A recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein and its preparation method

Abstract

This invention provides a recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein. The structure of this fusion protein is shown in formulas M1, M2, or M3 in Figure 1. Its amino acid sequence is included in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and its gene sequence is included in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:25. In NO:32, the linker peptide is (GGGGS)n (n = 0, 1, 2, 3, or 4), the amino acid sequence of the linker peptide includes SEQ ID NO:16, and the gene sequence includes SEQ ID NO:33; the amino acid sequence of the signal peptide includes SEQ ID NO:17, and the gene sequence includes SEQ ID NO:34. Compared with the prior art, the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein provided by this invention generally has higher activity than the corresponding monoclonal antibody, and it has good development and application prospects in the field of anti-tumor therapy.

Description

Technical Field

[0001] This invention belongs to the technical fields of molecular biology and biopharmaceuticals, and specifically relates to a recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein and its preparation method. Background Technology

[0002] The number of newly diagnosed cancer patients worldwide continues to rise. Statistics show that by 2030, the number of new cancer patients globally will reach 24 million, with over 5.8 million in China, accounting for nearly a quarter. Among numerous immune checkpoints, PD-1 / PD-L1 is an important target. Blocking immune checkpoints can reactivate T cells, helping the immune system to eliminate cancer cells. The tumor microenvironment mediates the induction of the programmed cell death protein 1 (PD-1) pathway and the accumulation of regulatory T (Treg) cells, which is believed to help suppress anti-tumor immunity. PD-1 is an inhibitory surface receptor expressed on T cells that can bind to the upregulated immunosuppressive programmed cell death ligand 1 (PD-L1) on tumor cells. Receptor-ligand interactions can inhibit the production of anti-tumor cytokines and the cytolytic activity of PD-1-highly expressed tumor-infiltrating T cells, thereby silencing the immune system. Blocking the binding between PD-1 and PD-L1 can enhance the immune response of T cells and mediate anti-tumor activity. PD-1 / PD-L1 inhibitors, as highly effective broad-spectrum anti-tumor drugs, have made epoch-making progress in the history of anti-tumor drugs. Since their launch, they have rapidly penetrated the global oncology drug market and their sales have continued to grow.

[0003] Currently, six PD-1 / PD-L1 products have been approved in overseas markets, covering more than 80 oncology indications. Four PD-1 drugs are from Merck (Keytruda), Bristol-Myers Squibb (Opdivo), Pfizer / Merck (Beta), and Sanofi / Regeneron (Leta), while two PD-L1 drugs are from Roche (Texas) and AstraZeneca (Italy). The NMPA has approved 12 PD-1 / PD-L1 products, covering 11 cancer types and 44 indications, including eight domestically produced and four imported drugs. 45% of the approved indications for marketed PD-1 / PD-L1 drugs were obtained through the accelerated approval pathway, typically involving only single-arm trials, with subsequent confirmatory randomized clinical trials showing inconsistent results. ClinicalTrials.gov shows 2909 PD-1 and 2318 PD-L1 clinical trials, indicating a crowded and highly competitive market. Furthermore, many patients lack response to checkpoint inhibitor monotherapy, with effectiveness limited to a small number of cancer patients. It is generally believed that combined immunotherapy can achieve the greatest anti-tumor efficacy.

[0004] Chemokine receptor 4 (CXC chemokine receptor 4, CXCR4) is a seven-transmembrane G protein-coupled receptor (GPCR) that mediates chemical interactions with target cells, playing biological roles such as chemotaxis of immune cells and maintaining immune cell homeostasis. CXCR4 consists of 352 amino acids and is expressed in most tissues and organs in vivo, including the surface of cells derived from bone marrow, umbilical cord blood, and mobilized peripheral blood, as well as on the surface of various non-hematopoietic stem cells. It is also abnormally highly expressed in many tumors. Current research indicates that CXCR4 is associated with more than 23 types of cancer and can promote angiogenesis, cell metastasis, growth, and survival. SDF-1, belonging to the CXC chemokine family, is a ligand for CXCR4. The role of the SDF-1 / CXCR4 receptor-ligand system is primarily studied in the field of immunology, where it plays a crucial role in mediating tumor-directed migration, invasion, and metastasis. Several CXCR4-developed drugs have shown great potential in clinical treatment. Studies have shown that CXCR4 can reduce the infiltration of regulatory T (Treg) cell tumors, and these drugs will open a new avenue for tumor immunotherapy. Currently, research targeting CXCR4 is quite mature. Besides the FDA-approved CXCR4 inhibitor plerixafor for hematologic malignancies, recent research has revealed several other CXCR4-targeting inhibitors. For example, Motixafortide, which has been approved for clinical trials in China and is currently in Phase III, is used to treat locally advanced or metastatic triple-negative breast cancer. Similarly, Mavorixafor, also in Phase III clinical trials, uses hematopoietic stem cell mobilization for autologous bone marrow transplantation in patients with multiple myeloma and plans to submit its NDA in the first half of this year, potentially becoming the world's second CXCR4-targeting drug. Among existing technologies, monoclonal antibody patents include Pierre Fabre Medicament's patent application US9388248B2 in 2008, Eli Lilly's patent application CN102027015A for CXCR4 monoclonal antibody in 2010 for cancer treatment, Bristol-Myers Squibb Company's patent application US20200231683A1 for CXCR4 monoclonal antibody in 2006, The General Hospital Corporation's patent application US20160128974A1 in 2010, and Pfizer Inc.'s patent application US10927178B2 in 2013.

