Chemical manipulation method for TCR bonding mode and use thereof

Abstract

Provided are a modified antigen peptide and a preparation method therefor. The modified antigen peptide is obtained by means of chemical manipulation for a T-cell receptor (TCR) bonding mode. Compared with an unmodified peptide, the bonding form between the modified antigen peptide and a TCR is changed. Also provided is a use of the modified antigen peptide in disease treatment.

Description

Chemical manipulation methods of TCR bonding and applications thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a modified antigen peptide and its application as an immunotherapy tool, which is obtained by chemical manipulation modification of TCR bonding. BACKGROUND

[0002] T cell receptors initiate downstream signaling pathways by recognizing major histocompatibility complex (MHC) and the antigen peptide (pMHC) presented thereby, further regulating the state and function of T cells, and ultimately affecting the fate of T cells [1-2] . The interaction of the TCR-pMHC ternary complex is the most upstream event to initiate T cell-based cellular immunity, and the strength of this interaction directly determines the fate of T cell development and functionalization in vivo [3-7] . Due to the TCR maturation mechanism in vivo, high-affinity TCR epitopes are eliminated under the condition of thymic "negative selection", and the mature and migrated TCR has a weak affinity, with an affinity threshold range of 1-100 μM [8-9] . Among them, the affinity of tumor-derived TCR is mainly distributed in 10-100 μM, and the affinity of virus-derived TCR is mainly distributed in 1-10 μM

[0010] . This is because tumor-derived antigen epitopes are mostly derived from autologous epitope mutations, and strong affinity TCRs recognizing them have been eliminated in the thymus, while virus epitope sequences differ greatly from human body self-epitopes, and can stimulate the production of strong affinity TCRs. In terms of its chemical nature, the amino acid sequence of the virus epitope is very different from that of the human body, and the chemical structure (space) based on the different arrangement of natural amino acids is very different from the chemical space of the human body's own antigen sequence, which can induce the body to produce strong affinity TCRs. This also suggests whether we can obtain stronger affinity by using a chemical space completely different from natural amino acids.

[0003] In recent years, many papers have reported that TCR avidity is positively correlated with T cell function, especially when the affinity of TCR and different pMHC is similar, the avidity of TCR bond after TCR and pMHC binding plays a decisive role in the function and fate of T cells [11-12] . Therefore, methods are needed to break through the weak binding of natural TCR and pMHC to achieve chemical regulation of T cell function and fate through TCR signaling pathways, and to expand the corresponding clinical application scenarios. The optimization of TCR-pMHC interaction can be carried out from both sides of TCR and pMHC. For example, by directed evolution or phage display to obtain TCR sequences with higher affinity and / or avidity for specific pMHC [13-14]On the other hand, antigen analogs (new amino acid sequences) with higher affinity are screened for specific TCRs, such as the Davis team screening alternative peptides for specific pathological environment TCRs by means of yeast display pMHC library, and using the recognition of alternative peptides to TCR to mobilize T cells to function

[0015] Although different large-scale screening methods (such as various surface display strategies) can achieve the enhancement of TCR-pMHC binding force (TCR bond) through rearrangement of natural chemical space (20 amino acids), the strategy of large-scale screening is often time-consuming and labor-intensive, and cannot adjust the TCR-pMHC binding force (affinity) while ensuring that the affinity does not change much. In addition, excessive engineering of TCRs has potential off-target risks. Therefore, it is urgent to develop a new method to improve the interaction of TCR-pMHC ternary complex, to enhance the binding force of the ternary complex (or reverse lock bond

[0016] ) without changing the affinity, in order to develop new immunotherapy tools to adapt to various clinical scenarios of autoimmune diseases and tumor treatment. In the strategy of chemical biology, the introduction of exogenous chemical space (i.e. chemical structures that do not exist in 20 amino acids) is an important method to regulate the activity of biological molecules. Non-natural chemical modification or modification of natural antigen peptides has been used to enhance the binding force of antigen and MHC, but the general view is that the region of the antigen peptide recognized by TCR to form TCR bond generally cannot introduce completely new exogenous chemical modification or chemical structure (such as larger combinations of heteroatoms or heavy atoms) to avoid destroying specific immune recognition or making the antigen completely lose the ability to be recognized by the original TCR. At present, only a few examples introduce chemical modification in the TCR recognition region of natural antigen, but almost all are chemical analogs of natural antigen (i.e. only introduce very small chemical changes, such as H to F), in order to comply with the above principles. SUMMARY

[0004] Unlike the prior art of modifying antigen peptides, or evolving sequences from the TCR side, the present application does not adhere to the bonding form that can be produced by natural amino acids. By introducing unnatural amino acid structures at the TCR recognition site of the antigen peptide, a non-natural chemical bonding space between the TCR and the antigen peptide-MHC (pMHC) is constructed. Surprisingly, it is found that this modification can induce strong interaction of TCR-pMHC, thereby specifically enhancing the functional avidity of TCR-pMHC, and ultimately changing the function and fate of T cells with a given TCR. Compared with before modification, the modified antigen peptide of the present application exhibits great T cell activation properties. In a specific embodiment, the modified antigen peptide of the present application can mediate strong killing effect of specific T cells at very low concentration (such as using 100 pM concentration of modified antigen peptide to induce 1G4 TCR-T cells specific to NY-ESO-1 epitope). In another specific embodiment, the modified antigen peptide of the present application can mediate effective expansion of specific T cells at very low concentration (such as using 10 pM or 100 pM concentration of modified antigen peptide to induce 1G4 TCR-T cells specific to NY-ESO-1 epitope). In another specific embodiment, the modified antigen peptide of the present application can induce specific T cell exhaustion at high concentration (such as using 100 nM or more concentration of modified antigen peptide to induce 1G4 TCR-T cells specific to NY-ESO-1 epitope). In summary, the present application proposes to introduce completely different new chemical space at the TCR recognition region of the antigen to change the chemical type of the TCR bond, thereby greatly enhancing the binding force (avidity) of TCR after recognizing pMHC, and controlling the function and fate of T cells.

[0005] In one embodiment, the modified antigen peptide of the present application can mediate pM level EC 50 of TCR functional avidity, which is comparable to or even exceeds the affinity level mediated by viruses or heterologous antigens (more than 400 times higher than unmodified). The modified antigen peptide of the present application can specifically stimulate T cells, thereby driving T cells to produce stronger phenotypes. For example, in the process of high concentration continuous stimulation of T cells, the modified antigen peptide of the present application can induce the exhaustion phenotype of specific T cells. On the contrary, when stimulated at low concentration, the modified antigen peptide of the present application can stimulate T cells to expand faster and more, while producing a strong activated effector phenotype. The above immunological phenotypes show that the modified antigen peptide of the present application, or the method of modifying antigen peptide of the present application, can be used to develop new immunotherapy tools for various clinical scenarios, such as autoimmune diseases and tumor treatment.

[0006] The present application provides a modified antigenic peptide, which has altered binding mode to a given T cell receptor (TCR) as compared to the binding mode of the unmodified antigenic peptide to the TCR.

[0007] In certain embodiments, the modified antigenic peptide does not substantially generate broad TCR reactivity, i.e., has higher TCR orthogonality (at a given concentration). In certain embodiments, the modified antigenic peptide does not exhibit higher cross-reactivity and immunogenicity than the unmodified antigenic peptide. In certain embodiments, the modified antigenic peptide does not substantially exhibit higher cross-reactivity and immunogenicity than the unmodified antigenic peptide.

[0008] In certain embodiments, the modified antigenic peptide does not substantially generate reactivity to TCRs other than the given TCR, but does not exclude the case of generating reactivity to individual TCRs.

[0009] In certain embodiments, the modified antigenic peptide has a recognition space for binding to the TCR that is a heterogeneous chemical space as compared to the chemical space for binding to the TCR by the unmodified antigenic peptide.

[0010] In certain embodiments, the heterogeneous chemical space is not the chemical space of natural amino acid side chains.

[0011] In certain embodiments, the modified antigenic peptide has a longer half-life of the TCR bond to the TCR as compared to the TCR bond to the TCR by the unmodified antigenic peptide. In certain embodiments, the half-life of the TCR bond is detected using one or more methods selected from the group consisting of biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, fluctuation method, and / or cell function assay related to the TCR bond.

[0012] In certain embodiments, the modified antigenic peptide has an enhanced bond energy of the TCR bond to the TCR as compared to the TCR bond to the TCR by the unmodified antigenic peptide. In certain embodiments, the bond energy of the TCR bond is detected using one or more methods selected from the group consisting of biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, fluctuation method, and / or cell function assay related to the TCR bond.

[0013] In certain embodiments, the modified antigenic peptide forms a ternary complex pMHC-TCR with a major histocompatibility complex (MHC), TCR that is more stable than the ternary complex pMHC-TCR formed by the unmodified antigenic peptide and the MHC, TCR. The stability of the TCR binding to pMHC is determined by the strength of the interactions between them, which includes chemical bonds and non-covalent interactions such as hydrogen bonds, van der Waals forces, and electrostatic interactions. Methods for detecting TCR bond half-life can also be used to detect the stability of the pMHC-TCR ternary complex. In certain embodiments, the stability of the pMHC-TCR is detected using one or more methods selected from the group consisting of biolistic force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method, and / or cell function assays related to TCR bond.

[0014] In certain embodiments, the modified antigenic peptide can bind to a T cell expressing a TCR via an antigen presenting cell (APC), and the avidity between the APC presenting the modified antigenic peptide and the T cell is stronger than the avidity between the APC presenting the unmodified antigenic peptide and the T cell.

[0015] In certain embodiments, the avidity between the APC and the T cell is detected by one or more methods selected from the group consisting of flow cytometry, cell proliferation assay, cell adhesion assay, enzyme-linked immunosorbent assay, single cell imaging technique, single molecule technique, surface plasmon resonance, thermal fluctuation method, molecular dynamics simulation, functional T cell response detection, and / or microfluidic chip technology.

[0016] In certain embodiments, the modified antigenic peptide activates a T cell to a degree that is at least 20% greater, for example, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, at least 100% greater, at least 120% greater, at least 150% greater, at least 180% greater, at least 200% greater, at least 250% greater, or greater than the degree of activation of the T cell by the unmodified antigenic peptide.

[0017] In certain embodiments, the activation of the T cell includes upregulation of the expression of proteins such as CD69, CD25, CD134, CD279, CD223, CD44, CD39, CD38, CD6, CCL3, XCL1, HLA-DR, and downregulation of the expression of CD62L, CCR7, TCF-1, CD127, and the like.

[0018] In certain embodiments, the activation of the T cell comprises T cell proliferation.

[0019] In certain embodiments, the activation of the T cell comprises secretion of a cytokine. In certain embodiments, the cytokine comprises IL-2, Gzmb, IFN-gamma, and TNF alpha.

[0020] In certain embodiments, the modification comprises introducing a chemical group to one or more amino acid residues of the antigenic peptide. For example, one or more amino acid residues located at a recognition site for binding of the antigenic peptide to the TCR.

[0021] In certain embodiments, the modification comprises replacing one or more amino acid residues of the antigenic peptide with a non-natural amino acid residue. For example, one or more amino acid residues located at a recognition site for binding of the antigenic peptide to the TCR.

[0022] In certain embodiments, the modification comprises replacing one or more amino acid residues of the antigenic peptide with an amino acid residue that is opposite in nature in a property selected from the group consisting of: acid-base character, electrical character, polar character, chemical reactivity, and / or hydrophilic-hydrophobic character. For example, one or more amino acid residues located at a recognition site for binding of the antigenic peptide to the TCR.

[0023] In certain embodiments, the modification is to one or more amino acid residues located at a recognition site for binding of the antigenic peptide to the TCR.

[0024] In certain embodiments, the modification is to one or more amino acid residues involved in forming a chemical space of the antigenic peptide and the TCR.

[0025] In certain embodiments, the modification comprises inserting one or more amino acid residues at a recognition site between the antigenic peptide and the TCR prior to the modification.

[0026] In certain embodiments, the one or more amino acid residues are involved in forming a chemical bond (i.e., a TCR bond) between the antigenic peptide and the TCR, the type of bond including but not limited to a covalent bond, an ionic bond (including positive-negative ion interaction, ion-dipole interaction, ion-induced dipole interaction), a halogen bond (halogen-heteroatom interaction), a coordinate bond, a hydrogen bond, a π-π interaction, a π-X interaction (X = C, F, CI, Br, I, S, etc.), a π-cation interaction (π-cation), a π-R interaction (R = alkyl), a hydrophobic interaction.

[0027] In certain embodiments, the modification comprises contacting the antigenic peptide with one or more metal ions, and the metal ions are involved in forming a chemical space of the modified antigenic peptide and the TCR.

