PD-1 variants and their uses

Abstract

High affinity PD-1 variants and uses thereof are provided. [Solution] The PD-1 variants of the present invention have high affinity for PD-L1 and do not bind to one or more anti-PD-1 monoclonal antibodies, preferably at least two anti-PD-1 monoclonal antibodies. The present invention also relates to fusion proteins comprising the PD-1 variants. The present invention further relates to nucleic acid molecules encoding the PD-1 variants and fusion proteins, and uses thereof.

Description

[Technical Field]

[0001] This application relates to the field of immunotherapy. Specifically, this application provides PD-1 variants. This application also provides nucleic acid molecules encoding the PD-1 variants, fusion proteins or compositions comprising the PD-1 variants, and uses of the PD-1 variants. [Background technology]

[0002] Programmed death-1 (PD-1) is a protein expressed primarily on the surface of activated lymphocytes. PD-1 is an immune checkpoint protein that suppresses immune responses. Therefore, inhibiting PD-1 can release the suppression of the immune system and attack tumors. Therefore, PD-1 has become a popular immunotherapy target in recent years. Various antibodies against PD-1 have been approved and are commercially available.

[0003] PD-1 binds to its ligand, PD-L1, to generate immunosuppressive signals. Tumor cells express PD-L1 and achieve immune evasion via the PD-1 / PD-L1 pathway. Blocking the interaction between PD-1 and PD-L1 can achieve PD-1 inhibition. To achieve this, the development of PD-1 analogs capable of binding to PD-L1 is being investigated. Furthermore, these PD-1 analogs with binding specificity to PD-L1 can also be used to identify and bind tumor cells.

[0004] For example, various PD-1 variant sequences are described in Stanford University's PCT Patent Publication WO2016 / 023001. These variants have higher affinity for the ligand PD-L1 compared to human wild-type PD-1, and at the same time, lack the transmembrane domain. However, all of the variants in this application have multiple amino acid differences compared to the wild-type. The application does not mention the affinity of the PD-1 variants with anti-PD-1 antibodies.

[0005] When PD-1 variants are used therapeutically, they may be combined with anti-PD-1 antibodies. In such cases, if the anti-PD-1 antibodies can recognize and bind to PD-1 variants, the two therapeutic pathways may interfere with each other, reducing their efficacy.

[0006] Therefore, further improvements in PD-1 polypeptide variants are needed in this field to accommodate a wider range of applications. Summary of the Invention

[0007] To solve the above problems, the present inventors constructed a library of PD-1 mutant plasmids and sorted them using flow cytometry to select a series of PD-1 variants. These PD-1 variants retain high affinity for their ligand, PD-L1, while not binding to multiple commercially available anti-PD-1 monoclonal antibodies, thereby completing the present invention.

[0008] Accordingly, a first aspect of the present application provides PD-1 variants that have a different amino acid sequence from wild-type human PD-1 polypeptide, and that bind to PD-L1 with high affinity (e.g., higher affinity than wild-type human PD-1 polypeptide), while simultaneously exhibiting substantial resistance to binding to multiple anti-PD-1 monoclonal antibodies.

[0009] A second aspect of the present application provides a fusion protein comprising the PD-1 variant of the first aspect. In one embodiment, the fusion protein is a transmembrane fusion protein having the PD-1 variant as an extracellular segment. In a further embodiment, the fusion protein is a fusion protein comprising an immunoglobulin Fc.

[0010] A third aspect of the present application provides a nucleic acid molecule encoding a PD-1 variant of the first aspect or a fusion protein of the second aspect.

[0011] A fourth aspect of the present application provides an expression vector for the nucleic acid molecule of the third aspect.

[0012] A fifth aspect of the present application provides a host cell comprising the nucleic acid molecule of the third aspect or the expression vector of the fourth aspect.

[0013] A sixth aspect of the present application provides a pharmaceutical composition comprising a PD-1 variant of the first aspect or a fusion protein of the second aspect.

[0014] A seventh aspect of the present application provides the use of a PD-1 variant of the first aspect or a fusion protein of the second aspect for the manufacture of a medicament. In a particular embodiment, the medicament is for the treatment of cancer.

[0015] An eighth aspect of the present application provides a drug combination comprising (1) a PD-1 variant of the first aspect or a fusion protein of the second aspect, and (2) an anti-PD-1 antibody. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows the structure of the plasmid vector used to construct the PD-1 variant library in Example 1. [Figure 2] FIG. 10 shows a schematic diagram of cells that bind to PD-L1 but do not bind to anti-PD-1 antibodies, sorted by flow cytometry in Example 3. [Figure 3] Figure 1 shows the results of SDS-PAGE of wild-type PD-1 or PD-1 variant / rabbit IgG Fc fusion proteins expressed in Example 5. (A) Non-reducing conditions, (B) reducing conditions. Lane 1 is wild-type PD-1, lanes 2-7 are PD-1 variants represented by SEQ ID Nos: 2-7, and lane 8 is a molecular weight marker. [Figure 4] FIG. 10 shows the inhibitory effect of PD-1 variant-Fc fusion protein on IL-2 release by T cells measured in Example 7. [Figure 5]This figure shows that when T cells are armed with the PD-1-CD28-enhanced receptor (108-CD28) constructed using the PD-1 variant in Example 8, these T cells are stimulated by target cells expressing PD-L1 and can secrete the cytokine IL-2. [Figure 6] FIG. 10 shows that the PD-1-CD28-enhanced receptor (108-CD28) constructed using a PD-1 variant in Example 9 increases T cell killing of the corresponding target cells. DETAILED DESCRIPTION OF THE INVENTION

[0017] definition Unless otherwise indicated, the practice of certain methods disclosed herein will rely on conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA technology, all of which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th ed. (2012).

[0018] The term "about" or "approximately" means within an acceptable error range for a particular value, which can be understood by one of ordinary skill in the art. In some cases, the acceptable error range may depend on the limitations of the measurement method or measurement system. For example, following common practice in the art, "about" may refer to a range of one or more standard deviations. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly in the context of biological systems or processes, the term may refer to within an order of magnitude of a value, preferably within 5-fold of a value, and more preferably within 2-fold of a value. In this specification and claims, when a specific value is described, the term "about" should be understood to mean within an acceptable error range for the particular value, unless otherwise specified.

[0019] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may also include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribose nucleic acid (RNA)). The term nucleotide may include adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP); and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, and dTTP; or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, or 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Representative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled using well-known techniques. Labeling may also be performed using quantum dots. Detectable labels may include radioisotopes, fluorescent markers, chemiluminescent markers, bioluminescent markers, enzyme markers, etc.

[0020] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, which may be single-stranded, double-stranded, or multiple-stranded deoxyribonucleotides or ribose nucleotides, or analogs thereof. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene, or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. A polynucleotide may contain one or more analogs (e.g., altered backbones, sugars, or nucleobases). Some non-limiting examples of such analogs include 5-bromidouracil, peptide nucleic acids, heterologous nucleic acids, morpholinos, locked nucleotides, glycol nucleic acids, threo-nucleotides, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine, sugar-linked fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouracil, pseudouridine, dihydrouracil, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, a locus defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cellular-free DNA (cfDNA) and cellular-free RNA (cfRNA)), nucleic acid probes and primers.The sequence of nucleotides may be interrupted by non-nucleotide components.

[0021] The term "expression" refers to the process or processes by which a polynucleotide from a DNA template is transcribed (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.

[0022] The terms "peptide," "polypeptide," and "protein" refer to a polymer of at least two amino acid residues linked by one or more peptide bonds and are used interchangeably herein. The terms are not intended to specify the length of the polymer, nor are they intended to imply or distinguish whether the peptide is prepared by recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The terms apply to naturally occurring amino acid polymers or amino acid polymers having at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length (including full-length proteins) and proteins with or without secondary and / or tertiary structure (e.g., structural domains). The terms also include amino acid polymers formed by, for example, disulfide bonds or amino acid polymers modified by glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation (e.g., conjugation with a labeling component). As used herein, the term "amino acid" generally refers to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids include both natural and unnatural amino acids, which are chemically modified to include non-naturally occurring groups or chemical moieties. Amino acid analogs may also refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0023] The term "variant" or "fragment," when referring to a polypeptide, refers to a polypeptide that is related to a wild-type polypeptide, for example, in amino acid sequence, (e.g., secondary and / or tertiary) structure, activity (e.g., enzymatic activity), and / or function. Polypeptide variants and fragments may contain one or more amino acid changes (e.g., mutations, insertions, deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide.

[0024] The term "fusion protein" refers to a protein produced by joining two or more independent genes or fragments thereof. A fusion protein may contain one or more non-naturally occurring amino acid sequences. A fusion protein may be a chimeric protein. A fusion protein may contain a peptide-affinity tag.

[0025] As used herein, the term "antigen" refers to a molecule or fragment thereof that can bind to a selective binding agent. For example, an antigen can be a ligand that can be bound by a selective binding agent (e.g., a receptor). As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent (e.g., an immunoprotein (e.g., an antibody). Furthermore, an antigen can also refer to a molecule or fragment thereof that can be used in an animal to produce antibodies that can bind to the antigen.

[0026] As used herein, the term "antibody" refers to a protein-binding molecule with immunoglobulin-like functions. The term "antibody" includes antibodies (e.g., monoclonal and polyclonal antibodies) as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Igs) of various classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). An antibody derivative, variant, or fragment may also refer to a functional derivative, variant, or fragment that retains the binding specificity (e.g., fully and / or partially) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibodies, dimerized V regions (diabodies), and single-domain antibodies ("sdAb," or "nanobodies" or "camelids"). The term antibody includes optimized, genetically engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity-matured antibodies. Examples of genetically engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized for fragment crystallizable regions) and multispecific antibodies (e.g., bispecific antibodies).

[0027] Sintilimab is an injectable PD-1 monoclonal antibody developed jointly by Innovent Biologics and Eli Lilly and Company.

[0028] "Nivolumab" is an anti-PD-1 monoclonal antibody jointly developed by Ono Pharmaceutical Co., Ltd. and Medarex Pharmaceuticals, Inc. (the latter acquired by Bristol-Myers Squibb). Nivolumab is sold under the trade name Opdivo and is also known as the "O drug." Opdivo is the world's first approved and most representative anti-PD-1 monoclonal antibody.

