TCR or fragment thereof with improved affinity and use thereof

Abstract

The present invention relates to the field of immunity, in particular to T cell receptors and their use in detecting, diagnosing, preventing, alleviating or treating KRAS G12V mutation-positive diseases and / or conditions. The present invention provides a TCR or a fragment thereof with improved affinity, wherein the CDR3α of the TCR with improved affinity comprises amino acid substitutions at positions V106 and / or I117 relative to the CDR3α of the parent TCR. The TCR provided by the present invention has the ability to bind to target antigen peptides with high affinity, significantly improves the target cell recognition ability, and can mediate the specific killing effect of effector cells on antigen-positive target cells, and has no allogeneic reaction to HLA of different types. It can be used to treat a variety of cancers that are positive for KRAS G12V, providing an important tool for cell therapy, and showing strong technical value and clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of immunity, in particular to T cell receptors (TCRs) and uses thereof, in particular to the detection, diagnosis, prevention, alleviation or treatment of KRAS G12V mutation-positive diseases and / or disorders. Background Art

[0002] The protein encoded by the KARS gene is a small GTPase that belongs to the RAS superprotein family.

[0003] In cells, KRAS protein switches between inactive and activated states. When KRAS binds to guanine nucleoside diphosphate (GDP), it is inactive. When it binds to guanine nucleoside triphosphate (GTP), it is in active state and can activate downstream signaling pathways, including MAPK signaling pathway, PI3K signaling pathway, and Ral-GEFs signaling pathway. These signaling pathways play an important role in promoting cell survival, proliferation and cytokine release.

[0004] KRAS is the most commonly mutated gene in the RAS family, accounting for 85% of the total RAS gene mutations (NRAS (12%) is second, and HRAS (3%) is the least). In human cancer, nearly 90% of pancreatic cancer, 30-40% of colon cancer, 17% of endometrial cancer, and 15-20% of lung cancer (mostly NSCLC) are caused by KRAS gene mutations. KRAS mutations have also been found in cancer types such as bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer. Among all KRAS mutations, 97% are G12D, G12V, and G13D mutations.

[0005] The applicant previously successfully isolated and identified a TCR sequence that specifically targets the KRAS-G12V mutation from G12V mutation-positive colorectal cancer tissues. This TCR restrictively recognizes the KRAS-G12V antigen peptide presented by HLA-A1101 and named it "KVA11-N01". A patent application was submitted in 2021 and was granted (CN116063511B). Summary of the invention

[0006] The applicant of the present invention uses KVA11-N01 as a template and optimizes the affinity of KVA11-N01, ultimately producing a series of mutant TCR sequences with enhanced affinity for target antigen epitopes and retaining the original specificity, which can mediate the specific killing effect of effector cells on antigen-positive target cells, thereby completing the present invention.

[0007] Accordingly, in a first aspect, the present invention provides a TCR or a fragment thereof with improved affinity, wherein the TCR with improved affinity comprises an α chain variable region comprising CDR1α, CDR2α and CDR3α and a β chain variable region comprising CDR1β, CDR2β and CDR3β; wherein the CDR3α of the TCR with improved affinity comprises amino acid substitutions at positions V106 and / or I117 relative to the CDR3α of the parent TCR;

[0008] wherein the amino acid substitution at position V106 is selected from V106P, V106G, V106A, V106C, V106S, V106T or V106M;

[0009] The amino acid substitution at position I117 is selected from I117C, I117S, I117T, I117M, I117R or I117K;

[0010] Wherein, the parent TCR comprises CDR1α with an amino acid sequence as shown in SEQ ID NO: 1, CDR2α with an amino acid sequence as shown in SEQ ID NO: 2, CDR3α with an amino acid sequence as shown in SEQ ID NO: 3, CDR1β with an amino acid sequence as shown in SEQ ID NO: 4, CDR2β with an amino acid sequence as shown in SEQ ID NO: 5, and CDR3β with an amino acid sequence as shown in SEQ ID NO: 6;

[0011] The amino acid positions are numbered according to the nomenclature of the International Immunogenetics Information System (IMGT).

[0012] In a second aspect, the present invention provides a nucleic acid encoding the TCR or a fragment thereof of the first aspect.

[0013] In a third aspect, the present invention provides a vector comprising the nucleic acid according to the second aspect.

[0014] In a fourth aspect, the present invention provides a cell comprising the TCR or fragment thereof of the first aspect, the nucleic acid of the second aspect, or the vector of the third aspect.

[0015] In a fifth aspect, the present invention provides a use of a nucleic acid comprising the TCR or fragment thereof of the first aspect, or a vector of the third aspect, in the preparation of a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12V-positive disease or condition.

[0016] The TCR provided by the present invention has the ability to bind target antigen peptides with high affinity, significantly improves the target cell recognition ability, and can mediate the specific killing effect of effector cells on antigen-positive target cells, and has no allogeneic reaction to HLA of different types. It can be used to treat a variety of cancers positive for KRAS G12V, providing an important tool for cell therapy, showing strong technical value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 and Figure 2 Activation results of screened high-affinity TCRs on reporter cells are shown.

[0018] Figure 3 The affinity-optimized TCR showed its ability to kill antigen target cells.

[0019] Figure 4 The cytokine secretion function of affinity-optimized TCRs to antigenic target cells was demonstrated.

[0020] Figure 5 Results of affinity-optimized TCR alloreactivity assays are shown.

[0021] Figure 6 The results of the off-target response detection of affinity-optimized TCR to non-antigenic peptides are shown.

[0022] Figure 7 X-SCAN detection results of affinity-optimized TCR KVA11-M01-231 are shown. DETAILED DESCRIPTION

[0023] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. For example, the terms used herein are as defined in Janeway CA Jr, Travers P, Walport M, et al., Immunobiology, Fifth Edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations), Leuenberger, HGW, Nagel, B. and Kölbl, H., eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0024] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" may be used interchangeably.

[0025] When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. For the purpose of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists of only these embodiments.

