Method for preparing recombinant canine pd-1 and canine sirpα double fusion protein and application thereof
By developing a canine PD-1 and SIRPα fusion protein, the challenge of tumor treatment caused by overexpression of CD47 and PD-L1 in dogs has been solved, achieving safe and efficient tumor cell inhibition and immune activation effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING VJT BIO CO LTD
- Filing Date
- 2022-11-04
- Publication Date
- 2026-07-21
AI Technical Summary
Currently, there is a lack of effective treatments for diseases caused by overexpression of CD47 and/or PD-L1 in dogs, especially tumors, particularly malignant tumors. Existing chemotherapy drugs have problems such as poor tumor specificity and significant toxic side effects.
A fusion protein was developed, comprising a mutant extracellular region of canine PD-1 protein and canine SIRPα protein, linked by an Fc fragment, to bind to PD-L1 and CD47 on tumor cells, blocking immune escape pathways, activating T cells, and promoting macrophage phagocytosis.
This fusion protein can safely and effectively inhibit the binding of tumor cells to T cells and macrophages, promote T cell activation and macrophage phagocytic activity, thereby killing tumor cells, and has no blood toxicity.
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Figure CN116023508B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of canine tumor treatment, specifically to a fusion protein, a nucleic acid encoding the fusion protein as described above, a biomaterial containing the gene encoding the fusion protein, a method for producing the fusion protein as described above, a pharmaceutical composition, a kit, and the use of the fusion protein, nucleic acid, or biomaterial in the preparation of a medicament for treating canine diseases caused by overexpression of CD47 and / or PD-L1. Background Technology
[0002] Cancer is a common and prevalent disease in humans and animals, with canine malignant tumors becoming a leading cause of death in dogs. International research reports show that one in four dogs is diagnosed with cancer; the rate is even higher, exceeding 50%, in dogs over 10 years old. Current clinical treatments for canine cancer primarily include surgery, chemotherapy, and radiation therapy. Common chemotherapy drugs include cyclophosphamide, doxorubicin, vincristine, and prednisolone, all of which have drawbacks such as poor tumor specificity and significant toxic side effects. While some small-molecule and protein drugs have been approved for treating canine cancers abroad, this market is currently unavailable in China.
[0003] Programmed cell death receptor 1 (PD-1) is a type I transmembrane glycoprotein of approximately 55 kDa, belonging to the CD28 superfamily of receptors, and is mainly expressed on the surface of T cells, B lymphocytes, and activated macrophages. PD-1 has two ligands: PD-L1 and PD-L2. Under normal physiological conditions, the binding of PD-1 to PD-L1 / PD-L2 inhibits T cell activation, thereby protecting the body from attack by the autoimmune system. However, various solid tumors and some hematological malignancies, including melanoma, breast cancer, various digestive system tumors, lymphoma, and leukemia, also express PD-L1 in large quantities. PD-L1 on the tumor cell membrane binds to PD-1 on T cells, inhibiting T cell activation, thus successfully evading recognition and attack by the body's immune system, achieving immune escape for tumor cells. Furthermore, studies have found that PD-L1 expression on tumor cells is associated with poor prognosis in several tumor types. Therefore, blocking the interaction between PD-1 and PD-L1 has become an effective approach for cancer treatment.
[0004] The CD47-SIRPα (signal-regulatory protein α) signaling pathway is also known as the "don't eat me" signal. SIRPα belongs to the SIRP receptor family of proteins and is mainly expressed on the surface of myeloid cells (monocytes, macrophages, granulocytes, and myeloid dendritic cells, etc.), and is also expressed in neurons of the nervous system. CD47 is the most important ligand of SIRPα. It is a transmembrane protein widely expressed on the surface of normal cells, with a molecular weight of approximately 50 kDa, and belongs to the immunoglobulin superfamily. Under normal physiological conditions, CD47 on the cell membrane binds to SIRPα on the surface of macrophages, thereby inhibiting the phagocytosis of macrophages. This function can be used to mark "self" and "non-self" to avoid "collateral damage"; however, CD47 is also highly expressed on the surface of various tumor cells, sending out the "don't eat me" signal, thereby inhibiting the phagocytosis of tumor cells by macrophages and achieving immune escape. Therefore, this target can also serve as an effective target for tumor therapy.
