KR20220007559A - Bispecific Antibody Capable of Binding to SARS-CoV-2 and Immune Cell Receptor and Pharmaceutical Composition Comprising Same - Google Patents

Abstract

The present invention relates to a bispecific antibody capable of effectively neutralizing SARS-CoV-2 through activated immune cells according to antibody-dependent cytotoxicity by simultaneously binding to a surface antigen of SARS-CoV-2 virus and CD16 or CD3. In the present invention, by applying an ACE2-derived amino acid sequence to a SARS-CoV-2 binding region of the bispecific antibody, immune cells can effectively recognize and kill the virus due to a high binding force between SARS-CoV-2 and ACE2. In addition, an amino acid sequence derived from a receptor rather than an antibody of SARS-CoV-2 is used to exhibit a neutralizing effect on various virus strains. In addition, It is possible to form a variant in an ACE2 sequence, thereby preventing side effects due to an enzymatic activity of ACE2 while maintaining antigen-binding ability, and forming a variant in an Fc region, thereby suppressing side effects caused by autoimmune reactions.

Description

Bispecific Antibody Capable of Binding to SARS-CoV-2 and Immune Cell Receptor and Pharmaceutical Composition Comprising Same

The present invention relates to a bispecific antibody capable of simultaneously binding to SARS-CoV-2 and an immune cell receptor CD16 or CD3, and a pharmaceutical composition comprising the same, and more particularly, to a human cell receptor for SARS-CoV-2 A bispecific antibody capable of simultaneously binding to the surface antigen of SARS-CoV-2 and CD16 or CD3, including an ACE2-derived antigen-binding region and an anti-CD16 or anti-CD3 antigen-binding region, and COVID-19 containing the same It relates to a pharmaceutical composition for prevention or treatment.

Coronavirus Infectious Disease-19 (COVID-19) has spread rapidly since it occurred in December 2019, and the World Health Organization (WHO) has declared COVID-19 a global pandemic following Hong Kong Flu and H1N1 flu.

The pathogen of COVID-19 is SARS-CoV-2, It is transmitted when droplets of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. COVID-19 is highly contagious and can be fatal if infected, such as the elderly or people with low immunity, and in severe cases, death. In addition, with the rapid spread of COVID-19, damage is occurring not only in the health field, but also in various fields such as society and economy. Therefore, the development of vaccines and therapeutics for COVID-19 is urgent.

As an example of a therapeutic agent for COVID-19, an antiviral agent using a nucleoside analog is being developed. For example, Korean Patent Publication No. 10-2145197 describes the use of treating a coronavirus infection by administering a drug containing a specific L-nucleoside compound. However, currently approved antiviral drugs have only been shown to be effective in patients with very severe conditions.

Another type of therapeutic agent is an antibody therapeutic using an antibody. Antibody therapy is a therapeutic agent with a mechanism that prevents the virus from entering human cells by binding the antibody injected from the outside to the spike protein on the surface of the virus.

Antibody treatment for SARS-CoV-2 binds to the SARS-CoV-2 spike protein to neutralize the virus, thereby blocking the binding of SARS-CoV-2 to the human cell receptor ACE2 receptor. Since SARS-CoV-2 has a very high binding affinity with the human cell receptor compared to other viruses, the antibody applied to the antibody treatment requires a very strong binding affinity to the SARS-CoV-2 surface antigen.

A representative antibody therapeutic for SARS-CoV-2 includes REGN-CoV-2. REGN-CoV-2 is an antibody generated by administering the receptor binding region of the SARS-CoV-2 spike protein to mice, and the SARS-CoV-2 receptor binding region from B cells of patients recovered after COVID-19 infection. A binding antibody is used. In addition, Korean Patent Publication No. 10-2205028 describes various antibody therapeutics that neutralize viruses by binding to the spike protein on the surface of SARS-CoV-2.

However, in the case of SARS-CoV-2, numerous mutated viruses have been reported since the first virus was discovered, and in particular, mutants such as B.1.1.7 and B.1.351 were found to have higher transmission power compared to existing viruses. However, since the antibody binds only to a specific antigen, there is a problem that the antibody therapeutic agent against SARS-CoV-2 and its variant virus cannot work properly against variants other than the target virus.

Therefore, there is a need to develop a therapeutic agent that has a strong SARS-CoV-2 virus neutralizing effect and can also exhibit effects on various virus strains.

In this situation, the inventors of the present invention prepared a bispecific antibody by conjugating a recombinant protein in which an amino acid sequence derived from a cell receptor for a virus and an Fc region of an antibody were fused with an antibody fragment for an immune cell receptor to produce a bispecific antibody. found that can be achieved, and completed the present invention.

It is an object of the present invention to provide a bispecific antibody capable of effectively neutralizing SARS-CoV-2 and exhibiting a neutralizing effect on mutant viruses.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19) comprising the bispecific antibody.

In order to achieve the above object, the present invention includes a first antigen-binding region comprising the amino acid sequence of SEQ ID NO: 1 as a region that specifically binds to the surface antigen of SARS-CoV-2, and a first Fc region linked thereto a first area to And it provides a bispecific antibody, comprising a second region comprising a second antigen-binding region and a second Fc region linked thereto that specifically binds to CD16 or CD3, an immune cell activation receptor.

In the present invention, the amino acid sequence of SEQ ID NO: 1 may include one or more mutations of H374N, H378N, E402N, H345A, E375N, R514A, Y510A, Y515A, H505A and R273A.

In the present invention, the second antigen-binding region, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (V _H ) and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 (V _L ); Or it may include a heavy chain variable region (V _{H ) comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region (V L} ) comprising the amino acid sequence of SEQ ID NO: 5.

