WO2022025129A1 - Production method of novel anti-hbs immunoglobulin - Google Patents

Abstract

The present invention addresses the problem of providing a safe and efficacious antibody preparation so as to fundamentally overcome the difficulty that the national self-sufficiency of starting blood for producing anti-HBs human immunoglobulin preparations is still on the way.　According to the present invention, the aforesaid problem is solved by providing a recombinant anti-HBs human immunoglobulin preparation that is produced by screening an anti-HBs antigen-specific monoclonal antibody capable of exhibiting a neutralizing activity against HBV from B cells in the peripheral blood circulating in the body of a human individual, said human individual having acquired anti-HBs antibody by HBV vaccination and showing an elevated antibody titer, and then isolating the gene of the antibody.

Description

New method for producing anti-HBs immunoglobulin

The present invention relates to providing a recombinant anti-HBs human immunoglobulin preparation.

It is estimated that there are about 300 million people with persistent hepatitis B virus (HBV) infection worldwide, and about 900,000 people die annually from HBV-related diseases, and the infection rate of HBV in Japan is about 1. It is estimated to be%. When infected with HBV during childbirth or infancy, more than 90% of cases shift to persistent infection, and about 90% of them become asymptomatic carriers in younger age, and the condition stabilizes in most cases. However, in the remaining 10%, the activity of the virus continues and the state of chronic hepatitis continues, and at an annual rate of about 2%, it shifts to liver cirrhosis and progresses to hepatocellular carcinoma and liver failure.

In recent years, vertical transmission of mother and child and HBV infection by blood transfusion have decreased sharply, but horizontal transmission as a sexually transmitted disease is occurring frequently mainly among young people. On the other hand, HBV reactivation in patients with immunodeficiency such as after transplantation is a new risk of developing hepatitis created by advanced medical treatment.

Currently, in Japan, anti-HBs human immunoglobulin preparations (HBIG) are generally used to prevent HBV mother-to-child transmission, HBV infection after medical accidents such as needle sticks, and to suppress recurrence and onset of hepatitis B after liver transplantation. in use. HBIG is an immunoglobulin preparation purified from blood donor plasma having extremely high titers against HBs antigen, which is a neutralizing epitope of HBV (anti-HBs antibody).

For blood products derived from human blood containing HBIG, including plasma fractionation products, it is stipulated by the "Act on Ensuring a Stable Supply of Safe Blood Products, etc." that the raw materials should be self-sufficient in Japan. Since the start of sales in Japan, the HBIG raw material blood self-sufficiency rate has remained at a very low rate of 2 to 3%, which is far from being completely self-sufficient in raw materials.

On the other hand, in order to solve this problem from 2013 (2013), the vaccine is additionally inoculated to human individuals who have already acquired anti-HBs antibody by HBV vaccine, and the anti-HBs antibody titer is increased. In addition, a project to secure raw material plasma by donating blood is being carried out, and the Japanese Red Cross Society has been entrusted with this project and has been implementing it so far [Sugauchi F, et al., Hepatol Res. 2006; 36: 107- 14].

However, even if the above project based on this additional vaccination is implemented, the amount of raw material plasma that can be secured is only partial, and it is far from being completely self-sufficient.

An object of the present invention is to provide a safe and effective antibody preparation by fundamentally solving the problem of unachieved domestic self-sufficiency of raw blood of anti-HBs human immunoglobulin preparation.

The present invention is an anti-HBs antigen-specific monoclonal that can have neutralizing activity against HBV from in-vivo peripheral blood circulation B cells of a human individual whose antibody titer is increased by acquiring anti-HBs antibody by inoculation with HBV vaccine. It was decided to solve the above-mentioned problems by screening an antibody, isolating the gene of the antibody, and providing a recombinant anti-HBs human immunoglobulin preparation.

More specifically, the present application provides the following aspects in order to solve the above-mentioned problems:
[1]: (1) Heavy chain complementarity determining regions, CDR1 (GFMFSGHS, SEQ ID No: 1), CDR2 (IGSTGEFI, SEQ ID No: 2), and CDR3 (AREQGTRGRYYYYGLDV, SEQ ID No: 3), and Light chain complementarity determining regions, CDR1 (SQSVSTY, SEQ ID No: 4), CDR2 (DAF, SEQ ID No: 5), and CDR3 (QQRGHWPLT, SEQ ID No: 6);
(2) Heavy chain complementarity determining regions, CDR1 (SGGSISGHY, SEQ ID No: 7), CDR2 (IHYSGIT, SEQ ID No: 8), and CDR3 (ARGDATYGY, SEQ ID No: 9), and light chain complementarity. Sex determining regions, CDR1 (SQSLLHRNGYNY, SEQ ID No: 10), CDR2 (LGS, SEQ ID No: 11), and CDR3 (MQALRTPWT, SEQ ID No: 12);
(3) Heavy chain complementarity determining regions, CDR1 (SGFSFSNYG, SEQ ID No: 13), CDR2 (IWRDGSHQ, SEQ ID No: 14), and CDR3 (AREDPAIVLPVLDH, SEQ ID No: 15), and light chain complementarity. Sex determining regions, CDR1 (QRINSY, SEQ ID No: 16), CDR2 (GAS, SEQ ID No: 17), and CDR3 (QQGYSTPLLS, SEQ ID No: 18);
Contains the complementarity determining regions of any heavy or light chain selected from the group consisting of, has binding to HBsAg of HBV, has neutralizing activity against HBV, is not derived from blood source, Antigens or antibody derivatives;
[2]: Recombinant antibody or antibody derivative according to [1];
[3]: The HBsAg is one or more selected from the group consisting of L antigen, M antigen, S antigen, HBsAg adr type, adw type, ayr type, and ayw type of HBsAg, [1]. Or the antibody or antibody derivative according to [2];
[4]: Any of [1] to [3], wherein the antibody derivative is selected from an antibody variant selected from humanized antibodies, chimeric antibodies, polyvalent antibodies, and multispecific antibodies or functional fragments thereof. Or the antibody or antibody derivative according to item 1;
[5]: The amino acid sequence of the heavy chain variable region VH domain of the antibody or antibody derivative is
(1) Of the amino acid sequence of SEQ ID No: 19 or the amino acid sequence of SEQ ID No: 19, CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
(2) Of the amino acid sequence of SEQ ID No: 21 or the amino acid sequence of SEQ ID No: 21, CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9) ), An amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions, and (3) the amino acid sequence of SEQ ID No: 23, or SEQ ID No: 23. Of the amino acid sequence of, one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15). Amino acid sequence, including substitutions), insertions, or deletions,
The antibody or antibody derivative according to any one of [1] to [4], which is selected from the above;
[6]: The amino acid sequence of the light chain variable region VL domain of the antibody or antibody derivative is
(1) Of the amino acid sequence of SEQ ID No: 20 or the amino acid sequence of SEQ ID No: 20, CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
(2) Of the amino acid sequence of SEQ ID No: 22 or the amino acid sequence of SEQ ID No: 22, CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12). ), An amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions, and (3) the amino acid sequence of SEQ ID No: 24, or SEQ ID No: 24. Of the amino acid sequence of, one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18). Amino acid sequence, including substitutions), insertions, or deletions,
The antibody or antibody derivative according to any one of [1] to [5], which is selected from the above;
[7]: The antibody or antibody derivative is
(1) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 25) and a light chain (SEQ ID No: 26);
(2) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 27) and a light chain (SEQ ID No: 28);
(3) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 29) and a light chain (SEQ ID No: 30);
Item 6. The antibody or antibody derivative according to any one of [1] to [6], which is selected from the group consisting of.
[8]: A pharmaceutical composition for neutralizing HBV, which comprises the antibody or antibody derivative according to any one of [1] to [7] and does not contain blood-derived components;
[9]: The pharmaceutical composition according to [8], which comprises a plurality of types of the antibody or antibody derivative according to any one of [1] to [7];
[10]: The pharmaceutical composition according to [8] or [9] for preventing reactivation of hepatitis B or preventing medical infection of HBV;
[11]: A step of bringing a biological sample collected from a subject into contact with the antibody or antibody derivative according to any one of [1] to [7] in vitro.
A step of detecting and measuring HBV in a sample bound to the antibody or antibody derivative.
A method for detecting and measuring the presence / abundance of HBV in a biological sample including
[12]: A kit for detecting and measuring the presence / abundance of HBV in a subject, which comprises the antibody or antibody derivative according to any one of [1] to [7].