[0005] There are currently some patents related to dual targets of human PD-(L)1 and CD47, but there are no research reports on dual targets of PD-(L)1 and CD47 in dogs. Summary of the Invention
[0006] The purpose of this invention is to provide a fusion protein targeting both PD-(L)1 and CD47 in dogs, suitable for the treatment of diseases caused by overexpression of CD47 and / or PD-L1 in dogs, with no hematologic toxicity and good safety profile. This invention also provides a nucleic acid encoding the fusion protein as described above, a biological material containing the encoding gene of the fusion protein, a method for producing the fusion protein as described above, a pharmaceutical composition, a kit, and the use of the fusion protein, nucleic acid, or biological material in the preparation of a medicament for treating canine diseases caused by overexpression of CD47 and / or PD-L1.
[0007] To achieve the above objectives, a first aspect of the present invention provides a fusion protein comprising a canine PD-1 protein extracellular region mutant and canine SIRPα protein. Preferably, the canine PD-1 protein extracellular region mutant is the canine PD-1 protein extracellular region T132L.
[0008] Preferably, the fusion protein includes an Fc fragment.
[0009] A second aspect of the present invention provides a nucleic acid encoding the fusion protein as described above.
[0010] A third aspect of the present invention provides a biological material containing a gene encoding a fusion protein as described above, wherein the biological material is an expression cassette, a transposon, a vector, or a host cell.
[0011] A fourth aspect of the present invention provides a method for preparing a fusion protein as described above, the method comprising culturing a host cell as described above under conditions suitable for expression of the fusion protein, and then recovering the expressed fusion protein from the culture medium.
[0012] A fifth aspect of the present invention provides a pharmaceutical composition comprising the fusion protein as described above, as well as a pharmaceutically acceptable carrier and optional other therapeutic agents.
[0013] A sixth aspect of the present invention provides a kit comprising the fusion protein, the nucleic acid, or the biological material as described above.
[0014] The seventh aspect of the present invention provides the use of the fusion protein, nucleic acid, or biological material described above in the preparation of a medicament for treating canine diseases caused by overexpression of CD47 and / or PD-L1.
[0015] The fusion protein described in this invention has no blood toxicity, good safety, and can simultaneously bind to PD-L1 and CD47 on tumor cells, inhibiting the binding of tumor cells to T cells and macrophages, thereby promoting T cell activation and macrophage phagocytic activity, and achieving the purpose of killing tumor cells. Attached Figure Description
[0016] Figure 1 A schematic diagram of the structure of the fusion protein described in this invention is shown; Figure 2 The SDS-PAGE electrophoresis images of the fusion protein described in this invention are shown, where A: 6% denaturing electrophoresis; B: 8% non-denaturing electrophoresis; where 1 is canine PD-1mu-SIRPα-Fc; 2 is canine SIRPα-PD-1mu-Fc; 3 is canine PD-1mu-Fc-SIRPα; 4 is canine SIRPα-Fc-PD-1mu; 5 is canine PD-1mu-SIRPαmu-Fc; 6 is canine SIRPαmu-PD-1mu-Fc; 7 is canine PD-1mu-Fc-SIRPαmu; 8 is canine SIRPαmu-Fc-PD-1mu. Figure 3 The results of the erythrocyte agglutination assay for the fusion protein are shown; Figure 4 The SDS-PAGE electrophoresis images of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc are shown, where A: 8% denaturing electrophoresis image; B: 8% non-denaturing electrophoresis image; 1 is the purified protein of canine PD-1mu-SIRPα-Fc; 2 is the purified protein of canine PD-1mu-SIRPαmu-Fc. Figure 5The results of the binding activity assays of canine PD-1mu-SIRPα-Fc with canine PD-L1 and human CD47 are shown. Figure 6 The results of biological activity assays for canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc are shown. Detailed Implementation
[0017] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0018] The first aspect of the present invention provides a fusion protein comprising a canine PD-1 protein extracellular region mutant (denoted as canine PD-1mu) and canine SIRPα protein.
[0019] Preferably, the fusion protein includes an Fc fragment.
[0020] The fusion protein of this invention comprises two symmetrical parts connected by disulfide bonds (located on the Fc fragment). Each part contains a canine PD-1 extracellular region mutant, canine SIRPα protein, and the Fc fragment. The connection order of the canine PD-1 extracellular region mutant, canine SIRPα protein, and Fc fragment is not particularly limited; from the amino terminus to the carboxyl terminus, the order of canine PD-1 mutant, canine SIRPα protein, and Fc fragment can be as follows: 1) Canine PD-1mu, canine SIRPα protein, and Fc fragment (corresponding to A); or 2) Canine SIRPα protein, canine PD-1mu, and Fc fragment (corresponding to B); or 3) Canine PD-1mu, Fc fragment, and canine SIRPα protein (corresponding to C); or 4) Canine SIRPα protein, Fc fragment and canine PD-1mu (corresponding to D).