[0029] "Tilelizumab" or "Tirelizumab" is an anti-PD-1 antibody developed by BeiGene.

[0030] "Camrelizumab" or "camrelizumab" is an anti-PD-1 antibody developed by Jiangsu Hengrui Co., Ltd.

[0031] "Pemrolizumab" or "pembrolizumab" is an anti-PD-1 antibody developed by Merck Sharp & Dohme. Pembrolizumab is sold under the trade name Keytruda and is also known as the "K drug."

[0032] "Toripalimab" or "Toriparimab" is an anti-PD-1 antibody developed by Junshi Biosciences, and is the first domestically developed anti-PD-1 antibody approved in China.

[0033] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to vertebrates, preferably mammals, particularly humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sport animals, and pets.

[0034] As used herein, the term "treatment" refers to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any treatment-related improvement or effect on one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the compositions can be administered to subjects at risk of developing a particular disease, condition, or symptom, or to subjects who exhibit one or more physiological indicators of a disease (even if the disease, condition, or symptom is not yet apparent).

[0035] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent (e.g., a variant, fusion protein, or composition comprising thereof) sufficient to exert a desired activity when administered to a subject in need thereof. As used herein, a "therapeutically effective amount" refers to an amount of an agent sufficient to delay the onset, inhibit the progression, or ameliorate or alleviate at least one symptom of the condition being treated.

[0036] "Synergistic effect" refers to the effect of two or more drugs being administered simultaneously to produce an additive or synergistic effect, preferably a synergistic effect, meaning that the effect when administered together is greater than the sum of the effects of each drug administered individually.

[0037] Mutation Naming Conventions In the present invention, when mutations contained in PD-1 variants are described, they are all based on the human PD-1 sequence shown in SEQ ID NO:1, unless otherwise specified. In the amino acid sequence of human PD-1 shown in SEQ ID NO:1, amino acids 1-20 are the signal peptide, amino acids 21-170 are the PD-1 extracellular domain, amino acids 171-191 are the transmembrane domain, and amino acids 192-288 are the intracellular domain. According to this numbering system, in human PD-1 shown in SEQ ID NO:1, the extracellular domain is a sequence of 150 amino acids consisting of proline at position 21 to valine at position 170, and the transmembrane domain is a sequence of 21 amino acids consisting of valine at position 171 to isoleucine at position 191.

[0038] In the present invention, when the position of an amino acid mutation is described, the mutated amino acid is indicated by its position in SEQ ID NO:1. When the type of amino acid mutation is described, the amino acid type at the corresponding amino acid position in SEQ ID NO:1 is described as the amino acid before the mutation, before (to the left of) the position number, and the amino acid present in the mutated PD-1 variant is described after (to the right of) the position number. When multiple different amino acids are mutated at the same position, these options are separated by a " / ".

[0039] Based on the above rules, for example, A129S mutation means that alanine (A) at position 129 in SEQ ID NO: 1 is mutated to serine (S), and A129S / H means that alanine (A) at position 129 may be mutated to serine (S) or histidine (H).

[0040] PD-1 variants A "PD-1 variant" of the present invention refers to a polypeptide sequence that differs by one or more amino acids compared to the amino acid sequence of wild-type human PD-1 as set forth in SEQ ID NO: 1. In a specific embodiment, the PD-1 variants of the present invention are variants of truncated fragments of wild-type human PD-1, such as variants of truncated fragments that do not include the intracellular domain, or variants that do not include both the intracellular domain and the transmembrane domain.

[0041] The PD-1 variants of the invention have one or more of the following characteristics compared to wild-type human PD-1: (1) Improved affinity with PD-L1 ligands (2) improved affinity for the PD-L2 ligand; and (3) It does not bind to two or more commercially available anti-PD-1 antibodies.

[0042] As used herein, the phrase "PD-1 variants do not bind to anti-PD-1 antibodies" may refer to PD-1 variants whose binding affinity for a particular anti-PD-1 antibody is below the instrument's detection limit, or to PD-1 variants whose affinity, expressed as a KD value, is above the detection limit. For example, when measuring affinity using a BIACORE T200 instrument, a detectable KD value is 10 -12 M~10 -3 The KD value should be in the range of 10 -3 If it is higher than M, the instrument will not detect the binding curve, which indicates that the PD-1 variant does not bind to the anti-PD-1 antibody in the present invention.

[0043] Because the most important characteristic of the PD-1 variants of the present invention is altered binding affinity, the mutations are primarily concentrated in the extracellular segment. Specifically, the PD-1 variants of the present invention contain mutations at one or more amino acid positions selected from the two regions E84-G90 and A129-Q133 relative to wild-type PD-1 polypeptide. Preferably, at least one mutation is selected from the region consisting of E84-G90, and at least one mutation is selected from the selected region consisting of A129-Q133. Preferably, the PD-1 variants do not contain any mutations within the region consisting of M70-K78 relative to wild-type PD-1 polypeptide, and have the same amino acid residues at the corresponding positions as the wild-type PD-1 polypeptide shown in SEQ ID NO:1.

[0044] In a preferred embodiment, the PD-1 variants of the invention contain one or more amino acid mutations, relative to the wild-type human PD-1 polypeptide set forth in SEQ ID NO:1, selected from the group consisting of: E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I; Q88L, Q88F, Q88N, Q88T; P89C, P89V, P89R; G90T, G90S; A129S, A129G, A129Q, A129Y; P130T; K131P; A132V, A132G; and Q133W.

[0045] The PD-1 variants of the present invention include combinations of mutations contained in variants having the amino acid sequence set forth in any of SEQ ID NOs: 2-26. Specifically, these combinations of mutations include: (1) A129S, P130T, K131P, A132V, and Q133W; (2) E84F, D85L, R86F, S87N, Q88L, A129S, P130T, K131P, A132V and Q133W; (3) E84W, D85M, R86F, S87C, Q88F, A129S, P130T, K131P, A132V and Q133W; (4) R86F, S87R, Q88N, P89C, G90T, A129S, P130T, K131P, A132V and Q133W; (5) R86F, S87F, Q88T, P89V, G90S, A129S, P130T, K131P, A132V and Q133W; (6) S87I, Q88N, P89R, G90S, A129S, P130T, K131P, A132V and Q133W; (7) E84F, D85L, R86F, S87N, Q88L, P130T, K131P and Q133W; (8) R86F, S87R, Q88N, P89C, G90T, A129G, P130T, K131P and Q133W; (9) R86F, S87R, Q88N, P89C, G90T, P130T, K131P, A132G and Q133W; (10) R86F, S87R, Q88N, P89C, G90T, A129Q, P130T, K131P, and Q133W; or (11) R86F, S87F, Q88T, P89V, G90S, A129Y, P130T, K131P and Q133W; (12) A129H, P130F, K131R, A132F and Q133N; (13) A129S, P130E, K131P, A132I and Q133V; (14)S87L, Q88C, P89V, G90F, A129H, P130F, K131R, A132F and Q133N; (15)R86S, S87F, Q88E, P89V, G90R, A129H, P130F, K131R, A132F and Q133N; (16)R86L, S87Y, Q88L, P89K, G90Y, A129S, P130E, K131P, A132I and Q133V; (17) R86F, S87R, Q88N, P89C, G90T, A129W, P130T, K131P, A132M and Q133W; (18)R86F, S87F, Q88T, P89V, G90S, A129H, P130T, K131P, A132F and Q133W; (19) R86F, S87F, Q88T, P89V, G90S, A129G, P130T, K131P, A132F and Q133W; (20)R86F, S87F, Q88T, P89V, G90S, A129I, P130T, K131P, A132G and Q133W; (21)S87I, Q88N, P89R, G90S, A129T, P130T, K131P, A132F and Q133W; (22)S87I, Q88N, P89R, G90S, A129V, P130T, K131P and Q133W; (23)S87I, Q88N, P89R, G90S, P130T, K131P, and Q133W; (24) R86F, S87R, Q88N, P89C, G90T, P130T, K131P and Q133W; (25) R86F, S87F, Q88T, P89V, G90S, P130T, K131P, and Q133W.

[0046] In a preferred embodiment, the PD-1 variants of the invention comprise or consist of the amino acid sequence set forth in any of SEQ ID NOs:2-26. In a preferred embodiment, the PD-1 variants of the invention are variants of the extracellular domain of wild-type human PD-1 set forth in SEQ ID NO:1, wherein the PD-1 variants comprise or consist of the amino acid sequence set forth in any of SEQ ID NOs:2-26; and further, the PD-1 variants do not include the intracellular domain of wild-type PD-1 or do not include both the intracellular domain and the transmembrane domain of wild-type PD-1.

[0047] It is acceptable for the PD-1 variant to contain conservative amino acid substitutions at one or more amino acid positions, depending on the presence or absence of specific mutations and / or combinations of mutations. At the same time, it should be understood that PD-1 variants containing these conservative amino acid substitutions retain the activity and binding specificity desired by the present application. The rules for conservative amino acid substitutions are well known to those skilled in the art. For example, as long as the PD-1 variant contains one of the amino acid combinations (1)-(25) above and has the same amino acids as the wild-type variant within the region consisting of M70-K78, it is acceptable for the variant to have one or more conservative amino acid substitutions relative to wild-type human PD-1 at other positions. For example, the variant may contain 1-20 conservative amino acid substitutions, preferably 1-15 conservative amino acid substitutions, more preferably 1-10 conservative amino acid substitutions, and even more preferably 1-5 conservative amino acid substitutions.

[0048] In another embodiment, the PD-1 variants of the present invention have at least 85% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:2-26, based on the presence or absence of specific mutations and / or combinations of mutations. Preferably, the extracellular domain has at least 90% sequence identity, more preferably at least 95% sequence identity, e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Methods for determining sequence identity between two amino acid sequences are well known to those of skill in the art. For example, amino acid sequences can be aligned using the BLAST program from NCBI, utilizing the BLOSUM62 scoring matrix.