[0026] T cell receptor (TCR)

[0027] The inventors of the present invention have found through research that introducing V106A, V106S or V106T amino acid mutations at position V106 in CDR1 (CDR3α) of the parental TCR α chain, and / or introducing I117T or I117R amino acid substitutions at position I117 can result in improved binding affinity to the KRAS G12V mutant antigen and ability to bind to the target antigen peptide, thereby completing the present invention.

[0028] It is understood by those skilled in the art that the 20 kinds of amino acid residues constituting proteins can be divided into four categories according to the polarity of the side chains: 1. Non-polar amino acids: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W) and proline (P); 2. Polar uncharged amino acids: serine (S), threonine (T), cysteine ​​(C), aspartic acid (N), methionine (M), glutamine (Q) and tyrosine (Y); 3. Positively charged amino acids: arginine (R), lysine (K) and histidine (H); 4. Negatively charged amino acids: aspartic acid (D) and glutamic acid (E) (see "Biochemistry" (Second Edition), Volume 1, Shen Tong, Wang Jingyan, pages 82-83, Higher Education Press, December 1990). Among them, the amino acids under each category can be further divided according to factors such as their size, acidity and base, complexity, etc., as shown in Table 1. If a substitution of amino acid residues belonging to the same category occurs in a protein, that is, an amino acid substitution in the same row of the second column in Table 1, such as R replacing K or L replacing I, the role of the residue in the protein domain (such as providing a positive charge or forming a hydrophobic pocket structure) does not change, so the three-dimensional structure of the protein will not be affected, so the function of the protein can still be achieved. For example, it is well known to those skilled in the art that V and I, D and E, S and T, A and G, A and P, K and R, L and I, L and V, are mutually substituted in pairs, which will not affect the three-dimensional structure and function of the protein. Therefore, conservative substitutions of V106A, V106S, V106T, I117T and I117R are also expected to improve the affinity of TCR.

[0029] Table 1

[0030]

[0031] Thus, the present invention provides a TCR or a fragment thereof with improved affinity, wherein the TCR with improved affinity comprises an α chain variable region comprising CDR1α, CDR2α and CDR3α and a β chain variable region comprising CDR1β, CDR2β and CDR3β; wherein the CDR3α of the TCR with improved affinity comprises amino acid substitutions at positions V106 and / or I117 relative to the CDR3α of the parent TCR;

[0032] wherein the amino acid substitution at position V106 is selected from V106P, V106G, V106A, V106C, V106S, V106T or V106M;

[0033] The amino acid substitution at position I117 is selected from I117C, I117S, I117T, I117M, I117R or I117K;

[0034] Wherein, the parent TCR comprises CDR1α with an amino acid sequence as shown in SEQ ID NO: 1, CDR2α with an amino acid sequence as shown in SEQ ID NO: 2, CDR3α with an amino acid sequence as shown in SEQ ID NO: 3, CDR1β with an amino acid sequence as shown in SEQ ID NO: 4, CDR2β with an amino acid sequence as shown in SEQ ID NO: 5, and CDR3β with an amino acid sequence as shown in SEQ ID NO: 6;

[0035] The amino acid positions are numbered according to the nomenclature of the International Immunogenetics Information System (IMGT).

[0036] The term "parent TCR" as used herein refers to the HLA-A1101 restricted human TCR targeting the KRAS G12V mutant antigen previously developed and patented by the applicant of the present invention (KVN01, authorization publication number: CN116063511B), which is incorporated herein by reference in its entirety. In some embodiments, the parent TCR comprises a TCR α chain variable region having an amino acid sequence as shown in SEQ ID NO: 10; and a TCR β chain variable region having an amino acid sequence as shown in SEQ ID NO: 11. In some embodiments, the parent TCR comprises a TCR α chain having an amino acid sequence as shown in SEQ ID NO: 17; and a TCR β chain having an amino acid sequence as shown in SEQ ID NO: 18.

[0037] The term "avidity" or "binding affinity" refers to the ability of T cells expressing the TCR of the present invention to respond to a given concentration of ligand in vitro, and is believed to be related to the in vivo effector capacity of cells expressing the TCR. By definition, cells expressing TCRs with high binding affinity respond to very low antigen doses in in vitro tests, while such cells with lower binding affinity require higher amounts of antigen before reaching an immune response similar to that of cells expressing TCRs with high affinity. Therefore, binding affinity can be considered as a quantitative determinant of the activation threshold of cells expressing TCRs. This is determined by exposing such cells to different amounts of cognate antigens in vitro. Cells with TCRs with high binding affinity respond to low antigen doses. In some embodiments, EC50 is used as a criterion for judging the affinity of a given TCR.

[0038] In the present invention, the term "affinity improvement" means that the ability of the mutant TCR provided by the present invention to bind to the target antigen peptide is improved relative to the parent TCR, for example, by at least 2 times, at least 3 times, at least 4 times, or at least 5 times.

[0039] In some embodiments of the present invention, the TCR provided herein is capable of binding to an epitope comprising the amino acid sequence SEQ ID NO: 22 or a complex of the epitope and an MHC molecule.

[0040] The term "epitope" generally refers to a site on an antigen, typically a (poly)peptide recognized by a binding domain. The term "binding domain" refers to an "antigen binding site" in its broadest sense, i.e., a domain that characterizes a molecule that binds / interacts with a specific epitope on an antigen target. An antigen target may contain a single epitope, but typically contains at least two epitopes, and depending on the size, conformation, and type of the antigen, the antigen target may include any number of epitopes. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained in an amino acid primary sequence, and it typically includes at least 2 amino acids or more amino acids. A conformational epitope is formed by non-continuous amino acids that are arranged side by side by the folding of a target antigen, and particularly a target (poly)peptide.

[0041] As used herein, the term "T cell receptor" or "TCR" can be used interchangeably to refer to a natural TCR or its variants, fragments and constructs. The term thus includes heterodimers, multimers and single-chain constructs comprising a TCR α chain and a TCR β chain; optionally comprising other domains and / or portions, as long as the TCR retains its ability to recognize an antigen target (preferably in a complex with HLA-A*11).

[0042] In its native form, the TCR exists as a complex of several proteins on the surface of the T cell. The T cell receptor consists of two (separate) protein chains, which are produced by independent T cell receptor α and β (TCRα and TCRβ) genes and are called α chain and β chain. Each chain of the TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region / domain, an Ig-constant (C) region / domain, a transmembrane / cell membrane spanning region that anchors the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminus.

