阅读说明：本技术 抗乙型肝炎病毒表面抗原前s1抗原的纳米抗体及其用途 (Nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen and application thereof ) 是由 张军方 于 2019-07-25 设计创作，主要内容包括：本发明公开了抗乙型肝炎病毒表面抗原前S1抗原(HBV-preS1)的纳米抗体。本发明通过用HBV-preS1免疫羊驼,筛选得到了抗HBV-preS1的纳米抗体。本发明还涉及这些抗体的片段及与Fc融合组成的完整抗体,该抗体用于乙型肝炎病毒及肝癌的治疗,和制备该抗体的方法。(The invention discloses a nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen (HBV-preS 1). The invention obtains the nano antibody of anti-HBV-preS1 by screening the alpaca immunized by the HBV-preS 1. The invention also relates to fragments of the antibodies and complete antibodies fused with Fc, the antibodies are used for treating hepatitis B virus and liver cancer, and a method for preparing the antibodies.)

1. A nano antibody and antibody fragment for resisting the pre-S1 antigen (HBV-preS1) of the surface antigen of hepatitis B virus is disclosed, which contains the amino acid sequence regions of CDR1, CDR2 and CDR 3.

2. The molecule of claim 1, wherein said antibody is an antibody against hepatitis b virus surface antigen pre S1 antigen (HBV-preS 1).

3. The molecule according to claim 1, characterized in that said antibody is a nanobody, also called single domain antibody or heavy chain antibody.

4. The molecule of claim 1, wherein the antibody fragment can be combined with an Fc to form a complete antibody.

5. The molecule of claim 1, wherein the antibody fragment can be combined with other antibodies to form a bi-or multi-specific antibody.

6. The molecule of claim 1, wherein said nanobody is an antibody drug conjugate comprised of a toxin, radionuclide combination.

7. The molecule of claim 1, wherein said antibody is useful for the diagnosis and treatment of hepatitis b virus.

8. The molecule of claim 1, wherein said antibody is useful for the diagnosis and treatment of liver cancer.

9. The molecule of claim 1, comprising the sequences of antibody CDR1, CDR2, CDR 3.

10. The antibody CDR1 sequence may be SEQ ID NO: 1 to SEQ ID NO: 12 or a sequence of any one of the above.

11. The antibody CDR2 sequence may be SEQ ID NO: 13 to SEQ ID NO: 21 any one of the sequences.

12. The antibody CDR3 sequence may be SEQ ID NO: 22 to SEQ ID NO: 30 or a sequence thereof.

13. The antibody comprises a sequence combination of CDR1, CDR2, CDR 3.

CDR1 sequence is SEQ ID NO: 1 to SEQ ID NO: 12, the amino acid sequences of which are: GFIFSIYV, GFISSIYD, GFTFSSYS, GSIFSRYT, GMFSIYD, GFTLDYYG, GSVFSRYT, GFTFSTYS, GNIFSRYT, GRIFSRYT, GSIFSRYV, GFILSIYT.

CDR2 sequence is SEQ ID NO: 13 to SEQ ID NO: 21, the amino acid sequences of which are: INNIGTT, IGRGGRYT, IVSNVGGG, ITSGVST, IGRGGGT, TNNIGTT, ITSGGST, ISGVGGTT, ISSSGGST.

CDR3 sequence is SEQ ID NO: 55 to SEQ ID NO: 80, the amino acid sequences of which are: SCKAYQTLEDGLGPGVLEY, RYYRSDDDFPEYAQDY, RYYRSDDDFPEHAYEY, KAYQTLEDGLGPGVLEY, RYYRSDDDFPEYAYDY, AARKNNYYCSGYLSSGGARHDY, VYYCNADFGLGL, NALFGIDGL, HADFGLDGL are provided.

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a nano antibody sequence of anti-hepatitis B virus surface antigen pre-S1 antigen (HBV-preS 1). The invention is used for diagnosing or treating hepatitis B and liver cancer.

Background

The invention relates to a nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen (HBV-preS1), which is used for diagnosing or treating hepatitis B and liver cancer.

After more than thirty years of development, therapeutic monoclonal antibody drugs have made great progress in the fields of tumors and autoimmune diseases, and are ready to become one of the important means for clinical treatment of such diseases. Development of therapeutic antibodies against HBV is expected to provide a new therapeutic strategy for chronic HBV infection.

