Anti-TIGIT / rhIL-21 bifunctional fusion protein and application thereof

Abstract

The invention belongs to the technical field of fusion proteins, and particularly relates to an anti-TIGIT / rhIL-21 bifunctional fusion protein and application thereof. According to the anti-TIGIT / rhIL-21 bifunctional fusion protein provided by the invention, the Fc segment is combined with the rhIL-21, so that the in-vivo stability of the IL-21 is remarkably enhanced, the half-life period of the IL-21 is successfully prolonged, the limitation that the half-life period of the rhIL-21 is short when the rhIL-21 is independently used is overcome, and a powerful guarantee is provided for continuous treatment. According to the anti-TIGIT / rhIL-21 bifunctional fusion protein provided by the invention, the antibody aiming at TIGIT and the recombinant human interleukin-21 are ingeniously fused, so that effective blocking of immune checkpoints and efficient activation of immune cells are synchronously carried out, the in-vivo pharmacokinetic characteristics of IL-21 are optimized, and the anti-TIGIT / rhIL-21 bifunctional fusion protein has the advantages that the anti-TIGIT / rhIL-21 bifunctional fusion protein can be used for preparing anti-TIGIT antibodies. And mobilization and function enhancement of immune cells are greatly promoted, an innovative strategy is provided for solving the problem of cold tumors and improving the immunotherapy effect, and wide application prospects are shown.

Description

Technical Field

[0001] The present invention belongs to the technical field of fusion proteins, and specifically relates to an anti-TIGIT / rhIL-21 bifunctional fusion protein and an application thereof. Background Art

[0002] TIGIT is a currently popular immune checkpoint with a similar structure to DNAM-1. It regulates the functions of various immune cells by competitively binding to PVR.

[0003] T cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor mainly expressed on the cell surface of NK cells, activated CD8+T cells and CD4+T cells, and Treg (regulatory T cells). TIGIT and the activating receptor DNAM-1 (CD226) both have similar immunoglobulin-like domains and competitively bind to PVR, but have opposite effects on the activation and function of NK cells. Competition experiments have confirmed that TIGIT has a stronger affinity for PVR and can effectively block the binding of DNAM-1 to PVR, thereby weakening the immune surveillance of immune cells against tumor cells. When TIGIT binds to CD155, a major inhibitory signal is initiated through an ITT-like motif, while the ITIM motif mediates a smaller inhibitory signal. The ITT-like motif is phosphorylated at Tyr225 and binds to the intracytoplasmic signaling molecules Grb2 and β-arrestin2 to recruit inositol-containing SH2 phosphatase-1 (SHIP-1). SHIP-1 blocks the signal transduction of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs), and also inhibits the activation of TRAF6 and NF-κB, thereby negatively regulating the cytokine secretion and cytotoxic activity of immune cells such as NK cells and T cells.

[0004] TIGIT has become an immune checkpoint that has attracted much attention after PD-1 / PD-L1 and CTLA-4. Currently, more than 10 TIGIT antibodies have entered clinical research worldwide, attracting the attention of many researchers in the field of tumor immunotherapy. Clinical studies and animal experiments based on solid tumors have confirmed that the combination of TIGIT monoclonal antibodies and PD-1 monoclonal antibodies can effectively reverse the immune exhaustion of T cells and NK cells in the tumor microenvironment (TME); however, the existing clinical research data of anti-TIGIT monoclonal antibodies in refractory and relapsed B-cell lymphoma are not satisfactory. Lymphoma TME research based on spatial multi-omics technology has revealed that a considerable number of patients with refractory and relapsed B-cell lymphoma have TMEs that are "cold tumors" with insufficient immune infiltration. Due to the lack of sufficient immune effector cells, immune checkpoint inhibitors cannot effectively function. The degree of immune infiltration in the TME has been shown to play a key role in tumor development and affect the patient's immunotherapy outcome. "Cold tumors" with insufficient immune cell infiltration have a low response rate to immune checkpoint blockers (ICIs). Therefore, remodeling the TME to increase the number of immune cells and anti-tumor activity will be an important strategy to improve the efficacy of ICIs such as TIGIT blockade. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention constructs an anti-TIGIT / rhIL-21 bifunctional fusion protein with immune checkpoint blocking and immune cell activation functions.