In the present invention, in one or more of the first Fc region and the second Fc region, (based on EU numbering) 1 to 10 residues including all or part of the A327 to P329 sequences and Y296 to S298 sequences are deleted; And one or more mutations among one or more substitutions selected from the group consisting of L234A, L235A, G236K and G237K may be formed.

The present invention also provides a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19) comprising the bispecific antibody and a pharmaceutically acceptable carrier.

Since the bispecific antibody of the present invention simultaneously binds to the surface antigen of SARS-CoV-2 virus and CD16 or CD3, SARS-CoV- 2 can be effectively neutralized.

In particular, by applying an ACE2-derived amino acid sequence to the SARS-CoV-2 binding region of the bispecific antibody, immune cells can effectively recognize and kill the virus due to the high binding force between SARS-CoV-2 and ACE2. In addition, since the amino acid sequence derived from the receptor, not the antibody of SARS-CoV-2, is used, it can exhibit a neutralizing effect on various virus strains.

In addition, by forming a variant in the ACE2 sequence, side effects due to the enzymatic activity of ACE2 can be prevented while maintaining antigen-binding ability, and by forming a variant in the Fc region, side effects caused by autoimmune reactions can be suppressed.

1 is an image conceptually showing the bispecific antibody of the present invention.
Figure 2 schematically shows the construction of the polypeptide chain constituting the bispecific antibody of the present invention.
3 (a) to (f) show the entire amino acid sequence of the ACE2-Fc-knob 1 to 6 mutant chain according to an embodiment of the present invention.
Figure 4 shows the entire amino acid sequence of the Anti-CD16-LC chain according to an embodiment of the present invention.
5 (a) to (c) show the entire amino acid sequence of the Anti-CD16-HC-hole 1 to 3 mutant chain according to an embodiment of the present invention.
6 (a) to (c) show the entire amino acid sequence of the Anti-CD16-scFv-Fc-hole 1 to 3 mutant chain according to an embodiment of the present invention.
7 shows the entire amino acid sequence of the Anti-CD3-LC chain.
8 (a) to (c) show the entire amino acid sequence of the Anti-CD3-HC-hole 1 to 3 mutant chain according to an embodiment of the present invention.
9 (a) to (c) show the entire amino acid sequence of the Anti-CD3-scFv-Fc-hole 1 to 3 mutant chain according to an embodiment of the present invention.
10 shows the results of SDS-PAGE of the heterodimeric protein prepared in Examples of the present invention.
11 is a graph showing the result of measuring fluorescence after treating SARS-CoV-2 spike protein-expressing lung cells with the bispecific antibody of the present invention.
12 is a graph showing changes in viral titer after administration of the bispecific antibody of the present invention to ACE2 transgenic mice infected with SARS-CoV-2.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

The present invention relates to a bispecific antibody capable of simultaneously binding to a surface antigen of SARS-CoV-2 and an immune cell activation receptor.

In the present invention, the term "antibody" includes immunoglobulin (Ig) molecules having immunological reactivity with a specific antigen, and includes both polyclonal and monoclonal antibodies. The term also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

In the present invention, the term "bispecific antibody" refers to an antibody capable of binding to two different types of antigens, and includes a form produced by genetic engineering. In the present invention, the bispecific antibody may refer to an antibody that induces apoptosis of the target antigen by antibody-dependent cytotoxicity by simultaneously binding to the target antigen and the immune cell activation receptor.

In the present invention, the term "antigen-binding region" refers to a part of an antibody comprising a region that specifically binds to part or all of an antigen and is complementary to part or all of an antigen. In the present invention, the first antigen-binding region can bind to an antigen by including an amino acid sequence derived from a cell receptor for the antigen, and the second antigen-binding region is a variable antibody against an activating receptor (CD16 or CD3) of an immune cell. By including the region, it can bind to an activating receptor of an immune cell.

In the present invention, the term "Fc region" includes the CH2 and CH3 domains of the constant region of an antibody. In the present invention, the Fc region may be connected to the antigen-binding region by a hinge or a linker.

In the present invention, the constant region may be appropriately selected from the constant region of an immunoglobulin, for example, a human immunoglobulin. For example, the heavy chain constant region may be an IgG1 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region, and the light chain constant region may be a kappa (κ) constant region or a lambda (λ) light chain constant region.

In the present invention, a variant means a substitution, deletion and/or addition of amino acid(s) in a native amino acid sequence.

The present invention relates to a bispecific antibody capable of simultaneously binding to a surface antigen of SARS-CoV-2 and an immune cell activation receptor.

The bispecific antibody of the present invention comprises: a first region comprising a first antigen-binding region that specifically binds to a surface antigen of SARS-CoV-2 and a first Fc region linked thereto; and a second antigen-binding region that specifically binds to CD16 or CD3, which is an immune cell activation receptor, and a second region comprising a second Fc region linked thereto.

1 schematically shows the bispecific antibody of the present invention. In FIG. 1 , the left side shows the first region, and includes a first antigen-binding region that binds to SARS-CoV-2 and a first Fc region linked thereto, wherein the first antigen-binding region binds to SARS-CoV-2 It may include an amino acid sequence derived from ACE2 for binding. In addition, the right side of FIG. 1 shows the second region, and includes a second antigen-binding region derived from an Anti-CD16 or Anti-CD3 antibody and a second Fc region linked thereto. In this structure, the first region and the second region may be joined by a knob-into-hole formed in the Fc region of each region to form a bispecific antibody.

In the bispecific antibody of the present invention, the first antigen-binding region can specifically bind to the surface antigen of SARS-CoV-2, including the amino acid sequence derived from ACE2.

The amino acid sequence derived from ACE2 may include the amino acid sequence of ACE2, fragments, homologues, and variants thereof, and in particular may include an extracellular domain (ectodomain) of ACE2.

