WO2023232003A1

WO2023232003A1 - Anti-hbsag antibody and use thereof

Info

Publication number: WO2023232003A1
Application number: PCT/CN2023/096996
Authority: WO
Inventors: 王铁军
Original assignee: 安徽荣航生物科技发展有限责任公司
Priority date: 2022-05-31
Filing date: 2023-05-30
Publication date: 2023-12-07

Abstract

The present invention relates to an anti-HBsAg antibody. In particular, the present invention relates to a tetravalent antibody bound to HBsAg and a corresponding heavy chain antibody. The present invention further relates to a pharmaceutical composition comprising the antibodies, and medical use thereof. The antibodies of the present invention have a strong binding force with the HBsAg, do not cause aggregation of HBsAg aggregates, can effectively reduce the HBsAg level in vivo, and can be used for treating and/or preventing hepatitis B related diseases.

Description

Anti-HBsAg antibodies and their applications

Technical field

The present invention relates to anti-HBsAg antibodies. More specifically, the present invention relates to tetravalent antibodies that bind to HBsAg and corresponding heavy chain antibodies. The invention also relates to pharmaceutical compositions comprising these antibodies and their medicinal uses.

Background technique

Hepatitis B is a disease caused by Hepatitis B Virus (HBV) infecting the body. Hepatitis B virus is a hepatotropic virus that mainly exists in liver cells and damages liver cells, causing liver cell inflammation, necrosis, and fibrosis. Hepatitis B virus is divided into two types: acute and chronic. Acute hepatitis B in adults can mostly resolve spontaneously through their own immune mechanisms. However, chronic hepatitis B (CHB) has become a great challenge to global health care and is also the main cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. WHO estimates that in 2019, 296 million people were living with chronic hepatitis B infection (defined as hepatitis B surface antigen positivity). Approximately one-third of patients with chronic hepatitis B develop cirrhosis, liver failure, and hepatocellular carcinoma, resulting in 600,000 deaths annually.

HBV consists of (i) an envelope containing three related surface proteins (collectively known as hepatitis B surface antigen, HBsAg) and lipids and (ii) an icosahedral nucleocapsid that surrounds the viral DNA genome and DNA polymerase. Three HBV envelope proteins, S-HBsAg, M-HBsAg and L-HBsAg, form complex transmembrane folds in the endoplasmic reticulum and form disulfide-linked homodimers and heterodimers. During budding at the intracellular membrane, a short linear domain in the cytoplasmic pre-S region interacts with binding sites on the capsid surface. Viral particles are then secreted into the blood. Alternatively, surface proteins can bud and aggregate to form subviral particles (SVPs) in the absence of capsids. SVP is non-infectious due to the lack of nucleocapsids. SVP is greatly involved in disease progression, especially the immune response to hepatitis B virus. In the blood of infected people, the amount of SVP is at least 10,000 times the amount of virus, trapping the immune system and weakening the body's immune response to hepatitis B virus. High levels of HBsAg can deplete HBsAg-specific T cell responses and are thought to be an important factor in viral immune tolerance in patients with chronic hepatitis B.

It is estimated that for patients with hepatitis B, if HBsAg is cleared before cirrhosis, the incidence of cirrhosis and hepatocellular carcinoma will be reduced by 60 times. The guidelines of the American Association for the Study of Liver Diseases (AASLD), the Asia-Pacific Association for the Study of the Liver (APASL), and the European Association for the Study of the Liver (EASL) all use HBsAg serum clearance as one of the treatment endpoint criteria. In addition, high levels of antigen induce immune tolerance, and the reduction of antigen HBsAg levels can restore immunological control of HBV infection.

The anti-HBV drugs currently approved for marketing are mainly immunomodulators (interferon-α and peginterferon-α-2α) and antiviral therapeutic drugs (lamivudine, adefovir dipivoxil, entecavir, tecavir, etc.). Bivudine, tenofovir, clavudine, etc.). Among them, antiviral therapeutic drugs are nucleoside drugs. Their mechanism of action is to inhibit the synthesis of HBV DNA and cannot directly reduce HBsAg levels. As with extended therapy, nucleoside agents showed HBsAg clearance rates similar to those observed in nature. Current clinical first-line drugs, including nucleoside and interferon drugs, do not have the effect of reducing the level of the antigen HBsAg, let alone clearing HBsAg.

Therefore, it is of great practical significance to develop antibody drugs that can effectively bind HBsAg and reduce or even eliminate HBsAg.

Contents of the invention

In a first aspect, the invention provides an IgG type tetravalent antibody that binds to hepatitis B surface antigen (HBsAg) or a subviral particle (SVP) containing HBsAg, wherein the tetravalent antibody comprises two heavy chains and two A light chain, each of the heavy chain and the light chain comprising as a variable region a VHH portion comprising CDR1, CDR2, CDR3, wherein:

(a) CDR1 includes the sequence shown in SEQ ID NO: 4, CDR2 includes the sequence shown in SEQ ID NO: 5, and CDR3 includes the sequence shown in SEQ ID NO: 6;

(b) CDR1 includes the sequence shown in SEQ ID NO: 11, CDR2 includes the sequence shown in SEQ ID NO: 12, and CDR3 includes the sequence shown in SEQ ID NO: 13;

(c) CDR1 includes the sequence shown in SEQ ID NO: 18, CDR2 includes the sequence shown in SEQ ID NO: 19, and CDR3 includes the sequence shown in SEQ ID NO: 20;

(d) CDR1 contains the sequence shown in SEQ ID NO: 25, CDR2 contains the sequence shown in SEQ ID NO: 26, and CDR3 contains the sequence shown in SEQ ID NO: 27; or

(e) CDR1 includes the sequence shown in SEQ ID NO: 32, CDR2 includes the sequence shown in SEQ ID NO: 33, and CDR3 includes the sequence shown in SEQ ID NO: 34.

