PD-1 IR2 Applications in immunoassay or tumor treatment drugs

Abstract

This invention belongs to the field of biomedical technology, specifically relating to PD-1. IR2 Applications in immunoassay or tumor treatment drugs. The second intron of the PD-1 isoform is retained, and the PD-1 isoform contains a signal peptide, an IgV domain, and a novel short cytoplasmic tail; its protein sequence is SEQ ID NO.1. This invention elucidates a novel PD-1 isoform and explores PD-1... IR2 Expression profile and immune function of the protein in T lymphocytes. In vitro and in vivo functional studies demonstrated its inhibition of CD8. + The function of T cells promotes tumor progression and mediates resistance to PD-L1 therapy, providing a new mechanism for resistance to PD-1 / PD-L1 inhibitors and a new target for tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to PD-1. IR2 Applications in immune detection or tumor treatment drugs. Background Technology

[0002] Cancer is a general term for a group of diseases in which cells grow out of control and have the ability to metastasize to distant sites. Cancer cells are characterized by continuous proliferation, escape from growth inhibition, antagonism of cell death, maintenance of permanent replication, induction of angiogenesis, activation of invasion and metastasis, reprogramming of energy metabolism, and evasion of immune surveillance. Although there has been some progress in the 5-year survival rate of patients after cancer intervention, cancer remains one of the leading causes of death worldwide.

[0003] Over the past decade, therapies targeting the immune system have revolutionized cancer treatment. Immune checkpoint inhibitors (ICIs) work by releasing the "brakes" on T cells, triggering a robust activation of the immune system and generating an anti-tumor immune response, leading to durable remissions in patients with a variety of tumor types. Despite these successes, immunotherapy remains ineffective for most cancer patients, and among those who initially respond, some develop resistance and experience tumor recurrence over time.

[0004] Programmed death receptor-1 (PD-1, also known as CD279), encoded by the PDCD1 gene, belongs to the CD28 receptor superfamily and is a representative immune checkpoint molecule. PD-1 regulates T cell function through interactions with two natural ligands, PD-L1 and PD-L2. When PD-1 binds to its ligands, it induces phosphorylation of tyrosine residues in the ITSM and ITIM domains of the PD-1 cytoplasmic tail, thereby inhibiting T cell activation and differentiation. Under physiological conditions, PD-1 suppresses excessive activation of the immune system, maintains immune tolerance, and protects tissues from damage. However, PD-L1 expressed by tumor cells can also bind to PD-1 on the surface of T cells, mediating T cell dysfunction and leading to tumor immune escape. Targeting PD-1 can restore CD8... +T cells function to kill cancer cells. In recent years, antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for cancer treatment, opening a new chapter in tumor immunotherapy. Preliminary clinical trials of PD-1 antibodies nivolumab and pembrolizumab have demonstrated potent and durable antitumor activity with limited immunotoxicity across a wide range of cancer types, including melanoma, non-small cell lung cancer, renal cell carcinoma, and colorectal cancer. However, some issues remain to be addressed, including a large number of non-responders and immune-related adverse events, indicating that the mechanisms of tumor immune escape are still poorly understood. Therefore, identifying novel immune checkpoints, exploring their mechanisms of action, and using them as potential therapeutic targets for new drug research may provide new approaches to antitumor immunotherapy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the objective of this invention can be achieved through the following technical solutions:

[0006] A PD-1 isoform, wherein the second intron is retained, the PD-1 isoform contains a signal peptide, an IgV domain and a novel short cytoplasmic tail, and the protein sequence thereof is SEQ ID NO.1.

[0007] Application of a reagent containing a PD-1 isomer in the preparation of a kit for detecting tumors.

[0008] Furthermore, the reagent for detecting PD-1 isoforms contains an antibody that specifically binds to the PD-1 isoform, specifically Mouse lgG2b.K.

[0009] Application of a reagent containing a knockdown PD-1 isomer in the preparation of drugs for treating tumors.

[0010] Furthermore, the reagent for knocking down the PD-1 isomer contains specific primers that bind to the PD-1 isomer. The specific primers include Sh-F1, Sh-R1, Sh-F2, and Sh-R2. The sequence of Sh-F1 is SEQ ID NO.2, the sequence of Sh-R1 is SEQ ID NO.3, the sequence of Sh-F2 is SEQ ID NO.4, and the sequence of Sh-R2 is SEQ ID NO.5.