Infection with Hepatitis B Virus (HBV) is a serious public health problem that faces globally. Statistics show that more than 20 million people worldwide are infected with HBV, of which about 3.5 million people are infected with Chronic HBV (CHB). Hepatitis b causes a serious economic burden on our country because of the extremely high infection rate of HBV infection and the mortality rate of liver cancer developed by patients with chronic hepatitis b ranked second among the total mortality rates of cancer in our country. Infection with HBV varies greatly from country to country and from region to region, with the majority of hepatitis b carriers concentrating in asia, africa and the western pacific. Therefore, hepatitis B virus infection and a series of liver diseases caused by hepatitis B virus infection, liver cirrhosis, liver cancer and the like become one of the public health problems all over the world.

Therefore, HBV infection, especially chronic HBV infection, and liver diseases such as chronic hepatitis, liver cirrhosis and primary liver cancer cause a heavy economic burden on patients. The currently approved drugs for clinical treatment of chronic hepatitis B include interferon and nucleoside analogs, which have positive effects on inhibiting virus replication and delaying disease progression, but still have the problems of long treatment period, low response rate, serious side effects (interferons) and potential drug resistance (nucleoside analogs), and the like, so that the development of anti-HBV drugs based on new targets or new mechanisms is urgently needed.

Small circular DNA (3182-3221) with HBV genome size of 3.2kb base pair (bp) is one of the smallest known eukaryotic cell DNA viruses. The HBV genome has 4 Open Reading Frames (ORFs): S-ORF, P-ORF, C-ORF, X-ORF. The S-ORF encodes three envelope proteins, SHBs (S), MHBs (preS2+ S) and LHBs (preS1+ preS2+ S). The C-ORF has a pre-C region (preC), a secreted protein (HBeAg) that plays an important role in the development of immune tolerance to chronic infections, and a C gene region that constitutes the viral Capsid and that envelops the viral genome, this segment of the HBV genome being the most conserved and targeted epitope for immune attack. The gene product HBx protein of X-ORF has transcription function of a transcription activation enhancer and a promoter, is a multifunctional trans-regulatory factor, and has important relation with virus infection, transcription, replication, apoptosis and HCC occurrence. The P-ORF is the longest ORF in the HBV genome, responsible for transcription of DNA polymerase. In addition, the respective open reading frames of HBV are highly overlapping, having a very compact structure.

The HBV genome contains 4 genes, 4 different mRNAs are transcribed and can encode and synthesize 9 different virus proteins, including outer membrane proteins (L-HBsAg, M-HBsAg and S-HBsAg) and structural proteins of nucleocapsid (HBcAg) of the assembled virus; 3P proteins (terminal protein, DNAp and RNase H) and HBx protein which regulates the replication function of the virus, and HBeAg protein encoded by another pc/C gene is also a functional protein.

Hepatitis B virus outer membrane protein (HBsAg), also known as Australian antigen, was first discovered by Blumberg in 1965 and was widely used to quantify the amount of HBsAg in serum to predict the level of HBV infection in patients. The HBV outer membrane protein is synthesized by S-ORF code, comprising 3 outer membrane components of L-HBsAg, M-HBsAg and S-HBsAg, 3 proteins have respective initial codes, but the termination codes of S-HBsAg are used together.

The core protein of HBV is encoded by the C-ORF (nucleotein), and has the form 2: one is the structural component of the virus nucleocapsid, HBcAg; the other is soluble, secreted HBeAg. The C gene of HBV has 2 related ORFs, in which two initiation codons are distinguished as Pre-C (Pre-C) and C region, but have a common stop codon. The Pre-C/P mRNA synthesizes the nucleocapsid protein HBcAg of P21, the Pre-C-mRNA generates P25Pre-C protein, and the protein becomes HBeAg after a series of treatments. HBcAg can be spontaneously assembled into a Capsid structure in different cell lines, such as bacteria, yeast, mammalian cells and the like, without other HBV related proteins or elements, and can be used as a vector of a genetic engineering vaccine.

According to the infection 2011 epidemic situation distribution report published by the ministry of health, the method comprises the following steps: 1.5 hundred million people break through in liver disease patients in China, wherein the chronic hepatitis B patients reach 4000-. At present, about 3000 thousands of liver cirrhosis patients in China exist, 4.7% of liver cirrhosis patients are finally transformed into liver cancer, about 30 thousands of people die of liver cancer every year, and China becomes a large country for liver disease occurrence.

The most major risk factor for the development of liver cancer is chronic HBV infection. In recent years, it has been discovered that viruses may play a direct role as mutagens, and researchers have discovered that viral DNA sequences are integrated into potential oncogenes from an increasing number of HCC cases. Several other factors are also associated with the risk of HCC development in patients with chronic hepatitis b, including alcohol, metabolic and environmental factors (e.g., aflatoxin), among others.

