WO2021109913A1 - Antimalarial dimer immunoadhesin, pharmaceutical composition, and use - Google Patents

Abstract

Provided is an antimalarial dimer immunoadhesin, comprising two dimerization polypeptide chains. The structural general formula of the first polypeptide chain is Z1-Z2. The structural general formula of the second polypeptide chain is Y1-Y2. Z1 is an extracellular domain of a first cell surface receptor, or a functional variant, or a fragment thereof. Z2 is a dimerization domain, or a functional variant, or a fragment thereof. Y1 is an extracellular domain of a second cell surface receptor, or a functional variant, or a fragment thereof. Y2 is a dimerization domain, or a functional variant, or a fragment thereof. The first cell surface receptor and the second cell surface receptor are respectively selected from any one of GYPA, GYPB, GYPC, CR1, and BSG. The dimer immunoadhesin can block the Plasmodium strain from invading the red blood cell and improve the antimalarial effect of the immunocyte.

Description

Antimalarial dimer immunoadhesin, pharmaceutical composition and use Technical field

The present invention relates to the technical field of biomedical engineering, in particular to a dimer immunoadhesin, a pharmaceutical composition using it as an active component and its medical use, especially its use for preventing and treating malaria.

Background technique

Malaria is a vector-borne disease caused by the infection of the malaria parasite through the bite of a mosquito or transfusion of the blood of a person carrying the malaria parasite. There are four kinds of Plasmodium parasitizing humans, namely Plasmodium vivax, Plasmodium vivax, Plasmodium falciparum and Plasmodium ovale. In my country, it is mainly Plasmodium vivax and Plasmodium falciparum; the other two are rare, and some cases imported from abroad have occasionally been seen in recent years. Different malaria parasites cause vivax, vivax, falciparum and ovale respectively. This disease mainly manifests as periodic regular attacks, chills, fever, and hyperhidrosis all over the body. After a long period of repeated attacks, it can cause anemia and splenomegaly.

In the field of antibody engineering, the use of fusion proteins to dimerize the extracellular domains of natural receptors can enhance the binding properties of these soluble receptors, making them useful therapeutically antagonists of the corresponding ligands. Representatives of such dimeric fusions are immunoadhesins (e.g., Sledziewski et al, U.S. Patent Nos. 5,155,027 and 5,567,584; Jacobs et al, U.S. Patent No. 5,605,690; Wallner et al, U.S. Patent No. 5,914,111; and Ashkenazi and Chamow, Curr. Opin. Immunol. 9:195-200, 1997).

The dimerized immunoadhesin overcomes the disadvantages of the instability of the extracellular domain of the soluble receptor and the loss of biological function, and has strong application value. Plasmodium invasion of red blood cells requires the participation of receptor proteins on the surface of red blood cells. However, there is no report on the use of receptor proteins on the surface of red blood cells to prepare immunoadhesins. Whether the biological activity of the prepared and prepared immunoadhesins can be achieved The existence, whether the prepared immunoadhesin has anti-disease effect, which receptor protein to choose for engineering, and which engineering method to choose are all unknown, and need to be further confirmed.

Summary of the invention

The purpose of the present invention is to rely on the above research background to study whether soluble dimer immunoadhesin can be used in anti-malarial applications, and to describe the specific structure, preparation method and application of dimer immunoadhesin, namely Provided are dimeric immunoadhesins, preparation methods and uses thereof.

The first aspect of the present invention provides a soluble antimalarial dimer immunoadhesin, comprising a dimerized first polypeptide chain and a second polypeptide chain, the first polypeptide chain has the general formula Z1-Z2, and the second The general structure of each polypeptide chain is Y1-Y2. Where Z1 is the extracellular domain of the first cell surface receptor or its functional variant or fragment; Z2 is the dimerization domain or its functional variant or fragment; Y1 is the extracellular domain of the second cell surface receptor Or a functional variant or fragment thereof, Y2 is a dimerization domain or a functional variant or fragment thereof.

The first cell surface receptor and or the second cell surface receptor are each selected from the group consisting of: GYPA (UniProtKB: P02724); GYPC (UniProtKB: P04921); GYPB (UniProtKB: P06028); CR1 (UniProtKB: P17927) ; BSG (UniProtKB: P35613).