ACE2 is an abbreviation of Angiotensin-converting enzyme 2, and is a carboxypeptidase related to angiotensin-converting enzyme (ACE). ACE2 is a protein consisting of 805 amino acids including a zinc-binding sequence, and is known as an enzyme that affects homeostasis of blood pressure and heart function.

In addition, ACE2 has been shown to be a functional receptor for coronaviruses associated with severe acute respiratory syndrome (SARS). When SARS-CoV-2 enters the human body, infection occurs when ACE2 binds to the SARS-CoV-2 spike protein.

The binding affinity between ACE2 and the surface antigen of SARS-CoV-2 virus is about 20 to 40 nM, which is several hundred times higher than that between other viruses and receptors, and it is known that the recently discovered variant viruses have higher binding affinity. .

In the bispecific antibody of the present invention, the first antigen-binding region may include an amino acid sequence derived from ACE2, and preferably include the amino acid sequence of SEQ ID NO: 1 corresponding to 1 to 615 of ACE2 .

[SEQ ID NO: 1] 1 to 615 of ACE2

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

In the bispecific antibody of the present invention, the first antigen-binding region can strongly bind to SARS-CoV-2 virus due to the high binding affinity between SARS-CoV-2 and ACE2. Accordingly, a large amount of immune cells activated by the CD16 or CD3 binding region of the opposite region can be induced to the periphery of the virus, and thus the virus can be very effectively neutralized.

In addition, even when a virus is mutated, since the site for recognizing the cell receptor on the surface antigen does not change, the first antigen-binding region is composed of an amino acid sequence derived from a cell receptor rather than an amino acid sequence derived from an antibody to the virus, thereby providing a variety of variants. It can also exhibit a virus-neutralizing effect against viruses.

In one embodiment of the present invention, a mutant may be formed and used in the amino acid sequence derived from ACE2 present in the first antigen-binding region.

Since ACE2 is an enzyme that modifies angiotensin peptide in the human body and performs blood pressure control, inflammation control, and blood coagulation control, etc. This can happen. In order to prevent this, in the present invention, while being able to bind to the target antigen, the enzymatic activity was disabled ACE2 inactivation mutant was designed.

The ACE2 inactivation mutant of the present invention may be one in which one or more mutations among H374N, H378N, E402N, H345A, E375N, R514A, Y510A, Y515A, H505A and R273A are formed in the ACE2-derived amino acid sequence. Preferably, the ACE2 inactivating variant may be a variant having mutations of H374N, H378N and E402N, or variants having mutations of H374N, H378N, E402N, H345A, E375N, R514A, Y510A, Y515A, H505A and R273A.

In the bispecific antibody of the present invention, the second antigen-binding region can specifically bind to CD16, an activating receptor for natural killer cells (NK cells), or CD3, which is an activating receptor for T cells.

Specifically, the second antigen-binding region may include a light chain variable region (V _L ) and a heavy chain variable region (V _H ) derived from an antibody against CD16 or CD3. The amino acid sequence derived from the antibody against CD16 or CD3 may include the amino acid sequence of the antibody against CD16 or CD3, fragments, homologues and variants thereof.

In one embodiment of the present invention, the second antigen-binding region may be a CD16-binding region of a natural killer cell, that is, an anti-CD16 region. In this case, the anti-CD16 region may include a light chain variable region (V _L ) and a heavy chain variable region (V _H ) derived from an antibody against CD16.

In the anti-CD16 region, the light chain variable region (V _L ) may include the amino acid sequence of SEQ ID NO: 2 below, and the heavy chain variable region (V _H ) may include the amino acid sequence of SEQ ID NO: 3 below. The amino acid sequence excludes the signal peptide of the heavy or light chain.

[SEQ ID NO: 2] Anti-CD16 V _L

DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGIPARFSASGSGTDFTLNIHPVEEEDTATYYCQQSNEDPYTFGGGTKLELKR

[SEQ ID NO: 3] Anti-CD16 V _H

QVTLKESGPGILQPSQTLSLTCSFSGFSLRTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSSNQVFLKIASVDTADTATYYCAQINPAWFAYWGQGTLVTVSA

In one embodiment of the present invention, the second antigen-binding region may be a CD3 binding region of a T cell, that is, an anti-CD3 region. In this case, the anti-CD3 region may include a light chain variable region (V _L ) and a heavy chain variable region (V _H ) derived from an antibody against CD3.

In the anti-CD3 region, the light chain variable region (V _L ) may include the amino acid sequence of SEQ ID NO: 4 below, and the heavy chain variable region (V _H ) may include the amino acid sequence of SEQ ID NO: 5 below. The amino acid sequence excludes the signal peptide of the heavy or light chain.

[SEQ ID NO: 4] Anti-CD3 V _L

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN

[SEQ ID NO: 5] Anti-CD3 V _H

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

The second antigen binding region is Fab, Fab', F(ab')2, Fv fragment, rIgG, single chain Fv fragment (scFv), tandem single chain Fv fragment (scFv)2, bispecific T-cell participant It may be selected from the group consisting of (BiTE), diabody (Diabody), tandem diabody (TandAb), triabody (Triabody) and tetrabody (Tetrabody), and preferably may be in the form of Fab or scFv.

The Fab form includes a variable region (V _H ) and a CH1 domain of a heavy chain, and a variable region (V _L ) and a CL domain of a light chain, and a disulfide bond is formed between the CH1 domain and the CL domain.

The scFv form refers to a single-chain variable fragment (scFv) in which Fv is continued, and refers to _{a recombinant domain in which V H} and V _L regions are connected by a peptide linker. The linker may be a peptide consisting of 5 to 20 amino acids. For example, the linker may be (GGGGX) _n , X is preferably A or S, and n is preferably a natural number of 1 to 4.