INDUSTRIAL APPLICABILITY According to the present invention, an antibody expected to have safe, effective and stable activity that can fundamentally solve the problems of unachieved domestic self-sufficiency and the problems of blood products related to anti-HBs human immunoglobulin preparations derived from blood raw materials. Formulations can be provided.

FIG. 1 is a diagram showing a virus infection neutralization experiment in HepG2-hNTCP-C4 cells infected with genotype D HBV. FIG. 2 is a diagram showing a virus infection neutralization experiment in primary human hepatocytes PXB cells infected with genotype C HBV. FIG. 3 is a diagram showing an electrophoretic image in which the binding characteristics of the recombinant anti-HBs antibody were evaluated. FIG. 4 is a diagram confirming that the recombinant anti-HBs antibody of the present invention also binds to the HBs antigen aggregate by the ELISA method. FIG. 5 is a diagram analyzing the interaction of the recombinant anti-HBs antibody candidate antibody of the present invention with the recombinant HBs antigen using surface plasmon resonance (SPR) technology. FIG. 6 is a diagram showing the results of confirming the non-specific binding (cross-reactivity) of the recombinant anti-HBs antibody to the human-derived component. FIG. 7 is a diagram showing the results of immunostaining of human tissues using recombinant anti-HBs antibody. Here, FIG. 7a shows a histological image in which HBsAg was detected. FIG. 7 is a diagram showing the results of immunostaining of human tissues using recombinant anti-HBs antibody. Here, FIG. 7b shows that no staining was observed on various normal human tissues.

The terms used in the present invention are defined as follows:
(A) HBsAg: An antigen that is one of the proteins that make up the outer shell of HBV and is tested when determining the presence or absence of HBV infection. Antibodies to this HBsAg are found in vivo when HBV has been infected in the past but the virus has been eliminated, or when HBV vaccine is given and the antibody becomes positive (+);
(B) HBIG: Human anti-HBs Immunoglobulin. Commercially available HBIG is a pharmaceutical product obtained by purifying IgG from human plasma, which has a high anti-HBs antibody titer. Although it has an immediate effect, its effect lasts for about 3 months, which is relatively short.

The present invention is not derived from a blood source, has binding to HBsAg of HBV, has neutralizing activity against HBV, contains complementarity determining regions of heavy and light chains having a specific amino acid sequence. It has been shown that the subject of the present invention can be solved by providing an antibody or an antibody derivative. This antibody or antibody derivative can be any antibody or antibody derivative as long as it has binding property to HBs antigen of HBV, has neutralizing activity against HBV, and is not derived from blood raw materials. It is included in the scope of the present invention.

<Antibody or antibody derivative>
The antibody or antibody derivative used in the present invention has various problems peculiar to blood preparations such as blood-derived antibody preparations conventionally used for the treatment of hepatitis virus (particularly, infectivity problems and immunity problems). Viruses such as HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) that may be contained in the blood (risk of being mixed in blood preparations) for the purpose of solving the problem. It is characterized by being free of pathogens such as, and free of blood-derived components that may cause immune abnormalities in the administered individual, such as antigenic proteins contained in blood and antibodies that bind to human proteins. ..

As an antibody or antibody derivative that does not contain blood-derived components, immortalized cells derived from antibody-producing cells are obtained under conditions that do not contain blood-derived components, and are prepared from the cells under culture conditions that do not contain blood-derived components. Antibodies and recombinant antibodies that can be prepared by recombinantly expressing the antibody protein using the DNA defining the antibody protein obtained from the immortalized cells can be used.

That is, in one embodiment, the antibody in the present invention is derived from antibody-producing cells by obtaining and immortalizing a cell clone that produces an antibody against HBs antigen derived from HBV from the blood of an individual to which the HBV vaccine has been administered in the past. It can be obtained by obtaining immortalized cells and selecting cells having an action of neutralizing HBV in vivo from the immortalized antibody-producing cells.

In another aspect, in order to realize the above-mentioned characteristics, it is also possible to provide a recombinant anti-HBs antibody or antibody derivative having neutralizing activity against HBV by using the gene recombination technique. More specifically, the present invention comprises obtaining an mRNA and producing a cDNA from an immortalized antibody-producing cell having an action of neutralizing HBV selected by the above-mentioned method according to a well-known method, and then producing an antibody protein. It has neutralizing activity obtained by obtaining the specified DNA sequence and expressing it in a mammalian expression system containing no blood-derived components using a vector to produce it as recombinant immunoglobulin (IgG). Monoclonal antibodies or antibody derivatives thereof can also be provided as recombinant HBIG.

In the present invention, derivatives of these antibodies can also be used. As the antibody derivative that can be used in the present invention, for example, an antibody variant selected from a humanized antibody, a chimeric antibody, a polyvalent antibody, and a multispecific antibody or a functional fragment thereof can be used. , Not limited to these. Of these, as the functional fragment, for example, F (ab') 2 can be used, but the functional fragment is not limited thereto.

Derivatives of these antibodies can be produced according to a method well known in the art after the antibody is obtained, and can be prepared as the recombinant antibody derivative described for the antibody of the present invention described above.

The antibody or antibody derivative obtained by the above-mentioned method has binding property to HBs antigen derived from HBV, but in the present invention, such an antibody having binding property to HBs antigen derived from HBV. Alternatively, it is characterized in that screening is performed based on having an action of further neutralizing HBV from among antibody derivatives, and an antibody or antibody derivative having the neutralizing action is provided.

Although a plurality of types of antibodies actually obtained are shown in the examples described later, the present invention is not limited to these.

<Target of antibody or antibody derivative>
HBV is a spherical virus with a diameter of 42 nm that forms a double structure of the envelope and core. As proteins constituting HBV, HBs antigen, HBc antigen, HBe antigen, X protein (HBx antigen) and the like are known. Since the HBc antigen is a protein in the inner core of the virus, its antibody is useful for confirming the state of infection, but it is known that it cannot be used to protect against HBV infection. It is known that an antibody against the HBe antigen (anti-HBe antibody), which is an overproduced protein when proliferating in, does not protect against HBV infection, and the X protein binds to enhancer and promoter regions. It is a trans-activated protein having a function of regulating the expression of various proteins, and is known not to have a function of protecting against HBV infection.

On the other hand, HBs antigen is a protein that constitutes the outer shell (envelope) of HBV, and it is known that the presence of anti-HBs antibody can protect against HBV infection. In Japan today, in order to prevent HBV infection when it is necessary to prevent HBV mother-to-child transmission, prevent HBV infection after medical accidents such as needle sticks, and suppress hepatitis B recurrence and onset after liver transplantation. HBIG is commonly used. Therefore, in the present invention, the antibody or antibody derivative needs to have binding property to HBsAg.