[0043] Antigen specificity is conferred by the variable regions of the α and β chains. Both variable regions of the TCR α and β chains contain three hypervariable or complementary determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by framework (FR) regions. CDR3 is the main determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 mainly interact with MHC molecules presenting antigenic peptides.

[0044] TCR recognizes antigenic peptides that are bound to major histocompatibility complex (MHC) molecules at the surface of antigen presenting cells ("presented / displayed on MHC molecules"). Antigenic peptides presented on MHC molecules are also referred to herein as "complexes of epitopes with MHC molecules", "epitope-MHC complexes" or "target antigen peptide-MHC complexes". There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cell compartments. MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptides or protein fragments from intracellular compartments to cytotoxic T cells. In humans, MHC is also known as human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B and HLA-C. Once TCR binds to its specific epitope-MHC complex, the T cell is activated and exerts biological effector functions.

[0045] The TCR provided herein can advantageously (specifically) recognize the KRAS G12V epitope or a complex of the epitope and an MHC molecule, such as a complex of the KRAS G12V epitope and HLA-A*11, and has a significantly improved binding affinity compared to its parental TCR.

[0046] In some embodiments, the MHC molecule is of HLA-A*11 type, preferably HLA-A*11:01.

[0047] In some embodiments, the CDR3α of the affinity-enhanced TCR comprises a substitution at V106 relative to the CDR3α of the parent TCR. Preferably, the amino acid substitution at position V106 is selected from V106P, V106G, V106A, V106C, V106S, V106T or V106M. More preferably, the amino acid substitution at position V106 is selected from V106A, V106S or V106T. Further preferably, the amino acid substitution at position V106 is V106A.

[0048] In some embodiments, the CDR3α of the affinity-enhanced TCR comprises an amino acid substitution at position I117 relative to the CDR3α of the parent TCR. Preferably, the amino acid substitution at position I117 is selected from I117C, I117S, I117T, I117M, I117R or I117K. More preferably, the amino acid substitution at position I117 is selected from I117T or I117R.

[0049] In some embodiments, the CDR3α of the affinity-enhanced TCR comprises amino acid substitutions at positions V106 and I117 relative to the CDR3α of the parent TCR. Preferably, the amino acid substitution at position V106 is selected from V106P, V106G, V106A, V106C, V106S, V106T or V106M, and the amino acid substitution at position I117 is selected from I117C, I117S, I117T, I117M, I117R or I117K. More preferably, the amino acid substitution at position V106 is selected from V106A, V106S or V106T, and the amino acid substitution at position I117 is selected from I117T or I117R. Further preferably, the CDR3α of the affinity-enhanced TCR comprises amino acid substitutions V106T and I117T; or V106S and I117R relative to the CDR3α of the parent TCR.

[0050] In some preferred embodiments, the affinity-enhanced TCR (KVA11-M01-002) comprises CDR1α as shown in SEQ ID NO: 1, CDR2α as shown in SEQ ID NO: 2, CDR3α as shown in SEQ ID NO: 7, CDR1β as shown in SEQ ID NO: 4, CDR2β as shown in SEQ ID NO: 5, and CDR3β as shown in SEQ ID NO: 6.

[0051] In some preferred embodiments, the affinity-enhanced TCR (KVA11-M01-231) comprises a CDR1α with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2α with an amino acid sequence as shown in SEQ ID NO: 2, a CDR3α with an amino acid sequence as shown in SEQ ID NO: 8, a CDR1β with an amino acid sequence as shown in SEQ ID NO: 4, a CDR2β with an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3β with an amino acid sequence as shown in SEQ ID NO: 6.

[0052] In some preferred embodiments, the affinity-enhanced TCR (KVA11-M01-254) comprises a CDR1α with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2α with an amino acid sequence as shown in SEQ ID NO: 2, a CDR3α with an amino acid sequence as shown in SEQ ID NO: 9, a CDR1β with an amino acid sequence as shown in SEQ ID NO: 4, a CDR2β with an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3β with an amino acid sequence as shown in SEQ ID NO: 6.

[0053] In some embodiments, the TCR comprises a TCR alpha chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 14-16.

[0054] In some embodiments, the TCR comprises a TCR β chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:11.

[0055] In some embodiments, the TCR comprises a TCR alpha chain variable region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NOs: 14-16, and a TCR beta chain variable region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 11.

[0056] In some embodiments, the TCR comprises a TCR alpha chain variable region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:14, and a TCR beta chain variable region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:11.

[0057] In some embodiments, the TCR comprises a TCR alpha chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:15, and a TCR beta chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:11.

[0058] In some embodiments, the TCR comprises a TCR alpha chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:16, and a TCR beta chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:11.

[0059] The term "sequence identity" as used herein refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.

[0060] Therefore, the amino acid sequences of SEQ ID NOs: 14-16 can be used as "subject sequences" or "reference sequences", while the amino acid sequences of TCR α chain or β chain variable regions that differ therefrom can be used as "query sequences".

[0061] In some embodiments, the TCR comprises a TCR alpha chain variable region having an amino acid sequence shown in any one of SEQ ID NOs: 14-16.

[0062] In some embodiments, the TCR comprises a TCR β chain variable region having the amino acid sequence shown in SEQ ID NO:11.

[0063] In some embodiments, the TCR comprises a TCR α chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 14-16, and the TCR comprises a TCR β chain variable region having an amino acid sequence as shown in SEQ ID NO:11.

[0064] In some embodiments, the TCR comprises a TCR α chain variable region having an amino acid sequence shown in SEQ ID NO:14, and the TCR comprises a TCR β chain variable region having an amino acid sequence shown in SEQ ID NO:11.

[0065] In some embodiments, the TCR comprises a TCR α chain variable region having an amino acid sequence as shown in SEQ ID NO:15, and the TCR comprises a TCR β chain variable region having an amino acid sequence as shown in SEQ ID NO:11.

[0066] In some embodiments, the TCR comprises a TCR α chain variable region having the amino acid sequence shown in SEQ ID NO:16, and the TCR comprises a TCR β chain variable region having the amino acid sequence shown in SEQ ID NO:11.

