists and CD4 T cells results in sustained regulation of the T cell response. (a) Schematic diagram of the experimental model. (b) Graph showing IL-2 production during activation of CD4 T cells from day 0 to day 3 in the presence (white) or absence (gray) of TCR-PD-1 agonists. Each point represents one donor, n=12, 5 independent experiments, paired t-test, *p≦0.05. (c) Graph showing IL-2 production from day 8 to day 11 by CD4 T cells pre-activated (white) or unactivated (gray) with TCR-PD-1 agonists. Each point represents one donor, n=8, 3 independent experiments, paired t-test, **p≦0.01. (d) Graph showing IL-2 production from day 8 to day 11 by CD4 T cells reactivated in the presence (white) or absence (gray) of TCR-PD-1 agonists. Each point represents one donor, n=8, 3 independent experiments, paired t-test, **p≦0.01. [Modes for carrying out the invention]

[0220] Array description HLA-A*02 restriction peptide The source protein gene is indicated in the parentheses below. SEQ ID NO: 1 (Preproinsulin) 15-24 ;Uniprotref: P01308):ALWGPDPAAA Sequence ID 67 (Basic Helix-Loop-Helix Family, Member e41, "Mim1"): ALLGPDPAAA Sequence ID 88 (Nuclear DEAF-1-related transcription regulatory protein 8, "Mim2"): ALPGPDEAAA Sequence ID 89 (Forkheadbox Protein F2, "Mim3"): ALMSPPPAAA Sequence ID 90 (Bone marrow zinc finger protein, "Mim4"): ALWDPGPEAA Sequence ID 91 (Histone Lysine N-methyltransferase 2D, "Mim5"): ALGSPPPAAA

[0221] Typical scaffolding TCR alpha chain (SEQ ID NO: 2) JPEG2026514992000002.jpg21132 Sequence ID No. 2 is the amino acid sequence of the alpha chain of an exemplary wild-type (e.g., “scaffolding”) TCR (including the alpha chain of Sequence ID No. 2 and the beta chain of Sequence ID No. 12) that binds to ALWGPDPAAA (Sequence ID No. 1) complexed with HLA-A*02. This TCR is referred to herein as “S2”. The alpha chain contains a variable domain (Sequence ID No. 3) and a constant domain (Sequence ID No. 4, italicized). CDRs (CDR1, CDR2, and CDR3) are underlined and designated as Sequence IDs No. 5, 6, and 7, respectively, and framework regions (FR1, FR2, FR3, and FR4) are normally lettered and designated as Sequence IDs No. 8, 9, 10, and 11, respectively. The constant domain contains the T48C mutation (numbered by Sequence ID No. 4), shown in bold (compared to the wild-type constant domain), which introduces a non-natural covalent disulfide bond between the alpha and beta chains.

[0222] Exemplary scaffold TCR beta chain (SEQ ID NO: 12) Sequence ID 12 is the amino acid sequence of the beta chain of an exemplary wild-type (e.g., "scaffold") TCR (including the alpha chain of Sequence ID 2 and the beta chain of Sequence ID 12) that binds to ALWGPDPAAA (Sequence ID 1) complexed with HLA-A*02. This TCR is referred to herein as "S2". The beta chain contains a variable domain (Sequence ID 13) and a constant domain (Sequence ID 14, italicized). CDRs (CDR1, CDR2, and CDR3) are underlined and designated as Sequence IDs 15, 16, and 17, respectively, and framework regions (FR1, FR2, FR3, and FR4) are normally lettered and designated as Sequence IDs 18, 19, 20, and 21, respectively. The constant domain includes the S57C mutation (numbered by SEQ ID NO: 14), shown in bold (compared to the wild-type constant domain), which introduces a non-natural covalent disulfide bond between the alpha and beta chains. The C75A mutation (numbered by SEQ ID NO: 14), also shown in bold, removes the natural cysteine ​​and reduces the formation of erroneous disulfide bonds.

[0223] Exemplary mutant TCR alpha chain variable domain The following sequence is an exemplary alpha-chain variable domain containing mutations compared to the wild-type sequence of Sequence ID No. 3, introduced to improve affinity, stability, and / or manufacturability. CDRs are underlined, and mutations are shown in bold.

[0224] Alpha-chain variable domain "a2" (sequence number 22) containing CDRs designated as sequence numbers 23, 6, and 7 (CDR1, CDR2, and CDR3 - underscores) and framework regions designated as sequence numbers 8, 9, 10, and 11 (FR1, FR2, FR3, and FR4 - normal characters): JPEG2026514992000004.jpg11131

[0225] Alpha-chain variable domain "a18" (sequence number 24) containing CDRs designated as sequence numbers 23, 6, and 7 (CDR1, CDR2, and CDR3 - underscores) and framework regions designated as sequence numbers 25, 9, 10, and 11 (FR1, FR2, FR3, and FR4 - normal characters): JPEG2026514992000005.jpg11131

[0226] Alpha-chain variable domain "a19" (sequence number 26) containing CDRs designated as sequence numbers 23, 27, and 7 (CDR1, CDR2, and CDR3 - underscores), and framework regions designated as sequence numbers 25, 9, 10, and 11 (FR1, FR2, FR3, and FR4 - normal characters): JPEG2026514992000006.jpg11131

[0227] Exemplary mutant TCR beta chain variable domain The following sequence is an exemplary beta-chain variable domain containing mutations compared to the wild-type sequence of Sequence ID No. 12, introduced to improve affinity, stability, and / or manufacturability. CDRs are underlined, and mutations are shown in bold.

[0228] The beta-chain variable domain "b3" (sequence number 68) contains CDRs designated as sequence numbers 28, 29, and 30 (CDR1, CDR2, and CDR3 - underscores) and framework regions designated as sequence numbers 18, 19, 20, and 21 (FR1, FR2, FR3, and FR4 - normal characters): JPEG2026514992000007.jpg11131

[0229] The beta-chain variable domain "b16" (sequence number 31) contains CDRs designated as sequence numbers 32, 29, and 30 (CDR1, CDR2, and CDR3 - underscores) and framework regions designated as sequence numbers 18, 19, 33, and 21 (FR1, FR2, FR3, and FR4 - normal characters): JPEG2026514992000008.jpg11131

[0230] The beta-chain variable domain "b19" (SEQ ID NO: 34) containing CDRs (CDR1, CDR2, and CDR3 - underlined) designated as SEQ ID NOs: 35, 29, and 30, respectively, and framework regions (FR1, FR2, FR3, and FR4 - normal text) designated as SEQ ID NOs: 18, 19, 33, and 21, respectively: JPEG2026514992000009.jpg11131

[0231] The beta-chain variable domain "b20" (SEQ ID NO: 74) containing CDRs (CDR1, CDR2, and CDR3 - underlined) designated as SEQ ID NOs: 35, 29, and 30, respectively, and framework regions (FR1, FR2, FR3, and FR4 - normal text) designated as SEQ ID NOs: 18, 19, 75, and 21, respectively: JPEG2026514992000010.jpg11131

[0232] The beta-chain variable domain "b21" (SEQ ID NO: 76) containing CDRs (CDR1, CDR2, and CDR3 - underlined) designated as SEQ ID NOs: 35, 29, and 30, respectively, and framework regions (FR1, FR2, FR3, and FR4 - normal text) designated as SEQ ID NOs: 18, 19, 77, and 21, respectively: JPEG2026514992000011.jpg11131

[0233] The beta-chain variable domain "b22" (SEQ ID NO: 78) containing CDRs (CDR1, CDR2, and CDR3 - underlined) designated as SEQ ID NOs: 35, 29, and 30, respectively, and framework regions (FR1, FR2, FR3, and FR4 - normal text) designated as SEQ ID NOs: 18, 19, 79, and 21, respectively: JPEG2026514992000012.jpg11131

[0234] Exemplary TCR The following sequences are TCRs containing exemplary combinations of the alpha and beta chain variable domains shown above. Constant domains are shown in italics. CDRs are underlined, and mutations compared to scaffold TCR sequences (i.e., SEQ ID NOs. 2 or 12) are shown in bold.

[0235] a2b3 TCR The TCR "a2b3" alpha chain sequence (sequence number 70) containing the aforementioned a2 variable domain (sequence number 22 - normal text) and the aforementioned stationary domain derived from the scaffold TCR (sequence number 4 - italics): JPEG2026514992000013.jpg21132

[0236] The TCR "a2b3" beta-chain sequence (sequence number 69) containing the b3 variable domain (sequence number 68 - normal text) and the stationary domain derived from the aforementioned scaffold TCR (sequence number 14 - italics): JPEG2026514992000014.jpg22132

[0237] a18b16 TCR The TCR "a18b16" alpha chain sequence (sequence number 36) containing the aforementioned a18 variable domain (sequence number 24 - normal text) and the variant constant domain (sequence number 37 - italics): JPEG2026514992000015.jpg22132

[0238] TCR "a18b16" beta-chain sequence (sequence number 38) containing the b16 variable domain (sequence number 31 - normal text) and the variant constant domain (sequence number 39 - italics): JPEG2026514992000016.jpg27132

[0239] a19b19 TCR The TCR "a19b19" alpha chain sequence (sequence number 40) containing the aforementioned a19 variable domain (sequence number 26 - normal text) and the variant constant domain (sequence number 37 - italics): JPEG2026514992000017.jpg22132

[0240] The TCR "a19b19" beta-chain sequence (sequence number 41) containing the b19 variable domain (sequence number 34 - normal text) and the variant constant domain (sequence number 39 - italics): JPEG2026514992000018.jpg27132