The HBsAg as a target of the antibody or antibody derivative of the present invention has three domains, S domain, Pre-S1 domain, and Pre-S2 domain, and from the combination of these, S antigen (consisting only of S domain). ,
-M antigen (composed of S domain and Pre-S2 domain) and-L antigen (composed of three domains, S domain, Pre-S2 domain, and Pre-S1 domain)
There are three types. The antibody or antibody derivative of the present invention is for the HBsAg, whether it is for the S antigen, the M antigen, or the L antigen, as long as it has a binding property to the HBs antigen. You may.

Further, it is known that this HBsAg has three types of antigenic determinants, an antigenic determinant a, an antigenic determinant d or y, and an antigenic determinant r or w, and the antigenic determinant a is all. It is common to subtypes and can be classified into four subtypes, adr type, adw type, ayr type, and ayw type, depending on the combination. In Japan, adr type accounts for 70 to 90%, adw type accounts for 10 to 30%, and ayr type and ayw type are rare. This evaluation of HBs subtypes is used to elucidate the infection route of HBV and analyze coinfection. The antibody or antibody derivative of the present invention targets any of these four subtypes, adr type, adw type, ayr type, and ayw type, as long as it has binding property to HBs antigen. Of these, a plurality of them may be targeted.

<Neutralizing action of HBV>
The antibody or antibody derivative of the present invention is any of the above-mentioned HBV-derived HBs antigens (for example, any of the S antigen, M antigen, and L antigen, and any of the four subtypes of adr, adw, ayr, and ayw. Anything that binds to (possibly) and has the effect of neutralizing HBV as a result can be used to achieve the object of the present invention.

As mentioned above, HBIG preparations have traditionally been used for the treatment of patients suffering from HBV. The antibody or antibody derivative of the present invention is characterized in that blood is superior in titer and quality stability as compared with HBIG as a raw material. Further, since the antibody or antibody derivative of the present invention is intended to be used as an HBV therapeutic agent in place of the conventional HBIG, it is preferably the same as, preferably the same as, more preferably the neutralizing activity of HBV. It can have the above neutralization activity of HBV. On the other hand, since the antibody or antibody derivative of the present invention is characterized in that a plurality of antibodies or antibody derivatives can be used as a cocktail, even if a single antibody or antibody derivative has low neutralizing activity, a plurality of antibodies or antibody derivatives can be used. Can be used as a therapeutic agent in combination with the antibody or antibody derivative of.

The neutralizing activity of HBV is obtained by infecting cultured human liver-derived cells or primary human liver cells with HBV and adding an antibody to the cells under culture conditions.
・ Suppression of HBV viral load,
-Inhibition of intracellular expression of HBV-derived proteins (eg, HBs antigen, HBc antigen, HBe antigen, X protein (HBx antigen), etc.),
-Semi-quantitative depending on whether or not the amount of HBV-derived DNA in the cell (for example, the amount of viral DNA (capsid-associated relaxed circular DNA [rcDNA)) covered with HBV capsid in the cell) is suppressed. Can be specified in.

<Example of antibody of the present invention>
In the present invention, cells producing an antibody that binds to an HBs antigen derived from HBV are collected from an individual vaccinated with a known HBV vaccine in the above-mentioned <antibody or antibody derivative> and obtained. The neutralizing activity against HBV was examined for the antibody or antibody derivative as described above <HBV neutralizing action>. As a result, a plurality of antibodies and cells producing the antibodies were obtained from a plurality of individuals.

Under such circumstances, as specific examples examined in the examples, the complementarity determining regions of heavy chains and light chains having the following specific amino acid sequences:
(1) Heavy chain complementarity determining regions, CDR1 (GFMFSGHS, SEQ ID No: 1), CDR2 (IGSTGEFI, SEQ ID No: 2), and CDR3 (AREQGTRGRYYYYGLDV, SEQ ID No: 3), and light chain complementarity. Sex determining regions, CDR1 (SQSVSTY, SEQ ID No: 4), CDR2 (DAF, SEQ ID No: 5), and CDR3 (QQRGHWPLT, SEQ ID No: 6);
(2) Heavy chain complementarity determining regions, CDR1 (SGGSISGHY, SEQ ID No: 7), CDR2 (IHYSGIT, SEQ ID No: 8), and CDR3 (ARGDATYGY, SEQ ID No: 9), and light chain complementarity. Sex determining regions, CDR1 (SQSLLHRNGYNY, SEQ ID No: 10), CDR2 (LGS, SEQ ID No: 11), and CDR3 (MQALRTPWT, SEQ ID No: 12);
(3) Heavy chain complementarity determining regions, CDR1 (SGFSFSNYG, SEQ ID No: 13), CDR2 (IWRDGSHQ, SEQ ID No: 14), and CDR3 (AREDPAIVLPVLDH, SEQ ID No: 15), and light chain complementarity. Sex determining regions, CDR1 (QRINSY, SEQ ID No: 16), CDR2 (GAS, SEQ ID No: 17), and CDR3 (QQGYSTPLLS, SEQ ID No: 18);
By way of example, it is possible to provide recombinant antibodies or antibody derivatives thereof containing the above, but the present invention is not limited to these.

The present invention, in one embodiment, as such an antibody or antibody derivative:
(1) Of the amino acid sequence of SEQ ID No: 19 or the amino acid sequence of SEQ ID No: 19, CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
(2) Of the amino acid sequence of SEQ ID No: 21 or the amino acid sequence of SEQ ID No: 21, CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9) ), An amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions, and (3) the amino acid sequence of SEQ ID No: 23, or SEQ ID No: 23. Of the amino acid sequence of, one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15). Amino acid sequence, including substitutions), insertions, or deletions,
The amino acid sequence of the heavy chain variable region VH domain selected from; and (1) the amino acid sequence of SEQ ID No: 20 or the amino acid sequence of SEQ ID No: 20, CDR1 (SEQ ID No: 4), CDR2 ( Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in moieties other than SEQ ID No: 5) and CDR3 (SEQ ID No: 6).
(2) Of the amino acid sequence of SEQ ID No: 22 or the amino acid sequence of SEQ ID No: 22, CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12). ), An amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions, and (3) the amino acid sequence of SEQ ID No: 24, or SEQ ID No: 24. Of the amino acid sequence of, one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18). Amino acid sequence, including substitutions), insertions, or deletions,
Amino acid sequence of light chain variable region VL domain selected from;
As a more specific example, an antibody or antibody derivative containing the above can be mentioned. Here, the binding property of the antibody is determined by 6 CDRs (that is, CDR1 to CDR3 in each of the heavy chain and the light chain), and the binding property of the antibody is determined by the CDRs other than the CDRs of the heavy chain variable region and the light chain variable region. In a region, one or several amino acid mutations (amino acid substitutions, insertions or deletions) do not lose their intended binding.

As a more detailed aspect of the present invention, in the following examples,
(1) M2-11 clone: An antibody or antibody derivative containing a heavy chain variable region (SEQ ID No: 19) and a light chain variable region (SEQ ID No: 20);
(2) 5A4 clone: antibody or antibody derivative containing heavy chain variable region (SEQ ID No: 21) and light chain variable region (SEQ ID No: 22); or (3) 3B6 clone: heavy chain variable region (SEQ ID) Antibodies or antibody derivatives containing No: 23) and light chain variable region (SEQ ID No: 24); or (1) M2-11 clones: heavy chain (SEQ ID No: 25) and light chain (SEQ ID No: 26). ) Containing antibodies or antibody derivatives;
(2) 5A4 clone: antibody or antibody derivative containing heavy chain (SEQ ID No: 27) and light chain (SEQ ID No: 28); or (3) 3B6 clone: heavy chain (SEQ ID No: 29) and light An antibody or antibody derivative containing a chain (SEQ ID No: 30);
Can be given as a more specific example.

