A bifunctional fusion protein based on CD24 and ACE2

Abstract

The present invention provides a CD24-hACE2-hFc bifunctional fusion protein, which combines the advantages of ACE2 and CD24, has the functions of both without affecting each other's activity, can not only overcome the problem of decreased antibody neutralization ability due to virus mutation, but also reduce the risk of body damage caused by inflammatory response. The CD24-hACE2-hFc bifunctional fusion protein of the present invention has good neutralizing activity against wild strains of the new coronavirus and its mutant strains (such as Alpha, Beta, Gama and Delta, etc.), and has a broad spectrum; it can inhibit inflammatory response and accelerate the recovery of new crown patients, which is more advantageous than existing antibody drugs. As a safe, broad-spectrum and effective new crown infection therapeutic drug that can both neutralize the virus and inhibit the "inflammatory storm", the present invention has great innovation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and immunology, and in particular to a bifunctional fusion protein that has a neutralizing effect on the novel coronavirus and relieves inflammation. Background Art

[0002] At present, the research and development of therapeutic drugs for new coronary infection mainly includes small molecule drugs for antiviral replication, antiviral neutralizing antibodies and ACE2 receptor fusion proteins, as well as protein or hormone drugs that can relieve the "inflammatory storm". Among them, the development of antiviral small molecule drugs is slow, and there has been no breakthrough progress for the time being. Although a number of antiviral neutralizing antibodies have been approved for clinical use, the emergence of viral variants may affect the neutralizing activity of antibodies, greatly increasing the uncertainty of the use of neutralizing antibodies. Glucocorticoids, a potent inflammatory inhibitor, can effectively relieve the "inflammatory storm" of new coronary patients and reduce tissue and organ damage, but serious side effects limit its widespread clinical application. As of recently, there is still no specific medicine and safe and effective treatment for new coronary infection.

[0003] The development of ACE2 fusion protein began with the outbreak of SARS. In theory, it can block the infection of SARS virus, new coronavirus and its variants, and is a potential new coronavirus drug with broad-spectrum neutralizing activity. The fastest progress in the ACE2 fusion protein drug is HLX71 independently developed by Henlius, and the US FDA has approved its clinical trial of new drugs for the treatment of new coronavirus infection. CD24 can inhibit the inflammatory response caused by infection by interacting with Siglec-10 on natural immune cells. Currently, there are few drug development institutions and companies targeting this target. Oncoimmune is at the forefront of the world in the development of CD24-Fc fusion protein new coronavirus drugs. The company's co-founders, Dr. Liu Yang and Dr. Zheng Pan, have nearly 30 years of history in the research and clinical application of CD24.

[0004] In April 2020, CD24-Fc fusion protein was approved by the U.S. FDA as a Class I new drug to directly enter Phase III clinical trials. In addition, Nadir Arber of Ichilov Hospital in Israel developed the exosome drug EXO-CD24 targeting CD24 and conducted clinical trials. Clinical data show that both new crown drugs targeting CD24 can accelerate the recovery of new crown patients and significantly reduce the critical illness and mortality of new crown infected patients. However, for therapeutic drugs for new crown infection, domestic and foreign institutions and companies mostly focus on the research and development of single-function drugs. For example, antiviral neutralizing antibodies and ACE2 receptor fusion proteins have the function of neutralizing viruses to block infection, while CD24-Fc fusion proteins and EXO-CD24 have the function of inhibiting inflammation. There are few reports on the development of new crown infection therapeutic drugs that can both neutralize viruses and inhibit the "inflammatory storm". Summary of the invention

[0005] The present invention is committed to developing a bifunctional fusion protein drug based on ACE2 and CD24, in the hope that the fusion protein has safe, broad-spectrum and effective antiviral and anti-COVID-19 infection functions. The smooth development and completion of this project will provide a highly potential clinical therapeutic drug for COVID-19 infection.

[0006] Based on the bifunctional fusion protein technology, the goal of this study is to develop safe, broad-spectrum and effective new crown infection therapeutic drugs that can both neutralize the virus and inhibit the "inflammatory storm". Angiotensin-converting enzyme 2 (ACE2) is the receptor for the invasion of the new coronavirus on the surface of human cells. Fusion proteins based on the ACE2 receptor can compete with the ACE2 receptor on the surface of human cells, thereby blocking the virus from entering human cells. In theory, compared with antiviral antibodies, ACE2 receptor fusion proteins are more broad-spectrum and can block the infection of the new coronavirus and its variants. CD24 is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein. Previous studies have shown that CD24 can inhibit inflammatory responses caused by infection, sepsis, liver damage and graft-versus-host disease by interacting with Siglec-10 (sialic-acid-binding Ig-like lectin10) on innate immune cells. Therefore, CD24 is a potential target for the treatment of new crown infection. In fact, CD24-Fc fusion protein has recently completed phase III clinical trials, and the results showed that patients with severe or critical COVID-19 infection recovered significantly faster and the chances of the disease progressing to death or respiratory failure were significantly reduced. In this project, we used ACE2 and CD24 as candidate targets, designed a bifunctional fusion protein, and completed its preclinical functional verification.