[0049] In the present invention, the terms "extracellular domain" and "extracellular structural domain" are used interchangeably and have the same meaning. These terms are generally used to describe PD-1 variants derived from the extracellular domain of wild-type human PD-1. In any of the above mutation patterns, the PD-1 variants of the present invention may simply be polypeptide fragments corresponding to all or part of the extracellular domain of wild-type PD-1, i.e., "extracellular domain variants." In this case, the PD-1 variants of the present invention can be considered as variant polypeptides that have both the transmembrane and intracellular domains of wild-type PD-1 removed and further contain the mutations. For example, the PD-1 variants may not contain any of the amino acids corresponding to both the intracellular and transmembrane domains of wild-type PD-1. For example, the PD-1 variants may contain a signal peptide present in wild-type PD-1, such as the signal peptide represented by amino acids 1-20 of SEQ ID NO:1. For example, the exemplary PD-1 variants of the present invention are all 155 amino acids in length, corresponding to amino acids 1-155 of wild-type human PD-1 as shown in SEQ ID NO: 1. However, as one of ordinary skill in the art will understand, this does not mean that the PD-1 variants of the present invention can be or are required to be only 155 amino acids in length, nor that they cannot contain a transmembrane domain or an intracellular domain. For example, if it is desired to utilize the binding properties of the PD-1 variants of the present invention and use them in the preparation of fusion proteins, particularly transmembrane proteins, the PD-1 variants of the present invention can still contain more amino acids corresponding to the PD-1 polypeptide. Specifically, the amino acids of the transmembrane domain of wild-type PD-1 can be incorporated as the transmembrane domain of the fusion protein.

[0050] Fusion proteins containing PD-1 variants The unique binding properties of the PD-1 variants of the present invention, particularly the PD-1 extracellular domain variants, give them broad potential applications, for example, the binding properties of the PD-1 variants can be exploited to use the PD-1 variants as part of a fusion protein (e.g., an extracellular binding domain) to target PD-L1 and / or PD-L2 (e.g., to target cells expressing PD-L1 and / or PD-L2).

[0051] In one embodiment, PD-1 variants can be constructed as antibody-like molecules. For example, the PD-1 variants can be fused to the Fc region of an immunoglobulin to form a functional polypeptide that specifically binds to PD-L1, such as an anti-PD-L1 antibody. The Fc region is preferably a mammalian, e.g., human, immunoglobulin Fc region, e.g., human IgG1 Fc region.

[0052] In another embodiment, the fusion protein containing the PD-1 variant is a chimeric stimulating molecule, also known as a potentiating receptor, switch molecule, or switch receptor. The chimeric stimulating molecule comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is a PD-1 variant of the invention, particularly a PD-1 extracellular domain variant (which can bind to PD-L1 and / or PD-L2), and the intracellular domain is an intracellular domain of a costimulatory molecule that mediates the activation of immune cells such as T cells. The costimulatory molecule is selected from the group consisting of interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-correlated antigen 1 (LFA-1), LIGHT, NKG2C, or OX40. In a preferred embodiment, the costimulatory molecule is selected from OX40, 4-1BB, CD28, ICOS, and CD27. In a particularly preferred embodiment, the costimulatory molecule is CD28.

[0053] The transmembrane domain of the fusion protein may be derived from wild-type PD-1 or a variant thereof. For example, it may be the transmembrane domain of wild-type PD-1 or a variant thereof, or a fragment of the transmembrane domain. In one embodiment, the transmembrane domain is derived from wild-type PD-1, for example, comprising amino acids 171-191 of the amino acid sequence set forth in SEQ ID NO:1, or a fragment thereof, or consisting of amino acids 171-191 of the amino acid sequence set forth in SEQ ID NO:1, or a fragment thereof. In one embodiment, the transmembrane domain comprises a sequence having at least 70% identity, at least 80% identity, or 90% identity to amino acids 171-191 of the amino acid sequence set forth in SEQ ID NO:1.

[0054] The transmembrane domain of the fusion protein can also be derived from other sources, such as a transmembrane domain derived from a costimulatory molecule. Immune cells can be modified to express the chimeric stimulating molecule. Immune cells containing the chimeric stimulating molecule generate an immune cell activating signal in the immune cell upon binding to PD-L1, rather than the immune cell inactivating signal normally associated with PD1 / PD-L1 binding. In this way, an immune suppressive signal is converted into an activating signal, thereby activating immune cell function.

[0055] Preparation method The PD-1 variants of the present invention can be prepared by molecular biological techniques.

[0056] For example, the PD-1 variants or fusion polypeptides of the present invention can be prepared by recombinant techniques. Nucleotide fragments, such as DNA or RNA, encoding the PD-1 variants or fusion polypeptides can be synthesized in vitro, constructed into appropriate expression vectors, and expressed in appropriate host cells. For example, the sequences to be expressed can be delivered to host cells by a viral carrier, such as a lentiviral carrier. The desired polypeptide can be obtained by harvesting and, optionally, purifying, the protein expressed by the host cells.

[0057] Methods for purifying polypeptides are known in the art and include, for example, chromatographic techniques such as column chromatography, HPLC, affinity chromatography, size exclusion chromatography, etc. Proteins can also be purified using magnetic beads.

[0058] Furthermore, PD-1 variants or fusion polypeptides can be prepared by cell-free polypeptide synthesis systems. Devices available for cell-free polypeptide synthesis are widely known in the art.

[0059] Purpose As discussed above, the PD-1 variants of the present invention have a variety of potential uses due to their unique binding properties.

[0060] The PD-1 variants of the present invention retain their ability to bind to the ligands PD-L1 and PD-L2, and therefore can be used to substitute for the functions of wild-type human PD-1 or its extracellular domain in a variety of applications, particularly those that utilize the binding function of PD-1 to PD-L1 and PD-L2. Because the PD-1 variants of the present invention have improved ligand-binding ability, they may provide superior results compared to wild-type PD-1.

[0061] In one embodiment, when expressed in immune cells, the chimeric stimulating molecule containing a PD-1 variant of the present invention converts inhibitory PD-1 / PD-L1 signals into stimulatory signals, thereby enhancing the function of immune cells such as T cells. Therefore, the chimeric stimulating molecule can be used in immune cell-based cell therapy and can be applied to modify therapeutic immune cells. Such therapeutic immune cells can be the subject's own immune cells or those derived from a half-identical donor. The immune cells can be derived from the subject's peripheral blood, for example, by selecting peripheral blood mononuclear cells. The immune cells can also be derived from tumor tissue, for example, tumor-infiltrating lymphocytes (TILs). Furthermore, when T cells expressing the chimeric stimulating molecule contact PD-L1-expressing tumor cells, they increase the secretion of interleukin-2 (IL-2), which in turn binds to a receptor on the T cells to further stimulate the T cells.

[0062] In one embodiment, antibody analogs containing the PD-1 variants of the present invention have functions similar to those of PD-1 monoclonal antibodies. For example, fusion proteins formed between the PD-1 variants of the present invention and Fc can inhibit the PD-1 / PD-L1 signaling pathway, thereby suppressing IL-2 release. Therefore, PD-1 variant Fc fusion proteins can play a role in releasing the "immune brake," similar to PD-1 or PD-L1 monoclonal antibodies.

[0063] Furthermore, because the PD-1 variants of the present invention do not bind to several commercially available PD-1 monoclonal antibodies, they can be used in combination with these PD-1 monoclonal antibodies without affecting the efficacy of each, providing the possibility of combination therapy, which is not possible when using wild-type human PD-1 or its extracellular domain. Furthermore, because the PD-1 variants of the present invention retain the ability to bind to specific PD-1 monoclonal antibodies, the monoclonal antibodies can be used for purification and other manipulations.

[0064] Based on the above uses, the PD-1 variants of the present invention or fusion proteins comprising said variants are particularly suitable for cancer therapy. "Cancer," in the context of the present invention, includes malignant tumors arising in different organs, tissues, and cell types, including both solid tumors and hematological cancers.

[0065] The present application also includes at least the following content: 1. A PD-1 variant, (1) the PD-1 variant has the ability to bind to the human PD-L1 polypeptide set forth in SEQ ID NO: 28; and (2) The PD-1 variant does not bind to one or more anti-PD-1 antibodies selected from the group consisting of sintilimab, nivolumab, camrelizumab, pembrolizumab, and toripalimab.

[0066] 2. The PD-1 variant of paragraph 1, wherein, relative to the wild-type human PD-1 polypeptide set forth in SEQ ID NO: 1, the PD-1 variant comprises a mutation at one or more amino acid positions selected from E84, D85, R86, S87, Q88, P89, G90, A129, P130, K131, A132, and Q133, wherein the amino acid positions are numbered with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0067] 3. Compared to the wild-type human PD-1 polypeptide set forth in SEQ ID NO: 1, the PD-1 variants are: E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I, S87L, S87Y; Q88L, Q88F, Q88N, Q88T, Q88C, Q88E; P89C, P89V, P89R, P89K; G90T, G90S, G90F, G90R, G90Y; A 3. The PD-1 variant of Item 1 or 2, comprising one or more amino acid mutations selected from the group consisting of: 129S, A129G, A129Q, A129Y, A129H, A129W, A129V, A129T, A129I; P130T, P130F, P130E; K131P, K131R; A132V, A132G, A132F, A132I, A132M; Q133W, Q133N, Q133V.

[0068] 4. The PD-1 variant of paragraph 3, wherein, compared to the wild-type human PD-1 polypeptide set forth in SEQ ID NO:1, the PD-1 variant contains one or more amino acid mutations selected from the group consisting of: E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I; Q88L, Q88F, Q88N, Q88T; P89C, P89V, P89R; G90T, G90S; A129S, A129G, A129Q, A129Y; P130T; K131P; A132V, A132G; and Q133W.

[0069] 5. The PD-1 variant of any of Items 2-4, wherein, compared to the wild-type human PD-1 polypeptide represented by SEQ ID NO:1, the PD-1 variant contains a mutation at one or more amino acid positions selected from E84, D85, R86, S87, Q88, P89, and G90, and also contains a mutation at one or more amino acid positions selected from A129, P130, K131, A132, and Q133.