As such, in the bispecific antibody of the present invention, the ACE2-derived first antigen-binding region binds to SARS-CoV-2, and the opposite second antigen-binding region binds to and activates the immune cell activation receptor CD16 or CD3. By allowing the infected immune cells to gather around SARS-CoV-2, it can effectively kill the virus.

In the present invention, the Fc region of the bispecific antibody can be divided into a first Fc region and a second Fc region, and the Fc region linked to the first antigen-binding region is referred to as a first Fc region, and the Fc region linked to the second antigen-binding region The Fc region is referred to as the second Fc region.

In the present invention, the first and second Fc regions may include an immunoglobulin Fc region (Ig-Fc). The immunoglobulin may be IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

In the present invention, the first and second Fc regions may include sequences 221 to 447 of the IgG1 heavy chain of SEQ ID NO: 6 below. Here, amino acid numbers are according to EU numbering.

[SEQ ID NO: 6] HC 221 to 447 of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKKNQVSLTCLDWSDNGQPQSDIKFSDKVSDELTKNQVSLKVVSDGSGPFNWYTKSKSPVWSDNGWFFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGG

In this case, a knob or a hole may be formed in the first Fc region and the second Fc region to form a heterodimer, and thus a knob-into-hole structure may be formed. can be combined to form

The knob-into-hole technology allows only heterodimerziation to be formed between the heavy chains of antibody fragments, but does not allow homodimerization. Here, the knob has a side chain protruding into the contralateral domain, and is inserted into a hole in the contralateral domain. Due to this, heavy chains cannot homodimerize due to side chain collisions, only heterodimerization is possible. In the present invention, a structure in which a knob or a hole is formed in the Fc may be referred to as an Fc-knob or an Fc-hole, respectively.

The Fc-knob may be formed by substituting one or more amino acids of Ig-Fc with a large amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F) and tyrosine (Y).

For example, Fc-knob may be the amino acid sequence of SEQ ID NO: 7 in which T366W mutation is formed in sequences 221 to 447 of the IgG1-Fc heavy chain.

[SEQ ID NO: 7] HC 221 to 447 knob of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The Fc-hole may be formed by substituting one or more amino acids of Ig-Fc with a small amino acid selected from the group consisting of alanine (A), serine (S), threonine (T), or valine (V).

For example, the Fc-holes are T366S, L368A and Y407V in the sequences 221-447 of the IgG1-Fc heavy chain. The mutation may be the amino acid sequence of SEQ ID NO: 8.

[SEQ ID NO: 8] HC 221 to 447 hole of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C V A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In one embodiment of the present invention, the first or second Fc region may be used by forming an immunocompromised mutant.

Wild-type Fc binds to various Fc receptors to induce an immune response. Among them, binding to a gamma III-type Fc receptor (FcRγIII) induces NK cells to cause self-killing of other NK cells and immune cells. According to this reaction, other NK cells may be induced while the bispecific antibody is bound to NK cells or T cells and an autoimmune reaction may occur, which may cause destruction of healthy NK cells or T cells in the human body. have.

The present invention can suppress the side effects caused by the autoimmune reaction by redesigning by forming an immunodisabled mutant in the receptor binding site of the Fc region.

The Fc immunodisabled mutant of the present invention comprises a deletion of 1 to 10 residues including all or part of the A327 to P329 sequences and Y296 to S298 sequences in the Fc region (based on EU numbering); and L234A, L235A, G236K, and G237K may be a mutant in which at least one mutation is formed among one or more substitutions selected from the group consisting of.

Specifically, the Fc immunodisabled mutant may be a variant in which one or more of A327 to P329, Y296 to S298, L234A, L235A, G236K and G237K mutations are formed in the sequences 221 to 447 of the IgG1-Fc heavy chain. In the present invention, by forming the mutation, it is possible to design an Fc immunodisabled mutant so as not to induce NK cells while maintaining the effect of extending the plasma retention time by Fc.

Figure 2 shows the structure of the polypeptide chain constituting the bispecific antibody of the present invention. After constructing a polypeptide chain corresponding to each region, a bispecific antibody is constructed by conjugating a knob-into-hole to form a heterodimer.

Referring to FIG. 2 , the first region may be configured to include sequences 1 to 615 of ACE2 and an Fc-knob linked thereto. At this time, inactivation mutants may be formed in sequence regions 1 to 615 of the ACE2.

The second region comprises a chain comprising a Fab heavy chain (i.e., the V _H and CH1 domains of the heavy chain) linked to a heavy chain signal peptide and an Fc-hole linked to said Fab heavy chain, and a Fab light chain linked to a light chain signal peptide (i.e., the V of the light chain) _L and CL domains).

Alternatively, the second region may be configured to include an scFv linked to a heavy chain signal peptide (ie, _{a structure in which V H} and V _L are linked by a linker) and an Fc-hole linked thereto.

At this time, immuno-disabled variants may be formed in each Fc region.

According to the bispecific antibody of the present invention, the first antigen-binding region comprising the amino acid sequence derived from ACE2 binds to the surface antigen of SARS-CoV-2 virus, and the amino acid sequence derived from the antibody against CD16 or CD3, that is, anti - A second antigen-binding region comprising a CD16 or anti-CD3 amino acid sequence binds to an activating receptor of an immune cell.

Accordingly, immune cells activated according to antibody dependent cell cytotoxicity (ADCC) can recognize and kill SARS-CoV-2. At this time, due to the high binding force between SARS-CoV-2 and the ACE2 receptor, immune cells can effectively recognize and kill SARS-CoV-2. In addition, even if a virus is mutated, since the site for recognizing cell receptors on the surface antigen does not change, there is an advantage in that the virus killing effect appears for various mutants.