At present, human beings mainly take prophylactic vaccines as a strategy for treating HBV, but for patients with chronic hepatitis B, effective antiviral drugs are required to be found. Seven drugs have been approved by FDA for treating chronic hepatitis b in the past 20 years, and the first two drugs for treating hepatitis b are Interferon (Interferon-alfa-2b) and Lamivudine (Lamivudine), which have been well used in the last 90 th century. At the beginning of this century, five drugs were approved by the FDA for the treatment of hepatitis b: adefovir dipivoxil (adefovir dipivoxil), interferon (alfa-2 apoeg interferon alfa-2a), tenofovir disoproxil fumarate (tenofovir), entecavir (entecavir), telbivudine (telbivudine).

Single domain antibodies (SDAb), also known as nanobodies or heavy chain antibodies (hcAb), were originally isolated from camelids (alpacas) as a mutant form of antibody with a very small molecular weight of only 12-15kDa, but only about 1/12 the molecular weight of conventional antibodies. A single domain antibody, consisting of only heavy chains and no light chains, differs from the constitutive structure of conventional antibodies in that the Fab region is only a single domain, linked to the Fc region by a hinge region. The single-domain antibody has stable conformation and good water solubility, and can still be combined with antigen with high affinity in gastric juice and viscera. The single domain antibody has small molecular weight, so that the single domain antibody can recognize certain unique antigen epitopes, for example, the single domain antibody can enter the active site of enzyme or enter the crack of a bacterial or virus surface receptor, and the single domain antibody can be used for simulating drugs to design agonists, enzyme inhibitors or antagonists and the like of small molecule receptors.

Disclosure of Invention

The invention uses HBV-preS1 antigen to immunize alpaca, collects PBMC after separation and immunization, extracts total RNA and obtains cDNA, obtains antibody library by phage display technology, and further uses HBV-preS1 to screen and obtain nano antibody specifically reacting with the antibody. The nano antibody prepared by the invention aiming at HBV surface antigen pre-S1 antigen and CDR1, CDR2 and CDR3 sequences of the antibody composition are disclosed, the nano antibody can specifically react with HBV-preS1, can effectively reduce the content of HBV virus in a cell model and a mouse in vivo model, has an obvious effect of treating hepatitis B, and can block the conversion of chronic hepatitis B to liver cancer.

According to a first aspect of the present invention, there is provided a nanobody against HBV-preS1, which is obtained by immunizing alpaca with HBV-preS1, and screening using phage display technology.

Further, the molecule is a nanobody against hepatitis b virus.

Further, the above molecule is a nanobody against HBV-preS 1.

Further, the above molecules are screened by phage display technology.

According to a second aspect of the invention, the invention provides a molecule comprising a CDR1, CDR2, CDR3 sequence.

Further, the sequence of the molecular CDR1 is SEQ ID NO: 1 to SEQ ID NO: 12 all sequences.

Further, the sequence of the molecular CDR2 is SEQ ID NO: 13 to SEQ ID NO: 21 all sequences.

Further, the sequence of the molecular CDR3 is SEQ ID NO: 22 to SEQ ID NO: 30 all sequences.

According to the third aspect of the present invention, the molecule provided by the present invention has a significant therapeutic effect on hepatitis b and liver cancer.

Further, the above molecule has a therapeutic effect on hepatitis B virus.

Further, the above molecule can reduce HBsAg levels.

Further, the above molecules can reduce HBeAg levels.

Furthermore, the molecule can have a therapeutic effect on liver cancer.

Drawings

FIG. 1 is an electrophoretogram of PCR amplification products in one embodiment of the present invention (M: DNA marker 2000; lanes 1 and 2: amplification products);

FIG. 2 is a diagram showing a double-cut electrophoresis of pHEN1 and VHH in FIG. 2.4 in one embodiment of the present invention (M: DNA marker 2000; lane 1: pHEN 1; lane 2: pHEN1 after sfil/notI cleavage; lane 3: VHH after sfil/notI cleavage);

FIG. 3 shows colony PCR verified library insertion rates in one embodiment of the invention (M: DNA marker 2000; lanes 1-48: randomly picked clones);

FIG. 4 is a sequence of the selected nanobody;

FIG. 5 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention can significantly reduce the serum HBsAg level in HBV transgenic mice compared to the Control nanobody (Control VHH);

FIG. 6 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention can significantly reduce the serum HBeAg level in the HBV transgenic mouse compared to the Control nanobody (Control VHH);

FIG. 7 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention can significantly reduce the HBV DNA level in the HBV transgenic mouse compared to the Control nanobody (Control VHH);

FIG. 8 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention significantly inhibits tumor growth of hepatoma carcinoma cells integrated with HBV genome in mice compared to the Control nanobody (Control VHH);

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.