In the case where both Z1 and Y1 are extracellular domains of cell surface receptors or functional variants or fragments thereof, the first and second cell surface receptors may be the same or different.

The Z2 and Y2 dimerization domains include the constant region of the immunoglobulin heavy chain. In a specific variation, the dimerization domains Z2 and Y2 are Fc fragments of IgG, such as human immunoglobulin γ1 Fc fragments. When Z1 and Y1 are different, the dimerization domains Z2 and Y2 can be engineered to increase the formation of specific heterodimerization, such as Knob-in-hole, ART-Ig, BiMab with altered charge polarity Bispecific antibody constant region construction methods and engineering methods (review literature Brinkmann U, Kontermann R E. mAbs, 2017, 9(2): 182-212.).

In addition, the dimerization domains Z2 and Y2 may also contain peptide linkers, which are composed of 15-32 amino acid residues, of which 1-8 (for example, 2) of these residues are cysteine Residues. In specific variations, Z2 and Y2 comprise an immunoglobulin hinge region or variants thereof. For example, in a specific embodiment, Z2 and Y2 comprise immunoglobulin hinge variants (e.g., human immunoglobulin γ1 hinge variants), in which the 220 corresponding cysteine residues of the Fc fragment are replaced by serine. Particularly suitable peptide linkers used in accordance with the aforementioned dimerization domains Z2 and Y2 include peptide linkers that comprise a plurality of glycine residues, and optionally at least one serine residue.

In certain embodiments of the present invention, the dimerization domains Z2 and Y2 may be active variants of the Fc fragment of human immunoglobulin, such as using the Fc domain of IgG2, IgG3, or IgG4. In some embodiments, Fc mutants can be further used to reduce the biological activities of immunoglobulins such as ADCC, complement fixation, etc., such as LALA-PG mutant, L235E; E318A; K320A; K322A mutant, etc.

In some preferred embodiments of the present invention, each of Z1 and Y1 is the extracellular domain of BSG or a functional variant or fragment thereof. The amino acid sequence of Z1 and Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, and the amino acid sequence shown in SEQ ID NO.1. Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.

At this time, the dimeric immunoadhesin comprises the amino acid sequence of the human BSG immunoadhesin shown in SEQ ID NO.2.

In some preferred embodiments of the present invention, Z1 is the extracellular domain of BSG or a functional variant or fragment thereof, and Y1 is the extracellular domain of GYPA or a functional variant or fragment thereof. The amino acid sequence of Z1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably the amino acid sequence of the extracellular domain of human BSG shown in SEQ ID NO.1 At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The amino acid sequence of Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably the amino acid sequence of human GYPA extracellular domain shown in SEQ ID NO.3 At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.

The two polypeptide chains of the soluble dimer immunoadhesin comprise an amino acid sequence selected from the following: (a) The Z1-Z2 polypeptide chain includes the amino acid sequence of the human BSG immunoadhesin Hole mutant shown in SEQ ID NO.4 , And (b) The Y1-Y2 polypeptide chain includes the amino acid sequence of the human GYPA extracellular domain Knob mutant shown in SEQ ID NO.5.

In some preferred embodiments of the present invention, Z1 is the extracellular domain of BSG or a functional variant or fragment thereof, and Y1 is the extracellular domain of GYPB or a functional variant or fragment thereof. The amino acid sequence of Z1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably the amino acid sequence of the extracellular domain of human BSG shown in SEQ ID NO.1 At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The amino acid sequence of Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably the amino acid sequence of human GYPB extracellular domain shown in SEQ ID NO.6 At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.

The two polypeptide chains of the soluble dimer immunoadhesin comprise an amino acid sequence selected from the following: (a) The Z1-Z2 polypeptide chain has the amino acid sequence of the human BSG immunoadhesin Hole mutant shown in SEQ ID NO: 4 , And (b) The Y1-Y2 polypeptide chain has the amino acid sequence of the Knob mutant of the extracellular domain of human GYPB shown in SEQ ID NO:7.

In some preferred embodiments of the present invention, Z1 is the extracellular domain of BSG or a functional variant or fragment thereof. Y1 is the extracellular domain of GYPC or a functional variant or fragment thereof. The amino acid sequence of Z1 and the amino acid sequence shown in SEQ ID NO.1 have at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more Preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The amino acid sequence of Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably the amino acid sequence of human GYPC extracellular domain shown in SEQ ID NO.8 At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.