[0021] Specifically, it can be as follows: Figure 1 The connection method shown.
[0022] Preferably, from the amino terminus to the carboxyl terminus, the order of canine PD-1mu, canine SIRPα protein, and Fc fragment is canine PD-1mu, canine SIRPα protein, and Fc fragment, respectively. Under this preferred condition, the performance of the fusion protein can be further improved.
[0023] Preferably, canine PD-1mu, canine SIRPα protein, and Fc fragment are directly or indirectly linked via linkers.
[0024] Preferably, the linker is a flexible polypeptide composed of 2-20 flexible amino acids, wherein the flexible amino acids are selected from at least one of Gly, Ser, Ala and Thr; more preferably, the linker is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer between 2 and 5 (e.g., it can be 2, 3, 4 or 5).
[0025] Preferably, the canine PD-1 protein extracellular region mutant is the canine PD-1 protein extracellular region T132L, that is, the canine PD-1 protein extracellular region mutant is derived from the canine PD-1 protein extracellular region protein and obtained by T132L mutation. For further explanation of the mutant, please refer to CN110590959A, which will not be repeated here.
[0026] Preferably, the canine PD-1 protein extracellular region mutant has (a) The amino acid sequence as shown in SEQ ID NO: 1; or (b) An amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% identity with SEQ ID NO: 1 and having the same function.
[0027] In this invention, the canine SIRPα protein can be a protein possessing the properties of canine SIRPα protein, and can be the full-length, truncated, or mutant form of the extracellular region of canine SIRPα protein. The mutant can be a mutant of the full-length extracellular region of canine SIRPα protein, or a mutant of the truncated form.
[0028] Preferably, the truncated body is a canine SIRPα D1 region truncated body.
[0029] Preferably, the canine SIRPα protein has (a) An amino acid sequence as shown in any one of SEQ ID NO: 2-4; or (b) An amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% identical to any one of SEQ ID NO: 2-4 and has the same function.
[0030] Preferably, the Fc fragment is of the IgG type, more preferably of the IgG1 subtype.
[0031] Preferably, the Fc fragment comprises an immunoglobulin hinge region, a CH2 region, and a CH3 region.
[0032] The source of the Fc fragment is not particularly limited and can be derived from mammals, such as humans, cats, mice, and dogs, with dogs being the preferred source.
[0033] Preferably, the Fc fragment has (a) The amino acid sequence as shown in SEQ ID NO: 5; or (b) An amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% identity with SEQ ID NO: 5 and having the same function.
[0034] Preferably, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 6-17; more preferably, it comprises an amino acid sequence as shown in SEQ ID NO: 6 or 10.
[0035] A second aspect of the present invention provides a nucleic acid encoding the fusion protein as described above.
[0036] Those skilled in the art can obtain the nucleic acid based on the sequence of the fusion protein and using techniques in the art. The nucleic acid comprises a corresponding nucleotide sequence encoding each part of the fusion protein.
[0037] Preferably, the nucleotide sequence encoding canine PD-1mu is shown in SEQ ID: 18.
[0038] Preferably, the nucleotide sequence encoding the canine SIRPα protein is shown in any one of SEQ ID: 19-21.
[0039] Preferably, the nucleotide sequence encoding the Fc fragment is shown in SEQ ID: 22.
[0040] Preferably, the nucleic acid encoding the fusion protein as described above comprises the nucleotide sequence shown in any one of SEQ IDs: 23-34, or a variant thereof with equivalent function. In this preferred embodiment, expression of the fusion protein is more favorable.
[0041] In a preferred embodiment of the present invention, the canine PD-1mu-SIRPα-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 6, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 23.
[0042] In a preferred embodiment of the present invention, the canine SIRPα-PD-1mu-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 7, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 24.
[0043] In a preferred embodiment of the present invention, the canine PD-1mu-Fc-SIRPα fusion protein comprises the amino acid sequence shown in SEQ ID NO: 8, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 25.
[0044] In a preferred embodiment of the present invention, the canine SIRPα-Fc-PD-1mu fusion protein comprises the amino acid sequence shown in SEQ ID NO: 9, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 26.
[0045] In a preferred embodiment of the present invention, the canine PD-1mu-SIRPαmu-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 10, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 27.
[0046] In a preferred embodiment of the present invention, the canine SIRPαmu-PD-1mu-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 11, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 28.