[0067] In some embodiments, the TCR α chain variable region is contained on a first polypeptide, and the TCR β chain variable region is contained on a different second polypeptide. In some embodiments, the TCR α chain variable region and the TCR β chain variable region are contained on a single polypeptide.

[0068] In some embodiments, the TCR is soluble or membrane-bound.

[0069] The TCR of the present invention can be provided in a soluble form, for example, in the form of a soluble TCR. Soluble TCR (sTCR) can be used as a diagnostic tool and a carrier or "adapter" for specifically targeting therapeutic agents or effector cells to, for example, cancer cells expressing an antigen target recognized by a soluble TCR. Soluble TCRs are generally fragments or constructs comprising TCR α chains and / or β chains or their variable regions or CDRs, and optionally they are stabilized by disulfide bonds or covalently linked by suitable linkers. Typically, soluble TCRs do not include, for example, a transmembrane region.

[0070] The TCR of the present invention is provided in the form of a membrane-bound TCR. Typically, a membrane-bound TCR includes a transmembrane region to anchor it to the cell membrane.

[0071] In some embodiments, the TCR further comprises a TCR constant region or a fragment thereof.

[0072] As used herein, the term "constant region" may be a human constant region or derived from another species, thereby creating a "chimeric" TCR. For example, the human α and / or β chains may be replaced ("murinization") with their murine counterparts, which have been found to enhance surface expression of the human TCR by supporting preferential pairing of the TCR α and β chains and to enhance more stable binding to the CD3 co-receptor.

[0073] In some embodiments, the TCR constant region is a murine constant region or a human constant region.

[0074] It is reported that adding a disulfide bond in the constant region can promote the correct pairing of TCR α and β chains (Kuball J et al. Blood. 2007 Mar 15; 109(6): 2331-8). Therefore, in some embodiments of the present invention, one or more cysteine ​​modifications are added to the constant region to form a disulfide bond between the TCR α chain and the TCR β chain.

[0075] In some embodiments, the TCR constant region includes a TCR α chain constant region and / or a TCR β chain constant region; preferably, the TCR α chain constant region and / or the TCR β chain constant region contains at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TCR α chain and the TCR β chain.

[0076] In some embodiments, the cysteine ​​mutation is at one or more of the following positions: position 48 of the wild-type human TCR α chain constant region, position 48 of the wild-type mouse TCR α chain constant region, position 57 of the wild-type human TCR β chain constant region, and position 57 of the wild-type mouse TCR β chain constant region.

[0077] The sequence of the wild-type TCR constant region can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the TCR molecule α chain is "TRAC*01", and the constant domain sequence of the TCR molecule β chain is "TRBC1*01" or "TRBC2*01".

[0078] To facilitate the description of the position of the cysteine ​​mutation, the position of the amino acid sequence of the wild-type TCR constant region in the present invention is numbered according to the naming rules of the International Immunogenetics Information System (IMGT). For example, an amino acid in the TCR α chain constant region (TRAC) is numbered 48 in the position listed in IMGT, and it is described herein as the 48th amino acid in the TCR α chain constant region (TRAC); an amino acid in the TCR β chain constant region (TRBC) is numbered 57 in the position listed in IMGT, and it is described herein as the 57th amino acid in the TCR β chain constant region (TRBC), and so on. Herein, the position numbering of the amino acid sequence of the variable region TRAV and TRBV follows the position numbering listed in IMGT. For example, an amino acid in TRAV is numbered 46 in the position listed in IMGT, and it is described herein as the 46th amino acid in TRAV, and so on. In the present invention, if the sequence position numbering of other amino acids is specially specified, it is in accordance with the special instructions.

[0079] In some embodiments, the TCR α chain constant region further comprises an LVL mutation or a LIV mutation, such that the constant region (and / or the transmembrane region) comprises the amino acid sequence LLVIVLRIL (SEQ ID NO: 30). For example, when the TCR α chain comprises a human constant region, the human constant region may comprise an LVL mutation, such that the constant region (and / or the transmembrane region) comprises the amino acid sequence SEQ ID NO: 30. When the TCR α chain comprises a mouse constant region, the mouse constant region may comprise an LIV mutation, such that the constant region (and / or the transmembrane region) comprises the amino acid sequence SEQ ID NO: 30.

[0080] In some embodiments, the TCR α chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the TCR β chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 13. In some embodiments, the TCR α chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 12, and the TCR β chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 13.

[0081] In some embodiments, the fragment of the TCR constant region is an extracellular segment of the TCR constant region.

[0082] In some embodiments, the TCR is a TCR comprising an α chain and a β chain. In some cases, the TCR α chain and / or the β chain may comprise a leader sequence. For example, the leader sequence of the TCR α chain may have an amino acid sequence as shown in SEQ ID NO: 23. The leader sequence of the TCR β chain may have an amino acid sequence as shown in SEQ ID NO: 24.

[0083] In some embodiments, the TCR further comprises a transmembrane region and / or a cytoplasmic region.

[0084] In some embodiments, the TCR comprises a TCR alpha chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 19-21.

[0085] In some embodiments, the TCR comprises a TCR beta chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO:18.

[0086] In some embodiments, the TCR comprises a TCR alpha chain having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NOs: 19-21, and a TCR beta chain having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 18.

[0087] In some embodiments, the TCR comprises a TCR alpha chain having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 19, and a TCR beta chain having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the TCR comprises a TCR alpha chain having the amino acid sequence set forth in SEQ ID NO: 19, and a TCR beta chain having the amino acid sequence set forth in SEQ ID NO: 18.

[0088] In some embodiments, the TCR comprises a TCR alpha chain having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 20, and a TCR beta chain having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the TCR comprises a TCR alpha chain having the amino acid sequence set forth in SEQ ID NO: 19, and a TCR beta chain having the amino acid sequence set forth in SEQ ID NO: 18.

[0089] In some embodiments, the TCR comprises a TCR alpha chain having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 21, and a TCR beta chain having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the TCR comprises a TCR alpha chain having the amino acid sequence set forth in SEQ ID NO: 19, and a TCR beta chain having the amino acid sequence set forth in SEQ ID NO: 18.

