Monkeypox virus bispecific antibody and its application

Abstract

This invention belongs to the field of biomedicine, specifically relating to a bispecific antibody against monkeypox virus. It is composed of a monoclonal neutralizing antibody targeting the monkeypox virus M1R antigen and a monoclonal neutralizing antibody targeting the monkeypox virus B6R antigen. They can broadly bind to the monkeypox virus M1R or B6R antigens and proteins homologous to M1R or B6R in vaccinia virus, smallpox virus, and cowpox virus. They can also treat orthopoxvirus infections, represented by vaccinia virus, and can be used for the prevention and treatment of orthopoxvirus infections, including but not limited to monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus, and have great application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to bispecific antibodies against monkeypox virus and their applications. Background Technology

[0002] Monkeypox, caused by the monkeypox virus (MPXV), is a zoonotic disease that was previously prevalent mainly in Africa. In May 2022, a global outbreak of monkeypox occurred, caused by MPXV clade IIb. In July 2022, the WHO declared the monkeypox outbreak a Public Health Emergency of International Concern, and the number of infections continues to rise. Furthermore, this year, several African countries have experienced monkeypox outbreaks caused by a new variant clade Ib, which is highly contagious, causes severe illness, and has a case fatality rate >3%; the incidence among children has increased significantly, with children under 5 years old accounting for 39% of all cases in the Democratic Republic of Congo, and a case fatality rate exceeding 7%. On August 14, 2024, the WHO again declared the monkeypox outbreak a Public Health Emergency of International Concern.

[0003] Studies have shown that although the smallpox vaccine provides some protection against monkeypox, with the eradication of smallpox and the cessation of vaccination, most people (born after 1980) lack immunity to orthopox viruses such as MPXV and Variola virus (VARV). Currently, although chemical drugs such as Tecovirimat and Brincidofovir have been approved for the treatment of smallpox, and vaccinia immunoglobulin (VIG) has been approved for complications caused by smallpox vaccination and can also be used for orthopox infections such as monkeypox, research on their efficacy against monkeypox and other orthopox virus infections remains limited. There are currently no approved drugs or vaccines specific to monkeypox virus.

[0004] MPXV belongs to the genus *Orthopoxvirus*, which also includes VARV, Vaccinia virus (VACV), and Cowpox virus (CPXV). Infection in humans can cause diseases of varying severity, endangering human health. MPXV possesses two different forms of infectious viral particles: intracellular mature virion (IMV) and extracellular enveloped virion (EEV), which have different envelope structures, modes of infection, and surface antigens. The M1R antigen on the surface of IMV and the B6R antigen on the surface of EEV indicate their ability to induce the production of neutralizing antibodies, thus making them important targets for research in therapeutic antibodies and vaccines.

[0005] Since MPXV has two different infectious particles, IMV and EEV, and these two different infectious particles have different surface antigens, antibody combinations targeting multiple antigens may exert a better antiviral effect. Our team's previous research yielded monoclonal neutralizing antibodies against the M1R antigen on the surface of MPXV IMV and the B6R antigen on the surface of EEV. The purpose of this invention is to design bispecific antibodies based on monoclonal antibodies against the M1R and B6R antigens. Summary of the Invention

[0006] This invention utilizes a humanized mouse anti-h40 targeting the monkeypox virus M1R antigen and a humanized antibody D21 targeting the B6R antigen, and based on the antibody conformation reported in the literature of inserting a LAIR1 domain between the antibody VH and CH1, successfully constructed a bispecific antibody. Its antiviral infection ability is significantly improved compared to the original h40 and D21.

[0007] Its antibody specificity makes it suitable for constructing bispecific antibodies, thus completing this invention.

[0008] The present invention provides a bispecific antibody against monkeypox virus, wherein the heavy chain variable region is formed by linking the heavy chain variable region of a first antibody (h40), the light chain variable region of a second antibody, the heavy chain variable region of a second antibody, and a linker; or the heavy chain variable region is formed by linking the heavy chain variable region of a second antibody (D21), the light chain variable region of a first antibody, the heavy chain variable region of a first antibody, and a linker.