[0047] In a preferred embodiment of the present invention, the canine PD-1mu-Fc-SIRPαmu fusion protein comprises the amino acid sequence shown in SEQ ID NO: 12, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 29.
[0048] In a preferred embodiment of the present invention, the canine SIRPαmu-Fc-PD-1mu fusion protein comprises the amino acid sequence shown in SEQ ID NO: 13, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 30.
[0049] In a preferred embodiment of the present invention, the canine PD-1mu-SIRPαD1-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 14, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 31.
[0050] In a preferred embodiment of the present invention, the canine SIRPαD1-PD-1mu-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 15, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 32.
[0051] In a preferred embodiment of the present invention, the canine PD-1mu-Fc-SIRPαD1 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 16, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 33.
[0052] In a preferred embodiment of the present invention, the canine SIRPαD1-Fc-PD-1mu fusion protein comprises the amino acid sequence shown in SEQ ID NO: 17, and the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID: 34.
[0053] The nucleic acid molecules described in this invention are not limited to the sequences disclosed herein, but also include variants and corresponding other nucleic acid forms, such as mRNA, cDNA, and their variants. Variants in this invention can be described with reference to their physical properties in hybridization. Those skilled in the art will recognize that nucleic acids can be used to identify their complements and equivalents or homologues using nucleic acid hybridization techniques.
[0054] A third aspect of the present invention provides a biological material containing a gene encoding a fusion protein as described above, wherein the biological material is an expression cassette, a transposon, a vector, or a host cell.
[0055] The vector can be a plasmid, bacteriophage, or viral vector, etc., and the nucleic acid molecule encoding the fusion protein of the present invention is inserted into the vector. It should be understood that the design of the vector is influenced by a variety of factors, such as the choice of host cell, the desired protein expression level, and whether the expression is constitutive or inducible.
[0056] The host cell can be transformed or transfected using the vector. The host cell can be bacteria (such as Escherichia coli, Bacillus subtilis, etc.), yeast, insect or mammalian cells (such as 293 cells, COS cells or CHO cells), etc.
[0057] Those skilled in the art can prepare the biomaterial using methods conventional in the field.
[0058] A fourth aspect of the present invention provides a method for preparing a fusion protein as described above, the method comprising culturing a host cell as described above under conditions suitable for expression of the fusion protein, and then recovering the expressed fusion protein from the culture medium.
[0059] Preferably, the host cell is a CHO cell; more preferably, it is a CHOK1 cell.
[0060] Those skilled in the art can culture host cells using conventional methods in the field. The preferred conditions for expressing the fusion protein include a temperature of 28-37°C and a rotation speed of 100-200 rpm.
[0061] Fusion proteins can be recovered and purified from host cell cultures using known methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, or high performance liquid chromatography.
[0062] A fifth aspect of the present invention provides a pharmaceutical composition comprising the fusion protein as described above, as well as a pharmaceutically acceptable carrier and optional other therapeutic agents, which may include, but are not limited to, paladin, cyclophosphamide, doxorubicin, vincristine, and prednisolone.
[0063] It should be understood that when the pharmaceutical composition is also combined with one or more other therapeutic agents, the resulting combination does not cause unacceptable adverse effects.
[0064] The pharmaceutical compositions of the present invention can be prepared in a manner known in the art, for example by conventional methods of mixing, dissolving, granulating, grinding, emulsifying, encapsulating, embedding, or lyophilizing.
[0065] A sixth aspect of the present invention provides a kit comprising the fusion protein, the nucleic acid, or the biological material as described above.
[0066] Preferably, the kit further includes a drug delivery device.
[0067] The seventh aspect of the present invention provides the use of the fusion protein, nucleic acid, or biological material as described above in the preparation of a medicament for treating canine diseases caused by overexpression of CD47 and / or PD-L1, preferably in the preparation of a medicament for treating canine malignant tumors.
[0068] The diseases caused by overexpression of CD47 and / or PD-L1 can include melanoma, lymphoma, mast cell tumor, sarcoma, head and neck tumors, non-small cell lung cancer, breast cancer, urothelial carcinoma, and bladder cancer.
[0069] Preferably, the dosage of the fusion protein is 5-20 mg / kg body weight per dose. The fusion protein can be administered, for example, by intravenous infusion, and there is no particular limitation on the dosing cycle, such as once a week, once every two weeks, or once a month.
[0070] The present invention will be described in detail below through embodiments.
[0071] Unless otherwise specified, the methods used in the following examples are conventional methods in the art, and the reagents and materials used are commercially available.