[0090] In some embodiments, the TCR further comprises an intracellular signaling region. In some embodiments, the TCR further comprises one or more antigen binding regions that bind to other antigens or epitopes.

[0091] In some embodiments, the TCR is isolated or purified.

[0092] As used herein, the term "isolated or purified" means that it has been identified, separated and / or recovered from the components of its production environment, such that the "isolated or purified" TCR is free or substantially free of other contaminant components from its production environment that may interfere with its therapeutic or diagnostic use. Contaminant components may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Thus, an "isolated or purified" TCR can be prepared by at least one purification step that removes or substantially removes these contaminant components.

[0093] Nucleic Acids

[0094] The present invention provides a nucleic acid encoding the TCR or a fragment thereof provided by the present invention.

[0095] It is well known in the art that among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are mostly more than one codon to determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acid, so the nucleotide sequence of the gene encoding the same protein can be different. According to the known codon table, those skilled in the art can completely deduce the nucleotide sequence of the gene that can encode them from the amino acid sequence disclosed in the present invention, and obtain the nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method, so this part of the nucleotide sequence should be included in the scope of the present invention. On the contrary, the DNA sequences disclosed in this article can also be used to modify the nucleic acid sequences provided by the present invention by methods known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989) to obtain an amino acid sequence consistent with the activity of the TCR or its fragment described in the present invention.

[0096] In some embodiments, the nucleic acid encoding the parental TCR or fragment thereof comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:25, encoding the TCR α chain variable region, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:26, encoding the TCR β chain variable region.

[0097] Therefore, those skilled in the art can easily obtain the nucleotide sequences encoding the TCR α chain variable region and the TCR β chain variable region with improved affinity of the present invention based on the nucleotide sequences encoding the TCR α chain variable region and the TCR β chain variable region provided above, and the amino acid sequence of the TCR with improved affinity provided by the present invention according to methods known in the art.

[0098] In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a TCR α chain constant region and / or encoding a TCR β chain constant region. In some embodiments, the nucleic acid encoding the TCR α chain constant region has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with SEQ ID NO: 27. In some embodiments, the nucleic acid encoding the TCR β chain constant region has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with SEQ ID NO: 28.

[0099] Carrier

[0100] The present invention provides a vector comprising the nucleic acid provided by the present invention.

[0101] The term "vector" as used herein is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule as a vector can, for example, be replicated and / or expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors and adenovirus-associated vectors (AAV)), bacteriophages, phagemids, cosmids and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton". Engineered vectors are typically included in the starting point of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally include a promoter, a genetic marker, a reporter gene, a targeting sequence and / or a protein purification tag. As known to those skilled in the art, a large number of suitable vectors are known to those skilled in the art, and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the vector is preferably selected from the group consisting of a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0103] cell

[0104] The present invention provides a cell comprising the TCR, nucleic acid, or vector provided by the present invention.

[0105] The term "cell" as used herein refers to any type of cell capable of expressing the TCR of the present invention. The cell can be a eukaryotic cell, for example, a plant (without the potential to develop into a plant), an animal, a fungus or an algae, or can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human. The cell can be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of producing the TCR of the present invention, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

[0106] In some embodiments, the cell is selected from lymphocytes (e.g., T cells, NK cells), monocytes (e.g., PBMCs), and stem cells. The term "stem cell" as used herein is a stem cell for expressing the TCR (particularly TCR) of the present invention. For example, a stem cell may be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC), or a hematopoietic stem cell (HSC). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos, and / or do not include omnipotent stem cells for development and formation of animal individuals. Gene transfer to stem cells generally does not result in expression of TCR on the cell surface because CD3 molecules are not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate expression of the introduced TCR molecules on the surface of thymocytes.

[0107] In some embodiments, the stem cell is a lymphoid progenitor cell or an induced pluripotent stem cell (iPSC).

[0108] In some embodiments, the cell is a T cell. The T cell can be any T cell, such as a cultured T cell, such as a primary T cell or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or liquids. The T cell can also be enriched or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell separated from a person. The T cell can be any type of T cell and can be a T cell at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD4+T cells, CD8+T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. In some embodiments, the T cell does not express endogenous TCR.

[0109] In some embodiments, the method of preparing the cells of the present invention comprises the step of transducing or transfecting the cells with the vector of the present invention.

[0110] As used herein, the term "transfection" is a process in which nucleic acid molecules or polynucleotides (including vectors) are intentionally introduced into target cells. An example is RNA transfection, which is the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into host cells. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides. The transfection of animal cells generally involves opening a transient hole or "hole" in the cell membrane to allow the uptake of materials. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0111] In some embodiments, the method further comprises the step of expanding and / or activating the cells either before or after the transduction or transfection.

[0112] use

[0113] The present invention provides use of the T cell receptor or a fragment thereof, nucleic acid, or vector provided by the present invention in preparing a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12V-positive disease or condition.

[0114] In some embodiments, the product is a drug, which comprises the T cell receptor or fragment thereof of the present invention, the nucleic acid of the present invention, or the vector of the present invention.

[0115] In some embodiments, the drug is in the form of a conjugate, which comprises a TCR of the present invention and an active agent coupled or conjugated to the TCR.

[0116] In some embodiments, the active agent is selected from detectable markers, immunostimulatory molecules and therapeutic agents. Preferably, detectable markers are selected from biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules. Preferably, immunostimulatory molecules are selected from cytokines (such as IL-2 and IFN-γ), chemokines (such as IL-8), platelet factors (such as platelet factor 4) and complement initiators. Preferably, therapeutic agents are selected from immunomodulators, radioactive compounds, enzymes, chemotherapeutic agents and toxins. Other suitable therapeutic agents include small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells with a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e. compounds that decay or are converted to release cytotoxic agents under physiological conditions. Examples of such agents include cisplatin, maytansine derivatives, rachlombycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, pyroximate sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronide, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells; for example, ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA e and RNase; radionuclides, i.e. unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying, for example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225 and astatine-213; chelating agents, which can be used to promote the binding of these radionuclides to molecules or their polymers; or heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0117] In some embodiments, the drug is in the form of a composition, which comprises the TCR, nucleic acid, vector, or cell of the present invention. Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0118] The term "composition" refers in particular to a composition suitable for administration to humans. However, the term also generally encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e., active agent and optional carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutically acceptable composition of the present invention may be, for example, sterile. Specifically, the term "pharmaceutically acceptable" may mean approved by a regulatory agency or other recognized pharmacopoeia for use in animals, more particularly in humans.

