Hepatitis B surface antigen (HBsAg) / CD3 dual-characteristic antibody and application thereof

Abstract

The invention provides a bispecific antibody capable of simultaneously and specifically targeting HBV (Hepatitis B Virus) surface antigen (HBsAg) and CD3 and a pharmaceutical composition of the bispecific antibody. Specifically, the invention provides a nucleic acid molecule for coding the bispecific antibody and an expression vector containing the nucleic acid molecule. The invention also provides application of the bicharacteristic antibody, the nucleic acid molecule and the expression vector in preparation of drugs for preventing or treating HBV infection and related diseases thereof.

Description

Technical Field

[0001] The present invention relates to a bispecific antibody, in particular to a bispecific homodimer antibody and use thereof in treating related diseases caused by HBV infection and related hepatocellular carcinoma. Background Art

[0002] Approximately 400 million people worldwide are chronically infected with the hepatitis B virus (HBV), accounting for 3.5% of the total population. According to WHO statistics, the Western Pacific and Africa are high-incidence areas for chronic hepatitis B (the incidence rate is 6%). Acute HBV infection may develop into chronic hepatitis B (the incidence rate in newborns and adults is 90% and 5% respectively), with a poor prognosis, leading to cirrhosis, liver failure and hepatocellular carcinoma, and causing 600,000 deaths each year.

[0003] The antiviral drugs currently used to treat HBV infection (such as tenofovir and entecavir) can inhibit viral replication, but cannot eliminate HBV covalently closed circular DNA (HBV cccDNA) carried by infected hepatocytes. Once the patient stops taking the medication, secondary infection of the HBV virus may be activated. Therefore, the complete cure of chronic hepatitis B depends on the elimination of the hepatocytes carrying HBVcccDNA. Studies have found that the clearance of HBV infection is related to the sustained control of the virus by effector T cells, and the progression of chronic hepatitis B infection is believed to be limited by virus-specific T cell responses. Therefore, there is an urgent need to develop a new immunotherapy method for chronic hepatitis B to restore HBV-specific T cell-mediated antiviral responses.

[0004] T cell redirecting bispecific antibodies refer to molecules containing more than two binding domains, wherein the first domain specifically binds to cell surface antigens on target cells / tissues (e.g., tumor-related / pathogenic specificity); the second domain specifically binds to T cell surface antigens (e.g., CD3). This dual-target binding molecule can redirect T cells to target cells / tissues, thereby eliminating target cells.

[0005] CD3 is a surface antigen of human T lymphocytes. It regulates T cell activation by forming a TCR / CD3 complex with TCRα / β or TCRγ / δ heterodimers co-expressed on the surface of T cells. Human CD3 mainly includes four subunits: CD3γ, CD3δ, CD3ε and CD3ζ. UniProtKB P07766 (CD3ε_HUMAN) describes the basic information of human CD3ε. The anti-CD3ε antibody SP34 (Yang SJ et al. The Journal of Immunology, 1986) has been reported in the literature. Another anti-CD3 antibody UCHT-1 is described in the prior art WO2000041474. Fred Hutchison Cancer Research Institute mentioned another anti-CD3 antibody BC-3 for Phase I / II trials of GvHD (Anasetti et al. Transplantation, 1992). The difference between SP34 and UCHT-1 and BC3 is that SP34 recognizes an antigenic epitope that only exists on CD3ε, while UCHT-1 and BC-3 recognize an antigenic epitope on the CD3εδ heterodimer (Salmeron et al., 1991). Antibody sequences having the same sequence as antibody SP34 are mentioned in WO2008119565, WO2008119566, WO2008119567, WO2010037836, WO2010037837 and WO2010037838. WO2007042261 mentions an antibody sequence with 96% homology to VH of antibody SP34. However, many existing CD3 antibodies, including SP34, have the problems of large toxic side effects and low drugability.