[0241] a19b20 TCR The TCR "a19b20" alpha chain sequence (sequence number 40) containing the aforementioned a19 variable domain (sequence number 26 - normal text) and the variant constant domain (sequence number 37 - italics): JPEG2026514992000019.jpg22132

[0242] TCR "a19b20" beta-chain sequence (sequence number 80) containing the b20 variable domain (sequence number 74 - normal text) and the variant constant domain (sequence number 39 - italics): JPEG2026514992000020.jpg27132

[0243] a19b21 TCR The TCR "a19b21" alpha chain sequence (sequence number 40) containing the aforementioned a19 variable domain (sequence number 26 - normal text) and the variant constant domain (sequence number 37 - italics): JPEG2026514992000021.jpg22132

[0244] The TCR "a19b21" beta-chain sequence (sequence number 81) containing the b21 variable domain (sequence number 76 - normal text) and the variant constant domain (sequence number 39 - italics): JPEG2026514992000022.jpg27132

[0245] a19b22 TCR The TCR "a19b22" alpha chain sequence (sequence number 40) containing the aforementioned a19 variable domain (sequence number 26 - normal text) and the variant constant domain (sequence number 37 - italics): JPEG2026514992000023.jpg22132

[0246] TCR "a19b22" beta-chain sequence (sequence number 82) containing the b22 variable domain (sequence number 78 - normal text) and the variant constant domain (sequence number 39 - italics): JPEG2026514992000024.jpg27132

[0247] Exemplary PD1 agonist VHH sequence Sequence ID 42 is the amino acid sequence of an exemplary camelid PD1 agonist VHH. CDRs (CDR1, CDR2, and CDR3) are underlined and designated as Sequence IDs 43, 44, and 45, respectively. Positions differing from the human IgHV3-23 consensus are shown in bold.

[0248] Sequence ID 42: JPEG2026514992000025.jpg11131

[0249] Sequence ID 71 is the amino acid sequence of the humanized version of the exemplary PD1 agonist VHH of Sequence ID 42. Mutations compared to Sequence ID 42 are shown in bold. JPEG2026514992000026.jpg11131

[0250] Exemplary TCR-PD1 agonist VHH sequence a2b3VHH "a2b3VH" is a binding molecule containing the above-mentioned TCR "a2" alpha chain (SEQ ID NO: 70) and TCR beta chain-PD1 agonist VHH fusion (SEQ ID NO: 72). The beta chain-PD1 agonist VHH fusion sequence (SEQ ID NO: 72) is shown below and contains the above-mentioned PD1 agonist VHH (SEQ ID NO: 71, italicized) fused to the above-mentioned TCR "b3" beta chain (SEQ ID NO: 69). The TCR beta chain and PD1 agonist VHH sequence are linked via a glycine-serine linker (underlined), designated SEQ ID NO: 73.

[0251] Sequence ID 72: JPEG2026514992000027.jpg37132

[0252] a18b16VHH "a18b16VH" is a binding molecule containing the above-mentioned TCR "a18" alpha chain (SEQ ID NO: 36) and TCR beta chain-PD1 agonist VHH fusion (SEQ ID NO: 46). The beta chain-PD1 agonist VHH fusion sequence (SEQ ID NO: 46) is as shown below and contains the above-mentioned PD1 agonist VHH (SEQ ID NO: 71, italicized) fused to the above-mentioned TCR "b16" beta chain (SEQ ID NO: 38). The TCR beta chain and PD1 agonist VHH sequence are linked via a glycine-serine linker (underlined), designated SEQ ID NO: 73.

[0253] Sequence ID 46: JPEG2026514992000028.jpg37132

[0254] a19b19VHH "a19b19VHH" is a binding molecule containing the above-mentioned TCR "a19" alpha chain (SEQ ID NO: 40) and TCR beta chain-PD1 agonist VHH fusion (SEQ ID NO: 47). The beta chain-PD1 agonist VHH fusion sequence (SEQ ID NO: 47) is as shown below and contains the above-mentioned PD1 agonist VHH (SEQ ID NO: 71, italicized) fused to the above-mentioned TCR "b19" beta chain (SEQ ID NO: 41). The TCR beta chain and PD1 agonist VHH sequence are linked via a glycine-serine linker (underlined), designated SEQ ID NO: 73.

[0255] Sequence ID 47: JPEG2026514992000029.jpg37132

[0256] a19b20VHH "a19b20VHH" is a binding molecule containing the above-mentioned TCR "a19" alpha chain (SEQ ID NO: 40) and TCR beta chain-PD1 agonist VHH fusion (SEQ ID NO: 83). The beta chain-PD1 agonist VHH fusion sequence (SEQ ID NO: 83) is as shown below and contains the above-mentioned PD1 agonist VHH (SEQ ID NO: 71, italicized) fused to the above-mentioned TCR "b20" beta chain (SEQ ID NO: 80). The TCR beta chain and PD1 agonist VHH sequence are linked via a glycine-serine linker (underlined) designated as SEQ ID NO: 73.

[0257] Sequence ID 83: JPEG2026514992000030.jpg37132

[0258] a19b21VHH "a19b21VHH" is a binding molecule containing the above-mentioned TCR "a19" alpha chain (SEQ ID NO: 40) and TCR beta chain-PD1 agonist VHH fusion (SEQ ID NO: 84). The beta chain-PD1 agonist VHH fusion sequence (SEQ ID NO: 84) is shown below and contains the above-mentioned PD1 agonist VHH (SEQ ID NO: 71, italicized) fused to the above-mentioned TCR "b21" beta chain (SEQ ID NO: 81). The TCR beta chain and PD1 agonist VHH sequence are linked via a glycine-serine linker (underlined), designated SEQ ID NO: 73.

[0259] Sequence ID 84: JPEG2026514992000031.jpg37132

[0260] a19b22VHH "a19b22VHH" is a binding molecule containing the above-mentioned TCR "a19" alpha chain (SEQ ID NO: 40) and TCR beta chain-PD1 agonist VHH fusion (SEQ ID NO: 85). The beta chain-PD1 agonist VHH fusion sequence (SEQ ID NO: 85) is as shown below and contains the above-mentioned PD1 agonist VHH (SEQ ID NO: 71, italicized) fused to the above-mentioned TCR "b22" beta chain (SEQ ID NO: 82). The TCR beta chain and PD1 agonist VHH sequence are linked via a glycine-serine linker (underlined), designated SEQ ID NO: 73.

[0261] Sequence ID 85: JPEG2026514992000032.jpg37132

[0262] Exemplary half-life prolonged TCR-PD1 agonist VHH sequence a2b3VHH-HLE "a2b3VHH-HLE" is a conjugation molecule comprising a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 72 mentioned above, a second polypeptide chain (SEQ ID NO: 48) containing the TCR "a2" alpha chain (SEQ ID NO: 70) fused to an Fc domain (SEQ ID NO: 49 - italicized) via a hinge (SEQ ID NO: 50 - underlined), and a third polypeptide chain (SEQ ID NO: 51) containing an Fc domain (SEQ ID NO: 52 - italicized) fused to a hinge (SEQ ID NO: 50 - underlined).

[0263] Sequence ID 48: JPEG2026514992000033.jpg42132

[0264] Sequence ID 51: JPEG2026514992000034.jpg22132

[0265] a2b3VHH-HLE(YTE) "a2b3VHH-HLE(YTE)" is a binding molecule identical to the aforementioned "a2b3VHH-HLE," except that the Fc region sequence includes M252Y, S254T, and T256E substitutions (EU numbering system) to enhance binding to FcRn. a2b3VHH-HLE(YTE) comprises a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 72, a second polypeptide chain (SEQ ID NO: 97) containing the aforementioned TCR "a2" alpha chain (SEQ ID NO: 70) fused to the Fc domain (SEQ ID NO: 93 - italicized) via a hinge (SEQ ID NO: 50 - underlined), and a third polypeptide chain (SEQ ID NO: 98) containing the Fc domain (SEQ ID NO: 94 - italicized) fused to a hinge (SEQ ID NO: 50 - underlined).

[0266] Sequence ID 97: JPEG2026514992000035.jpg42132

[0267] Sequence ID 98: JPEG2026514992000036.jpg22132

[0268] a18b16VHH-HLE "a18b16VHH-HLE" is a conjugation molecule comprising a first polypeptide chain containing the TCR beta-chain-PD1 agonist of Sequence ID No. 46, a second polypeptide chain (Sequence ID No. 53) containing the TCR "a18" alpha chain (Sequence ID No. 36) fused to an Fc domain (Sequence ID No. 49 - italicized) via a hinge (Sequence ID No. 50 - underlined), and a third polypeptide chain (polypeptide third chain) (Sequence ID No. 51) containing an Fc domain (Sequence ID No. 52) fused to a hinge (Sequence ID No. 50).

[0269] Sequence ID 53: JPEG2026514992000037.jpg42132

[0270] a18b16VHH-HLE(YTE) "a18b16VHH-HLE(YTE)" is a binding molecule identical to the aforementioned "a18b16VHH-HLE," except that the Fc region sequence includes M252Y, S254T, and T256E substitutions (EU numbering system) to enhance binding to FcRn. a18b16VHH-HLE(YTE) comprises a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 46, a second polypeptide chain (SEQ ID NO: 99) containing the aforementioned TCR "a18" alpha chain (SEQ ID NO: 36) fused to the Fc domain (SEQ ID NO: 93 - italicized) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 98) containing the aforementioned Fc domain (SEQ ID NO: 94) fused to a hinge (SEQ ID NO: 50).