In a second aspect, the invention provides a heavy chain antibody that binds to hepatitis B surface antigen (HBsAg) or a subviral particle (SVP) comprising HBsAg, wherein the heavy chain antibody comprises a VHH as a heavy chain variable region part, the VHH part includes CDR1, CDR2, CDR3, where:

In a third aspect, the invention provides a nucleotide sequence comprising a polynucleotide encoding any of the above antibodies. On the other hand, the present invention also provides a vector comprising the aforementioned nucleotide sequence. On the other hand, the present invention also provides a host cell comprising the aforementioned vector. In addition, the present invention also provides cell lines that produce the antibodies of the present invention, recombinant expression vectors containing the nucleotides of the present invention, and methods for preparing antibodies by culturing antibody-producing cell lines.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned antibodies, nucleotide sequences, vectors or host cells, and a pharmaceutically acceptable carrier.

In a fifth aspect, the present invention provides the use of any of the above-mentioned antibodies, nucleotide sequences, vectors or host cells in the preparation of medicaments for treating and/or preventing hepatitis B or for preventing diseases caused by hepatitis B.

In a sixth aspect, the present invention provides any of the above antibodies, nucleotide sequences, vectors or host cells for treating and/or preventing hepatitis B or for preventing diseases caused by hepatitis B.

In a seventh aspect, the present invention provides a method for treating and/or preventing hepatitis B or preventing diseases caused by hepatitis B, which method includes administering an effective amount of any of the above antibodies, nucleotide sequences, and vectors to a subject. or host cells.

In any of the above aspects, the hepatitis B is chronic hepatitis B, and the disease caused by hepatitis B is cirrhosis, liver failure or hepatocellular carcinoma caused by hepatitis B.

The antibody of the present invention has strong binding force with HBsAg, does not cause the aggregation of HBsAg aggregates, and can effectively reduce HBsAg levels in the body, and can be used to treat and/or prevent hepatitis B-related diseases.

Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.

Description of the drawings

Figure 1: Schematic structural comparison of the antibodies constructed in the present invention and control conventional antibodies.

Figure 2: Conventional ELISA test to test the binding activity of antibodies to HBsAg.

Figure 3: Direct HBsAg ELISA assay tests the effect of antibodies on HBsAg aggregation.

Figure 4: Schematic diagram of the experimental process of the HDI-HBV model.

Detailed ways

definition

As used herein, "about" means that the value is within an acceptable error range for the specific value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, "about" in every practice in the art may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times a value. Unless stated otherwise, when a specific value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

A composition or method described herein as "comprising" one or more of the recited elements or steps is open-ended, meaning that the recited element or step is required but may be within the scope of the recited composition or method. Add other components or steps. It will also be understood that any composition or method described as "comprising" one or more of the recited elements or steps also describes a corresponding, more limited composition or method that "consists essentially of" the recited elements or steps, meaning that The composition or method includes such essential elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

As used herein, the term "antibody" refers to any form of antibody that exhibits a desired biological activity, such as inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling. "Antibody fragment" and "antigen-binding fragment" refer to antigen-binding fragments of antibodies and antibody analogs, which generally include at least part of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies making up the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. The modifier "monoclonal" indicates the character of an antibody obtained from a substantially homogeneous population of antibodies and is not to be understood as requiring that the antibody be prepared by any particular method. For example, monoclonal antibodies for use in the present invention can be prepared by hybridoma or recombinant DNA methods.

Monoclonal antibodies may include "chimeric" antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the antibody forms part of a larger biomolecule, such as a fusion protein or an antibody drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the binding activity of the parent when activity is expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target.

As used herein, the term "multivalent antibody" refers to an antibody that contains more than one antigen recognition site (i.e., antigen-binding domain). For example, a "bivalent" antibody has two antigen recognition sites, while a "tetravalent" antibody has four antigen recognition sites. In the present invention, multivalent antibodies particularly refer to tetravalent antibodies, which are usually engineered to have four antigen-binding sites and are generally not naturally occurring antibodies. For example, for HBsAg, a quadrivalent antibody has four binding parts for HBsAg, each of which is capable of binding HBsAg alone.

As used herein, the term "heavy chain antibody" refers to an antibody lacking a light chain and consisting only of a heavy chain, which contains two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (i.e. VHH). Examples include, but are not limited to, native heavy chain antibodies, antibodies naturally lacking light chains, heavy chain antibodies derived from conventional 4-chain antibodies, and engineered antibodies. Heavy chain antibodies may be derived from species of the family Camelidae, such as those produced in camels, llamas, dromedaries, alpacas, and draft horses. Species other than Camelidae may produce heavy chain antibodies that naturally lack light chains; such heavy chain antibodies are within the scope of the invention.

As used in this article, the term "single domain antibody" refers to a single domain antibody composed only of the heavy chain variable region obtained by cloning the variable region of a heavy chain antibody, also known as VHH (Variable domain of heavy chain of heavy chain antibody) or nanobody (nanobody), is the smallest functional antigen-binding fragment. Single-domain antibodies recognize antigens with high specificity and affinity similar to IgG antibodies, but can better penetrate tumor tissue due to their smaller size (~15 kD). In addition, single domain antibodies are resistant to pH extremes, thermal denaturation, proteolysis, solvents, and detergents. They can be expressed and produced in high yields and high solubility.

As used herein, the term "humanized antibody" refers to an antibody that contains the CDRs of an antibody derived from a mammal other than human, and the framework regions (FR) and constant regions of a human antibody.

As used herein, the term "chimeric antibody" will be considered to refer to any antibody in which the immunoreactive region or site is obtained or derived from a first species and a constant region (which may be complete, partial or in accordance with this openly modified) was obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (eg, mouse or primate) and the constant region is human.

An "equivalent" of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity with the amino acid sequence of the antibody or polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, equivalents have one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to a reference antibody or polypeptide. In some aspects, equivalents of the antibody or polypeptide retain the activity (eg, epitope binding) or structure (eg, salt bridges) of the reference sequence.

As used herein, a "variant" of a sequence refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

As used herein, "% identity" between two sequences refers to a function of the number of equivalent positions shared by the sequences (i.e., % identity = number of equivalent positions/total number of positions x 100), taking into account gaps The number and length of each gap, which needs to be introduced when optimally aligning the two sequences. Sequence comparison and determination of % identity between two sequences can be accomplished using mathematical algorithms.

"Conservative substitutions" refer to amino acid substitutions known to those skilled in the art, which substitutions are made generally without altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change or do not substantially alter the biological activity. "Does not change or does not substantially change" means that one or more aspects have no more than about 20%, about 15%, about 10%, about 9 %, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% difference.