[0009] in,

[0010] The three complementarity-determining regions (CDRs) of the variable region of the heavy chain of the first antibody (h40) have amino acid sequences selected from the group consisting of:

[0011] As shown in SEQ ID NO:1, CDR1(GYIFTRYW),

[0012] As shown in SEQ ID NO:2, CDR2(INPSTGYT), and

[0013] As shown in SEQ ID NO:3, CDR3(ARSDYTNYVFEY);

[0014] The three complementarity-determining regions (CDRs) of the h40 light chain variable region have amino acid sequences selected from the following group:

[0015] As shown in SEQ ID NO:4, CDR1(QSLVHSNGNTY),

[0016] As shown in SEQ ID NO:5, CDR2(KVS), and

[0017] CDR3(SQSTHVPYT) as shown in SEQ ID NO:6;

[0018] The three complementarity-determining regions (CDRs) of the heavy chain variable region of the second antibody (D21) have amino acid sequences selected from the following group:

[0019] As shown in SEQ ID NO:7, CDR1(GFSISTYP),

[0020] As shown in SEQ ID NO:8, CDR2 (ISHDGRNK), and

[0021] As shown in SEQ ID NO:9, CDR3 (ARAYPYAFDV);

[0022] The three complementarity-determining regions (CDRs) of the D21 light chain variable region have amino acid sequences selected from the following group:

[0023] As shown in SEQ ID NO:10, CDR1(QSVRND),

[0024] As shown in SEQ ID NO:11, CDR2(GAS), and

[0025] As shown in SEQ ID NO:12, CDR3(QQYKDWPPWT);

[0026] More preferably, the amino acid sequence of the heavy chain variable region of the first antibody (h40) is SEQ ID NO:13; and the amino acid sequence of the light chain variable region is SEQ ID NO:14.

[0027] The amino acid sequence of the heavy chain variable region of the second antibody (D21) is SEQ ID NO:15; the amino acid sequence of the light chain variable region is SEQ ID NO:16.

[0028] Specifically, the variable regions of the bispecific antibody heavy chain are connected in the following manner: the variable region of the first antibody heavy chain, the variable region of the second antibody light chain, the linker, and the variable region of the second antibody heavy chain are connected sequentially, wherein the positions of the variable regions of the second antibody light chain and the variable regions of the second antibody heavy chain can be interchanged; or the connection method is that the variable regions of the second antibody heavy chain, the variable regions of the first antibody light chain, the linker, and the variable regions of the second antibody heavy chain are connected sequentially, wherein the positions of the variable regions of the first antibody light chain and the variable regions of the first antibody heavy chain can be interchanged.

[0029] Preferably, the amino acid sequence of the linker is SEQ ID NO:17;

[0030] Preferably, the bispecific antibody has the amino acid sequence of the heavy chain variable region as SEQ ID NO:18 and the amino acid sequence of the light chain variable region as SEQ ID NO:14; or the amino acid sequence of the heavy chain variable region as SEQ ID NO:19 and the amino acid sequence of the light chain variable region as SEQ ID NO:16.

[0031] More preferably, the bispecific antibody further includes a heavy chain constant region and a light chain constant region; in a specific example, the amino acid sequence of the heavy chain constant region is SEQ ID NO:20, and the amino acid sequence of the light chain constant region is SEQ ID NO:21;

[0032] This invention provides a polynucleotide encoding the aforementioned bispecific antibody;

[0033] This invention provides an expression vector comprising the aforementioned polynucleotides;

[0034] This invention provides a host cell comprising the above-described expression vector;

[0035] This invention provides the use of the above-mentioned bispecific antibody in the preparation of drugs for the treatment and prevention of orthopoxvirus infections, including but not limited to monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus;

[0036] The present invention provides a pharmaceutical composition comprising the aforementioned bispecific antibody and a pharmaceutical carrier.

