PD-L1 nano antibody and sialidase bifunctional antibody with anti-tumor activity and application of PD-L1 nano antibody and sialidase bifunctional antibody
By constructing a bifunctional antibody of PD-L1 nanoantibody and sialidase, the problem of low response rate of existing PD-1/PD-L1 antibody treatment was solved, effective targeted treatment of tumors was achieved, and tumor growth was significantly inhibited.
Patent Information
- Application Number
- CN202510024490.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-03-07
- Filing Date
- 2025-01-07
- Publication Date
- 2025-09-09
AI Technical Summary
Existing PD-1/PD-L1 antibody drugs have a low response rate in treating tumors. It is difficult to completely reverse the exhausted state of T cells by inhibiting a single immune checkpoint molecule, and other immune checkpoint inhibitors need to be used in combination. In addition, traditional monoclonal antibodies have problems with poor tissue penetration and difficulty in modification in tumor treatment.
A bifunctional antibody of PD-L1 nanobody (Nb16) and sialidase (SiaT) was constructed, and Nb16 and SiaT were fused and expressed through recombinant DNA technology to form a fusion protein that can simultaneously block PD-1/PD-L1 signaling and hydrolyze cell surface sialic acid, thereby achieving targeted treatment of tumors.
It significantly inhibits tumor cell proliferation and effectively inhibits tumor growth in mice, has a significant anti-tumor effect, and can significantly inhibit tumor growth under the optimized dosage regimen.
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Figure CN120607622A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of antibody technology, and specifically relates to a bifunctional antibody targeting PD-L1 and sialic acid, and a preparation method and application thereof. Background Art
[0002] Under the influence of various tumorigenic factors, normal cells in the human body mutate into uncontrolled tumor cells. However, because the normal human immune system monitors, identifies, and eliminates tumor cells, these mutated tumor cells are eliminated and ultimately do not develop into tumors. Within tumor tissue, however, tumor cells can evade clearance and killing by immune cells in various ways, such as expressing various immune negative regulatory molecules that transmit "brake" signals to immune cells, causing them to lose function or directly inducing apoptosis.
[0003] The strategy of blocking immune checkpoints to activate immune cells and thus fight tumors has become an effective option in clinical cancer treatment. PD-1 / PD-L1 are the "star molecules" among immune checkpoints, and antibody drugs targeting the PD-1 / PD-L1 axis have achieved tremendous success in clinical practice, effectively increasing clinical benefits for patients. However, as PD-1 / PD-L1 antibody drugs become more widely used in clinical practice, some of the shortcomings of this therapy have gradually become apparent.
[0004] Currently, the clinical response rate for PD-1 / PD-L1 antibody therapy is approximately 20-40%. This low response rate is attributed to compensatory regulation by other immune checkpoints. Many immune checkpoints are independent pathways, and inhibiting a single checkpoint molecule alone is insufficient to completely reverse T cell exhaustion. Simultaneous inhibition of multiple immune checkpoint pathways is necessary. Therefore, combining PD-1 / PD-L1 antibody therapy with other immune checkpoint inhibitors is an option. The sialic acid-Siglecs pathway is considered a potential next-generation immune checkpoint. Under normal physiological conditions, sialic acid on the surface of one's own cells can transmit inhibitory signals to lectins on the surface of immune cells, achieving self-tolerance and immune homeostasis. However, when this effect is exploited by pathogens or tumor cells, immune escape can occur, rendering the immune system unable to kill and eliminate tumor cells and pathogens.
[0005] Based on monoclonal antibodies, a new type of antibody drug—bifunctional antibodies—has been developed to further improve efficacy and reduce toxic side effects. Bifunctional antibodies can simultaneously recognize two different targets, thereby generating more biological mechanisms of action and opening up new application pathways. Bifunctional antibodies do not exist naturally but are produced through cell fusion or recombinant DNA technology. Nanobodies possess many characteristics not possessed by traditional monoclonal antibodies, such as small molecular weight, good tissue penetration, and ease of modification and humanization. Therefore, nanobodies have unique advantages in the development of bifunctional antibody drugs. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to construct a bifunctional antibody that is a fusion of PD-L1 nanobody (Nb16) and sialidase (SiaT), which can simultaneously block PD-1 / PD-L1 signal transmission and hydrolyze sialic acid on the cell surface. The two do not interfere with each other and can effectively exert anti-tumor effects in vivo.
