A PD1 variant and its use

Abstract

The present invention provides a PD1 variant containing an ITSM motif mutation; 1) the ITSM motif of the PD1 variant is an activating ITSM motif; or 2) the ITSM motif of the PD1 variant contains partial amino acid residues or fragments of an activating ITSM motif. Wild-type PD1 mediates downstream inhibitory signals, which are not conducive to CAR-T or TCR-T tumor therapy; PD1 knockout only reduces PD1-mediated inhibitory signals. The PD1 variant of the present invention can enhance the anti-tumor function of CAR-T or TCR-T when co-expressed with EAT-2.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a PD1 variant and uses thereof. Background Art

[0002] Programmed death receptor 1 (PD1) is an important inhibitory immune receptor in T cells. The T cell activation process upregulates the expression of PD1, and the PD1-mediated downstream signaling pathway can inhibit activation-related signaling pathways (such as TCR and CD28 signaling pathways), thereby inhibiting T cell activation. In the tumor microenvironment, tumor cells often highly express the PD1 ligand PDL1 and inhibit the anti-tumor activity of T cells by activating the PD1 signal of T cells. Clinically, blocking the binding of PD1 and PDL1 through PD1 or PDL1 antibodies can generally restore the anti-tumor activity of T cells in the tumor microenvironment, thereby achieving the effect of tumor treatment. There have been no reports of restoring T cell anti-tumor activity or further activating T cells by changing its inhibitory nature through mutation of PD1. Summary of the Invention

[0003] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide PD1 variants and uses thereof.

[0004] To achieve the above objectives and other related objectives, the present invention provides a PD1 variant in a first aspect, wherein the PD1 variant contains an ITSM motif mutation:

[0005] 1) The ITSM motif of the PD1 variant is an activated ITSM motif; or

[0006] 2) The ITSM motif of the PD1 variant contains partial amino acid residues or fragments of an activated ITSM motif.

[0007] The second aspect of the present invention provides a polynucleotide encoding the aforementioned PD1 variant.

[0008] The third aspect of the present invention provides a nucleic acid construct, which contains the aforementioned polynucleotide and is capable of expressing the PD1 variant.

[0009] A fourth aspect of the present invention provides a lentiviral vector system, comprising the aforementioned nucleic acid construct and lentiviral vector auxiliary components.

[0010] A fifth aspect of the present invention provides a T cell, wherein the T cell expresses the aforementioned PD1 variant.

[0011] The use of the aforementioned PD1 variants or polynucleotides, or nucleic acid constructs, or lentiviral vector systems in the preparation of any one or more of the following products: (1) preparing T cells; (2) reducing the inhibition of T cells by the PD-L1 / PD1 signaling pathway in the tumor microenvironment; (3) enhancing the anti-tumor function of T cells.

[0012] The use of the aforementioned PD1 variants or polynucleotides, or nucleic acid constructs, or lentiviral vector systems, or T cells in the preparation of tumor treatment products or anti-chronic infection products.

[0013] As described above, the PD1 variants and uses thereof of the present invention have the following beneficial effects:

[0014] Wild-type PD1 mediates downstream inhibitory signals, which is not conducive to CAR-T or TCR-T tumor treatment; PD1 knockout only reduces PD1-mediated inhibitory signals. The PD1 variant of the present invention can enhance the anti-tumor function of CAR-T or TCR-T when co-expressed with EAT-2.

[0015] Considering that the Immunoreceptor Tyrosine Switch Motif (ITSM) is the main motif for PD1 to mediate downstream inhibitory signals, tyrosine phosphorylation of ITSM can recruit SHP2, which, as a phosphatase, can dephosphorylate TCR or CD28 downstream signaling molecules, thereby inhibiting the immune function of T cells. The present invention attempts to replace the ITSM motif of PD1 with the ITSM motif of other immune receptors (mainly the SLAM family). It is unexpectedly found that the complete or partial replacement of the ITSM motif of PD1 with an activating ITSM motif can change the immunosuppressive properties of PD1, and even reverse its immunosuppressive properties, turning it into an immune receptor with a certain degree of activation, enabling it to mediate downstream activation signals when EAT2 is expressed, further activating T cells, thereby achieving the purpose of anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Raji cells incubated with SEE (30ng / ml) stimulated Jurkat cells overexpressing vector control, wild-type PD1, and PD1 variants. 24 hours later, the concentration of IL-2 in the supernatant was detected by ELISA kit. The IL-2 produced by Raji cells expressing PD-L1 was normalized according to the amount of IL-2 produced by Raji cells that did not express PD-L1. The relative amount of IL-2 produced can characterize the inhibitory ability of PD1 and variants. In the figure, vector control is Jurkat cells expressing vector control, and the others are Jurkat cells expressing wild-type PD1 and different PD1 variants.

