WO2021051661A1 - Recombinant canine pd-1 fusion protein and preparation method therefor and application thereof - Google Patents

Abstract

A recombinant canine PD-1 fusion protein and a preparation method therefor and an application thereof. The fusion protein is formed by directly or indirectly linking the extracellular domain of a canine PD-1 protein or the mutant of the extracellular domain of the canine PD-1 protein to an Fc fragment. Compared with wild-type PD-1, the recombinant canine PD-1 fusion protein has higher affinity and a better effect, and provides more options for the immunotherapy of canine tumors.

Description

Recombinant canine PD-1 fusion protein and its preparation method and application

cross reference

This application claims the priority of the Chinese patent application No. 201910888315.7 with the patent title "Recombinant Canine PD-1 Fusion Protein and Its Preparation Method and Application" filed on September 19, 2019, the entire disclosure of which is incorporated herein by reference in its entirety. .

Technical field

The invention relates to the field of biomedicine, in particular to two novel recombinant canine PD-1 fusion proteins and their preparation methods and applications.

Background technique

PD-1, called Programmed Cell Death Receptor 1 (also known as PDCD1), is a type I membrane glycoprotein with a size of 50-55KD. It belongs to the CD28 superfamily of receptors and is an important immunosuppressive molecule. PD-1 protein is mainly expressed on the surface of T cells (including T cells activated by antigen stimulation), B lymphocytes and activated macrophages, and bone marrow cells also express PD-1. PD-1 has two ligands—PD-L1 and PD-L2. Under normal physiological conditions, the combination of PD-1 and PD-L1/PD-L2 inhibits the activation of T cells and the production of cytokines, thereby protecting the body from the immune system. However, a variety of solid tumors and some hematological malignancies, including melanoma, Hodgkin’s lymphoma cell line, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer PD-L1 is also abundantly expressed on the cell surface. PD-L1 on the tumor cell membrane binds to PD-1 on T cells, down-regulating T cell activation and cytokine secretion, successfully evading the recognition and attack of the body's immune system, leading to immune escape of tumor cells. At the same time, studies have found that the expression of PD-L1 on tumor cells is related to the poor prognosis of multiple tumor types.

Blocking the interaction of PD-1 and PD-L1 has become an effective way to treat tumors. At present, most of the anti-tumor drugs targeting PD-1/PD-L1 interactions are anti-PD-1 or PD-L1 monoclonal antibodies, including PD-1 monoclonal antibodies, such as Bristol-Myers Squibb (BMS) company’s Niwu Monoclonal antibody (Nivolumab), Merck (Merck) Pembrolizumab (Pembrolizumab), PD-L1 monoclonal antibody drugs, such as Roche Genentech's Atezolizumab (Atezolizumab), AstraZeneca's dervaluzumab (Durvalumab), Avelumab from Pfizer and Merck. These monoclonal antibodies are mainly used to treat human malignant tumors, including melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, head and neck squamous cell carcinoma, etc. The monoclonal antibody treatment has achieved certain therapeutic effects, but there are also certain limitations. For example, the average treatment efficiency is low (single-drug effective rate is about 20%), high immunogenicity, individual variability, and limited indications. Therefore, there is an urgent need for the development of new and highly effective candidate tumor drugs.

PD-1 fusion protein is a new tumor drug targeting the PD-1/PD-L1 signaling pathway. It can competitively bind to the PDL-1/PD-L2 ligand of tumor cells and block the binding of tumor cells and T cells. , So as to restore the activation of T cells and increase the level of cytokine secretion, and enhance the immune response. There are few reports on the application and research of PD-1 fusion protein related drugs in humans, and the research on canine tumors is still at a blank stage.