[0070] 6. The PD-1 variant has one of the following structures (1)-(25) relative to the wild-type human PD-1 polypeptide shown in SEQ ID NO:1: (1) A129S, P130T, K131P, A132V, and Q133W; (2) E84F, D85L, R86F, S87N, Q88L, A129S, P130T, K131P, A132V, and Q133W; (3) E84W, D85M, R86F, S87C, Q88F, A129S, P130T, K131P, A132V, and Q133W; (4) R86F, S87R, Q88N, P89C, G90T, A129S, P130T, K131P, A132V, and Q133W; (5) R86F, S87F, Q88T, P89V, G90S, A129S, P130T, K131P, A132V, and Q133W; (6) S87I, Q88N, P89R, G90S, A129S, P130T, K131P, A132V, and Q133W; (7) E84F, D85L, R86F, S87N, Q88L, P130T, K131P, and Q133W; (8) R86F, S87R, Q88N, P89C, G90T, A129G, P130T, K131P, and Q133W; (9) R86F, S87R, Q88N, P89C, G90T, P130T, K131P, A132G, and Q133W; (10) R86F, S87R, Q88N, P89C, G90T, A129Q, P130T, K131P, and Q133W; or (11) R86F, S87F, Q88T, P89V, G90S, A129Y, P130T, K131P, and Q133W; (12) A129H, P130F, K131R, A132F, and Q133N; (13) A129S, P130E, K131P, A132I, and Q133V; (14)S87L, Q88C, P89V, G90F, A129H, P130F, K131R, A132F, and Q133N; (15)R86S, S87F, Q88E, P89V, G90R, A129H, P130F, K131R, A132F, and Q133N; (16)R86L, S87Y, Q88L, P89K, G90Y, A129S, P130E, K131P, A132I, and Q133V; (17)R86F, S87R, Q88N, P89C, G90T, A129W, P130T, K131P, A132M, and Q133W; (18)R86F, S87F, Q88T, P89V, G90S, A129H, P130T, K131P, A132F, and Q133W; (19)R86F, S87F, Q88T, P89V, G90S, A129G, P130T, K131P, A132F, and Q133W; (20)R86F, S87F, Q88T, P89V, G90S, A129I, P130T, K131P, A132G, and Q133W; (21)S87I, Q88N, P89R, G90S, A129T, P130T, K131P, A132F, and Q133W; (22)S87I, Q88N, P89R, G90S, A129V, P130T, K131P, and Q133W; (23)S87I, Q88N, P89R, G90S, P130T, K131P, and Q133W; (24)R86F, S87R, Q88N, P89C, G90T, P130T, K131P, and Q133W; (25)R86F, S87F, Q88T, P89V, G90S, P130T, K131P, and Q133W, 6. The PD-1 variant of any one of items 1 to 5, comprising or containing only a combination of amino acid mutations selected from one set of:

[0071] 7. The PD-1 variant of paragraph 6, wherein, compared to the wild-type human PD-1 polypeptide shown in SEQ ID NO:1, the PD-1 variant comprises a combination of amino acid mutations from one set of (1)-(11), or comprises only these.

[0072] 8. The PD-1 variant of paragraph 7, wherein, compared to the wild-type human PD-1 polypeptide set forth in SEQ ID NO:1, the PD-1 variant comprises a combination of, or comprises only, one of the sets of amino acid mutations (1)-(6).

[0073] 9. The PD-1 variant of any of paragraphs 1-8, wherein the PD-1 variant does not contain the intracellular domain or does not contain both the transmembrane domain and the intracellular domain.

[0074] 10. The PD-1 variant of any of paragraphs 1-9, wherein the variant comprises or consists of an amino acid sequence set forth in any of SEQ ID NOs:2-26, or an amino acid sequence having at least 85% sequence identity thereto.

[0075] 11. The PD-1 variant of clause 10, wherein the variant comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs:2-12, or an amino acid sequence having at least 85% sequence identity thereto.

[0076] 12. The PD-1 variant of clause 11, wherein the variant comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs:2-7, or an amino acid sequence having at least 85% sequence identity thereto.

[0077] 13. The PD-1 variant of any of paragraphs 1-12, wherein the PD-1 variant has the same amino acids as the wild-type human PD-1 polypeptide set forth in SEQ ID NO:1 at the following positions: M70, S871, P72, S873, N74, Q75, T76, D77, and K78.

[0078] 14. The PD-1 variant of any of paragraphs 1-13, wherein the PD-1 variant has a signal peptide represented by amino acids 1-20 of SEQ ID NO:1.

[0079] 15. The PD-1 variant of any of paragraphs 1-14, wherein the PD-1 variant binds to the PD-L1 polypeptide with higher affinity compared to wild-type human PD-1 polypeptide.

[0080] 16. The PD-1 variant is 10 -7 16. The PD-1 variant of any of items 1-15, which binds to the PD-L1 polypeptide with a KD value of M or less.

[0081] 17. The PD-1 variant is 10 -6 17. The PD-1 variant of any of paragraphs 1-16, which binds to human PD-L2 polypeptide with a KD value of M or less.

[0082] 18. The PD-1 variant of any of paragraphs 1-17, wherein the PD-1 variant binds to the PD-L2 polypeptide with higher affinity compared to wild-type human PD-1 polypeptide.

[0083] 19. The PD-1 variant according to any of Items 1 to 18, which does not bind to at least two, preferably at least three, anti-PD-1 antibodies selected from the group consisting of sintilimab, nivolumab, camrelizumab, pembrolizumab, and toripalimab.

[0084] 20. The PD-1 variant does not bind to the following anti-PD-1 antibodies: sintilimab, nivolumab, and pembrolizumab. The PD-1 variant according to item 19.

[0085] 21. The PD-1 variant according to clause 20, wherein the PD-1 variant does not bind to any of sintilimab, nivolumab, camrelizumab, pembrolizumab, and toripalimab.

[0086] 22. The PD-1 variant according to any one of items 1 to 21, wherein the PD-1 variant binds to tilelizumab.

[0087] 23. The binding affinity of the PD-1 variant to tilelizumab is KD value 10 -9 The PD-1 variant of clause 22, wherein the PD-1 variant is M or less.

[0088] 24. A fusion protein comprising the PD-1 variant of any one of paragraphs 1 to 23.

[0089] 25. The fusion protein of paragraph 24, further comprising an immunoglobulin Fc fragment.

[0090] 26. The fusion protein of paragraph 25, wherein the immunoglobulin Fc fragment is derived from a mammal, preferably a human.

[0091] 27. The fusion protein comprises the PD-1 variant as an extracellular domain, and the PD-1 variant does not comprise an intracellular domain; 25. The fusion protein of claim 24, further comprising an intracellular signaling domain derived from a costimulatory molecule.

[0092] 28. The fusion protein of paragraph 27, wherein the costimulatory molecule is any of OX40, 4-1BB, CD28, ICOS, and CD27.

[0093] 29. The fusion protein of paragraph 27 or 28, further comprising the transmembrane domain of PD-1.

[0094] 30. An isolated nucleic acid molecule encoding a PD-1 variant according to any one of paragraphs 1-23.

[0095] 31. The nucleic acid molecule of paragraph 30, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 56-61 or a nucleotide sequence having at least 85% homology to the nucleotide sequence set forth in any one of SEQ ID NOs: 56-61.

[0096] 32. An isolated nucleic acid molecule encoding a fusion polypeptide according to any one of paragraphs 24-29.

[0097] 33. An expression vector comprising the isolated nucleic acid molecule of any of paragraphs 30-32.

[0098] 34. A host cell comprising the isolated nucleic acid molecule of any of paragraphs 30-32, or the expression vector of paragraph 33.

[0099] 35. A pharmaceutical composition comprising the PD-1 variant according to any one of items 1 to 23 and a pharmaceutically acceptable carrier.

[0100] 36. A pharmaceutical composition comprising the fusion protein according to any one of paragraphs 24 to 29 and a pharmaceutically acceptable carrier.

[0101] 37. Use of a PD-1 variant according to any one of paragraphs 1-23 or a fusion protein according to any one of paragraphs 24-29 for the preparation of a medicament for the treatment of cancer.

[0102] 38. A drug combination comprising (1) a PD-1 variant described in any one of paragraphs 1-23 or a fusion protein described in any one of paragraphs 24-29, and (2) an anti-PD-1 antibody.

[0103] 39. The drug combination of clause 38, wherein the anti-PD-1 variant does not bind to the anti-PD-1 antibody.

[0104] 40. The drug combination of paragraph 39, wherein the anti-PD-1 antibody is selected from the group consisting of sintilimab, nivolumab, camrelizumab, pembrolizumab, and toripalimab.

[0105] 41. A drug combination according to any of paragraphs 38-40 used in the treatment of cancer.

[0106] 42. A cell modified to express the fusion protein of paragraphs 24-29.

[0107] 43. The cell of paragraph 42, which is an immune cell.

[0108] 44. The cell of paragraph 43, wherein the immune cell, before being modified, is derived from a peripheral blood mononuclear cell or is a tumor-infiltrating lymphocyte.

[0109] 45. The cell of paragraph 44, wherein the immune cell is a T cell.

[0110] 46. ​​The cell of any one of paragraphs 42-45, modified for the treatment of cancer. [Example]

[0111] In order to fully understand and apply the present invention, the present invention will be described in detail below with reference to embodiments and drawings. However, the following embodiments are shown as examples of the present invention and are not intended to limit the scope of the present invention. The scope of the present invention is particularly limited by the appended claims.

[0112] Example 1. Construction of a PD-1 variant library This example describes the process of constructing a PD-1 variant library.

[0113] In brief, random mutations were introduced into PD-1 (SEQ ID NO:1) at sites involved in the interaction between PD-1 and PD-L1 or PD-L2. These regions typically contain antibody-binding epitopes. A variant plasmid library was constructed and further transfected into 293T cells for analysis.

[0114] Specifically, random mutations were introduced into three regions of SEQ ID NO:1 (the first region consisting of amino acids 70-78, the second region consisting of amino acids 84-90, and the third region consisting of amino acids 127-133). Six sets of random mutation primers were designed to introduce random mutations, with each primer pair randomly mutating the codons corresponding to five consecutive amino acids in each of the above regions. Specifically, the six sets of primers contained codons used to introduce random mutations into amino acids 70-74 (7074-For / Rev), amino acids 74-78 (7478-For / Rev), amino acids 84-88 (8488-For / Rev), amino acids 86-90 (8690-For / Rev), amino acids 127-131 (12731-For / Rev), and amino acids 129-133 (12933-For / Rev). The six sets of primers used to introduce random mutations contained codons corresponding to these amino acids in the upstream primer. These codons were designed as five consecutive NNK codons, where N represents any of the bases A, T, C, or G, and K represents any of the bases G or T. The sequences of the six sets of primers (SEQ ID NOs: 27-40) are shown in Table 1 below. Two additional primers, P1-For and P1-Rev (SEQ ID NOs: 35 and 36), were also designed, and their sequences are also shown in Table 1. P1-For and P1-Rev were used in combination with the other six mutation primer pairs to amplify the non-mutated and mutated regions and were used to amplify the entire PD-1 DNA sequence by overlapping PCR.