[0072] Example 1 This example illustrates the expression and purification of fusion proteins.
[0073] The amino acid sequences of canine PD-1 protein (UniProtKB: A0A024FCJ9), canine SIRPα protein (UniProtKB: F1PK00), and canine IgG1-Fc fragment (GenBank: AF354264) were searched in the UniProt and GenBank libraries. The Thr mutated at position 132 of canine PD-1 to Leu, forming canine PD-1mu (as shown in SEQ ID NO: 1). The D1 region of canine SIRPα (as shown in SEQ ID NO: 2) was truncated to obtain canine SIRPαD1 (as shown in SEQ ID NO: 3). Mutation treatment of canine SIRPα (as shown in SEQ ID NO: 2) (mutating Asn at position 80 to Ala) yielded canine SIRPαmu (as shown in SEQ ID NO: 4).
[0074] Based on the canine PD-1 protein extracellular region mutant (canine PD-1mu), canine SIRPα protein, and Fc fragment, according to... Figure 1 The fusion protein was designed according to the sequence shown. Eight artificially synthesized molecules (specific amino acid sequences and corresponding nucleotide sequences are shown in Table 1) were constructed into the pcDNA3.1 vector and transiently transfected into 293 cells. Cell supernatant was collected 4-5 days after transfection, and the protein was obtained through purification. The SDS-PAGE electrophoresis image of the fusion protein is shown below. Figure 2 As shown.
[0075] Table 1
[0076] Note: PD-1mu: PD-1 protein T132L mutant; SIRPαmu: SIRPα protein N80A mutant.
[0077] Example 2 This example illustrates the determination of the affinity of the fusion protein for canine PD-L1 and CD47.
[0078] Affinity analysis was performed using a BIAcore 3000 instrument. The experiment consisted of 11 sample groups: fusion proteins 1-8 from Example 1, canine PD-1mu-Fc (denoted as D1), canine SIRPα-Fc (denoted as D2), and canine PD-1-SIRPα-Fc (denoted as D3).
[0079] (1) Affinity determination of fusion protein with canine PD-L1 First, fusion proteins 1-8, D1, and D3 were coupled to channel 2, and then channel 1 was blocked as a control channel. The analyte (canine PD-L1-His) was serially diluted (1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM) and then infused into the chip. The binding curves of the samples coupled to the chip surface were measured, and the signal Fc2-1 was recorded using the Wizard software. The affinity constant KD was determined using a 1:1 binding model analysis, and the results are shown in Table 2 below.
[0080] Table 2
[0081] (2) Affinity determination of fusion protein with canine CD47 First, fusion proteins 1-8, D2, and D3 were coupled to channel 2, and then channel 1 was blocked as a control channel. The analyte (canine CD47-His) was serially diluted (1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM) and then infused into the chip. The binding curves of the samples coupled to the chip surface were measured, and the signal Fc2-1 was recorded using the Wizard software. The affinity constant KD was determined using a 1:1 binding model analysis, and the results are shown in Table 3 below.
[0082] Table 3
[0083] The results in Table 2 show that fusion proteins numbered 1 and 5, namely canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc, have significantly higher affinity for canine PD-L1 than fusion proteins D1, D3, and the other six combinations. This indicates that linking the canine PD-1mu, canine SIRPα protein, and Fc fragment in that order is more conducive to improving the affinity of the fusion protein for canine PD-L1. A comparison of the results for numbers 1 and 5 shows that using canine PD-1mu instead of canine PD-1 further increases the affinity of the fusion protein for canine PD-L1, and is better than D3 (canine PD-1-SIRPα-Fc).
[0084] Table 3 shows that among fusion proteins with the same canine SIRPα element, fusion proteins numbered 1 and 5, namely canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc, exhibited significantly higher affinity for canine CD47 than fusion proteins with the same element linkage sequences (D2, D3, and other sequences). This indicates that linking the canine PD-1mu, canine SIRPα protein, and Fc fragment in that order is more conducive to improving the affinity of the fusion protein for canine CD47. A comparison of the results for numbers 1 and 5 shows that using canine SIRPαmu instead of canine SIRPα further increases the affinity of the fusion protein for canine CD47.
[0085] Example 3 This embodiment is used to illustrate the blood toxicity detection of canine PD-1mu / SIRPα dual fusion protein.
[0086] Since CD47 is expressed on the surface of red blood cells, CD47-targeted drugs need to be considered for potential side effects such as red blood cell agglutination. An in vitro red blood cell agglutination test should be performed, and the specific steps are as follows.