[0028] In certain embodiments, the T cells comprise cytotoxic T lymphocytes, Th cells, and / or Treg cells.

[0029] In certain embodiments, the bonded form is verified by methods such as crystal structure.

[0030] In certain embodiments, the modified antigenic peptide comprises, prior to modification, an amino acid sequence as set forth in SEQ ID NO: 1 (SLLMWITQV), SEQ ID NO: 2 (SIIYFEKL), SEQ ID NO: 3 (EIINFEKL), SEQ ID NO: 4 (SLLAWITQV), SEQ ID NO: 5 (SIIFFEKL), SEQ ID NO: 8 (SIICFEK{-N3}L), SEQ ID NO: 13 (KYNKANVFL), SEQ ID NO: 16 (KAVYNFATM), SEQ ID NO: 17 (KAVYNWATM), SEQ ID NO: 20 (EAAGIGILTV), and SEQ ID NO: 21 (SIINFEKL).

[0031] In another aspect, the present application provides a modified antigenic peptide, wherein the amino acid at the fifth position (W) is replaced with a bromo-, fluoro- or iodo-modified tryptophan, e.g., an iodo-modified tryptophan (5-iW), as compared to the amino acid sequence as set forth in SEQ ID NO: 1.

[0032] In another aspect, the present application provides a modified antigenic peptide, wherein the amino acid at the fourth position (Y) is replaced with a sulfonyl fluoride-modified tyrosine (FSY), as compared to the amino acid sequence as set forth in SEQ ID NO: 2.

[0033] In another aspect, the present application provides a modified antigenic peptide, wherein the amino acid at the fourth position (N) is replaced with a sulfonyl fluoride-modified tyrosine (FSY), as compared to the amino acid sequence as set forth in SEQ ID NO: 3.

[0034] In another aspect, the present application provides a modified antigenic peptide, wherein the amino acid at the fifth position (W) is replaced with a bromo-, fluoro- or iodo-modified tryptophan, e.g., an iodo-modified tryptophan (5-iW), as compared to the amino acid sequence as set forth in SEQ ID NO: 4.

[0035] In another aspect, the present application provides a modified antigenic peptide, wherein the amino acid at the fourth position (F) is replaced with a sulfonyl fluoride-modified tyrosine (FSY), as compared to the amino acid sequence as set forth in SEQ ID NO: 5.

[0036] In another aspect, the present application provides a method of modifying an antigenic peptide, the method comprising (1) providing an antigenic peptide, and (2) introducing to the antigenic peptide a chemical structure that is capable of changing the bonding pattern between the antigenic peptide and a TCR.

[0037] In certain embodiments, the modification changes the recognition space for the binding between the antigenic peptide and the TCR to a heterogeneous chemical space, as compared to the chemical space for the binding between the antigenic peptide and the TCR before the modification.

[0038] In certain embodiments, the heterogeneous chemical space is not the chemical space between natural amino acids.

[0039] In certain embodiments, the modification changes the half-life of the TCR bond between the antigenic peptide and the TCR to be longer, as compared to the half-life of the TCR bond between the antigenic peptide and the TCR before the modification. In certain embodiments, the half-life of the TCR bond is detected using one or more methods selected from the group consisting of biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method and other biophysical methods and / or cell function assays related to the TCR bond.

[0040] In certain embodiments, the modification changes the stability of the ternary complex pMHC-TCR formed by the antigenic peptide and the TCR to be stronger, as compared to the stability of the ternary complex pMHC-TCR formed by the antigenic peptide and the TCR before the modification. The stability of the TCR binding to pMHC is determined by the strength of the interaction between them, which includes chemical bonds and non-covalent interactions such as hydrogen bonds, van der Waals forces and electrostatic interactions. The methods used to detect the half-life of the TCR bond can also be used to detect the stability of the pMHC-TCR ternary complex. In certain embodiments, the stability of the pMHC-TCR is detected using one or more methods selected from the group consisting of biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method and other biophysical methods and / or cell function assays related to the TCR bond.

[0041] In certain embodiments, the modified antigenic peptide can bind to a T cell expressing a TCR via an antigen presenting cell (APC) and the modification results in a stronger avidity between the APC presenting the modified antigenic peptide and the T cell as compared to the avidity between the APC presenting the unmodified antigenic peptide and the T cell. In certain embodiments, the avidity between the APC and the T cell is detected by one or more methods selected from the group consisting of flow cytometry, cell proliferation assay, cell adhesion assay, enzyme-linked immunosorbent assay, single cell imaging technique, single molecule technique, surface plasmon resonance, thermal fluctuation method, molecular dynamics simulation, functional T cell response detection, and / or microfluidic chip technique.

[0042] In certain embodiments, the modification results in at least a 20% increase in the degree of activation of the T cell by the antigenic peptide as compared to the degree of activation of the T cell by the unmodified antigenic peptide, for example, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 150% increase, at least a 180% increase, at least a 200% increase, at least a 250% increase, or more.

[0043] In certain embodiments, the activation of the T cell comprises upregulation of expression of markers CD69, CD25, CD134, CD279, CD223, CD44, CD39, CD38, CD6, CCL3, XCL1, HLA-DR, and the like, and downregulation of expression of CD62L, CCR7, TCF-1, CD127, and the like.

[0044] In certain embodiments, the activation of the T cell comprises T cell proliferation.

[0045] In certain embodiments, the activation of the T cell comprises secretion of cytokines. In certain embodiments, the cytokines comprise IL-2, Gzmb, IFN-γ, and TNFα.

[0046] In certain embodiments, the modification comprises introducing a chemical group to one or more amino acid residues of the antigenic peptide. For example, one or more amino acid residues located at a recognition site for binding of the antigenic peptide to the TCR.

[0047] In certain embodiments, the modification comprises replacing one or more amino acid residues of the antigenic peptide with a non-natural amino acid residue. For example, one or more amino acid residues located at a recognition site for binding of the antigenic peptide to the TCR.

[0048] In certain embodiments, the modification comprises replacing one or more amino acid residues of the antigenic peptide with an amino acid residue that is opposite in nature selected from the group consisting of: acid-base, electrical, polarity, chemical reactivity, and / or hydrophilic-hydrophobic. For example, one or more amino acid residues located at the recognition site of the antigenic peptide binding to the TCR.

[0049] In certain embodiments, the modification is made to one or more amino acid residues located at the recognition site of the antigenic peptide binding to the TCR.

[0050] In certain embodiments, the modification is made to one or more amino acid residues involved in forming the chemical space of the antigenic peptide and the TCR.

[0051] In certain embodiments, the modification comprises inserting one or more amino acid residues at the recognition site between the antigenic peptide and the TCR before the modification.

[0052] In certain embodiments, the one or more amino acid residues are involved in forming a chemical bond (i.e., TCR bond) between the antigenic peptide and the TCR, types of bonds include but are not limited to covalent bond, ionic bond (including positive-negative ion interaction, ion-dipole interaction, ion-induced dipole interaction), halogen bond (halogen-heteroatom interaction), coordinate bond, hydrogen bond, π-π interaction, π-X interaction (X = C, F, Cl, Br, I, S, etc.), π-cation interaction (π-cation), π-R interaction (R = alkyl), hydrophobic interaction.

[0053] In certain embodiments, the modification comprises contacting the antigenic peptide with one or more metal ions, and the metal ions are involved in forming the chemical space of the modified antigenic peptide and the TCR.

[0054] In certain embodiments, the T cell comprises cytotoxic T lymphocyte, Th cell, and / or Treg cell.

[0055] In certain embodiments, the bonding form is verified by methods such as crystal structure detection.

[0056] In another aspect, the present application provides a fusion protein comprising the modified antigenic peptide described herein.

[0057] In another aspect, the present application provides an immunoconjugate comprising the modified antigenic peptide described herein and an antigen-binding protein. In certain embodiments, the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0058] In another aspect, the present application provides an isolated nucleic acid molecule comprising the modified antigenic peptide described herein, the fusion protein described herein, and / or the immunoconjugate described herein.

[0059] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.

[0060] In another aspect, the present application provides a nanocompound comprising the modified antigenic peptide, the fusion protein, the immunoconjugate, and / or the nanocarrier in a nanoform.

[0061] In certain embodiments, the nanocarrier comprises any antigen- loadable carrier such as a virus, a polymeric microsphere, and the like.

[0062] In another aspect, the present application provides a pharmaceutical composition comprising the modified antigenic peptide described herein, the fusion protein described herein, the immunoconjugate described herein, the nanocompound described herein, the isolated nucleic acid molecule described herein and / or the vector described herein and optionally a pharmaceutically acceptable carrier.

[0063] In another aspect, the present application provides a kit comprising the modified antigenic peptide described herein, the fusion protein described herein, the immunoconjugate described herein, the isolated nucleic acid molecule described herein, the nanocompound described herein and / or the vector described herein.

[0064] In another aspect, the present application provides a method of activating T cells, the method comprising administering an effective amount of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the nanocompound described herein and / or the pharmaceutical composition described herein. In certain embodiments, the method is an in vitro method. In certain embodiments, the method is an ex vivo method. In certain embodiments, the method is an in vivo method.

[0065] In another aspect, the present application provides a method of causing T cell exhaustion, the method comprising administering an effective amount of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the nanocompound described herein and / or the pharmaceutical composition described herein. In certain embodiments, the method is an in vitro method. In certain embodiments, the method is an ex vivo method. In certain embodiments, the method is an in vivo method.

[0066] In another aspect, the present application provides a method for regulating T cell fate based on TCR signaling pathway, which utilizes the modified peptide to induce specific T cells to directionally expand and maintain stemness and function at a certain dose and frequency after screening, or to drive specific T cells to produce exhausted phenotype and loss of function under high dose (e.g., about 1 nM to about 500 nM, about 100 nM to about 500 nM, about 100 nM to about 1000 nM, high frequency (e.g., 1 time per day, 1 time per 2 days or 1 time per 3 days) stimulation, the method comprising administering an effective amount of the modified antigen peptide described in the present application, the immunoconjugate described in the present application, the fusion protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the nano compound described in the present application and / or the pharmaceutical composition described in the present application. In certain embodiments, the method is an in vivo method.

[0067] In another aspect, the present application provides a method for regulating T cells, the method comprising contacting an effective amount of the modified antigen peptide described in the present application, the immunoconjugate described in the present application, the fusion protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the nano compound described in the present application and / or the pharmaceutical composition described in the present application with T cells. In certain embodiments, the method is an in vitro method. In certain embodiments, the method is an ex vivo method. In certain embodiments, the method is an in vivo method. In certain embodiments, the T cells are TCR-T, CAR-T or tumor infiltrating lymphocyte (TIL) cells. The method enhances the functional response of TCR-T, CAR-T, TILs cells in tumor therapy by specifically activating and expanding TCR-T, CAR-T or TILs cells through the strong interaction between the modified peptide and its corresponding TCR.

[0068] In another aspect, the present application provides a pharmaceutical combination comprising the modified antigen peptide described in the present application, and engineered cells. In certain embodiments, the engineered cells are T cells. In certain embodiments, the engineered cells comprise TCR-T, CAR-T or tumor infiltrating lymphocyte (TIL) cells.

[0069] In another aspect, the present application provides a pharmaceutical combination comprising the modified antigenic peptide described herein, and one or more immune checkpoint inhibitors. In certain embodiments, the immune checkpoint inhibitors comprise a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a TIM-3 inhibitor, and / or a VISTA inhibitor. In certain embodiments, the immune checkpoint inhibitors comprise a PD-1 antibody, a PD-L1 antibody, a CTLA4 antibody, a LAG-3 antibody, a TIGIT antibody, a TIM-3 antibody, and / or a VISTA antibody. In another aspect, the present application provides a method of preventing, treating, and / or ameliorating a disease or a disorder, the method comprising administering to a subject in need thereof an effective amount of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the nanocompound described herein, the pharmaceutical combination described herein, and / or the pharmaceutical composition described herein.

[0070] In another aspect, the present application provides the use of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the nanocompound described herein, the pharmaceutical combination described herein, and / or the pharmaceutical composition described herein in the manufacture of a medicament for preventing, treating, and / or ameliorating a disease or a disorder.

[0071] In another aspect, the present application provides the use of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the nanocompound described herein, the pharmaceutical combination described herein, and / or the pharmaceutical composition described herein in the manufacture of a medicament for preventing, treating, and / or ameliorating a disease or a disorder.

[0072] In certain embodiments, the disease comprises a tumor. For example, the tumor can comprise colon cancer. For example, the tumor can comprise bladder cancer. For example, the tumor can comprise melanoma, lung cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, bladder cancer, esophageal cancer, pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, colorectal cancer, head and neck cancer, prostate cancer, osteosarcoma, lymphoma, leukemia, or multiple myeloma.