[0006] HBsAg refers to the hepatitis B virus envelope antigen present on the surface of virus-infected liver cells. HBV surface antigens include small, medium and large surface antigens, which are encoded and transcribed and translated by the same open reading frame (ORF), but differ from each other in the N-terminal part. The large surface antigen contains peptides that are neither present in the medium nor in the small surface antigen, and the medium surface antigen contains peptides that are present in the large surface antigen but not in the small surface antigen. The small surface antigen is the C-terminal part present in the medium and large surface antigens. It is speculated that although the virus particles are released into intracellular vesicles, many HBV surface antigens will still be integrated into the inner membrane of the endoplasmic reticulum. During vesicle transport, the inner membrane can fuse with the cell membrane, resulting in the display of HBV surface antigens on the surface of infected cells. Therefore, HBsAg provides an ideal therapeutic target for targeting viruses for the treatment of chronic hepatitis B and related diseases. Summary of the invention

[0007] On the one hand, the present application provides a structural form suitable for HBsAg / CD3 bispecific antibodies, wherein one antigen binding domain is an antibody in the form of IgG, and the other antigen binding domain is in the form of scFv single-chain antibody or VHH nanobody. Preferably, the CD3 antibody is in the form of IgG1 structure, and the anti-HBsAg antibody is in the form of scFv and fused to the N-terminus or C-terminus of the CD3 antibody heavy chain. On the other hand, the present application provides a humanized CD3 antibody or its antigen binding fragment with significantly reduced affinity, and a bispecific antigen binding molecule or multispecific antigen binding molecule comprising the CD3 antibody or its antigen binding fragment of the present application.

[0008] In a first aspect, the present application provides a HBsAg / CD3 bispecific antigen binding molecule, wherein the bispecific antigen binding molecule comprises:

[0009] (a) a first polypeptide chain, comprising a heavy chain of an anti-human CD3 antibody (anti-CD3 VH-CH1-Fc), wherein the heavy chain is fused to a single-chain variable fragment of an anti-hepatitis B surface antigen antibody (HBsAg scFv), and the hepatitis B surface antigen comprises a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B virus; and

[0010] (b) A second polypeptide chain comprising a light chain of an anti-human CD3 antibody (anti-CD3 VL-CL).

[0011] In some embodiments, the HBsAg scFv is fused to the N-terminus or C-terminus of the anti-CD3 antibody heavy chain; preferably, fused to the C-terminus of the CD3 antibody heavy chain. In some embodiments, the HBsAg scFv is fused to the N-terminus or C-terminus of the anti-CD3 antibody heavy chain via a glycine-serine linker. In some embodiments, the glycine-serine linker is selected from one or a combination of (GGGGS)n as shown in SEQ ID NO:33, (GGGSG)n as shown in SEQ ID NO:34, (GSGGG)n as shown in SEQ ID NO:35, (GSGGGP)n as shown in SEQ ID NO:36, (GSEPS)n as shown in SEQ ID NO:37, (GGEGGGP)n as shown in SEQ ID NO:38, (GGEGGGSEGGGS)n as shown in SEQ ID NO:39, and (GGGSGGGG)n as shown in SEQ ID NO:40, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0012] In some embodiments, the HBsAg scFv comprises HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6.

[0013] In some embodiments, the anti-human CD3 antibody comprises HCDR1 of SEQ ID NO:11, HCDR2 of SEQ ID NO:12, HCDR3 of SEQ ID NO:13, LCDR1 of SEQ ID NO:14, LCDR2 of SEQ ID NO:15, and LCDR3 of SEQ ID NO:16.

[0014] In some embodiments, the HBsAg scFv comprises a VH domain and a VL domain; the VH domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7, and the VL domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:8.

[0015] In some embodiments, the VH domain and VL domain of the HBsAg scFv are connected by a YOL linker, and the YOL linker comprises the amino acid sequence shown in SEQ ID NO:9.

[0016] In some embodiments, the HBsAg scFv comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:10.

[0017] In some embodiments, the CD3 antibody of the HBsAg / CD3 bispecific antibody is a humanized antibody, and its affinity for CD3 is significantly reduced. In some embodiments, the humanized anti-human CD3 antibody comprises a VH domain and a VL domain: the VH domain amino acid sequence is as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, or an amino acid sequence having at least 90% sequence identity thereto; in some embodiments, the VL domain amino acid sequence is as shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or an amino acid sequence having at least 90% sequence identity thereto.

[0018] In some preferred embodiments, the VL domain amino acid sequence of the CD3 antibody is as shown in SEQ ID NO: 25. In other preferred embodiments, the VH domain amino acid sequence of the CD3 antibody is as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and the VL domain amino acid sequence of the CD3 antibody is as shown in SEQ ID NO: 25 or an amino acid sequence having at least 90% sequence identity thereto.