[0119] The term "excipient" includes fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. It is known to those skilled in the art to select suitable excipients to prepare the compositions of the present invention. Exemplary carriers for the compositions of the present invention include saline, buffered saline, glucose and water. Typically, the selection of suitable excipients depends particularly on the desired dosage form of the active agent used, the disease to be treated and the compositions.

[0120] Depending on the active agent used (e.g., soluble TCR), the composition of the invention can be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. Known processes for producing drugs are shown in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes.

[0121] Compositions of host cells or soluble TCRs as described herein are typically provided in liquid form, and preferably contain a pharmaceutically acceptable buffer.

[0122] In some embodiments, the composition of the present invention further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0123] Preferred examples of the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, E), vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet factor 4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides.

[0124] In some embodiments, the product is a kit comprising the TCR or conjugate of the present invention, and the kit is used to detect the presence of a positive epitope in a sample to be tested, wherein the epitope comprises a KRAS G12V mutation epitope.

[0125] In some embodiments, the kit is a kit for detecting (eg, diagnosing) a KRAS G12V mutation-positive disease and / or condition in a subject, comprising a TCR or conjugate of the present invention.

[0126] In some embodiments, the conjugate comprises a detectable label. Examples of detectable labels include, but are not limited to, biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule; preferably an enzyme or a catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent or chemiluminescent molecule. In some embodiments, the kit may further comprise instructions for how to use the kit.

[0127] In an embodiment of the present invention, the disease and / or condition may include a KRAS G12V mutation-positive disease and / or condition. In some embodiments, the KRAS G12V mutation may be an epitope of the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the KRAS G12V mutation-positive disease may include a tumor. In some embodiments, the tumor may include a solid tumor and / or a hematological tumor. In some embodiments, the tumor includes: at least one of colorectal cancer, pancreatic cancer, lung adenocarcinoma, and endometrial cancer.

[0128] The present invention is further described by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are carried out according to the conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0129] Example 1 This example is used to illustrate the screening of TCRs with enhanced affinity

[0130] The inventors used the human KVA11-N01 sequence (CN116063511B) as a template, and used an in vitro substitution method to perform single-site substitutions on the complementary determining region (CDR) of KVA11-N01, and obtained substitution clones with improved affinity by in vitro positive screening of the substituted TCR. Through this method, the preferred TCR screened was a clone with an amino acid substitution in V106 or I117 of the CDR 3α complementary determining region, and the specific amino acid substitutions included: V106A, V106S, V106T, I117T or I117R.

[0131] The above mutations were further combined to screen for mutant TCRs with additive affinity. Representative substituted TCRs are shown in Table 2 below.

[0132] In order to increase the correct pairing and expression of exogenous TCR on the surface of human T cells, the variable regions of the α and β chains of TCR were fused to the mouse TCR constant region, and the classic α chain C region "LIV" substitution was introduced into the mouse TCR constant region (i.e., amino acid substitutions were introduced into the mouse TCR α chain constant region so that the constant region contained the amino acid sequence L L V IVLRIL; in bold) and the classical α chain T48C and β chain S57C. Specifically, the amino acid sequence of the α chain constant region is shown in SEQ ID NO:25, and the encoding nucleic acid sequence is shown in SEQ ID NO:27; the amino acid sequence of the β chain constant region is shown in SEQ ID NO:26, and the encoding nucleic acid sequence is shown in SEQ ID NO:28.

[0133] Through the 2A self-cleavage peptide SEQ ID NO: 29, the α chain and β chain of TCR (the N-termini of the α chain and β chain contain signal peptide sequences SEQ ID NO: 23 and SEQ ID NO: 24, respectively) are constructed in series into a lentiviral expression vector in the order of β-P2A-α.

[0134] In order to determine the degree of improvement of the affinity of the TCR provided by the present invention compared with the parent TCR, the mutant TCR sequence was cloned into the TCR tandem expression lentiviral vector, the lentiviral transduction reporter cell JKR9 was packaged, and the TCR expression positive cells were sorted, and then the target cell T2 was loaded with different concentrations of KRAS-G12V polypeptide (HLA-A1101 affinity peptide: VVGAVGVGK) and co-incubated with the transduced natural TCR sequence KVA11-N01 and the mutant TCR sequence for activation. After 16 hours of activation, the reporter gene was stained and flow cytometry was performed to analyze the activation positive ratio of different mutant clones under the same conditions, and the half activation concentration EC50 of different mutant TCRs was calculated, and the EC50 of KVA11-N01 was used as the benchmark to calculate the EC50 enhancement multiple, and the results are shown in Table 2. Figure 1 and Figure 2 The activation results of the selected high-affinity TCRs on reporter cells are shown.

[0135] Table 2 Representative replacement TCRs

[0136]

[0137] It can be seen from Table 2 that the activation function of the TCR provided by the present invention is stronger than that of the wild type compared with the parent TCR; among them, M1001, M1002, and M1010 are significantly activated at -8M.

[0138] Example 2 This example is used to illustrate the specific function verification of mutant TCR

[0139] In order to preliminarily verify whether the mutant TCR retains the specific recognition function of the target, this example expresses the high-affinity mutant TCR on the reporter cell JKR9 to verify whether it has a non-specific activation reaction to the target cells with HLA typing matching and negative antigen expression. The off-target reaction and ALLO reaction show that all high-affinity mutant TCRs retain the specificity of target antigen recognition. The verification process is briefly described as follows:

[0140] a) First, screen a variety of cell lines derived from different tissues, which can express the vast majority of human genes.

[0141] b) Combine RNAseq, RT-PCR and other methods to detect and clarify the expression level of target antigens in different cell lines.

[0142] c) Expressing a restricted HLA (here HLA-A0201) that can be acted upon by the mutant TCR into the selected cell line.