[0007] The present invention provides a fusion protein against severe acute respiratory syndrome type II coronavirus SARS-COV-2, which comprises three parts, namely, human CD24 part, human angiotensin converting enzyme 2 (hACE2 part) and hFc part, from N-terminus to C-terminus, or a polypeptide of any combination order of the three structural units;

[0008] The human CD24 portion comprises a human CD24 fragment, the hACE2 portion is a human ACE2 extracellular segment, and the hFc portion is a human antibody Fc segment protein;

[0009] Preferably, the human CD24 part and the hACE2 part are connected via a connecting peptide, more specifically, the connecting peptide is (GGGGS)3.

[0010] Specifically, the amino acid sequence of the human CD24 portion is shown in SEQ ID No.1.

[0011] The amino acid sequence of hACE2 is shown in SEQ ID No.3; preferably, the hACE2 is a mutant having 5 amino acid mutations, namely T27F, K31Y, R273Q, L79W and N330Y, based on the sequence shown in SEQ ID No.3.

[0012] Specifically, the amino acid sequence of the hFc part is shown in SEQ ID No.5.

[0013] More specifically, the amino acid sequence of the connecting peptide is shown in SEQ ID No.7.

[0014] The present invention also provides the encoding nucleic acid of the fusion protein, preferably, the encoding nucleotide sequence of the human CD24 part is as shown in SEQ ID No.2; the encoding nucleotide sequence of hACE2 is as shown in SEQ ID No.4; the encoding nucleotide sequence of the hFc part is the sequence shown in SEQ ID No.6; the encoding nucleotide sequence of the connecting peptide is as shown in SEQ ID No.8.

[0015] The present invention also provides an expression vector containing the encoding nucleic acid of the fusion protein.

[0016] The present invention also provides a host cell, such as a mammalian cell, containing an expression vector of a nucleic acid encoding the fusion protein.

[0017] The present invention further provides a pharmaceutical composition comprising the fusion protein as an effective ingredient.

[0018] The present invention also provides the use of the fusion protein in the preparation of a drug for treating or preventing diseases caused by the new coronavirus.

[0019] The CD24-hACE2-hFc bifunctional fusion protein of the present invention is an antibody-like protein, which can be widely used in novel coronavirus strains with mutations in the spike protein, and is expected to be used in the vast majority of existing novel coronavirus strains, thereby avoiding the decrease in the ability of antibodies to neutralize mutants due to viral mutations, and can also reduce the risk of damage to the body caused by inflammatory responses. The CD24-hACE2-hFc bifunctional fusion protein of the present invention can inhibit inflammatory responses while treating infections of novel coronavirus wild strains and their mutants (such as Alpha, Beta, Gama, and Delta, etc.), and can be widely used in clinical treatment, epidemiological surveys, and corresponding drugs for such uses, including the preparation of vaccines, the detection of biological samples, the presence of novel coronavirus on the surface or inside of non-biological samples, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .Structural model diagram of CD24-hACE2-hFc bifunctional fusion protein.

[0021] Figure 2 .SDS-PAGE and affinity chromatography purification verified the expression of CD24-hACE2-hFc bifunctional fusion protein.

[0022] Figure 3 .Octet assay for the binding of CD24-hACE2-hFc to siglec10. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0024] The relevant sequence information involved in the embodiments of the present invention is as follows:

[0025] 1. Unit component sequence:

[0026] SEQ ID NO.1: Human TNFR2 amino acid sequence (CD24)

[0027] SETTTGTSSNSSQSTSNSGLAPNPTNATTKAAG

[0028] SEQ ID NO.2: Human TNFR2 nucleotide sequence (CD24)

[0029] agtgaaacaacaactggaacttcaagtaactcctcccagtacttccaactctgggttggccccaaatccaactaa tgccaccaccaaggcggctggt

[0030] SEQ ID NO.3: Amino acid sequence of human ACE2 (hACE2)

[0031] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS

[0032] SEQ ID NO.4: Nucleotide sequence of human ACE2 (hACE2)

[0033]

[0034] SEQ ID NO.5: Human Fc amino acid sequence (hFc)

[0035] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0036] SEQ ID NO.6: Human Fc nucleotide sequence (hFc)

[0037] acatgtccaccgtgcccagctccggaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcacgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctcccgggaaa

[0038] SEQ ID NO.7: (GGGGS)3 Amino Acid Sequence

[0039] GGGGSGGGGSGGGGS

[0040] SEQ ID NO.8: (GGGGS)3 Nucleotide Sequence

[0041] ggcggcggcggatccggcggaggcggctccggcggcggcggcagc。

[0042] The CD24-hACE2-hFc bifunctional fusion protein of the present invention can be obtained by methods commonly used in the art.