<Pharmaceutical composition>
In one aspect of the present invention, it is also possible to provide a pharmaceutical composition containing the antibody or antibody derivative described above and for neutralizing HBV. This pharmaceutical composition is used to prevent or treat the onset of symptoms caused by HBV, to prevent reactivation of hepatitis B, or to prevent the reactivation of hepatitis B, or to the mother and child of HBV, for individuals infected or suspected of being infected with HBV. It can be used to stop infections and medical infections.

Since the antibody or antibody derivative of the present invention contained in this pharmaceutical composition is not derived from a blood raw material, this pharmaceutical composition may be contained in blood (there is a risk of contamination with blood preparations). Blood that does not contain pathogens such as viruses such as HBV, HCV, and HIV, and has a risk of causing immune abnormalities in administered individuals, such as antigenic proteins contained in blood and antibodies that bind to human proteins. It is characterized by not containing derived components.

The pharmaceutical composition in the present invention may contain one type of the above-mentioned antibody or antibody derivative, or may contain a plurality of types.

<Other uses of antibodies or antibody derivatives>
The antibody or antibody derivative produced in the present invention can be used for the purpose of detecting and measuring the presence / absence of HBV in a biological sample. When used for such a purpose, a biological sample collected from a subject is brought into contact with the antibody or antibody derivative of the present invention in vitro, and the antibody or antibody derivative of the present invention bound to HBV is obtained by a secondary antibody. By detecting, the presence / abundance of HBV in a biological sample can be detected / measured by detecting / measuring HBV in a sample bound to the antibody or antibody derivative.

Since HBV in a biological sample can be detected and measured by the antibody or antibody derivative of the present invention as described above, an existing anti-HBs human immunoglobulin preparation (for example, a commercially available HBIG preparation (Japan Blood Product Organization) Alternatively, it is possible to compare the performance with Nihon Pharmaceutical Co., Ltd. (sold by Takeda Pharmaceutical Co., Ltd.)).

The antibody or antibody derivative of the present invention can also be used as a research reagent in applications such as ELISA and Western blotting for detecting and measuring HBV in a sample. By using such applications, it is possible to confirm the infection of subjects suspected of being infected with HBV, and it is also used for HBV vaccine testing and infection control control in infection experiments for anti-HBV drug evaluation. be able to.

Further, it can be used as a kit for detecting and measuring the presence / abundance of HBV in a subject body containing the antibody or antibody derivative of the present invention. Such a kit may include a labeled secondary antibody for detecting the antibody or antibody derivative.

Hereinafter, the present invention will be specifically shown with reference to examples. The examples shown below do not limit the invention in any way.

Example 1: Antigen-specific single cell sorting method; AgS-SCS method)
The purpose of this example was to isolate the gene for anti-HBs antibody from a human individual vaccinated with HBV vaccine.

Of the B cells circulating in the peripheral blood, about 60% are naive B cells that have not been stimulated with antigen, and 40% are memory B cells that express any of membrane-bound IgG, IgA, IgM, or IgE. It is known to be a cell. It is considered that the antibody sequence in Memory B cells already contains somatic hypermutation, and the binding affinity to the antigen is enhanced. Therefore, as a method for isolating the target antibody gene, only cells expressing a high-affinity antibody that specifically binds to the HBs antigen are selectively sorted from the memory B cell population that has been class-switched to IgG. We decided to isolate the anti-HBs antibody gene by adopting the antigen-specific single cell sorting method (AgS-SCS method) that isolates the antibody gene using cell cloning technology.

HBV vaccine booster vaccination was given to human individuals who had been vaccinated with HBV vaccine in the past. The boosted HBV vaccine includes beamgen (KM biologics, Japan), which is a recombinant HBs antigen vaccine derived from yeast with HBV genotype C and serotype adr sequence, or yeast-derived group with HBV genotype A and serotype adr sequence. A replacement HBsAg vaccine, Heptavax II (Merck, German), was used. The details of the vaccine at the time of initial inoculation of human individuals were unknown.

Approximately 30 mL of whole blood was collected from a human individual with a plasma anti-HBs antibody titer of 4,000 IU / L or higher. The antibody titer measurement in the screening was performed by the enzyme-linked immunosorbent assay (ELISA).

On the 6th and 28th days after the additional inoculation of the HBV vaccine to the human individuals who underwent the experiment, 30 mL of whole blood was collected and magnetic beads (MACSxpress Whole Blood B Cell Isolation Kit (Miltenyi Biotec, Germany)). )) Was used for one-step purification of B cells by the negative selection method.

The purified B cells 5.0 × 10 ⁷ cells were stained under the following conditions, and single-cell sorted was performed on a 96-well microplate in which 8 μL of buffer for reverse transcription reaction was dispensed.

After blocking non-specific reactions using mouse serum diluted with purified B cells, yeast-derived recombinant HBs antigen (HBsAg) that retains a virus-like particle (hereinafter referred to as VLP) structure with HBs antigen. -XT, Beacle Inc, Japan) was reacted with blocking-treated B cells on ice for 30 minutes at a concentration of 200 ng / mL.

100 μL antibody cocktail (anti-HBs polyclonal rabbit antibody (Beacle Inc), anti-rabbit IgG-Alexa488 (Jackson ImmunoReserach Laboratories, USA), anti-human IgG-APC (BD Bioscience), Anti-CD27-PE (BD Bioscience), Anti-CD19-ACP-Cy7 (BD Bioscience), Anti-CD38-PE-Cy7 (BioLegend, USA), 7-AAD7-AAD (BD Bioscience) It was mixed at the specified dose and stained with 100 μL of FACS buffer) on ice for 30 minutes.

Using the cell sorting device BD FACSAria (BD Bioscience, USA), using the gates of CD19 +, CD27 +, CD38-, IgG +, 7-ADD-, HBsAg +, in a 96-well plate containing 8 μL of RT buffer per well. One cell was dispensed into each well.

Specifically, cell sorting is first of all
・ Lymphocytes are selected from all blood cells based on the cell size by forward scattered light (FSC), cell morphology by lateral scattered light (SSC), and cell internal structure such as nuclei and granules.
-A live cell population (7AAD-) contained in the cells was obtained.
・ After that, IgG + and CD19 + cells were selected and selected.
-Furthermore, IgG memory B cells were obtained by selecting CD27 + and CD38- cells. From these cells, HBs antigen bound to the anti-HBs antibody expressed on the memory B cell membrane was detected by HBs antigen ^high gate using the anti-HBs polyclonal antibody for detection, and as a result, about 300 cells were obtained. rice field.

Sorted single cells are placed in individual wells of a 96-well plate containing 8 μl of reverse transcriptase buffer per well and reverse transcriptase containing reverse transcriptase primers (ATATGGATCC GGCGCGCCGT CGACTTTTTT TTTTTTTTTT TTTTTTTT; SEQ ID No: 32). 2 μL of the liquid was dispensed into each well, and a reverse transcription reaction was carried out.

Using 1 μL of the reverse transcribed cDNA solution, 1st PCR was performed on the variable region of the heavy chain and the variable region and the constant region of the light chain. The primer sequences and their amounts are shown in Table 1 below.

 

Furthermore, using 2.5 μL of each PCR product after the 1st PCR, 2nd PCR was also performed on the variable region of the heavy chain and the variable region and the constant region of the light chain. The sequences and their usage are as shown in Table 2 below.

 

After purifying the PCR product of the 2nd PCR (QIAquick PCR Purification Kit, QIAGEN, Germany), for the heavy chain, IgG ₁ expression cloning vector pIgG ₁ H7 (pcDNA3.1 (+) Hyg (Invitrogen) 962 to 991) A plasmid vector in which the IgG heavy chain secretion signal peptide sequence gene, the restriction enzyme recognition sequence for variable region cloning (NotI-XhoI), and the IgG ₁ constant region gene were artificially synthesized and inserted as the skeleton lacking 29 bp. ) Was cloned into the restriction enzyme recognition sequence (NotI-XhoI) site for cloning to obtain the desired antibody heavy chain expression vector.