[0019] In some embodiments, the bispecific antibody comprises a heavy chain constant region selected from IgG1 subtype, IgG2 subtype, IgG3 subtype or IgG4 subtype and conventional variants thereof, and comprises a light chain constant region selected from κ subtype or λ subtype and conventional variants thereof. Preferably, the constant region is IgG1-κ subtype. In some embodiments, the bispecific antibody further comprises an alanine at position 234 of the heavy chain and an alanine at position 235, wherein the residue numbering is according to the EU index.

[0020] In some embodiments, the bispecific antibody of the present application comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises from the N-terminus to the C-terminus: i. Domain I, comprising three subdomains, wherein subdomain IA comprising a heavy chain variable region (VH) can bind to a recognition epitope of a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B virus, and comprises an amino acid sequence as shown in SEQ ID NO:7; wherein subdomain IB comprises a YOL linker, and the amino acid sequence of the YOL linker is as shown in SEQ ID NO:9; wherein subdomain IC comprising a light chain variable region (VL) can bind to a recognition epitope of a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B virus, and comprises an amino acid sequence as shown in SEQ ID NO:8; ii. Domain II, comprising a glycine-serine linker selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; iii. domain III, comprising an antibody heavy chain variable region (VH) that can bind to human CD3 and comprising at least one of the amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24; iv. domain IV, wherein the amino acid sequence of the constant region of the heavy chain is as shown in SEQ ID NO 30 or SEQ ID NO:31; and the second polypeptide chain comprises, from N-terminus to C-terminus: i. domain I, comprising an antibody light chain variable region (VL) that can bind to human CD3 and comprising at least one of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID At least one of the amino acid sequences shown in NO:29; ii. Domain II, wherein the sequence of the light chain constant region is as shown in SEQ ID NO:32.

[0021] In some embodiments, the bispecific antibody of the present application comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: i. Domain I, comprising an antibody heavy chain variable region (VH) that can bind to human CD3 and comprises at least one of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; ii. Domain II, wherein the constant region sequence of the heavy chain comprises at least one of the amino acid sequences shown in SEQ ID NO: 30 and SEQ ID NO: 31; iii. Domain III, comprising a glycine-serine linker selected from SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40. NO:40, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; iv. domain IV, comprising three subdomains, wherein subdomain IVA comprising a heavy chain variable region (VH) can bind to a recognition epitope of a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B virus, and comprises an amino acid sequence as shown in SEQ ID NO:7; wherein subdomain IVB comprises a YOL linker, the amino acid sequence of the YOL linker is as shown in SEQ ID NO:9; wherein subdomain IVC comprising a light chain variable region can bind to a recognition epitope of a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B virus, and comprises an amino acid sequence as shown in SEQ ID NO:8; and the second polypeptide chain comprises from the N-terminus to the C-terminus: i. domain I, comprising an antibody light chain variable region (VL) that can bind to human CD3 and a linker sequence, the amino acid sequence comprising SEQ ID NO:25, SEQ ID One of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29; ii. domain II, wherein the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:32.

[0022] In some embodiments, the bispecific antibody of the present application comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from the N-terminus to the C-terminus: i. domain I, comprising a single-chain variable fragment as shown in SEQ ID NO: 10, wherein the scFv can bind to a recognition epitope of a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B; ii. domain II, comprising a glycine-serine linker selected from one of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; iii. domain III, wherein the heavy chain variable region can bind to a recognition epitope of human CD3, and comprises SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 NO:23 and one of the amino acid sequences shown in SEQ ID NO:24; iv. domain IV, wherein the heavy chain constant region includes one of the amino acid sequences shown in SEQ ID NO:30 and SEQ ID NO:31; and the second polypeptide chain comprises from N-terminus to C-terminus: i. domain I, including an antibody light chain variable region (VL) that can bind to an antigenic epitope of human CD3, and including one of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29; ii. domain II, wherein the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:32.