In addition, by forming a variant in the ACE2 sequence, side effects due to enzyme activity can be prevented while maintaining the antigen-binding ability of ACE2, and side effects caused by autoimmune reactions can be suppressed by forming a variant in the Fc region. Accordingly, it is possible to produce a bispecific antibody with minimal side effects while effectively neutralizing the virus.

Accordingly, the present invention also relates to a pharmaceutical composition for preventing or treating SARS-CoV-2 virus infection disease comprising the bispecific antibody.

The bispecific antibody of the present invention binds to the SARS-CoV-2 surface antigen and at the same time binds to CD16 or CD3, an activating receptor of NK cells or T cells, whereby the activated immune cells recognize SARS-CoV-2 This can effectively kill the virus. Accordingly, it is possible to prevent or treat coronavirus infection-19 (COVID-19), a SARS-CoV-2 virus infectious disease.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the bispecific antibody.

The pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition comprising the bispecific antibody of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, It can be administered by intrapulmonary administration and rectal administration.

The pharmaceutical composition comprising the bispecific antibody of the present invention is a sterile injection solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, an external application, or a suppository according to a conventional method. It can be formulated in the form of Since the protein or peptide is digested upon oral administration, oral compositions may be formulated to coat the active agent or to protect it from degradation in the stomach.

The pharmaceutical composition comprising the bispecific antibody of the present invention may further comprise at least one other therapeutic or diagnostic agent. For example, interferon, anti-S protein monoclonal antibody, anti-S protein polyclonal antibody, nucleoside analog, DNA polymerase inhibitor, siRNA agent or therapeutic vaccine together with the bispecific antibody as an antiviral drug may additionally include.

By administering the pharmaceutical composition of the present invention to mammals including humans, it is possible to prevent or treat SARS-CoV-2 infection and diseases caused by SARS-CoV-2 infection. In this case, the dosage of the bispecific antibody depends on the subject to be treated, the severity of the disease or condition, the rate of administration, and the judgment of the prescribing physician.

Example

The present invention will be described in more detail with reference to the following examples. However, these Examples show some experimental methods and compositions for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples.

Preparation Example 1: Preparation of bispecific antibody

1-1. Vector construction for the expression of each region of the antibody

The expression vector was constructed to have a polyhistidine tag at the C-terminus of the protein based on pcDNA 3.1 / Myc His-A (Invitrogen).

The polynucleotide encoding the amino acid sequence of the target protein was amplified by PCR and cloned using restriction enzymes and ligases. Through this, anti-CD16-LC, anti-CD3-LC expression vectors and Fc expression vectors expressing human immunoglobulin G1 (IgG1) heavy chain (sequences 221 to 447) were constructed.

For additional vector construction, ACE2 (sequences 1 to 615), anti-CD16-scFv (3G8), anti-CD3-scFv (OKT3), anti-CD16-Fab-HC at the N-terminus of the protein expressed in the Fc expression vector, respectively , nucleotides encoding each site were inserted so that anti-CD3-Fab-HC was fused. Through this, ACE2-Fc, anti-CD16-scFv-Fc, anti-CD3-scFv-Fc, anti-CD16-HC, and anti-CD3-HC expression vectors were constructed.

1-2. Introduction of knobs and holes for heterodimeric protein production

Based on the prepared vector, a mutant for forming a knob and a hole capable of producing a heterodimeric protein was introduced.

In the ACE2-Fc expression vector, an ACE2-Fc-knob expression vector was prepared by changing the nucleotide sequence to form a knob due to T366W mutation in the IgG1 heavy chain sequence portions 221 to 447 of the Fc region.

Anti-CD16-scFv-Fc, anti-CD3-scFv-Fc, anti-CD16-HC and anti-CD3-HC expression vectors are caused by T366S, L368A and Y407V mutations in the IgG1 heavy chain sequence region 221 to 447 of the Fc region. By changing the nucleotide sequence to form a hole, anti-CD16-scFv-Fc-hole, anti-CD3-scFv-Fc-hole, anti-CD16-HC-hole, and anti-CD3-HC-hole expression vectors were constructed, respectively. did In the polypeptide chain expressed by each vector, the Fc-knob region in which T366W mutation is formed is shown in SEQ ID NO: 7, and the Fc-hole region in which T366S, L368A and Y407V mutations are formed is shown in SEQ ID NO: 8.

1-3. Introduction of variants for disabling the ACE2 active site and ligand binding site

A variant of ACE2 was designed to maintain the binding force to the surface protein while disabling the active site and ligand binding site of ACE2.

The ACE2 variant is based on sequences 1 to 615 of ACE2, H374N, H378N and E402N variants (ACE2-mutant 1, SEQ ID NO: 9); Or H374N, H378N, E402N, H345A, E375N, R514A, Y510A, Y515A, H505A and R273A variants (ACE2-mutant 2, SEQ ID NO: 10) was constructed.

[SEQ ID NO: 9] H374N, H378N and E402N variants 1 to 615 of ACE2

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAH N EMG N IQYDMAYAAQPFLLRNGANEGFH N AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

[SEQ ID NO: 10] H374N, H378N, E402N, H345A, E375N, R514A, Y510A, Y515A, H505A and R273A variants of ACE2 from 1 to 615

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWG A FWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVC A PTAWDLGKGDFRILMCTKVTMDDFLTAH NN MG N IQYDMAYAAQPFLLRNGANEGFH N AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLF A VSND A SFI AA YTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

1-4. Introduction of variants for autoimmune disabling of the Fc region

A variant was designed for autoimmune disabling of the Fc region.