[0017] Figure 2: Detection of the relative IL-2 production of Jurkart cells expressing PD1-WT, PD1-tailless and PD1-M2 under the action of PD-L1.

[0018] Figure 3 : The relative IL-2 production of Jurkat cells expressing PD1 variants obtained by partial mutation of the ITSM motif of PD1-WT was measured in response to PD-L1. Panel A measures the relative IL-2 production of Jurkat cells in response to PD-L1 after replacing four amino acid residues on the left (PD1-M2_L) or right (PD1-M2_R) sides of the PD1 ITSM with the corresponding amino acid residues from the second ITSM of SLAMF1. Panel B shows the relative IL-2 production of Jurkat cells in response to PD-L1 after further replacing any two amino acid residues at the left non-conserved sites.

[0019] Figure 4 : The relative IL-2 production of Jurkart cells expressing vector control (vectorcontrol), PD1-WT, PD1-M1 and PD1-M2 under the action of PD-L1 was detected in the case of co-expression of vector control (VC) and EAT-2.

[0020] Figure 5 A shows the recruitment of SHP2 by PD1-WT, PD1-M1, and PD1-M2 in Jurkat cells under the influence of PD-L1. B shows the recruitment of SHP2 and EAT-2 by PD1-WT and PD1-M2 in Jurkat cells co-expressing EAT-2 at different time points under the influence of PD-L1. C shows the effects of expression vector control (VC), PD1-WT, and PD1-M2 on the phosphorylation of downstream signaling molecules in Jurkat cells co-expressing EAT-2.

[0021] In the accompanying figures, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, no significant difference. DETAILED DESCRIPTION

[0022] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0024] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0025] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0026] The PD1 variant according to one embodiment of the present invention contains an ITSM motif mutation:

[0027] 1) The ITSM motif of the PD1 variant is an activated ITSM motif; or

[0028] 2) The ITSM motif of the PD1 variant contains partial amino acid residues or fragments of an activated ITSM motif.

[0029] The activating ITSM motif may be derived from NTRK2, CXADR, CD244, SLAMF1, CD84, SLAMF6, SLAMF7 or KDR.

[0030] The amino acid sequence of the activated ITSM motif is shown in any one of SEQ ID NOs: 2-16.

[0031] The details are shown in Table 1:

[0032] Table 1

[0033]

[0034]

[0035] The PD1 variant is of human origin and has PD-L1 binding activity.

[0036] Compared with wild-type PD1, the PD1 variant weakens the inhibitory effect on IL-2 secretion produced by immune cells after stimulation with PD-L1; alternatively, the PD1 variant promotes IL-2 secretion by immune cells after stimulation with PD-L1.

[0037] In one embodiment, the amino acid sequence of the PD1 variant is shown in any one of SEQ ID NOs: 18-32.

[0038] PD1-NTRK2 (SEQ ID NO: 18):

[0039] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP YSTDYYRVG FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0040] PD1-CXADR (SEQ ID NO: 19):

[0041] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCV PSKTQYNQVP FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0042] PD1-CD244-ITSM1 (SEQ ID NO: 20):

[0043] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP FLTIYEDVK FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0044] PD1-CD244-ITSM2(SEQ ID NO:21):

[0045] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP GSTIYSMIQ FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0046] PD1-CD244-ITSM3(SEQ ID NO:22):

[0047] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP AYTLYSLIQ FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0048] PD1-CD244-ITSM4(SEQ ID NO:23):

[0049] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP NSTIYEVIG FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0050] PD1-SLAMF1-ITSM1(SEQ ID NO:24):

[0051] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP SLTIYAQVQ FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0052] PD1-SLAMF1-ITSM2(SEQ ID NO:25):

[0053] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP SITVYASVT FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0054] PD1-CD84-ITSM1(SEQ ID NO:26):

[0055] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP KKTIYTYIM FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0056] PD1-CD84-ITSM2(SEQ ID NO:27):

[0057] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP VNTVYSEVQ FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0058] PD1-SLAMF6-ITSM1(SEQ ID NO:28):

[0059] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP NNTVYASVT FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0060] PD1-SLAMF6-ITSM2(SEQ ID NO:29):

[0061] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP TITIYSTIN FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0062] PD1-SLAMF7-ITSM1(SEQ ID NO:30):

[0063] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP ENTEYDTIP FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0064] PD1-SLAMF7-ITSM2(SEQ ID NO:31):

[0065] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP ANTVYSTVE FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0066] PD1-KDR(SEQ ID NO:32):

[0067] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP LKTGYLSIV FPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0068] Furthermore, in the scheme 2), based on the wild-type PD1 sequence, the mutation site of the ITSM motif of the PD1 variant is selected from one or more of the following sites: 244, 245, 246, 247, 249, 250, 251, and 252.