Generally, CDR3 and CDR3 are considered to play a more important role in antigen recognition than other CDRs. Therefore, in the case of substitution, the present invention preferably performs conservative substitutions on CDRs other than CDR3. In some embodiments, CDR3 is not substituted. Preferred amino acid substitutions include, but are not limited to: (1) reducing proteolytic sensitivity, (2) reducing oxidative sensitivity, (3) altering the binding affinity of protein complexes formed, (4) providing or modifying other physical properties of these analogs. Substitution of chemical or functional properties. Analogues may include mutations of various sequences in addition to naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence, preferably in portions of the polypeptide outside regions where intermolecular contacts are formed. Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to disrupt helices present in the parent sequence, or be characteristic of other secondary structure types that disrupt the parent sequence).

It is expected that the binding domain of the antibody or antigen-binding fragment thereof of the present invention may carry a signal peptide, which is usually located at the N-terminus of the secreted protein and generally consists of 15 to 30 amino acids. When the signal peptide sequence is synthesized and recognized by the signal recognition particle (SRP), protein synthesis is paused or slowed down. The signal recognition particle carries the ribosomes to the endoplasmic reticulum, and protein synthesis restarts. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity, and the signal peptide sequence is removed under the action of signal peptidase. If the termination transport sequence exists at the C-terminus of the nascent peptide chain, it may not be cleaved by signal peptidase. For example, ovalbumin contains an internal signal peptide. Neither its precursor nor its mature form is cleaved by signal peptidase.

According to the present invention, the term "binding" preferably relates to specific binding. When referring to a ligand/receptor, antibody/antigen or other binding pair, "specific" binding refers to a binding response that determines the presence or absence of the protein in a heterogeneous population of proteins and/or other biological agents. Thus, under specified conditions, a specific ligand/antigen binds to a specific receptor/antibody and does not bind in significant amounts to other proteins present in the sample. "Specific binding" means that the monoclonal antibody or antigen-binding fragment thereof of the present invention can specifically bind to at least two, three, four, five, six, seven, eight or more of each human target molecule. More amino acids interact. The "specific binding" of an antibody is mainly characterized by two parameters: qualitative parameters (binding epitope or antibody binding position) and quantitative parameters (binding affinity or binding strength). The antibody-binding epitope can be determined by FACS method, peptide spot epitope mapping method, mass spectrometry or peptide ELISA method. The Biacore method and/or ELISA method can determine the binding strength of an antibody to a specific epitope. The signal-to-noise ratio is often calculated as a representative measure of binding specificity. In such a signal-to-noise ratio, the signal represents the strength of the antibody binding to the target epitope, while the noise represents the strength of the antibody binding to other non-target epitopes. Preferably, the antibody being evaluated is considered to bind to the target epitope in a specific manner, ie, "specifically binds" when the signal-to-noise ratio for the target epitope is about 50. An antigen-binding protein (including an antibody) "specifically binds" to an antigen if it binds to the antigen with a high binding affinity as determined by the affinity constant (KD) value. In some embodiments, the affinity constant KD is less than 10 ^-9 M. The term "KD" as used herein refers to the affinity constant for a specific antibody-antigen interaction.

As used herein, the term "patient" or "subject" refers to any organism to which a provided antibody, antigen-binding fragment thereof, or pharmaceutical composition is or can be administered for experimental, diagnostic, prophylactic, cosmetic and /or therapeutic purposes. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the subject is a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. A patient may exhibit one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions. In some embodiments, the patient is receiving or has received certain therapy for the diagnosis and/or treatment of such diseases, disorders or conditions.

In the present invention, the term "treatment" refers to therapeutic and preventive measures that prevent or slow down the occurrence or progression of undesirable physiological changes or conditions in a subject. Beneficial or desired clinical effects include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization of disease state (i.e., no worsening), delay or slowing of disease progression, alleviation or alleviation of disease state, and partial or complete disease progression. Cure, regardless of whether the above effects are detectable. "Treatment" may also refer to prolongation of survival compared with no treatment. Subjects in need of treatment include subjects who already suffer from the disease or condition, subjects who are likely to suffer from the disease or condition, or subjects who want to prevent the disease or condition.

The term "prevention" as used herein includes preventing or slowing the onset of the development of clinically significant disease or preventing or slowing the onset of a preclinically significant stage of disease in an at-risk individual. This includes preventive treatment of individuals at risk of developing the disease.

When "administer" and "treat" or "prevent" are used to refer to an animal, human, experimental subject, cell, tissue, organ or biological fluid, it means that the exogenous drug, therapeutic agent, diagnostic agent or composition is administered to the animal Contact with , persons, subjects, cells, tissues, organs or biological fluids. "Administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods and experimental methods. Treating the cells includes contacting an agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells. "Administration" and "treatment" also mean in vitro and ex vivo treatment of cells, for example by agents, diagnostic agents, binding compositions or by other cells.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an antibody that binds to HBsAg of the invention when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject. An amount effective to prevent or slow down the disease or condition to be treated. A therapeutically effective dose further refers to an amount of the compound sufficient to cause alleviation of symptoms, such as treatment, cure, prevention, or alleviation of the associated medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of the condition. . When an active ingredient is administered to an individual, the therapeutically effective amount refers to that ingredient alone. When a combination is administered, a therapeutically effective amount refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered jointly, sequentially, or simultaneously. A therapeutically effective amount will reduce symptoms usually by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

As used herein, "pharmaceutically acceptable carrier" includes materials that, when combined with the active ingredients of the composition, allow the ingredients to remain biologically active and not cause a destructive reaction with the subject's immune system. These carriers may include stabilizers, preservatives, salts or sugar complexes or crystals, and the like. "Pharmaceutically acceptable" refers to molecules and ingredients that do not produce allergic reactions or similar undesirable reactions when administered to humans.

anti-HBsAg antibody

The present invention obtains alpaca-derived heavy chain antibodies and VHH by immunizing alpacas with HBsAg antigen, and uses VHH to construct quadrivalent antibodies for the treatment and/or prevention of hepatitis B and related diseases.