[0023] In some embodiments, the bispecific antibody of the present application comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises in the N-terminal to C-terminal direction: i. domain I comprises an antibody heavy chain variable region that can bind to human CD3, and the amino acid sequence thereof is one of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; ii. domain II, wherein the amino acid sequence of the constant region of the heavy chain comprises one of SEQ ID NO: 30 and SEQ ID NO: 31; iii. domain III, comprising a glycine-serine linker selected from one of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; iv. domain IV, comprising SEQ ID NO: NO:10 shows a single-chain variable fragment (scFv), wherein the scFv can bind to a recognition epitope of a small surface antigen of hepatitis B virus, a medium surface antigen of hepatitis B virus or a large surface antigen of hepatitis B virus; and the second polypeptide chain comprises, from N-terminus to C-terminus: i. Domain I, comprising an antibody light chain variable region (VL) that can bind to human CD3 and comprises one of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29; ii. Domain II, wherein the sequence of the light chain constant region is as shown in SEQ ID NO:32.

[0024] In some embodiments, the sequence of the glycine-serine linker is as shown in SEQ ID NO: 31, wherein n=4.

[0025] In some embodiments, the first polypeptide chain of the present application includes one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52. In some embodiments, the second polypeptide chain includes one of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57.

[0026] In some embodiments, the first polypeptide chain comprises one of the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID: NO 53. In an exemplary embodiment, the bispecific antibody is BsAb2-5-011.

[0027] In some embodiments, the first polypeptide chain comprises one of the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 54. In an exemplary embodiment, the bispecific antibody is BsAb2-5-030.

[0028] In some embodiments, the first polypeptide chain comprises one of the amino acid sequences shown in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52; and the second polypeptide chain comprises SEQ ID NO: 54. In an exemplary embodiment, the bispecific antibody is BsAb2-6-017.

[0029] In some embodiments, the first polypeptide chain comprises one of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52; and the second polypeptide chain comprises SEQ ID NO: 53. In an exemplary embodiment, the bispecific antibody is one of BsAb2-6-001, BsAb2-6-006, BsAb2-6-011 and BsAb2-6-016.

[0030] The second aspect of the present application provides a nucleic acid molecule encoding the bispecific antibody described in the first aspect of the present application.

[0031] The third aspect of the present application provides an expression vector comprising the nucleic acid described in the second aspect of the present application.

[0032] The fourth aspect of the present application provides a pharmaceutical composition, which comprises the bispecific antibody described in the first aspect of the present application, the nucleic acid described in the second aspect of the present application or the expression vector described in the third aspect of the present application, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0033] The fifth aspect of the present application provides the use of the bispecific antibody described in the first aspect of the present application, the nucleic acid described in the second aspect of the present application, the expression vector described in the third aspect of the present application, and the pharmaceutical composition described in the fourth aspect of the present application in the preparation of a drug for treating or preventing HBV infection and / or a disease caused by HBV infection, wherein the disease caused by HBV infection is selected from one or more of liver fibrosis, cirrhosis or liver cancer.

[0034] In the sixth aspect of the present application, a CD3 antibody or an antigen-binding fragment thereof is provided. Surprisingly, it is found that the affinity level of the CD3 antibody of the present application for binding to the CD3 antigen is significantly reduced. The use of the same to prepare bispecific molecules can overcome the excessive activation of T cells caused by the excessively high affinity of the CD3 antibody in the prior art, which in turn causes side effects such as T cell exhaustion and cytokine storm.

[0035] In some embodiments, the CD3 antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain: the VH domain amino acid sequence is as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or an amino acid sequence thereof having at least 90% sequence identity; and / or

[0036] The VL domain amino acid sequence is shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or an amino acid sequence thereof having at least 90% sequence identity.

[0037] In some embodiments, the CD3 antibody VH domain amino acid sequence is shown as SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24; and the VL domain amino acid sequence is shown as SEQ ID NO:25.

[0038] The present application also provides a bispecific antigen-binding molecule and a multispecific antigen-binding molecule comprising the CD3 antibody or antigen-binding fragment thereof described in the sixth aspect of the present application.

[0039] In some embodiments, the bispecific antigen-binding molecule comprising the CD3 antibody or antigen-binding fragment thereof according to the sixth aspect of the present application further comprises a second antigen-binding domain in addition to the CD3 antibody or antigen-binding fragment thereof.

[0040] In some embodiments, the second antigen binding domain comprises a virus-associated antigen binding domain, a tumor-associated antigen binding domain.