[0069] The ITSM motif of wild-type PD1 is shown in SEQ ID NO: 1. Specifically, EQTEYATIV.

[0070] Furthermore, the amino acid sequence of the wild-type PD1 is shown in SEQ ID NO: 17. Specifically,

[0071] PD1-WT:

[0072] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL.

[0073] Specifically, the ITSM motif of the wild-type PD1 sequence is shown in SEQ ID NO: 1, which is: EQTEYATIV, which is at positions 244-252 of the amino acid sequence of the wild-type PD1.

[0074] Further,

[0075] The mutation at position 244 is selected from any one of E244A, E244R, E244N, E244D, E244C, E244Q, E244G, E244H, E244I, E244L, E244K, E244M, E244F, E244P, E244S, E244T, E244W, E244Y, and E244V.

[0076] The mutation at position 245 is selected from any one of Q245A, Q245R, Q245N, Q245D, Q245C, Q245E, Q245G, Q245H, Q245I, Q245L, Q245K, Q245M, Q245F, Q245P, Q245S, Q245T, Q245W, Q245Y, and Q245V.

[0077] The mutation at position 246 is selected from any one of T246A, T246R, T246N, T246D, T246C, T246Q, T246G, T246H, T246I, T246L, T246K, T246M, T246F, T246P, T246S, T246E, T246W, T246Y, and T246V.

[0078] The mutation at position 247 is selected from any one of E247A, E247R, E247N, E247D, E247C, E247Q, E247G, E247H, E247I, E247L, E247K, E247M, E247F, E247P, E247S, E247T, E247W, E247Y, and E247V.

[0079] The mutation at position 249 is selected from any one of A249E, A249R, A249N, A249D, A249C, A249Q, A249G, A249H, A249I, A249L, A249K, A249M, A249F, A249P, A249S, A249T, A249W, A249Y, and A249V.

[0080] The mutation at position 250 is selected from any one of T250A, T250R, T250N, T250D, T250C, T250Q, T250G, T250H, T250I, T250L, T250K, T250M, T250F, T250P, T250S, T250E, T250W, T250Y, and T250V.

[0081] The mutation at position 251 is selected from any one of I251A, I251R, I251N, I251D, I251C, I251Q, I251G, I251H, I251E, I251L, I251K, I251M, I251F, I251P, I251S, I251T, I251W, I251Y, and I251V.

[0082] The mutation at position 252 is selected from any one of V252A, V252R, V252N, V252D, V252C, V252Q, V252G, V252H, V252I, V252L, V252K, V252M, V252F, V252P, V252S, V252T, V252W, V252Y, and V252E.

[0083] In a preferred embodiment, the mutations include at least Q245I and E247V mutations.

[0084] In one embodiment, the amino acid sequence of the PD1 variant is shown in any one of SEQ ID NOs: 33-34.

[0085] Specifically:

[0086] PD1-M2_L (SEQ ID NO: 33):

[0087] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP SITVY ATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0088] PD1-Q245I / E247V (SEQ ID NO: 34):

[0089] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYL CGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE I T V YATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL.

[0090] The polynucleotide according to one embodiment of the present invention encodes the aforementioned PD1 variant.

[0091] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0092] The polynucleotides described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order.

[0093] The nucleic acid construct of one embodiment of the present invention comprises the aforementioned polynucleotide and is capable of expressing the PD1 variant.

[0094] The nucleic acid construct also includes one or more regulatory sequences operably linked to the aforementioned polynucleotide sequence. The coding sequence of the PD1 variant described herein can be manipulated in a variety of ways to ensure expression of the protein. Prior to insertion into a vector, the nucleic acid construct can be manipulated according to the specific expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0095] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0096] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0097] The regulatory sequence may also be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0098] Preferably, the nucleic acid construct is a vector.

[0099] Expression of a polynucleotide encoding a PD1 variant is typically achieved by operably linking the polynucleotide encoding the PD1 variant to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0100] The polynucleotide sequence encoding the PD1 variant of the present invention can be cloned into many types of vectors. For example, it can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Furthermore, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0101] More preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3'LTR, a 5'LTR and the aforementioned polynucleotide.