[0115] Table 1. Primer design used to introduce random mutations JPEG2025529002000001.jpg94170

[0116] Polymerase chain reaction (PCR) was performed using the seven primer pairs listed in Table 1 with PrimeStar DNA polymerase (Takara, R045B) to amplify fragments containing and excluding the PD-1 mutation region. For example, the primers used to mutate amino acids 70-74 were 7074-For + P1-Rev and P1-For + 7074-Rev. The amplification conditions were 98°C for 10 seconds, 58°C for 5 seconds, and 72°C for 5 seconds, for a total of 30 cycles.

[0117] Next, overlapping PCR was performed. For example, to mutate amino acids 70-74, an equimolar mixture of the products amplified by the above two PCRs was used as a template. The full-length PD-1 sequence containing random mutations at amino acids 70-74 was amplified using primers P1-For and P1-Rev. The amplification conditions were 98°C for 10 seconds, 58°C for 5 seconds, and 72°C for 5 seconds, for a total of 30 cycles. The PCR product of the full-length sequence and the lentiviral carrier pLV2-PD1-CD28 (the plasmid structure is shown in Figure 1; the complete sequence of the plasmid is shown in SEQ ID NO: 63, and it is 8417 bp in length, of which 1368-1877 corresponds to the PD1 extracellular domain, 1878-1940 corresponds to the PD1 transmembrane domain, and 1941-2063 corresponds to the CD28 intracellular domain) were digested with SalI and XbaI restriction endonucleases, respectively. The digested full-length sequence containing random mutations was ligated to the carrier using T4 DNA ligase to prepare a PD-1 random mutation plasmid library.

[0118] E. coli Trans1-T1 strain (Beijing Quanshi Jinbio, product number CD501-03) was transformed with each ligation product. Specifically, the ligation product was added to thawed E. coli Trans1-T1 chemical transformation-competent cells and left on ice for 30 minutes. The cells were then incubated at 42°C for 45 seconds, then placed on ice for 2 minutes, and LB medium was added. The cells were then incubated at 37°C in a shaker for 45 minutes. After incubation, the bacterial culture was spread onto LB agar plates containing ampicillin and cultured overnight at 37°C. After incubation, the total number of library clones was counted, and the library plasmids were extracted.

[0119] Example 2. Transient transfection of mammalian 293T cells with PD-1 random mutation library plasmids This example describes the construction of a 293T cell line expressing PD-1 variants by transfecting the PD-1 random mutation library plasmid constructed in Example 1 into 293T cells.

[0120] 293T cells were cultured as follows: Human embryonic kidney 293T cell line cells with less than 20 generations were removed from liquid nitrogen and quickly placed in a 37°C water bath until completely thawed. The thawed cells were added to 10 ml of D10 complete medium (DMEM containing 10% fetal bovine serum). After thorough mixing, the cells were centrifuged at 400 g for 5 min and the medium was discarded. The cells were resuspended in an appropriate amount of medium and added to a T25 or T75 cell culture flask for culture. The starting cell density was approximately 2 x 10 5The concentration was 1 / ml. The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours, after which the medium was aspirated and discarded. The adherent cells were washed with phosphate-buffered saline (PBS), and the buffer was aspirated and discarded. The cells were digested with 0.5% trypsin-EDTA solution for 3-5 minutes until most of the cells had detached from the bottom of the culture flask. 2 mL of D10 complete medium was then added, mixed, and centrifuged at 400 g for 5 minutes. The supernatant was then aspirated and resuspended in 1 mL of D10 complete medium. An appropriate amount of cells was collected for flow cytometry analysis or subculture.

[0121] The constructed variant plasmid library containing randomly mutated PD-1 genes was transfected into 293T cells using the PEI (Poly(ethylenimine)) transfection method. Each library plasmid was mixed with PEI at a mass ratio of 1:3 and transiently transfected into 293T cells. As a result, 293T cells carrying the randomly mutated PD-1 variant library plasmids were obtained.

[0122] Example 3. Flow cytometric screening of cells that bind to PD-L1 but not to anti-PD-1 antibodies In this example, a process for selecting a cell population with a desired binding ability using flow cytometry is described.

[0123] Forty-eight hours after transient transfection, cells were digested with trypsin-EDTA solution and centrifuged at 400 g for 5 minutes. The supernatant was aspirated and resuspended in phosphate buffered saline. A portion of the cell suspension was incubated with PD-L1-Fc (R&D Systems, 156-B7-100) and Dylight 650-conjugated goat anti-human IgG Fc secondary antibody (Abcam, ab97006) at 4°C for 30 minutes. Another portion of the cell suspension was incubated with biotin-conjugated anti-PD-1 antibody (including sintilimab, nivolumab, tirelizumab, camrelizumab, pembrolizumab, or toripalimab) and PE-conjugated streptavidin (BD Biosciences, 554061).

[0124] Cell populations that bind to PD-L1 but not to anti-PD-1 antibodies were analyzed and collected by flow cytometry sorting. First, we performed flow cytometry sorting using pembrolizumab (see Figure 2). Specifically, we sorted the cells indicated by the red box in the flow cytometry map in Figure 2. Next, we subjected these cells to binding assays with five other anti-PD-1 antibodies, and selected cell populations that did not bind to four or five of the antibodies. As a result of the initial screening, a total of 5,000 cells were collected.

[0125] Example 4. Isolation and identification of plasmids in cells obtained by sorting This example describes the process of isolating and identifying the PD-1 variant plasmid contained in the cells obtained in Example 3.

[0126] Sequence Using a small volume plasmid extraction kit (EM101-02, manufactured by TransGen biotech), plasmids were extracted from 5,000 cells that had been sorted by flow cytometry and recovered in Example 3.

[0127] The extracted plasmid was transformed into Escherichia coli Trans1-T1 strain using the same method as in Example 1. After incubation, monoclonal E. coli were selected and sequenced using EF-1α oligonucleotide primers. The nucleotide sequence of the PD-1 variant contained in each clone was obtained, and the corresponding amino acid sequence was identified based on this. Of the 5,000 cells used, approximately 300 clones were obtained and sequenced. Sequencing analysis of the sequenced clones revealed that approximately 60 clones (approximately 20%) contained unrelated nucleotide sequences, approximately 30 clones (approximately 10%) contained clones that had successfully mutated PD-1 but contained a stop codon, and approximately 210 clones (approximately 70%) were clones that had successfully mutated PD-1 and were capable of efficient expression.

[0128] Table 2 shows the amino acid positions and amino acid types where mutations occurred in the PD-1 variants obtained by screening compared to wild-type PD-1 (not all sequences of the sequenced clones are shown). In Table 2, SEQ ID NO: 1 is wild-type PD-1, and SEQ ID NOs: 2-26 are the sequences of representative PD-1 variants obtained.

[0129] Table 2 JPEG2025529002000002.jpg255168

[0130] Table 3 As can be seen from Table 2, the mutation sites of the variants of the present invention were concentrated in 12 locations: E84, D85, R86, S87, Q88, P89, G90, A129, P130, K131, A132, and Q133. When aligned with the three regions where random mutations were performed during the initial design (see Example 1), these mutations were primarily concentrated in the second region (E84 to G90) and the third region (S127 to Q133, more specifically, A129 to Q133). In each representative variant, the region from M70 to K78 contained no amino acid mutations, and no mutations occurred at S127 and L128.

[0131] Flow cytometry analysis using 293T cells was used to verify the binding ability of various variants to PD-L1, PD-L2, and various antibodies. Based on the sequencing results, clones were selected that had successfully mutated the corresponding sites within the three regions predicted to be mutated and contained no stop codons. Plasmids from these clones were extracted and used for transient transfection of 293T cells, respectively. The transfection method was as described in Example 2.

[0132] Forty-eight hours after transfection, cells were digested, and a portion of the cells was incubated with PD-L1 expressed in fusion with human IgG1-derived Fc (R&D Biosystems) and Dylight650-conjugated goat anti-human IgG Fc secondary antibody (Abcam) for 30 minutes at 4°C. After 5 minutes of centrifugation at 400 x g, the cells were resuspended and washed in PBS phosphate buffer. This centrifugation and washing process was repeated once. Another cell sample was incubated with PD-L2 expressed in fusion with human IgG1-derived Fc (R&D Biosystems, PD2-H5251) and Dylight650-conjugated goat anti-human IgG Fc secondary antibody (Abcam, ab97006), followed by two washes with phosphate buffer. Another cell sample was then incubated with biotin-conjugated anti-PD-1 antibodies and PE-conjugated streptavidin.

[0133] The binding of each variant and wild-type PD-1 to PD-L1, PD-L2, and anti-PD-1 antibodies was analyzed by flow cytometry. The results are shown in Table 2 (raw data maps are not shown). In Table 2, "+" indicates binding, and "-" indicates no binding (i.e., below detection). The wild-type PD-1 used was a fragment of wild-type PD-1 that was the same length as the variant PD-1 and corresponded to amino acid positions 1 to 155 of the wild-type human PD-1 amino acid sequence shown in SEQ ID NO:1.

[0134] Of the 210 clones that successfully mutated the PD-1 target sequence and did not contain a stop codon, approximately 60 clones (approximately 20%) were unable to bind to PD-L1, approximately 120 clones (approximately 40%) were able to bind to PD-L1 but also bound to 4-6 test antibodies, and approximately 30 clones (approximately 10%) fulfilled the condition of being able to bind to PD-L1 and only binding to 1-3 test antibodies. Ultimately, 25 variants with the sequences shown in Table 2 were identified.