[0041] In some embodiments, the second antigen binding domain comprises an antibody or fragment thereof that can bind to a second antigen, a TCR or a soluble fragment thereof, a receptor or receptor extracellular domain corresponding to any antigen, a ligand or a ligand extracellular domain, and one or more of "derivatives" and "analogs" of the above domains.

[0042] In some embodiments, the second antigen binding domain is a hepatitis B surface antigen binding domain, and the hepatitis B surface antigen includes hepatitis B virus small surface antigen, hepatitis B virus medium surface antigen or hepatitis B virus large surface antigen.

[0043] In some embodiments, the hepatitis B surface antigen binding domain is an anti-hepatitis B surface antigen single chain antibody (HBsAg scFv).

[0044] On the other hand, the present application provides a polynucleotide molecule encoding the CD3 antibody or antigen-binding fragment thereof described in the sixth aspect of the present application. On the other hand, the present application provides a polynucleotide molecule encoding a bispecific antigen-binding molecule or a multispecific antigen-binding molecule comprising the CD3 antibody or antigen-binding fragment thereof described in the sixth aspect of the present application. Compared with the prior art, the beneficial effects of the present application are:

[0045] On the one hand, the present application constructs a structural form of a bispecific antibody that specifically binds CD3 and HBsAg, with HBsAgscFv fused to the C-terminus of the heavy chain of the IgG-form CD3 antibody, and has the performance of simultaneously targeting target cells and T cells infected with HBV, bridging T cells to target cells and forming immune synapses, activating T cells, and then completing the killing of target cells through cytotoxic and non-cytotoxic effects; at the same time, it has extremely strong HBV virus neutralization performance. Therefore, the bispecific antibody of the present application can efficiently neutralize free HBV virus particles in the blood, efficiently target and remove HBV-infected hepatocytes, and the released cytokines have both viral clearance functions, and effectively treat HBV virus-induced infections and liver cancer through multiple mechanisms. At the same time, the bispecific antibody of the present application has a stable structure, is easy to express and purify, and has good pharmacological development.

[0046] On the other hand, the present application provides a CD3 antibody or its antigen-binding fragment with significantly reduced binding affinity to the CD3 antigen. The use of the same to prepare bispecific antigen-binding molecules and multispecific antigen-binding molecules can overcome the excessive activation of T cells caused by the excessively high affinity of CD3 antibodies in the prior art, which in turn induces side effects such as T cell exhaustion and cytokine storm, and effectively improves the limitations of T cell engager drugs such as the narrow dosing window.

[0047] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form further embodiments of the invention.

[0048] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 is a schematic diagram of four bispecific HBsAg / CD3 antibodies of the present invention. It should be noted that the antibodies corresponding to the IgG and scFv domains can be exchanged (for example, the anti-CD3 binding molecule is scFv, and the anti-HBsAg binding molecule is IgG). FIG. 1 (A) and (B) contain a single-chain fragment (scFv) of the hepatitis B virus surface antigen (HBsAg) fused to the N / C terminus of the heavy chain of the anti-CD3 IgG antibody; FIG. 1 (C) and (D) contain a single-chain fragment (scFv) binding to the human CD3 antigen fused to the N / C terminus of the heavy chain of the anti-HBsAg IgG antibody.

[0050] Figure 2 shows the ELISA test results of HBsAg / CD3 bispecific antibodies binding to HBsAg. Figures 2 (A) and (B) show the ELISA test results of BsAb2-5 binding to HBsAg, and Figures 2 (C) and (D) show the ELISA test results of BsAb2-6 binding to HBsAg, respectively.

[0051] Figure 3 shows the ELISA test results of HBsAg / CD3 bispecific antibody binding to human CD3ε. Figures 3 (A) and (B) show the ELISA test results of BsAb2-5 binding to human CD3ε, and Figures 3 (C) and (D) show the ELISA test results of BsAb2-6 binding to human CD3ε.

[0052] Figure 4 shows the ELISA test results of HBsAg / CD3 bispecific antibody binding to rhesus monkey CD3ε. Figures 4(A) and (B) show the ELISA test results of BsAb2-5 binding to rhesus monkey CD3ε, and Figures 4(C) and (D) show the ELISA test results of BsAb2-6 binding to rhesus monkey CD3ε, respectively.