[0011] A method for constructing a PD-1 isoform knock-in mouse model, wherein the method comprises inserting a complete DNA sequence unit, including a promoter and PDCD1, into the safe site of the mouse ROSA26 gene. IR2The CDS-Flag region and PolyA tail are used; specifically, Cre recombinase is used to act on the Loxp fragment. Cre recombinase recognizes specific sites, causing deletion or recombination of gene sequences between Loxp fragments; Cd4cre tool mice have the Cre gene inserted after the CD4 cell promoter, and after hybridization with Loxp mice, mice with specific gene knock-in or knock-out only in T cells are obtained; PDCD1 is used. IR2 By crossing flox / flox mice with Cd4cre mice and using Cre recombinase, a mouse model was obtained that specifically knocks into PDCD in T cells. 1IR2 Mice.

[0012] Furthermore, the gene sequence of Loxp is SEQ ID NO.6.

[0013] A PD-1 isoform knock-in mouse model is constructed using the PD-1 isoform knock-in mouse model construction method described above.

[0014] The PD-1 isoform knock-in mouse model described above is used for research on tumor diseases.

[0015] The beneficial effects of this invention: This invention elucidates a novel PD-1 isomer and explores PD-1 IR2 Expression profile and immune function of the protein in T lymphocytes. In vitro and in vivo functional studies demonstrated its inhibition of CD8. + The function of T cells promotes tumor progression and mediates resistance to PD-L1 therapy, providing a new mechanism for resistance to PD-1 / PD-L1 inhibitors and offering a new target for tumor immunotherapy. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0017] Figure 1 For the identification of the PD-1 isomer in this invention;

[0018] Figure 2 PD-1 in this invention IR2 Preparation of monoclonal antibodies and detection of endogenous proteins;

[0019] Figure 3 PD-1 in this invention IR2 Inducible expression;

[0020] Figure 4 PD-1 in this invention IR2Expression status in T cell subsets and in cancer patients;

[0021] Figure 5 PD-1 in this invention IR2 Knock down the function of immune cells;

[0022] Figure 6 PD-1 in this invention IR2 Overexpression inhibits the function of immune cells;

[0023] Figure 7 PD-1 in this invention IR2 Construction of gene knock-in mice;

[0024] Figure 8 PD-1 in this invention IR2 Gene knock-in causes mouse CD8 + Impaired T-cell anti-tumor ability;

[0025] Figure 9 PD-1 in this invention IR2 Gene knock-in leads to resistance to PD-L1 antibody therapy;

[0026] Figure 10 PD-1 in this invention IR2 Humanized mice exhibit reduced antitumor activity. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1

[0029] Peripheral blood mononuclear cells (PBMCs) were sorted by flow cytometry. PDCD1 was amplified using reverse transcription PCR (RT-PCR) in PBMCs, T cells, B cells, and monocytes. A larger band was observed above wild-type PDCD1. Based on Sanger sequencing, this band retained a second intron, totaling 267 bp, compared to wild-type PDCD1. Figure 1 AB), we named it PD-1 IR2 Its gene diagram is shown below ( Figure 1 C). Because the retained introns contain an early termination cipher, PD-1 IR2Translated as a truncated protein. PD-1 IR2 Containing a signal peptide, an IgV domain, and a novel short cytoplasmic tail, its protein sequence is SEQ ID NO.1, and its protein structure diagram is shown below (). Figure 1 D), PD-1 IR2 Protein sequences such as ( Figure 1 .E). To further verify PD-1 IR2 Expression spectrum, design PD-1 across the second intron IR2 Specific primers ( Figure 1 F), PD-1 expression was detected in multiple human leukemia cell lines using quantitative RT-PCR (qRT-PCR), including Jurkat (T-cell leukemia cell line), Raji and Jeko-1 (B-cell lymphoma cell lines), U937 (monocytic leukemia cell line), K562 (myeloid leukemia cell line), and HL-60 (human promyelocytic leukemia cell line). The analysis showed that PD-1... IR2 It is expressed in all of these human leukemia cell lines, especially in the Jurkat and K562 cell lines where the expression level is relatively high. Figure 1 G).