[0073] In certain embodiments, the disease comprises an autoimmune disease. For example, the autoimmune disease can be systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), graft versus host disease (GVHD), inflammatory bowel disease (IBD, including Crohn’s disease and ulcerative colitis), type 1 diabetes, psoriasis, or Sjogren’s syndrome.

[0074] Other aspects and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings. The detailed description only shows and describes exemplary embodiments of the present application. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the application as described. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0075] The specific features of the application are shown in the appended claims. The features and advantages of the application can be better understood from the exemplary embodiments described in detail below and from the accompanying drawings. A brief description of the drawings is as follows:

[0076] Figure 1 shows the construction strategy of the modified antigen peptides described in the present application. One is to "add" non-natural chemical groups to the original (native) antigen, i.e. based on chemical or biological reactions, such as chemical coupling reaction shown in strategy 1, biological enzymatic labeling reaction shown in strategy 2 and metal coordination / chelation reaction shown in strategy 3; the other is to completely remodel the structure of the original (native) antigen, such as introducing non-natural amino acids by solid-phase synthesis of polypeptides shown in strategy 4 and constructing antigen proteins or antigen peptides with non-natural amino acids based on the expansion of genetic code shown in strategy 5.

[0077] Figures 2 and 3 show the construction of chemically modified peptides by introducing non-natural amino acids by solid-phase synthesis of polypeptides. The non-natural amino acid codes in the lower brackets, "p-fF", "p-ffF", "5,6-dfW", "X", etc. are used to replace the corresponding positions of the native antigen peptide with non-natural amino acids. After the replacement of the non-natural amino acids at the corresponding positions of the native antigen peptide, the non-natural antigen peptide is named as "non-natural amino acid code + number representing the residue position", for example, "X4" indicates that the 4th amino acid of the native peptide is replaced with FSY (represented by X).

[0078] Figures 4 and 5 show that the activation of T cells by the modified antigen peptides of the present application is enhanced.

[0079] Figure 6 shows the comparison of the activation of T cells by OT-1 antigen peptides X4 and Y4, 1G4 antigen peptides 5-iW5 and W5.

[0080] Figure 7 shows the comparison of the activation of T cells by OT-1 antigen peptides E1X4 and E1N4, 1G4 antigen peptides 4A5-iW5 and 4AW5.

[0081] Figure 8 shows the comparison of the activation of T cells by OT-1 antigen peptides X4 and Y4.

[0082] Figures 9 and 10 show a comparison of the fluorescence intensity of tetramers of OT-1 antigenic peptides X4 and N4, 1G4 antigenic peptides 5-iW5 and W5.

[0083] Figure 11 shows a comparison of the extent of binding of OT-1 antigenic peptides X4, Y4, Q4 and V4 to T cells.

[0084] Figure 12 shows a comparison of the extent of binding of 1G4 antigenic peptides 5-iW5 and W5 to T cells.

[0085] Figure 13 shows that antigenic peptide X4 mediates cytokine release by OT-1 T cells.

[0086] Figure 14 shows that antigenic peptide 5-iW5 mediates cytokine release by 1G4 T cells.

[0087] Figure 15 shows that antigenic peptide X4 mediates killing of cancer cells expressing H2-Kb by OT-1 T cells.

[0088] Figure 16 shows that antigenic peptide 5-iW5 mediates killing of cancer cells expressing HLA-A*0201 by 1G4 T cells.

[0089] Figure 17 shows that antigenic peptide X4 stimulates expansion of T cells.

[0090] Figures 18 and 19 show that antigenic peptide 5-iW5 stimulates expansion of T cells.

[0091] Figure 20 shows that antigenic peptide X4 stimulates expansion of specific T cells in vivo in mice.

[0092] Figure 21 shows that antigenic peptide 5-iW5 mediates specific expansion and activation of TCR-Ts.

[0093] Figure 22 shows that antigenic peptide X4 induces T cell exhaustion in vitro.

[0094] Figure 23 shows that antigenic peptide 5-iW5 induces T cell exhaustion in vitro.

[0095] Figure 24 shows that antigenic peptide X4 induces T cell exhaustion in vivo.

[0096] Figure 25 shows that antigenic peptide X4 induces more exhausted T cells than antigenic peptide N4.

[0097] Figure 26 shows the structure of OT-1 antigenic peptides Y4 and F4 before modification.

[0098] Figure 27 shows the structure of OT-1 antigenic peptides after modification, with exemplary modification types of tyrosine, as modified antigenic peptide X4 when R is -OSO2F.

[0099] Figure 28 shows exemplary modifications based on chemical coupling reactions on the OT-1 antigenic peptide C4 (SIICFEK{-N3}L, SEQ ID NO: 8).

[0100] Figure 29 shows the 1G4 antigenic peptide W5 before modification.

[0101] Figure 30 shows the structure of the 1G4 antigenic peptide after modification, where exemplary modification types of amino acids are listed, and when R is iodine, it is the diluted antigenic peptide 5-iW5.

[0102] Figure 31 shows the structure of the NY8.3 antigenic peptide before modification, with the sequence KYNKANVFL

[0017] (SEQ ID NO: 13).

[0103] Figure 32 shows the structure of the NY8.3 antigenic peptide after modification, where exemplary modification types of phenylalanine are listed, and when R is bromine or iodine (position 2 of the benzene ring), it is the modified antigenic peptide F8-2Br or F8-2I.

[0104] Figure 33 shows the structure of the P14 antigenic peptide before modification, with the sequence KAVYNFATM

[0018] (SEQ ID NO: 16) and KAVYNWATM (SEQ ID NO: 17).

[0105] Figure 34 shows the structure of the P14 antigenic peptide after modification, where exemplary modification types of phenylalanine and tryptophan are listed, and when R is methoxy (position 7 of the indole ring), it is the modified antigenic peptide W6(7-OMe).

[0106] Figure 35 shows the structure of the Mel8 antigenic peptide before modification, with the sequence EAAGIGILTV

[0019] (SEQ ID NO: 20).

[0107] Figure 36 shows the structure of the Mel8 antigenic peptide after modification, where exemplary modification types of leucine are listed, and when R is cyclopentyl, it is the modified antigenic peptide L8-cycloPEN.

[0108] Figure 37 shows the comparison of the degree of activation of T cells by the P14 antigenic peptides W6 and W6(7-OMe).

[0109] Figure 38 shows the comparison of the degree of activation of T cells by the NY8.3 antigenic peptides F8 and F8-2I, F8-2Br.

[0110] Figure 39 shows the comparison of the degree of activation of T cells by the Mel 8 antigenic peptides L8 and L8-cycloPEN.

[0111] Figure 40 shows the bonding mode between TCR and unnatural strong antigen predicted by the method of AI prediction + molecular docking.

[0112] Figure 41 shows the enhanced effect of unnatural strong antigen on T cell killing tumor in vivo evaluated in tumor-bearing B16-OVA mice.

[0113] Figure 42 shows the reversal of large tumor (V>1000 mm 3 ) growth trend by the method of unnatural antigen in the scenario of ICB therapy.

[0114] Figure 43 shows the ability of unnatural strong antigen OT-1 TCR to enhance P14 T cell killing of tumor expressing antigen (GP33) in vivo. DETAILED DESCRIPTION

[0115] The following examples illustrate the embodiments of the present application by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the specification.

[0116] Terminology

[0117] In the present application, the term "bonding mode" generally refers to the interaction mode (types of bonds including but not limited to covalent bond, ionic bond (including positive-negative ion interaction, ion-dipole interaction, ion-induced dipole interaction), halogen bond (halogen-heteroatom interaction), coordination bond, hydrogen bond, π-π interaction, π-X interaction (X C, F, Cl, Br, I, S, etc.), π-cation interaction (π-cation), π-R interaction (R = alkyl), hydrophobic interaction) between a given T cell receptor (TCR) and antigen peptide. When referring to the change of "bonding mode", it generally refers to the change of the interaction mode between TCR and antigen peptide. The change of interaction mode can include but is not limited to changing from no one or more interactions to one or more interactions (e.g., no covalent interaction to covalent interaction or any of the bond types mentioned above), one or more interactions being enhanced (e.g., weak π-π stacking becomes stronger or any of the bond types mentioned above is enhanced).

[0118] In this application, the term "TCR bond" or "TCR bond" refers to the binding between the T-cell receptor (TCR) and the peptide-major histocompatibility complex (pMHC). This binding is a key step in T cell activation, differentiation, proliferation, and function. The interaction of TCR with pMHC can significantly increase the sensitivity of antigens and amplify the ability of antigen recognition by the application of mechanical force. In this interaction, there are two types of binding modes: catch-slip bonds and slip-only bonds. Among them, in the interaction with strong pMHC, TCR can form catch-slip bonds with pMHC, and the binding lifetime of such bonds will increase to a peak under the action of a certain force, and then decrease with the further increase of force; in the interaction with weak agonist or antagonist pMHC, TCR forms slip-only bonds with pMHC, and the binding lifetime of such bonds decreases with the increase of force. The distinction of these binding modes is crucial for understanding how T cells regulate their signaling and antigen recognition according to the binding strength. For more information on TCR bonds, please refer to Choi, HK., Cong, P., Ge, C. et al. Catch bond models may explain how force amplifies TCR signaling and antigen discrimination. Nat Commun 14, 2616 (2023).

[0119] Detecting TCR bonds, i.e. the interaction between T cell receptors and their ligands pMHC (peptide-major histocompatibility complex), usually uses the following techniques, including but not limited to:

[0120] 1. Biomembrane Force Probe (BFP): This is a single-molecule force spectroscopy technique that can be used to measure the binding strength and dynamics between cell surface molecules. BFP works by fixing a cell on a microbead and then applying force with another microbead to simulate intercellular interactions.

[0121] 2. Optical Tweezers (OT): Optical tweezers use the radiation pressure generated by a focused laser beam to manipulate small objects such as microbeads. By connecting microbeads to T cells and pMHC molecules, the interaction force between them can be measured.

[0122] 3. Flow Cytometry: While flow cytometry cannot directly measure force, it can be used to measure the frequency and strength of T cell binding to pMHC molecules by detecting labeled T cell receptors or pMHC molecules.

[0123] 4. Surface Plasmon Resonance (SPR): SPR is a technique for real-time monitoring of biomolecular interactions that can be used to analyze the binding kinetics between T cell receptors and their ligands.

[0124] 5. Fluorescence Resonance Energy Transfer (FRET): FRET can be used to measure the change in molecular distance when T cell receptors bind to their ligands, providing dynamic information about the binding event.

[0125] 6. Molecular Dynamics (MD): Through computer simulations, the interactions and structural changes of T cell receptors and pMHC molecules at the atomic level can be observed.

[0126] 7. Thermal Fluctuation Assay: This method uses the thermal fluctuations of microbeads to measure the binding lifetime of T cell receptors and pMHC molecules under zero force.

[0127] 8. Cell Proliferation Assay: By measuring the proliferation of T cells after encountering specific pMHC ligands, the activity of TCR bonds can be indirectly assessed.

[0128] Mathematical models can also be combined to analyze and interpret experimental data. Through these methods, those skilled in the art can quantitatively describe the behavior of TCR bonds and explore their role in T cell activation.

[0129] In the present application, the term "half-life" of TCR bond can also be referred to as the lifetime of TCR bond, or bond lifetime, which refers to the time for half dissociation of T cell receptor (TCR) after binding to its ligand peptide-major histocompatibility complex (pMHC). The half-life of TCR bond is closely related to the activation of T cells, signal transduction and the initiation of immune response. Longer bond lifetime can allow more signal transduction events, thereby enhancing the response of T cells to antigen. The half-life of TCR bond can be measured by single-molecule techniques such as biological force probe (BFP) or optical tweezers (OT). These techniques can monitor the binding and dissociation process between TCR and pMHC in real time at the molecular level. By analyzing the experimental data of TCR binding to pMHC, the statistical distribution of half-life or bond lifetime can be obtained, and further study the relationship between T cell function.

[0130] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linked by a linker molecule) an active agent (e.g., a chemotherapeutic agent, a radioactive element, a cell growth inhibitor, and a cytotoxic agent) to an antigen-binding protein, which can specifically bind to an antigen on a target cell (e.g., a tumor cell) through an antibody or an antigen-binding fragment thereof, thereby delivering the other agent to the target cell. In the present application, the modified antigenic peptide of the present application can be fused to the antigen-binding protein, or conjugated to the active agent (e.g., covalently linked by a linker molecule).