[0007] The present invention screened the nanoantibody Nb16, which has PD-L1 binding activity, from a camel antibody library immunized with mouse PD-L1. The sialidase SiaT was isolated from the genome of oral actinomycetes. Using recombinant DNA technology, Nb16 and SiaT were fused and expressed via a linker peptide (G4S)3. After isolation and purification, the bifunctional antibody Nb16-SiaT with anti-tumor activity was obtained.
[0008] The present invention provides a bifunctional antibody targeting PD-L1 and sialic acid, wherein the bifunctional antibody has PD-L1 binding activity and sialic acid hydrolysis activity. The structure of the bifunctional antibody comprises a nanobody Nb16 that binds to mouse PD-L1, a connecting peptide, and a sialidase SiaT, wherein the N-terminus of the sialidase is connected to the C-terminus of the PD-L1 nanobody via a connecting peptide; the PD-L1 nanobody Nb16 has an amino acid sequence as shown in SEQ ID NO.1, and the sialidase SiaT has an amino acid sequence as shown in SEQ ID NO.2; the amino acid sequence of the connecting peptide is (GGGGX)n, wherein X is Gly or Ser, and n is a natural number from 1 to 5.
[0009] Preferably, the amino acid sequence of the bifunctional antibody Nb16-SiaT is shown in SEQ ID NO:3.
[0010] The present invention also provides a nucleic acid molecule encoding the aforementioned bifunctional antibody targeting PD-L1 and sialic acid. In a preferred embodiment of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO: 4. This sequence can express an active bifunctional antibody in Escherichia coli.
[0011] Preferably, the expression vector of the bifunctional antibody Nb16-SiaT may include pET-28a.
[0012] The host strain containing the above expression vector may be an optimized Escherichia coli BL21 (DE3).
[0013] The present invention also provides a recombinant plasmid for expressing the bifunctional antibody Nb16-SiaT, wherein the recombinant plasmid at least comprises the amino acid sequence shown in SEQ ID NO: 3.
[0014] The present invention also provides a recombinant strain for expressing the bifunctional antibody Nb16-SiaT, wherein the recombinant strain comprises the recombinant plasmid. The present invention also provides a biological material containing the above nucleic acid molecule, wherein the biological material is any one of the following: recombinant DNA, expression cassette, vector, host cell, engineered bacteria or cell line.
[0015] The method of the present invention for producing a bifunctional antibody targeting PD-L1 and sialic acid comprises:
[0016] Introducing a nucleic acid encoding the bispecific antibody into a host cell to obtain a host cell that stably expresses the bispecific antibody; and / or
[0017] The host cells are cultured and the bifunctional antibody is obtained through purification.
[0018] Accordingly, the present invention provides a product comprising the above-mentioned bifunctional antibody targeting PD-L1 and sialic acid, wherein the product is a fusion protein, an immunotoxin, a drug, a detection reagent or a detection kit.
[0019] The present invention also provides applications of the aforementioned bifunctional antibody targeting PD-L1 and sialic acid or nucleic acids encoding the same, wherein the applications are selected from one or more of the following:
[0020] (1) Use in the preparation of drugs for diagnosing, preventing or treating diseases related to PD-L1 expression;
[0021] (2) Use in the preparation of drugs for diagnosing, preventing or treating diseases targeting PD-L1;
[0022] (3) Use in the preparation of drugs that kill PD-L1-expressing cells;
[0023] (4) Application in the preparation of PD-L1 detection reagents or kits;
[0024] (5) Application in the preparation of reagents for CAR-T therapy;
[0025] (6) Application in the preparation of immunotoxins or labeled antibodies;
[0026] (7) Application in the preparation of antibody-drug conjugates.
[0027] Preferably, the bifunctional antibody targeting PD-L1 and sialic acid is used in the preparation of an anti-tumor drug; the tumor may be derived from lung cancer, colon cancer, gastric cancer, liver cancer or breast cancer, etc.
[0028] The bifunctional antibody Nb16-SiaT of the present invention can be used for anti-tumor purposes. Experimental results show that the bifunctional antibody of the present invention can inhibit tumor cell proliferation and significantly suppress tumor growth in mice. The optimized in vivo dosing regimen in mice is: intraperitoneal injection, 5 mg / kg, once every two days.