[0005] Studies have found that combining the blocking of chemokine receptor CXCR4 and immune checkpoint PD-1 significantly reduces specific cells and functional elements in the immunosuppressive tumor microenvironment, enhancing tumor-specific cell-mediated immune responses and generating stronger control. Therefore, the combined use of PD-1 / L1 inhibitors and CXCR4 inhibitors exhibits synergistic effects. Summary of the Invention

[0006] This invention provides a recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein and its preparation method.

[0007] The specific technical solution of the present invention is as follows:

[0008] The recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein provided by this invention is characterized in that,

[0009] The structure of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein is shown in formula M1, M2 or M3 as follows:

[0010]

[0011] The amino acid sequence of this recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein is included in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and the gene sequence is included in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In ID NO:32; the linker peptide of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein is (GGGGS)n (n = 0, 1, 2, 3 or 4), the amino acid sequence of the linker peptide includes SEQ ID NO:16, and the gene sequence includes SEQ ID NO:33; the signal peptide amino acid sequence of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein includes SEQ ID NO:17, and the gene sequence includes SEQ ID NO:34.

[0012] The present invention also provides an expression vector, characterized in that the expression vector can highly express the amino acid sequence of the above-mentioned recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein.

[0013] The present invention also provides a host cell, characterized in that the host cell can highly express the amino acid sequence of the above-mentioned recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein, and the host cell includes Escherichia coli, yeast, CHO cells, HEK293 or at least one of them.

[0014] The present invention also provides a method for preparing the above-mentioned recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein, characterized by comprising the following steps: step S1, culturing the above-mentioned host cells to obtain a culture medium containing the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein; step S2, separating the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein from the culture medium.

[0015] The role and effect of invention

[0016] Compared with existing technologies, the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) provided by this invention generally has higher activity than the corresponding monoclonal antibody, and it has good development and application prospects in the field of anti-tumor therapy. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (biantibody) according to an embodiment of the present invention.

[0018] Figure 2 This is an ELISA diagram of the binding of recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) and its contrast agent PD-1Mab to the human PD-1 receptor, according to an embodiment of the present invention.

[0019] Figure 3 This is a flow cytometry data (Facs) of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) and its comparative CXCR4 monoclonal antibody with HEK293-CXCR4-luc cells, according to an embodiment of the present invention. Figure 4 This is a reporter gene assay diagram showing the activation of the CD3 signaling pathway caused by the blocking of PD-1 / PD-L1 binding by the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (dual antibody) and its comparative 221-PD-1 monoclonal antibody in the embodiment of this invention. Detailed Implementation

[0020] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.

[0021] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.

[0022] The reagents used in the following examples were obtained through common commercial channels. Experimental procedures and conditions not specified are in accordance with conventional procedures and conditions in the art.

[0023] The specific embodiments of the present invention will be described below with reference to the examples and accompanying drawings.

[0024] <Example>

[0025] This embodiment provides a recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) and its preparation method.

[0026] Figure 1 This is a schematic diagram of the structure of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (biantibody) according to an embodiment of the present invention.

[0027] like Figure 1 As shown, the structure of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) in this embodiment is as follows: Figure 1 As shown in Chinese style M1, M2, or M3.

[0028] The amino acid sequence of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein is included in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and the gene sequence is included in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32.

[0029] The linker peptide of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein is (GGGGS)n (n = 0, 1, 2, 3 or 4), the amino acid sequence of the linker peptide includes SEQ ID NO:16, and the gene sequence includes SEQ ID NO:33.

[0030] The signal peptide amino acid sequence of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein includes SEQ ID NO:17, and the gene sequence includes SEQ ID NO:34.