[0087] Peripheral blood was collected from healthy dogs, centrifuged, washed, and prepared into a 1% erythrocyte suspension. This suspension was added to 96-well U-bottom plates. Simultaneously, fusion protein 1 (canine PD-1mu-SIRPα-Fc), 5-7 (canine PD-1mu-SIRPαmu-Fc, canine SIRPαmu-PD1mu-Fc, canine PD-1mu-Fc-SIRPαmu), a positive control (phytohemagglutinin PHA), and a negative control (canine IgG1-Fc) were diluted at 500 g / mL, resulting in six concentration gradients (1.5-500 μg / mL). These were added to the 96-well plates, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator for 4 hours. The plates were then removed for observation and photographed using a gel imaging analyzer. Figure 3 ).
[0088] The results showed that PHA, as a positive control, exhibited significant erythrocyte agglutination at the first three concentration gradients; the four proteins, canine PD-1mu-SIRPα-Fc, canine PD-1mu-SIRPαmu-Fc, canine SIRPαmu-PD1mu-Fc, and canine PD-1mu-Fc-SIRPαmu, did not induce erythrocyte agglutination under various concentration conditions.
[0089] Experiments were conducted using the same method described above, and it was found that the four fusion proteins 2-4 (canine SIRPα-PD-1mu-Fc, canine PD-1mu-Fc-SIRPα, canine SIRPα-Fc-PD-1mu) and 8 (canine SIRPαmu-Fc-PD-1mu) did not induce erythrocyte agglutination at the above concentrations.
[0090] Example 4 This example illustrates the expression and purification of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc fusion proteins.
[0091] The successfully constructed stable expression plasmids of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc were electroporated into CHOK1 cells, and monoclonal cell lines were obtained through two rounds of limiting dilution screening. The cell culture expression medium was obtained by fed-batch culture, and the cell culture supernatant was obtained by deep filtration to remove cells and cell debris.
[0092] Cell culture supernatant was subjected to anion exchange chromatography: first, equilibration was performed with equilibration buffer (10 mM Tris-HCl, 40 mM NaCl, pH 7.2) to baseline, followed by elution with elution buffer (10 mM Tris-HCl, 200 mM NaCl, pH 7.2), and the eluent was collected; then, hydrophobic chromatography was performed: first, equilibration was performed with equilibration buffer (10 mM Tris-HCl, 1 M (NH4)2SO4, pH 7.2) to baseline, followed by washing with wash buffer (10 mM Tris-HCl, 0.1 M (NH4)2SO4, pH 7.2), followed by elution with elution buffer (10 mM Tris-HCl, 0.1 M (NH4)2SO4, pH 7.2), and the eluent was collected; finally, combined-mode anion exchange chromatography was performed: first, equilibration was performed with equilibration buffer (10 mM Tris-HCl, 30 mM NaCl, pH 7.2) to baseline, followed by elution with elution buffer (10 mM Tris-HCl, 500 mM NaCl, pH 7.2), followed by elution with elution buffer (10 mM Tris-HCl, 500 mM NaCl, pH 7.2), and the eluent was collected. Elute with NaCl (pH 7.2), collect the eluent; finally, concentrate by ultrafiltration, then replace the eluent with the solution to obtain the target protein that meets the requirements. Figure 4 ).
[0093] like Figure 4 As shown, the purity of the canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc fusion proteins can reach over 95%.
[0094] Example 5 This embodiment is used to illustrate the detection of the binding activity of the canine PD-1mu-SIRPα-Fc fusion protein with canine PD-L1 and human CD47.
[0095] Full-length canine PD-L1 was constructed and transfected into CHOK1 cells. High-expression monoclonal cell lines (hereinafter referred to as canine PD-L1 / CHOK1 cells) were obtained through screening. The ability of canine PD-1mu-SIRPα-Fc and canine PD-1mu-Fc to block the binding of canine PD-1 to canine PD-L1 / CHOK1 cells was compared by flow cytometry.
[0096] 500 nM canine PD-1-His-Biotin was mixed with serially diluted canine PD-1mu-SIRPα-Fc and canine PD-1mu-Fc, respectively, and the mixture was added to 96-well plates containing canine PD-L1 / CHOK1 cells. Cells were incubated at 4°C for 1 hour, washed twice with cold PBS, and then incubated at 4°C for 30 minutes with streptavidin bound to DyLight 650 (Invitrogen, 84547). Cells were washed twice with cold PBS and resuspended in 200 mL PBS. FACS analysis was then performed using a flow cytometer (Beckman Coulter, CytoFLEX).