[0271] Sequence ID 99: JPEG2026514992000038.jpg42132

[0272] a19b19VHH-HLE "a19b19VHH-HLE" is a conjugation molecule comprising a first polypeptide chain containing the TCR beta-chain-PD1 agonist of Sequence ID No. 47 mentioned above, a second polypeptide chain (Sequence ID No. 54) containing the TCR "a19" alpha chain (Sequence ID No. 40) fused to an Fc domain (Sequence ID No. 49 - italicized) via a hinge (Sequence ID No. 50 - underlined), and a third polypeptide chain (Sequence ID No. 51) containing an Fc domain (Sequence ID No. 52) fused to a hinge (Sequence ID No. 50).

[0273] Sequence ID 54: JPEG2026514992000039.jpg42132

[0274] a19b19VHH-HLE(YTE) "a19b19VHH-HLE(YTE)" is a binding molecule identical to the aforementioned "a19b19VHH-HLE," except that the Fc region sequence includes M252Y, S254T, and T256E substitutions (EU numbering system) to enhance binding to FcRn. a19b19VHH-HLE(YTE) comprises a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 47, a second polypeptide chain (SEQ ID NO: 100) containing the aforementioned TCR "a19" alpha chain (SEQ ID NO: 40) fused to the Fc domain (SEQ ID NO: 93 - italicized) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 98) containing the aforementioned Fc domain (SEQ ID NO: 94) fused to the hinge (SEQ ID NO: 50).

[0275] Sequence ID 100: JPEG2026514992000040.jpg42132

[0276] a19b20VHH-HLE "a19b20VHH-HLE" is a conjugation molecule comprising a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 83, a second polypeptide chain (SEQ ID NO: 54) containing the TCR "a19" alpha chain (SEQ ID NO: 40) fused to an Fc domain (SEQ ID NO: 49) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 51) containing an Fc domain (SEQ ID NO: 52) fused to a hinge (SEQ ID NO: 50).

[0277] a19b20VHH-HLE(YTE) "a19b20VHH-HLE(YTE)" is a binding molecule identical to the aforementioned "a19b20VHH-HLE," except that the Fc region sequence includes M252Y, S254T, and T256E substitutions (EU numbering system) to enhance binding to FcRn. a19b20VHH-HLE(YTE) comprises a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 83, a second polypeptide chain (SEQ ID NO: 100) containing the aforementioned TCR "a19" alpha chain (SEQ ID NO: 40) fused to the Fc domain (SEQ ID NO: 93) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 98) containing the aforementioned Fc domain (SEQ ID NO: 94) fused to a hinge (SEQ ID NO: 50).

[0278] a19b21VHH-HLE "a19b21VHH-HL" is a conjugation molecule comprising a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 84, a second polypeptide chain (SEQ ID NO: 54) containing the TCR "a19" alpha chain (SEQ ID NO: 40) fused to an Fc domain (SEQ ID NO: 49) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 51) containing an Fc domain (SEQ ID NO: 52) fused to a hinge (SEQ ID NO: 50).

[0279] a19b21VHH-HLE(YTE) "a19b21VHH-HLE(YTE)" is a binding molecule identical to the aforementioned "a19b21VHH-HLE," except that the Fc region sequence includes M252Y, S254T, and T256E substitutions (EU numbering system) to enhance binding to FcRn. a19b21VHH-HLE(YTE) comprises a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 84, a second polypeptide chain (SEQ ID NO: 100) containing the aforementioned TCR "a19" alpha chain (SEQ ID NO: 40) fused to the Fc domain (SEQ ID NO: 93) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 98) containing the aforementioned Fc domain (SEQ ID NO: 94) fused to a hinge (SEQ ID NO: 50).

[0280] a19b22VHH-HLE "a19b22VHH-HL" is a conjugation molecule comprising a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 85, a second polypeptide chain (SEQ ID NO: 54) containing the TCR "a19" alpha chain (SEQ ID NO: 40) fused to an Fc domain (SEQ ID NO: 49) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 51) containing an Fc domain (SEQ ID NO: 52) fused to a hinge (SEQ ID NO: 50).

[0281] a19b22VHH-HLE(YTE) "a19b22VHH-HLE(YTE)" is a binding molecule identical to the aforementioned "a19b22VHH-HLE," except that the Fc region sequence includes M252Y, S254T, and T256E substitutions (EU numbering system) to enhance binding to FcRn. a19b22VHH-HLE(YTE) comprises a first polypeptide chain containing the TCR beta-chain-PD1 agonist of SEQ ID NO: 85, a second polypeptide chain (SEQ ID NO: 100) containing the aforementioned TCR "a19" alpha chain (SEQ ID NO: 40) fused to the Fc domain (SEQ ID NO: 93) via a hinge (SEQ ID NO: 50), and a third polypeptide chain (SEQ ID NO: 98) containing the aforementioned Fc domain (SEQ ID NO: 94) fused to a hinge (SEQ ID NO: 50).

[0282] Exemplary Fc domain sequence Human IgG1 Fc region (CH2 and CH3 domains), unmodified (SEQ ID NO: 92): JPEG2026514992000041.jpg16161

[0283] Another exemplary IgG1 Fc region sequence is shown below (SEQ ID NO: 49). This sequence has four substitutions (in bold) compared to the unmodified IgG1 Fc sequence (SEQ ID NO: 92) described above. These are the N297G substitution, which inhibits binding to FcγR, and the T366S, L368A, and Y407V substitutions (hole-forming substitutions), which promote dimerization with other Fc regions (e.g., SEQ ID NO: 52), including the T366W substitution (knob-forming substitution). The substitution numbering in this sequence follows the EU numbering system. JPEG2026514992000042.jpg22131

[0284] Another exemplary IgG1 Fc region sequence is shown below (SEQ ID NO: 52). This sequence has two substitutions (double underlined) compared to the unmodified IgG1 Fc sequence (SEQ ID NO: 92) described above. These are the N297G substitution, which inhibits binding to FcγR, and the T366W substitution (knob-forming substitution), which promotes dimerization with other Fc regions (e.g., SEQ ID NO: 49), including the T366S, L368A, and Y407V substitutions (hole-forming substitutions). The substitution numbering in this sequence follows the EU numbering system. JPEG2026514992000043.jpg22131

[0285] Another exemplary IgG1 Fc region sequence is shown below (Sequence ID 93). This sequence is identical to Sequence ID 49, except that it further includes the M252Y, S254T, and T256E substitutions (EU numbering system) shown in bold to enhance binding to FcRn. JPEG2026514992000044.jpg22131

[0286] Another exemplary IgG1 Fc region sequence is shown below (SEQ ID NO: 94). This sequence is identical to SEQ ID NO: 52, except that it further includes the M252Y, S254T, and T256E substitutions (EU numbering system) shown in bold to enhance binding to FcRn. JPEG2026514992000045.jpg22131

[0287] Exemplary IgG hinge sequence The following sequence number 95 is an exemplary IgG1 hinge sequence (compared to the natural human IgG1 sequence, it includes a C-to-S substitution at position 5, numbered according to sequence number 95; double underlined): JPEG2026514992000046.jpg531

[0288] The following sequence number 50 is another example IgG1 hinge sequence (a shortened version of the above IgG1 hinge sequence): JPEG2026514992000047.jpg651

[0289] The following sequence number 96 is an exemplary IgG4 hinge sequence. JPEG2026514992000048.jpg525

[0290] Exemplary amino acid linker sequences GGGGS (SEQ ID NO: 73), GGGSG (SEQ ID NO: 55), GGSGG (SEQ ID NO: 56), GSGGG (SEQ ID NO: 57), GSGGGP (SEQ ID NO: 58), GGEPS (SEQ ID NO: 59), GGEGGGP (SEQ ID NO: 60), GGEGGGSEGGGS (SEQ ID NO: 61), GGGSGGGG (SEQ ID NO: 62), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), EAAAK (SEQ ID NO: 65), and EAAAKEAAAKEAAAK (SEQ ID NO: 66)

[0291] Amino acid sequences of human PD-1 and PD-L1 Sequence ID 101 is the amino acid sequence of the extracellular region of human PD-1 (bold residues are located in the VHH epitopes described in Sequence IDs 42 and 71): JPEG2026514992000049.jpg11161

[0292] Sequence ID 102 is the amino acid sequence of a soluble fragment of human PD-L1 that can bind to PD-1: JPEG2026514992000050.jpg11161 [Examples]

[0293] The present invention will be better understood by referring to the following examples. However, these should not be construed as limiting the scope of the invention. The examples and embodiments described herein are for illustrative purposes only, and those skilled in the art will be able to propose various modifications or changes thereon, which should be understood to be within the scope of this application and the appended claims. [Example 1]

[0294] Isolation of a soluble native TCR that binds to a preproinsulin (PPI)-derived HLA-A*02 restriction peptide but does not bind to a mimetic peptide. a) Production of soluble natural TCRs TCRs binding to the preproinsulin-derived HLA-A*02 restriction peptide ALWGPDPAAA were isolated by panning of a TCR phage library, and the amino acid sequences of the corresponding TCR alpha and beta variable regions were determined. The construction and panning of the natural TCR phage library have been previously described (International Publication Nos. 2015136072, 2017046201, and 2017046198). Soluble TCRs were prepared by fusing the variable regions to cleaved versions of the constant domains of the alpha and beta chains, respectively, and incorporating unnatural interchain disulfide bonds between the constant domain residues, as previously reported (International Publication No. 2003020763). To purify the soluble TCRs, the alpha and beta chains were expressed separately in E. coli inclusion cells. Subsequently, the solubilized inclusion cells containing the alpha and beta chains were mixed. The refolded soluble TCR was further purified by anion exchange and size exclusion chromatography using established methods (Boulter, et al. (2003), Protein Eng. 16, 707-711; Liddy, et al. (2012), Nature medicine vol. 18,6: 980-7). The yield was calculated from the concentration of the purified material, determined from the absorbance at 280 nm using a Nanodrop spectrophotometer.