[0029] The bifunctional antibody of the present invention, which is a fusion of the PD-L1 nanobody (Nb16) and sialidase (SiaT), has the functions of blocking PD-1 / PD-L1 signaling and hydrolyzing sialic acid on the cell surface, and can effectively exert an anti-tumor effect in vivo. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, each drawing described below is for some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.
[0031] Figure 1 Schematic diagram of the plasmid construction of the bifunctional antibody Nb16-SiaT and the SDS-PAGE results of expression and purification.
[0032] Figure 2 This is the identification result of the bifunctional antibody Nb16-SiaT's PD-L1 binding activity and sialic acid hydrolysis activity.
[0033] Figure 3 These are the results of in vivo anti-tumor activity assay of the bifunctional antibody Nb16-SiaT.
[0034] Figure 4 The anti-tumor dose optimization results of the bifunctional antibody Nb16-SiaT. DETAILED DESCRIPTION
[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0036] Example 1 Expression and purification of the bifunctional antibody Nb16-SiaT in Escherichia coli
[0037] The Nb16 sequence was obtained from the camel antibody library immunized with mouse PD-L1, and the sialidase SiaT sequence was obtained from the Uniprot website. The N-terminus of sialidase SiaT was connected to the C-terminus of PD-L1 nanobody Nb16 via a connecting peptide. The construction strategy is as follows: Figure 1 shown.
[0038] After the target gene is synthesized, it is connected to the vector pET-28 (a) plasmid through molecular cloning, and then the above plasmid is transformed into the chemically competent cell DH5α by the 42°C heat shock method. The single clone is screened with LB plates containing kanamycin resistance, and the plasmid sequencing is entrusted to the Anshengda Suzhou Laboratory. The plasmid with correct sequencing is saved for subsequent experiments. The constructed vector is heat-shocked and transformed into the chemically competent cell BL21 (DE3). The bacterial solution is amplified at 37°C and 220rpm overnight and then transferred to a new LB culture medium at a ratio of 1:100. After shaking for 12 hours, IPTG with a final concentration of 1mM is added to induce the expression of the target protein. After shaking at 37°C and 220rpm for 12-16 hours, the bacterial solution is collected, the bacteria are broken by high-pressure homogenization, and the supernatant is centrifuged. After affinity purification by nickel column, the target protein with high purity is obtained. The protein molecular weight and purity are identified by SDS-PAGE, and the results are as follows. Figure 1 shown.
[0039] Example 2 Identification of the PD-L1 binding activity and sialic acid hydrolysis activity of the bifunctional antibody Nb16-SiaT MaxiSorp96Immunoplates were coated with 1 μg / mL mPD-L1 solution (50 μL per well) at 4°C overnight. The next day, 5% BSA was added to block the plate at 37°C and washed three times with PBS solution containing 0.5% Tween-20. Nanobody Nb16 and bifunctional antibody Nb16-SiaT were added at a starting concentration of 3.8 μM and 8-fold gradient dilution, respectively, and incubated at room temperature for 1.5 hours and then washed. Alkaline phosphatase-labeled mouse anti-His antibody was added and incubated at 37°C for 1.5 hours and then washed. Alkaline phosphatase colorimetric solution TMB was added to react for 10 minutes, and then 50 μL 2M H2SO4 was added to terminate the reaction. The absorbance value was detected at 450 nm, and the EC value of Nb16 binding to mouse PD-L1 was calculated after 4-parameter fitting using GraphPad. 50 =2.92nM, EC for Nb16-SiaT binding to mouse PD-L1 50 =2.73nM, the results are as follows Figure 2 (Left) shows that the fusion modification does not affect the activity of Nb16 binding to PD-L1.
[0040] The purified protein obtained in Example 1 was subjected to preliminary enzyme activity identification. The total volume of the enzyme cleavage reaction system was 60 μL, including 1 μL of purified enzyme solution (2 mg / mL), 6 μL of pNP-α-N-acetylneuraminicaci (10 mM) and 53 μL of PBS buffer. The reaction system was incubated at 37°C for 5 minutes, and then 90 μL of 1M Na2CO3 solution was added to terminate the reaction. The absorbance of the solution at 405 nm was then measured on a microplate reader. The results showed that both SiaT and Nb16-SiaT had high monosaccharide cleavage activity. The results are shown in FIG. Figure 2 (Right) shows that the fusion modification does not affect the sialic acid hydrolysis activity of SiaT.