[0097] To determine whether canine PD-1mu-SIRPα-Fc can bind to tumor cells, preliminary affinity assays showed that canine SIRPα binds to human CD47 (data not shown). Therefore, flow cytometry was used to detect the binding ability of canine PD-1mu-SIRPα-Fc to Jurkat (human T-lymphocytic leukemia cells) and Raji (human Burkitt's lymphoma cells) cells. Canine PD-1-SIRPα-Fc was added to 1.5 mL EP tubes containing Jurkat or Raji cells. Cells were incubated at 4°C for 1 hour, washed twice with cold PBS, and then incubated at 4°C for 30 minutes with a FITC-bound secondary antibody against canine IgG (Southernbiotech, 6070-02). Cells were washed twice with cold PBS and resuspended in 200 mL PBS. Subsequently, the cells were analyzed by flow cytometry using FACS.
[0098] Figure 5 The results of the binding activity assays of canine PD-1mu-SIRPα-Fc with canine PD-L1 and human CD47 are shown (*: p <0.05; **: p <0.01), FACS analysis results of canine PD-1mu-SIRPα-Fc and canine PD-1mu-Fc blocking the binding of canine PD-1 to canine PD-L1 / CHO cells are shown in […]. Figure 5 FACS analysis results for whether canine PD-1mu-SIRPα-Fc can bind to tumor cells (A and 5B) are shown below. Figure 5 C and 5D.
[0099] The results showed that canine PD1mu-SIRPα-Fc exhibited significantly higher competitive binding activity to canine PD-L1 / CHOK1 cells than canine PD-1mu-Fc; canine PD1mu-SIRPα-Fc could bind to Jurkat and Raji cells.
[0100] Experiments conducted using the same method as described above revealed that the competitive binding activity of canine PD1mu-SIRPαmu-Fc to canine PD-L1 / CHOK1 cells was comparable to that of canine PD1mu-SIRPα-Fc; canine PD1mu-SIRPαmu-Fc could also bind to Jurkat and Raji cells.
[0101] Example 6 This embodiment is used to illustrate the detection of the biological activity of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc.
[0102] Since canine PD-1 can bind to human PD-L1 (affinity data not shown), the biological activity of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc dual fusion proteins in blocking the PD-1 / PD-L1 signaling pathway was detected using the human PD-1 / PD-L1 fluorescent reporter gene cell line assay. Human PD-1-NFAT / Jurkat cells and human PD-L1 / CHO APC cells were added to black transparent 96-well plates at a 5:1 ratio. The canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc fusion proteins were added to the same 96-well plates after serial dilutions. The plates were incubated at 37°C in a 5% CO2 incubator for 6 h. Then, 50 µLone-glo enzyme substrate (Promega, E6120) was added, and the plates were incubated at room temperature in the dark for 15 min before fluorescence signal detection. The IC50 was calculated using Prism software.
[0103] To determine the effects of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc on macrophage phagocytic activity, we extracted peripheral blood mononuclear cells (PBMCs) from human whole blood, obtained CD14+ monocytes by magnetic bead sorting, added M-CSF and IFN-γ to induce them into macrophages, and then co-cultured them with Raji cells labeled with the fluorescent dye CFSE. Canine PD-1mu-SIRPα-Fc, canine PD-1mu-SIRPαmu-Fc, negative control (canine IgG1-Fc), and canine SIRPα-Fc were added. Flow cytometry was used to detect the phagocytic activity of macrophages on tumor cells.
[0104] in, Figure 6 The results of biological activity assays for canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc are shown, among which... Figure 6 A shows the results of the fusion protein blocking the PD-1 / PD-L1 signaling pathway. Figure 6B shows the results of macrophage phagocytosis of tumor cells. The results showed that both canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc effectively blocked the PD-1 / PD-L1 signaling pathway, with IC50 values of 1.86 nM and 1.54 nM, respectively. Both canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc promoted macrophage phagocytosis of tumor cells, with phagocytosis rates higher than those of canine SIRPα-Fc, and the phagocytosis rate of canine PD-1mu-SIRPαmu-Fc was higher than that of canine PD-1mu-SIRPα-Fc.
[0105] Example 7 This embodiment is used to illustrate the therapeutic effects of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc on canine malignant tumors.
[0106] Thirty dogs with cancer (including 15 with melanoma and 15 with lymphoma) were selected from a pet hospital in Beijing to study the efficacy of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc in treating dogs with malignant tumors. The test dogs included Golden Retrievers, Toy Poodles, Chihuahuas, Schnauzers, Dachshunds, Bichon Frises, Labradors, and mixed breeds, with an average age of 13 years (range 11-16 years), and all were dogs with advanced malignant tumors.