[0295] b) Bond characterization To evaluate the ability of soluble TCRs to recognize target peptide-MHC complexes, binding parameters were obtained by surface plasmon resonance (SPR). SPR measurements were performed on BIAcore 8K, BIAcore 3000, or BIAcore T200 instruments. Briefly, biotinylated class I HLA-A*02 molecules were refolded with the target peptide and purified using available methods (O'Callaghan et al. (1999), Anal Biochem 266(1): 9-15; Garboczi, et al. (1992), Proc Natl Acad Sci USA 89(8): 3429-3433). Biotinylated peptide-MHC monomers were immobilized on streptavidin-coupled CM-5 sensor chips. Equilibrium binding constants were determined using serial dilutions of soluble TCRs. D The values ​​were obtained by nonlinear curve fitting using Prism software and the Langmuir binding isotherm (binding = C * Max / (C + KD), where "binding" is the equilibrium binding of the response unit at the injected TCR concentration C, and Max is the maximum binding). Unless otherwise noted, measurements were performed at 25°C in Dulbecco's PBS buffer supplemented with 0.005% P20.

[0296] The following table details two soluble TCRs identified from the library. Both TCRs bind to the target peptide-MHC complex with low micromolar concentration range affinity and can be purified from E. coli in high yield. The complete amino acid sequences of the alpha and beta chains of the soluble S2 TCR are described in SEQ ID NO: 2 and SEQ ID NO: 12, respectively.

[0297] [Table 2]

[0298] c) Evaluation of specificity To determine the specificity of target recognition, the binding of the natural TCR to the alternative peptide-MHC complex was evaluated using the same Biacore procedure as described above.

[0299] First, peptide mimetic molecules of the target sequence ALWGPDPAAA were identified that had up to three mismatches and were confirmed by mass spectrometry to form complexes with HLA-A*02 and be spontaneously presented on the cell surface. Five mimetic peptides were identified, which are shown in the table below.

[0300] [Table 3]

[0301] Figure 1 shows the relative binding of soluble TCR to each mimetic peptide. The data shows that TCR S2 did not show any detectable binding to any of the above mimetic peptides that are highly similar to the reference peptide ALWGPDPAAA (SEQ ID NO: 1). On the other hand, binding to Mim1 was detected for TCR S1.

[0302] Next, we evaluated TCR binding to a mixture containing 20 HLA-A*O2-binding peptides that were determined to be naturally presented in high quantities on cells by mass spectrometry. In this case, no binding for TCR S1 and S2 was detected.

[0303] d) Generation of binding motifs To further investigate the interaction between soluble TCR and peptide ALWGPDPAAA (SEQ ID NO: 1), each binding motif was determined using an approach similar to the previously described method (International Publication No. 2014096803). Briefly, each amino acid in the peptide was sequentially substituted with serine, and TCR binding was evaluated using Biacore. The position within the peptide was considered essential for recognition if the corresponding serine substitution mutant showed a 50% or greater decrease in binding affinity compared to the WT peptide, and was therefore considered part of the binding motif. The more essential residues in the motif, the higher the specificity of the TCR.

[0304] Figure 2 shows the binding motifs of S1 and S2 TCRs. For TCR S1, five residues were shown to be essential for recognition, while for TCR S2, seven residues were essential. [Example 2]

[0305] Generation of soluble TCR mutants with high pM affinity and specificity after affinity maturation. a) Affinity maturation by phage display As previously described, we identified mutations with higher affinity using a naturally occurring soluble S2 TCR as a template (Li et al. (2005), Nat. Biotechnol. 23, 349-354). In short, we constructed a TCR phage library using NNK oligonucleotides to generate mutations in the complementarity-determining regions (CDRs) of TCR alpha and beta. Typically, multiple affinity maturation cycles are required to achieve picomolar (pM) affinity. We monitored the binding of mimetic peptides during affinity maturation.

[0306] b) Bond characterization For high-affinity interactions, binding parameters were determined by single-cycle kinetic analysis. Five different concentrations of the binding molecule were injected onto a flow cell coated with approximately 100–200 RU (or 50–100 RU for the Biacore 8K instrument) of peptide-MHC complexes, using a flow rate of 50–60 μl / min. Typically, 60–120 μl (or approximately 240 μl for the Biacore 8K instrument) of the binding molecule was injected at a maximum concentration of 50–100 nM (or 2–50 nM for the Biacore 8K instrument), with the other four injections using sequential 2-fold dilutions. The lowest concentration was injected first. To measure the dissociation phase, buffer was injected until more than 10% dissociation occurred, usually after 1–3 hours. Dynamic parameters were calculated using BIAevaluation® software. The dissociation phase was fitted to a single exponential decay equation, which allowed for the calculation of the half-life. The equilibrium constant KD was calculated from koff / kon. Unless otherwise specified, measurements were performed at 25°C in Dulbecco's PBS buffer supplemented with 0.005% P20.

[0307] Surprisingly, a mutation exhibiting pM affinity target binding was identified while maintaining a high level of binding specificity. The resulting TCR was named "a2b3". Binding to mim1 was detected in the nM range. No binding to other mimetic molecules was detected.

[0308] [Table 4]

[0309] These data demonstrate that TCR a2b3 possesses high specificity and can adequately distinguish mimics with only a single residue mismatch. The affinity window between recognition of (SEQ ID NO: 1) and the off-target (Mim1) is 4400-fold, which provides a large potential safety window and indicates that TCR is particularly suitable for the development of therapeutic agents for the treatment of type 1 diabetes (T1D). [Example 3]

[0310] Soluble TCRs exhibit improved specificity and increased yield compared to previously disclosed TCRs that bind to the same peptide. A soluble, high-affinity TCR that recognizes the same ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex is disclosed in International Publication No. 2015092362. All disclosed TCRs are variants of surrogate native TCRs isolated from T cell clones obtained from human donors. Further analyses were performed to determine the production yield and specificity of the previously disclosed TCRs.

[0311] Soluble TCRs having alpha and beta variable domains corresponding to SEQ ID NO: 58 and SEQ ID NO: 90, respectively, in International Publication No. 2015092362, were evaluated using the methods described in Examples 1 and 2 above. The binding affinity of this TCR to the ALWGPDPAAA(SEQ ID NO: 1)-HLA-A*02 complex was found to be 240 pM. When expressed in E. coli using the method described above, this TCR had a yield of less than 400 μg / L. Furthermore, the same TCR was shown to bind to Mim1 with an affinity window of less than 3x using the Biacore method, similar to the method described above. These data indicate that the TCRs previously disclosed in International Publication No. 2015092362 have substantially lower yields and specificity compared to the TCRs disclosed herein, and are therefore less suitable for the development of therapeutic agents for T1D. [Example 4]

[0312] The binding molecule, which includes a TCR variant fused to the PD-1 agonist-binding domain, retains target specificity and is functional in vitro. a) Sequence optimization The soluble TCR a2b3 was further mutated to remove a potential glycosylation site and modify other residues that were thought to be potentially harmful to the manufacturing process. The sequences of the resulting soluble TCR mutants are shown.

[0313] [Table 5]

[0314] b) PD1 agonist immunosuppressive factors V of PD-1 agonist antibodies HH The domain was fused to the N-terminus of the beta chain of a soluble TCR via a short linker to produce a "TCR PD-1" agonist-binding molecule.

[0315] PD-1-specific antibodies were generated after immunizing llamas with recombinant human PD-1-His protein (Acro Biosystems #PD1-H5221) and performing 2-3 phage display panning with immobilized recombinant human PD-1-Fc protein (Acro Biosystems #PD1-H5257). Selected PD-1 agonists in monomeric form V HH Domain specificity was confirmed using Retrogenix Cell Microarray Technology, which includes a panel of over 5000 cell-binding antigens (Figure 9). Biacore measurements confirmed that the TCR PD-1 agonist molecule binds to PD-1 non-competitively with its native ligand (PD-L1) and is therefore additive to the native PD-L1 response. Briefly, a biotinylated PPI peptide HLA-A*02 complex was immobilized on a streptavidin-coated CM5 chip. The TCR PD-1 agonist molecule is an affinity-enhanced PPI 15-24 TCR-PPI 15-24 The peptide was captured on the chip via HLA-A*O2 interaction. Excess PD-1 was allowed to pass through the chip (1 μM or 10 × K for each PD-1 antibody). D ), followed by passing an excess amount of PD-1 and PD-L1-Fc (15 μM). The PD-1 agonist VHH was then subjected to a K in the range of 50-70 nM. D It was confirmed that it binds to PD-1 with a binding half-life in the range of 15-20 seconds. The sequence of the PD-1 VHH domain is described in SEQ ID NO: 42, and a humanized variant of the same VHH is described in SEQ ID NO: 71.