The two polypeptide chains of the soluble dimer immunoadhesin comprise an amino acid sequence selected from the following: (a) the Z1-Z2 polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 4, and (b) the Y1-Y2 polypeptide The chain includes the amino acid sequence of the Knob mutant of the extracellular domain of human GYPC shown in SEQ ID NO: 9.

In some preferred embodiments of the present invention, Z1 is the extracellular domain of BSG or a functional variant or fragment thereof. Y1 is the extracellular domain of CR1 or a functional variant or fragment thereof. The amino acid sequence of Z1 and the amino acid sequence shown in SEQ ID NO.1 have at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more Preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The amino acid sequence of Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably the amino acid sequence of human CR1 extracellular domain shown in SEQ ID NO.10 At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.

The two polypeptide chains of the soluble dimer immunoadhesin comprise an amino acid sequence selected from the following: (a) the Z1-Z2 polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 4, and (b) the Y1-Y2 polypeptide The chain includes the amino acid sequence of human CR1 extracellular domain Knob mutant shown in SEQ ID NO: 11.

The second aspect of the present invention provides a polynucleotide encoding the above-mentioned anti-malarial dimer immunoadhesin, a vector for carrying the nucleotide, and a cell containing the vector.

The expression vector provided by the present invention includes the following operably linked elements: a transcription promoter, a DNA region encoding the dimeric immune fusion protein, and a transcription terminator.

By culturing a cell containing a vector for the production of the polypeptide or dimeric protein as disclosed above, including: (i) culturing a cell containing the expression vector as disclosed above, wherein the cell expresses the dimer immunofusion encoded by the DNA segment Protein and produce the encoded dimer immune fusion protein; (ii) recover the soluble dimer immune fusion protein.

Similarly, in some variations of the method of preparing dimeric proteins, the method includes: (i) culturing a cell containing an expression vector as disclosed above, wherein the cell expresses the dimeric immune fusion protein encoded by the DNA segment, And produce the encoded dimeric immune fusion protein as a dimeric protein; and (ii) recover the dimeric protein.

The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned soluble antimalarial dimer immunoadhesin and at least one pharmaceutically acceptable carrier. The pharmaceutical composition uses soluble antimalarial dimer immunoadhesin as the main or only active ingredient, and the auxiliary material can ensure the conformational integrity of the amino acid core sequence of the TIGIT immunoadhesin disclosed in the present invention, and at the same time protect the protein content. The functional group prevents its degradation (including but not limited to agglomeration, deamination or oxidation), so as to achieve a more stable therapeutic effect.

In the form of the drug, it can be a suspension, water injection, freeze-dried preparation commonly used in the pharmaceutical field, and preferably a water injection or freeze-dried preparation. Liquid formulations can be stored at 2°C-8°C for at least one year, and lyophilized formulations can be stored at 30°C for at least six months.

For the water injection or lyophilized preparation of the dimeric immunoadhesin disclosed in the present invention, the pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic regulators, and buffers. Among them, surfactants include non-ionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); lauric sulfuric acid Sodium; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount added should minimize the tendency of bifunctional bispecific antibody protein to granulate; solution stabilizers can be sugars, Including reducing sugars and non-reducing sugars, amino acids include monosodium glutamate or histidine, alcohols include triols, higher sugar alcohols, propylene glycol, polyethylene glycol, one or a combination of them, solution stabilizers The amount added should enable the final formulation to remain stable within the time considered by those skilled in the art to reach a stable state; the isotonicity regulator can be one of sodium chloride and mannitol; the buffer can be TRIS, histidine buffer , One of the phosphate buffer.

The fourth aspect of the present invention provides the use of the anti-malarial dimer immunoadhesin, pharmaceutical composition, polynucleotide, vector or host cell of the present invention in the treatment or prevention of malaria, especially: 1) in the prevention of malaria To cut off the effect of Plasmodium invading red blood cells, as in Examples 3-5; 2) Mediate the anti-malarial effect of immune cells, as in Example 6; 3) Regulate immune disorders during chronic infection and reduce bone damage caused by chronic malaria infection, As in Example 7.