[0131] In the present application, the term "avidity" between APC and T cell is generally determined by the affinity of the interaction between TCR and pMHC molecules. The strength of the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) molecules. The following are some exemplary methods for detecting the avidity between APC and T cell:

[0132] 1. Flow Cytometry: Flow cytometry can be used to measure the avidity of T cell binding to pMHC molecules expressed on the surface of APC, and the binding of TCR or pMHC is detected by fluorescently labeled antibodies.

[0133] 2. Cell Proliferation Assays: By measuring the proliferation of T cells after co-culturing with APC, the avidity between T cells and APC can be indirectly reflected. The higher the degree of proliferation, the stronger the avidity may be.

[0134] 3. Cell Adhesion Assays: Under in vitro conditions, the avidity between T cells and APCs can be assessed by observing their adhesion. The longer the adhesion time, the higher the potential avidity.

[0135] 4. Enzyme-Linked Immunosorbent Assay (ELISA): Using ELISA technology, the amount of T cell binding to pMHC molecules on the surface of APCs can be detected, indirectly assessing avidity.

[0136] 5. Single-Cell Imaging Techniques: Using imaging techniques such as confocal microscopy, the interaction between T cells and APCs can be observed, and the formation and stability of contact areas can be analyzed.

[0137] 6. Single-Molecule Techniques: Techniques such as atomic force microscopy (AFM) and optical tweezers (OT) can be used to measure the binding strength between individual TCRs and pMHC molecules.

[0138] 7. Surface Plasmon Resonance (SPR): SPR technology can monitor the kinetic parameters of T cell binding to pMHC molecules on the surface of APCs in real time, including binding affinity and dissociation rate.

[0139] 8. Thermal Fluctuation Assay: Using thermal fluctuations of microbeads, the stability of T cell binding to APCs can be measured, assessing avidity.

[0140] 9. Molecular Dynamics Simulations: Through computational simulations, the stability and affinity of the interaction between T cells and APCs can be predicted at the molecular level.

[0141] 10. Functional T Cell Response Detection: By detecting activation markers and cytokine secretion in T cells after binding to APCs, functional avidity between T cells and APCs can be assessed.

[0142] 11. Microfluidic Chip Technology: Using microfluidic chips, the dynamic interaction and affinity between T cells and APCs can be studied by simulating in vivo environments.

[0143] The above methods can be used individually or in combination to obtain comprehensive information about the avidity between T cells and APCs.

[0144] In the present application, the term "recognition site" between an antigenic peptide and a TCR (T cell receptor) generally refers to a specific region of the antigenic peptide molecule and the TCR molecule that interact with each other, such interaction being through non-covalent bonds or hydrogen bonds, etc. The space where the recognition site is located is called "recognition space", or the recognition site constitutes "recognition space". The "recognition space" is a "chemical space". The recognition site between an antigenic peptide and a TCR can be confirmed by crystallographic methods, biochemical methods, and / or functional experiments, for example, X-ray crystallography, nuclear magnetic resonance (NMR), point mutation analysis, mutation scanning, biosensor technology (e.g. SPR), or cell function experiments.

[0145] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable substance, composition or vehicle involved in carrying or transporting a chemical agent. Such a substance can routinely contain a salt, a buffer, a preservative, a compatible carrier, and optionally other therapeutic agents. Such a pharmaceutically acceptable preparation can also contain a compatible solid or liquid filler, diluent or encapsulating substance suitable for administration to a human.

[0146] In the present application, the term "heterogeneous chemical space" generally refers to a chemical space formed by the side chain structure of a non-natural amino acid other than the 20 natural amino acids. Among them, the 20 natural amino acids refer to Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartate, Histidine, Asparagine, Glutamate, Lysine, Glutamine, Methionine, Arginine, Serine, Threonine, Cysteine and Proline. If the chemical space between the 20 natural amino acids is changed by any means to become a heterogeneous chemical space.

[0147] In the present application, the term "tumor" generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0148] In the present application, the term "autoimmune disease" generally refers to a disorder or condition resulting from an autoimmune response against self-antigens. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens.

[0149] In the present application, the term "pMHC-TCR" generally refers to a ternary complex formed by a T cell receptor (TCR) and a peptide-major histocompatibility complex (pMHC) complex.

[0150] In the present application, the term "activation of T cells" generally refers to the process by which T cells transition from a resting state (G0 phase) to an active state, which involves the recognition of antigens by T cell receptors (TCRs) and the initiation of a series of downstream signaling events. Activated T cells acquire various effector functions, including proliferation, differentiation, cytokine production, and direct killing of infected or abnormal cells. T cell activation can be observed at the molecular, cellular, and tissue levels. Some common indicators and manifestations of T cell activation include, but are not limited to, upregulation of surface markers (e.g., CD69 and CD25 (IL-2 receptor alpha chain)), cytokine production (e.g., interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2)), cell proliferation, changes in cell morphology (e.g., increased cell volume, formation of pseudopods, and reorganization of the cytoskeleton), enhanced metabolic activity, enhanced cell migration ability, formation of immunological synapses, changes in gene expression (including upregulation of effector molecules and cytokines), enhanced cytotoxic T cell killing ability, formation of memory T cells, regulatory T cell function, resistance to apoptosis. T cell activation can be detected by various experimental techniques, including flow cytometry, enzyme-linked immunosorbent assay (ELISA), cell proliferation assays, gene expression analysis, etc. Through these detections, one skilled in the art can assess the activation status of T cells and their roles in immune responses.

[0151] In the present application, the term "T cell exhaustion" generally refers to a state of T cell functional decline in which T cells can gradually lose their original effector functions due to persistent exposure to high levels of antigens and / or inflammatory environments.

[0152] DETAILED DESCRIPTION

[0153] Modified antigenic peptides

[0154] In one aspect, the application provides a modified antigenic peptide that has an altered binding mode with a TCR as compared to the binding mode of the unmodified antigenic peptide with the TCR. A TCR can specifically recognize and / or bind to an antigenic peptide having a particular amino acid sequence, but not to an antigenic peptide that does not have the particular amino acid sequence. Thus, for a given TCR, the antigenic peptide epitope that it can specifically recognize and / or bind to is essentially determined. The modification described herein is made to the antigenic peptide having the epitope. In other embodiments, the unmodified antigenic peptide can not be specifically recognized and / or bound by the TCR, but after the modification described herein, the binding mode of the modified antigenic peptide with the TCR is altered such that the modified antigenic peptide can be specifically recognized and / or bound by the TCR.

[0155] In the present application, the modified antigenic peptide has an altered interaction mode with a TCR as compared to the interaction mode of the unmodified antigenic peptide with the TCR.

[0156] For example, for a given TCR, there is no covalent interaction between the unmodified antigenic peptide and the TCR, but a covalent interaction is generated between the modified antigenic peptide and the TCR.

[0157] For example, for a given TCR, the interaction between the modified antigenic peptide and the TCR is enhanced (e.g., becomes stronger from weaker π-π stacking) as compared to the interaction between the unmodified antigenic peptide and the TCR. For example, the enhancement is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250%, or more.

[0158] For example, for a given TCR, the type of interaction between the modified antigenic peptide and the TCR is changed from a weak type of interaction to a strong type of interaction (e.g., from weak hydrogen bonding or hydrophobic interaction to strong ionic bonding (charge-charge interaction) or π-X interaction (X = F, CI, Br, I, S, etc. heteroatom)) as compared to the interaction between the unmodified antigenic peptide and the TCR.

[0159] In the present application, the recognition space of the binding between the modified antigenic peptide and the TCR is a heterogeneous chemical space compared to the unmodified antigenic peptide. The heterogeneous chemical space is a concept relative to the chemical space of 20 natural amino acid side chain structures. In one specific example, the heterogeneous chemical space includes the filling of the chemical space, for example, with a chemical group to a single atom filling of the chemical space.

[0160] In the present application, the change in the binding form between the antigenic peptide and a given TCR before and after modification can bring about the heterogeneity of the chemical space. The change in the binding form can be embodied in, for example, the half-life of the TCR bond between the antigenic peptide and the TCR becoming longer. For example, the half-life becomes longer by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more.

[0161] The half-life of the TCR bond between the antigenic peptide and the TCR can be detected by various methods, including but not limited to physical and chemical methods, biological methods such as biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method and / or cell function experiments related to the TCR bond.

[0162] In an immune response, the antigenic peptide usually needs to be presented by an antigen presenting cell, binds to the TCR on the surface of a T cell to form a pMHC-TCR ternary complex. The change in the binding form can also be embodied in the stability of the pMHC-TCR formed by the antigenic peptide before and after modification becoming stronger. For example, the stability becomes stronger by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more.

[0163] The stability of the pMHC-TCR can be detected by various methods, including but not limited to physical and chemical methods, biological methods such as biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method and / or cell function experiments related to the TCR bond.

[0164] In the present application, the change in the binding form can also be reflected in that the avidity between the antigen peptide before and after modification can be stronger. For example, the avidity is stronger by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more.

[0165] The avidity between the antigen peptide and the T cell can be detected by various methods, including but not limited to flow cytometry, cell proliferation experiment, cell adhesion experiment, enzyme-linked immunosorbent assay, single cell imaging technology, single molecule technology, surface plasmon resonance, thermal fluctuation method, molecular dynamics simulation, functional T cell response detection and / or microfluidic chip technology.

[0166] In a specific example, the kinetic parameters of the binding between the antigen peptide and the TCR are detected using techniques such as SPR, for example, K d and K off , so as to compare the binding affinity between the antigen peptide before and after modification and the TCR.

[0167] For another example, the avidity can be characterized using the strategy of pMHC tetramer staining. For another example, the avidity can be characterized by destroying the binding force between the antigen peptide and the T cell by ultrasonic.

[0168] In the present application, the modified antigen peptide can enhance the activation of T cells by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more compared with before modification.

[0169] In the present application, the modified antigen peptide enhances the level of expression of surface protein markers (for example, CD25 and / or CD69) of T cells compared with before modification. For example, the enhancement is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more.

[0170] In a specific example, the dendritic cells are used to present the antigen peptide, and are co-incubated with CD8 T cells, and then the expression amount of CD69 on the surface of the T cells is detected, so as to compare the activation of T cells by the antigen peptide before and after modification.

[0171] In another specific example, the antigen peptide described herein is presented by a cell line expressing HLA-A*0201, T2 cells, and co-incubated with CD8 Jurkat cells harboring the corresponding TCR, and then the expression level of CD69 on the surface of Jurkat cells is detected, so as to compare the activation degree of T cells by the antigen peptide before and after modification.

[0172] In the present application, the modified antigen peptide can increase the proliferation ability of T cells compared with before modification. For example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more. For example, the proliferation of T cells can be detected by flow cytometry (e.g., CFSE staining).

[0173] In the present application, the modified antigen peptide can make T cells secrete more cytokines (e.g., Gzmb, IL-2, IFN-γ and TNFα) compared with before modification. For example, the secretion amount of cytokines is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 250% or more.

[0174] In one specific example, the antigen peptide described herein is loaded into tumor cells, co-incubated with CD8 T cells, and the cytokines released by T cells are detected, so as to compare the degree of activation of T cells secreting cytokines by the antigen peptide before and after modification.

[0175] In another specific example, the antigen peptide described herein is presented by tumor cells, co-incubated with CD8 T cells, and the killing effect of T cells on cancer cells (e.g., quantified by luciferase luminescence) is detected, so as to compare the degree of activation of T cells killing target cells by the antigen peptide before and after modification.

[0176] In one specific example, the degree of activation of T cells can be reflected by detecting the expression level of activation-related genes (e.g., IL2, CD160, IFNG, XCL2, XCL1, NR4A3, CSF2, CCL4, NR4A2, CCL3, GZMB, MYC, KDM6B, TNFSF14, REL, SLAMF1, IL2RA, CD69, CD44, TNF, IL21R, CD109, CD55, TNFRSF9, CD200, ICAM1, IRF4, CD82, BATF, TRAF3, etc.).

[0177] In the present application, the modified antigenic peptide can be immunologically heterogeneous compared to the unmodified one. That is, for a given TCR, the activation of the antigenic peptide before and after modification is enhanced, but for other TCRs that cannot specifically recognize the antigenic peptide before modification, the recognition and / or binding ability of the modified antigenic peptide can be substantially unchanged.

[0178] Methods of modifying antigenic peptides

[0179] In another aspect, the present application provides a method for obtaining the modified antigenic peptide.

[0180] In another aspect, the present application provides a method for modifying an antigenic peptide, comprising (1) providing an antigenic peptide, and (2) introducing a modification to the antigenic peptide that can change the bonding form between the antigenic peptide and a TCR.

[0181] The modified antigenic peptide described in the present application can be prepared in various ways as long as it can introduce a non-natural chemical bond.