[0041] Example 3 Determination of the in vivo anti-tumor activity of the bifunctional antibody Nb16-SiaT
[0042] Resuscitate mouse CT26 tumor cells and passage them to ensure that the tumor cells have been passaged for at least 3 generations. CT26 tumor cells were subcutaneously inoculated into female BALB / c mice at an inoculation volume of 1×10 6 The mice were randomly divided into five groups (8 mice per group): negative control (NC), nanobody Nb16 (Nb16), sialidase SiaT (SiaT), combination therapy (Combo), and bifunctional antibody Nb16-SiaT (Nb16-SiaT). Each group received the same molar dose after conversion based on molar concentration: 45 μg / mouse for Nb16, 150 μg / mouse for SiaT, and 200 μg / mouse for Nb16-SiaT. Administration was intraperitoneal for a total of four times, on days 4, 7, 9, and 11 after tumor loading.
[0043] Starting from the 4th day, the tumors were measured and weighed every 2 days. The weight of the mice and the long diameter (L) and short diameter (W) of the tumors were recorded until the end of the dissection. The tumor volume (V) was calculated as (L×W×W) / 2, and the tumor growth curve was drawn to calculate the tumor inhibition rate. When the subcutaneous tumor volume of the animals reached 1000mm 3 After euthanasia, the animals were dissected and the subcutaneous tumors were removed.
[0044] During the experimental period, the mouse tumor growth curve was as follows Figure 3 As shown in (left), Nb16-SiaT significantly inhibited tumor growth. The tumor volume and tumor weight at the anatomical endpoints also suggested that Nb16-SiaT significantly inhibited tumor growth. Figure 3 (right) Nb16-SiaT has a significantly better anti-tumor effect than the single-dose group and the combined-dose group.
[0045] The above results indicate that Nb16-SiaT is a bifunctional antibody drug with potential development value.
[0046] Example 4: Optimization of the anti-tumor dose of the bifunctional antibody Nb16-SiaT
[0047] The establishment of an animal tumor model was the same as described in Example 3. Tumor-bearing mice were randomly divided into three groups (8 mice per group), namely: a negative control group (0 mg / kg), a low-dose group (2.5 mg / kg), and a high-dose group (5 mg / kg). The mice were administered intraperitoneally four times on days 4, 7, 9, and 11 after tumor bearing. Starting from day 4, the tumors were measured and weighed every two days. The weight of the mice and the long diameter (L) and short diameter (W) of the tumors were recorded until the end of the autopsy. The tumor volume (V) was calculated as (L×W×W) / 2, and the tumor growth curve was plotted to calculate the tumor inhibition rate. When the subcutaneous tumor volume of the animals reached 1000 mm 3 Afterwards, the animals were euthanized, dissected, and the subcutaneous tumors were removed. During the experimental period, the tumor growth curve of the mice was as follows: Figure 4 (Left) shows the photos of mouse tumors after the end point dissection. Figure 4 (right) The results showed that the anti-tumor effect of the bifunctional antibody Nb16-SiaT was dose-dependent, with a dose of 5 mg / kg achieving the best anti-tumor effect.
[0048] The sequence involved in the present invention is as follows:
[0049] >SEQ ID NO.1
[0050] QVQLQESGGGSVQAGGSLRLSCAASGYTDRNYWMGWFRQAPGKEREGVAALYTRTGSTYYADSVKGRFTISHDNAKNTLYLQMNSLKPEDTAVYYCAADSNAYGLAPLHHYWGQGTQVTVSS
[0051] >SEQ ID NO.2
[0052] GDHPQATPAPAPDASTELPASMSQAQHVAPNTATDNYRIPAITTAPNGDLLISYDERPKDNGNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSQAGTDPDNRGIIQAEVSTSTDNGWTWTHRTITADITKDNPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDQNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPKPWSRDRGTISMSCDDGASWTTSKIFHEPFVGYTTIAVQSDGSIGLLSEDAHDGANYGGIWYRNFTMNWLGEQCGQKPAE
[0053] >SEQ ID NO.3
[0054] QVQLQESGGGSVQAGGSLRLSCAASGYTDRNYWMGWFRQAPGKEREGVAALYTRTGSTYYADSVKGRFTISHDNAKNTLYLQMNSLKPEDTAVYYCAADSNAYGLAPLHHYWGQGTQVTVSSGGGGSGGGGSGGGGSGDHPQATPAPAPDASTELPASMSQAQHVAPNTATDNYRIPAITTAPNGDLLISYDERPKDNGNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSQAGTDPDNRGIIQAEVSTSTDNGWTWTHRTITADITKDNPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDQNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPKPWSRDRGTISMSCDDGASWTTSKIFHEPFVGYTTIAVQSDGSIGLLSEDAHDGANYGGIWYRNFTMNWLGEQCGQKPAE
[0055] >SEQ ID NO:4
[0056]
[0057] In the description of the present invention, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0058] The embodiments described above are merely specific implementations of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be imagined by any person skilled in the art within the technical scope disclosed in the present application without resorting to creative effort should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims in the present application.