[0102] An example of a suitable promoter is an early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence high-level expression that can be operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, it is also possible to consider the use of inducible promoters. The use of inducible promoters provides a molecular switch that can open the expression of the polynucleotide sequence that can be operably connected to an inducible promoter when the deadline is expressed, and close expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0103] To assess expression of a PD1 variant or portion thereof, the expression vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by a viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0104] The reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of the regulatory sequence. After DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known technology to prepare or commercially obtain.

[0105] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0106] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0107] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0108] The aforementioned nucleic acid construct also contains a polynucleotide encoding the EAT-2 protein, and is capable of simultaneously expressing the PD1 variant and the EAT-2 protein.

[0109] The two sequences can be linked by an internal ribosome entry site (IRES) to express two proteins simultaneously in one vector.

[0110] The polynucleotide sequence encoding the EAT-2 protein is shown in SEQ ID NO: 35; specifically:

[0111] atggatctgccttactaccatggacgtctgaccaagcaagactgtgagaccttgctgctcaaggaaggggtggatggcaactttcttttaagagacagcg agtcgataccaggagtcctgtgcctctgtgtctcgtttaaaaatattgtctacacataccgaatcttcagagagaaacacgggtattacaggatacagac tgcagaaggttctccaaaacaggtctttccaagcctaaaggaactgatctccaaatttgaaaaaccaaatcaggggatggtggttcaccttttaaagcca ataaagagaaccagccccagcttgagatggagaggattgaaattagagttggaaacatttgtgaacagtaacagcgattatgtggatgtcttgccttga.

[0112] The lentiviral vector system according to one embodiment of the present invention comprises the aforementioned nucleic acid construct and lentiviral vector auxiliary components.

[0113] The lentiviral auxiliary components include lentiviral packaging plasmids and cell lines.

[0114] The lentiviral vector system is formed by viral packaging of the aforementioned nucleic acid construct with the assistance of a lentiviral packaging plasmid and a cell line. The method for constructing the lentiviral vector system is a commonly used method in the art.

[0115] The T cells according to one embodiment of the present invention express the aforementioned PD1 variant.

[0116] Optionally, the T cells contain the aforementioned polynucleotide, or contain the aforementioned nucleic acid construct, or are infected with the aforementioned lentiviral vector system.

[0117] The use of the aforementioned PD1 variants or polynucleotides, or nucleic acid constructs, or lentiviral vector systems in the preparation of any one or more of the following products: (1) preparing T cells; (2) reducing the inhibition of T cells by the PD-L1 / PD1 signaling pathway in the tumor microenvironment; (3) enhancing the anti-tumor function of T cells.

[0118] The use of the aforementioned PD1 variants or polynucleotides, or nucleic acid constructs, or lentiviral vector systems, or T cells in the preparation of tumor treatment products or anti-chronic infection products.

[0119] The present invention also provides a method for treating tumors, comprising administering the aforementioned PD1 variant or polynucleotide, or nucleic acid construct, or lentiviral vector system, or T cells to a subject.

[0120] The subject can be a mammal or a mammalian cancer cell. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like. The primate is preferably a monkey, an ape, or a human. The cancer cell can be an ex vivo cancer cell.

[0121] The subject may be a patient suffering from cancer or an individual expecting treatment for cancer, or the subject may be an ex vivo cancer cell of a patient suffering from cancer or an individual expecting treatment for cancer.

[0122] The aforementioned PD1 variants or polynucleotides, or nucleic acid constructs, or lentiviral vector systems, or T cells can be administered to a subject before, during, or after receiving lung cancer treatment.

[0123] The present invention also provides a method for combating chronic infection, comprising administering the aforementioned PD1 variant or polynucleotide, or nucleic acid construct, or lentiviral vector system, or T cells to a subject.

[0124] The subject can be a mammal or a mammalian infected cell. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like. The primate is preferably a monkey, an ape, or a human. The infected cell can be an in vitro infected cell.

[0125] The subject may be a patient suffering from a chronic infection or an individual suffering from a chronic infection who is awaiting treatment for the chronic infection, or the subject may be an ex vivo infected cell of a patient suffering from a chronic infection or an individual suffering from a chronic infection who is awaiting treatment for the chronic infection.

[0126] The aforementioned PD1 variants or polynucleotides, or nucleic acid constructs, or lentiviral vector systems, or T cells can be administered to a subject before, during, or after receiving treatment for chronic infection.

[0127] The T cells described in the present invention may be CAR-T cells or TCR-T cells.