In one aspect of the invention, the invention provides an IgG type tetravalent antibody that binds to hepatitis B surface antigen (HBsAg) or a subviral particle (SVP) containing HBsAg, wherein the tetravalent antibody comprises two heavy chains and two light chains, each of said heavy and light chains comprising as a variable region a VHH portion comprising CDR1, CDR2, CDR3, wherein:

(a) CDR1 includes the sequence shown in SEQ ID NO: 4 or its equivalent, CDR2 includes the sequence shown in SEQ ID NO: 5 or its equivalent, and CDR3 includes the sequence shown in SEQ ID NO: 6 or its equivalent. equivalent;

(b) CDR1 includes the sequence shown in SEQ ID NO: 11 or its equivalent, CDR2 includes the sequence shown in SEQ ID NO: 12 or its equivalent, and CDR3 includes the sequence shown in SEQ ID NO: 13 or its equivalent. equivalent;

(c) CDR1 includes the sequence shown in SEQ ID NO: 18 or its equivalent, CDR2 includes the sequence shown in SEQ ID NO: 19 or its equivalent, and CDR3 includes the sequence shown in SEQ ID NO: 20 or its equivalent. equivalent;

(d) CDR1 includes the sequence shown in SEQ ID NO: 25 or its equivalent, CDR2 includes the sequence shown in SEQ ID NO: 26 or its equivalent, and CDR3 includes the sequence shown in SEQ ID NO: 27 or its equivalent. equivalent; or

(e) CDR1 includes the sequence shown in SEQ ID NO: 32 or its equivalent, CDR2 includes the sequence shown in SEQ ID NO: 33 or its equivalent, and CDR3 includes the sequence shown in SEQ ID NO: 34 or its equivalent. equivalent.

In some embodiments, the VHH portion comprises any or each sequence selected from SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, and SEQ ID NO: 29 equivalent.

In some embodiments, the tetravalent antibody is selected from any of the following antibodies:

(a) The heavy chain includes the sequence shown in SEQ ID NO: 2 or its equivalent, and the light chain includes the sequence shown in SEQ ID NO: 3 or its equivalent;

(b) The heavy chain includes the sequence shown in SEQ ID NO: 8 or its equivalent, and the light chain includes the sequence shown in SEQ ID NO: 9 or its equivalent;

(c) The heavy chain includes the sequence shown in SEQ ID NO: 16 or its equivalent, and the light chain includes the sequence shown in SEQ ID NO: 17 or its equivalent;

(d) The heavy chain includes the sequence shown in SEQ ID NO: 23 or its equivalent, and the light chain includes the sequence shown in SEQ ID NO: 24 or its equivalent; and

(e) The heavy chain includes the sequence shown in SEQ ID NO: 30 or its equivalent, and the light chain includes the sequence shown in SEQ ID NO: 31 or its equivalent.

In some embodiments, the VHH portion includes CDR1, CDR2, CDR3, wherein:

(a) CDR1 includes the sequence shown in SEQ ID NO: 4 or a variant thereof, which variant has a single substitution, deletion or insertion, and CDR2 includes the sequence shown in SEQ ID NO: 5 or a variant thereof, which variant has A single substitution, deletion or insertion, CDR3 contains the sequence shown in SEQ ID NO: 6 or a variant thereof, which variant has a single substitution, deletion or insertion;

(b) CDR1 includes the sequence shown in SEQ ID NO: 11 or a variant thereof, which variant has a single substitution, deletion or insertion, and CDR2 includes the sequence shown in SEQ ID NO: 12 or a variant thereof, which variant has A single substitution, deletion or insertion, CDR3 contains the sequence shown in SEQ ID NO: 13 or a variant thereof, which variant has a single substitution, deletion or insertion;

(c) CDR1 includes the sequence shown in SEQ ID NO: 18 or a variant thereof, which variant has a single substitution, deletion or insertion, and CDR2 includes the sequence shown in SEQ ID NO: 19 or a variant thereof, which variant has A single substitution, deletion or insertion, CDR3 contains the sequence shown in SEQ ID NO: 20 or a variant thereof, which variant has a single substitution, deletion or insertion;

(d) CDR1 includes the sequence shown in SEQ ID NO: 25 or a variant thereof, which variant has a single substitution, deletion or insertion, and CDR2 includes the sequence shown in SEQ ID NO: 26 or a variant thereof, which variant has or

(e) CDR1 includes the sequence shown in SEQ ID NO: 32 or a variant thereof, which variant has a single substitution, deletion or insertion, and CDR2 includes the sequence shown in SEQ ID NO: 33 or a variant thereof, which variant has Single substitution, deletion or insertion, CDR3 contains the sequence shown in SEQ ID NO: 34 or a variant thereof, which variant has a single substitution, deletion or insertion.

In some embodiments, the VHH portion comprises any or each sequence selected from SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, and SEQ ID NO: 29 A variant that has at least 75%, 80%, or 85%, 90%, 95%, 98% or 99% sequence identity.

(a) The heavy chain includes the sequence shown in SEQ ID NO: 2 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98 % or 99% sequence identity, the light chain comprising the sequence shown in SEQ ID NO: 3 or a variant thereof having at least 75%, 80%, 85%, 90% identity with SEQ ID NO: 3 %, 95%, 98% or 99% sequence identity;

(b) The heavy chain includes the sequence shown in SEQ ID NO: 8 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98 % or 99% sequence identity, the light chain comprising the sequence shown in SEQ ID NO: 9 or a variant thereof having at least 75%, 80%, 85%, 90% identity with SEQ ID NO: 9 %, 95%, 98% or 99% sequence identity;

(c) The heavy chain includes the sequence shown in SEQ ID NO: 16 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98 % or 99% sequence identity, the light chain comprising the sequence shown in SEQ ID NO: 17 or a variant thereof having at least 75%, 80%, 85%, 90% identity with SEQ ID NO: 17 %, 95%, 98% or 99% sequence identity;

(d) The heavy chain includes the sequence shown in SEQ ID NO: 23 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% similarity with SEQ ID NO: 23 % or 99% sequence identity, the light chain comprising the sequence shown in SEQ ID NO: 24 or a variant thereof, which has at least 75%, 80%, 85%, 90% identity with SEQ ID NO: 24 %, 95%, 98% or 99% sequence identity; and

(e) The heavy chain includes the sequence shown in SEQ ID NO: 30 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% similarity with SEQ ID NO: 30 % or 99% sequence identity, the light chain comprising the sequence shown in SEQ ID NO: 31 or a variant thereof, which has at least 75%, 80%, 85%, 90% identity with SEQ ID NO: 31 %, 95%, 98% or 99% sequence identity.