Canine neoplastic diseases are relatively common diseases, including melanoma, breast cancer, mast cell tumors, lipomas, etc. About a quarter of dogs will develop neoplastic diseases at some stage in their lives, and older dogs are more common. Ten In dogs over the age of almost 50%, tumors will occur. The main treatment methods for canine tumors are surgery, chemotherapy, and radiotherapy. The main therapeutic drugs used abroad include chemotherapeutics such as lomustine, vinblastine, and glucocorticoid drugs such as prednisolone. However, these drugs have serious side effects and are harmful The self-injury is serious; at present, there are no canine tumor drugs on the market in China, which is a blank in the market.

The present invention provides canine PD-1 fusion protein, which is specifically aimed at canine tumor diseases, by enhancing the anti-tumor function of T cells, assisting the autoimmune system to clear tumor cells, with high effectiveness and less damage to the body.

Summary of the invention

The purpose of the present invention is to provide two novel recombinant canine PD-1 fusion proteins and their preparation methods and applications.

In order to achieve the purpose of the present invention, in the first aspect, the present invention provides two novel recombinant canine PD-1 fusion proteins. One fusion protein is formed by directly or indirectly connecting the extracellular region of canine PD-1 protein to the Fc fragment, and the other This fusion protein canine PD-1 protein extracellular region mutant is directly or indirectly connected to the Fc fragment.

The extracellular domain of the canine PD-1 protein is:

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 5; or

(b) The sequence shown in SEQ ID NO: 5 is a protein derived from (a) that has been substituted, deleted or added one or several amino acids and has the same function.

The mutants of the extracellular region of the canine PD-1 protein are:

(c) A protein consisting of the amino acid sequence shown in SEQ ID NO: 6; or

(d) The sequence shown in SEQ ID NO: 6 is substituted, deleted or added one or several amino acids and has the same function as a protein derived from (c).

The Fc fragment includes an immunoglobulin hinge region, CH2 and CH3 regions.

The immunoglobulin is derived from humans or mammals, preferably humans or mammals such as dogs, cats, pigs, mice, horses, and cattle, and more preferably canine IgG1 immunoglobulins.

The fusion protein is formed by linking the extracellular region of the canine PD-1 protein or the mutant of the extracellular region of the canine PD-1 protein directly or indirectly with the Fc fragment through a Linker. Wherein, the Linker is a flexible polypeptide composed of 2-20 flexible amino acids, and the flexible amino acid is selected from at least one of Gly, Ser, Ala, and Thr.

Preferably, the Linker is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer between 2-5, more preferably n is 2.

Further, the two new recombinant canine PD-1 fusion proteins are cPD1-Fc and cPD1mu-Fc;

The fusion protein cPD1-Fc is:

(e) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or

(f) A protein derived from (e) that has more than 90% homology with the amino acid sequence shown in SEQ ID NO:1 and has the same function.

The fusion protein cPD1mu-Fc is:

(g) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3; or

(h) A protein derived from (g) that has more than 90% homology with the amino acid sequence shown in SEQ ID NO: 3 and has the same function.

In the second aspect, the present invention provides a biological material containing the gene encoding the recombinant canine PD-1 fusion protein, and the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, an engineered bacteria, or a transgenic cell line Wait.

In the third aspect, the present invention provides a method for preparing the recombinant canine PD-1 fusion protein, the method comprising: artificially synthesizing the coding gene of the fusion protein, and performing codon optimization, and constructing the optimized gene into eukaryotic The expression vector is transformed into eukaryotic cells and expressed in eukaryotic cells to separate and purify the target protein.

Preferably, the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cell is a CHO cell.

The optimized nucleotide sequences of cPD1-Fc and cPD1mu-Fc are shown in SEQ ID NO: 2 and 4, respectively.

In a fourth aspect, the present invention provides any of the following applications of the recombinant canine PD-1 fusion protein:

1) For the treatment of canine tumors and related diseases, including promoting tumor shrinkage or effectively controlling tumor growth, and improving the quality of life of the dog;

2) For the preparation of canine anti-tumor drugs or compositions.

In the fifth aspect, the present invention provides a canine anti-tumor drug or composition, the active ingredient is the recombinant canine PD-1 fusion protein.