[0182] In the present application, the basic strategy for modifying an antigenic peptide is to change the bonding form between the antigenic peptide and a TCR. Therefore, a feasible modification strategy can be to modify the recognition site of the antigenic peptide and the TCR.

[0183] One or more amino acid residues in an antigenic peptide interact with a T cell receptor (TCR) when the antigenic peptide is presented on a major histocompatibility complex (MHC) molecule. Identifying the recognition site of the antigenic peptide and the TCR is a known technique in the art. Such techniques can include substituting each amino acid in the peptide sequence one by one, for example, using alanine scanning mutagenesis or constructing a library of peptide analogs, and detecting the recognition or activation ability of each substituted peptide on T cells. By comparison, it can be determined that when a certain residue is substituted, the recognition of T cells is significantly reduced, and the residue is a TCR contact residue. Alternatively, structural analysis techniques such as X-ray crystallography or cryo-electron microscopy can be used to obtain atomic level structural information of the TCR-peptide-major histocompatibility complex (MHC) ternary complex, thereby directly identifying the peptide residues in contact with the TCR. In addition, biophysical methods such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), etc. can be used to determine the binding affinity of different peptide variants to the TCR, so as to identify the important residues for the interaction with the TCR.

[0184] In certain embodiments, a chemical moiety can be introduced to one or more amino acid residues of the recognition site at which the antigenic peptide binds to the TCR. For example, the chemical moiety that can be introduced to the amino acid residues can include, but is not limited to, the following: a phosphate group, an acetyl group, a methyl group, a methoxy group (-OCH3), a sugar group, a hydroxyl group, a carboxamide group, a disulfide bond, a nitro group (-NO2), a halogen (including one or more of fluorine, chlorine, bromine, and iodine), and -OSO2F.

[0185] In certain embodiments, a chemical moiety can be introduced to one or more phenylalanine, tyrosine, tryptophan, or leucine residues of the recognition site at which the antigenic peptide binds to the TCR.

[0186] For example, the benzene ring of a phenylalanine or tyrosine of the antigenic peptide prior to modification can be substituted with a substituent selected from the group consisting of: a phosphate group, an acetyl group, a methyl group, a methoxy group (-OCH3), a sugar group, a hydroxyl group, a carboxamide group, a disulfide bond, a nitro group (-NO2), a halogen (including one or more of fluorine, chlorine, bromine, and iodine), and -OSO2F.

[0187] For example, a phenylalanine or tyrosine of the TCR recognition site of the antigenic peptide prior to modification can be replaced with an amino acid selected from the group consisting of:

[0188] For example, a phenylalanine or tyrosine of the TCR recognition site of the antigenic peptide prior to modification can be replaced with an amino acid selected from the group consisting of:

[0189] For example, a tryptophan of the TCR recognition site of the antigenic peptide prior to modification can be replaced with an amino acid selected from the group consisting of:

[0190] For example, a tryptophan of the TCR recognition site of the antigenic peptide prior to modification can be replaced with an amino acid selected from the group consisting of:

[0191] For example, a leucine of the TCR recognition site of the antigenic peptide prior to modification can be replaced with an amino acid selected from the group consisting of:

[0192] In certain embodiments, one or more amino acid residues of the recognition site at which the antigenic peptide binds to the TCR can be replaced with a non-natural amino acid residue. For example, the chemical moiety that can be replaced of the amino acid residues can include, but is not limited to, the following: a phosphate group, an acetyl group, a methyl group, a methoxy group (-OCH3), a sugar group, a hydroxyl group, a carboxamide group, a disulfide bond, a nitro group (-NO2), a halogen (including one or more of fluorine, chlorine, bromine, and iodine), and -OSO2F.

[0193] In certain embodiments, one or more amino acid residues of the recognition site of the antigenic peptide bound to the TCR can be replaced with an amino acid residue of opposite property, which can be selected from the group consisting of acid-base property, electric property, polarity, and / or hydrophilic-hydrophobic property. For example, an acidic amino acid residue can be replaced with a basic or neutral amino acid residue, or vice versa. For example, a polar amino acid residue can be replaced with a non-polar amino acid residue, or vice versa. For example, a negatively charged amino acid residue can be replaced with a positively charged amino acid residue, or vice versa. For example, a hydrophilic amino acid residue can be replaced with a hydrophobic amino acid residue, or vice versa.

[0194] In the present application, the modification of the binding mode of the antigenic peptide to the TCR can also include insertion of one or more amino acid residues into the recognition site of the antigenic peptide to the TCR, or deletion of one or more amino acid residues from the recognition site of the antigenic peptide to the TCR. The inserted amino acid residues can be natural amino acid residues, or non-natural amino acid residues with chemical group modifications.

[0195] In the present application, the modification of the binding mode of the antigenic peptide to the TCR can also include insertion of one or more substances other than amino acid residues, such as metal ions or other atoms or ions, into the recognition site of the antigenic peptide to the TCR.

[0196] In certain embodiments, the modification includes contacting the antigenic peptide with one or more metal ions, and the metal ions participate in forming the chemical space of the modified antigenic peptide to the TCR.

[0197] In one specific example, the modification can include replacement of a neutral amino acid (e.g., asparagine) of the recognition site of the antigenic peptide with an aromatic amino acid (e.g., phenylalanine or tyrosine). In another specific example, the modification can include introduction of a non-natural chemical group, such as fluorine, chlorine, bromine, iodine, methyl, -OMe, -NO2, -OSO2F, to an amino acid of the recognition site of the antigenic peptide. For example, one or more (e.g., 2, 3, 4, or 5) substituents of fluorine, chlorine, bromine, iodine, methyl, -OMe, -NO2, and -OSO2F are substituted on the benzene ring of the amino acid.

[0198] In one embodiment, the application employs a construction strategy based on solid-phase synthesis of unnatural amino acids insertion. In addition, the application can include, but is not limited to, the following specific methods: unnatural amino acid insertion, chemical labeling reaction, enzymatic catalytic reaction and / or metal coordination. In some embodiments, unnatural chemical groups can be added to the original antigenic peptide to obtain the modified antigenic peptide described in the application, i.e., a method based on chemical or biological reaction. The method based on chemical or biological reaction includes, but is not limited to: chemical coupling reaction shown in strategy A, biological enzymatic labeling reaction shown in strategy B and chelation reaction of metal coordination peptide shown in strategy C (Figure 1). In other embodiments, the modified antigenic peptide described in the application can be obtained by a method that completely remodels the structure of the original antigenic peptide. Such examples can include, but are not limited to: the method of polypeptide solid-phase synthesis of unnatural amino acids shown in strategy D and the strategy of constructing new peptide structure by unnatural amino acid insertion shown in strategy E (Figure 1).

[0199] As an example, the antigenic epitope peptides of 1G4, OT-1, NY8.3, P14 and Mel8 were modified according to the application. Among them, the amino acid sequences of the alpha chain and beta chain of 1G4 TCR are shown in SEQ ID NO: 10 and 9, respectively, the amino acid sequences of the alpha chain and beta chain of OT-1 TCR are shown in SEQ ID NO: 7 and 6, respectively, the amino acid sequences of the alpha chain and beta chain of NY8.3 TCR are shown in SEQ ID NO: 12 and 11, respectively, the amino acid sequences of the alpha chain and beta chain of 1G4 TCR are shown in SEQ ID NO: 10 and 9, respectively, the amino acid sequences of the alpha chain and beta chain of P14 TCR are shown in SEQ ID NO: 17 and 16, respectively, and the amino acid sequences of the alpha chain and beta chain of Mel8 TCR are shown in SEQ ID NO: 19 and 18, respectively.

[0200] For example, the antigenic peptide before modification can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, 8, 16, 17, 20 and 21.

[0201] Pharmaceutical composition

[0202] In another aspect, the application provides a fusion protein comprising the modified antigenic peptide described in the application.

[0203] In another aspect, the application provides an immunoconjugate comprising the modified antigenic peptide described in the application and an antigen binding protein. In certain embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0204] In another aspect, the present application provides an isolated nucleic acid molecule comprising the modified antigenic peptide described herein, the fusion protein described herein, and / or the immunoconjugate described herein.

[0205] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.

[0206] In another aspect, the present application provides a pharmaceutical composition comprising the modified antigenic peptide described herein, the fusion protein described herein, the immunoconjugate described herein, the isolated nucleic acid molecule described herein and / or the vector described herein and optionally a pharmaceutically acceptable carrier.

[0207] In another aspect, the present application provides a kit comprising the modified antigenic peptide described herein, the fusion protein described herein, the immunoconjugate described herein, the isolated nucleic acid molecule described herein and / or the vector described herein.

[0208] Therapeutic methods

[0209] In another aspect, the present application provides a method of activating T cells, comprising administering an effective amount of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein and / or the pharmaceutical composition described herein. In certain embodiments, the method is an in vitro method. In certain embodiments, the method is an ex vivo method. In certain embodiments, the method is an in vivo method.

[0210] In another aspect, the present application provides a method of causing T cell exhaustion, comprising administering an effective amount of the modified antigenic peptide described herein, the immunoconjugate described herein, the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein and / or the pharmaceutical composition described herein. In certain embodiments, the method is an in vitro method. In certain embodiments, the method is an ex vivo method. In certain embodiments, the method is an in vivo method.

[0211] In another aspect, the present application provides a method for regulating T cell fate based on TCR signaling pathway, which utilizes the modified peptide to induce specific T cells to directionally expand and maintain stemness and function at a certain dose and frequency after screening, or to drive specific T cells to produce exhausted phenotype and lose function under high dose and high frequency stimulation, the method comprising administering an effective amount of the modified antigenic peptide described in the present application, the immunoconjugate described in the present application, the fusion protein described in the present application, the nano compound described in the present application, the pharmaceutical combination described in the present application and / or the pharmaceutical composition described in the present application. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vivo method.

[0212] In another aspect, the present application provides a method for regulating T cells based on “TCR-non-natural peptide” molecular pairs, which utilizes the strong interaction between the modified peptide and its corresponding TCR to specifically activate and expand TCR-T, CAR-T or TILs cells, thereby enhancing the functional response of TCR-T, CAR-T, TILs cells in tumor treatment, the method comprising administering an effective amount of the modified antigenic peptide described in the present application, the immunoconjugate described in the present application, the fusion protein described in the present application, the nano compound described in the present application, the pharmaceutical combination described in the present application and / or the pharmaceutical composition described in the present application. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vivo method.

[0213] In another aspect, the present application provides a method for preventing, treating and / or alleviating a disease or a disorder, the method comprising administering to a subject in need thereof an effective amount of the modified antigenic peptide described in the present application, the immunoconjugate described in the present application, the fusion protein described in the present application, the nano compound described in the present application, the pharmaceutical combination described in the present application and / or the pharmaceutical composition described in the present application.

[0214] In another aspect, the present application provides the use of the modified antigenic peptide described in the present application, the immunoconjugate described in the present application, the fusion protein described in the present application, the nano compound described in the present application, the pharmaceutical combination described in the present application and / or the pharmaceutical composition described in the present application in the preparation of a medicament for preventing, treating and / or alleviating a disease or a disorder.

[0215] In some embodiments, the disease comprises a tumor. For example, the tumor can comprise colon cancer. For example, the tumor can comprise bladder cancer.

[0216] In some embodiments, the disease comprises an autoimmune disease.

[0217] In the methods of the present application, the modified antigenic peptides described herein can be co-administered with one or more anti-cancer therapies. For example, the anti-cancer therapies can include, but are not limited to, immune checkpoint inhibitors (such as antibodies against PD-1, PD-L1, or CTLA-4) to relieve the immune suppression of T cells by the tumor microenvironment; cellular therapies (such as CAR-T cells, TCR-T cells, or TIL cells) to provide additional specific immune effector cells; radiotherapy or chemotherapy to release tumor antigens, alter the tumor microenvironment, and enhance the immunogenicity of the antigenic peptides; and tumor vaccines, oncolytic viruses, immune stimulants (such as TLR agonists, cytokines), and other immunotherapeutic means. In the above-mentioned regimens, the modified antigenic peptides can be administered simultaneously, sequentially, or at specific time intervals with the one or more anti-cancer therapies to achieve synergistic or complementary therapeutic effects, to enhance the tumor-specific immune response, to reduce the tumor burden, and to improve the prognosis of the patient. In certain embodiments, the modified antigenic peptides can be administered via the same route of administration as the one or more anti-cancer therapies. In certain embodiments, the modified antigenic peptides can be administered via a different route of administration than the one or more anti-cancer therapies. In one embodiment of the present application, the modified antigenic peptides are administered to the patient by injection to ensure their effective delivery to the body and to induce a specific immune response against the tumor antigen. The injection can include, but is not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intradermal injection, or other suitable injection routes. The specific route of administration can be determined based on the physicochemical properties, immunogenicity, patient condition, and tolerability of the modified antigenic peptides. In certain embodiments, the modified antigenic peptides described herein, the immunoconjugates described herein, the fusion proteins described herein, the nanocomposites described herein, the pharmaceutical combinations described herein, and / or the pharmaceutical compositions described herein can also be used in conjunction with suitable adjuvants, slow-release carriers, or delivery systems for administration by injection to enhance their immunogenicity and prolong their duration of action.