The beneficial guarantees and effects of the present invention:

Through cell experiments and animal model experiments, the dimer immunoadhesin provided by the present invention can effectively block the invasion of red blood cells by four Plasmodium strains, and the invasion inhibition rate is close to 100%; in addition, through in vivo stability experiments, the The half-life of the antimalarial dimer immunoadhesin in vivo is close to that of the antimalarial antibody cetuximab on the market, and the in vivo stability is excellent. In addition, in a chronic infection model, the dimeric immunoadhesin provided by the present invention can effectively regulate immune disorders in the process of chronic infection and reduce bone damage. Therefore, when used alone or in combination with other related disease drugs, it can effectively prevent and treat malaria, and has broad clinical application prospects.

Description of the drawings

Figure 1 is a schematic diagram of the structure of the antimalarial dimer immunoadhesin of the present invention;

Figure 2 shows the experimental results of anti-malarial dimer immunoadhesin against the invasion of erythrocytes by Plasmodium in vitro;

Figure 3 shows the experimental results of in vivo infection treatment with antimalarial dimer immunoadhesin;

Figure 4 shows the results of the anti-malarial dimer immunoadhesin preventing infection in vivo.

Detailed ways

The following examples and experimental examples further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and quality-pulling methods, inserting protein-encoding genes into such vectors and quality-pulling methods or methods of introducing plasmids into host cells. Such methods It is well known to those of ordinary skill in the art and is described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 ^nd edition , Cold spring Harbor Laboratory Press.

Example 1. Construction and expression of soluble dimeric immunoadhesin

As shown in Figure 1, soluble antimalarial dimer immunoadhesin is a dimer with antibody IgG Fc. The method of constructing and expressing dimer immunoadhesin itself is a conventional experimental technique in the field. , A brief description is as follows:

(1) Full-gene synthetic soluble dimeric immunoadhesin BSG-Fc (comprising two polypeptide chains, the amino acid sequence and nucleotide sequence of each polypeptide chain are shown in SEQ ID NO. 2 and SEQ ID NO. 12) ; BSG/GYPA-Fc (contains two polypeptide chains, the amino acid sequence and nucleotide sequence of the first polypeptide chain are shown in SEQ ID NO. 4 and SEQ ID NO. 13, and the amino acid sequence of the second polypeptide chain and The nucleotide sequence is shown in SEQ ID NO. 5 and SEQ ID NO. 14); BSG/GYPB-Fc (comprising two polypeptide chains, the amino acid sequence and nucleotide sequence of the first polypeptide chain are shown in SEQ ID NO. 4 and SEQ ID NO. 13, the amino acid sequence and nucleotide sequence of the second polypeptide chain are shown in SEQ ID NO. 7 and SEQ ID NO. 15). BSG/GYPC-Fc (contains two polypeptide chains, the amino acid sequence and nucleotide sequence of the first polypeptide chain are shown in SEQ ID NO. 4 and SEQ ID NO. 13, and the amino acid sequence and core of the second polypeptide chain The nucleotide sequence is shown in SEQ ID NO. 9 and SEQ ID NO. 16).

(2) Expression and purification of fusion protein

According to the literature (Finck B K. Science, 265.; Mihara M et al.. Journal of Clinical Investigation. 2000; 106: 91-101; Yu X, et al. Nature Immunology. 2009; 10: 48-57. Liu S ,et al.Clin Immunol.2019Jun;203:72-80.) method for the expression of soluble dimeric immunoadhesin.

Example 2. In vivo stability test of soluble dimeric immunoadhesin

The literature (Hu S, et al. Science translational medicine, 2017, 9(380): eaag0339.) method was used to evaluate the half-life of antimalarial dimeric immunoadhesin in NOG mice. The results showed that the in vivo half-lives of BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, and BSG/GYPC-Fc were 8.9 days, 8.1 days, 7.5 days and 8.4 days respectively; the positive control cetuximab was 8.6 The half-life of the three BSG antigen peptides (amino acid sequence TNINTLENSDHTCFAR; SNPYFIVGSR; ENYYNSDIAGPAR) is too short to detect, and the half-life of BSG extracellular protein half-life is too short to detect.