Based on the sequences 221 to 447 of the heavy chain of IgG1, A327-P329 deletion mutant (Fc-mutant 1); A327-P329 and Y296-S298 deletion mutants (Fc-mutant 2); Alternatively, L234A, L235A, G236K and G237K variants (Fc-mutant 3) were constructed. At this time, the variant Fc-hole-mutants 1 to 3 formed in the Fc hole may be represented by the amino acid sequence of SEQ ID NOs: 9 to 11, and the variant Fc-knob-mutants 1 to 3 formed in the Fc knob are SEQ ID NOs: 12 to 14 It can be represented by the amino acid sequence of

[SEQ ID NO: 11] HC 221 to 447 knob A327-P329 deletion of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[SEQ ID NO: 12] HC 221 to 447 knob A327-P329 and Y296-S298 deletion of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[SEQ ID NO: 13] HC 221 to 447 knobs L234A, L235A, G236K and G237K of IgG1

DKTHTCPPCPAPE AAKK PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[SEQ ID NO: 14] HC 221 to 447 hole A327-P329 deletion of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C V A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[SEQ ID NO: 15] HC 221 to 447 hole A327-P329 and Y296-S298 deletion of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C V A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[SEQ ID NO: 16] HC 221 to 447 holes of IgG1 L234A, L235A, G236K and G237K

DKTHTCPPCPAPE AAKK PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C V A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1-5. Design of the amino acid sequence expressed by the vector

In this way, the vector is expressed in a form in which ACE2 or a variant thereof is attached to Fc-knob or a variant thereof, and an anti-CD16 or anti-CD3 Fab or scFv is attached to Fc-hole or a variant thereof. composed.

In this case, in the case of preparing an Fc-hole mutant to which anti-CD16 or anti-CD3 Fab is attached, the heavy chain (HC) and light chain (LC) were co-expressed.

The amino acid sequence for each polypeptide chain expressed using the vector is shown below.

Seq # below means the amino acid sequence number of the present invention.

Linkages between the described amino acid sequences may be formed via linkers or hinges. For example, the two variable regions of the scFv may be joined through a linker, and the antigen-binding region and the Fc region may be connected through a hinge or a linker.

(1) Chain design of each area where Mutants are not formed

The ACE2-Fc-knob chain of the first region can be designed as shown in Table 1 below.

division first antigen binding region first Fc region ACE2-Fc-knob Seq 1 Seq 7

Anti-CD16-Fc-hole in the second region can be designed as shown in Table 2 below, and Anti-CD3-Fc-hole can be designed as shown in Table 3. Here, IgG1 CL and CH1 refer to the light chain constant region (CL) and the heavy chain constant region (CH1) of the Fab region of IgG1, respectively.

Anti-CD16-Fc-hole second antigen binding region second Fc region Fab form LC Seq 2 IgG1 CL - HC-hole Seq 3 IgG1 CH1 Seq 8 scFv form scFv-Fc-hole Seq 2 - Seq 3 - Seq 8

Anti-CD3-Fc-hole second antigen binding region second Fc region Fab form LC Seq 4 IgG1 CL - HC-hole Seq 5 IgG1 CH1 Seq 8 scFv form scFv-Fc-hole Seq 5 - Seq 4 - Seq 8

(2) Chain design of each area where Mutants are formed

The ACE2-Fc-knob mutant chain of the first region can be designed as shown in Table 4 below.

division first antigen binding region first Fc region ACE2-Fc-knob 1 Seq 9 seq 11 ACE2-Fc-knob 2 Seq 9 Seq 12 ACE2-Fc-knob 3 Seq 9 Seq 13 ACE2-Fc-knob 4 Seq 10 seq 11 ACE2-Fc-knob 5 Seq 10 Seq 12 ACE2-Fc-knob 6 Seq 10 Seq 13

3 (a) to (f) show the entire amino acid sequence of the ACE2-Fc-knob 1 to 6 mutant chain, respectively. In the sequence of Figure 3, the part marked in red corresponds to the mutant sequence of ACE2 1 to 615 of the first antigen-binding region, and the part indicated in blue is the knob in the mutant of IgG1 221 to 447 of the first Fc region. corresponding to the formed sequence.

Anti-CD16-Fc-hole in the second region can be designed as shown in Table 5 below.

Anti-CD16-Fc-hole second antigen binding region second Fc region Fab form LC Seq 2 IgG1 CL - HC-hole 1 Seq 3 IgG1 CH1 Seq 14 HC-hole 2 Seq 3 IgG1 CH1 Seq 15 HC-hole 3 Seq 3 IgG1 CH1 Seq 16 scFv form scFv-Fc-hole 1 Seq 2 - Seq 3 - Seq 14 scFv-Fc-hole 2 Seq 2 - Seq 3 - Seq 15 scFv-Fc-hole 3 Seq 2 - Seq 3 - Seq 16

4 shows the entire amino acid sequence of the Anti-CD16-LC chain. In FIG. 4 , the portion indicated in red _{corresponds to the V L} region derived from the Anti-CD16 antibody, and the portion indicated in green corresponds to the light chain constant region (CL) sequence of IgG1.

5 (a) to (c) show the entire amino acid sequence of the Anti-CD16-HC-hole 1 to 3 mutant chain, respectively. In FIG. 5 , the portion indicated in red _{corresponds to the V H} region derived from the Anti-CD16 antibody, and the portion indicated in green corresponds to the sequence including the heavy chain constant region (CH1) and hinge of IgG1. The part marked in blue corresponds to the sequence in which a hole is formed in the mutant of IgG1 221 to 447 of the first Fc region.

6 (a) to (c) show the entire amino acid sequence of the Anti-CD16-scFv-Fc-hole 1 to 3 mutant chain, respectively. In FIG. 6 , the first part indicated in red _{corresponds to the V L} region derived from the Anti-CD16 antibody, and the part indicated in the second red color corresponds to the V _H region derived from the Anti-CD16 antibody. The part marked in blue corresponds to the Fc sequence in which a hole is formed in the mutant of IgG1 221 to 447 of the first Fc region.