As for the light chain, the light chain secretion signal is obtained by deleting the PCR product from the light chain expression cloning vector pLight (pcDNA3.1 (+) Hyg (Invitrogen) from 962 to 991 at 29 bp). A plasmid vector in which a sequence gene and an artificially synthesized restriction enzyme recognition sequence for light chain cloning (NotI-XhoI) were inserted), and an antibody light chain expressed by incorporating it into the restriction enzyme recognition sequence for light chain cloning (NotI-XhoI) site. Obtained a vector. Subsequently, among the prepared heavy chain expression vector and light chain expression vector, the one in which the entire base sequence was determined and the open reading frame was confirmed to be normal was selected as a heavy chain + light chain pair of human kidney-derived cell line 293T cells. Transient gene transfer (Lipofectamin LTX, Thermo Fisher scientific) into (ATCC CRL-3216), and the amount of target antibody in the culture supernatant after 5 days is determined by a commercially available ELISA kit (Enzygnost anti-HBs II, SIEMENS, Germany) or in. Measured by house ELISA. As a result, a strongly positive antibody (M2-11) was obtained. This was used as a candidate for recombinant HBIG, and the heavy chain expression vector of this M2-11 antibody was used as a pIgG ₁ H7-M2-11 and M2-11 antibody. The light chain expression vector was identified as pL-M2-11.

Example 2: Antibody gene isolation (EBV-hybridoma method)
The purpose of this example was to isolate the anti-HBs antibody gene from a human individual vaccinated with the HBV vaccine by a method different from that of Example 1.

Peripheral blood mononuclear cells (PBMC) are purified by density gradient centrifugation from whole blood collected from human individuals previously vaccinated with HBV vaccine, and Epstein-Barr virus (EBV) persists in the culture medium. EBV was infected and B cells were immortalized by adding a culture supernatant of B95-8 cells, which are infected human B cell lines.

After mixing 1.0 × 10 ⁶ EBV-infected immortalized B cells and JMS-3 myeloma cells, collect them and add 1 mL of 50% PEG (PEG 1540, FUJIFILM Wako Pure Chemical Corporation, Japan) over 1 minute. The cells were added dropwise, 1 mL of RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) was added over 1 minute, and 10 mL of RPMI 1640 medium was added over 1 minute, mixed, and then centrifuged for recovery.

Cells were harvested, suspended in RPMI 1640 medium supplemented with 20% FCS at a cell concentration of 2.5 × 10 ⁵ cells / mL, dispensed 0.1 mL each into 96-well plates, and the next day, 0.1 mL HAT selective medium (0.5). The culture was continued with the addition of μM vavine), and it was confirmed that the antibody was produced in the culture supernatant after 7 to 10 days.

Antibody-producing cells were collected from wells where antibody production was confirmed, and the antibody-producing cells were cloned by the limiting dilution method. For the antibody in the culture supernatant of each hybridoma, a passive hemagglutination reaction method (Passive) was performed. High-throughput screening was performed using an automatic measuring device (PK7300, Beckman Coulter, USA) by Hemmaglutination: PHA) (Mycel II antibody-HBs, Special Immunology Laboratory). Since the PHA method is characterized by the fact that the strength of antibody avidity (binding ability to specific antigens) is reflected in the antibody titer, an antibody with virus neutralizing activity can be obtained by adopting screening by this method. It is more likely to be obtained. As a result, 12 strains of HBsAg aggregation-positive hybridomas were established from PBMCs of 3 individuals.

RNA was purified from PHA-positive hybridomas (NucleoSpin RNA (TaKaRa)) to obtain antibody heavy chain variable regions, light chain variable regions and constant regions, and carried out using the same primers as those in Example 1 above. It was cloned into a vector by the same method as in Example 1 and expressed as a recombinant antibody by transient gene transfer (Lipofectamin LTX, Thermo Fisher scientific) into human kidney-derived cell line 293T cells (ATCC CRL-3216).

For the antibody confirmed to be IgG ₁ by the analysis of the base sequence, ELISA was performed in the same manner as in Example 1. As a result, three types of antibodies (5A4, 3D1 and _3B6 ) became strongly positive in ELISA, and these were designated as recombinant HBIG candidates. Chain expression vector is pL-5A4, 3D1 antibody heavy chain expression vector is pIgG ₁ H7-3D1, 3D1 antibody light chain expression vector is pL-3D1, 3B6 antibody heavy chain expression vector is pIgG ₁ H7-3B6, 3B6 antibody The light chain expression vector was identified as pL-3B6.

Example 3: Recombinant antibody expression and purification The purpose of this example was to express and purify four types of recombinant HBIG candidate antibodies obtained in Examples 1 and 2.

Heavy chain expression vector and light chain expression vector (pIgG ₁ H7-M2-11 and pL-M2-11) of M2-11 antibody obtained in Example 1, and three kinds of antibodies obtained in Example 2 (pIgG 1 H7-M2-11 and pL-M2-11). Heavy chain expression vector and light chain expression vector for 5A4, 3D1 and 3B6) (pIgG ₁ H7-5A4 and pL-5A4 for 5A4 antibody, pIgG ₁ H7-3D1 and pL-3D1 for 3D1 antibody, pL-3D1 for 3B6 antibody pIgG ₁ H7-3B6 and pL-3B6) were transiently transfected into antibody-expressing cells. Specifically, 45 μg of heavy chain expression was performed using the gene transfer reagent GIBCO Expifectamine 293 (Thermo Fisher Scientific) for 1.5 × 10 ⁸ cells of the floating human kidney-derived cell line GIBCO Expi 293F cell (Thermo Fisher Scientific). A vector and a 15 μg light chain expression vector were introduced into the gene, and the cells were cultured in a serum-free medium (Gibco Expi293 Expression Medium, Thermo Fisher Scientific) for 7 days (125 rpm, 37 ° C., 8% CO ₂ ), and then the culture supernatant. Was recovered as an antibody stock solution.

The culture supernatant was centrifuged to remove cell debris, and filtered through hydrophilic membranes (Durapore, Merck, USA) with pore sizes of 0.45 μm and 0.22 μm to adjust the pH to 7.4. After affinity purification of the antibody from the culture supernatant using HiTrap Protein G HP Columns (GE Healthcare, USA) equilibrated with PBS, gel filtration using Sephadex G-25 (PD-10 column, GE Healthcare) is performed. Buffer exchange to PBS and concentration were performed by the extrafiltration method (Centricon Plus-70 Centrifugal Filter, Merck). As a result, purified antibodies were obtained for the four antibodies M2-11, 5A4, 3D1 and 3B6 that were strongly positive for ELISA in Example 1 and Example 2.

Example 4: Evaluation of Neutralizing Activity of Recombinant Antibody This Example was performed to confirm that the purified antibody obtained in Example 3 has neutralizing activity against HBV.

(4-1) First screening using cells infected with genotype D HBV When evaluating neutralization activity, first screening was performed using cells infected with genotype D HBV. rice field. Human hepatocyte line HepG2 cells (HepG2-hNTCP-C4 cells) expressing the sodium-taurocholate co-transporting polypeptide (NTCP), which is a cell receptor for HBV, were transferred to 12 well dishes 2.5 × 10 Sown at ⁵ cells / well.

The next day, the culture medium of the above cells was infected with 1,000 genomic equivalents (GEq) / cell (MOI = 1,000) containing HBV of genotype D and the four purified antibodies obtained in Example 3. Replace with medium (DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 400 μg / ml G418 (Nacalai Tesque), 10% fetal bovine serum (FBS), and 4% PEG 8000 (Nacalai Tesque)). The cells were infected by culturing for 1 day.