[0135] As shown in Table 2, flow cytometry analysis demonstrated that wild-type human PD-1 could bind to PD-L1 and PD-L2, and all six anti-PD-1 antibodies tested. In contrast, all 25 representative PD-1 variants of the present invention could bind to PD-L1, and most variants (except PD1_49#_93 and 56#-2AA) could also bind to PD-L2. All variants could bind to tilizumab but did not bind to at least three or more anti-PD1 monoclonal antibodies. Of note, variants with mutations only in the third domain (PD1_56#, PD1_49#, PD1_110#) retained the ability to bind to three antibodies (including tilizumab), whereas other variants with mutations in both the second and third domains retained the ability to bind to only one antibody (i.e., tilizumab).

[0136] Using human T cells, the binding ability of various variants to PD-L1, PD-L2, and various antibodies was analyzed and verified using flow cytometry.

[0137] In the above, various variants were expressed in 293T cells and their binding ability to PD-L1, PD-L2, and various antibodies was tested. Because the effector function of various variants is primarily mediated by T cells, the variants were further expressed in human T cells using lentivirus, and the binding ability of the screened variants to PD-L1, PD-L2, and various antibodies was examined by flow cytometry. The results are shown in Table 3 (raw data maps are not shown).

[0138] The isolation, viral infection, and culture of human T cells were performed as follows: 1 × 10 8Peripheral blood mononuclear cells (PBMCs) were isolated and rapidly thawed in a 37°C water bath. Then, they were added to a 5x volume of pre-warmed T cell medium (X-VIVO15 + 0.5% HSA + 300 U / ml IL-2) and mixed thoroughly. The mixture was then centrifuged at 400g for 5 minutes, and the supernatant was removed. The cells were resuspended in 20 ml of the same T cell medium, transferred to a T175 culture flask, and cultured in an incubator at 37°C with 5% CO2.

[0139] One day later, CD3 / CD28 magnetic beads (manufactured by Thermo) in an amount three times the number of cells were added to the cell suspension, and the culture was continued.

[0140] After one more day, lentivirus was added to the T cell culture at an MOI of 3 and continued to incubate. The lentivirus was prepared as follows: 0.75 mL of DMEM medium (Biological Industries, 06-1055-57-1A) and a plasmid solution (VSVG 2.5 μg + dR 8.91 7.5 μg + PLV2-PD1-CD28 10 μg, total 20 μg) were added to tube 1, and 0.75 mL of DMEM medium and 60 μl of polyethyleneimine (PEI) solution were added to tube 2. The solutions in tubes 1 and 2 were mixed and left for 30 minutes before being added to a T75 culture flask containing 293T cells. After 24 hours, the viral supernatant was collected and centrifuged at 100,000 g for 2 hours. The supernatant was discarded. The virus was resuspended in 500 μl of X-VIVO15 medium and used to infect T cells.

[0141] After 5 days, the cell-magnetic bead complex was blown thoroughly (pipetting) to separate the beads and cells as much as possible. The centrifuge tube was then placed on a magnetic rack for adsorption. After 1 minute, the cell suspension was transferred to a new centrifuge tube, and the above blowing and adsorption steps were repeated three times. The cells were resuspended in T cell medium and continued to be cultured.

[0142] T cells cultured for 7–14 days were harvested and incubated with PD-L1 expressed via fusion with human IgG1-derived Fc (R&D Biosystems) and Dylight650-conjugated goat anti-human IgG Fc secondary antibody (Abcam). A portion of the cells was incubated with PD-L2 expressed via fusion with human IgG1-derived Fc (R&D Biosystems, PD2-H5251-100µg) and Dylight650-conjugated goat anti-human IgG Fc secondary antibody (Abcam, ab97006), then washed twice with phosphate buffered saline. Another portion of the cells was incubated with biotin-conjugated anti-PD-1 antibody and PE-conjugated streptavidin.

[0143] The binding of the variants to PD-1 and anti-PD-1 antibodies was analyzed by flow cytometry, and the results are shown in Table 3. In Table 3, "+" indicates binding and "-" indicates no binding (i.e., below detection). The binding characteristics were found to be identical to those obtained with 293T cells.

[0144] Example 5. Preparation of PD-1 variant-rabbit IgGFc fusion protein Some PD-1 variants (PD-1 variants with SEQ ID NOs: 2-7) were fused with rabbit IgG Fc and expressed and purified. Wild-type PD-1 (SEQ ID NO: 1) was used as a control. The specific steps are as follows:

[0145] The nucleotide sequences (SEQ ID NOs: 55-61) corresponding to the wild-type (SEQ ID NO: 1) and six variants (variants with amino acid sequences SEQ ID NOs: 2-7) shown in Table 2 were amplified using PCR. The pLV2-PD1-CD28 vector containing wild-type PD-1 and PD-1 variants constructed in Example 1 was used as a template, and the primers used were P32-For and P32-Rev (SEQ ID NOs: 43 and 44) ​​shown below. The amplification reagents, system, and conditions were the same as those used in Example 1.

[0146] P32-For:TCAGTAGCTAGCGGTACCGCCGCCACCatgcagatcccacagg P32-Rev:GTTGAGGATCCgtgggctgtgggcacttctg The PCR product and the protein expression vector, pcDNA 3.4-RFc (pcDNA 3.4 vector (Thermo) containing the rabbit IgGFc (RFc) gene), were digested with Nhe I and BamH I restriction endonucleases, respectively. The digested PD-1 coding sequence was ligated into the vector using T4 DNA ligase to construct the pcDNA 3.4-PD1-RFc expression vector. This expression vector was used for cell transfection. Before transfection, HEK293 cells (Kairui Biotech) were cultured at 37°C, 5% CO2, and 120 rpm at 1 × 10 6 The cells were cultured to a density of 1.5 mg / ml. The constructed pcDNA3.4-PD1-RFc vector was transfected into the HEK293 cells using 5 mL / L of transfection reagent TA-293 (Kairui Biotech) to a transfection plasmid concentration of 1.5 mg / L. After transfection, the cells were cultured at 37°C, 8% CO2, and 120 rpm for 7 days. After transfection, the culture was centrifuged at 4000 rpm and the supernatant was collected. The protein was purified using Protein A beads and eluted with 500 μL of 0.1 M Gly-HCl eluent, pH 2.6-3.0. The eluate was collected to obtain the purified fusion protein.

[0147] The purified fusion proteins were subjected to SDS-PAGE under reducing and non-reducing conditions. Figure 3 shows the protein electrophoresis. Lanes 1–7 show fusion proteins containing wild-type PD-1 and PD-1 variants 1–6, respectively, and lane 8 shows protein molecular weight standards. Figure 3A shows electrophoresis under non-reducing conditions, and Figure 3B shows electrophoresis under reducing conditions. The predicted molecular weight of the fusion protein was approximately 43 kDa. Under reducing conditions, the band size was close to the predicted value. Under non-reducing conditions, the fusion protein formed a dimer and underwent modifications such as glycosylation, resulting in a molecular weight higher than 86 kDa. Under non-reducing conditions, multiple distinct bands were observed in lanes 3, 4, and 6, with the bands at higher molecular weights. This may indicate different forms of polymerization, such as the formation of multimers, or different degrees or types of protein modifications.

[0148] Example 6. Affinity measurement with Biacore T200 The affinities of wild-type PD-1 and representative PD-1 variants of the present invention (variants having SEQ ID NOs: 2-7) to PD-L1, PD-L2, and the six PD-1 monoclonal antibodies described in Example 3 were measured using a Biacore T200.

[0149] The affinity statistical results are shown in Tables 4A-4B. In Table 4A, "unbound" indicates that the measurement was below the lower limit of detection of the Biacore T200 instrument, i.e., the affinity KD value was 10 -3 Indicates higher than M.

[0150] Table 4A. Affinity assays of PD-1 variants to PD-L1, PD-L2, and six PD-1 monoclonal antibodies JPEG2025529002000004.jpg48170

[0151] Table 4B. Raw data from Table 4A. JPEG2025529002000005.jpg217170

[0152] As can be seen from the results in Table 4, the Biacore experiments verified the various binding properties determined by flow cytometry in the previous examples and further provided specific binding affinity values. All six variants had high binding affinity to PD-L1 and PD-L2 and tilelizumab, with KD values ​​all less than 10-6. The KD values ​​of all six variants were at least two orders of magnitude lower than those of wild-type PD-1, indicating significantly higher binding affinity to PD-L1. Variant 2 (SEQ ID NO:3), variant 5 (SEQ ID NO:6), and variant 6 (SEQ ID NO:7) also had higher binding affinity to PD-L2 than wild-type PD-1. However, variants 1, 3, and 4 were found to have lower binding affinity to PD-L2 than wild-type PD-1. The binding affinity of variant 1 (SEQ ID NO: 2) and variant 6 (SEQ ID NO: 7) to the anti-PD-1 antibody tilelizumab was comparable to that of wild-type PD-1.

[0153] Example 7.108 - Functional assay of inhibition of cytokine secretion by CD28Fc When PD1-CD28-expressing T cells (PD1-CD28-T) were cocultured with PD-L1-positive tumor cells (J82-PD-L1), the T cells secreted IL-2. Addition of an anti-PD-1 antibody blocked PD-1-mediated T cell binding to tumor cells, thereby suppressing IL-2 release. Similarly, addition of a PD-1 variant Fc fusion protein bound to PD-L1 on target cells, preventing PD-L1 from binding to PD-1 on the T cell membrane, inhibiting IL-2 release in the same way as anti-PD-1 antibodies.

[0154] PD1-CD28-T cell preparation process The pLV2-PD1-CD28 plasmid (wherein PD-1 is human wild-type PD-1) described in Example 1 was prepared into a lentiviral vector as described in Example 4, and human T cells were infected with it to obtain PD1-CD28- T cells. The T cell culture method was as described in Example 4.

[0155] Preparation of J82-PDL1 cells The pLV2-PDL1 plasmid (where PD-L1 is human wild-type PD-L1) was prepared into a lentiviral vector as described in Example 4 and then infected into a human bladder cancer cell line (purchased from the Chinese Academy of Medical Sciences, product code 1101HUM-PUMC000346) to establish the J82-PDL1 cell line.