Example 3. In vitro invasion experiment of soluble dimeric immunoadhesin

Use O-type human red blood cells to carry out the Plasmodium invasion experiment, the test method is the same as the literature (Zhang M Y, et al. blood, 2018, 131(10): 1111-1121.). Four kinds of Plasmodium beads Dd2, 3D7, FCC1, Nf54 were used for this experiment. Healthy human IgG was used as a control group, and BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc and control were administered respectively. IgG, the dosage is 10μg/ml. The calculation method of the invasion inhibition rate is also the same as in the literature, and the results are shown in Figure 2: It shows that BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc have a strong ability to inhibit invasion, and the invasion inhibition rate is close to 100%.

Example 4. In vivo infection treatment experiment with soluble dimeric immunoadhesin

This experiment was carried out using the NOG mouse model of Plasmodium infection, and the model construction method was the same as that in the literature (Zhang M Y, et al. blood, 2018, 131(10): 1111-1121.). The mice were divided into BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc, control IgG and blank groups. The blank group was given an equal volume of PBS, the other groups were given 10 mg/kg, once on the second day of infection, N=8 in each group. After 7 days of infection, the mouse infection rate was calculated. The detection method and calculation method are the same as those in the literature. The results are shown in Figure 3: BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc have a strong in vivo Inhibiting invasion ability, the infection rate of the BSG/GYPA-Fc group was single digit, and the infection rate of the other three groups were all zero.

Example 5. In vivo infection prevention experiment with soluble dimeric immunoadhesin

The method of constructing this experimental model using the NOG mouse model of Plasmodium infection is the same as that in the literature (Zhang M Y, et al. blood, 2018, 131(10): 1111-1121.). Using a preventive administration method, the mice were divided into BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc, control IgG and blank groups one day before the mice were infected with Plasmodium. The blank group was given an equal volume of PBS, and the remaining groups were given 10 mg/kg. N=8 in each group. It was administered again on the 7th day of infection, and the infection rate of mice was calculated 15 days after infection. The detection method and calculation method were the same as those in the literature. The results are shown in Figure 4, BSG-Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc have a strong ability to prevent infection, and none of the mice in the administration group was infected with Plasmodium.

Example 6. Soluble dimeric immunoadhesin mediates the anti-Plasmodium effect of NK cells

The acquisition of NK cells from the peripheral mononuclear cells of healthy volunteers, the establishment of human red blood cells (iRBC) infected with Plasmodium bead Dd2, and the hemoglobin release test were all in accordance with the literature (Arora G, et al. Elife, 2018, 7: e36806.). A brief description is as follows: iRBC nucleus and NK cells were washed twice with RPMI 1640 by centrifugation, mixed and cultured in RPMI 1640 medium without serum according to the ratio of NK cells: iRBC cells at a ratio of 5:1; then grouped and divided into BSG -Fc, BSG/GYPA-Fc, BSG/GYPB-Fc, BSG/GYPC-Fc, control IgG and blank group, each group has 3 replicate wells, the administration concentration is 10μg/ml. After incubating for 4 hours at 37°C, collect the clear on the wells, and use QuantiChrom Hemoglobin Analysis Kit (BioAssay) for hemoglobin using a 96-well plate reader (Enspire, Perkin Elmer). The release of hemoglobin is based on iRBC dissolved in 1% Triton-X-100 as the standard. At the end of 4 hours, the hemoglobin release volume of each group was calculated with standard products and blank wells. The results are shown in Table 1:

Table 1 Hemoglobin release

Group	Release (%)	SD	p value
Standard	100	8.87	To
Blank group (medium only)	1.55	0.15	To

Control IgG	1.38	0.09	p＜0.05
BSG-Fc	65.33	8.74	p＜0.05
BSG/GYPA-Fc	85.67	7.55	p＜0.05
BSG/GYPB-Fc	55.48	9.15	p＜0.05
BSG/GYPC-Fc	59.81	8.81	p＜0.05

It can be seen from the results that the soluble dimeric immunoadhesin effectively mediates NK cells to lyse the red blood cells infected with Plasmodium, and is effective against malaria.

Example 7. Soluble dimeric immunoadhesin reduces inflammation disorders and bone damage in Plasmodium infection

According to Example 5, the NOG mouse model of Plasmodium infection was prepared, and the infection event was extended to 14 days after grouping. The grouping was the same as that of Example 5, and the recombinant protein BSG, GYPA, GYPB, GYPC group and other control groups were added. The dose of each treatment group was 3 mg/kg once. According to the literature method [Lee MSJ, et al. Sci Immunol. 2017Jun2; 2(12): eaam8093.], the serum cytokine level was detected. The results are shown in Table 2-4.