[0031] The method for preparing the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) provided in this embodiment includes the following steps:

[0032] Step S1: Culturing the above host cells to obtain a culture medium containing the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein. The specific process is as follows:

[0033] Step S1-1: Prepare the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) expression vector. The specific process is as follows:

[0034] The linker peptide used in this embodiment is (GGGGS)n (where n = 0, 1, 2, 3 or 4), the amino acid sequence of the linker peptide is SEQ ID NO:16, and the gene sequence is SEQ ID NO:33. The codons of the target molecule were optimized to facilitate expression in human species and host cells. Finally, the gene plasmid was synthesized to obtain the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (dual antibody) expression vector. This expression vector can highly express the amino acid sequence of the above-mentioned recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein.

[0035] Step S1-2 involves the expression of recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody). The specific process is as follows:

[0036] Using Expi CHO-S (Gibco, A29133) as the host cell (which can highly express the amino acid sequence of the above-mentioned recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein), the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (biantibody) was transiently expressed using the chemical transfection reagent Polyplus-FectoPRO (polyplus, 116-010), and a culture medium containing the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (biantibody) was obtained.

[0037] Step S2 involves isolating the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) from the culture medium. The specific process is as follows:

[0038] The culture medium obtained in step S1 was subjected to two-stage centrifugation (first stage: 3000g, 30min; second stage: 12000g, 20min), and the supernatant was collected and filtered through a 0.2μm filter for later use; Protein A affinity chromatography: the filtrate was loaded onto a Protein A column pre-equilibrated with 20mM PB (pH 7.2) and 150mM NaCl (pH 7.2) for 5min; reequilibration: elution was performed using 50mM NaAc-HAc (pH 4.5), 50mM NaAc-HAc (pH 4.0), and 50mM NaAc-HAc (pH 3.5), respectively, with the peak range being 50mAU-peak-50mAU, to obtain recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (biantibody) with SEC purity meeting the requirements (>98.0).

[0039] In this embodiment, nine recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion proteins (biantibodies) were obtained, and their molecular sequences are shown in Table 1.

[0040] Table 1

[0041] Molecular serial number amino acid sequence structure gene sequence 221-S1 SEQ ID NO:8, SEQ ID NO:9 M2 SEQ ID NO:25, SEQ ID NO:26 221-S2 SEQ ID NO:10, SEQ ID NO:11 M2 SEQ ID NO:27, SEQ ID NO:28 221-S3 SEQ ID NO:12, SEQ ID NO:13 M2 SEQ ID NO:29, SEQ ID NO:30 221-S4 SEQ ID NO:4, SEQ ID NO:5 M2 SEQ ID NO:21, SEQ ID NO:22 221-S5 SEQ ID NO:6, SEQ ID NO:7 M2 SEQ ID NO:23, SEQ ID NO:24 221-S6 SEQ ID NO:1, SEQ ID NO:2 M1 SEQ ID NO:18, SEQ ID NO:19 221-S7 SEQ ID NO:1, SEQ ID NO:3 M1 SEQ ID NO:18, SEQ ID NO:20 221-S8 SEQ ID NO:1, SEQ ID NO:14 M3 SEQ ID NO:18, SEQ ID NO:31 221-S9 SEQ ID NO:1, SEQ ID NO:15 M3 SEQ ID NO:18, SEQ ID NO:32

[0042] <Test Example 1>

[0043] This test case uses an ELISA assay to test the binding activity of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) obtained in the above examples to the human PD-1 receptor.

[0044] Detection method: 100 ng / well of human PD-1 receptor was coated into each well of a 96-well cell plate and incubated overnight at 4°C. Serially diluted concentrations (10000-0.009537 ng / mL) of PD-1Mab, 221-S1, 221-S2, 221-S3, 221-S4, 221-S5, 221-S6, 221-S7, 221-S8, and 221-S9 were added and incubated at 37°C for 1 hour. HRP-anti-human IgG-Fc was added and incubated at 37°C for 1 hour. Finally, TMB was added for color development for 10 minutes. The absorbance value at 450 nm was read using a microplate reader. Curve fitting was performed using the four-parameter method to calculate the EC50. 50 value.

[0045] Figure 2 This is an ELISA diagram of the binding of recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) and its contrast agent PD-1Mab to the human PD-1 receptor, according to an embodiment of the present invention.

[0046] The results of the binding activity assays of recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) and its counterpart PD-1Mab (corresponding monoclonal antibody) to the human PD-1 receptor are listed in Table 2.

[0047] Table 2

[0048]

[0049] Depend on Figure 2 As shown in Table 2, the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) binds to EC. 50 Low value, similar to PD-1Mab EC 50 The similarity indicates that it has strong binding activity to the human PD-1 receptor.

[0050] <Test Example 2>

[0051] This test case uses FACS technology to test the flow cytometry binding activity of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) and its comparative CXCR4 monoclonal antibody to the human CXCR4 receptor obtained in the above examples.