In some embodiments, the VHH portion includes CDR1, CDR2, CDR3, wherein:

(a) CDR1 is the sequence shown in SEQ ID NO: 4, CDR2 is the sequence shown in SEQ ID NO: 5, and CDR3 is the sequence shown in SEQ ID NO: 6;

(b) CDR1 is the sequence shown in SEQ ID NO: 11, CDR2 is the sequence shown in SEQ ID NO: 12, and CDR3 is the sequence shown in SEQ ID NO: 13;

(c) CDR1 is the sequence shown in SEQ ID NO: 18, CDR2 is the sequence shown in SEQ ID NO: 19, and CDR3 is the sequence shown in SEQ ID NO: 20;

(d) CDR1 is the sequence shown in SEQ ID NO: 25, CDR2 is the sequence shown in SEQ ID NO: 26, and CDR3 is the sequence shown in SEQ ID NO: 27; or

(e) CDR1 is the sequence shown in SEQ ID NO: 32, CDR2 is the sequence shown in SEQ ID NO: 33, and CDR3 is the sequence shown in SEQ ID NO: 34.

In some embodiments, the VHH portion includes any sequence selected from SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, and SEQ ID NO: 29.

(a) The heavy chain includes the sequence shown in SEQ ID NO: 2, and the light chain includes the sequence shown in SEQ ID NO: 3;

(b) The heavy chain includes the sequence shown in SEQ ID NO: 8, and the light chain includes the sequence shown in SEQ ID NO: 9;

(c) The heavy chain includes the sequence shown in SEQ ID NO: 16, and the light chain includes the sequence shown in SEQ ID NO: 17;

(d) The heavy chain includes the sequence shown in SEQ ID NO: 23, and the light chain includes the sequence shown in SEQ ID NO: 24; and

(e) The heavy chain includes the sequence shown in SEQ ID NO: 30, and the light chain includes the sequence shown in SEQ ID NO: 31.

In some embodiments, any of the above antibodies is of the IgG type, such as IgGl, IgG2, IgG3 or IgG4 type. In some embodiments, any of the above-described antibodies is of the IgG1 type.

In another aspect of the invention, the invention provides a heavy chain antibody that binds to hepatitis B surface antigen (HBsAg) or a subviral particle (SVP) comprising HBsAg, wherein the heavy chain antibody comprises as a heavy chain variable The VHH part of the region, the VHH part includes CDR1, CDR2, CDR3, where:

In some embodiments, the heavy chain antibody comprises a VHH portion comprising CDR1, CDR2, CDR3, wherein:

In any of the above embodiments, the CDR1, CDR1 and CDR3 are defined based on any one of IMGT, Kabat, Chothia, Contact or Martin definition schemes. In some embodiments, the CDR1, CDR1 and CDR3 are defined based on the IMGT definition scheme.

In some embodiments, the substitutions described herein are conservative substitutions.

"Conservative (amino acid) substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine ) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Therefore, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be substituted with a structurally similar string that differs in the order and/or composition of the side chain family members.

Those skilled in the art will also appreciate that the antibodies disclosed herein can be modified so that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a given protein may be similar to the starting sequence, e.g., have a certain percentage of identity, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95 %, 98%, 99%, or a range between any two of these values is the same as the starting sequence.

In some embodiments, antibodies comprise an amino acid sequence or one or more portions not typically associated with antibodies. Exemplary modifications are described in greater detail herein. For example, the antibodies disclosed herein can include flexible linker sequences, or can be modified to add functional moieties (eg, polyethylene glycol (PEG), drugs, toxins, or labels).

The antibodies, variants or derivatives thereof of the present invention include derivatives that are modified, ie, by covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, and not by way of limitation, antibodies can be modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolysis Cleavage, attachment to cellular ligands or other proteins, etc. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, antibodies may contain one or more non-canonical amino acids.

In some embodiments, the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

Antibodies can be conjugated or fused to therapeutic agents, which can include detectable labels (e.g., radioactive labels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which can be drugs or toxins), ultrasound enhancers, non-radioactive labels, combinations thereof, and other such agents known in the art.

Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermostable acridinium esters, imidazole, acridinium salts and oxalate esters.

Antibodies can also be detectably labeled using fluorescent emitting metals such as Eu or other metals of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known.

Humanized and Chimeric Antibodies

In any aspect of the invention, the tetravalent antibody or heavy chain antibody is preferably a humanized antibody or a chimeric antibody.

Humanized antibodies are antibody molecules derived from antibodies of a non-human species that bind a desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Typically, framework residues in the human framework regions will be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. base. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting, veneering or resurfacing and chain shuffling.

Fully human antibodies are particularly ideal for therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but that express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells either randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be rendered functional separately or simultaneously upon introduction of human immunoglobulin loci via homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal manner with a selected antigen, eg, all or part of the desired target polypeptide. Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. Transgenic mice carry human immunoglobulin transgenes that rearrange during B cell differentiation and subsequently undergo class switching and somatic mutations. Therefore, using this technology, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies.

Fully human antibodies that recognize selected epitopes can also be generated using a technique called "guided selection." In this approach, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of fully human antibodies that recognize the same epitope.

The DNA encoding the desired monoclonal antibody can be readily isolated and sequenced using routine procedures (eg, by using oligonucleotide probes capable of binding specifically to the genes encoding the murine antibody heavy and light chains). Isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or bone marrow that does not produce immunoglobulins. tumor cells. More specifically, the isolated DNA can be used to clone constant and variable region sequences for use in making antibodies. Essentially, this requires RNA to be extracted from selected cells, converted to cDNA, and amplified by PCR using Ig-specific primers. As described herein, transformed cells expressing the desired antibodies can be grown in relatively large quantities to provide clinical and commercial supplies of immunoglobulins.