The next day, cells were added with 400 μg / ml G418 (Nacalai Tesque) and 10% fetal bovine serum (FBS) to a growth medium (DMEM (Nacalai Tesque, Kyoto, Japan)) containing any of the four purified antibodies. The amount of viral DNA (capsid-associated relaxed circular DNA [rcDNA]) covered with HBV capsid in the cells was collected on the 14th day of infection by exchanging with () and subculturing every 2 days. Was quantified by quantitative PCR (qPCR) to confirm whether the purified antibody had neutralizing activity.

The qPCR reaction uses nucleic acid recovered from infected cells as a template, Fast SYBER Green Master Mix (Applied Biosystems, USA), primer set (forward primer 5'-gagtgtggattcgcactcc-3'(SEQ ID No: 79), and reverse primer 5'. -This was done using gaggcgagggagttcttct-3'(SEQ ID No: 80).

As a result, the three types of purified antibodies, M2-11, 5A4, and 3B6, showed good neutralization activity by reducing the copy number of HBV in a concentration-dependent manner, but showed good neutralization activity in 3D1. The evaluation of the 3D1 antibody was terminated here because no decrease in DNA copy number was observed and no neutralizing activity was observed (Fig. 1).

(4-2) Second screening with primary human hepatocytes infected with genotype C HBV Next, a second screening using primary human hepatocytes infected with genotype C HBV. Was done. In an infection experiment using primary human hepatitis, a 2 × 10 ⁶ genome equivalent (GEq) was obtained against chimeric mouse-derived fresh human hepatitis PXB cells (PhoenixBio) cultured at 4 × 10 ⁵ cell / well in a 24-well dish. ) (MOI = 5) genotype C HBV, one of the recombinant anti-HBs antibodies (3 types of purified antibodies M2-11, 5A4, 3B6) or commercially available HBIG (Hebsbrin intramuscular injection 1000 units, Japan) It was preincubated and infected with a growth medium (dHCGM medium, PhoenixBio, Japan) containing blood preparation mechanism).

16 hours after infection, cell culture was started in growth medium, and the cells were cultured for 11 days in the presence of each antibody while exchanging the medium every 5 days. The culture supernatant on the 11th day was collected, and the amount of HBe antigen derived from HBV was measured by ELISA (CSB-E13557h, Cusabio, USA).

As a result, it was shown that all three types of recombinant anti-HBs antibodies (purified antibodies of M2-11, 5A4, 3B6) reduced the amount of HBe antigen in a concentration-dependent manner, when the antibody concentration was 20 μg / mL. It was found that all three types of antibodies reduced the amount of HBe antigen below the detection limit. In addition, all three purified antibodies, M2-11, 5A4, and 3B6, are more effective in reducing HBe antigen at 0.02 μg / mL than commercially available HBIG at 0.2 μg / mL (ie, at least 10-fold or more). It means neutralization activity) (Fig. 2). Thus, the three anti-HBs human monoclonal antibodies showed strong neutralizing activity, indicating that either alone or a mixture of these antibodies is promising as recombinant HBIG.

Example 5: Evaluation of binding characteristics of recombinant antibody In this example, 3 types of human anti-HBs recombinant monoclonal antibodies (M2-11, 5A4, 3B6 3) whose neutralizing activity was confirmed in the screening of Example 4 This was done to confirm the binding properties of the purified antibody) to HBV.

Recombinant HBs antigen (HBsAg-XT) was heated at 100 ° C for 10 minutes and then slowly cooled to 20 ° C at a rate of 1 ° C / min to induce virus-like particles (VLP). For SDS-PAGE, adjust the heat-treated antigen to 0.1 mg / mL, add a reducing agent (50 mM DTT, GE Healthcare) to half of the samples, heat at 100 ° C for 5 minutes, and 10% Bolt. Electrophoresis was performed on a Bis-Tris Plus gel (Thermo Fisher Scientific).

On the other hand, for Native PAGE, the Blue Native-PAGE method was used. That is, the above HBs antigen was adjusted to 0.1 mg / mL with Native sample buffer (Thermo Fisher Scientific), a reducing agent (50 mM DTT) was added to half of the samples, and NativePAGE 4-16% Bis-Tris. Electrophoresis was performed on Protein Gels (Thermo Fisher Scientific). The protein after electrophoresis is transferred to a PVDF membrane (iBlot 2 Transfer Stacks, PVDF, mini, Thermo Fisher Scientific), blocked at room temperature for 1 hour, and then 2 μg / mL of the antibody to be evaluated (3 types of human anti-HBs recombination). A primary reaction was performed overnight at 4 ° C. with any of the monoclonal antibodies (three purified antibodies of M2-11, 5A4, 3B6) or the comparative control HBIG or horse polyclonal antibody).

Next, secondary antibodies (peroxidase-labeled anti-human IgG (709-035-149, Jackson Reserach Laboratories) or HRP-labeled anti-horse IgG polyclonal antibody (ab6921 abcam, UK)) and chemical luminescent reagents (ECL Select, Thermo Fisher Scientific) ) Was used to detect the binding property of the evaluation antibody to the HBs antigen on the gel.

As a result, under the reduction conditions of SDS-PAGE, the HBs antigen is detected as a monomer (about 25 kDa) and a dimer (about 50 kDa) by the anti-HBs horse polyclonal antibody used as a control, but it is not reduced. In the state, a signal was observed near the sample well, indicating that VLP, which is a polymer multimer, was detected under non-reducing conditions (Fig. 3, top).

In addition, the three monoclonal antibodies of the present invention did not bind to the monomeric and dimeric HBs antigens at all under the reducing conditions SDS-PAGE, and hardly bound to VLP even in the non-reducing state. However, 3B6 bound weakly to the non-reduced VLP (Fig. 3, top).

On the other hand, in Native-PAGE, where the protein is expected to maintain its original structure from SDS-PAGE, all three monoclonal antibodies bound to VLP, but in M2-11 and 5A4, in the non-reduced state, It was shown that it binds more strongly to VLP than in the reduced state, and 3B6 is almost unaffected by the reducing agent (Fig. 3, middle and bottom). Moreover, when the binding strength to VLP in the non-reduced state was compared, it was 3B6> M2-11> 5A4.

From the above results, the epitope recognized by the three monoclonal antibodies is a conformational epitope consisting of discontinuous amino acids, is not defined as a continuous peptide sequence, and at least M2-11 /. It was shown that 5A4 and 3B6 recognize different epitopes.

HBsAg has 8 cysteine residues in the extracellular space, and intramolecular and intermolecular disulfide bonds by these residues greatly contribute to the VLP multimer structure. Therefore, the VLP binding of M2-11 and 5A4 is reducing agent sensitive, which strongly supports that these antibodies recognize the multimeric structure itself consisting of HBsAg.

Example 6: Evaluation of binding properties of candidate antibodies to antigen aggregates (ELISA)
Since the HBsAg may exist as an aggregate in the living body and the higher-order structure of the antigen changes in such a case, in this example, the antibody of the present invention also binds to the HBsAg aggregate. It was decided to confirm whether or not to do so by the ELISA method.

Recombinant HBsAg (HBsAg-XT) was heated at 100 ° C for 10 minutes and then rapidly cooled to 4 ° C to induce HBsAg aggregates. The antigen aggregate 5 μg / mL thus prepared was immobilized on a 96-well plate (Corning, 9018) and allowed to stand overnight at 4 ° C. It was then washed with PBS-10xT (PBS + 0.05% Tween20) solution and then blocked with skim milk solution (adjusted with PBS-10xT to a final concentration of 5%). As a control, the recombinant HBs antigen prepared by the method of Example 5 was used.