[0316] Crystallographic analysis was used to map the epitopes recognized by the PD-1 agonist VHH. All three CDR loops were shown to mediate contact with PD-1. Molecular modeling showed that the epitopes are located away from the membrane proximal region and are adjacent to but do not overlap with the PD-L1 binding site, which is consistent with Biacore competitive measurements. The key residues to which the PD-1 agonist VHH (SEQ ID NOs. 42 and 71) bind are shown in bold in the following sequence of the extracellular domain of human PD-1 (SEQ ID NO: 101). JPEG2026514992000055.jpg11161

[0317] c) Half-life extension (Fc) domain To extend the in vivo half-life, a functionally silent Fc domain (including SEQ ID NOs. 49 and 52) was added to the C-terminus of the TCR alpha chain via a cleaved hinge region (SEQ ID NOs. 50).

[0318] Figure 3 shows a schematic diagram of the obtained half-life extended TCR PD-1 agonist binding molecule.

[0319] d) Mammalian expression The molecules were expressed in CHO cells using the Thermo ExpiCHO® transient expression protocol, and then purified by immobilized metal affinity chromatography and size exclusion chromatography.

[0320] e) Biophysical characterization TCR PD-1 agonist-binding molecules containing an Fc domain were tested for binding to target peptides and mimetic peptides. The experiments were performed using the single-cycle kinetic analysis described above, except that the measurements were taken at 37°C.

[0321] [Table 6]

[0322] The data demonstrate that TCR PD-1 agonist-binding molecules containing an Fc domain can be produced in high yield in mammalian cells, maintaining high-affinity recognition of the target and an appropriate window for binding to mim1. This high level of specificity suggests that the molecule is particularly well-suited for therapeutic development as a potential treatment for T1D.

[0323] f) In vitro function-Jurkat NFAT cell reporter assay To determine the ability of TCR PD-1 agonist-binding molecules to inhibit signaling in activated T cells, we developed an NFAT reporter assay. Briefly, Jurkat cells expressing i) a TCR specific to the Melan A-derived HLA-A*02 restriction peptide (ELAGIGILTV), ii) PD-1, and iii) a luciferase reporter driven by an NFAT-responsive element were incubated with the PPI-positive beta cell line ECN90 pulsed with the Melan A-derived peptide, thereby inducing TCR signaling and NFAT promoter-mediated luminescence. Control experiments were performed using a PPI-negative target cell line (Mel624, NCI-H1703) instead of ECN90.

[0324] Target cells were harvested and seeded at 50,000 cells / well in Optiβ3 medium into the inner 60 wells of a white 96-well cell culture plate pre-coated with β-coat (Univercell Biosolutions). After incubation at 37°C and 5% CO2 for 16–20 hours, the medium was removed and assay buffer containing Melan-A peptide was added. No peptide was added to the Mel624 melanoma cell line, which spontaneously displays Melan-A peptide. After pulsed at 37°C and 5% CO2 for 2 hours, assay buffer alone or assay buffer containing TCR PD-1 agonist-binding molecules at serial dilutions was added to each well. The assay was initiated by immediately adding 50,000 Jurkat NFL Mel5 PD-1 effector cells and incubating at 37°C and 5% CO2 for 16–20 hours. Bioluminescence signals were detected and quantified using the Bio-Glo® Luciferase Assay System (Promega) and a luminometer (CLARIOstar). NFAT activity was normalized to a TCR-stimulated control, and dose-response data were analyzed using Prism (GraphPad) with a four-parameter nonlinear least squares method. IC 50 The value was determined.

[0325] The resulting IC 50The values ​​are shown for each TCR-PD1 agonist-binding molecule in the table below. The values ​​are based on the average of two independent experiments. Figure 4 shows the data from one experiment for two of the molecules tested.

[0326] [Table 7]

[0327] Fc domain is IC 50 To evaluate the impact on the values, a similar NFAT-based reporter assay was used.

[0328] In this case, HLA-A*02 human B lymphoblastoid cells (Raji) pulsed with 20 μM PPI peptide at 37°C and 5% CO2 for 2 hours were used as target cells. The cells were harvested and seeded at 50,000 cells / well in assay medium (antibiotic-free R10) in the inner 60 wells of a white 96-well cell culture plate. Subsequently, the cells were treated with 2 μg / ml SEB (Staphylococcal enterotoxin B) at 37°C and 5% CO2 for 1 hour. Assay buffer alone or assay buffer containing TCR PD-1 agonist-binding molecules at serial dilutions was added to each well. The assay was initiated by immediately adding 50,000 Jurkat NFL Mel5 PD-1 effector cells and incubating at 37°C and 5% CO2 for 16–20 hours. Bioluminescence signals were detected by the method described above.

[0329] The data were obtained using the TCR-PD1 agonist-binding molecule a18b16 having an Fc domain as described in Example 4, and glycosylated variants regardless of the presence or absence of the Fc domain.

[0330] The data in Figure 4b shows that including the Fc domain has minimal effect on in vitro efficacy.

[0331] Reporter assay data demonstrate that TCR PD-1 agonist-binding molecules can potently inhibit T cell activation, indicating molecular therapeutic potential in the treatment of T1D. [Example 5]

[0332] TCR-PD1 agonist-binding molecules extend the half-life in vivo. The pharmacokinetic properties of the TCR PD-1 agonist-binding molecule a18b16 (described in Example 4), which contains an Fc domain, were evaluated in SCID mice. The test substance was administered intravenously (IV) or subcutaneously (SC) at a dose of 1 mg / kg, and blood samples were collected continuously for 21 days. Samples were taken from four mice at each time point and for each administration route. The binding molecule was detected in serum using a bifunctional MSD (Meso Scale Diagnostics) assay. PK parameters were extracted by non-compartmental analysis.

[0333] The average PK parameters are shown in the table below. Figure 5 shows the serum concentrations of the test substance over a three-week period.

[0334] [Table 8]

[0335] In this study, terminal t of TCR PD-1 agonists with Fc 1 / 2 The incubation period was approximately 7 days, and the bioavailability after subcutaneous administration was shown to be over 80%. These characteristics indicate therapeutic potential for simplifying the administration schedule in the treatment of T1D. [Example 6]

[0336] TCR-PD-1 agonist-binding molecules show potent efficacy in in vitro models. a) Primary human T cell IL-2 assay The TCR PD-1 agonist binding molecules described in Example 4 were tested to determine their ability to inhibit the activation of primary human CD4+ T cells by antigen-presenting cells (APCs). As a control, free PD-1 agonists were used along with untargeted TCR PD-1 agonist controls that do not bind to PPI peptides.

[0337] Raji cells transduced with HLA-A*02 β2-microglobulin were used as the APC (Raji-A2). Primary human CD4+ T cells were isolated from PBMCs using a pan-T cell isolation kit (Miltenyi). T cells were pre-activated by incubation with irradiated Raji A2 cells pre-loaded with 1 μg / ml SEB (Sigma). After pre-activation, the proliferated T cells were mainly CD4+ T cells, and typically 60-70% were PD-1 positive. Raji A2 cells were sterilized using 20 μM PPI peptide in a 2 × 10⁶ IVF test. 6 Raji A2 cells were pulsed in R10 medium at a concentration of cells / ml at 37°C and 5% CO2 for 2 hours, or left unpulsed. Subsequently, Raji A2 cells were loaded with 31.6 ng / ml of SEB at 37°C and 5% CO2 for 1 hour, followed by 33 Gy of radiation. Raji A2 cells were seeded at 100,000 cells / well, and the test molecule was added. After 1 hour of pre-incubation, washed pre-activated T cells were added to the Raji A2 cells at 100,000 cells / well, and incubated at 37°C and 5% CO2 for 48 hours. The supernatant was collected, and IL-2 levels were measured by ELISA (IL2 Ready-SET-Go! ELISA, Invitrogen). IL-2 release was normalized to the SEB-stimulated control, and dose-response data were analyzed using Prism (GraphPad) with a 4-parameter nonlinear least squares method. IC 50 The value was determined.

[0338] The results showed that, in the presence of APCs pulsed with PPI peptides, TCR PD-1 agonist molecules, when present at picomolar concentrations, reduced IL-2 production from activated T cells by 40-50% (Figure 6). Furthermore, no reduction in IL-2 levels was observed with PD-1 agonist alone or in control with untargeted TCR PD-1 agonist, indicating that targeting of PD-1 agonists to immune synapses is necessary for functional activity.

[0339] These data demonstrate that targeted TCR PD-1 agonist molecules are potent inhibitors of primary CD4+ T cells. Furthermore, the lack of activity observed with untargeted molecules suggests the possibility of avoiding the risk of systemic activation in vivo.

[0340] b) Protection of pancreatic β-cells co-cultured with autoreactive T cells The TCR PD-1 agonist binding molecules described in Example 4 were tested to determine their ability to inhibit the killing of pancreatic β-cell line EndoCβH2-A2 and cytokine release by autoreactive CD8+ T cells.