[0030] Example 2

[0031] To verify endogenous PD-1 IR2 The actual existence of protein levels, using PD-1 IR2 A 10-amino acid fragment specific to the cytoplasmic tail was used as an antigen to immunize mice to prepare monoclonal antibodies. Mouse lgG2b.K was used as the antibody. The antibody was first verified to specifically bind to PD-1. IR2 Overexpression of PD-1 in HEK293T cells IR2 -GFP, using IgG, PD-1 and prepared PD-1 respectively. IR2 Monoclonal antibodies were incubated with cells, and PD-1 was detected by flow cytometry (FACS). IR2 The expression level of PD-1. Flow cytometry results showed that PD-1... IR2 Monoclonal antibodies can specifically recognize this isoform, with a detection efficiency of approximately 20%. Figure 2 A), where IgG and PD-1 antibodies served as negative and positive controls, respectively.

[0032] Wild-type Jurkat cells and a previously established PDCD1 knockout (KO) Jurkat cell line were used. After stimulation with 5 μg / ml PHA for 48 h, PD-1 was administered. IR2Monoclonal antibodies were incubated with cells, and FACS was used to detect the endogenous expression of this isoform. The results showed that PD-1 was detectable in activated Jurkat cells compared to the Jurkat-KO strain. IR2 Protein level expression ( Figure 2 B). Due to PD-1 IR2 The lack of a transmembrane segment suggests that it may be located in the cytoplasm or secreted extracellularly. To test this hypothesis, PD-1 was overexpressed in Jurkat's KO cells. IR2 Simultaneously, cell lysates and cell supernatants were collected and subjected to Western blotting experiments. Experimental results showed that PD-1... IR2 It is mainly located inside cells and is almost undetectable in the supernatant. Figure 2 C).

[0033] Example 3

[0034] Jurkat cells were activated using three classic methods. Specifically, cells were stimulated with CD3 and CD28 (5 μg / ml) for 72 h, with PMA (50 ng / ml) and iodine (1 μM) for 5 h, and with PHA (5 μg / ml) for 48 h. After stimulation, qRT-PCR was used to detect PDD-1 in Jurkat cells. IR2 mRNA levels, FACS detection of PD-1 IR2 The protein levels of PD-1 in activated Jurkat cells were observed. IR2 Both mRNA and protein levels were elevated. Figure 3 AF).

[0035] Example 4

[0036] To evaluate PD-1 IR2 The expression of T cell subsets in healthy individuals and cancer patients was analyzed using RNA-seq data downloaded from the GEO database. Results were presented as TPM (Transcripts Per Million), where TPM represents the percentage of RNA measured per million RNA molecules. The analysis showed that PD-1... IR2 CD4 counts from PBMCs from 24 healthy donors + and CD8 + PD-1 is expressed in all T cells, and PD-1 is also expressed in T cells. IR2 In CD8 + The expression level in T cells is higher than that in CD4. + T cells ( Figure 4 A). In addition, PD-1 IR2 The expression of PD-1 is correlated ( Figure 4 B). Analysis of the CD8+ T cell subsets in this database showed that PD-1IR2 In CD8 + All T cell subsets were expressed, but the expression level was lower than that of PD-1. Figure 4 CD). Compared with naive T cells, PD-1 IR2 In central memory (CM) T cells, effector memory 1 (EM1) T cells, and PD-1 + CD39 + Increased expression in T cells ( Figure 4 C), which is related to PD-1 in CD8 + The expression components in T cell subsets are consistent ( Figure 4 D). Compared to naive T cells, PD-1 IR2 Expression was significantly increased in follicular helper T cells (Tfh). Figure 4 E), which is related to PD-1 in CD4 + The expression components in the T subgroup are similar ( Figure 4 F), the above analysis results suggest that PD-1 IR2 It may regulate T cell function. In addition, another set of sequencing data analyzed PD-1 in healthy individuals and patients with cutaneous T-cell lymphoma (CTCL). IR2 The expression of PD-1 was found. IR2 The expression level of PD-1 was higher in CTCL patients than in healthy individuals, and PD-1 IR2 The expression level of PD-1 is highly correlated with tumor stage; that is, as the stage increases, the expression level of PD-1 decreases. IR2 Increased expression levels ( Figure 4 G), prompting PD-1 IR2 It may be associated with poor prognosis in patients with CTCL. Further research is needed on PDCD1 in T cells. IR2 To investigate PDCD1 expression, T-cell RNA sequencing data from 364 patient samples representing 10 different solid tumor types were analyzed. Results showed that PDCD1... IR2 PDCD1 is expressed in all of these tumor types. Figure 4 H). Tumor-infiltrating lymphocytes (TILs) were further isolated from clinical lung cancer samples, and the experimental results showed PDCD1. IR2 Both PDCD1 and PDCD1 are expressed in TILs, but there is no difference in expression. Figure 4 I).