[0037] This invention also provides pharmaceutical compositions containing the bispecific antibody against monkeypox virus of this invention. To prepare the pharmaceutical compositions, various desired dosage forms can be prepared by mixing the bispecific antibody with a pharmaceutical carrier or excipient. Examples of dosage forms for the pharmaceutical compositions of this invention include, for example, oral dosage forms such as tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, small pellets, sublingual tablets, and ointments; and non-oral dosage forms such as injections, suppositories, transdermal preparations, ointments, plasters, and topical liquids. Those skilled in the art can select appropriate dosage forms based on the route of administration and the target population.

[0038] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0039] The beneficial effects of this invention are:

[0040] This invention has constructed two bispecific antibodies against monkeypox virus, which are composed of a monoclonal neutralizing antibody targeting the monkeypox virus M1R antigen and a monoclonal neutralizing antibody targeting the monkeypox virus B6R antigen. They can broadly bind to the monkeypox virus M1R or B6R antigens and proteins homologous to M1R or B6R in vaccinia virus, smallpox virus, and cowpox virus, and can treat orthopoxvirus infections represented by vaccinia virus. They have the potential to treat and prevent orthopoxvirus infections including but not limited to monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus. Attached Figure Description

[0041] Figure 1 Kinetic curves of the binding of antibody 40# to MPXV M1R antigen before and after humanization.

[0042] Figure 2 The effect of antibody #40 on in vitro neutralization of vaccinia virus infection, represented by VCV, before and after humanization.

[0043] Figure 3A The effect of bispecific antibodies on in vivo resistance to orthopoxvirus infection, represented by VCV, is shown on the graph. The horizontal axis represents the number of days after challenge, and the vertical axis represents the percentage change in body weight of mice.

[0044] Figure 3B The effect of bispecific antibodies against orthopoxvirus infection, represented by VCV, in vivo. The horizontal axis shows the number of days after challenge, and the vertical axis shows the survival percentage of mice.

[0045] Figure 3C The effect of bispecific antibodies against orthopoxvirus infection, represented by VCV, in vivo. The horizontal axis shows the names of the bispecific antibodies and controls, and the vertical axis shows the viral load in mouse lungs. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Example 1: Humanization of antibody #40

[0048] The CDR regions of the light and heavy chains of the antibody were predicted using the IMGT or Kabat database. The CDR regions of the light and heavy chains of the 40# mouse antibody were then transplanted into the light and heavy chain frameworks of the human antibody with the highest homology to the mouse antibody, respectively, to form humanized antibodies.

[0049] Example 2: Detection of antibody-antigen binding ability using surface plasmon resonance technology

[0050] Surface plasmon resonance (SPR) analysis was performed using Biacore 8K (GE Healthcare). The specific steps were as follows: a protein A chip (GE Healthcare) was used. Protein A binds to the antibody Fc, immobilizing the antibody protein on the chip. The antibody immobilization amount was approximately 500 RU. The antigen protein was serially diluted with PBST solution (pH 7.4), and the sample was loaded onto the chip surface. Changes in the response values ​​were recorded. The kinetic curves and kinetic constants of the antibody-antigen binding were analyzed using BIAevaluation software 8K (GE Healthcare). Figure 1 As shown in the figure. The results showed that the affinity of antibody 40# for MPXV M1R before (40) and after (h40) humanization was 0.01 nM and 0.02 nM, respectively, with no significant change.

[0051] Example 3: Plaque assay for detecting the neutralizing effect of antibodies against VCV virus

[0052] VACV IMV Neutralization: The pre-humanized h40 antibody and the post-humanized h40 antibody were serially diluted 2-fold starting at 100 μg / mL, for a total of 10 dilutions. The diluted virus solution (approximately 100 PFU / well) was added to each well. The mixture was incubated at 37°C for 2 hours. The incubated antibody-virus mixture was added to cells, and the cells were infected at 37°C for 1 hour. The mixture was discarded, and the cells were washed once with PBS in each well. Then, 1 mL of a 1:1 mixture of 2x DMEM and carboxymethyl cellulose was added, and the cells were cultured for another 48 hours. 4% paraformaldehyde fixative was added to each well, and the cells were fixed for at least 2 hours. The paraformaldehyde was discarded, and crystal violet staining solution was added to each well. The results were observed after staining for 2 hours. The neutralizing activity value (PRNT) of the antibody was calculated based on the number of plaques. 50 The result is as follows: Figure 2 As shown, the humanized 40# antibody can still effectively neutralize VCV virus infection, with an activity of 0.3576 μg / mL.