[0053] Figure 5 shows the dose-dependent binding of HBsAg / CD3 bispecific antibodies to CD3-positive cells and HBsAg-positive cells, respectively. Figures 5(A) and (B) are the average fluorescence intensities of bsAb2-5 and bsAb2-6 binding to CD3+T cells, respectively, and Figure 5(C) is the average fluorescence intensities of bsAb2-5 and bsAb2-6 binding to HepG2-LMS-660 cells.

[0054] Figure 6 shows that HBsAg / CD3 bispecific antibody-induced T cell activation is dose-dependent. Figure 6 (A) and (B) show the expression of NFAT reporter gene induced by BsAb2-5 and BsAb2-6, respectively, with the HBsAg-uncoated group as a negative control.

[0055] Figure 7 shows that the T cell proliferation induced by HBsAg / CD3 bispecific antibody is dose-dependent. Figure 7 (A) and (B) respectively analyze the T cell proliferation induced by BsAb2-5 and BsAb2-6 by CSFE labeling flow cytometry.

[0056] Figure 8 The cytokine release of human PBMC stimulated by HBsAg / CD3 bispecific antibody. The huTh1 / Th2CBA kit II (BD, 551809) was used to detect the cytokine levels in the supernatant of human PBMC stimulated by BsAb2-6, where Figure A: IFN-γ; Figure B: TNF-α; Figure C: IL-10; Figure D: IL-6; Figure E: IL-4; Figure F: IL-2.

[0057] Fig. 9It is shown that HBsAg / CD3 bispecific antibodies mediate cytotoxic effects of human T cells on HBs+ cell lines. HBs+ HepAD38 cells were co-cultured with bsAb2-6-001, bsAb2-6-006, bsAb3-6-011, bsAb6-016, bsAb2-16-017 or bsAb2-5-011 and activated T cells as effector cells (E:T=3:1).

[0058] Fig.10 Shown are the binding affinities of HBsAg / CD3 bispecific antibodies and recombinant human CD3ε measured by surface plasmon resonance (SPR).

[0059] Fig.11 Shown is the binding affinity of HBsAg / CD3 bispecific antibody to cynomolgus monkey CD3ε measured by surface plasmon resonance (SPR).

[0060] Fig.12 Shown is the binding affinity of the HBsAg / CD3 bispecific antibody and HBsAg measured by surface plasmon resonance (SPR).

[0061] Fig.13 Shown is the SEC-HPLC characterization of the anti-HBsAg / anti-CD3 bispecific antibody.

[0062] Fig.14 The in vitro HBV neutralization efficacy of HBsAg / CD3 bispecific antibody in Huh7-NTCP cells is shown. The HBeAg level in the supernatant was measured using an HBeAg ELISA kit.

[0063] Figure 15 shows the anti-tumor activity of HBsAg / CD3 bispecific antibodies on the HBs+HepAD38-CDX model. Figure 15 (A) shows the average tumor volume of mice in the vehicle group, control antibody or BsAb2-6-011 treatment group, mean ± SEM. Figure 15 (B) shows the characteristics of human leukocytes in the peripheral blood of mice treated with BsAb2-6-011 or control antibody. Peripheral blood cells were collected at the end of the experiment, stained with human CD45 antibodies and measured by flow cytometry. Figure 15 (C) shows the average body weight of mice in the vehicle group, control antibody or bsAb2-6-011 treatment group, mean ± SEM.

[0064] Figure 16 shows the pharmacokinetic characteristics of HBsAg / CD3 bispecific antibodies in cynomolgus monkeys. Figure 16 (A) Pharmacokinetic characteristics of blood samples at different time points before and after administration of BsAb2-6-011. Figure (B) Body weight changes of cynomolgus monkeys after administration of 5 mpk BsAb2-6-011. DETAILED DESCRIPTION

[0065] the term:

[0066] In order to make the present invention more easily understood, some technical and scientific terms of the present invention are explained before describing the embodiments. Unless otherwise clearly defined elsewhere in the present application documents, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0067] The "bispecific antigen binding molecule" of the present application refers to a molecule capable of binding to two different antigens or epitopes, i.e., comprising two different antigen binding domains, which are functionally connected or conjugated by chemical coupling, gene fusion, non-covalent binding or other means. The two different antigen binding domains are selected from a specific antibody or a fragment thereof, a TCR or a soluble fragment thereof, a receptor or a receptor extracellular domain corresponding to any antigen, a ligand or a ligand extracellular domain, and one or more "derivatives" and "analogs" of the above domains.