[0037] Example 5

[0038] To investigate PD-1 IR2 Potential molecular functions of PD-1 were detected using CFSE staining and FACS techniques. IR2 Effects on Jurkat cell proliferation. Reagents were used to knock down PDCD1. IR2 The reagent used to knock down the PD-1 isomer contains specific primers that bind to the PD-1 isomer. These specific primers include Sh-F1, Sh-R1, Sh-F2, and Sh-R2. The sequence of Sh-F1 is SEQ ID NO.2, the sequence of Sh-R1 is SEQ ID NO.3, the sequence of Sh-F2 is SEQ ID NO.4, and the sequence of Sh-R2 is SEQ ID NO.5. Results showed that, compared with the control group, PD-CD1... IR2 Knockdown (KD) promoted the proliferation of Jurkat cells. Figure 5 A). Next, the secretion level of IL2 was detected using an ELISA kit, and the results showed that PDCD1 IR2 KD promotes the production of IL-2. Figure 5 B). Furthermore, immune cells were co-cultured with human non-small cell lung cancer (NCI-H1299) tumor cells, and PDCD1 was evaluated using an apoptosis detection kit and FACS technology. IR2 The knockdown of immune cells reduced their ability to kill tumor cells. Results showed that, compared to the control group, PDCD1... IR2 Knockdown Jurkat cells showed enhanced killing ability against NCI-H1299 tumor cells. Figure 5 C).

[0039] Sh-F1: 5-GATCCGcatctctgtcctctagctctTTCAAGAGAagagctagaggacagagatgCTTTTTTG-3;

[0040] Sh-R1: 5-AATTCAAAAAAGcatctctgtcctctagctctTCTCTTGAAagagctagaggacagagatgCG-3;

[0041] Sh-F2: 5-GATCCCctttgtgcccttccagagagTTCAAGAGActctctggaagggcacaaagGTTTTTTG-3;

[0042] Sh-R2: 5-AATTCAAAAAACctttgtgcccttccagagagTCTCTTGAActctctggaagggcacaaagGG-3;

[0043] Example 6

[0044] Constructing PD-1 and PD-1 IR2 Overexpression of PD-1 and PD-1 were performed in two KO strains of PD-1 via plasmid. IR2 PD-1 was used as a positive control. PD-1 and PD-2 were detected using CFSE staining and FACS techniques. IR2 The effect of PD-1 on the proliferation capacity of Jurkat cells showed that, compared with the control group, PD-1 IR2 Overexpression of [a substance] inhibited the proliferation of Jurkat cells. Figure 6 (A and D). The secretion level of IL2 was detected using an ELISA kit, indicating that PD-1... IR2 Overexpression of IL-2 reduces IL-2 production. Figure 6 (B and E). Similarly, immune cells were co-cultured with NCI-H1299 tumor cells, and PD-1 was evaluated using an apoptosis detection kit and FACS technology. IR2 The ability of overexpressed immune cells to kill tumor cells. Results showed that, compared to the control group, PD-1... IR2 Overexpression leads to a decrease in the killing ability of Jurkat cells against NCI-H1299. Figure 6 C and F).

[0045] Example 7

[0046] The three amino acid sites (K78, I126, E136) of PD-1 are located in the IgV domain and are crucial for the binding of PD-1 to PD-L1. Given that PD-1... IR2 It retains the IgV domain that binds to PD-L1, therefore it may exhibit inhibitory function by interacting with PD-L1, similar to PD-1. Although it does not bind to PD-1 in mice... IR2 Similar to the mouse Pd-1 subtype, but because mouse Pd-l1 and human PD-L1 have similar molecular structures, we speculate that the knock-in PD-1 IR2 The gene can bind to Pd-l1 in mice to exert its biological effects.