[0156] Assay of IL-2 secretion inhibition by PD-1 variant-Fc fusion proteins 1. J82-PDL1 cells were digested with 0.05% trypsin, washed, and resuspended at a density of 1 x 10 / mL according to the method described in Example 2. PD1-CD28- T cells were harvested, washed, resuspended, and adjusted to a density of 1 x 10 / mL according to Example 4. 6 After adjusting the concentration to 1 / mL, 6 ng / mL of PMA (Phorbol 12-tetradecanonate 13-acetate, Sigma, product code P8139-1MG) was added.

[0157] 2. 100 μl of J82-PDL1 cells and 100 μl of PD1-CD28-T cells were added to a 96-well plate, and final concentrations of 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 nM of Anti-IgG4-RFc (negative control, does not bind to PD-1 or PD-L1), tirelizumab (positive control), and the two PD1-RFc (variants 56#-16 and 56#-108) fusion proteins prepared in Example 5 were added, respectively, and the mixture was incubated at 37°C for 16 hours.

[0158] 3. The 96-well plate was centrifuged at 500 g for 3 minutes, and 50 μL of the supernatant was taken and the IL-2 concentration was measured using an IL-2 ELISA kit (Dakewe Biotech Co., Ltd., product number 1110203).

[0159] Table 5 shows the measured levels of IL-2 secreted by T cells after the addition of different concentrations of antibody or fusion protein. Figure 5 shows a trend chart of the data in Table 5. The data in Table 5 indicate that tilelizumab did not significantly affect IL-2 secretion at concentrations below 6.4 nM, but significantly inhibited IL-2 secretion at concentrations above 6.4 nM. The 56#-16 fusion protein showed a decreasing trend in IL-2 secretion with increasing concentrations, but did not completely inhibit IL-2 secretion. The 56#-108 fusion protein showed a decreasing trend in IL-2 secretion with increasing concentrations, which was more pronounced than the 56#-16 fusion protein group but less pronounced than the tilelizumab group. However, at concentrations above 25.6 nM, the inhibitory effect on IL-2 secretion was stronger than that of the tilelizumab group. These results suggest that PD-1 variant fusion proteins have a similar inhibitory effect on PD-1 / PD-L1 signaling as PD-1 monoclonal antibodies.

[0160] Table 5 JPEG2025529002000006.jpg110170

[0161] Example 8. Cytokine secretion assay In this example, the effect of the enhanced receptor based on the PD1 variant of the present application on T cell function was verified by an IL-2 secretion assay.

[0162] When PD1-CD28-expressing T cells (PD1-CD28-T) are co-cultured with PD-L1-positive tumor cells, the T cells release the cytokine IL-2 through the interaction between PD-1 and PD-L1. Therefore, the effect of PD1-CD28 in promoting IL-2 secretion can be determined by measuring the amount of IL-2 in the co-culture medium, which can be detected by enzyme-linked immunosorbent assay (ELISA).

[0163] As shown in Figure 5, a co-culture experiment was performed using a 96-well plate. Malme-3M tumor cells overexpressing human PD-L1 (3M-PDL1-OE) and Malme-3M tumor cells in which human PD-L1 was knocked out (3M-PDL1-KO) were used as target cells, and T cells expressing an enhanced receptor composed of a PD1 variant (56#-108 variant) and CD28 (shown as 108T in Figure 5, also referred to as 108-CD28-T) and unmodified T cells (T) were used as effector cells. The ratio of target cells to 108T was 10:1, i.e., the target cells were 1 × 10 5 / well, 10 T cells = 1 x 10 4 The cells were co-cultured in a medium containing 3 ng / ml PMA (phorbol ester) at 1000 x g / well. The supernatants after overnight incubation were used as test samples and analyzed by IL-2 ELISA together with assay standards.

[0164] ELISA was performed using a human IL-2 detection kit (ELISA method) (Dakewe Biotech Co., Ltd., product number 1110203) as follows: A microtiter plate was precoated with anti-human IL-2 capture antibody. Standards or samples and the detection antibody (biotin-labeled anti-human IL-2 antibody) were simultaneously added to the microtiter plate, followed by horseradish peroxidase-conjugated streptavidin (SA-HRP), which specifically binds to biotin in the immune complex. The enzyme substrate, tetramethylbenzidine (TMB), was added to produce a blue color, the intensity of which correlates with the IL-2 concentration in the standard or sample. After 5–10 minutes of color development, the reaction was stopped by adding a reaction stop solution, and the optical absorbance (OD) at 450 nm was measured using a multimode microplate reader. The concentration of human IL-2 was proportional to the OD value within a certain range, and a standard curve was created using ELISA with an IL-2 standard. The standard curve was used to calculate the IL-2 concentration in each co-culture.

[0165] As shown in Figure 5, when PD-L1 was knocked down on target cells, neither control T cells nor T cells armed with the PD-1 variant-enhanced receptor (108T, i.e., 108-CD28-T) secreted IL-2 due to the absence of a second stimulatory signal (red bars). On the other hand, when cocultured with target cells highly expressing PD-L1, T cells armed with the 108T-enhanced receptor released large amounts of IL-2 upon stimulation. This result suggests that the fusion protein of the PD1 variant 108 and CD28 (108-CD28) functions to transmit signals that stimulate T cells.

[0166] Example 9. Tumor cell killing assay In this example, we demonstrate a cell killing experiment in which T cells were made to express a PD1-C28-enhanced receptor containing the PD-1 variant of the present invention, and demonstrated that the PD-1 variant-enhanced receptor enhanced the killing ability of T cells against PD-L1-expressing tumor cells.

[0167] Tumor cells can express HLA-peptide complex antigens on their surface, and T cells express T cell receptors (TCRs) that target these antigens. When these tumor cells are co-incubated with the corresponding T cells, contact between the TCR on the T cell and the complex antigen on the tumor cell triggers a series of T cell killing responses that lyse the tumor cells.

[0168] In this experiment, J82 cells expressing Eso1 antigen and PD-L1 (J82Eso1PDL1) were used as tumor cells. To generate the J82Eso1PDL1 tumor cell line, lentiviral vectors expressing the human PD-L1 gene and the ESO1 gene were prepared according to the method described in Example 4 and named pLV2-PDL1 and pLV2-ESO1, respectively. As described in Example 4, the pLV2-PDL1 lentivirus was used to infect a human bladder cancer cell line (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, product number 1101HUM-PUMC000346) to generate J82-PDL1 cells expressing human PD-L1. The J82-PDL1 cells were then infected with the pLV2-ESO1 lentivirus prepared as described above. The ESO1 antigen of these cells is expressed and processed intracellularly, then delivered extracellularly as the ESO1 peptide, where it forms an antigen complex with the HLA*A0201 protein expressed on the surface of J82 cells, and this antigen complex is recognized by a specific T cell receptor (T cell receptor, TCR).

[0169] Three types of T cells were prepared: (1) T cells expressing a TCR targeting the HLA-Eso1 peptide (TCR-T), (2) T cells expressing an enhanced receptor consisting of a PD1 variant (56#-108 variant, i.e., variant 6) and CD28, and a TCR targeting the HLA-Eso1 peptide (108-TCRT), and (3) T cells expressing GFP (green fluorescent protein) as a control (GFP-T).

[0170] First, PD1+ T cells were prepared. Frozen whole blood cells from healthy donors were collected, rapidly thawed in a 37°C water bath, and added to 5 volumes of prewarmed T cell culture medium. The medium was X-VIVO15 (Lonza; BE02-053Q) containing 0.5% human serum albumin (Xin Ji Er, Beijing SL Pharmaceutical Co. Ltd.). After thorough mixing, the cells were centrifuged at 400g for 5 minutes, and the supernatant was removed. The cells were resuspended in PBS-0.5% HSA and diluted to 10 8 The cells were adjusted to a density of 100 total cells / ml. Biotin-labeled anti-PD1 antibody Biotin-anti-PD1 (Biolegend, product code 329934) was added and incubated at 4°C for 20 minutes, followed by washing twice with PBS-0.5% HSA. Anti-Biotin Microbeads (Miltenyi Biotec, product code 130-090-485) were added, mixed well, and incubated at 4°C for 20 minutes. The cells were washed twice with PBS-0.5% HSA. PD1 was isolated using an LS sorting column (Miltenyi Biotec, product code 130-042-401). + T cells were sorted. The obtained PD1 + T cells were cultured with reference to the method described in Example 4.

[0171] To prepare TCR-T cells, a lentiviral vector pLV2-TCR containing a TCR coding sequence was prepared by the method described in Example 4. The TCR is a T cell receptor that specifically recognizes the HLA*A0201 / ESO1 peptide complex, and the coding sequence is shown in SEQ ID NO: 41. Next, the PD1 + T cells were infected with the lentivirus to obtain TCR-T cells.

[0172] To prepare T cells expressing the enhanced receptor, a lentiviral vector containing the coding sequence of the PD1 variant enhanced receptor PD1-CD28 (abbreviated as 108-CD28) and the coding sequence of a TCR was prepared according to the method described in Example 4. The lentivirus was then used to transfect the T cells with the PD1 variant enhanced receptor PD1-CD28 (abbreviated as 108-CD28). + T cells were infected using the method described in Example 4 to obtain 108-TCR T cells. The gene sequence of 108-TCR is shown in SEQ ID NO: 42. The transmembrane domain of the enhanced receptor was the transmembrane domain derived from PD1.

[0173] To detect the survival of target cells in the killing assay, we used real-time label-free cell analysis (RTCA). Specifically, J82Eso1PDL1 target cells were seeded into a 96-well culture plate (100 μl per well, target cell concentration 8 x 10). 4 After 18 hours, TCR-T cells or TCR-T cells modified with enhanced receptors (108 TCR-T) were added to the corresponding wells, and the proliferation of target cells was observed and recorded. The results are shown in Figure 6.

[0174] In Figure 6, the cell index on the ordinate reflects the adhesion status of cells, with a higher value indicating a higher number of adherent cells, which also means a higher number of live cells and a better proliferation status. The abscissa represents time.

[0175] The data in Figure 6 show that all TCR-T cells expressing the enhanced receptors exhibited stronger tumor cell suppression than TCR-T cells alone. J82Eso1PDL1 cells cocultured with GFP-T cells, a control cell line lacking a TCR, exhibited some nonspecific killing but maintained a proliferative state (Figure 6, blue curve); in contrast, 82Eso1PDL1 cells cocultured with Eso1-TCR-T cells exhibited significant inhibition of proliferation (green curve). The number of target cells cocultured with TCR-T cells expressing the enhanced receptors of the present invention (108-TCRT) was significantly reduced, indicating target cell killing (Figure 6, red curve).