The tumors include, but are not limited to, melanoma (such as malignant melanoma), lymphoma, mast cell tumor, sarcoma, head and neck tumor, non-small cell lung cancer, breast cancer, urothelial cancer, bladder cancer.

Further, the present invention also provides the canine PD-1 fusion protein or its preparation alone or in combination with surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy, gene therapy, angiogenesis inhibition therapy, palliative care, etc. One or several treatment methods are used to treat canine tumor-related diseases.

In the sixth aspect, the present invention provides a canine PD-1 protein mutant or a canine PD-1 protein truncated mutant, which contains a T→ at residue 132 of the amino acid sequence of the canine PD-1 protein. L mutation site.

It should be understood that the engineered proteins, including canine cPD1-Fc and cPD1mu-Fc fusion proteins or canine PD-1 extracellular domain and its mutant protein are fused with canine Fc or with other proteins to form a fusion that does not change the activity of the protein All proteins belong to the protection scope of the present invention.

Modified proteins, including two fusion proteins cPD1-Fc, cPD1mu-Fc, or canine PD-1 and their mutants are glycosylated, pegylated, acetylated or combined with BSA, etc., all belong to the protection of the present invention range.

With the above technical solutions, the present invention has at least the following advantages and beneficial effects:

Compared with wild-type PD-1, the two novel recombinant fusion proteins cPD1-Fc and cPD1mu-Fc provided by the present invention have higher PD-L1 affinity and better pharmacological effects, and provide immunotherapy for canine tumors. More choices.

Description of the drawings

Figure 1 is an SDS-PAGE electrophoresis diagram of cPD1-Fc and cPD1mu-Fc in Example 2 of the present invention. Among them, A: denaturing electrophoresis of cPD1-Fc; B: non-denaturing electrophoresis of cPD1-Fc, C: denaturing electrophoresis of cPD1mu-Fc; D: non-denaturing electrophoresis of cPD1mu-Fc. MK: Protein Marker; 1: Clarified fermentation broth; 2: Protein A collection broth; 3: Capto Q collection broth.

Fig. 2 shows the application of cPD1-Fc and cPD1mu-Fc in the treatment of canine melanoma, stromal sarcoma, and breast cancer in Example 4 of the present invention. Among them, A, B, C, D, E are respectively No. 6 (melanoma), No. 4 (melanoma), No. 13 (breast cancer), No. 25 (stromal sarcoma), No. 7 (melanoma) canine tumor Changes in load.

detailed description

The technical scheme of the present invention is as follows: the amino acid sequence of the extracellular region of canine PD-1 or its mutant is directly or indirectly fused to the Fc fragment through a connecting element to synthesize recombinant canine PD-1 fusion proteins: cPD1-Fc and cPD1mu-Fc.

The cPD1-Fc fusion protein is a fusion protein of the extracellular domain of canine PD-1, and is a protein composed of the amino acid sequence shown in SEQ ID NO:1; or has a homology of more than 90% with SEQ ID NO:1 , And a protein composed of amino acid sequences with equivalent functions.

The cPD1mu-Fc fusion protein is a fusion protein of a mutant of the extracellular region of canine PD-1, and is a protein composed of the amino acid sequence shown in SEQ ID NO: 3; or it has 90% homology with SEQ ID NO: 3 A protein composed of the above and having the same functional amino acid sequence. The fusion protein contains a mutant part of the canine PD-1 protein. The Thr of position 132 of canine PD-1 participates in the binding interface of cPD-1 and cPD-L1. The Thr is mutated to Leu, which enhances the binding to PD-L1 ligand. Affinity.

The fusion protein includes an Fc fragment, the Fc fragment includes an immunoglobulin hinge region and CH2 and CH3 regions; the immunoglobulin comes from mammals such as dogs, cats, humans, and pigs.

The connection relationship between the canine PD-1 extracellular region or its mutant and Fc is direct splicing connection or connection via Linker, preferably via Linker; wherein the Linker is a flexible polypeptide composed of 2-20 flexible amino acids, so The flexible amino acid is selected from at least one of Gly, Ser, Ala and Thr; preferably, the Linker is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer between 2-5, and more Preferably n is 2.