[0047] To further study PD-1 IR2 Based on the physiological functions, we prepared PDCD1. IR2Flox / Flox mice. A complete DNA sequence unit, including the promoter and PDCD1, was inserted securely into the ROSA26 gene site in C57BL / 6J mice. IR2 The CDS-Flag region and PolyA tail. Cre recombinase is often used in conjunction with loxP sites to form the Cre / loxP recombination system. This system is widely used in gene knockout, gene knock-in, and spatiotemporal control of gene expression. The loxP sequence is a DNA fragment with a specific order that Cre recombinase can recognize and cleave. The gene sequence of the LoxP is SEQ ID NO. 6. The function of Cre recombinase: Cre enzymes mediate DNA recombination by recognizing sequences between loxP sites. For example, it can cause deletion, flipping, or insertion of DNA sequences between loxP sites. In Cd4cre tool mice, after inserting the Cre gene into the CD4 cell promoter, mice that only knock in or knock out specific genes in T cells can be obtained when crossed with Loxp mice. We used PDCD1... IR2 When flox / flox mice are crossed with Cd4cre mice, the stop unit is deleted under the action of Cre recombinase, and the target gene begins to be expressed, thereby obtaining PDCD1 knock-in specifically in T cells. IR2 The mice, namely PDCD1 IR2 Conditional key-in (PDCD1) IR2 CKI mice were used. However, the target gene was not expressed in the absence of the Cd4cre gene. The genotype of the mice was identified by tail PCR, and PD-1 mice were selected. IR2+ / + Cd4cre + / - (PD-1 IR2 Homozygous and Cre) were used as the experimental group, with PD-1 from the same littermate. IR2+ / + Cd4cre - / - (PD-1 IR2 homozygous) as the control group ( Figure 7 A). Using EasySep TM mouse CD8 + T-cell isolation kit to isolate mouse spleen T cells, PD-1 IR2 After incubation with specific antibodies, mouse PD-1 was detected using FACS. IR2 The expression situation and the results show PDCD1 IR2CKI Mouse isoform gene knock-in successful ( Figure 7 B).

[0048] Example 8

[0049] Exploring PD-1 using the MC38 colorectal cancer model IR2 Functions of PD-1 inhibitors in tumors. Patients aged 6-8 weeks were selected. IR2+ / +Cd4cre + / - Experimental group and PD-1 from the same litter IR2+ / + Cd4cre - / - Five male mice were used as the control group. MC38 tumor cells were subcutaneously injected into the back of each mouse at a dose of 1×10⁶. Figure 8 A). Mice were sacrificed on day 15, and tumor tissue was removed and weighed. The measurement results showed PDCD1. IR2 CKI The tumors in the mice grew faster than those in the WT mice, as evidenced by the tumor volume ( Figure 8 B) and tumor weight ( Figure 8 Increased CD1 levels were observed. Tumor tissue was minced and ground, mouse TILs were isolated using density gradient centrifugation, and stained with anti-CD45, CD3, CD4, CD8, CD25, CD44, FOXP3, Ki-67, Gzmb, and IFN-γ. Immunocyte phenotype was analyzed using FACS. PDCD1 was observed. IR2 CKI and WT mouse tumor-infiltrating CD8 + The proportion of CD44 in T cells was similar, and CD44 is a T cell activation marker, indicating that the two groups had similar antigen presentation processes. Figure 8 E). PDCD1 IR2 The knock-in of the gene did not affect CD4 in TILs + T cells, CD8 + The percentage of T cells, relative to CD4 + With CD8 + The ratio of T cells, Treg levels, and Ki-67 expression were also unaffected. Figure 8 FH). However, compared with WT mice, PDCD1 IR2 CKI GzmB and IFN-γ were decreased in mouse TILs ( Figure 8 IJ), indicating that CD8 + These results indicate impaired T-cell cytotoxicity. IR2 Impaired cytotoxic CD8 in the tumor microenvironment + The function of T cells is to promote tumor growth.