[0143] d) Finally, JKR9 expressing high-affinity mutant TCR is reacted with antigen-negative target cells (HLA matching) from different tissue sources, and mutant TCRs with non-specific reactions are excluded based on the reaction results.

[0144] Example 3. This example is used to illustrate the efficient and specific killing of antigen-positive tumor cells by affinity-optimized TCR-T cells

[0145] TCR-T cells expressing KVA11-N01, KVA11-M01-002, KVA11-M01-231, KVA11-M01-254 or TK34 were used as effector cells, and PBMCs without TCR transduction were expanded and cultured in parallel as effector cell controls; SW480-B2M-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive), Hela-A1101-G12V (KRASG12V positive, HLA-A*11:01 positive), and PANC-1-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive) were used as HLA-antigen peptide-matched positive target cells; HUCCT-1 (KRAS G12V negative, HLA-A*11:01 positive) as HLA-antigen peptide mismatch negative target cells; all target cells stably express Luciferase gene. Effector cells and different target cells are incubated for 16 hours according to the effector-target ratio (E:T) ratio shown in the figure, and Luciferase substrate is added to detect the surviving target cells, and the proportion of target cells killed is calculated based on the remaining target cells.

[0146] The results showed that both TK34 and KVA11-N01 had clear killing activity, but it was clearly observed that KVA11-N01 had stronger killing ability against adherent positive target cells SW480, Hela and PANC-1 compared with TK34; the killing activity of the optimized TCR was even stronger than that of KVA11-N01, and was positively correlated with affinity within a certain range, while there was no obvious killing activity against the negative target cell HUCCT-1 ( Figure 3), indicating that the affinity-enhanced TCR optimized based on KVA11-N01 has better killing activity and specificity.

[0147] Example 4 This example is used to illustrate the specific INF-γ secretion of affinity-optimized TCR-T cells against antigen-positive tumor cells

[0148] TCR-T cells expressing KVA11-N01, KVA11-M01-002, KVA11-M01-231, KVA11-M01-254 or TK34 were used as effector cells, and PBMCs without TCR transduction were expanded and cultured in parallel as effector cell controls; SW480-B2M-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive), Hela-A1101-G12V (KRASG12V positive, HLA-A*11:01 positive), and PANC-1-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive) were used as HLA-antigen peptide-matched positive target cells; HUCCT-1 (KRAS G12V negative, HLA-A*11:01 positive) were used as HLA-antigen peptide mismatched negative target cells. The number of TCR-T positive effector cells was 1E5, and they were incubated with different target cells at a 1:1 effector-target ratio for 20 hours. The IFN-γ secretion in the supernatant was detected using IFN-γ ELISA Kits (ExCell Bio, Cat# EH008-96).

[0149] The results showed that each TCR-T could be activated by adherent cells Hela-A1101-G12V, PANC-1-A11N04-G12V and SW480-A1101-B2M-TAP-G12V to release IFN-γ; all TCR-T could not be activated by antigen-negative adherent cells HUCCT-1. Compared with TK34, the IFN-γ secretion of KVA11-N01 was significantly increased, and the secretion of cytokines by TCR-T after affinity optimization was also significantly higher than that of KVA11-N01, and the response to KRAS G12V-positive cell lines was stronger ( Figure 4 ), the above results indicate that as the affinity increases, the secretion of cytokines increases.

[0150] Example 5 This example is used to illustrate that the affinity-enhanced TCR does not have non-specific activation for T2KO-TAP1 cell lines expressing different HLA typing

[0151] To detect whether the TCR based on KVA11-N01 affinity optimization can produce alloreactivity, that is, antigen-independent cross-reaction with other mismatched HLA, cell lines expressing different HLAs were constructed by T2KO-TAP1 (HLA knockout and TAP1 expression), and the selected high-frequency HLA-A, HLA-B, and HLA-C can cover more than 90% of the population. After T2KO-TAP1 cells were transduced with different HLA genes and sorted by flow cytometry, the HLA expression rate was higher than 95%. 1E5 TCR-T cells expressing KVA11-N01 were used as effector cells and co-cultured with 1E5 target cells for 16-24 hours. The supernatant was collected for IFN-γ detection, and T2-A1101 cells loaded with 10^-6M KRAS G12V 8~16 peptide were used as positive controls of the experiment.

[0152] The results showed that target cells did not significantly stimulate the secretion of IFN-γ by KVA11-N01, KVA11-M01-002, KVA11-M01-231, and KVA11-M01-254 ( Figure 5 ), indicating that the TCR optimized based on KVA11-N01 as a template has no allogeneic reaction to HLA of different types, indicating that no allogeneic reaction will occur.

[0153] Example 6 This example is used to illustrate that the affinity-enhanced TCR does not produce non-specific activation on cells from different tissues.

[0154] In order to test the safety of TCR based on KVA11-N01 affinity optimization, tumor cell lines from different human tissues were first selected to transduce the HLA-A*11:01 gene and sorted by flow cytometry to make the HLA-A*11:01 expression rate higher than 90%. They were used as non-specific antigen presenting cells to detect whether KVA11-N01 could respond to non-specific antigens. TCR-T cells expressing KVA11-N01 were used as effector cells, and PBMCs without TCR transduction were expanded and cultured in parallel as effector cell controls; Hela-G12V-1101 (overexpressing KRAS G12V and HLA-A*11:01) was used as positive target cells; tumor cell lines transduced with HLA-A*11:01 were used as target cells; the number of positive effector cells was 1E5 and incubated with different target cells at a 1:1 effector-target ratio for 24 hours, and the supernatant IFN-γ secretion was detected using IFN-γ ELISA Kits (ExCell Bio, Cat# EH008-96).

[0155] The results showed that the TCR-T based on KVA11-N01 affinity optimization did not secrete IFN-γ to all non-specific antigen presenting target cells more than the control PBMC, indicating that KVA11 did not have a non-specific activation response to HLA-A*11:01-positive tumor cell lines from different tissues, showing excellent antigen specificity ( Figure 6 ).