Further, the present invention also provides a biological material encoding the DNA sequence of the fusion protein, the biological material being an expression cassette, an expression vector, a cloning vector, an engineered bacteria or a transgenic cell line.

The present invention provides a method for preparing recombinant canine PD-1 fusion protein. The coding genes of cPD1-Fc and cPD1mu-Fc are artificially synthesized, and the codons are optimized. The optimized genes are cloned into a eukaryotic expression vector; cPD1-Fc And cPD1mu-Fc recombinant vectors were transformed into eukaryotic cells and expressed in eukaryotic cells to separate and purify the target protein; preferably, the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cells include CHO cells.

The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the examples follow conventional experimental conditions, such as Sambrook J & Russell DW (Molecular Cloning: a Laboratory Manual, 2001), or the conditions suggested by the manufacturer's instructions.

Example 1 Construction of eukaryotic expression vector for canine cPD1-Fc and cPD1mu-Fc fusion protein

Search for the amino acid sequences of canine PD-1 (UniProtKB: A0A024FCJ9) and canine Fc (GenBank: AF354264) in UniProt and GenBank libraries; mutate the Thr at position 132 of canine PD-1 to Leu to form cPD1mu.

Codon optimization was performed on cPD1-Fc and cPD1mu-Fc respectively. The nucleotide sequence of cPD1-Fc is shown in SEQ ID NO: 2, and the nucleotide sequence of cPD1mu-Fc is shown in SEQ ID NO: 4. The above-mentioned nucleotides were artificially synthesized and constructed in pcDNA3.1 vectors to obtain recombinant expression vectors pcDNA3.1-cPD1-Fc and pcDNA3.1-cPD1mu-Fc. The pcDNA3.1-cPD1-Fc and pcDNA3.1-cPD1mu-Fc recombinant vectors were linearized and electrotransformed into CHO cells respectively to obtain stable transfected cell lines of cPD1-Fc and cPD1mu-Fc.

Example 2 Purification of canine cPD1-Fc and cPD1mu-Fc fusion protein

The stable transfected cells expressing canine cPD1-Fc and cPD1mu-Fc are fermented and cultured in a fermentor. The fermentation broth is first passed through a two-stage deep filtration membrane package to remove cells and cell debris, and then filtered with a 0.22μm filter membrane to obtain a clarified fermentation. liquid. The fermentation broth is first purified by affinity chromatography Protein A (MabSelect SuReTM, GE Healthcare): first equilibrate to the baseline with a balance solution (50mM glycine, 0.15M NaCl, pH 7.2), and then use the eluent (50mM glycine, pH 3.0) Elute and collect the eluate. The Protein A collection solution was refined by anion exchange Capto Q (GE Healthcare) column chromatography: the collection solution was adjusted to pH 8.0 with 1M NaOH, balanced to the baseline with the balance solution (50mM glycine pH 8.0), and then the eluent ( 50mM glycine, 0.2M KCl, pH 8.0) eluted and collected purified protein. Concentrate by ultrafiltration first, and then replace the buffer. SDS-PAGE gel electrophoresis was performed on the purified target protein (Figure 1).

Example 3 Detection of affinity constants of canine cPD1-Fc and cPD1mu-Fc fusion proteins