[0006] The objective of the present invention can be achieved through the following technical scheme: an anti-TIGIT / rhIL-21 bifunctional fusion protein, wherein the bifunctional fusion protein connects rhIL-21 to the FC terminal segment of the TIGIT antibody via a linker.

[0007] In the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein, the amino acid sequence of the linker is shown in SEQ ID NO:1.

[0008] In the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein, the amino acid sequence of rhIL-21 is shown in SEQ ID NO:2.

[0009] In the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein, the TIGIT antibody comprises a sequence of any one constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD of human or mouse antibodies.

[0010] Preferably, the TIGIT antibody is a humanized IgG1 antibody.

[0011] In the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein, the V H The complementarity determining region CDR has an amino acid sequence of a CDR selected from the group consisting of CDR1 shown in SEQ ID NO: 3, CDR2 shown in SEQ ID NO: 4, and CDR3 shown in SEQ ID NO: 5;

[0012] And, its V L The complementarity determining region CDR has an amino acid sequence selected from the group consisting of CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8.

[0013] In the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein, the V H and V L The amino acid sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0014] The present invention also provides a nucleic acid molecule, which encodes the TIGIT antibody according to claim 1 or 4.

[0015] The present invention also provides an expression vector, which contains the nucleic acid molecule according to claim 7.

[0016] The present invention also provides a pharmaceutical composition for treating or inhibiting diseases or conditions related to tumor cell proliferation or tumor cell metastasis, wherein the pharmaceutical composition comprises the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein.

[0017] The present invention also provides a detection kit, which contains the above-mentioned anti-TIGIT / rhIL-21 bifunctional fusion protein, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned pharmaceutical composition.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The anti-TIGIT / rhIL-21 bifunctional fusion protein provided by the present invention cleverly combines antibodies against TIGIT and recombinant human interleukin-21, achieving simultaneous effective blocking of immune checkpoints and efficient activation of immune cells. It not only optimizes the in vivo pharmacokinetic properties of IL-21, but also greatly promotes the mobilization and functional enhancement of immune cells, providing an innovative strategy for solving the "cold tumor" problem and improving the effect of immunotherapy, and showing broad application prospects.

[0020] 2. The present invention provides an anti-TIGIT / rhIL-21 bifunctional fusion protein, whose variable region can bind to the TIGIT protein on the surface of immune cells. This mechanism effectively blocks the interaction between TIGIT and PVR, thereby interrupting the TIGIT-mediated immunosuppressive signaling pathway, effectively reversing the exhaustion of immune cells in the anti-tumor process, and restoring the anti-cancer activity of the immune system.

[0021] 3. The present invention provides an anti-TIGIT / rhIL-21 bifunctional fusion protein, whose Fc segment binds to rhIL-21, significantly enhancing the in vivo stability of IL-21 and successfully extending its half-life, overcoming the limitation of the short half-life of rhIL-21 when used alone, and providing a strong guarantee for continuous treatment.

[0022] 4. In the anti-TIGIT / rhIL-21 bifunctional fusion protein provided by the present invention, the structure of the anti-TIGIT antibody gives the fusion protein a unique immune orientation. The antigen binding site of its variable region can not only bind to key immune effector cells such as NK cells and T cells expressing TIGIT, but also its Fc end can interact with Fc receptors (FcR) on the surface of other immune cells such as NK cells and macrophages, which not only promotes the enrichment of IL-21 in the tumor microenvironment, but also promotes the efficient binding of IL-21 to IL-21 receptor (IL-21R), thereby significantly enhancing the proliferation and activation of these immune cells and stimulating a stronger anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the molecular structure of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1.

[0024] Figure 2 This is a diagram of the construction of the anti-TIGIT / rhIL-21 bifunctional fusion protein expression vector prepared in Example 1; Figure 2 A is the amino acid sequence of the anti-TIGIT light chain, anti-TIGIT heavy chain and rhIL-21 concatemer after optimization design using the codon optimization software MaxCodon TM Optimization Program (V13); Figure 2 B is a schematic diagram of the structure of the light chain gene vector for synthesizing the anti-TIGIT / rhIL-21 bifunctional fusion protein using the whole gene; Figure 2 C is a schematic diagram of the heavy chain gene structure for synthesizing the anti-TIGIT / rhIL-21 bifunctional fusion protein; Figure 2 D is a schematic diagram of constructing the pcDNA3.1-DT7532 (TIGIT-LC) expression vector; Figure 2F is a schematic diagram of constructing the pcDNA3.1-DT7532 (TIGIT-HC) expression vector.