[0218] For example, the modified antigenic peptides described herein, the immunoconjugates described herein, the fusion proteins described herein, the nanocomposites described herein, the pharmaceutical combinations described herein, and / or the pharmaceutical compositions described herein can be incorporated into a water-in-oil emulsion, a liposome, a nanoparticle, a polymeric microsphere, or other delivery system for slow release by intradermal or subcutaneous injection, thereby continuously stimulating the immune system and increasing the intensity and duration of the immune response. Thus, by selecting the appropriate injection route and formulation for administration of the modified antigenic peptides, it is possible to effectively stimulate a specific immune response while reducing systemic toxicity and local adverse reactions, improving patient tolerance, and increasing treatment compliance.

[0219] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the antigenic peptides, methods of preparation and uses of the present application and are not intended to limit the scope of the present application.

[0220] Examples

[0221] Methods

[0222] The methods employed by the examples of the present application are as follows:

[0223] 1. Cell line culture

[0224] Cell lines MC38, T2-A2, Jurkat, UM-UC-3 (unmodified or expressing GFP / LUC) were cultured in RPMI1640 (Gibco) with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (Gibco). Jurkat cells were lentivirally transduced to overexpress OT-1 TCR cells or 1G4 TCR cells. Cell lines K562 cells and primary cells mouse DC cells, mouse OT-1 CD8 T cells, human CD8 T cells, etc. were cultured in RPMI1640 (Gibco) with 10% fetal bovine serum (FBS, Gibco), 1% antibiotics (Gibco), 1% HEPES (Gibco), 1% NEAA (Gibco), 1% sodium pyruvate (Gibco) and 0.5% β-Me (Gibco). K562 cell line was transduced to express HLA-A0201, and human CD8 T cells were transduced to express 1G4 TCR.

[0225] 2. Construction of 1G4 TCR transduced human CD8 T cells

[0226] The 1G4 TCR sequence, including the alpha and beta chains of 1G4 TCR, connected by P2A, and the constant regions of the two chains were replaced by murine constant regions to reduce mismatches, was cloned into the pFG12 lentivirus vector using standard molecular biology techniques. Lentivirus particles were obtained by packaging plasmids pMD2G, pRSV-Rev, pMDLg / p and 293T cells, and titered by A TCR Jurkat cells.

[0227] Human CD8+T cells were isolated from peripheral mononuclear cells (PBMCs) by EasySep Human CD8+T Cell Isolation Kit, and after activation of CD8+T cells with human CD3 / CD28 Dynabeads (Invitrogen) at a ratio of 1:1 for 48 hours, transduction was performed using 1G4 TCR virus at MOI = 15, and finally 1G4 TCR T cells were obtained. Dynabeads were removed at 5 days of activation and rested until baseline activation state was recovered before use.

[0228] 3. In vitro short-term activation assay of T cells by different antigenic peptides

[0229] Using antigen presenting cells (e.g. murine DC cells with H-2kb epitope or human T2-A2 cells with HLA-A0201 epitope), after presenting different kinds and concentrations of antigenic peptides in vitro, the antigenic peptides were washed away. Then, these antigen presenting cells were co-incubated with T cells with corresponding TCR (e.g. OT-1 CD8 T cells or CD8 Jurkat cells transduced with 1G4 TCR) at a ratio of 1:1 for 2 hours. Subsequently, the positive expression rate of CD69 on T cells was detected by flow cytometry using anti-CD8 and anti-CD69 antibodies, to evaluate the activation degree of T cells by different kinds of antigenic peptides, and the EC50 value of different antigenic peptides was calculated according to the positive rate of CD69.

[0230] 4. In vitro long-term activation assay of T cells by antigenic peptides

[0231] Single cell suspension was prepared from the spleen of OT-1 TCR transgenic mice and plated at a cell density of 1 million / mL. After adding different antigenic peptides, co-incubation was performed for 72 hours. Subsequently, the long-term activation degree of T cells was detected by flow cytometry using anti-CD8, anti-CD69 and anti-CD25 antibodies.

[0232] 5. Measurement of antigenic peptide avidity

[0233] In the OT-1 TCR related experiment, MC38 cells were attached to the surface of a temperature-controlled microfluidic chip (LUMICKS C A B.V.) and different antigenic peptides were added for presentation. The chip was placed in a z-Movi cell avidity analyzer (LUMICKS) and the experiment was performed at 37°C. Subsequently, OT-1 CD8 T cells were allowed to flow into the microfluidic chip and interact with the MC38 monolayer cells, after which an acoustic force ramp (0-1000 pN relative force, over 150 seconds) was applied. All experiments were performed in triplicate.

[0234] ​In 1G4 TCR related experiments, K562 cells transduced with HLA-A0201 were attached on the surface of a temperature controlled microfluidic chip (LUMICKS CA B.V.) and different antigen peptides were added for presentation. The chip was placed in a z-Movi cell avidity analyzer (LUMICKS) and the experiment was performed at 37°C. Subsequently, CD8 Jurkat cells transduced with 1G4 TCR were flown into the microfluidic chip and interacted with the K562 monolayer cells, after which an acoustic force ramp (0-1000 pN relative force, over 150 seconds) was applied. All experiments were performed in triplicate.

[0235] 6. Strategy of pMHC tetramer staining for avidity characterization

[0236] After Jurkat cells overexpressing OT-1 TCR were constructed, different kinds and concentrations of antigen peptide-tetramer were used for binding staining and the binding ability of different antigen peptides was detected by flow cytometry, so as to obtain the Kd value of the antigen peptide. Similarly, Jurkat cells overexpressing 1G4 TCR were constructed, and different concentrations of antigen peptide-tetramer were used for staining and flow cytometry detection to evaluate the binding ability of different antigen peptides, and finally the Kd value was obtained.

[0237] 7. Bulk-seq analysis of short activated T cells

[0238] After DC cells were activated in vitro with different antigen peptides, they were co-incubated with OT-1 CD8 T cells at a ratio of 1:1 for 2 hours. Subsequently, staining and flow cytometry sorting were performed with Zombie and anti-CD8 antibody, and T cells were collected for bulk-seq experiment. Similarly, after T2-A2 cells were activated with different antigen peptides, they were co-incubated with human CD8 T cells transduced with 1G4 TCR at a ratio of 1:1 for 2 hours, and then staining and flow cytometry sorting were performed with Zombie and anti-CD8 antibody, and T cells were collected for bulk-seq experiment.

[0239] Bulk-seq experiment collects RNA and builds library by using new grid reagent kit, and then obtains data by sequencing. R language is used for analysis of sequencing data, correlation analysis is performed by scran package; differential expression analysis is performed by DESeq2; data visualization is performed by heatmap package.

[0240] 8. Cytokine detection of T cells mediated by antigen peptides

[0241] The MC38 cell line (with H-2kb epitope) was co-cultured with OT-1 CD8 T cells at a ratio of 1:1 for 2 hours, and then staining and flow cytometry sorting were performed with Zombie and anti-CD8 antibody, and T cells were collected for bulk-seq experiment. Similarly, T2-A2 cells were co-cultured with human CD8 T cells transduced with 1G4 TCR at a ratio of 1:1 for 2 hours, and then staining and flow cytometry sorting were performed with Zombie and anti-CD8 antibody, and T cells were collected for bulk-seq experiment. OT-1 CD8 T cells were co-incubated overnight at a 1:1 ratio with different kinds of antigen peptides. Subsequently, extracellular staining was performed using anti-CD8, anti-CD 107a antibodies, fixation was performed using BD Fixation / Permeabilization Kit, and intracellular staining was performed using anti-GZMB, TNF-a, IL-2, and IFN-g antibodies.

[0242] Similarly, T2-A2 cell line was co-incubated overnight at a 1:1 ratio with CD8 T cells transduced with 1G4 TCR, and different concentrations and kinds of antigen peptides were added. Subsequently, extracellular staining was performed using anti-CD8, anti-CD 107a antibodies, fixation was performed using BD Fixation / Permeabilization Kit, and intracellular staining was performed using anti-GZMB, TNF-a, IL-2, and IFN-g antibodies.

[0243] 9. Detection of antigen peptide-induced T cell killing function

[0244] Antigen-presenting target cells expressing GFP / LUC (e.g., MC38-GFP / Luc, UM-UC-3-GFP / Luc) were pre-plated in 96-well flat-bottom plates 24 hours in advance. Subsequently, mouse or human TCR-T cells were added to the wells at different effector-to-target ratios, and different kinds of antigen peptides were added for co-incubation. Three technical replicates were performed for each group, and wells with only target cells were used as background controls to determine the baseline luminescence. Luciferase detection reagents were then used for detection, and a microplate reader was used for fluorescence quantification. Specific lysis was determined by analyzing the luminescence difference of each well relative to the target cell-only well, so as to evaluate the killing effect induced by different antigen peptides. % Lysis of cells = 1 - (luminescence signal of experimental group / luminescence signal of background control group) x 100%.

[0245] 10. Detection of low-concentration antigen peptide-induced expansion of primary TCR-T cells

[0246] In the OT-1 TCR-related experiment, the spleen of the OT-1 TCR transgenic mouse was obtained and prepared into a single cell suspension. After the spleen cells were labeled with CFSE in the dark, the labeled spleen cells were plated in a 48-well plate (400 μl per well), antigen peptides and IL-2 were added for incubation, and then the antigen peptides were washed away and resuspended with new IL-2-containing medium. After 48 hours, anti-CD8 antibody flow cytometry staining was used, flow cytometry was used to analyze the absolute number of cells and the change in CFSE, and FlowJo was used for data analysis.

[0247] In the 1G4 TCR-related experiment, T2-A2 cells were treated with 20 μg / ml mitomycin C for 1 hour, and the treated T2-A2 cells were co-incubated with 1G4 TCR-positive T cells at a 1:1 ratio, while antigen peptides, IL-2, and IL-15 were added. The medium was changed and flow cytometry detection was performed every two days to analyze the change in the absolute number of cells.

[0248] 11. Detection of antigen peptide-induced TCR proliferation and activation using patient tumor cells.

[0249] Tumor cells from patients were co-incubated with human CD8 T cells transduced with 1G4 TCR, and different types of antigenic peptides were added. The 1G4 TCR positivity rate was then detected by flow cytometry to assess T cell proliferation, while CD25 expression levels and intracellular cytokine expression levels were detected to assess T cell activation.

[0250] 12. Detection of primary TCR-T cell exhaustion induced in vitro by high concentrations of antigenic peptides

[0251] In OT-1 TCR-related experiments, spleens were obtained from OT-1 TCR transgenic mice, and single-cell suspensions were prepared. Antigen peptides and IL-2 were added for activation, followed by washing away the antigen peptides and continued culturing in fresh IL-2-containing medium. After 5-7 days, cells were harvested for flow cytometry analysis to detect exhaustion markers such as PD-1, Lag3, CD39, Tim3, Tigit, and 2B4, in order to assess T cell exhaustion phenotype differences.

[0252] In the 1G4 TCR-related experiments, human CD8 T cells transduced with 1G4 TCR were first prepared according to the above method, and T2-A2 cells were treated with 20 μg / ml mitomycin C for 1 hour. Subsequently, the treated T2-A2 cells were co-incubated with 1G4 TCR-positive T cells at a 1:1 ratio, with the antigen peptide added simultaneously. The culture medium was changed every two days, and a number of mitomycin C-treated T2-A2 cells and the antigen peptide were added continuously for stimulation. Eight days later, T cells were collected for flow cytometry analysis to assess differences in T cell exhaustion phenotypes.

[0253] 13. Detection of T cell exhaustion induced in vivo by high concentrations of antigenic peptides

[0254] Using C57 / B6 mice, transfer was performed via tail vein injection. OT-1CD8 T cells were collected. Twenty-four hours later, the antigen peptide and MnJ adjuvant were injected intraperitoneally for five consecutive days. On day 6, the spleens of mice were harvested, and cells were collected for flow cytometry analysis to determine the exhaustion phenotype of T cells. Simultaneously, single-cell suspensions were prepared for single-cell sequencing, and library preparation and sequencing were performed using a Synergeton assay kit. Data analysis was conducted using the R language environment, with subpopulation analysis and differential gene analysis performed using Seurat.