Claims
1. A bifunctional antibody targeting PD-L1 and sialic acid, characterized in that: The bifunctional antibody has PD-L1 binding activity and sialic acid hydrolysis activity, and its structure includes a nanobody Nb16 that binds to mouse PD-L1, a connecting peptide, and a sialidase SiaT, wherein the N-terminus of the sialidase is connected to the C-terminus of the PD-L1 nanobody via a connecting peptide; The PD-L1 nanobody Nb16 has the amino acid sequence shown in SEQ ID NO.1, and the sialidase SiaT has the amino acid sequence shown in SEQ ID NO.2; The amino acid sequence of the connecting peptide is (GGGGX)n, wherein X is Gly or Ser, and n is a natural number of 1-5.
2. The bifunctional antibody targeting PD-L1 and sialic acid according to claim 1, characterized in that The amino acid sequence of the bifunctional antibody Nb16-SiaT is shown in SEQ ID NO:
3.
3. A nucleic acid molecule, characterized in that Encodes the bifunctional antibody targeting PD-L1 and sialic acid according to claim 1 or 2.
4. The nucleic acid molecule according to claim 3, characterized in that The sequence of the nucleic acid molecule is shown in SEQ ID NO:
4.
5. A biomaterial, characterized in that The biological material is any one of the following: recombinant DNA, expression cassette, vector, host cell, engineered bacteria or cell line; The biological material contains the nucleic acid molecule according to claim 3 or 4.
6. A method for preparing the bifunctional antibody targeting PD-L1 and sialic acid according to claim 1 or 2, characterized in that: The method includes: Introducing a nucleic acid encoding the bispecific antibody into a host cell to obtain a host cell that stably expresses the bispecific antibody; and / or The host cells are cultured and the bifunctional antibody is obtained through purification.
7. A product, characterized in that The product comprises the bifunctional antibody targeting PD-L1 and sialic acid according to claim 1 or 2, and the product is a fusion protein, an immunotoxin, a drug, a detection reagent or a detection kit.
8. Use of the bifunctional antibody targeting PD-L1 and sialic acid according to claim 1 or 2, characterized in that: The application is selected from one or more of the following: (1) Use in the preparation of drugs for diagnosing, preventing or treating diseases related to PD-L1 expression; (2) Use in the preparation of drugs for diagnosing, preventing or treating diseases targeting PD-L1; (3) Use in the preparation of drugs that kill PD-L1-expressing cells; (4) Application in the preparation of PD-L1 detection reagents or kits; (5) Application in the preparation of reagents for CAR-T therapy; (6) Application in the preparation of immunotoxins or labeled antibodies; (7) Application in the preparation of antibody-drug conjugates.
9. The use of the bifunctional antibody targeting PD-L1 and sialic acid according to claim 8, characterized in that: The use of the bifunctional antibody targeting PD-L1 and sialic acid in the preparation of an anti-tumor drug; the tumor is derived from lung cancer, colon cancer, gastric cancer, liver cancer or breast cancer.
10. Use of the nucleic acid molecule according to claim 3 or 4, characterized in that: The application is selected from one or more of the following: (1) Use in the preparation of drugs for diagnosing, preventing or treating diseases related to PD-L1 expression; (2) Use in the preparation of drugs for diagnosing, preventing or treating diseases targeting PD-L1; (3) Use in the preparation of drugs that kill PD-L1-expressing cells; (4) Application in the preparation of PD-L1 detection reagents or kits; (5) Application in the preparation of reagents for CAR-T therapy; (6) Application in the preparation of immunotoxins or labeled antibodies; (7) Application in the preparation of antibody-drug conjugates.