[0053] Example 4: Antibody efficacy against VCV virus infection in vivo

[0054] The in vivo protective effect of the antibody was evaluated using a BALB / c mouse model. A lethal dose of VCV was administered via nasal drops, and the antibody was administered intraperitoneally 4 hours before and after challenge, at a dose of 5 mg / kg. Control mice were given either an unrelated antibody (targeting SARS-CoV-2) or PBS, and were euthanized when their body weight decreased by more than 20%. Results are as follows: Figure 3A , Figure 3B and Figure 3C As shown, the bispecific antibody can significantly reduce the viral load in the lungs of mice, demonstrating a better protective effect.

Claims

1. A bispecific antibody against monkeypox virus, characterized in that, The variable region of the heavy chain is connected in the following ways: the variable region of the first antibody heavy chain, the variable region of the second antibody light chain, the linker, and the variable region of the second antibody heavy chain are connected in sequence; or the variable region of the first antibody heavy chain, the variable region of the second antibody heavy chain, the linker, and the variable region of the second antibody light chain are connected in sequence. Alternatively, the connection method may be formed by sequentially connecting the variable region of the second antibody heavy chain, the variable region of the first antibody light chain, the linker, and the variable region of the first antibody heavy chain, or by sequentially connecting the variable region of the second antibody heavy chain, the variable region of the first antibody heavy chain, the linker, and the variable region of the first antibody light chain. The first antibody heavy chain contains a heavy chain variable region as shown in SEQ ID NO:13 and a light chain variable region as shown in SEQ ID NO:

14. The second antibody contains a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO:16; The amino acid sequence of the linker is SEQ ID NO:

17.

2. The monkeypox virus bispecific antibody as described in claim 1, characterized in that, The amino acid sequence of its heavy chain variable region is SEQ ID NO:18, and the amino acid sequence of its light chain variable region is SEQ ID NO:14; or the amino acid sequence of its heavy chain variable region is SEQ ID NO:19, and the amino acid sequence of its light chain variable region is SEQ ID NO:

16.

3. The monkeypox virus bispecific antibody as described in claim 2, characterized in that, The antibody type is IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

4. The monkeypox virus bispecific antibody as described in claim 3, characterized in that, The antibody, excluding the CDR region, has a sequence source species selected from one or more of the following: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

5. The monkeypox virus bispecific antibody as described in claim 4, characterized in that, The antibody also includes a heavy chain constant region and a light chain constant region.

6. The monkeypox virus bispecific antibody as described in claim 5, characterized in that, The antibody contains a sequence of a constant region of one of the human antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

7. The monkeypox virus bispecific antibody as described in claim 6, characterized in that, The amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO:20; the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO:

21.

8. A polynucleotide encoding a bispecific antibody against monkeypox virus as described in any one of claims 1 to 7.

9. An expression vector comprising the polynucleotide as described in claim 8.

10. A host cell comprising the expression vector as described in claim 9.

11. Use of the monkeypox virus bispecific antibody as described in any one of claims 1 to 8 in the preparation of a medicament for treating and preventing orthopoxvirus infection.

12. The use as described in claim 11, characterized in that, The orthopox virus is selected from monkeypox virus, vaccinia virus, smallpox virus, or cowpox virus.

13. A pharmaceutical composition comprising a bispecific antibody against monkeypox virus as described in any one of claims 1 to 8 and a pharmaceutical carrier, and further comprising an excipient.

14. The pharmaceutical composition according to claim 13, characterized in that, The dosage form of the pharmaceutical composition is an oral dosage form or a non-oral dosage form.

15. The pharmaceutical composition according to claim 14, characterized in that, The oral dosage form is a tablet, powder, pill, granule, soft capsule, hard capsule, film-coated, or ointment; the non-oral dosage form is an injection, suppository, transdermal preparation, ointment, plaster, or topical liquid.

Images