Add an antibody solution (200 nM to 0.0033 nM, 3-fold dilution series, adjusted with PBS-T) of human anti-HBs recombinant monoclonal antibody (M2-11, 5A4) whose neutralizing activity was confirmed in the screening of Example 4. Then, after allowing to stand at room temperature for 1 hour, an HRP-labeled secondary antibody (anti-human IgG mouse antibody, diluted 10000 times) was added, a coloring agent TMB (SCY, TM4500) was added, and a plate reader (PHERAstar) was added. The color intensity was measured with PlusHTS Microplate Reader).

The results are shown in Fig. 4. In FIG. 4, control recombinant HBsAg and heat-recombinant HBsAg detected by M2-11 antibody are referred to as “M2-11” and “M2-11 heat”, and control recombinant HBs by 5A4 antibody. Antigens and heat-recombinant HBs antigens were detected as "5A4" and "5A4 heat". Both the M2-11 antibody and the 5A4 antibody showed the same degree of binding with or without heating. The binding activity concentration of each antibody at this time was as shown in Table 3 below. From this, it was shown that the antibody of the present invention binds to the aggregate of recombinant HBs antigens as well as to the recombinant HBs antigens that are not associated.

Example 7: Intermolecular interaction of the candidate antibody with the recombinant HBs antigen In this example, the interaction of the candidate antibody with the recombinant HBs antigen was analyzed using surface plasmon resonance (SPR) technology.

As the recombinant HBs antigen, the heated recombinant HBs antigen (aggregate) prepared in Example 6 and the recombinant HBs antigen prepared in Example 5 were used, and the antibody was neutralized by the screening of Example 4. Human anti-HBs recombinant monoclonal antibody (M2-11, 5A4) with confirmed activity was used.

Fill the surface plasmon resonance (SPR) device (Cytiva, Biacore T200) with PBS-T (PBS + 0.005% Tween20) solution, and immobilize each antibody on the sensor chip (Cytiva, Series S Sensor Chip Protein A), about 9000. I made it capture with RU. Antigen solutions of each concentration (271 nM, 813 nM, 2439 nM (2.4 μM), 7317 nM (7.3 μM), 21951 nM (22 μM)) were added to this chip stepwise from low concentration (0 seconds, respectively). Addition at 200 seconds, 400 seconds, 600 seconds, and 800 seconds, added at the time indicated by the arrow in FIG. 5), and the interaction between the antigen and the antibody was investigated.

The results are shown in Fig. 5. In all the antibodies, the binding reaction was increased after the addition of the antigen, and it was considered that the binding activities of both antibodies were almost the same. On the other hand, it was clarified that the binding strength differs between the M2-11 antibody and the 5A4 antibody, which is due to the molecular state in which the recombinant HBs antigen used as the antigen has activity on the M2-11 antibody and the activity on the 5A4 antibody. It is an antigen sample in which at least two different molecular states are mixed, and it is speculated that the epitope sites of both antibodies are different. In addition, it has been shown that the heat treatment of the antigen reduces the binding strength with the antibody, which indicates that the heating reduces the antigenicity.

Example 8: Evaluation of cross-reactivity of candidate antibody In this example, three human anti-HBs recombinant monoclonal antibodies (M2-11, 5A4, 3B6) whose neutralizing activity was confirmed in the screening of Example 4 were used. This was done to confirm that there was no non-specific binding to human-derived components (no cross-reactivity).

The most feared side reaction to the use of antibody drugs is
-If the administered antibody has a sequence derived from an animal species other than human, it must be antigenic to humans.
-The antibody used is non-specific binding to normal human tissue. Since the three monoclonal antibodies isolated this time are fully human antibodies derived from human B cells obtained from the peripheral blood of vaccinated healthy human individuals, the possibility of the former side reaction is considered to be extremely low. Also, regarding the latter, since the onset of inflammation, autoimmune diseases, etc. has not been reported at all in human individuals who provided B cells derived from these three types of monoclonal antibodies, these antibodies are self-reactive. Is unlikely to indicate.

To be on the safe side, three anti-HBs monoclonal antibodies were used in ELISA and tissue immunostaining to verify that these monoclonal antibodies did not bind non-specifically to normal human tissue (no cross-reactivity). The binding of the antibody to human-derived components was analyzed.

(8-1) ELISA
ELISA was performed using the Antigen-Down ELISA Development Kit (Immunochemistry technologies, Australia). In addition to the HBs antigen (HBAg-XT), the components targeted by ELISA are human insulin (Fujifilm Wako Pure Drug), which is used to evaluate non-specific binding of antibody drugs to human-derived components, and patients with autoimmune diseases. Genome DNA (used as human genomic DNA extracted from human cultured cell line HeLa cells (ATCC CCL-2)), which is a typical autoantibody antigen in the above, was immobilized on a 96-well plate for ELISA.

Add 100 μL / well of each of the three diluted recombinant anti-HBs antibodies to the plate, react with anti-human IgG-HRP (709-035-149, Jackson), and add a peroxidase substrate to develop color. rice field. The color reaction results were measured using an absorbance measurement microplate reader (MTP-300, CORONA Electric, Japan) as the absorbance at a measurement wavelength of 450 nm and a target wavelength of 630 nm.

The three human HBs monoclonal antibodies show non-specific binding to the human-derived component immobilized on the ELISA plate even at an antibody concentration of 10 times or more the concentration at which the binding to the specific antigen HBs is saturated. There was no (Fig. 6).

(8-2) Tissue immunostaining HBV-positive human liver tissue paraffin section array (LV1401, US Biomax Inc, USA) and normal human tissue paraffin section array (T8234708-5, BioChain, USA) after deparaffinization, 3% H ₂ The endogenous peroxidase was inactivated by reacting with O ₂ , and a blocking reaction was carried out overnight at 4 ° C. with 5% BSA.

Then, it was reacted with a recombinant anti-HBs antibody mixed solution (mixed solution of M2-11, 5A4, 3B6) diluted to 10 μg / mL, and then reacted with a secondary antibody (709-035-149, Jackson ImmunoReserach Laboratories). rice field. A peroxidase substrate (ImmPACTDAB, VECTORlaboratorie, USA) was added to develop color, and counterstaining was performed with Mayer's Hematoxylin.

As a result, the three monoclonal antibody mixed solutions clearly detected HBsAg in the liver tissue of HBV-positive individuals at an antibody concentration of 10 μg / mL (Fig. 7a), but at the same concentration, various normal human tissues were found. No staining was observed (Fig. 7b). From these results, it was clarified that the three monoclonal antibodies that are candidates for recombinant HBIG have extremely low cross-reactivity with human-derived components.

Example 9: Characteristics of candidate antibody CDR sequences In this example, the characteristics of the amino acid sequences of the heavy chain and light chain of each HBs monoclonal antibody obtained in the present invention were analyzed.

When the amino acid sequences of the heavy and light chains of the HBs monoclonal antibodies M2-11, 5A4, and 3B6 were determined, the amino acid sequences of the variable regions (VH region and VL region) of each chain were as follows. rice field.

The amino acid sequences of the heavy chain variable regions and light chain variable regions of each of these HBs monoclonal antibodies M2-11, 5A4, and 3B6 were analyzed in more detail, and the amino acids in the complementarity determining regions (CDRs) of each chain were analyzed. The sequences are underlined in the following amino acid sequences (CDR1, CDR2, CDR3 in each amino acid sequence, respectively). IMTG (the international ImMunoGeneTics information system) antibody sequence analysis software IMTG / V-QUEST was used to extract the CDR sequences.