Additionally, one or more CDRs of an antigen-binding polypeptide of the present disclosure can be inserted into a framework region using conventional recombinant DNA techniques, for example, into a human framework region to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and are preferably human framework regions. Preferably, the polynucleotide generated from the combination of framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of the desired polypeptide (eg, LIGHT). Preferably, one or more amino acid substitutions may be made within the framework region, and preferably the amino acid substitutions increase the binding of the antibody to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bonds to generate antibody molecules lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotides are included in this disclosure and are within the skill of the art.

In addition, techniques developed for the production of "chimeric antibodies" by splicing genes from mouse antibody molecules with appropriate antigen specificity, and genes from human antibody molecules with appropriate biological activity, can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a molecule having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

Another highly efficient method for generating recombinant antibodies results in the generation of primate antibodies containing monkey variable domains and human constant sequences.

For camelid antibodies, in particular heavy chain antibodies and single domain antibodies (VHH), humanizing the polypeptide according to the present invention involves using one or more camelid amino acids with their human counterparts found in the human consensus sequence substitution without the polypeptide losing its typical characteristics, that is, the humanization does not significantly affect the antigen-binding ability of the resulting polypeptide. This method is well known to the skilled person.

In one embodiment, "humanization" of a VHH refers to the replacement of one or more amino acid residues in the amino acid sequence (and specifically in the framework sequence) of a naturally occurring VHH sequence with conventional 4 amino acid residues from human One or more amino acid residues present at corresponding positions in the VH domain of the chain antibody. This can be done using humanization techniques known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions can be determined by methods known in the art, for example, by comparing the sequence of a VHH with the sequence of a naturally occurring human VH domain. Compare. In some embodiments, the humanizing substitutions are selected such that the resulting humanized VHH retains advantageous functional properties. Generally speaking, as a result of humanization, the VHH of the present application can become more "human-like" compared to the corresponding naturally occurring VHH domain, while still retaining advantageous properties, such as reduced immunogenicity. In various embodiments, the humanized VHH of the present application can be obtained by any suitable means known in the art, and is therefore not strictly limited to those that have been obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material. Peptides.

In various embodiments, "humanization" and "camelization" can be performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then using a method known in the art to One or more codons in the nucleotide sequence are altered in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. This nucleic acid can then be expressed in a manner known in the art to provide the desired VHH of the present application. Alternatively, the amino acid sequence of the desired humanized or camelized VHH of the present application can be designed based on the amino acid sequence of the naturally occurring VHH domain or VH domain, respectively, and then synthesized using peptides known in the art Technology synthesized from scratch. Furthermore, based on the amino acid sequence or nucleotide sequence of the naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized VHH can be designed, and then used as known in the art. The nucleic acid synthesis technology is de novo synthesized, and the nucleic acid thus obtained can thereafter be expressed in a manner known in the art to provide the desired VHH of the present application. Other suitable methods and techniques for obtaining VHHs of the present application and/or nucleic acids encoding them starting from naturally occurring VH sequences or VHH sequences are known in the art and may, for example, include combinations in a suitable manner One or more portions of one or more naturally occurring VH sequences (e.g., one or more FR sequences and/or CDR sequences), one or more portions of one or more naturally occurring VHH sequences (e.g., a or multiple FR sequences or CDR sequences) and/or one or more synthetic or semi-synthetic sequences to provide the VHH of the present application or the nucleotide sequence or nucleic acid encoding it.

Accordingly, the present invention provides a humanized IgG type tetravalent antibody that binds to hepatitis B surface antigen (HBsAg) or HBsAg-containing subviral particles (SVP), wherein the tetravalent antibody includes two heavy chains and two light chains, each of said heavy and light chains comprising as a variable region a VHH portion comprising CDR1, CDR2, CDR3, wherein:

Accordingly, the present invention provides humanized heavy chain antibodies that bind to hepatitis B surface antigen (HBsAg) or subviral particles (SVP) containing HBsAg, wherein the heavy chain antibodies comprise VHH as the heavy chain variable region part, the VHH part includes CDR1, CDR2, CDR3, where:

Accordingly, the present invention provides a chimeric IgG type tetravalent antibody that binds to hepatitis B surface antigen (HBsAg) or HBsAg-containing subviral particles (SVP), wherein the tetravalent antibody includes two heavy chains and Two light chains, each of the heavy chain and the light chain comprising as a variable region a VHH portion comprising a portion selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15 , any sequence of SEQ ID NO: 22 and SEQ ID NO: 29 or the equivalent of each thereof.

Accordingly, the present invention provides a chimeric heavy chain antibody that binds to hepatitis B surface antigen (HBsAg) or a subviral particle (SVP) containing HBsAg, wherein the heavy chain antibody comprises as a heavy chain variable region A VHH portion comprising any sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, and SEQ ID NO: 29, or the equivalent of each of them things.

The above-mentioned humanized or chimeric tetravalent antibody may comprise a heavy chain constant region and a light chain constant region, and the humanized or chimeric heavy chain antibody may comprise a heavy chain constant region, which is, for example, SEQ ID NO: The human IgG1 heavy chain constant region shown in 38, the light chain constant region is, for example, the human Igκ light chain constant region shown in SEQ ID NO: 39.

In another aspect of the invention, the invention provides a nucleotide sequence comprising a polynucleotide encoding an antibody of any of the above aspects. In some embodiments, the polynucleotide comprises any sequence selected from SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28, and SEQ ID NO: 35, or each sequence thereof. The equivalent of a.

Treatment/prevention methods and applications

One aspect of the invention provides a method of treating and/or preventing hepatitis B, the method comprising administering to a subject a therapeutically effective amount of any one of the above antibodies.

Another aspect of the invention provides a method of preventing diseases caused by hepatitis B, the method comprising administering to a subject a therapeutically effective amount of any one of the above antibodies.

Accordingly, another aspect of the present invention provides the use of any one of the above antibodies in the preparation of a medicament for the treatment and/or prevention of hepatitis B.

Accordingly, another aspect of the present invention provides the use of any one of the above antibodies in the preparation of a medicament for preventing diseases caused by hepatitis B.

Accordingly, another aspect of the present invention provides any of the above antibodies for use in the treatment and/or prevention of hepatitis B.