[0128] The genetically modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may include genetically modified T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0129] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0130] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that a pharmaceutical composition comprising the genetically modified T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6 The T cell composition can be administered at a dose of 10 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical field by monitoring the patient's disease signs and adjusting treatment accordingly.

[0131] Administration of the subject compositions can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0132] In some embodiments of the present invention, the genetically modified T cells of the present invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment can be combined with various radiotherapy agents, including cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin, and mycophenolic acid. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

[0133] As used herein, "tumor treatment" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in tumor cell count, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer. The tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, melanoma, kidney cancer, pancreatic cancer, skin cancer, glioma, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, and leukemia.

[0134] As used herein, "anti-chronic infection" refers to a biological effect that can be expressed as a reduction in chronic viral load, alleviation of damage caused by chronic viral infection, or improvement in various physiological symptoms associated with chronic viral infection. Chronic infection-related diseases may include hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV).

[0135] "Patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, in which an immune response can be elicited. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0136] Example 1 Construction of expression vectors for PD1 variants

[0137] Using the second ITSM motif of SLAMF1 (SEQ ID NO: 9) as an example, a PD1 variant was generated by gene synthesis, with its open coding frame sequence shown in SEQ ID NO: 36 (PD1-SLAMF1-ITSM2). Compared to the 9-amino acid ITSM motif of wild-type PD1 (centered around tyrosine and extending four amino acid residues before and after, EQTEYATIV; SEQ ID NO: 1), the ITSM motif of this variant is the second ITSM motif of SLAMF1 (SEQ ID NO: 9). The resulting PD1 variant sequence was further inserted into the pHAGE expression vector (pHAGE-fullEF1a-MCS-IRES-ZsGreen) by enzyme digestion (NheI and BamHI sites) and ligation.

[0138] ATGGTACCGTGCACGCTGCTCCTGCTGTTGGCGGCCGCCCTGGCTCCGACTCAGACCCGCGCGAAGTACCCCTACGACGTGCCCGACTACGCCAGCCTGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTTCGATCACGGTGTATGCCAGCGTTACCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA(SEQ ID NO:36)。

[0139] The construction method of other PD1 variants is the same as that of PD1-SLAMF1-ITSM2, with the only difference being that, compared with the 9-amino acid ITSM motif of wild-type PD1 (centered on tyrosine and extending 4 amino acid residues before and after, SEQ ID NO: 1), the ITSM motif of this variant is another activated ITSM motif in Table 1, thereby obtaining the ability to express the various PD1 variants described in SEQ ID NOs: 18-32.

[0140] Example 2 Construction and transformation of PD1 and EAT-2 co-expression vector

[0141] The human EAT-2 open reading frame sequence (SEQ ID NO: 35) was obtained by reverse transcription and PCR amplification. The EAT-2 open reading frame sequence was constructed into the expression vector of the PD1 variant (PD1-SLAMF1-ITSM2) obtained in Example 1. The two sequences were connected by the internal ribosome entry site (IRES) in the pHAGE vector, and the two proteins were expressed simultaneously in one vector.

[0142] Example 3 Verification of the Effect of PD1 Variants on Jurkat Cell Function in the Jurkat T Cell Line

[0143] Wild-type PD1 and the PD1 variants described in SEQ ID NOs: 18-32 were constructed into a pHAGE vector. The PD1 expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2G at a ratio of 10:7.5:3.5, respectively, and then transformed into 293FT cells using calcium phosphate transfection to produce lentiviral particles. The lentiviral supernatant was used to infect Jurkat cells to generate Jurkat cell lines expressing the modified PD1.

[0144] Jurkat cell lines with similar cell surface PD1 expression levels were obtained by sorting, and Jurkat cell function experiments were performed. 100,000 Jurkat cells expressing PD1 were co-cultured with 50,000 Raji cells expressing PD-L1 or vector control cells that were pre-incubated with superantigen SEE in a round-bottom 96-well plate for 24 hours. The cell supernatant was collected and the concentration of IL-2 in the supernatant was detected using an IL-2 ELISA kit. The IL-2 produced by Raji cells expressing PD-L1 was normalized according to the amount of IL-2 produced by Raji cells that did not express PD-L1. The relative amount of IL-2 produced can characterize the inhibitory ability of PD1 and variants. (The higher the relative amount of IL-2 production, the weaker the inhibitory ability of PD1 and variants.) The results are as follows Figure 1As shown in the figure, after the ITSM motif of wild-type PD1 (WT) was replaced with different ITSM motifs, the inhibitory function of PD-L1 on the secretion of IL-2 by Jurkat cells was weakened to varying degrees, among which the inhibitory function of the PD1 variant transformed into the second ITSM sequence of SLAMF1 (PD1-SLAMF1-ITSM2) was most significantly weakened.