[0043] Example 1. Preparation of CD24-hACE2-hFc bifunctional fusion protein

[0044] 1. Design of bifunctional fusion proteins

[0045] The bifunctional fusion protein consists of three structural parts (see Figure 1 ), the first structural part is CD24, the second structural part is the extracellular segment of ACE2, and the third structural part is immunoglobulin Fc, and CD24 and ACE2 are connected by a connecting peptide (GGGGS) 3.

[0046] 2. Construction of CD24-hACE2-hFc recombinant expression plasmid

[0047] First, the gene sequence encoding CD24 protein (sequence as shown in SEQ ID No. 2), the gene sequence encoding human ACE2 protein (sequence as shown in SEQ ID No. 4), the gene encoding human antibody hFc protein (sequence as shown in SEQ ID No. 6), and the gene encoding connecting peptide (GGGGS) 3 (sequence as shown in SEQ ID No. 8) were artificially synthesized (synthesis service provided by Abbotson Bio), connected and cloned into the pEE12.4 eukaryotic expression vector to obtain the pEE12.4-CD24-hACE2-hFc vector.

[0048] 3. Expression and purification of CD24-hACE2-hFc fusion protein

[0049] (1) Transfect HEK293F suspension cells and collect fusion protein

[0050] The pEE12.4-CD24-hACE2-hFc plasmid was transfected into HEK293F suspension cells by PEI, and the cell density was about 2.5×10 6 / mL, add feed solution once every two days, and collect cell supernatant every 5-7 days. The cell supernatant containing CD24-hACE2-hFc fusion protein is pumped to the Protein A affinity chromatography column at 4 degrees Celsius by a peristaltic pump to fully bind the supernatant and affinity chromatography column. Wash the impurities with binding buffer (PBS, pH7.4) until there is no protein in the buffer; elute the fusion protein with eluent (0.1M glycine, pH2.7), and add neutralization buffer (1M Tris-HCl, pH9.0) to the collection tube at a ratio of 1:10 to prevent the protein from being inactivated in an overly acidic environment. Finally, use a 50kDa ultrafiltration tube to concentrate the fusion protein and replace the solution with PBS to collect the protein.

[0051] (2) Purification of CD24-hACE2-hFc bifunctional fusion protein

[0052] Protein concentration was determined using Nanodrop and Superdex TM The eluted protein was purified by 200Increase 10 / 300GL gel filtration column (cytiva, No. 28990944), and the protein collection solution with the correct peak position was collected and concentrated. The results are shown in Figure 2 .

[0053] Example 2. Octet assay for binding of CD24-hACE2-hFc to siglec10

[0054] After biotinylation of siglec10-mFc using ForteBio's octet RED96e, it was diluted to 10 ng / μL with PBST to bind to SAsensor, and the binding value reached about 0.5-1.0 nm. CD24-hFc, CD24-hACE2-hFc diluted to 1 μM and NA protein were combined for 120 seconds to detect whether they were bound. The results are shown in Figure 3 .

[0055] The experimental results show that after the binding value of SAsensor combined with biotinylated siglec10-mFc reaches about 1.0nm, 1μM CD24-hFc, CD24-hACE2-hFc and NA protein are combined for 120 seconds, CD24-hFc and CD24-hACE2-hFc are shifted by about 0.06nm, and the irrelevant protein NA is only shifted by about 0.01nm. This shows that CD24 can bind to siglec10, but its affinity is low.

[0056] Example 3. Pseudovirus neutralization test of CD24-hACE2-hFc bifunctional fusion protein

[0057] 1. Sample preparation: Transfer the wild-type and mutant novel coronavirus pseudoviruses (VSV-△GS-GFP) from -80°C to a 4°C refrigerator or thaw on ice in advance, and dilute the virus to 1000 transducing units (TU) with DMEM medium containing 10% FBS before use.

[0058] 2. Preparation of protein stock solution: CD24-hACE2-hFc bifunctional fusion protein was dissolved in DMEM under sterile conditions and prepared into a solution with a concentration of 100μg / mL. Take a new 96-well cell culture plate for sample dilution. In the wells of the second column, the concentration of CD24-hACE2-hFc protein was 100μg / mL, and 120μL was added. 60μL of serum-free DMEM was added to columns 3-11, and then 60μL of dilution was taken from column 2 with a spray gun and added to column 3 for multiple dilution until it was diluted to column 9. The excess 60μL of liquid in the last column was discarded, and 3 replicates were set for each concentration. Then 60μL of diluted pseudovirus solution was added to each well. A mixture of 60μL pseudovirus and 60μL serum-containing DMEM medium was added to the virus control group, and only 120μL serum-containing DMEM medium was added to the cell control group. The 96-well cell culture plate was placed in a 37°C incubator and incubated for 1h.