Accordingly, another aspect of the present invention provides any of the above antibodies for use in preventing diseases caused by hepatitis B.

In some embodiments, the hepatitis B is chronic hepatitis B. In some embodiments, the disease caused by hepatitis B is cirrhosis, liver failure or hepatocellular carcinoma caused by hepatitis B.

Suitable routes of administration include parenteral (eg, intramuscular, intravenous or subcutaneous) and oral administration. Other conventional modes of administration include administration via tracheal intubation, oral ingestion, inhalation, topical application, or transdermal, subcutaneous, intraperitoneal, or intraarterial injection.

Appropriate dosages are determined by the clinician based on parameters or factors known or suspected in the art to affect treatment or expected to affect treatment. Typically, the dose is started slightly lower than the optimal dose and is increased by small amounts until the desired or optimal effect relative to any adverse side effects is achieved. Important monitoring indicators include measuring, for example, inflammatory symptoms or the levels of inflammatory cytokines produced.

pharmaceutical composition

One aspect of the present invention provides a pharmaceutical composition, which includes a therapeutically effective amount of any one of the above antibodies, and a pharmaceutically acceptable carrier.

In any of the above aspects, the pharmaceutical composition is used to treat and/or prevent hepatitis B, such as chronic hepatitis B. In some embodiments, the pharmaceutical composition is used to prevent diseases caused by hepatitis B, such as cirrhosis, liver failure, or hepatocellular carcinoma caused by hepatitis B.

To prepare a pharmaceutical or sterile composition, the drug is mixed with a pharmaceutically acceptable carrier or excipient. Formulations in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers. Pharmaceutically acceptable carriers are well known in the art. It is known in the art how to prepare aqueous compositions containing as active ingredient. Typically, these compositions are prepared as injectables or sprays, such as liquid solutions or suspensions; solid forms suitable for solution or suspension prior to injection or spraying may also be prepared.

sequence list

Example

Example 1: Screening and construction of antibodies

Screening of HBV heavy chain antibodies from alpacas immunized with HBsAg. The screened antibodies are then confirmed using Elisa, and the antibodies are sequenced to confirm their VHH portion.

Five anti-HBsAg IgG type quadrivalent monoclonal antibodies of the present invention were constructed on the human IgG1 antibody, named C1, C2, C3, C4 and C5 antibodies respectively, for further characterization and experiments.

Figure 1 is a schematic structural comparison of the antibody constructed as above and a conventional control antibody. As shown in the figure, the antibody constructed in this example has a generally similar structure to the conventional IgG1 antibody, both consisting of two heavy chains and two light chains. The difference lies in the light chain variable region and heavy chain corresponding to the conventional antibody. As part of the chain variable region, the antibody of the present invention has a VHH portion, thereby having 4 identical VHH portions on a single antibody molecule, forming a quadrivalent antibody.

The sequences of the five antibodies C1, C2, C3, C4, C5 and the control RH antibody constructed in this example can be found in the "Sequence Listing" section above. The control antibody RH is Rachel Eren et al. in Preclinical Evaluation of Two Human Anti-Hepatitis B Virus(HBV)Monoclonal Antibodies in the HBV-Trimera Mouse Model and in HBV Chronic Carrier Chimpanzees.Hepatology, 2000, 32(3): 588-596 The antibody 17.1.41 disclosed in is used in subsequent examples of this application.

Example 2: Binding activity of the antibody of the present invention to HBsAg

Conventional ELISA test: The ELISA method was performed to determine the interaction between the antibody constructed in Example 1 and HBsAg. Briefly, plates were coated with 0.5ug/ml HBsAg. Block and then add different concentrations of antibodies. Wash, then add secondary antibody with HRP. The plate was read at 420 nm after adding TMB substrate. The detection principle is shown in Figure 2.

Results: As shown in Table 1, all antibodies had strong binding to HBsAg. Compared with control RH, antibodies C1, C2, C3, C4, and C5 can all bind to HBsAg with similar or even stronger intensity.

Table 1: Binding activity of antibodies to HBsAg (OD420)

Example 3: Effect of the antibody of the present invention on HBsAg aggregation

The kit used in this example is Antu Bio's HBsAg ELISA kit.

Direct HBsAg ELISA test: A direct HBsAg ELISA method was performed to determine the effect of the antibodies constructed in Example 1 on HBsAg aggregation. Briefly, plates were coated with HBsAg. Block and then add different concentrations of antibodies. Wash, then add HBsAg with HRP. The plate was read at 420 nm after adding TMB substrate. The detection principle is shown in Figure 3.

Results: As shown in Table 2, considerable readings were obtained using the control RH antibody, indicating that such conventional IgG antibodies can continue to bind other HBsAg after binding HBsAg, causing the aggregation of HBsAg aggregates. HBsAg in the blood usually exists in the form of subviral particles (SVP) (aggregates of HBsAg). The results of this experiment show that conventional IgG antibodies will bind to more than one SVP, causing cross-linking and aggregation of SVP, thereby forming larger aggregates, preventing neutrophils, macrophages and other phagocytes from phagocytosis and clearing the SVP. Aggregates.

In contrast, the readings of antibodies C1, C2 and C5 are significantly lower, indicating that the VHH-based IgG antibodies constructed in the present invention will not continue to bind to other HBsAg after binding to HBsAg, and will not cause the aggregation of HBsAg aggregates. The results of this experiment show that the VHH-based IgG antibody constructed in the present invention only binds to a single SVP and does not cause cross-linking and aggregation of SVP, allowing macrophages and the like to perform phagocytosis against a single SVP and clear the aggregates.

Table 2: Effect of antibodies on HBsAg aggregation

Example 4: HDI-HBV model testing the in vivo activity of the antibody of the present invention

HBV plasmid preparation

Prepare HBV plasmid according to general procedures. Check DNA quality and concentration via NanoDrop. The A260/A280 ratio of high-quality plasmid DNA should be ≥1.8. It is important that the purified plasmid DNA is of high quality and free of proteins, endotoxins, DNase, and RNase to prevent adverse or toxic effects on the animal.

hydrodynamic injection

Measure the body weight of mice. Prepare 10 μg of HBV plasmid DNA and dissolve it in PBS equivalent to 8% of the mouse body weight.