[0145] To further examine the extent of reduced inhibitory function of the PD1 variant engineered as the second ITSM of SLAMF1 (SEQ ID NO: 25, labeled PD1-M2 in the figure), the aforementioned method of this example was used to detect the relative IL-2 production of Jurkart cells expressing PD1-WT, PD1-tailless, and PD1-M2 under the action of PD-L1, using a non-functional intracellular truncated PD1 (PD1-tailless) (SEQ ID NO: 37) as a control; Figure 2 The results showed that the inhibition of IL-2 secretion by PD1-M2 in Jurkat cells was almost the same as that of the non-functional PD1-tailless control.

[0146] PD1-tailless (SEQ ID NO: 37):

[0147] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLP NGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIG.

[0148] Using the method described above in this example, the present invention also located the key amino acid residues in PD1-M2 that affect the inhibitory function. Taking tyrosine as the zero point, the four amino acid residues on the left or right of the PD1 ITSM were detected to be replaced with the corresponding amino acid residues in the second ITSM of SLAMF1 to obtain PD1-M2_L (SEQ ID NO: 33) and PD1-M2_R (i.e., the wild-type PD1 was mutated as follows: T250S, I251V, V252T). The relative IL-2 production of Jurkat cells under the action of PD-L1 was then detected. The results are as follows: Figure 3 As shown in A, PD1-M2_L significantly reduces the inhibitory function.

[0149] Using the method described above in this example, the present invention also mutated the sites in the ITSM motif of wild-type PD1, taking tyrosine as the 0 point, and replaced any two amino acid residues in the non-conserved sites on the left side of the ITSM motif of wild-type PD1 with the corresponding amino acid residues in the second ITSM of SLAMF1, respectively obtaining PD1-E244S / Q245I, PD1-E244S / E247V, and PD1-Q245I / E247V, and then detecting the relative IL-2 production of Jurkat cells under the action of PD-L1. The results are shown in FIG. Figure 3 As shown in Figure 2, the -3 and -1 positions (with tyrosine as the 0 position) in the ITSM motif of wild-type PD1 were replaced with the corresponding amino acid residues in the second ITSM of SLAMF1. The resulting PD1 variant PD1-Q245I / E247V significantly reduced the inhibitory function.

[0150] Example 4: Verification of the Effect of Co-expression of Modified PD1 and EAT-2 on Jurkat Cell Function in the Jurkat T Cell Line

[0151] Lentiviral particles were produced by mixing EAT-2 and PD1 expression plasmids with packaging plasmids pSPAX2 and pMD2G at a ratio of 10:7.5:3.5, respectively, and then transfected into 293FT cells using calcium phosphate transfection. Lentiviral supernatant was used to infect Jurkat cells to generate Jurkat cell lines co-expressing EAT-2 and PD1 variants.

[0152] Jurkat cell lines with similar cell surface PD1 expression levels were obtained by sorting, and Jurkat cell function experiments were performed. 100,000 Jurkat cells were co-cultured with 50,000 Raji cells expressing PD-L1 or vector control cells that had been pre-incubated with superantigen SEE in a round-bottom 96-well plate for 24 hours. The cell supernatant was collected and the IL-2 concentration in the supernatant was detected using an IL-2 ELISA kit. The results are shown in the figure. Figure 4 As shown, replacing the wild-type PD1 ITSM motif with the first ITSM (PD1-M1 in the figure, i.e., SEQ ID NO: 24) or the second ITSM sequence (PD1-M2 in the figure, i.e., SEQ ID NO: 25) of SLAMF1 can weaken the inhibition of PD1 on IL-2 secretion of Jurkat cells, while adding EAT-2 expression can convert the inhibition of PD1 variants on IL-2 secretion of Jurkat cells into enhancement.

[0153] In order to study the mechanism of the reduced inhibitory function of PD1-M2 and the mediation of positive signals under the co-expression of EAT-2, the present invention uses immunoprecipitation and immunoblotting to detect the recruitment function of SHP2 and EAT-2 of PD1-M2 and the downstream signaling pathway. 10 million Jurkat cells and 10 million Raji cells expressing PD-L1 that were pre-incubated with superantigen SEE were mixed in an EP tube, centrifuged to allow the two cells to fully contact, and then stimulated at different time points in a 37-degree Celsius water bath. After lysing the cells, immunoprecipitation and immunoblotting experiments were performed. The results are as follows. Figure 5 As shown in A, the ability of PD1-M2 to recruit SHP2 was significantly reduced compared to PD1-WT in the absence of EAT-2 co-expression. In the case of co-expression of EAT-2, PD1-M2 recruited EAT-2 while PD1-WT recruited SHP2 ( Figure 5 B). Further detection of downstream signaling molecules revealed that under the co-expression of EAT-2, PD1-WT significantly reduced the phosphorylation of PLCγ1, while PD1-M2 significantly enhanced the phosphorylation of PLCγ1 ( Figure 5 C).