A BIAcore 3000 instrument was used for affinity analysis. Three sample groups (cPD-1, cPD1-Fc, cPD1mu-Fc) were set up in the experiment. First, the recombinant canine PD-L1 (the preparation method of recombinant canine PD-L1 was the same as in Example 2, and its amino acid sequence was as SEQ ID NO: 7 (Shown) is coupled to channel 2, and then channel 1 is sealed as a control channel. Analytes (cPD-1, cPD1-Fc, cPD1mu-Fc) were diluted (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) after inflow, and the binding curve of PD-L1 coupled to the chip surface was measured. Use the software Wizard program to record the signal Fc2-1. Analyze with 1:1 binding model to determine the affinity constant KD. The measurement results show: cPD1-Fc binding constants of PD-L1 recombinant canine (K _a) of 1.60 × 10 ^5, the dissociation constant (K _d) of 5.12 × 10 ^-1, the affinity constant (KD) of 3.2 × 10 ^{- 6} M; ^cPD1mu-Fc recombinant canine K _a of PD-L1 was 2.60 × 10 ^5, K _d of 1.33 × 10 ^-2, KD of 5.1 × 10 ^-8 M; cPD-1 and PD-L1 recombinant canine of K _a is 1.05×10 ⁵ , K _d is 9.45×10 ^-1 , and KD is 9.0×10 ^-6 M. It shows that both canine cPD1-Fc and cPD1mu-Fc can increase the affinity to cPD-L1, and the effect of cPD1mu-Fc is more obvious.

Example 4 Therapeutic effect of canine cPD1-Fc and cPD1mu-Fc fusion protein on canine malignant tumors

Thirty tumor dogs (including 12 melanoma dogs, 9 breast cancer dogs, and 9 mesenchymal sarcoma dogs) treated in a pet hospital in Beijing were screened for preliminary clinical effect research. The test dog breeds include: Golden Retriever, Toy Poodle, Chihuahua, Schnauzer, Pug, Dachshund, Chow Chow, Bichon Frise, Labrador, and Crossbreed Dogs, with an average age of 13 years (11- 16 years old), all dogs with advanced malignant tumors.

Table 1 Situation of test dogs

Divide all dogs into three groups: cPD1-Fc, cPD1mu-Fc and the negative control group. Each group includes 4 melanoma dogs, 3 breast cancer dogs, and 3 mesenchymal sarcoma dogs. The test group is based on 2mg/kg body weight. The doses were intravenous infusion of cPD1-Fc and cPD1mu-Fc (diluted with saline for injection). The negative control group was intravenously injected with saline, which was administered once every two weeks for four consecutive administrations. The test was terminated one week after the last administration. In the trial, the tumor burden was assessed through gross examination and computed tomography, and the tumor size was measured every two weeks to evaluate the efficacy and compare the objective remission rate.

Objective remission rate = dogs with complete remission and partial remission/total dogs × 100%

Among them, complete remission refers to the disappearance of all detectable tumors, and partial remission refers to a reduction of at least 30% in the sum of the maximum diameter of the target lesion.

The results of the experiment showed that during the whole experiment, all the animals in the test group were in good mental state, no animals died, routine physical examination, complete blood count, serum chemistry tests were normal, no allergic reactions or autoimmune diseases were found, and the overall state of the negative control group was poor. , Most canine tumors are enlarged or new tumors appear. In the cPD1-Fc group, 2 cases of dogs in the cPD1-Fc group had partial remission (the sum of the maximum diameter of the target lesion was reduced by ≥30%, maintained for at least 4 weeks). After two weeks of treatment, obvious tumor regression was observed, and the tumor suppression effect was obvious; partial remission The tumor size of dogs other than the case was not significantly reduced or slightly increased, but the overall state of the dog (good spirit, normal diet, increased activity) was significantly improved compared to before treatment, and the overall treatment objective remission rate was 20% (2/10). In the cPD1mu-Fc group, 3 cases of dogs in the cPD1mu-Fc group had partial remission. The tumor size of other dogs other than the partial remission cases was not significantly reduced or slightly increased, but the overall condition was significantly improved compared with before treatment, the tumor did not continue to deteriorate, and the overall treatment objective remission rate It was 30% (3/10). Most of the tumors of the dogs in the negative control group were enlarged or new tumors appeared, and the status was poor. The specific results are shown in Table 2 and Figure 2.

Table 2 The effect of cPD1-Fc and cPD1mu-Fc fusion protein in the treatment of tumors

Note: partial response (PR, partial response): the sum of the maximum diameter of the target lesion is reduced by ≥30%, and maintained for at least 4 weeks; stable disease (SD, stable disease): the sum of the maximum diameter of the target lesion is smaller than PR, or increases Less than PD; Progressive disease (PD, progressive disease): The sum of the maximum diameter of the target lesion increases by at least ≥20%, or new lesions appear.