[0176] Information about キーショック SEQ ID NO: 1 (PD1-wt) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDPRWNPPTFSPALLVVTEGDNATFCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFVRTVQLPNGRDHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQTLVVGVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPVPPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL SEQ ID NO: 2 (Mut1 / 56#) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDPRWNPPTFSPALLVVTEGDNATFCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFVRTVQLPNGRDHMSVVRARRNDSGTYLCGAISLSTPVWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 3 (Mut2 / 56#-2) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFCSFSNTSESFVLNWYRMSPSNQTDKLAAFPLFNLPGQDCRFRVTQLPNGRDHMSVVRARRNDSGTYLCGAISLSTPVWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 4 (Mut3 / 56#-3) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFCSFSNTSESFVLNWYRMSPSNQTDKLAAFPWMFFCPGQDCRFRVTQLPNGRDHMSVVRARRNDSGTYLCGAISLSTPVWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 5 (Mut4 / 56#-15) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFRNCTQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLSTPVWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 6 (Mut5 / 56#-16) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFFTVSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLSTPVWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 7 (Mut6 / 56#-108) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRINRSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLSTPVWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 8 (Mut7 / 56#-2-129A132A) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPFLFNLPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLATPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 9 (Mut8 / 56#-15-38) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFRNCTQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLGTPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 10 (Mut9 / 56#-15-61) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFRNCTQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLATPGWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 11 (Mut10 / 56#-15-69) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFRNCTQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLQTPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 12 (Mut11 / 56#-16-68) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFFTVSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLYTPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 13 PD1_49# MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLHFRFNIKESLRAELRVTERRAEVPTAH SEQ ID NO: 14 PD1_110# MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLSEPIVIKESLRAELRVTERRAEVPTAH SEQ ID NO: 15 PD1_49#_93 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRLCVFQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLHFRFNIKESLRAELRVTERRAEVPTAH SEQ ID NO: 16 PD1_49#_260 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDSFEVRQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLHFRFNIKESLRAELRVTERRAEVPTAH SEQ ID NO: 17 PD1_110#_48 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDLYLKYQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLSEPIVIKESLRAELRVTERRAEVPTAH SEQ ID NO: 18 56#-15-7 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFRNCTQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLWTPMWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 19 56#-16-12 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFFTVSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLHTPFWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 20 56#-16-13 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFFTVSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLGTPFWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 21 56#-16-50 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFFTVSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLITPGWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 22 56#-108-51 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRINRSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLTTPFWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 23 56#-108-60 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRINRSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLVTPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 24 56#-108AA MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRINRSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLATPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 25 56#-15AA MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFRNCTQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLATPAWIKESLRAELRVTERRAEVPTAH SEQ ID NO: 26 56#-16AA MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDFFTVSQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLATPAWIKESLRAELRVTERRAEVPTAH

Claims

1. A PD-1 variant, (1) the PD-1 variant has the ability to bind to the human PD-L1 polypeptide set forth in SEQ ID NO: 28; and (2) The PD-1 variant does not bind to one or more anti-PD-1 antibodies selected from the group consisting of sintilimab, nivolumab, camrelizumab, pembrolizumab, and toripalimab; A PD-1 variant characterized by:

2. 2. The PD-1 variant of claim 1, wherein, compared to a wild-type human PD-1 polypeptide set forth in SEQ ID NO: 1, the PD-1 variant comprises a mutation at one or more amino acid positions selected from E84, D85, R86, S87, Q88, P89, G90, A129, P130, K131, A132, and Q133, wherein the amino acid positions are numbered with reference to the amino acid sequence set forth in SEQ ID NO:

1.

3. Relative to the wild-type human PD-1 polypeptide set forth in SEQ ID NO: 1, the PD-1 variants include: E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I, S87L, S87Y; Q88L, Q88F, Q88N, Q88T, Q88C, Q88E; P89C, P89V, P89R, P89K; G90T, G90S, G90F, G90R, G90Y; A129S, The PD-1 variant of claim 1 or 2, comprising one or more amino acid mutations selected from the group consisting of: A129G, A129Q, A129Y, A129H, A129W, A129V, A129T, A129I; P130T, P130F, P130E; K131P, K131R; A132V, A132G, A132F, A132I, A132M; Q133W, Q133N, Q133V.

4. 4. The PD-1 variant of claim 2 or 3, wherein, compared to a wild-type human PD-1 polypeptide having SEQ ID NO: 1, the PD-1 variant contains a mutation at one or more amino acid positions selected from E84, D85, R86, S87, Q88, P89, and G90, and simultaneously contains a mutation at one or more amino acid positions selected from A129, P130, K131, A132, and Q133.

5. Compared to the wild-type human PD-1 polypeptide shown in SEQ ID NO: 1, the PD-1 variants have the following structures: (1)-(25): (1) A129S, P130T, K131P, A132V, and Q133W; (2) E84F, D85L, R86F, S87N, Q88L, A129S, P130T, K131P, A132V, and Q133W; (3) E84W, D85M, R86F, S87C, Q88F, A129S, P130T, K131P, A132V, and Q133W; (4) R86F, S87R, Q88N, P89C, G90T, A129S, P130T, K131P, A132V, and Q133W; (5) R86F, S87F, Q88T, P89V, G90S, A129S, P130T, K131P, A132V, and Q133W; (6) S87I, Q88N, P89R, G90S, A129S, P130T, K131P, A132V, and Q133W; (7) E84F, D85L, R86F, S87N, Q88L, P130T, K131P, and Q133W; (8) R86F, S87R, Q88N, P89C, G90T, A129G, P130T, K131P, and Q133W; (9) R86F, S87R, Q88N, P89C, G90T, P130T, K131P, A132G, and Q133W; (10) R86F, S87R, Q88N, P89C, G90T, A129Q, P130T, K131P, and Q133W; or (11) R86F, S87F, Q88T, P89V, G90S, A129Y, P130T, K131P, and Q133W; (12) A129H, P130F, K131R, A132F, and Q133N; (13) A129S, P130E, K131P, A132I, and Q133V; (14) S87L, Q88C, P89V, G90F, A129H, P130F, K131R, A132F, and Q133N; (15) R86S, S87F, Q88E, P89V, G90R, A129H, P130F, K131R, A132F, and Q133N; (16) R86L, S87Y, Q88L, P89K, G90Y, A129S, P130E, K131P, A132I, and Q133V; (17) R86F, S87R, Q88N, P89C, G90T, A129W, P130T, K131P, A132M, and Q133W; (18) R86F, S87F, Q88T, P89V, G90S, A129H, P130T, K131P, A132F, and Q133W; (19) R86F, S87F, Q88T, P89V, G90S, A129G, P130T, K131P, A132F, and Q133W; (20) R86F, S87F, Q88T, P89V, G90S, A129I, P130T, K131P, A132G, and Q133W; (21) S87I, Q88N, P89R, G90S, A129T, P130T, K131P, A132F, and Q133W; (22) S87I, Q88N, P89R, G90S, A129V, P130T, K131P, and Q133W; (23) S87I, Q88N, P89R, G90S, P130T, K131P, and Q133W; (24) R86F, S87R, Q88N, P89C, G90T, P130T, K131P, and Q133W; (25) R86F, S87F, Q88T, P89V, G90S, P130T, K131P, and Q133W, The PD-1 variant of any one of claims 1 to 4, characterized in that it contains or only contains a combination of amino acid mutations selected from one set of:

6. The PD-1 variant of any one of claims 1 to 5, characterized in that the PD-1 variant does not contain an intracellular domain or does not contain both a transmembrane domain and an intracellular domain.

7. The PD-1 variant of any one of claims 1 to 6, characterized in that the variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-26, or an amino acid sequence having at least 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 2-26, or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 2-26, or an amino acid sequence having at least 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 2-26.

8. The PD-1 variant does not bind to at least two or at least three anti-PD-1 antibodies selected from the group consisting of sintilimab, nivolumab, camrelizumab, pembrolizumab, and toripalimab. The PD-1 variant according to any one of claims 1 to 7.

9. A fusion protein comprising a PD-1 variant according to any one of claims 1 to 8.

10. 10. The fusion protein according to claim 9, further comprising an immunoglobulin Fc fragment, preferably said immunoglobulin Fc fragment being of mammalian, preferably human origin.

11. 10. The fusion protein of claim 9, wherein the fusion protein comprises the PD-1 variant as an extracellular domain, wherein the PD-1 variant does not comprise an intracellular domain, and further wherein the fusion protein comprises an intracellular signaling domain derived from a costimulatory molecule, and optionally, the fusion protein comprises a transmembrane domain of PD-1.

12. An isolated nucleic acid molecule, characterized in that it encodes a PD-1 variant according to any one of claims 1 to 8 or a fusion protein according to any one of claims 9 to 11.

13. The nucleic acid molecule of claim 12, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 56-61 or a nucleotide sequence having at least 85% homology to the nucleotide sequence set forth in any one of SEQ ID NOs: 56-61.

14. An expression vector comprising the isolated nucleic acid molecule of claim 12 or 13.

15. A host cell comprising an isolated nucleic acid molecule according to claim 12 or 13, or an expression vector according to claim 14.

16. A pharmaceutical composition comprising (1) a PD-1 variant described in any one of claims 1 to 8 or a fusion protein described in any one of claims 9 to 11, and (2) a pharmaceutically acceptable carrier.

17. Use of a PD-1 variant according to any one of claims 1 to 8 or a fusion protein according to any one of claims 9 to 11 for the preparation of a medicament for the treatment of cancer.

18. A drug combination comprising: (1) a PD-1 variant described in any one of claims 1-8, or a fusion protein described in any one of claims 9-11, and (2) an anti-PD-1 antibody, wherein the anti-PD-1 variant does not bind to the anti-PD-1 antibody.

19. A cell modified to express the fusion protein of any one of claims 9 to 11.

20. The cell of claim 9, wherein the cell is an immune cell, and the immune cell is derived from a peripheral blood mononuclear cell or a tumor-infiltrating lymphocyte before being modified.

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