[0154] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention. Sequence Listing <110> Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences <120> A PD1 variant and its use <160> 37 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Glu Gln Thr Glu Tyr Ala Thr Ile Val 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Tyr Ser Thr Asp Tyr Tyr Arg Val Gly 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Ser Lys Thr Gln Tyr Asn Gln Val Pro 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Phe Leu Thr Ile Tyr Glu Asp Val Lys 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Gly Ser Thr Ile Tyr Ser Met Ile Gln 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Ala Tyr Thr Leu Tyr Ser Leu Ile Gln 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Asn Ser Thr Ile Tyr Glu Val Ile Gly 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Ser Leu Thr Ile Tyr Ala Gln Val Gln 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <400> 9 Ser Ile Thr Val Tyr Ala Ser Val Thr 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Lys Lys Thr Ile Tyr Thr Tyr Ile Met 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Val Asn Thr Val Tyr Ser Glu Val Gln 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Asn Asn Thr Val Tyr Ala Ser Val Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Thr Ile Thr Ile Tyr Ser Thr Ile Asn 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Glu Asn Thr Glu Tyr Asp Thr Ile Pro 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Ala Asn Thr Val Tyr Ser Thr Val Glu 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Leu Lys Thr Gly Tyr Leu Ser Ile Val 1 5 <210> 17 <211> 288 <212> PRT <213> Artificial Sequence <400> 17 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 18 <211> 288 <212> PRT <213> Artificial Sequence <400> 18 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 19 <211> 288 <212> PRT <213> Artificial Sequence <400> 19 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Ser Lys Thr Gln Tyr Asn Gln Val Pro Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 20 <211> 288 <212> PRT <213> Artificial Sequence <400> 20 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15[[ID=X]] Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Phe Leu Thr Ile Tyr Glu Asp Val Lys Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 21 <211> 288 <212> PRT <213> Artificial Sequence <400> 21 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Gly Ser Thr Ile Tyr Ser Met Ile Gln Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 22 <211> 288 <212> PRT <213> Artificial Sequence <400> 22 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Ala Tyr Thr Leu Tyr Ser Leu Ile Gln Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 23 <211> 288 <212> PRT <213> Artificial Sequence <400> 23 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Asn Ser Thr Ile Tyr Glu Val Ile Gly Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270<o000752>Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu<o000753>275 280 285<o000754><210> 24<o000755><211> 288<o000756><212> PRT<o000757><213> Artificial Sequence<o000758><400> 24<o000759>Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln<o000760>1 5 10 15<o000761>Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp<o000762>20 25 30<o000763>Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp<o000764>35 40 45<o000765> It should be noted that there seems to be a small error in the original text where " " and " " are misspelled as "<o000752>" and "<o000753>" in the translation above. The corrected translation would be: 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Asn Ser Thr Ile Tyr Glu Val Ile Gly Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 24 <211> 288 <212> PRT <213> Artificial Sequence <400> 24 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Ser Leu Thr Ile Tyr Ala Gln Val Gln Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 25 <211> 288 <212> PRT <213> Artificial Sequence <400> 25 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Ser Ile Thr Val Tyr Ala Ser Val Thr Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 26 <211> 288 <212> PRT <213> Artificial Sequence <400> 26 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Lys Lys Thr Ile Tyr Thr Tyr Ile Met Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 27 <211> 288 <212> PRT <213> Artificial Sequence <400> 27 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15<00。00884>Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Val Asn Thr Val Tyr Ser Glu Val Gln Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 28 <211> 288 <212> PRT <213> Artificial Sequence <400> 28 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Asn Asn Thr Val Tyr Ala Ser Val Thr Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 29 <211> 288 <212> PRT <213> Artificial Sequence <400> 29 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Thr Ile Thr Ile Tyr Ser Thr Ile Asn Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 30 <211> 288 <212> PRT <213> Artificial Sequence <400> 30 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Asn Thr Glu Tyr Asp Thr Ile Pro Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 31 <211> 288 <212> PRT <213> Artificial Sequence <400> 31 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Ala Asn Thr Val Tyr Ser Thr Val Glu Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 32 <211> 288 <212> PRT <213> Artificial Sequence <400> 32 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Leu Lys Thr Gly Tyr Leu Ser Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 33 <211> 288 <212> PRT <213> Artificial Sequence <400> 33 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 / Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Ser Ile Thr Val Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 34 <211> 288 <212> PRT <213> Artificial Sequence <400> 34 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45<00,01175>Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Ile Thr Val Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 35 <211> 399 <212> DNA <213> Artificial Sequence <400> 35 atggatctgc cttactacca tggacgtctg accaagcaag actgtgagac cttgctgctc 60 aaggaagggg tggatggcaa ctttctttta agagacagcg agtcgatacc aggagtcctg 120 tgcctctgtg tctcgtttaa aaatattgtc tacacatacc gaatcttcag agagaaacac 180 gggtattaca ggatacagac tgcagaaggt tctccaaaac aggtctttcc aagcctaaag 240 gaactgatct ccaaatttga aaaaccaaat caggggatgg tggttcacct tttaaagcca 300 ataaagagaa ccagccccag cttgagatgg agaggattga aattagagtt ggaaacattt 360 gtgaacagta acagcgatta tgtggatgtc ttgccttga 399 <210> 36 <211> 906 <212> DNA <213> Artificial Sequence <400> 36 atggtaccgt gcacgctgct cctgctgttg gcggccgccc tggctccgac tcagacccgc 60 gcgaagtacc cctacgacgt gcccgactac gccagcctgc caggatggtt cttagactcc 120 ccagacaggc cctggaaccc ccccaccttc tccccagccc tgctcgtggt gaccgaaggg 180 gacaacgcca ccttcacctg cagcttctcc aacacatcgg agagcttcgt gctaaactgg 240 taccgcatga gccccagcaa ccagacggac aagctggccg ccttccccga ggaccgcagc 300 cagcccggcc aggactgccg cttccgtgtc acacaactgc ccaacgggcg tgacttccac 360 atgagcgtgg tcagggcccg gcgcaatgac agcggcacct acctctgtgg ggccatctcc 420 ctggccccca aggcgcagat caaagagagc ctgcgggcag agctcagggt gacagagaga 480 agggcagaag tgcccacagc ccaccccagc ccctcaccca ggccagccgg ccagttccaa 540 accctggtgg ttggtgtcgt gggcggcctg ctgggcagcc tggtgctgct agtctgggtc 600 ctggccgtca tctgctcccg ggccgcacga gggacaatag gagccaggcg caccggccag 660 cccctgaagg aggacccctc agccgtgcct gtgttctctg tggactatgg ggagctggat 720 ttccagtggc gagagaagac cccggagccc cccgtgccct gtgtcccttc gatcacggtg 780 tatgccagcg ttacctttcc tagcggaatg ggcacctcat cccccgcccg caggggctca 840 gctgacggcc ctcggagtgc ccagccactg aggcctgagg atggacactg ctcttggccc 900 ctctga 906 <210> 37 <211> 201 <212> PRT <213> Artificial Sequence <400> 37 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly 195 200