Anti-CD3-Fc-hole in the second region can be designed as shown in Table 6 below.

Anti-CD3-Fc-hole second antigen binding region second Fc region Fab form LC Seq 4 IgG1 CL - HC-hole 1 Seq 5 IgG1 CH1 Seq 14 HC-hole 2 Seq 5 IgG1 CH1 Seq 15 HC-hole 3 Seq 5 IgG1 CH1 Seq 16 scFv form scFv-Fc-hole 1 Seq 5 - Seq 4 - Seq 14 scFv-Fc-hole 2 Seq 5 - Seq 4 - Seq 15 scFv-Fc-hole 3 Seq 5 - Seq 4 - Seq 16

7 shows the entire amino acid sequence of the Anti-CD3-LC chain. In FIG. 7 , the portion indicated in red _{corresponds to the VL} region derived from the Anti-CD3 antibody, and the portion indicated in green corresponds to the light chain constant region (CL) sequence of IgG1.

8 (a) to (c) show the entire amino acid sequence of the Anti-CD3-HC-hole 1 to 3 mutant chain, respectively. In FIG. 8 , the portion indicated in red _{corresponds to the V H} region derived from the Anti-CD3 antibody, and the portion indicated in green corresponds to the sequence including the heavy chain constant region (CH1) and hinge of IgG1. The part marked in blue corresponds to the sequence in which a hole is formed in the mutant of IgG1 221 to 447 of the first Fc region.

9 (a) to (c) show the entire amino acid sequence of the Anti-CD3-scFv-Fc-hole 1 to 3 mutant chain, respectively. In FIG. 9 , the first part indicated in red _{corresponds to the V H} region derived from the Anti-CD3 antibody, and the part indicated in the second red color corresponds to the _{VL region derived from the Anti-CD3 antibody.} The part marked in blue corresponds to the sequence in which a hole is formed in the mutant of IgG1 221 to 447 of the first Fc region.

Preparation Example 2: Protein expression and purification

2-1. protein expression

Each protein was transiently expressed by transfection of the expression vector into ExpiCHO™ cells.

The transfection was carried out under the conditions of an initial cell density of 6 x 10 ⁶ cells/mL and an expression vector concentration of 0.8 μgDNA/mL. was used according to

After culturing the transfected cells in a carbon dioxide incubator for 8 days, centrifugation was performed to separate the cells into a cell culture medium containing each protein and cells.

2-2. refine

Affinity chromatography using a Ni-NTA (Qiagen) column was performed to separate target proteins from a culture medium containing each recombinant protein.

Specifically, after filling the Ni-NTA column with a buffer solution containing 50 mM Tris-HCl pH 7.5 and 200 mM NaCl, the culture solution obtained through centrifugation was filtered with a 0.45 μm filter paper and added. After completion of the reaction with the culture solution, the column was sequentially washed with a buffer solution containing 50 mM Tris-HCl pH 7.5 and 500 mM NaCl and a buffer solution containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl and 30 mM imidazole. Finally, using a buffer solution containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, and 500 mM imidazole, the protein bound to the column was eluted. After elution, the composition of the buffer solution containing the protein was replaced with 20 mM Tris-HCl pH 7.5 and 150 mM NaCl using a Hitrap™ Desalting (GE healthcare) column.

Preparation Example 3: Heterodimeric Protein Combination

After quantification of purified ACE2-Fc-knob protein and anti-CD16-Fc-hole protein, they were mixed at the same molecular ratio. A reagent was added to the mixed protein solution so that the final concentration became 0.1M arginine and 5mM L-glutathione (reduced), and the reaction was performed at 37°C for 2 hours to form a heterodimeric protein. After the reaction was completed, it was confirmed that a heterodimer was formed with a purity of about 80% through the non-reducing SDS-PAGE technique.

10 shows the results of SDS-PAGE of Preparation Example 3. In FIG. 10 , column 1 is anti-CD16-Fc-hole, column 2 is ACE2-Fc-knob, and column 3 is the result for the ACE2/anti-CD16 heterodimer. Here, the yield of anti-CD16-Fc-hole was 46 mg/L and that of ACE2-Fc-knob was 33 mg/L, and it was confirmed that the heterodimer was formed in a yield of 81%.

Experimental Example 1: Measurement of the ability to remove SARS-CoV-2 spike protein expression lung cells

Using the ACE2/anti-CD16 bispecific antibody, the ability to remove lung cells expressing SARS-CoV-2 spike protein on the cell surface was measured.

To mimic SARS-CoV-2 virus-infected cells, the A549 human lung cancer cell line (Korea Cell Line Bank) was used. A549 cells were seeded in a 96-well plate at ^{a concentration of 2 x 10 4} cells/well, and transiently transfected with a SARS-CoV-2 spike expression vector (Addgene) through Lipofectamine 3000 (Invitrogen).

After staining the transfected A549 cells with 5 μM Calcein Blue AM (eBioscience), 200 U/mL IL-2, heterodimeric protein and peripheral blood mononuclear cells (PBMC, peripheral blood mononuclear cell, STEMCELL Technologies) were added to the experimental conditions. mixed appropriately.

To measure the maximum fluorescence emission, a buffer solution containing 90% DMSO was added to the wells to lyse the cells. After 4 hours, the supernatant of each well was separated by centrifugation, and fluorescence was measured at a wavelength of 355 nm / 460 nm using a Victor X2 (Perkin-Elmer) plate meter, and the results are shown in FIG. 11 .

Referring to FIG. 11 , when 20 nM Ct-BiAB was added, an excellent cytolytic effect was observed in relative fluorescence unit (RFU) comparable to that of the avg positive control. On the other hand, when Ct-BiAB was not added, the cytolytic effect was significantly lower.