<M2-11 clone>
Amino acid sequence of heavy chain variable region (SEQ ID No: 19, CDR1, CDR2, CDR3 underlined)
AAAVLSQVQL VESGGGLVKP GESLRLSCAA S GFMFSGHS M VWVRQAPGKG LEWVSF IGST GEFI SYADSV RGRFTISRDN AKNSLYLQMN SLRADDTAVY YC AREQGTRG RYYYYGLDV W GQGTTVTVSS ASRTK
Amino acid sequence of light chain variable region (SEQ ID No: 20, CDR1, CDR2, CDR3 are underlined)
AAAMTQTPPS LSLSPGESAT LSCRA SQSVS TY LAWYQQKP GQAPRLLIY D AF NRATGIPA RFSGSGSGTD FTLTISGLEP EDFAVYYC QQ RGHWPLT FGG GTKVEIK
<5A4 clone>
Amino acid sequence of heavy chain variable region (SEQ ID No: 21, CDR1, CDR2, CDR3 are underlined)
AAAVLSQVQL QESGPGLVKP SDTLSLTCTV SGGSISGHY W SWIRRLPGGG LEWIGY IHYS GIT NYNPSLK SRVTMSVDMS KNQFSLRLRS VTATDTAVYY C ARGDATYGY WGQGAPVTVS SASRTK
Amino acid sequence of light chain variable region (SEQ ID No: 22, CDR1, CDR2, CDR3 are underlined)
AAAMTQSPFT LPVTPGEPAS ISCRS SQSLL HRNGYNY VNW YLRKPGQSPQ LLIS LGS DRA SGVPDRFRGS GAGTDFTLKI SRVEAEDVGV YYC MQALRTP WT FGQGTKVE IK
<3B6 clone>
Amino acid sequence of heavy chain variable region (SEQ ID No: 23, CDR1, CDR2, CDR3 are underlined)
AAAVQCEVQL VESGGGVVQP GKSLRVSCAA SGFSFSNYG M HWVRQAPGKG LEWVSV IWRD GSHQ YYADSV KGRFTVSRDN ARDTLFLQMD GLRAEDTAVY YC AREDPAIV LPVLDH WGRG TLVTVSSASR TK
Amino acid sequence of light chain variable region (SEQ ID No: 24, CDR1, CDR2, CDR3 are underlined)
AAAMTQSPVS LSASVGDRVT ITCRAS QRIN SY LNWYQQKP GKAPKLLIY G AS NLPSGVPS RFSGSGSGTY FTLTISSLQP EDLATYYC QQ GYSTPLLS FG PGTKVEIK

The SEQ ID NOs were assigned to each CDR amino acid sequence as shown in Table 4 below.

INDUSTRIAL APPLICABILITY According to the present invention, an antibody expected to have safe, effective and stable activity that can fundamentally solve the problems of unachieved domestic self-sufficiency and the problems of blood products related to anti-HBs human immunoglobulin preparations derived from blood raw materials. Formulations can be provided.

Claims (12)

1. (1) Heavy chain complementarity determining regions, CDR1 (GFMFSGHS, SEQ ID No: 1), CDR2 (IGSTGEFI, SEQ ID No: 2), and CDR3 (AREQGTRGRYYYYGLDV, SEQ ID No: 3), and light chain complementarity. Sex determining regions, CDR1 (SQSVSTY, SEQ ID No: 4), CDR2 (DAF, SEQ ID No: 5), and CDR3 (QQRGHWPLT, SEQ ID No: 6);
   (2) Heavy chain complementarity determining regions, CDR1 (SGGSISGHY, SEQ ID No: 7), CDR2 (IHYSGIT, SEQ ID No: 8), and CDR3 (ARGDATYGY, SEQ ID No: 9), and light chain complementarity. Sex determining regions, CDR1 (SQSLLHRNGYNY, SEQ ID No: 10), CDR2 (LGS, SEQ ID No: 11), and CDR3 (MQALRTPWT, SEQ ID No: 12);
   (3) Heavy chain complementarity determining regions, CDR1 (SGFSFSNYG, SEQ ID No: 13), CDR2 (IWRDGSHQ, SEQ ID No: 14), and CDR3 (AREDPAIVLPVLDH, SEQ ID No: 15), and light chain complementarity. Sex determining regions, CDR1 (QRINSY, SEQ ID No: 16), CDR2 (GAS, SEQ ID No: 17), and CDR3 (QQGYSTPLLS, SEQ ID No: 18);
   It has binding to the HBsAg of hepatitis B virus (HBV) and has neutralizing activity against HBV, including the complementarity determining regions of any of the heavy and light chains selected from the group consisting of. Antigens or antibody derivatives that are not derived from blood sources.
2. The antibody or antibody derivative according to claim 1, which is a recombinant type.
3. The invention according to claim 1 or 2, wherein the HBsAg is one or a plurality selected from the group consisting of L antigen, M antigen, S antigen, HBsAg adr type, adw type, ayr type, and ayw type of HBsAg. Antigen or antibody derivative of.
4. The invention according to any one of claims 1 to 3, wherein the antibody derivative is selected from an antibody variant selected from a humanized antibody, a chimeric antibody, a polyvalent antibody, and a multispecific antibody or a functional fragment thereof. Antibodies or antibody derivatives.
5. The amino acid sequence of the heavy chain variable region VH domain of an antibody or antibody derivative,
   (1) Of the amino acid sequence of SEQ ID No: 19 or the amino acid sequence of SEQ ID No: 19, CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
   (2) Of the amino acid sequence of SEQ ID No: 21 or the amino acid sequence of SEQ ID No: 21, CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9) ), An amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions, and (3) the amino acid sequence of SEQ ID No: 23, or SEQ ID No: 23. Of the amino acid sequence of, one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15). Amino acid sequence, including substitutions), insertions, or deletions,
   The antibody or antibody derivative according to any one of claims 1 to 4, which is selected from the above.
6. The amino acid sequence of the light chain variable region VL domain of an antibody or antibody derivative,
   (1) Of the amino acid sequence of SEQ ID No: 20 or the amino acid sequence of SEQ ID No: 20, CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
   (2) Of the amino acid sequence of SEQ ID No: 22 or the amino acid sequence of SEQ ID No: 22, CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12). ), An amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions, and (3) the amino acid sequence of SEQ ID No: 24, or SEQ ID No: 24. Of the amino acid sequence of, one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18). Amino acid sequence, including substitutions), insertions, or deletions,
   The antibody or antibody derivative according to any one of claims 1 to 5, which is selected from the above.
7. Antibodies or antibody derivatives
   (1) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 25) and a light chain (SEQ ID No: 26);
   (2) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 27) and a light chain (SEQ ID No: 28);
   (3) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 29) and a light chain (SEQ ID No: 30);
   The antibody or antibody derivative according to any one of claims 1 to 6, which is selected from the group consisting of.
8. A pharmaceutical composition for neutralizing HBV, which comprises the antibody or antibody derivative according to any one of claims 1 to 7 and does not contain blood-derived components.
9. The pharmaceutical composition according to claim 8, which comprises a plurality of types of the antibody or antibody derivative according to any one of claims 1 to 7.
10. The pharmaceutical composition according to claim 8 or 9, for preventing reactivation of hepatitis B or preventing medical infection of HBV.
11. A step of in vitro contacting a biological sample collected from a subject with the antibody or antibody derivative according to any one of claims 1 to 7.
    A step of detecting and measuring HBV in a sample bound to the antibody or antibody derivative.
    A method for detecting and measuring the presence / abundance of HBV in a biological sample including.
12. A kit for detecting and measuring the presence / absence of HBV in a subject, which comprises the antibody or antibody derivative according to any one of claims 1 to 7.
    
    
    
    
    

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