[0052] Detection method: Add 50 μL of 2×10⁻⁶ cells to each well of a 96-well cell plate. 6 HEK293-CXCR4-luc cells (which highly express human CXCR4) were incubated at concentrations (100,000-0.0954 ng / mL) with CXCR4 monoclonal antibody, 221-S1, 221-S2, 221-S3, 221-S4, 221-S5, 221-S6, 221-S7, 221-S8, and 221-S9, respectively. The cells were incubated at 4°C for 1 hour, followed by incubation at 37°C for 1 hour with PE-anti-human IgG-Fc. Flow cytometry was used to detect the fluorescence signal values, and the EC50 was calculated using a four-parameter method for curve fitting. 50 value.

[0053] Figure 3 This is a flow cytometry data (Facs) of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) and its comparative CXCR4 monoclonal antibody with HEK293-CXCR4-luc cells, according to an embodiment of the present invention.

[0054] The results of flow cytometry assays of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) and its comparative CXCR4 monoclonal antibody with human CXCR4 receptor are listed in Table 3.

[0055] Table 3

[0056]

[0057] Depend on Figure 3 As shown in Table 3, the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) binds to EC. 50 Low value, compared with CXCR4 monoclonal antibody EC 50 The similarity indicates that it has strong binding activity to the human CXCR4 receptor.

[0058] <Test Example 3>

[0059] This test case uses the reporter gene assay to detect the PD-1 / PD-L1 blocking activity of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein (dual antibody) obtained in the above examples and its comparative 221-PD-1 monoclonal antibody.

[0060] Detection method: Prepare 2×10 5Target cells CHO-K1-OS8-PD-L1 (F12 medium, containing 10% FBS) were added at 50 μL / well to a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. The next day, 25 μL of serially diluted (100,000-0.095 ng / mL) 221-PD-1 monoclonal antibody, 221-S1, 221-S2, 221-S3, 221-S4, 221-S5, 221-S6, 221-S7, 221-S8, and 221-S9 were added to each well of the 96-well cell culture plate. The plate was then incubated for another 6 hours at 37°C with 5% CO2. Finally, 100 μL of One-Glo Luciferase Assay (Promega, E6120) was added to each well. After 10 minutes of color development, the chemiluminescence signal was detected using a microplate reader. The signal values ​​are fitted using the four-parameter method, and EC is calculated. 50 The value is used to reflect the effect of PD-1 antibody.

[0061] Figure 4 This is a reporter gene assay diagram showing the activation of the CD3 signaling pathway caused by the blocking of PD-1 / PD-L1 binding by the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) and its comparative 221-PD-1 monoclonal antibody in the embodiment of this invention.

[0062] The recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (dual antibody) and its comparative 221-PD-1 monoclonal antibody blocking PD-1 / PD-L1 activity are listed in Table 4.

[0063] Table 4

[0064]

[0065] Depend on Figure 4 As shown in Table 4, the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) can block the binding between PD-1 and PD-L1, restore the CD3 activating signaling pathway, and thus activate T cells. Therefore, the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein (biantibody) has a strong function of blocking PD-1 / PD-L1 activity and activating T cell activity.

[0066] The above is a detailed description of the embodiments, which is intended to enable those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to technical solutions obtained by those skilled in the art based on the present invention and on the existing technology, without innovative effort but only through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.

Claims

1. A recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein, characterized in that, The structure of the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein is shown in formula M3 below: The recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein is 221-S8 or 221-S9, wherein the amino acid sequence of 221-S8 is SEQ ID NO:1 and SEQ ID NO:14, and the gene sequence is SEQ ID NO:18 and SEQ ID NO:31; the amino acid sequence of 221-S9 is SEQ ID NO:1 and SEQ ID NO:15, and the gene sequence is SEQ ID NO:18 and SEQ ID NO:

32. The signal peptide amino acid sequence of the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein includes SEQ ID NO:17, and the gene sequence includes SEQ ID NO:

34.

2. An expression carrier, characterized in that, The expression vector can highly express the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein as described in claim 1.

3. A host cell, characterized in that, The host cell can highly express the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein as described in claim 1, and the host cell includes Escherichia coli, yeast, CHO cells, HEK293, or at least one of them.

4. A method for preparing the recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein as described in claim 1, characterized in that, Includes the following steps: Step S1: Cultivate the host cells as described in claim 3 to obtain a culture medium containing the recombinant long-acting human CXCR4&PD-1 dual-target antibody fusion protein; Step S2: The recombinant long-acting human CXCR4 & PD-1 dual-target antibody fusion protein is isolated from the culture medium.

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