[0341] EndoC-βH2 target cells labeled with mKate 2 (EndoC-βH2 Red) were generated by transducing EndoC-βH2 cells with the HLA-A*02 β2-microglobulin lentivirus construct and the NucLight red lentivirus reagent (Sartorius). Target cells were placed in Optiβ3 medium in 5 × 10⁶ wells of a 96-well plate. 4 Cells were seeded and incubated overnight at 37°C and 5% CO2. TCR PD-1 agonist molecules or control molecules were added at different concentrations and incubated for 2 hours. To initiate the assay, one of two β-cell-specific CD8+ T cell clones with high / low affinity to target cells was added to EndoC-βH2 red target cells in a 5×10⁶ dose. 4Cells were added per well. As an additional control, PD-L1 transduced EndoC-βH2 red target cells + / - anti-PD-L1 blocking antibody were used. Cell toxicity was determined by quantifying the number of EndoC-βH2 red cells over time using the IncuCyte S3 imaging system (Sartorius). The number of red nucleus-labeled cells at each time point was normalized to the initial number of objects to account for fluctuations in cell density within the visible region. The number of events was obtained from four images and averaged. Cytokine release was measured using the V-PLEX Plus Proinflammatory Panel 1 (human) kit (MSD, Meso Scale Diagnostics) according to the manufacturer's instructions, using the culture supernatant from the IncuCyte toxicity assay 24 hours after each time point. Unstimulated T cells alone were also evaluated as an additional control in the cytokine assay. Cytokine release was normalized to the stimulated control, and dose-response data were analyzed using the 4-parameter nonlinear least squares method in Prism (GraphPad). IC 50 The value was determined.

[0342] The data showed that when co-cultured in the presence of autoreactive T cells, the relative number of β cells increased in a dose-dependent manner with increasing concentrations of the TCR PD-1 agonist-binding molecule, indicating that the molecule can protect β cells from being killed by autoreactive T cells. No effect was observed with PPI TCR alone or with an untargeted control (Figure 7a).

[0343] When the cell culture supernatants from both co-culture assays were evaluated for cytokine production, it was shown that the TCR PD-1 agonist strongly inhibited IFNγ production by autoreactive T cells (Figure 7b).

[0344] These data demonstrate that the TCR PD-1 agonist-binding molecule inhibits killing and cytokine release by T cells located at both ends of the predicted affinity range of the innate repertoire of autoreactive T cells, indicating the molecule's therapeutic potential.

[0345] c) PD-1 +veSuppression of NK cell stimulation The TCR PD-1 agonist-binding molecule described in Example 4 is used with PD-1 +ve Further investigations were conducted to determine the ability to inhibit NK cell stimulation. To investigate whether TCR PD-1 agonists could specifically suppress PD-1+ NK cells, NK cells were activated in the pancreatic β cell line EndoC-βH2. Activation was monitored by the expression of the cytotoxic marker CD107a and IFNγ production.

[0346] Primary human NK cells were isolated from PBMCs using an NK cell isolation kit (Miltenyi Biotec 130-092-657). The NK cells were incubated for 6 days in R10 medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate) containing dexamethasone (500 ng / mL, Merck, D2915), IL-12 (10 ng / mL, Miltenyi Biotec 130-096-704), IL-15 (25 ng / mL, Peprotech), and IL-18 (100 ng / mL, R&D systems, 9124-IL-050). Six days later, NK cells were washed with R10 and incubated with EndoC-βH2 HLA-A*02+ cells in a 1 / 4 (effector / target) ratio, with or without TCR-PD1 agonist-binding molecules, in R10 containing monensin, brefeldin A (GolgiPlug and GolgiStop BD), and anti-CD107a antibody for 4 hours (37°C, 5% CO2). After activation, NK cells were subjected to surface staining (anti-CD56, anti-CD3, anti-PD1, and dead cell markers) for 30 minutes, followed by fixation and permeabilization for intracellular staining of IFNγ (eBioscience Foxp3 Transcription Factor Staining Buffer Set Cat: 00-5523-00).

[0347] The data showed that CD107 and IFNγ expression levels in PD-1+ve NK cells were reduced in the presence of a TCR PD-1 agonist. No effect was observed in PD-1-ve NK cells. Therefore, the TCR PD-1 agonist specifically reduces PD-1+ NK cell activation (Figure 8). The data presented were obtained from two independent experiments.

[0348] These data show that the TCR PD-1 agonist binding molecule is PD-1 +ve This demonstrates the inhibition of NK cell stimulation. This could offer a potential additional therapeutic mechanism and differentiate it from other approaches.

[0349] Overall, these data obtained from various disease-related models support the therapeutic potential of TCR-PD-1 agonist molecules for the treatment of T1D. [Example 7]

[0350] Further evidence that TCR-PD-1 agonist molecules suppress the effector function of PD-1+ NK cells NK cells infiltrate the pancreas of type 1 diabetic patients and kill human pancreatic β-cells. To further investigate the mechanism of action of TCR-PD-1 agonist molecules, an established in vitro NK effector functional model was used. NK cell line NK92, which expresses PD-1, was activated by pulsed PPI peptides along with K562 lymphoblast cells transduced with HLA-A*02 (Figure 10a). NK cell activation was measured in the presence of either the TCR-PD-1 agonist molecule described in Example 4, or a control molecule containing an unrelated TCR fused to a PD-1 agonist. NK cell activation was evaluated by monitoring IFNγ production and CD107 degranulation marker expression.

[0351] In short, HLA-A*02+ve K562 target cells were used as target cells. They were labeled with Cell Tracker Orange (Invitrogen) for 30 minutes, washed, and resuspended in SCGM medium containing 20% ​​FBS. The target cells were loaded with the PPI peptide ALWGPDPAAA (SEQ ID NO: 1) at a concentration of 20 μM for 1 hour, and then incubated with either a TCR-PD-1 agonist molecule, a18b16, or a control molecule at the indicated final concentration for 1 hour. TCR-PD-1 agonist binding was confirmed with PE-conjugated goat anti-human IgG Fc. The number of bound molecules was calculated using the PE Quantitation Kit (BD Bioscience) (Figure 10b). NK92-PD-1+ cells were added to target cells along with monensin, brefeldin A (GolgiPlug 1 / 500 and GolgiStop 1 / 750 BD), and anti-CD107a antibody, and incubated for 4 hours (37°C, 5% CO2). After activation, NK92-PD-1+ cells were surface-stained (anti-CD56-BB700, anti-PD-1-PE-Cy7, and dead cell marker-Pacific-Orange) for 30 minutes, followed by fixation and permeabilization for intracellular staining of IFNγ (eBioscience Foxp3 Transcription Factor Staining Buffer Set Cat: 00-5523-00).

[0352] TCR-PD-1 agonist molecules were confirmed to suppress PD-1+ NK cell activation in a dose-dependent manner, while control molecules showed no effect (Figures c and d). TCR-PD-1 agonists significantly reduced IFNγ production (29% to 19%) and CD107 expression (35% to 24%) at a concentration of 10 nM, just 4 hours after the start of activation.

[0353] These data, obtained using an established NK cell effector function model, further demonstrate that TCR-PD-1 agonist molecules can specifically and concentration-dependently downregulate NK cell effector function, thus potentially providing an additional therapeutic mechanism for the treatment of T1D. [Example 8]

[0354] TCR-PD-1 agonist molecules confer a persistent wasting-like phenotype to CD4 T cells. To further investigate the effects of TCR-PD-1 agonists on the modulation of T cell responses, purified human CD4 T cells were activated with irradiated Raji-HLA-A2 loaded with SEB superantigen and PPI peptide, and incubated with the TCR-PD-1 agonist molecules described in Example 4. Decreased IL2 release was used as a marker of T cell depletion (Figure 11a).

[0355] In short, primary human CD4 T cells were isolated from frozen PBMCs using a CD4 T cell isolation kit (Miltenyi Biotec) and incubated in R10 medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate) at a rate of 2 × 10⁶ times. 6 The cells were resuspended at a concentration of cells / mL. Raji-HLA-A2 was loaded with SEB peptide (100 ng / mL List Labs) and PPI peptide (40 μM ALWGPDPAAA) in R10 medium at a concentration of 2.106 cells / mL for 1 hour (37°C, 5% CO2), and then irradiated with 60 Gray. The irradiated Raji-HLA-A2 was then incubated for 1 hour (37°C, 5% CO2) in or without a 20 nM TCR-PD-1 agonist, a19b20. CD4 T cells were activated for 8 days using irradiated Raji-HLA-A2 (effector cell to Raji cell ratio 1:1, with 10 nM TCR-PD-1 agonist added). On day 8, the activated CD4 T cells were washed twice with R10 medium and re-incubated for 3 days with freshly irradiated Raji-HLA-A2 prepared using the method described above. Culture supernatant was collected on days 3 and 11 during CD4 T cell activation. IL-2 produced by CD4+ T cells from days 0 to 3 and from days 8 to 11 was measured by ELISA (Invitrogen).

[0356] TCR-PD-1 agonists activated the PD-1 pathway on interacting CD4 T cells, achieving immunosuppression during T cell priming, as indicated by a decrease in IL-2 secretion on day 3 (Figure 11b). Sustained activation of CD4 T cells in the absence of TCR-PD-1 agonists induced a depletion-like phenotype, as indicated by a decrease in IL-2 concentration at reactivation from 15,000 pg / mL to 38 pg / mL on day 3 (Figures 11b and c, mean, untreated). Importantly, the decrease in IL-2 was further enhanced in the presence of TCR-PD-1 agonists prior to reactivation (Figure 11c). Furthermore, the addition of TCR-PD-1 agonists after reactivation also enhanced the decrease in IL-2 production, suggesting the potential for sustained immunomodulation (Figure 11d).

[0357] These data suggest that the TCR-PD-1 agonist molecule of the present invention may result in enhanced and sustained downregulation of the CD4 T cell response in vivo, which could offer a novel mechanism of action for the treatment of T1D.