[0255] 14. Treatment of solid tumors in mice

[0256] A mouse solid tumor model was constructed using C57 / B6 mice by subcutaneous injection of B16-OVA tumor cells. After the mice were tumor-bearing, they were randomly divided into groups. The negative control group was intravenously transfused with PBS solution, the positive control group was transfused with CARD11-PI3K3 modified OT-1 T cells (CAP+OT1), and the experimental group was transfused with CARD11-PI3K3 modified OT-1 cells and intraperitoneally injected with a non-natural strong antigen. The formula for calculating the volume of the mouse tumor was V = 0.5 x long diameter x short diameter 2 A mouse solid tumor model was constructed using C57 / B6 mice by subcutaneous injection of B16-GP33 tumor cells. After the mice were tumor-bearing, they were randomly divided into groups. The negative control group was intravenously transfused with PBS solution, the positive control group was transfused with CARD11-PI3K3 modified OT1+P14 T cells (CAP+OT1+P14), and the experimental group was transfused with CARD11-PI3K3 modified OT1+P14 T cells (CAP+OT1+P14) and intraperitoneally injected with a non-natural strong antigen. The formula for calculating the volume of the mouse tumor was V = 0.5 x long diameter x short diameter 2 Note: CAP is the abbreviation of CARD11-PI3K3, which is a gene reported to enhance T cell function.

[0257] Example 1 Construction of modified antigen peptides by solid-phase synthesis of polypeptides

[0258] In this example, we constructed chemically modified peptides by introducing unnatural amino acids using the method of solid-phase synthesis of polypeptides (as shown in Figures 2 and 3). For the screening of mouse OT-1 TCR, we constructed modified peptides with different unnatural chemical structures as shown in Figure 2; for the screening of human 1G4 TCR, we constructed modified peptides with different unnatural chemical structures as shown in Figure 3. Specifically, we introduced a variety of unnatural chemical structures at the site recognized by the OT-1 TCR (F at the 4th amino acid residue in Y4, SIIYFEKL (SEQ ID NO: 2), or Y in SIIFFEKL (SEQ ID NO: 5, F4)) to modify the benzene ring of F or Y with substituents including fluorine, bromine, iodine halogen atoms, different electronegative nitro groups, methoxy and trifluoromethoxy groups, and chemically reactive sulfonyl fluoride groups. We introduced a variety of unnatural chemical structures at the site recognized by the 1G4 TCR (W in W5, SLLMWITQV (SEQ ID NO: 1)) to modify the benzene ring of W with substituents including fluorine, chlorine, iodine halogen atoms, and methyl or methoxy groups at different substitution positions.

[0259] Example 2 Modified antigen peptides activate T cells at extremely low concentrations

[0260] In this example, the response of the TCR signaling pathway was used as an indicator to screen various non-natural modified peptides that can form a strong TCR bond with a given TCR. Specifically, mouse dendritic cells expressing H2-Kb and T2 cell lines expressing HLA-A*0201 were used as antigen-presenting cells to cross-present various modified peptides, and were co-incubated with T cells expressing OT-1 TCR and 1G4 TCR, and the modified peptides were screened by detecting the downstream activation marker CD69 of the T cell receptor (as shown in Figures 4 and 5). As shown in Figure 4, the methoxy-modified antigen peptide had a partial increase in the degree of activation compared to Y4 before modification; the bromo-, iodo-, nitro-, trifluoromethoxy- and sulfonyl fluoride-modified antigen peptides had a complete response of activation compared to Y4 or F4 before modification. As shown in Figure 5, the fluorinated, brominated and iodinated antigen peptides had a significant increase in activation compared to the natural antigen peptide W5; while the methoxy- or methyl-modified peptides resulted in weaker activation. The above results indicate that the activation of T cells by antigen peptides is enhanced after replacing the natural amino acids at the recognition site of the TCR with non-natural amino acids.

[0261] Example 3 Comparison of the degree of T cell activation by antigen peptides before and after modification

[0262] We directly compared the modified non-natural strong antigen with the antigen peptide before modification (as shown in Figure 6). For a specific OT-1 TCR, the non-natural strong antigen X4 (i.e., replacing the 4th amino acid Y of Y4 with FSY) was used as an example, which achieved a breakthrough from no response to complete response (with a half maximal effective concentration EC50 of ~10^(-9) M) compared to Y4 before modification; for a specific 1G4 TCR, the activation of the non-natural strong antigen 5-iW5 was significantly enhanced compared to W5, and the half maximal effective concentration was increased by more than 400 times (with a half maximal effective concentration EC50 of ~10^(-11.8) M) compared to before modification.

[0263] We further used the strong recognition model of OT-1 TCR recognizing the natural epitope N4 (SIINFEKL, SEQ ID NO: 21) as a control, and the activation of X4 on a specific OT-1 TCR was also more than 3 times higher than that of N4 (as shown in Figure 8). It is worth emphasizing that 5-iW5 only needs to be at a concentration of pM (10^(-12) M) to activate T cells to produce a response when activating a specific 1G4 TCR-T cell, which is much lower than the common natural TCR-antigen peptide (nM, 10^(-9) M) recognition model.

[0264] Example 4 The modification of the present application plays a decisive role in the function of antigen peptides and T cells

[0265] Based on the modification of the TCR recognition region chemical space, the construction of the strong recognition TCR bond form plays a decisive role in the activation of specific TCR. As shown in Figure 7, the E1N4 (EIINFEKL, SEQ ID NO: 3) antigen peptide is reported as the "antagonist" of the OT-1 TCR, which cannot effectively activate OT-1 T cells; but the antigen peptide E1X4 (compared with E1N4 of SEQ ID NO: 3, N at position 4 is replaced by FSY) based on the specific chemical space modification of this peptide can cause complete activation of OT-1 T cells. Similarly, the 4AW5 (SLLAWITQV, SEQ ID NO: 4) antigen peptide cannot effectively activate the 1G4 TCR-T cell; but the antigen peptide 4A5-iW5 (compared with SEQ ID NO: 4, W at position 5 is replaced by 5-iW) modified by specific chemical space can cause complete activation of 1G4 T cells. This shows that the chemical bond space of the specific TCR obtained by the method of the present application has a decisive role in the recognition of TCR, that is, the introduction of this non-natural structure in the sequence of a weak agonist or antagonist can cause a qualitative change, that is, induce complete activation of T cells.

[0266] Example 5 Chemical modification of non-natural antigen peptides has improved avidity compared to natural antigen peptides

[0267] Specifically, the avidity test method includes pMHC tetramer staining (as shown in Figures 9, 10) and cell-cell pulling test (as shown in Figures 11, 12). As shown in Figure 9, synthetic X4-tetramer and N4-tetramer were used to stain Jurkat cells overexpressing OT-1 TCR, and the results showed that X4-tetramer had stronger staining. As shown in Figure 10, synthetic 5-iW5-tetramer and W5-tetramer were used to stain Jurkat cells overexpressing 1G4 TCR, and the results showed that 5-iW5-tetramer had stronger staining. This shows that the "multivalency" binding of non-natural strong antigen to specific TCR is stronger than that of natural antigen. Further, we used the commercial instrument z-Movi of LUMICKS company to characterize the force of cell-cell stability test. As shown in Figure 11, the cell-cell binding strength of non-natural strong antigen X4 mediated antigen presenting cells and OT-1 T cells is higher than that of N4 and a series of natural APL (altered peptide ligand); as shown in Figure 12, the cell-cell binding strength of non-natural strong antigen 5-iW5 mediated antigen presenting cells and 1G4 T cells is higher than that of W5.

[0268] Example 6 Non-natural strong antigen mediated specific T cells can produce stronger effector release on cancer cells

[0269] To test the effect of T cell killing effector factors, OT-1 T cells were co-incubated with cancer cells expressing H2-Kb cross-presenting X4 or Y4, as shown in Figure 13, X4 mediated OT-1 T cells to release large amount of Gzmb, IFNg, TNFa and CD107a at low concentration 100 pM. Similarly, 1G4 T cells were co-incubated with cancer cells expressing HLA-A*0201 cross-presenting 5-iW5 or W5, as shown in Figure 14, 5-iW5 mediated 1G4 T cells to release large amount of Gzmb, IFNg and CD107a at lower concentration.

[0270] Example 7 Non-natural superantigen mediated specific T cells can produce stronger killing effect on cancer cells

[0271] To test the viability of cancer cells, OT-1 T cells were co-incubated with cancer cells over-expressing luciferase cross-presenting X4 or Y4, as shown in Figure 15, X4 mediated OT-1 T cells to effectively kill cancer cells expressing H2-Kb. Similarly, 1G4 T cells were co-incubated with cancer cells over-expressing luciferase cross-presenting 5-iW5 or W5, as shown in Figure 16, 5-iW5 mediated 1G4 T cells to effectively kill cancer cells expressing HLA-A*0201 at lower concentration. It is worth noting that we introduced LY-1G4 TCR, an engineered high-affinity TCR, as a reference (its affinity Kd = 1.1 μΜ) here, we reported that non-natural superantigen mediated killing is more effective than engineered high-affinity TCR mediated killing.

[0272] Example 8 Non-natural superantigen mediated specific T cells efficiently expand

[0273] The T cells were stimulated with the unnatural strong antigen or the natural antigen, and the dilution ratio of CFSE and the absolute count of specific T cells were detected to characterize the expansion of T cells. As shown in FIGS. 17, 18 and 19, X4 and 5-iW5 can stimulate the expansion of T cells more strongly than the unmodified antigen peptide, and the T cells expand more for 5-iW5 to help better killing. Further, we adoptively transferred OT-1 T cells to recipient mice, and stimulated the T cells to expand in vivo with the unnatural strong antigen or the natural antigen. As shown in FIG. 20, X4 can expand specific T cells more strongly in mice than Y4. We used cancer cells (expressing HLA-A*0201) of a clinical bladder cancer patient as antigen presenting cells, and cross-presented the unnatural strong antigen 5-iW5 or the natural antigen W5, and then co-incubated with 1G4 TCR-T to detect the expansion of human TCR-T in patient samples. As shown in FIG. 21, the unnatural strong antigen 5-iW5 can mediate the specific expansion and activation of TCR-T, which provides a basis for the clinical application of the unnatural strong antigen.

[0274] Example 9 Unnatural strong antigen can induce specific T cell exhaustion under certain conditions

[0275] We used the model of in vitro high concentration of antigen peptide to stimulate T cells, and the results are shown in FIGS. 22 and 23. X4 and 5-iW5 can induce specific T cells to differentiate into a phenotype with a higher degree of exhaustion in vitro. Further, we adoptively transferred OT-1 T cells to recipient mice, and repeatedly stimulated the T cells in vivo with the unnatural strong antigen or the natural antigen. Flow cytometry analysis is shown in FIG. 24, and the results show that X4 induces more OT-1 T cell exhaustion in vivo than Y4 and N4, the unmodified antigen. Single cell sequencing further analyzes the subgroups of exhausted T cells in vivo, and the results are shown in FIG. 25. X4 induces more exhausted T cells and fewer memory precursor T cells than N4. This indicates that the unnatural strong antigen induces the fate of specific T cells to change from memory to exhaustion in vivo.

[0276] Example 10 Unnatural strong antigen examples for different TCRs

[0277] We further display unnatural strong antigens for other TCRs, including P14 (as shown in FIG. 37), NY 8.3 (as shown in FIG. 38) and Mel 8 (as shown in FIG. 39). Specifically, for P14 TCR, we display that the modified antigen peptide modified with methoxy at position 7 of tryptophan is significantly improved compared to the unmodified antigen (as shown in FIG. 37). For NY 8.3 TCR, we display that the modified antigen peptide modified with bromo or iodo at position 2 of phenylalanine is improved compared to the unmodified antigen (as shown in FIG. 38). For Mel 8 TCR, we display that the modified peptide with cyclopentane structure replacing the leucine side chain is significantly improved compared to the unmodified antigen (as shown in FIG. 39).

[0278] Example 11 Chemically modified unnatural antigenic peptides form new TCR bonds with given TCRs

[0279] Based on the AI prediction + molecular docking method, the bonding form between TCR and unnatural strong antigen was predicted, as shown in Figure 40. In the predicted complex structure of 1G4 TCR-5-iW5 peptide, we found that the halogen bond / σ-hole interaction between W-5I---O=C-Y94 (TCR β chain) and the π-π interaction between W and Y were formed. In the complex of OT-1 TCR-Y-OCF3 peptide, we found that the halogen bond / σ-hole interaction between Y-OCF3---O=C-Y96 (TCR α chain) and the π-π interaction between Y and Y were formed. This result shows that new intermolecular interactions between these unnatural amino acids and TCRs, i.e., TCR bonds, are formed.