Table 2 Relative levels of IL-4

Group	Relative level (% blank control)	SD	p value
Uninfected blank control group	100	6.65	To
Infection group (negative control)	144.160	26.243	p＜0.05
Reorganization of BSG	139.998	27.884	p＜0.05
Reorganize GYPA	142.635	15.616	p＜0.05
Restructuring GYPB	175.392	17.170	p＜0.05
Restructuring GYPC	153.251	26.740	p＜0.05
Control IgG	176.726	16.929	p＜0.05
BSG-Fc	105.393	10.002	P>0.05
BSG/GYPA-Fc	92.403	6.786	p>0.05
BSG/GYPB-Fc	95.570	7.972	p>0.05
BSG/GYPC-Fc	91.888	14.367	p>0.05

Table 3 Relative level of INF-γ

Group	Relative level (% blank control)	SD	p value
Uninfected blank control group	100	5.085	To
Infection group (negative control)	284.162	63.719	p＜0.05
Reorganization of BSG	286.751	52.210	p＜0.05
Reorganize GYPA	299.973	60.020	p＜0.05
Restructuring GYPB	248.631	46.649	p＜0.05
Restructuring GYPC	255.472	67.622	p＜0.05

Control IgG	318.807	85.566	p＜0.05
BSG-Fc	111.794	11.897	P>0.05
BSG/GYPA-Fc	101.110	8.347	p>0.05
BSG/GYPB-Fc	96.393	11.636	p>0.05
BSG/GYPC-Fc	99.782	17.077	p>0.05

The bone morphology of each group of mice was further analyzed 30 days after infection, and the detection method was the same as the non-patent literature [Lee MSJ,et al.Sci Immunol.2017Jun 2; 2(12):eaam8093.] The ratio of bone volume to total volume is shown in the table 4

Table 4 Bone morphology analysis

Group	Bone volume/total volume (%)	SD	p value
Uninfected blank control group	14.210	1.726	To
Infection group (negative control)	5.815	1.205	p＜0.05
Reorganization of BSG	6.115	0.704	p＜0.05
Reorganize GYPA	4.829	0.571	p＜0.05
Restructuring GYPB	6.069	1.235	p＜0.05
Restructuring GYPC	6.855	1.295	p＜0.05
Control IgG	7.184	0.483	p＜0.05
BSG-Fc	13.884	2.538	P>0.05
BSG/GYPA-Fc	16.379	2.362	p>0.05
BSG/GYPB-Fc	15.930	3.100	p>0.05
BSG/GYPC-Fc	14.669	1.885	p>0.05

These experiments show that the dimeric immunoadhesin of the present invention can effectively reduce animal immune disorders and reduce bone injury syndrome during chronic infection, while the recombinant protein does not have similar immune regulation functions.

In summary, in the malaria model, the dimeric immunoadhesin has a good preventive and therapeutic effect on malaria, which is conducive to the development of subsequent clinical trials.

The unspecified parts involved in the present invention are the same as the prior art or implemented by the prior art. The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above-mentioned detailed methods, which does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims (9)

1. An anti-malarial dimer immunoadhesin, which is characterized in that it comprises a dimerized first polypeptide chain and a second polypeptide chain, and the structural formula of the first polypeptide chain is Z1-Z2, so The general structural formula of the second polypeptide chain is Y1-Y2,

   Wherein, Z1 is the extracellular domain of the first cell surface receptor or its functional variant or fragment, Z2 is the dimerization domain or its functional variant or fragment; Y1 is the extracellular structure of the second cell surface receptor Domain or a functional variant or fragment thereof, Y2 is a dimerization domain or a functional variant or fragment thereof,

   The first cell surface receptor and the second cell surface receptor are respectively selected from any one of GYPA, GYPB, GYPC, CR1, BSG.
2. The anti-malarial dimer immunoadhesin according to claim 1, wherein:

   Among them, Z2 and Y2 are Fc fragments of IgG or Fc mutants that change its biological activity, or a heterodimeric IgG-Fc constructed using Knob-in-hole technology, ART-Ig technology that changes charge polarity, or BiMab technology Fragment.
3. The anti-malarial dimer immunoadhesin according to claim 1, wherein:

   Wherein, the dimerization domains Z2 and Y2 also contain a peptide linker, the peptide linker is composed of 15-32 amino acid residues, the amino acid residues include multiple glycine residues, one to eight cysteine residues And at least one serine residue.
4. The anti-malarial dimer immunoadhesin according to claim 1, wherein:

   Wherein, Z1 and Y1 are of the same origin, and both are the extracellular domain of BSG or functional variants or fragments thereof. The amino acid sequences of Z1 and Y1 are at least 95% identical to the amino acid sequence shown in SEQ ID NO.1;

   The anti-malarial dimer immunoadhesin has the amino acid sequence of human BSG immunoadhesin shown in SEQ ID NO.2.
5. The anti-malarial dimer immunoadhesin according to claim 4, characterized in that:

   Among them, Z1 and Y1 have different sources, and are either of the following situations:

   (1) Z1 is the extracellular domain of BSG or its functional variants or fragments, Y1 is the extracellular domain of GYPA or its functional variants or fragments; the amino acid sequence of Z1 is the same as the amino acid sequence shown in SEQ ID NO.1 At least 95% identity, the Y1 amino acid sequence has at least 95% identity with the amino acid sequence shown in SEQ ID NO.3; the Z1-Z2 polypeptide chain sequence is shown in SEQ ID NO.4, and the Y1-Y2 polypeptide chain sequence As shown in SEQ ID NO.5;

   (2) Z1 is the extracellular domain of BSG or its functional variants or fragments, Y1 is the extracellular domain of GYPB or its functional variants or fragments; the amino acid sequence of Z1 is the same as the amino acid sequence shown in SEQ ID NO.1 At least 95% identity, the Y1 amino acid sequence has at least 95% identity with the amino acid sequence shown in SEQ ID NO.6; the Z1-Z2 polypeptide chain sequence is shown in SEQ ID NO.4, and the Y1-Y2 polypeptide chain sequence is shown in SEQ ID NO.4. As shown in SEQ ID NO.7;

   (3) Z1 is the extracellular domain of BSG or its functional variants or fragments, Y1 is the extracellular domain of GYPC or its functional variants or fragments; the amino acid sequence of Z1 is the same as the amino acid sequence shown in SEQ ID NO.1 At least 95% identity, the Y1 amino acid sequence has at least 95% identity with the amino acid sequence shown in SEQ ID NO. 8; the Z1-Z2 polypeptide chain sequence is shown in SEQ ID NO. 4, and the Y1-Y2 polypeptide chain sequence As shown in SEQ ID NO.9;

   Z1 is the extracellular domain of BSG or a functional variant or fragment thereof, Y1 is the extracellular domain of CR1 or a functional variant or fragment thereof; the amino acid sequence of Z1 has at least 95% of the amino acid sequence shown in SEQ ID NO.1 The Y1 amino acid sequence has at least 95% identity with the amino acid sequence shown in SEQ ID NO.10; the Z1-Z2 polypeptide chain sequence is shown in SEQ ID NO.4, and the Y1-Y2 polypeptide chain sequence is shown in SEQ ID. Shown in NO.11.
6. A pharmaceutical composition, which is characterized by comprising an active ingredient and a pharmaceutically acceptable drug carrier, the active ingredient comprising the antimalarial dimer immunoadhesin according to any one of claims 1 to 5, encoding The nucleotide of the anti-malarial dimer immunoadhesin or the carrier carrying the anti-malarial dimer immunoadhesin or the nucleotide.
7. Use of the antimalarial dimer immunoadhesin according to any one of claims 1 to 5 in the preparation of antimalarial drugs.
8. The use of the antimalarial dimer immunoadhesin in the preparation of antimalarial drugs according to claim 7, characterized in that:

   Wherein, the anti-malarial drugs are drugs that block the invasion of erythrocytes by malaria parasites, drugs that mediate the lysis of immune cells infecting erythrocytes, and drugs that alleviate immune disorders and bone damage in chronic malaria infection.
9. The use of the antimalarial dimer immunoadhesin in the preparation of antimalarial drugs according to claim 8, characterized in that:

   Wherein, the drug that mediates immune cell lysis of red blood cells infected with Plasmodium is a drug that mediates NK cell lysis of red blood cells infected with malaria.

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