Although the present invention has been described in detail with general descriptions and specific implementations above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.

Industrial applicability

The invention provides a recombinant canine PD-1 fusion protein and a preparation method and application thereof. The fusion protein is formed by directly or indirectly connecting the extracellular domain of the canine PD-1 protein or the mutant of the extracellular domain of the canine PD-1 protein to the Fc fragment. Compared with wild-type PD-1, the recombinant canine PD-1 fusion protein provided by the present invention has higher affinity and better efficacy, provides more options for the immunotherapy of canine tumors, and has better economic value And application prospects.

Claims (10)

1. The recombinant canine PD-1 fusion protein is characterized in that the fusion protein is formed by directly or indirectly connecting the extracellular region of the canine PD-1 protein or the mutant of the extracellular region of the canine PD-1 protein to the Fc fragment;

   The extracellular domain of the canine PD-1 protein is:

   (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 5; or

   (b) The sequence shown in SEQ ID NO: 5 is substituted, deleted or added with one or several amino acids and has the same function as a protein derived from (a);

   The mutants of the extracellular region of the canine PD-1 protein are:

   (c) A protein consisting of the amino acid sequence shown in SEQ ID NO: 6; or

   (d) The sequence shown in SEQ ID NO: 6 is substituted, deleted or added one or several amino acids and has the same function as a protein derived from (c).
2. The fusion protein of claim 1, wherein the Fc fragment comprises an immunoglobulin hinge region, CH2 and CH3 regions;

   The immunoglobulin is from human or mammal, preferably canine, cat, human, pig, mouse, horse, bovine IgG1 immunoglobulin.
3. The fusion protein according to claim 1, wherein the fusion protein is directly or indirectly connected to the Fc fragment through a Linker connection between the extracellular region of the canine PD-1 protein or the extracellular region of the canine PD-1 protein. Into; the Linker is a flexible polypeptide composed of 2-20 flexible amino acids, the flexible amino acids selected from at least one of Gly, Ser, Ala, Thr;

   Preferably, the Linker is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer between 2-5, more preferably n is 2.
4. The fusion protein of claim 1, wherein the fusion protein is cPD1-Fc or cPD1mu-Fc;

   The fusion protein cPD1-Fc is:

   (e) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or

   (f) A protein derived from (e) that has more than 90% homology with the amino acid sequence shown in SEQ ID NO:1 and has the same function;

   The fusion protein cPD1mu-Fc is:

   (g) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3; or

   (h) A protein derived from (g) that has more than 90% homology with the amino acid sequence shown in SEQ ID NO: 3 and has the same function.
5. The biological material containing the gene encoding the fusion protein of any one of claims 1 to 4, the biological material being an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, an engineered bacteria or a transgenic cell line.
6. The preparation method of the fusion protein according to any one of claims 1 to 4, characterized in that the method comprises: artificially synthesizing the coding gene of the fusion protein, and performing codon optimization, and constructing the optimized gene into eukaryotic In the expression vector, it is transformed into eukaryotic cells and expressed in eukaryotic cells to separate and purify the target protein;

   Preferably, the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cell is a CHO cell.
7. Use of the fusion protein of any one of claims 1 to 4 in the preparation of canine anti-tumor drugs or compositions.
8. The application according to claim 7, wherein the tumor is melanoma, lymphoma, mast cell tumor, sarcoma, head and neck tumor, non-small cell lung cancer, breast cancer, urothelial cancer, bladder cancer.
9. A canine anti-tumor drug or composition, characterized in that the active ingredient is the fusion protein according to any one of claims 1-4.
10. Canine PD-1 protein mutant or canine PD-1 protein truncated mutant, characterized in that the mutant contains a T→L mutation site located at residue 132 of the canine PD-1 protein amino acid sequence .

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