[0025] Figure 3 This is a verification report of the light chain gene vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1. The light chain gene was inserted into the expression vector pcDNA3.1 by full gene synthesis and double enzyme digestion, and the accuracy of the constructed light chain vector was verified by enzyme digestion.

[0026] Figure 4 This is a verification report of the heavy chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1. The heavy chain gene was inserted into the expression vector pcDNA3.1 by full gene synthesis and double enzyme digestion, and the accuracy of the constructed heavy chain expression vector was verified by enzyme digestion.

[0027] Figure 5 This is the sequencing result of the light chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1. Figure 6 This is the sequencing result of the heavy chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1.

[0028] Figure 7 These are the SDS-PAGE detection results of different steps in the purification process of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1.

[0029] Figure 8 This is the SDS-PAGE detection result of the final product obtained by purifying the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1.

[0030] Fig. 9 This is a diagram of affinity determination parameters between the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1 and the target proteins human IL-21R protein and human TIGIT protein.

[0031] Fig.10 This is a graph showing the affinity measurement results of the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1 and the target protein human TIGIT protein.

[0032] Fig.11 This is a graph showing the affinity determination results between the anti-TIGIT / rhIL-21 bifunctional fusion protein prepared in Example 1 and the target protein human IL-21R protein. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with specific embodiments and drawings. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not used to limit the scope of the present invention. And the drawings used herein are only for better illustrating the disclosed content of the present invention and do not have a limiting effect on the scope of protection. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0034] Example 1

[0035] (1) Construction of genetically engineered humanized TIGIT antibody

[0036] The mouse anti-TIGIT antibody obtained by mouse hybridoma in the previous laboratory was sequenced by amino acid sequencing, and the light chain and heavy chain variable region sequences of the TIGIT binding part were selected. The wild-type human IgG1 with ADCC effect was selected as the antibody constant region for humanization. The codon optimization software MaxCodon TM Optimization Program (V13) was used to optimize the amino acid sequence of anti-TIGIT as shown in SEQ ID NO: 9-10, and the DNA sequence of the humanized TIGIT antibody in the recombinant protein was obtained.

[0037] (2) Construction of expression vector for anti-TIGIT / rhIL-21 bifunctional fusion protein

[0038] The gene sequence of rhIL-21 encoding the amino acid sequence shown in SEQ ID NO: 2 was linked to the Fc terminal segment (C-terminus of the two heavy chains) of the DNA sequence of the above humanized TIGIT antibody by linking the DNA sequence with the linker G4S encoding the amino acid sequence shown in SEQ ID NO: 1. H The complementarity determining region CDR has an amino acid sequence of a CDR selected from the group consisting of CDR1 shown in SEQ ID NO: 3, CDR2 shown in SEQ ID NO: 4, and CDR3 shown in SEQ ID NO: 5;

[0039] And, its V L The complementarity determining region CDR has an amino acid sequence selected from the group consisting of CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8.

[0040] Table 2: Specific amino acid sequence information of anti-TIGIT / rhIL-21 bifunctional fusion protein

[0041]

[0042]

[0043] Table 3: Specific DNA sequence information of anti-TIGIT / rhIL-21 bifunctional fusion protein

[0044]

[0045]

[0046]

[0047]

[0048] The light chain gene sequence and heavy chain gene sequence of the bifunctional fusion protein were synthesized by whole gene, and the light chain gene and heavy chain gene of the anti-TIGIT / rhIL-21 fusion protein were inserted into the expression vector pcDNA3.1 by double restriction enzyme digestion to construct pcDNA3.1-DT7532 (TIGIT-LC) ( Figure 2 D) and pcDNA3.1-DT7532(TIGIT-HC) ( Figure 2 F) Two expression vectors, the accuracy of the final expression vector was confirmed by restriction digestion and sequencing.