[0156] Example 7 This example is used to illustrate the study of non-specific reactions of TCR target antigen peptides with improved affinity after affinity optimization

[0157] By using a mutant peptide library to load antigen presenting cells and stimulate TCR-T cells to secrete interferon / activate reporter cell lines, the peptide reaction fingerprint characteristics of candidate TCRs are summarized. Based on the summary results, query the peptides that may produce reactions. Determine the ability of peptides to bind to MHC based on the software; detect the ability of synthetic peptides to activate TCR-T cells; construct gene expression vectors, transduce antigen presenting cells, and detect the ability to be presented; query the expression of antigens in different tissues. Based on the results, obtain the non-specific reaction characteristics of TCR-T.

[0158] In order to more comprehensively detect the non-specific reaction of TCR (taking KVA11-M01-231 as an example) after affinity optimization of KVA11-N01, we performed single-site mutation on its target antigen peptide KARS G12V 8~16, VVGAVGVGK. Mutation principle: While keeping other amino acids in the target polypeptide unchanged, the amino acid at the mutation site is mutated into 19 other amino acids in sequence, and finally a 19×9 (number of polypeptide amino acids) mutant polypeptide is obtained. The polypeptide position is marked as P1, P2, P3, etc. starting from the N-terminus. Taking the G12V 8-16 peptide VVGAVGVGK as an example, the P1 amino acid V mutated to G, A, L, I, S, P, F, M, W, Q, T, C, N, Y, D, E, K, R, H, and 19 P1 mutant peptides were obtained, grouped and named KV1-P1X (here X refers to the mutated amino acids as a whole, and has no other meaning), the sequence is XVGAVGVGK (here X refers to the mutated amino acids as a whole, used to refer to any mutated amino acids), and the individual peptides are named KV1-P1G, KV1-P1A, etc. Adaptimmune uses 90% pure peptides, and the conventional purity we ordered from GenScript is 95%, in order to fully judge the specificity of TCR. A total of 191 mutant peptides were tested, and T2 cells were loaded with 10 -7M mutant peptides were co-incubated with 1E5 TCR-T cells at a ratio of 1:1 overnight, and IFN-γ ELISA Kits were used to detect the release of IFN-γ. The reactivity of the peptides after single-site mutations was summarized, and the mutations that could not stimulate TCR-T cells to produce IFN-γ were considered key amino acids, and the amino acids at other positions were considered non-key amino acids ( Figure 7 Figure A in the figure).

[0159] Summary of positive amino acid results for each site: P1[AVIQSCGMRKLT]-P2[VITAQLSM]-P3[G]-P4[ASCGL]-P5[HVMIQTA]-P6[GN]-P7[VSWFKYEHAILM]-P8[AGQHRSMENCFTL]-P9[K]( Figure 7 Figure B in the figure). After searching the human protein database https: / / myhits.sib.swiss / cgi-bin / pattern_search for amino acid patterns, 0 potential positive peptides were retrieved. The more comprehensive evaluation results of X-Scan clearly indicate that the TCR KVA11-M01-231 based on KVA11-N01 affinity optimization will not produce non-specific reactions with peptides in the human body, and has good specificity.

[0160] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications. Including the combination of various specific technical features in any suitable manner. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A TCR with improved affinity, characterized in that: The affinity-enhanced TCR comprises an α chain variable region comprising CDR1α, CDR2α and CDR3α and a β chain variable region comprising CDR1β, CDR2β and CDR3β; wherein the CDR3α of the affinity-enhanced TCR has an amino acid substitution at the V106 and / or I117 position relative to the CDR3α of the parent TCR; wherein the amino acid substitution at position V106 is selected from V106A, V106S or V106T; The amino acid substitution at position I117 is selected from I117T or I117R; Wherein, the parent TCR comprises CDR1α with an amino acid sequence as shown in SEQ ID NO: 1, CDR2α with an amino acid sequence as shown in SEQ ID NO: 2, CDR3α with an amino acid sequence as shown in SEQ ID NO: 3, CDR1β with an amino acid sequence as shown in SEQ ID NO: 4, CDR2β with an amino acid sequence as shown in SEQ ID NO: 5, and CDR3β with an amino acid sequence as shown in SEQ ID NO: 6; Wherein, the affinity-enhanced TCR can specifically recognize the KRAS G12V epitope or a complex of the epitope and the MHC molecule; The amino acid positions are numbered according to the nomenclature of the International Immunogenetics Information System (IMGT).

2. The TCR according to claim 1, wherein: The amino acid substitutions present in the CDR3α of the TCR with improved affinity relative to the CDR3α of the parent TCR are selected from V106A, V106TI117T and V106SI117R.

3. The TCR according to claim 2, wherein: The amino acid sequence of the α chain variable region is shown in any one of SEQ ID NOs: 14-16; and the amino acid sequence of the β chain variable region is shown in SEQ ID NO:

11.

4. The TCR according to any one of claims 1 to 3, wherein: The TCR further comprises a TCR constant region, which is a mouse constant region or a human constant region.

5. The TCR according to claim 4, wherein: The TCR constant region includes a TCR α chain constant region and a TCR β chain constant region; wherein the TCR α chain constant region and / or the TCR β chain constant region comprises at least one cysteine ​​substitution relative to the wild-type sequence to form a disulfide bond between the TCR α chain and the TCR β chain; and / or The TCR α chain constant region comprises an LVL substitution or a LIV substitution, such that the α chain constant region comprises the amino acid sequence LLVIVLRIL.

6. The TCR according to claim 4, wherein: The amino acid sequence of the TCR α chain is shown in any one of SEQ ID NOs: 19-21; and the amino acid sequence of the TCR α chain is shown in SEQ ID NO:

18.

7. A nucleic acid encoding a TCR according to any one of claims 1 to 6. A vector comprising the nucleic acid according to claim 7 .

9. A cell comprising a TCR according to any one of claims 1 to 6, a nucleic acid according to claim 7, or a vector according to claim 8.

10. Use of a TCR according to any one of claims 1 to 6, a nucleic acid according to claim 7, or a vector according to claim 8 in the preparation of a product for alleviating or treating a disease or condition that is KRAS G12V positive and HLA-A*11 positive; in, The disease or condition is selected from colon cancer, cervical cancer and pancreatic cancer.

Images