[0107] All affected dogs were divided into three groups: canine PD-1mu-Fc group, canine PD-1mu-SIRPα-Fc group, and canine PD-1mu-SIRPαmu-Fc group, with each group including 5 dogs with melanoma and 5 dogs with lymphoma. Each group received intravenous infusion of canine PD-1mu-Fc, canine PD-1mu-SIRPα-Fc, and canine PD-1mu-SIRPαmu-Fc (diluted with saline for injection) at a dose of 10 mg / kg body weight, once every two weeks for 8 consecutive weeks. The trial ended one week after the last administration. Tumor burden was assessed by gross examination and computed tomography (CT) scans. Tumor size was measured every two weeks to assess efficacy. Objective response rate (ORR) was compared. ORR = (number of dogs achieving complete or partial remission / total number of dogs) × 100%. Complete remission (CR) was defined as the disappearance of all detectable tumors; partial remission (PR) was defined as a reduction of at least 30% in the sum of the maximum diameters of the target lesions. The treatment results are shown in Table 4 below. Table 4. Summary of clinical antitumor efficacy of canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc
[0108] Note: ORR: Objective Remission Rate; CR: Complete Remission Rate Preliminary clinical results show that canine PD-1mu-SIRPα-Fc and canine PD-1mu-SIRPαmu-Fc have better anti-tumor efficacy than canine PD-1mu-Fc, and canine PD-1mu-SIRPαmu-Fc has better anti-tumor efficacy than canine PD-1mu-SIRPα-Fc.
[0109] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A fusion protein, characterized in that, The fusion protein is composed of a canine PD-1 protein extracellular region mutant PD-1mu, canine SIRPα protein, and an Fc fragment directly or indirectly linked by a linker. The amino acid sequence of the canine PD-1 protein extracellular region mutant is shown in SEQ ID NO: 1, the amino acid sequence of the canine SIRPα protein is shown in SEQ ID NO: 2 or 4, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO:
5. From the amino terminus to the carboxyl terminus, the order of canine PD-1mu, canine SIRPα protein, and Fc fragment is canine PD-1mu, canine SIRPα protein, and Fc fragment, respectively.
2. The fusion protein according to claim 1, wherein, The linker is a flexible polypeptide composed of 2-20 flexible amino acids, wherein the flexible amino acids are selected from at least one of Gly, Ser, Ala and Thr.
3. The fusion protein according to claim 2, wherein, The connector is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer between 2 and 5.
4. The fusion protein according to any one of claims 1-3, wherein, The amino acid sequence of the fusion protein is shown in SEQ ID NO: 6 or 10.
5. A nucleic acid encoding the fusion protein according to any one of claims 1-4.
6. The nucleic acid according to claim 5, wherein, The nucleotide sequence of the nucleic acid is shown in SEQ ID: 23 or 27.
7. A biomaterial containing the gene encoding the fusion protein according to any one of claims 1-4, characterized in that, The biomaterial is an expression cassette, vector, or host cell.
8. A method for preparing the fusion protein according to any one of claims 1-4, characterized in that, The method includes culturing the host cells of claim 7 under conditions suitable for the expression of the fusion protein, and then recovering the expressed fusion protein from the culture medium.
9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein according to any one of claims 1-4, as well as a pharmaceutically acceptable carrier and optional other therapeutic agents.
10. The pharmaceutical composition according to claim 9, wherein, The other therapeutic agents are selected from at least one of paladin, cyclophosphamide, doxorubicin, vincristine, and prednisolone.
11. A reagent kit, characterized in that, The kit comprises the fusion protein of any one of claims 1-4, the nucleic acid of claim 5 or 6, or the biological material of claim 7.
12. The kit according to claim 11, wherein, The kit also includes a drug delivery device.
13. The use of the fusion protein of any one of claims 1-4, the nucleic acid of claim 5 or 6, or the biomaterial of claim 7 in the preparation of a medicament for treating canine malignant tumors caused by overexpression of CD47 and / or PD-L1, wherein the canine malignant tumors caused by overexpression of CD47 and / or PD-L1 are melanoma, lymphoma, mast cell tumor, sarcoma, head and neck tumors, non-small cell lung cancer, breast cancer, urothelial carcinoma, and bladder cancer.
14. The application according to claim 13, wherein, The dosage of the fusion protein is 5-20 mg / kg body weight / dose.