Claims

1. A PD1 variant, characterized in that The amino acid sequence of the PD1 variant is shown in SEQ ID NO. 25, 33 or 34.

2. A polynucleotide encoding the PD1 variant according to claim 1.

3. A nucleic acid construct comprising the polynucleotide according to claim 2 and capable of expressing the PD1 variant.

4. The nucleic acid construct according to claim 3, wherein The nucleic acid construct also contains a polynucleotide encoding the EAT-2 protein, and is capable of simultaneously expressing the PD1 variant and the EAT-2 protein.

5. The nucleic acid construct according to claim 4, wherein The polynucleotide sequence encoding the EAT-2 protein is shown in SEQ ID NO:

35.

6. A lentiviral vector system, comprising the nucleic acid construct according to any one of claims 3 to 5 and lentiviral vector auxiliary components.

7. A T cell, characterized in that The T cells express the PD1 variant of claim 1.

8. The T cell according to claim 7, wherein The T cell contains the polynucleotide according to claim 2, or contains the nucleic acid construct according to any one of claims 3 to 5, or is infected with the lentiviral vector system according to claim 6.

9. The T cell according to claim 7 or 8, wherein The T cells co-express EAT-2 protein.

10. Use of the PD1 variant according to claim 1, the polynucleotide according to claim 2, the nucleic acid construct according to any one of claims 3 to 5, or the lentiviral vector system according to claim 6 in preparing a T cell product.

Images