[0050] Example 9

[0051] Transplanting MC38 tumor cells into WT and PDCD1 IR2 CKI In mice, the mice were treated with either PD-L1 or IgG antibodies. Results showed that, compared to IgG treatment, PD-L1 antibody treatment significantly delayed tumor growth in WT mice, manifested as reduced tumor volume and weight, and increased GZMB, while PDCD1... IR2 CKI Mice showed resistance to anti-PD-L1 treatment. Figure 9AD). In fact, we still observed that anti-PD-L1 therapy in PDCD1 IR2 CKI It showed some effect on tumors in mice, manifested as a reduction in tumor volume and weight, and an increase in GZMB. Figure 9 AD, CKI+anti-PD-L1 vs CKI+IgG). However, PDCD1 IR2 CKI The tumor volume in the mice was still larger than that in the WT mice. Figure 9 A, CKI+anti-PD-L1 vs WT+anti-PD-L1), this indicates that PD-1 IR2 It can still exert immunosuppressive effects after anti-PD-L1 treatment. Following anti-PD-L1 treatment, WT and PDCD1... IR2 CKI The number of Treg cells in mice is reduced ( Figure 9 E), which is consistent with previous reports of using checkpoint blockade therapy.

[0052] Example 10

[0053] A mouse model in which human genes, cells, tissues, organs, or the immune system are transplanted into a mouse is called a humanized mouse. Immune system humanization mice are mice in which the human immune system is reconstructed within the mouse. This is achieved by transplanting human hematopoietic stem cells, immune cells, or tissues into immunodeficient mice, giving them human immune function. Common immune system humanization methods include PBMC humanization and hematopoietic stem cell (HSC) humanization. To further confirm PD-1... IR2 To investigate the intrinsic role of PBMCs in antitumor activity, a humanized PBMC model (humanized PBMC-NOG, huPBMC-NOG) was constructed using NOG (NOD.Cg-PrkdcsidIL2rgtm1Sug / Jaccard) mice. NOG mice were subcutaneously inoculated with NCI-H1299 tumor cells in their backs. One week later, human PBMCs were injected via the tail vein. These PBMCs overexpressed both the empty vector control and PD-1. IR2 ( Figure 10 A). Tumor size was measured periodically and tumor growth curves were plotted. Mice were sacrificed on day 23, and tumor tissue was removed and weighed. Compared with the control group, PD-1 IR2 huPBMC-NOG mice exhibited impaired tumor growth inhibition, manifested as increased tumor volume and weight. Figure 10 BD). Tumor tissue was minced and ground, TILs were isolated, and after fluorescent antibody staining, FACS analysis was performed. The activation level of CD44 in tumor-infiltrating CD8+ T cells was not significantly different from that in control mice, indicating that they underwent the same antigen presentation (BD). Figure 10 E). Furthermore, no CD4 was observed in huPBMC-NOG mice.+ T cells and CD8 + The frequency of T cells changed significantly. Figure 10 F), Ki-67 expression in TILs was also unaffected. Figure 10 G). However, we found that CD8 in PD-1IR2 huPBMC-NOG mice + Impaired T-cell immune function is manifested in reduced production of cytokines GmzB and IFN-γ. Figure 10 H), this conclusion is consistent with PDCD1 IR2 CKI Consistent with mice.

[0054] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0055] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A PD-1 isomer, characterized in that, The PD-1 isoform contains a signal peptide, an IgV domain, and a novel short cytoplasmic tail, and its amino acid sequence is SEQ ID NO.

1.

2. A method for constructing a PD-1 isoform knock-in mouse model as described in claim 1, characterized in that, The construction method is as follows: a complete DNA sequence unit is inserted into the safe site of the mouse ROSA26 gene to obtain the PD-1 isoform flox / flox mouse. The DNA sequence unit includes a promoter, a terminator located between two Loxp fragments, a PD-1 isoform CDS-Flag region and a PolyA tail. The PD-1 isoform flox / flox mice were crossed with Cd4cre mice, which are mice in which the Cre gene was inserted after the CD4 cell promoter. Cre recombinase acts on the Loxp fragment. By recognizing a specific site, the terminator between Loxp fragments is deleted, and the target gene begins to be expressed, resulting in mice with PD-1 isoforms specifically knocked into T cells.

3. The method for constructing a PD-1 isoform knock-in mouse according to claim 2, characterized in that, The gene sequence of the CDS-Flag region of the PD-1 isoform is SEQ ID NO.

6.

4. The PD-1 isoform knock-in mouse model constructed by the method described in claim 3 is used for the study of tumor diseases.

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