[0059] 3. Transfer the sample-pseudovirus mixture and two groups of controls in the 96-well cell culture plate to the Vero cells (ATCC CCL81) plated on the 96-well plate the day before, 100 μL / well, and incubate in a 37°C incubator.

[0060] 4. After 15 hours, the number of TUs was counted on a CQ1 confocal image cytometer (Yokogawa). The data were analyzed using GraphPadPrism 8.0 (GraphPad Software Inc.), and the results are shown in Table 1.

[0061] As shown in Table 1, the IC values ​​of ACE2-hFc and CD24-hACE2-hFc bifunctional fusion proteins and different novel coronavirus pseudoviruses 50 Both are at the nM level, but the neutralizing activity of the CD24-hACE2-hFc bifunctional fusion protein is slightly lower than that of ACE2-hFc, which may be due to the effect of CD24 on the N-terminus of ACE2. Therefore, it is considered possible to mutate ACE2 to enhance its affinity; or change the structure to hACE2-hFc-CD24.

[0062] Table 1. IC of CD24-hACE2-hFc bifunctional fusion protein neutralizing different novel coronavirus pseudoviruses 50 (Unit: nM)

[0063] S protein type WT alpha beta gama delta omicron lambda ACE2-Fc 1.445 0.1574 0.1975 0.1244 0.1702 0.2015 0.205 CD24-ACE2-Fc 3.593 0.5578 0.3722 0.2307 0.3978 0.288 0.5217

[0064] Example 4. Pseudovirus neutralization test of ACE2 mutant CD24-hACE2-hFc bifunctional fusion protein

[0065] In this example, based on the existing document CN202110578921.6, five amino acids of ACE2 were mutated to T27F, K31Y, R273Q, L79W, and N330Y. According to the same design as in Example 1 (wherein the connecting peptide is also (GGGGS)3), the ACE2 part in pEE12.4-CD24-hACE2-hFc was enzymatically removed, and the ACE2 (mutant) fragments were connected by homologous recombination to construct

[0066] pEE12.4-CD24-hACE2(mutant)-hFc vector. 293F cells were transfected and CD24-hACE2(mutant)-hFc bifunctional fusion protein was expressed to test its effect. The specific experimental method was the same as that in Example 3, and pseudovirus neutralization test was performed. Results The IC of different novel coronavirus pseudoviruses 50 As follows: WT: 0.1729nM; alpha: 0.06214nM; beta: 0.05712nM; gama: 0.01634nM; delta: 0.05155nM; omicron: 0.01604nM; lambda: 0.08278nM.

[0067] Mutating ACE2 to T27F, K31Y, R273Q, L79W, and N330Y can increase affinity to the pm level. Therefore, mutating ACE2 can enhance the neutralizing activity of the CD24-hACE2-hFc bifunctional fusion protein and avoid the effect of CD24 on ACE2.

Claims

1. A fusion protein against severe acute respiratory syndrome type II coronavirus SARS-COV-2, comprising, from N-terminus to C-terminus, a human CD24 portion with an amino acid sequence as shown in SEQ ID NO: 1, a connecting peptide, a hACE2 portion with an amino acid sequence as shown in SEQ ID NO: 3, and an hFc portion with an amino acid sequence as shown in SEQ ID NO:

5.

2. The fusion protein according to claim 1, characterized in that The connecting peptide is (GGGGS) 3, and its amino acid sequence is shown in SEQ ID NO:

7.

3. A nucleic acid encoding the fusion protein according to claim 1 or 2.

4. The coding nucleic acid according to claim 3, characterized in that The encoding nucleotide sequence of the human CD24 part is shown as SEQ ID No.2; the encoding nucleotide sequence of the hACE2 part is shown as SEQ ID No.4; the encoding nucleotide sequence of the hFc part is the sequence shown as SEQ ID No.6; and the encoding nucleotide sequence of the connecting peptide is shown as SEQ ID No.

8.

5. An expression vector containing the encoding nucleic acid according to claim 3 or 4.

6. A host cell containing an expression vector encoding the nucleic acid according to claim 5.

7. A pharmaceutical composition comprising the fusion protein according to claim 1 or 2 as an active ingredient.

8. Use of the fusion protein according to claim 1 or 2 in the preparation of a drug for inhibiting SARS-CoV-2 infection.

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