[0280] Example 12 Unnatural strong antigens enhance T cell solid tumor treatment effect

[0281] We evaluated the enhancement effect of unnatural strong antigens on T cell killing of tumors in vivo. We infused genetically enhanced CAP+OT-1 T cells into tumor-bearing B16-OVA mice and administered unnatural antigen peptides by intraperitoneal injection. We found that the group administered with unnatural strong antigens (CAP+OT-1+BOOST) could more significantly inhibit tumor growth, as shown in Figure 41, which shows that the co-administration of unnatural antigen peptides and T cell therapy can achieve better tumor inhibition.

[0282] In the scenario of combined therapy with immune checkpoint inhibitors, as shown in Figure 42, the method of unnatural antigens can effectively reverse the growth trend of large tumors (V>1000 mm 3 ) and delay the progression of solid tumors, which shows that the co-administration of unnatural antigen peptides and immune checkpoint inhibitors can achieve better tumor inhibition.

[0283] Example 13 Unnatural peptides enhance the solid tumor treatment effect of weak TCR-T

[0284] Weak affinity TCR-T and CAR-T have poor effects on solid tumors. Therefore, in addition to directly using OT-1 TCR, which has strong affinity, to kill tumors, we further tried to use unnatural strong antigens and their corresponding TCRs as a pair of “switches” to regulate T cells. Specifically, we infected P14 mouse T cells with OT-1 TCR using retroviruses. We found that unnatural strong antigens can enhance the killing of P14 T cells on tumors expressing antigens (GP33) in vivo (as shown in Figure 43).

[0285] The references of the present application are as follows:

[0286] [1] Huppa, J. B. et al. T-cell-antigen recognition and the immunological synapse. Nat Rev Immunol 3, 973-983 (2003).

[0287] [2] Lever, M. et al. Phenotypic models of T cell activation. Nat Rev Immunol 14, 619-629 (2014).

[0288] [3] Palmer, E. et al. Affinity threshold for thymic selection through a T-cell receptor-co-receptor zipper. Nat Rev Immunol 9, 207-213 (2009).

[0289] [4] Gascoigne, N. R. J. et al. TCR Signal Strength and T Cell Development. Annu. Rev. Cell Dev. Biol. 32, 327-348 (2016).

[0290] [5] Achar, S. R. et al. Universal antigen encoding of T cell activation from high-dimensional cytokine dynamics. Science 376, 880-884 (2022).

[0291] [6] Sellars, M. C. et al. Cancer vaccines: Building a bridge over troubled waters. Cell 185, 2770-2788 (2022).

[0292] [7] Shakiba, M. et al. TCR signal strength defines distinct mechanisms of T cell dysfunction and cancer evasion. J. Exp. Med. 219, e20201966 (2022).

[0293] [8] Hoffmann, M. M. et al. T-cell receptor affinity in the age of cancer immunotherapy. Molecular Carcinogenesis 59, 862-870 (2020).

[0294] [9] Stone, J. D. et al. T-cell receptor binding affinities and kinetics: impact on T-cell activity and specificity. Immunology 126, 165-176 (2009).

[0295]

[0010] Aleksic, M. et al. Different affinity windows for virus and cancer-specific T-cell receptors: Implications for therapeutic strategies. Eur J Immunol 42, 3174-3179 (2012).

[0296]

[0011] Hebeisen, M. et al. Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens. Front. Immunol. 6, (2015).

[0297]

[0012] Zhong, S. et al. T-cell receptor affinity and avidity defines antitumor response and autoimmunity in T-cell immunotherapy. Proc. Natl. Acad. Sci. U.S.A. 110, 6973-6978 (2013).

[0298]

[0013] Holler, P.D. et al. TCRs with high affinity for foreign pMHC show self- reactivity. Nat Immunol 4, 55-62 (2003).

[0299]

[0014] Zhao, X. et al. Tuning T cell receptor sensitivity through catch bond engineering. Science 376, eabl5282 (2022).

[0300]

[0015] Saligrama, N. et al. Opposing T cell responses in experimental autoimmune encephalomyelitis. Nature 572, 481-487 (2019).

[0301]

[0016] Choi, H.-K. et al. Catch bond models may explain how force amplifies TCR signaling and antigen discrimination. Nat Commun 14, 2616 (2023).

[0302]

[0017] Gearty, S.V. et al. An autoimmune stem-like CD8 T cell population drives type 1 diabetes. Nature 602, 156-161 (2022).

[0303]

[0018] Utzschneider, D.T. et al. Early precursor T cells establish and propagate T cell exhaustion in chronic infection. Nat Immunol 21, 1256-1266 (2020).

[0304]

[0019] Dolton, G. et al. Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy. Cell 186, 3333-3349.e27 (2023).

Claims

1. A modified antigenic peptide, which has an altered binding mode with a given T cell receptor (TCR) as compared to the unmodified peptide.

2. The modified antigenic peptide of claim 1, which does not generate a broad spectrum of TCR reactivity.

3. The modified antigenic peptide of claim 1, which has an altered recognition space with the TCR as compared to the unmodified peptide.

4. The modified antigenic peptide of claim 3, wherein the altered recognition space is not the chemical space of natural amino acid side chains.

5. The modified antigenic peptide of any one of claims 1-4, which has an increased half-life of the TCR bond as compared to the unmodified peptide.

6. The modified antigenic peptide of any one of claims 1-5, which has an increased bond energy of the TCR bond as compared to the unmodified peptide.

7. The modified antigenic peptide of claims 5-6, wherein the half-life of the TCR bond is detected using biophysical methods such as biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method, and / or cell function assays related to the TCR bond.

8. The modified antigenic peptide of any one of claims 1-7, which has an increased stability of the ternary complex pMHC-TCR formed by the modified antigenic peptide and the MHC and the TCR as compared to the unmodified peptide.

9. The modified antigenic peptide of claim 8, wherein the stability of the pMHC-TCR is detected using biophysical methods such as biomembrane force probe, optical tweezers, flow cytometry, surface plasmon resonance, single molecule fluorescence resonance energy transfer, molecular dynamics simulation, thermal fluctuation method, and / or cell function assays related to the TCR bond.

10. The modified antigenic peptide of any one of claims 1-9, which can bind to a T cell expressing the TCR via an antigen presenting cell (APC), and has an increased avidity between the APC presenting the modified antigenic peptide and the T cell as compared to the unmodified peptide.

11. The modified antigenic peptide of claim 10, wherein the avidity between the APC and the T cell is detected by flow cytometry, cell proliferation assay, cell adhesion assay, enzyme-linked immunosorbent assay, single cell imaging technique, single molecule technique, surface plasmon resonance, thermal fluctuation method, molecular dynamics simulation, functional T cell response detection, and / or microfluidic chip technology.

12. The modified antigenic peptide of any one of claims 1-11, which has an at least 10% increased degree of activation of the T cell as compared to the unmodified peptide.

13. The modified antigenic peptide of claim 12, wherein the activation of the T cell comprises upregulation of expression of markers CD69, CD25, CD134, CD279, CD223, CD44, CD39, CD38, CD6, CCL3, XCL1, HLA-DR, and the like, and downregulation of expression of CD62L, CCR7, TCF-1, CD127, and the like.

14. The modified antigenic peptide of claim 12, wherein the activation of the T cell comprises T cell proliferation.

15. The modified antigenic peptide of claim 12, wherein the activation of the T cell comprises secretion of cytokines.

16. The modified antigenic peptide of claim 15, wherein the cytokines comprise IL-2, Gzmb, IFN-gamma, and TNFa.

17. The modified antigenic peptide of any one of claims 1-16, wherein the modification comprises introducing a chemical group to one or more amino acid residues of the antigenic peptide.

18. The modified antigenic peptide of any one of claims 1-17, wherein the modification comprises replacing one or more amino acid residues of the antigenic peptide with a non-natural amino acid residue.

19. The modified antigenic peptide of any one of claims 1-17, wherein the modification comprises replacing one or more amino acid residues of the antigenic peptide with an amino acid residue that is opposite in nature selected from the group consisting of: acid-base nature, electrical nature, polarity, chemical reactivity, and / or hydrophilic-hydrophobic nature.

20. The modified antigenic peptide of any one of claims 17-19, wherein the one or more amino acid residues are located at a recognition site of the antigenic peptide binding to the TCR.

21. The modified antigenic peptide of any one of claims 17-20, wherein the one or more amino acid residues are involved in forming a TCR bond of the antigenic peptide to the TCR.

22. The modified antigenic peptide of any one of claims 1-21, wherein the modification comprises inserting one or more amino acid residues at a recognition site between the antigenic peptide before modification and the TCR.

23. The modified antigenic peptide of any one of claims 1-22, wherein the modification comprises contacting the antigenic peptide with one or more metal ions, and the metal ions are involved in forming a chemical space of the modified antigenic peptide to the TCR.

24. The modified antigenic peptide of any one of claims 1-23, wherein the T cell comprises a cytotoxic T lymphocyte, a Th cell, and / or a Treg cell.

25. The modified antigenic peptide of any one of claims 1-24, wherein the bonded form is verified by molecular level methods such as crystal structure.

26. The modified antigenic peptide of any one of claims 1-25, which comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-5, 8, 13, 16, 17, 20, and 21 before modification.

27. A modified antigenic peptide, wherein the amino acid at the fifth position is a bromo-, fluoro- or iodo-modified tryptophan, as compared to the amino acid sequence set forth as SEQ ID NO:

1.

28. A modified antigenic peptide, wherein the amino acid at the fourth position is a sulfonyl fluoride-modified tyrosine, as compared to the amino acid sequence set forth as SEQ ID NO: 2 or 5.

29. A modified antigenic peptide, wherein the amino acid at the fourth position is a sulfonyl fluoride-modified tyrosine, as compared to the amino acid sequence set forth as SEQ ID NO:

3.

30. A modified antigenic peptide, wherein the amino acid at the fifth position is a bromo-, fluoro- or iodo-modified tryptophan, as compared to the amino acid sequence set forth as SEQ ID NO:

4.

31. A method of modifying an antigenic peptide, the method comprising (1) providing an antigenic peptide, and (2) introducing to the antigenic peptide a chemical structure capable of altering the bonding pattern between it and a TCR.

32. A fusion protein comprising the modified antigenic peptide of any one of claims 1-30.

33. An immunoconjugate comprising the modified antigenic peptide of any one of claims 1-30 and an antibody conjugate.

34. A nano-compound comprising the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, and / or a nanocarrier, in a nano-form.

35. A pharmaceutical combination comprising the modified antigenic peptide described herein, and one or more other anti-tumor agents.

36. The pharmaceutical combination of claim 35, wherein the other anti-tumor agent comprises an immune checkpoint inhibitor and / or an engineered cell.

37. The pharmaceutical combination of claim 36, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a TIM-3 inhibitor, and / or a VISTA inhibitor.

38. The pharmaceutical combination of claim 36, wherein the engineered cell comprises a T cell, such as a chimeric antigen receptor (CAR)-T cell, a TCR-T cell, and / or a tumor infiltrating lymphocyte (TIL).

39. A pharmaceutical composition comprising the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, the nano-compound of claim 34, and / or the pharmaceutical combination of any one of claims 35-38, and optionally a pharmaceutically acceptable carrier.

40. A method of activating a T cell, the method comprising administering an effective amount of the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, the nano-compound of claim 34, the pharmaceutical combination of any one of claims 35-38, and / or the pharmaceutical composition of claim 39.

41. A method of inducing T cell exhaustion, the method comprising administering an effective amount of the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, the nano-compound of claim 34, the pharmaceutical combination of any one of claims 35-38, and / or the pharmaceutical composition of claim 39.

42. A method of regulating T cell fate based on TCR signaling pathway, using the modified peptide to induce specific T cells to expand and maintain stemness and function at certain dosage and frequency after screening, or to drive specific T cells to produce exhaustion phenotype and loss of function at high dosage and high frequency stimulation, the method comprising administering an effective amount of the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, the nano-compound of claim 34, the pharmaceutical combination of any one of claims 35-38, and / or the pharmaceutical composition of claim 39.

43. A method of regulating T cells, the method comprising contacting an effective amount of the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, the nano-compound of claim 34, the pharmaceutical combination of any one of claims 35-38, and / or the pharmaceutical composition of claim 39 with T cells.

44. A method of preventing, treating, and / or ameliorating a disease, the method comprising administering to a subject in need thereof an effective amount of the modified antigenic peptide of any one of claims 1-30, the fusion protein of claim 32, the immunoconjugate of claim 33, the nano-compound of claim 34, the pharmaceutical combination of any one of claims 35-38, and / or the pharmaceutical composition of claim 39.

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