Accordingly, it was confirmed that when the bispecific antibody of the present invention was added, peripheral blood mononuclear cells (PBMC) could effectively kill the spike protein-expressing lung cells according to the ADCC mechanism.

Experimental Example 2: Covid-19 treatment effect in ACE2 transgenic mice by ACE2/anti-CD16 bispecific antibody

Using ACE2 transgenic mice, the effect of reducing viral titer after SARS-CoV-2 virus infection was measured.

Specifically, a 10-week-old female ACE2 TG mouse was infected with SARS-CoV-2 virus. After 24 hours, the ACE2/anti-CD16 bispecific antibody (Ct-BiAB) was administered once intraperitoneally at a low dose (10 nM) or a high dose (100 nM), and then the viral titer was measured, and the results are shown in FIG. 12 . it was For comparison, the results for Mock and control (virus only) are also shown. In each experiment, 3 mice were used for mock, 6 mice for virus only, 6 mice for low dose, and 6 mice for high dose.

As can be seen in Figure 12, as a result of treatment with Ct-BiAB at a low dose (10 nM) and a high dose (100 nM) in ACE2-TG mice, respectively, in day post infection (dpi) 3, the viral load compared to the control group (virus only) There was almost no change in titer, but there was a significant difference at dpi 5, showing a dose dependency.

In particular, at dpi 5, the viral titers of the control (virus only), low-dose (10nM) experimental group, and high-dose (100nM) experimental group were 7.14, 6.74, and 6.61 on a log scale, respectively, and the viral titer of the low-dose experimental group was reduced by half compared to the control group. In the high-dose experimental group, the result was significantly reduced to 30% or less compared to the control group.

Accordingly, it was confirmed that the bispecific antibody of the present invention exhibits an excellent therapeutic effect against SARS-CoV-2 infection.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Bispecific Antibody Capable of Binding to SARS-CoV-2 and Immune Cell Receptor and Pharmaceutical Composition Comprising Same <130> HPN21024 <150> KR 10-2020- 0085432 <151> 2020-07-10 <150> KR 10-2020-0116249 <151> 2020-09-10 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 615 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Me t Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Al a His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gl y Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gl y Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp 610 615 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD16 variable fragment light chain <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp 20 25 30 Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp T hr Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence < 220> <223> Anti-CD16 variable fragment heavy chain <400> 3 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Arg Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Gln Ile Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD3 variable fragment light chain <400> 4 Gln Ile Val Leu Thr Gln S er Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn 100 105 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD3 variable fragment heavy chain <400> 5 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln L eu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 6 <211 > 227 <212> PRT <213> Homo sapiens <400> 6 Asp Lys Thr His Thr Cys Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 7 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc knob <400> 7 Asp Lys Thr His Thr Cys Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Le u Ser 210 215 220 Pro Gly Lys 225 <210> 8 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc hole <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 9 <211> 615 <212> PRT <213> Artificial Sequence <220> <223> Ace2 615 inactivation mutant 1 <400> 9 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 7 5 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Ly s Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His Asn Gl u Met Gly Asn Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Asn Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Ty r Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp 610 615 <210> 10 <211> 615 <212> PRT <213> Artificial Sequence <220> <223> Ace2 615 inactivation mutant 2 < 400> 10 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Le u Phe Tyr Gln Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Ala Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys Ala Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His Asn Asn Met Gly Asn Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Asn Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Glu Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe Ala Val Ser Asn Asp Ala Ser Phe 500 505 510 Ile Ala Ala Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp 610 615 <210> 11 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc knob inactivation mutant 1 <400> 11 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr T hr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 12 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc knob inactivation mutant 2 <400> 12 Asp Lys Thr His Thr Cys Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Pro Ile Glu Lys Thr Ile Ser Lys 100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120 125 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys 130 135 140 Gly Phe Tyr Pr o Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165 170 175 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185 190 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 13 <211> 227 <212 > PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc knob inactivation mutant 3 <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Lys 1 5 10 15 Lys Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 14 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc hole inactivation mutant 1 <400> 14 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 15 <211> 221 <212> PRT <213> Artificial Sequence <220> <223 > IgG1 221-447 Fc hole inactivation mutant 2 <400> 15 Asp Lys Thr His Thr Cys Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gl n Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Pro Ile Glu Lys Thr Ile Ser Lys 100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120 125 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165 170 175 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185 190 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 16 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> IgG1 221-447 Fc ho le inactivation mutant 3 <400> 16 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Lys 1 5 10 15 Lys Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pr o Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gin Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys 225

Claims (5)

a first region comprising a first antigen-binding region comprising the amino acid sequence of SEQ ID NO: 1 as a region specifically binding to a surface antigen of SARS-CoV-2, and a first Fc region linked thereto; and
A second region comprising a second antigen-binding region that specifically binds to CD16 or CD3, which is an immune cell activation receptor, and a second Fc region linked thereto
comprising, a bispecific antibody. The method of claim 1,
The amino acid sequence of SEQ ID NO: 1 comprises a mutation of one or more of H374N, H378N, E402N, H345A, E375N, R514A, Y510A, Y515A, H505A and R273A, a bispecific antibody. The method of claim 1,
The second antigen-binding region, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (V _H ) and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 (V _L ); Or a bispecific antibody _{comprising a heavy chain variable region (V H} ) comprising the amino acid sequence of SEQ ID NO: 4 _{and a light chain variable region (V L} ) comprising the amino acid sequence of SEQ ID NO: 5. The method of claim 1,
deletion of residues 1 to 10 including all or part of the sequences A327 to P329 and Y296 to S298 (based on EU numbering) in at least one of the first Fc region and the second Fc region; and at least one mutation selected from the group consisting of L234A, L235A, G236K and G237K. A pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19) comprising the bispecific antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

Images