Claims

1. A binding molecule comprising a peptide-major histocompatibility complex (pMHC) binding domain having the property of binding to the ALWGPDAAA (SEQ ID NO: 1) HLA-A*02 complex, wherein the pMHC binding domain comprises (i) an alpha chain containing at least a TCR alpha chain variable domain, and (ii) a beta chain containing at least a TCR beta chain variable domain, where, (a) The TCR alpha chain variable domain has the following sequence: A sequence CDR1-DKHSQG (Sequence ID 23) having any one, two, or three mutations, CDR2-IYSQGD (Sequence ID 27) is a sequence having one, two, or three mutations, A sequence of CDR3-AVRGNEKLT (Sequence ID 7) having any one, two, or three mutations. Includes CDR1, CDR2 and CDR3, and / or (b) The TCR beta chain variable domain has the following sequence: A sequence CDR1-LQHSY (Sequence ID 35) having one, two, or three mutations, CDR2-SVGVGF (Sequence ID 29) is a sequence having one, two, or three mutations, A sequence of CDR3-ASAYMTGELF (Sequence ID 30) having one, two, or three mutations. A binding molecule containing CDR1, CDR2, and CDR3.

2. The binding molecule according to claim 1, wherein the mutation(s) in the TCR alpha chain variable domain CDR are selected from K28R(CDR1), H29G(CDR1), G32S(CDR1), and Q53N(CDR2) numbered according to SEQ ID NO: 26, and / or the mutation(s) in the beta chain CDR are selected from L27M(CDR1), Q28N(CDR1), S30N(CDR1), V52A(CDR2), F54I(CDR2), and A104S(CDR3) numbered according to SEQ ID NO:

74.

3. The following combinations of TCR alpha-chain variable domain CDRs and TCR beta-chain variable domain CDRs: (a) The amino acid sequences of alpha chain CDR1, CDR2, and CDR3, respectively, DRGSQS (SEQ ID NO: 5), IYSNGD (SEQ ID NO: 6), and AVRGNEKLT (SEQ ID NO: 7), and the amino acid sequences of beta chain CDR1, CDR2, and CDR3, respectively, MNHNY (SEQ ID NO: 15), SVGAGI (SEQ ID NO: 16), and ASSYMTGELF (SEQ ID NO: 17); (b) The alpha-chain CDR1, CDR2, and CDR3 amino acid sequences, respectively DKHSQG (SEQ ID NO: 23), IYSNGD (SEQ ID NO: 6), and AVRGNEKLT (SEQ ID NO: 7), and the beta-chain CDR1, CDR2, and CDR3 amino acid sequences, respectively MNHSY (SEQ ID NO: 28), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30); (c) The alpha chain CDR1, CDR2, and CDR3 amino acid sequences, respectively DKHSQG (SEQ ID NO: 23), IYSNGD (SEQ ID NO: 6), and AVRGNEKLT (SEQ ID NO: 7), and the beta chain CDR1, CDR2, and CDR3 amino acid sequences, respectively MQHSY (SEQ ID NO: 32), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30), or (d) The alpha chain CDR1, CDR2, and CDR3 amino acid sequences of DKHSQG (SEQ ID NO: 23), IYSQGD (SEQ ID NO: 27), and AVRGNEKLT (SEQ ID NO: 7), respectively, and the beta chain CDR1, CDR2, and CDR3 amino acid sequences of LQHSY (SEQ ID NO: 35), SVGVGF (SEQ ID NO: 29), and ASAYMTGELF (SEQ ID NO: 30), respectively. A binding molecule according to claim 1 or claim 2, comprising one of the above.

4. The TCR alpha chain variable domain has the following sequence: FR1-AKEVEQNSGPPLSVPEGAIASLQCTYS (Sequence ID 25), a sequence having one, two, or three mutations, FR2-FFWYRQYSGKSPELIMS (Sequence ID 9), a sequence having one, two, or three mutations, FR3-KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLC (Sequence ID 10), a sequence having one, two, or three mutations, FR4-FGTGTRLTIIP (Sequence ID 11) is a sequence having one, two, or three mutations. Includes framework regions FR1, FR2, FR3 and FR4, And / or, the TCR beta chain variable domain has the following sequence: FR1-NAGVTQTPKFRILKIGQSMTLQCAQD (Sequence ID 18), a sequence having one, two, or three mutations, FR2-MYWYRQDPGMGLKPIYY (Sequence ID 19), a sequence having one, two, or three mutations, FR3-TDKGEVPQGYQVSRSTTEDFPLRLESAAPSQTSVYFC (Sequence ID 75), a sequence having one, two, or three mutations, A sequence of the form FR4-FGEGSRLTVL (Sequence ID 21) having one, two, or three mutations. The binding molecule according to any one of the claims, comprising framework regions FR1, FR2, FR3, and FR4.

5. The binding molecule according to claim 4, wherein the mutation(s) in the TCR alpha chain variable domain framework region are selected from A1Q and Q22N numbered according to SEQ ID NO: 26, and / or the mutation(s) in the TCR beta chain variable domain framework region are selected from Q62N, Q62E, Q62D, and Q65N numbered according to SEQ ID NO:

74.

6. (a) The TCR alpha chain variable domain comprises an amino acid sequence described in any one of SEQ ID NOs: 3, 22, 24, and 26, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with any one of SEQ ID NOs: 3, 22, 24, and 26, and (b) The TCR beta chain variable domain comprises an amino acid sequence described in any one of SEQ ID NOs: 13, 68, 31, 34, 74, 76, and 78, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with any one of SEQ ID NOs: 13, 68, 31, 34, 74, 76, and 78. The binding molecule according to any one of the above claims.

7. The following combinations of TCR alpha and beta chain variable domains: (a) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 22, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 68; (b) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 24, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 31; (c) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 34; (d) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 74; (e) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO: 76; or (f) A TCR alpha chain variable domain containing the amino acid sequence described in SEQ ID NO: 26, and a TCR beta chain variable domain containing the amino acid sequence described in SEQ ID NO:

78. A binding molecule according to any one of the above claims, comprising one of the above.

8. The binding molecule according to any one of the claims, wherein the TCR alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 26, and the TCR beta chain variable domain comprises the amino acid sequence of SEQ ID NO:

74.

9. The binding molecule according to any one of the claims, wherein the alpha chain comprises a TCR alpha chain constant domain and / or the beta chain comprises a TCR beta chain constant domain.

10. The binding molecule according to claim 9, wherein a non-natural disulfide bond links a residue of the TCR alpha chain constant domain to a residue of the TCR beta chain constant domain.

11. The TCR alpha chain constant domain comprises the amino acid sequence described in SEQ ID NO: 37, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 37; and / or The TCR beta chain constant domain includes the amino acid sequence described in SEQ ID NO: 39, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO:

39. The binding molecule according to claim 9 or claim 10.

12. The binding molecule according to any one of the claims, comprising two or more polypeptide chains, wherein the alpha chain and the beta chain are contained in separate polypeptide chains.

13. (a) The alpha chain comprises an amino acid sequence described in any one of SEQ ID NOs: 2, 70, 36, and 40, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with an amino acid sequence described in any one of SEQ ID NOs: 2, 70, 36, and 40, and (b) The beta chain comprises an amino acid sequence described in any one of SEQ ID NOs: 12, 69, 38, 41, 80, 81, and 82, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with an amino acid sequence described in any one of SEQ ID NOs: 12, 69, 38, 41, 80, 81, and 82. The binding molecule according to any one of the above claims.

14. (a) The alpha chain contains the amino acid sequence of SEQ ID NO: 70, and the beta chain contains the amino acid sequence of SEQ ID NO: 69; (b) The alpha chain contains the amino acid sequence of SEQ ID NO: 36, and the beta chain contains the amino acid sequence of SEQ ID NO: 38; (c) The alpha chain contains the amino acid sequence of SEQ ID NO: 40, and the beta chain contains the amino acid sequence of SEQ ID NO: 41; (d) The alpha chain contains the amino acid sequence of SEQ ID NO: 40, and the beta chain contains the amino acid sequence of SEQ ID NO: 80; (e) The alpha chain contains the amino acid sequence of SEQ ID NO: 40 and the beta chain contains the amino acid sequence of SEQ ID NO: 81; or (f) The alpha chain contains the amino acid sequence of SEQ ID NO: 40, and the beta chain contains the amino acid sequence of SEQ ID NO:

82. The binding molecule according to any one of the above claims.

15. The binding molecule according to any one of claims 1 to 11, wherein the pMHC binding domain is in the form of a single polypeptide chain of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, or Vα-L-Vβ-Cβ, where Vα and Vβ are TCRα and β variable regions, respectively, Cα and Cβ are TCRα and β constant regions, respectively, and L is a linker sequence.

16. The binding molecule according to any one of the claims, further comprising an immunosuppressant.

17. The binding molecule according to claim 16, wherein the immunosuppressive factor is an immune checkpoint agonist, and optionally a PD-1 agonist.

18. The binding molecule according to any one of the claims, wherein the immunosuppressive factor includes an antigen-binding portion capable of binding to an antigen.

19. The binding molecule according to claim 18, wherein the antigen is PD-1 and the antigen-binding portion is a PD-1 agonist.

20. The binding molecule according to claim 18 or claim 19, wherein the antigen-binding portion comprises an antibody or an antigen-binding fragment thereof.

21. The binding molecule according to any one of claims 18 to 20, wherein the antigen-binding portion comprises a single-domain antibody, optionally VHH.