[0049] Figure 2 This is a diagram for the construction of the anti-TIGIT / rhIL-21 bifunctional fusion protein expression vector; Figure 2 B is a schematic diagram of the light chain gene structure of the anti-TIGIT / rhIL-21 bifunctional fusion protein synthesized using whole gene;

[0050] Figure 2 C is a schematic diagram of the heavy chain gene structure for synthesizing the anti-TIGIT / rhIL-21 bifunctional fusion protein; Figure 2 D is a schematic diagram of constructing the pcDNA3.1-DT7532 (TIGIT-LC) light chain gene expression vector; Figure 2 F is a schematic diagram of the construction of the pcDNA3.1-DT7532 (TIGIT-HC) heavy chain gene expression vector.

[0051] Figure 3 PCR diagram for enzyme cutting verification of the light chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein. As can be seen from the figure, the light chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein was successfully synthesized. The length of the DT7532-1 light chain target sequence is 734bp, the enzyme cutting site is BglII / HindIII, and the double enzyme cutting bands are located at 1611bp and 4444bp respectively.

[0052] Figure 4 PCR diagram for enzyme cutting verification of the heavy chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein. As can be seen from the figure, the heavy chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein was successfully synthesized. The length of the DT7532-1 heavy chain target sequence is 1862bp, the restriction sites are NdeI / HindIII, and the double restriction bands are 2267bp and 4916bp respectively.

[0053] Figure 5 and Figure 6 These are the sequencing results of the light chain gene expression vector and the heavy chain gene expression vector of the anti-TIGIT / rhIL-21 bifunctional fusion protein.

[0054] (3) Transfection-grade plasmid amplification and extraction

[0055] 80-100 ng of two expression plasmids containing pcDNA3.1-DT7532 (TIGIT-LC) and pcDNA3.1-DT7532 (TIGIT-HC) were respectively added to the prepared DH5α competent cells, and the competent cells after adding the plasmids were transformed by heat shock. The transformed competent cells were added to LB liquid culture medium and placed in a shaker at 37°C and 200 rpm for shaking and culture for about 30 minutes; the competent cells after culture were taken out, part of the suspension was applied to a plate containing ampicillin resistance, and placed in an incubator for overnight culture at 37°C; a single clone was picked from a fresh culture plate and placed in 2-5 mL LB culture medium, and cultured at 37°C and 200 rpm for 8 hours; it was inoculated into 200 mL LB culture medium at a ratio of 1 / 500, and cultured at 37°C and 200 rpm for 16 hours; the cultured bacterial liquid was collected and centrifuged, and the supernatant was removed; the plasmid was extracted using a Qiagen transfection-grade plasmid extraction kit, and the extracted plasmid was analyzed by 1% agarose gel electrophoresis.

[0056] (4) Transfect the vector carrying two target genes into HEK293 cells

[0057] One day before transfection, HEK293 cells were suspended and cultured to 1 L with a seeding density of 1×10 6 cells / mL, and cultured in an incubator at 110 rpm, 37°C, 5% CO2. On the day of transfection, the cell density was adjusted to 1-1.5×10 6 Add plasmid and transfection reagent to transfection buffer and mix well. Add DNA-transfection reagent mixture to cells to be transfected and place in incubator at 110rpm, 37℃, 5% CO 2 About 4 to 6 days after transfection, remove the cell culture, centrifuge and collect the supernatant.

[0058] (5) Purification of bifunctional fusion protein

[0059] The cell culture fluid after 5 days of transfection culture was centrifuged, the supernatant was filtered with a 0.22 μm filter, and dialyzed into 1×PBS with a buffer solution of pH 7.4 at 4°C. After the dialysis, it was purified with a protein A column, purified by Ni-IDA affinity chromatography, and then purified by SP Beads 6FF ion exchange chromatography. Lane 3 with relatively high purity was collected and dialyzed into 1×PBS with a pH of 7.4. After the dialysis, it was filtered with a 0.22 μm filter and divided into two portions, one for lyophilization and the other for freezing at -80°C.