Before injection, heat the mouse tail with a safe and effective heat source (such as a heating lamp (120W bulb)) for 5-10 minutes to dilate the tail blood vessels. This step helps visualize the tail vein and ensures optimal injection. As the mouse's tail warms, the veins should dilate and become more visible. Keep mice warm before hydrodynamic injection.

Mice were anesthetized by intramuscular injection using Imalgene (ketamine, 60 mg/kg) and Rompun ^™ (xylazine, 12 mg/kg). Immobilize the mouse with a restraint device before hydrodynamic injection. While working under a light source, position the dilated vein on the ventral side of the mouse's tail, preferably near the distal (tip) end of the tail. Wipe the area with an alcohol pad and allow it to air dry to further increase vein visibility and disinfect the injection site.

Attach the needle to the syringe and inject the entire injection solution, making sure there are no air bubbles in the needle or syringe. Place the syringe needle almost parallel to the tail, with the bevel facing downward (toward the tail). Insert the needle into the tail vein. The entire volume of solution was injected into the mouse tail vein at a constant rate over 5-7 seconds. Release the mouse from the restraint device. Use medical gauze to stop bleeding at the injection site.

Mice generally tolerate hydrodynamic injections well, but may remain motionless and exhibit dyspnea lasting approximately 5 minutes immediately following injection. The observed apnea may be due to a vasovagal response induced by rapid administration of large amounts of HBV DNA solution. Gently massaging the mouse's chest is enough to stimulate breathing and promote recovery. The heart rate may slow or increase rapidly during the first minutes after the injection, but this should normalize. Mice should recover within 5 minutes of injection. If a mouse appears to have seizures after an injection, this may indicate that air bubbles or impurities have entered the circulatory system and the mouse may not survive. Careful monitoring of mice after injection is necessary.

Administration and sampling

According to the scheme shown in Figure 4, the GTA/GTD HDI model C57BL/6 male mice treated by the above method were administered on day 0 and collected on days 1, 2, 3, 5, 7, 10 and 14 Serum was analyzed. Measure HBsAg in blood using the HBsAg Direct ELISA kit.

results and analysis

As shown in Table 3 and Table 4, the antibody constructed in Example 1 can effectively reduce HBsAg in vivo. In particular, the C5 antibody can significantly reduce HBsAg at a concentration of 0.3 mg/kg until the third day after administration, and at a concentration of 3 mg/kg, it can significantly reduce HBsAg until the fifth day after administration. C3 antibody appears to have the best effect, and can significantly reduce HBsAg at a concentration of 0.3mg/kg until the 7th day after administration.

Table 3: Serum 1:25 dilution-Antu HBsAg detection kit-measured value (IU/ml)

Table 4: Serum 1:10 dilution-Antu HBsAg detection kit-measured value (IU/ml)

It should be understood that, although the present invention has been specifically disclosed in terms of preferred embodiments and optional features, those skilled in the art will be able to make modifications, improvements and variations to the invention disclosed herein, which modifications, improvements and variations are considered within the scope of the invention. The materials, methods, and examples provided herein are representative and exemplary of preferred embodiments and are not intended as limitations on the scope of the invention.

Claims

An IgG type tetravalent antibody or heavy chain antibody that binds to hepatitis B surface antigen (HBsAg) or a subviral particle (SVP) containing HBsAg, wherein the tetravalent antibody contains two heavy chains and two light chains, each of said heavy chain and light chain comprises a VHH portion as a variable region, or wherein said heavy chain antibody comprises a VHH portion as a heavy chain variable region, said VHH portion comprising CDR1, CDR2, CDR3, in:

(a) CDR1 includes the sequence shown in SEQ ID NO:4, CDR2 includes the sequence shown in SEQ ID NO:5, and CDR3 includes the sequence shown in SEQ ID NO:6;

(b) CDR1 includes the sequence shown in SEQ ID NO:11, CDR2 includes the sequence shown in SEQ ID NO:12, and CDR3 includes the sequence shown in SEQ ID NO:13;

(c) CDR1 includes the sequence shown in SEQ ID NO:18, CDR2 includes the sequence shown in SEQ ID NO:19, and CDR3 includes the sequence shown in SEQ ID NO:20;

(d) CDR1 contains the sequence shown in SEQ ID NO:25, CDR2 contains the sequence shown in SEQ ID NO:26, and CDR3 contains the sequence shown in SEQ ID NO:27; or

(e) CDR1 includes the sequence shown in SEQ ID NO:32, CDR2 includes the sequence shown in SEQ ID NO:33, and CDR3 includes the sequence shown in SEQ ID NO:34.
The antibody of claim 1, wherein the VHH portion comprises any sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22 and SEQ ID NO: 29 .
The antibody of claim 1, wherein said antibody is a humanized antibody or a chimeric antibody.
The antibody of any one of claims 1-3, wherein the tetravalent antibody is an IgG1 antibody.
The antibody of claim 1, wherein the tetravalent antibody is selected from any of the following antibodies:

(a) The heavy chain includes the sequence shown in SEQ ID NO:2, and the light chain includes the sequence shown in SEQ ID NO:3;

(b) The heavy chain includes the sequence shown in SEQ ID NO:8, and the light chain includes the sequence shown in SEQ ID NO:9;

(c) The heavy chain includes the sequence shown in SEQ ID NO:16, and the light chain includes the sequence shown in SEQ ID NO:17;

(d) The heavy chain includes the sequence shown in SEQ ID NO:23, and the light chain includes the sequence shown in SEQ ID NO:24; and

(e) The heavy chain includes the sequence shown in SEQ ID NO:30, and the light chain includes the sequence shown in SEQ ID NO:31.
A polynucleotide comprising a polynucleotide encoding the antibody of any one of claims 1 to 5.
The polynucleotide of claim 6, wherein the polynucleotide comprises a polynucleotide selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28 and SEQ ID NO: 35 any sequence.
A vector comprising the polynucleotide of claim 6 or 7.
A non-human host cell comprising the vector of claim 8.
A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any one of claims 1 to 5, the polynucleotide of claim 6 or 7, the vector of claim 8 or the vector of claim 9 The above-mentioned host cells, and pharmaceutically acceptable carriers.