22. A single-domain antibody binds to PD-1, and the following amino acid sequence occurs: CDR1-GFTFSSYA (Sequence ID 43) is a sequence having one, two, or three mutations, CDR2-IASDGAST (Sequence ID 44) is a sequence having one, two, or three mutations, and A sequence of CDR3-CARGGYLTYDRY (Sequence ID 45) having one, two, or three mutations. The binding molecule according to claim 21, comprising CDR1, CDR2, and CDR3, which are CDRs having [a specific characteristic].

23. The conjugation molecule according to claim 21 or 22, wherein the single-domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 42, a humanized version thereof, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO:

42.

24. The conjugation molecule according to claim 21 or 22, wherein the single-domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO:

71.

25. The binding molecule according to any one of claims 16 to 24, wherein an immunosuppressant is covalently linked to a pMHC binding domain via the C-terminus or N-terminus of an alpha or beta chain, optionally via a linker sequence, and further optionally the linker sequence is selected from SEQ ID NOs. 55 to 66 or 73.

26. (a) A first polypeptide chain comprising an immunosuppressant and a beta chain of a pMHC-binding domain, and (b) The binding molecule according to any one of claims 16 to 25, comprising a second polypeptide chain containing an alpha chain of a pMHC binding domain.

27. The binding molecule according to claim 26, wherein the C-terminus of an immunosuppressive factor is optionally covalently linked to the N-terminus of a TCR beta chain via the linker sequence of SEQ ID NO:

73.

28. (a) The first polypeptide comprises an amino acid sequence described in any one of SEQ ID NOs: 72, 46, 47, 83, 84, and 85, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with an amino acid sequence described in any one of SEQ ID NOs: 72, 46, 47, 83, 84, and 85, (b) The binding molecule according to claim 26 or claim 27, wherein the second polypeptide comprises an amino acid sequence described in any one of SEQ ID NOs: 70, 36, and 40, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence described in any one of SEQ ID NOs: 70, 36, and 40.

29. (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70; (b) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36; (c) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (d) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 83, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (e) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; or (f) The binding molecule according to any one of claims 26 to 28, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

40.

30. The binding molecule according to any one of the claims, further comprising a half-life extension domain and optionally comprising a first portion (FC1) and a second portion (FC2) of the IgGFc region.

31. Further comprising a half-life extension domain including a first portion (FC1) of the IgG Fc region and a second portion (FC2) of the IgG Fc region, (a) A first polypeptide chain comprising an immunosuppressant and either (i) an alpha chain or (ii) a beta chain of a pMHC-binding domain; (b) FC1 and a second polypeptide chain comprising (i) the alpha chain and (ii) the other of the beta chain; and (c) The binding molecule according to any one of claims 16 to 29, comprising a third polypeptide chain containing FC2.

32. The binding molecule according to claim 31, wherein the C-terminus of an immunosuppressant is covalently linked to the N-terminus of either the (i) alpha chain or the (ii) beta chain of the pMHC binding domain, optionally via the linker sequence of Sequence ID No.

73.

33. The binding molecule according to claim 31 or 32, wherein (i) the C-terminus of the alpha chain and (ii) the other C-terminus of the beta chain are covalently linked to the N-terminus of FC1 via an IgG hinge sequence, and / or a third polypeptide comprises an IgG hinge sequence at the N-terminus of FC2, and optionally the IgG hinge comprises the amino acid sequence of SEQ ID NO:

50.

34. The binding molecule according to any one of claims 31 to 33, wherein the first polypeptide chain comprises a beta chain of the pMHC binding domain and the second polypeptide chain comprises an alpha chain of the pMHC binding domain.

35. Half-life extension domain (a) One or more amino acid substitutions that promote dimerization of FC1 and FC2; and / or (b) One or more amino acid substitutions that prevent or reduce binding to FcγR; and / or (c) One or more amino acid substitutions that promote binding to FcRn A binding molecule according to any one of claims 30 to 34, including the following:

36. (a) Either FC1 or FC2 contains an amino acid sequence described in SEQ ID NO: 49 or 93, or an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the amino acid sequence described in SEQ ID NO: 49 or 93, and (b) The binding molecule according to any one of claims 30 to 35, wherein the other of FC1 and FC2 comprises an amino acid sequence described in SEQ ID NO: 52 or 94, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence described in SEQ ID NO: 52 or 94.

37. (a) The first polypeptide comprises an amino acid sequence described in any one of SEQ ID NOs: 72, 46, 47, 83, 84, and 85, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with an amino acid sequence described in any one of SEQ ID NOs: 72, 46, 47, 83, 84, and 85, (b) The second polypeptide comprises an amino acid sequence described in any one of SEQ ID NOs: 48, 53, 54, 97, 99, or 100, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with an amino acid sequence described in any one of SEQ ID NOs: 48, 53, 54, 97, 99, or 100, and (c) The binding molecule according to any one of claims 31 to 36, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51 or 98, or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO: 51 or 98.

38. (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51; (b) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 53, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51; (c) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 54, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51; (d) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 83, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 54, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51; (e) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 54, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51; (f) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 85, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 54, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 51; (g) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 72, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 97, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 98; (h) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 46, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 99, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 98; (i) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 47, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 100, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 98; (j) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 83, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 100, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 98; (k) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 98; or (l) The binding molecule according to any one of claims 31 to 37, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

98.

39. The binding molecule according to any one of claims 31 to 37, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 83, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 54 or 100, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51 or 98.

40. The binding molecule according to any one of the claims, wherein the pMHC binding domain binds to the ALWGPDPAAAA (SEQ ID NO: 1)-HLA-A*02 complex with an affinity at least 10 times, at least 100 times, at least 500 times, or at least 1000 times higher than the affinity for the ALLGPDPPAAAA (SEQ ID NO: 67)-HLA-A*02 complex.

41. The following amino acid sequence: A sequence of CDR1-GFTFSSYA (Sequence ID 43) having any one, two, or three mutations; CDR2-IASDGAST (Sequence ID 44) is a sequence having any one, two, or three mutations; and A sequence of CDR3-CARGGYLTYDRY (Sequence ID 45) having one, two, or three mutations. A single-domain antibody that binds to PD-1, comprising CDR1, CDR2, and CDR3 having the following properties.

42. (a) Single-domain antibodies are isolated; and / or (b) The single-domain antibody is VHH; and / or (c) The single-domain antibody is a PD-1 agonist; and / or (d) A single-domain antibody against PD-1 in the range of approximately 1 nM to approximately 500 nM, or in the range of approximately 50 nM to approximately 70 nM. Ｄ Join with; and / or (e) A single-domain antibody binds to an epitope of PD-1 containing one or more or all of the following amino acids numbered according to Sequence ID No. 101: E38, F59, P60, E61, T75, Q76, L77, P78, N79, and G80; and / or (f) The single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 42, or a humanized version thereof, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 42; and / or (g) The single-domain antibody according to claim 41, wherein the single-domain antibody comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO:

71.

43. (a) A single-domain antibody according to claim 41 or claim 42, (b) a pMHC binding domain optionally comprising (i) an alpha chain containing at least a TCR alpha chain variable domain and (ii) a beta chain containing at least a TCR beta chain variable domain; and (c) Optionally, half-life extension domain A binding molecule containing this molecule.

44. (a) A binding molecule according to any one of claims 1 to 30 or 40, wherein the alpha chain and the beta chain are encoded in a single open reading frame or in two different open reading frames; (b) A binding molecule according to any one of claims 31 to 39, wherein the first, second and third polypeptide chains are encoded in a single open reading frame or in different open reading frames; and / or (c) A single-domain antibody according to claim 41 or claim 42, or a binding molecule according to claim 43. Nucleic acids that code for something.

45. An expression vector comprising the nucleic acid described in claim 44.

46. (a) The expression vector according to claim 45; (b) A first expression vector comprising a nucleic acid encoding a first polypeptide comprising the beta chain of the binding molecule according to any one of claims 1 to 30 or 40, and a second expression vector comprising a nucleic acid encoding a second polypeptide comprising the alpha chain of the binding molecule according to any one of claims 1 to 30 or 40; or (c) A first expression vector comprising a nucleic acid encoding a first polypeptide of the binding molecule according to any one of claims 31 to 39; a second expression vector comprising a nucleic acid encoding a second polypeptide of the binding molecule according to any one of claims 31 to 39; and a third expression vector comprising a nucleic acid encoding a third polypeptide of the binding molecule according to any one of claims 31 to 39. Cells that possess [certain properties].

47. Non-natural and / or purified and / or manipulated cells, preferably T cells, that present the binding molecule described in any one of claims 1 to 14.

48. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 40 or 43, a single-domain antibody according to claim 41 or 42, a nucleic acid according to claim 44, an expression vector according to claim 45, and / or a cell according to claim 46 or 47, together with one or more pharmaceutically acceptable carriers or excipients.

49. Preferably, for use in a human subject in a pharmaceutical, a binding molecule according to any one of claims 1 to 40 or 43, a single-domain antibody according to claim 41 or claim 42, a nucleic acid according to claim 44, an expression vector according to claim 45, and / or a cell according to claim 46 or 47.

50. Preferably, a binding molecule according to any one of claims 1 to 40 or 43 for use in a method for treating diabetes in human subjects, a single-domain antibody according to claim 41 or claim 42, a nucleic acid according to claim 44, an expression vector according to claim 45, and / or a cell according to claim 46 or 47.

51. A method for producing a binding molecule according to any one of claims 1 to 40 or 43, or a single-domain antibody according to claim 41 or 42, comprising: a) maintaining the cells according to claim 46 or 47 under conditions optimal for the expression of the binding molecule or single-domain antibody; and b) isolating the binding molecule or single-domain antibody.

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