[0060] Figure 7 This is a control diagram of the purification process of the anti-TIGIT / rhIL-21 bifunctional fusion protein. As can be seen from the figure, lane 1 represents that the concentration of the target protein in the culture supernatant before purification is low, and the content of other non-target proteins is high; lane 2 represents that there is no target protein band in the effluent after the protein A column binds the recombinant protein; lanes 3-6 represent that the recombinant protein bands in the non-reduced state after glycine elution are clear, with a molecular weight of about 180KDa; and lane 7 represents that there are two recombinant protein bands in the reduced state after glycine elution, representing the heavy chain (molecular weight of about 65KDa) and the light chain (molecular weight of about 25KDa).

[0061] (6) Identification of bifunctional fusion proteins

[0062] The bifunctional fusion protein was obtained after purification and buffer exchange in the culture supernatant, and the molecular weight and structural integrity of the bifunctional fusion protein were identified by reducing and non-reducing SDS-PAGE.

[0063] Figure 8 This is a diagram of the purification of the anti-TIGIT / rhIL-21 bifunctional fusion protein analyzed by SDS-PAGE. As can be seen from the figure, in lane 3 of non-reducing SDS-PAGE, the band of the recombinant protein with a complete structure is located near 180KDa; in lane 2 of reducing SDS-PAGE, the light chain and heavy chain bands of the recombinant protein are located near 25KDa and 65KDa, respectively.

[0064] (7) Affinity determination of bifunctional fusion protein and target protein TIGIT

[0065] from Figure 9-10 It can be seen that the affinity KD value of the anti-TIGIT / rhIL-21 bifunctional fusion protein and the human TIGIT protein determined by the surface plasmon resonance (SPR) method was 7.23E-9 (M), indicating a high affinity with the target protein.

[0066] (9) Affinity determination of bifunctional fusion protein and target protein human IL-21R:

[0067] Fig.11 The results of affinity determination of anti-TIGIT / rhIL-21 bifunctional fusion protein and target protein human IL-21R protein are shown in the figure. As can be seen from the figure, the affinity KD value of the recombinant fusion protein and human IL-21 receptor protein (IL-21R) determined by SPR method is 3.84E-9 (M), indicating a high affinity with the target protein.

[0068] The various aspects, embodiments, and features of the present invention should be considered to be illustrative in all aspects and not limiting of the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0069] In the preparation method of the present invention, the order of each step is not limited to the order listed. For those skilled in the art, without creative work, the order of each step is also within the protection scope of the present invention. In addition, two or more steps or actions can be performed simultaneously.

[0070] Finally, it should be noted that the specific embodiments described herein are merely examples of the present invention, and are not intended to limit the implementation methods of the present invention. A person skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described, or replace them in a similar manner. It is not necessary and impossible to provide all examples of all implementation methods here. However, these obvious changes or modifications derived from the essential spirit of the present invention still fall within the scope of protection of the present invention, and interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. An anti-TIGIT / rhIL-21 bifunctional fusion protein, characterized in that: The bifunctional fusion protein connects rhIL-21 to the FC terminal segment of the TIGIT antibody via a linker.

2. The anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1, characterized in that: The amino acid sequence of the linker is shown in SEQ ID NO:

1.

3. The anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1, characterized in that: The amino acid sequence of rhIL-21 is shown in SEQ ID NO:

2.

4. The anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1, characterized in that: The TIGIT antibody comprises a sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD of a human or mouse antibody.

5. An anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1 or 4, characterized in that: V of TIGIT antibody H The complementarity determining region CDR has an amino acid sequence of a CDR selected from the group consisting of CDR1 shown in SEQ ID NO: 3, CDR2 shown in SEQ ID NO: 4, and CDR3 shown in SEQ ID NO: 5; And, its V L The complementarity determining region CDR has an amino acid sequence selected from the group consisting of CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:

8.

6. An anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1 or 4, characterized in that: V of TIGIT antibody H and V L The amino acid sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the TIGIT antibody according to claim 1 or 4.

8. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 7.

9. A pharmaceutical composition for treating or inhibiting diseases or conditions related to tumor cell proliferation or tumor cell metastasis, characterized in that: The pharmaceutical composition comprises the anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1.

10. A kit, characterized in that: The detection kit contains the anti-TIGIT / rhIL-21 bifunctional fusion protein according to claim 1, or the nucleic acid molecule according to claim 7, or the expression vector according to claim 8, or the pharmaceutical composition according to claim 9.

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