[0068] The “antibodies” mentioned in the present invention include not only complete antibodies, but also fragments, polypeptide sequences, and derivatives and analogs thereof with antigen binding activity.

[0069] The "antigen-binding fragment" refers to one or more portions of a full-length antibody that retain the ability to bind to an antigen (e.g., HER2) and compete with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology (Paul W, 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, which contain at least a portion of an antibody sufficient to confer specific antigen binding ability on a polypeptide. The antigen binding portion of an antibody (e.g., the above-mentioned antibody fragment) can be obtained from a given antibody (e.g., monoclonal antibody 2E12) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen binding portion of the antibody can be screened for specificity in the same manner as for intact antibodies. The term "Fd fragment" means an antibody fragment consisting of a VH and CH1 domain; the term "Fv fragment" means an antibody fragment consisting of a single arm of an antibody VL and VH domain; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0070] The terms "derivatives" and "analogs" refer to polypeptides that substantially retain the same biological function or activity as an antibody, TCR or a soluble fragment thereof, an antigen receptor or a receptor extracellular domain, an antigen ligand or a ligand extracellular domain. The derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a fusion protein formed with a 6His tag). According to the teachings herein, these derivatives and analogs are within the scope known to those skilled in the art.

[0071] The "virus-related antigens" of this application refer to antigens related to a variety of known viruses, including DNA viruses, RNA viruses, protein viruses (such as prions) classified from the perspective of genetic material; true viruses (Euvirμs, referred to as viruses) and subviruses (Subvirμs, including viroids, pseudoviruses, and prions) classified from the perspective of virus structure; classified from the perspective of host type: bacteriophages (bacterial viruses), plant viruses (such as tobacco mosaic viruses), animal viruses (such as avian influenza viruses, smallpox viruses, HIV, etc.); classified from the perspective of morphology: spherical viruses, baculoviruses, brick viruses, coronaviruses (such as SARS-CoV-2), filoviruses, rotaviruses, enveloped spherical viruses, viruses with spherical heads, and insect viruses enclosed in inclusion bodies. It can be an antigen presented by the virus itself or an antigen formed after the virus infects a cell.

[0072] As used herein, the term "hepatitis B virus" is used interchangeably with the term "HBV" and refers to a well-known non-cytopathic hepatotropic DNA virus belonging to the family Hepadnaviridae.

[0073] The HBV genome is a partially double-stranded circular DNA with overlapping reading frames. Based on size, there are four transcripts encoded by the HBV genome (which may be referred to herein as "genes" or "open reading frames"). These include open reading frames called C, X, P and S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S is the gene encoding surface antigen (HBsAg). The HBsAg gene is a long open reading frame containing three in-frame "start" (ATG) codons, thereby producing three different sizes of polypeptides, called large, medium and small S antigens, i.e. large surface antigen, medium surface antigen and small surface antigen, pre-S1+pre-S2+S, pre-S2+S or S. In addition to decorating the HBV envelope, surface antigens are also part of subviral particles, which are produced in much larger amounts than viral particles and play a role in immune tolerance and sequestering anti-HBsAg antibodies, thereby allowing infectious particles to escape immune detection.

[0074] The "tumor antigen" in this application generally refers to an antigenic substance that appears or is overexpressed during the occurrence and development of a tumor. Tumor antigens are divided into tumor-specific antigens and tumor-associated antigens according to their specificity.

[0075] Tumor specific antigen (TSA) is a new antigen that is specific to tumor cells or exists only in certain tumor cells but not in normal cells. This type of antigen is confirmed by transplanting tumors between animals of the same species, so it is also called tumor specific transplantation antigen (TSTA) or tumor rejection antigen (TRA). Tumor antigens induced by chemical or physical factors, spontaneous tumor antigens, and virus-induced tumor antigens mostly belong to this category.

[0076] Tumor-associated antigen (TAA) refers to antigens that are not specific to tumor cells but also exist in normal cells and other tissues, but their content increases significantly when the cells become cancerous. This type of antigen only shows quantitative changes and has no strict tumor specificity. Embryonic antigens are a typical example.

[0077] In this application, "CD3e" and "CD3ε" have the same meaning.

[0078] The term "fusion" when referring to a polypeptide or polynucleotide refers to a form of the polypeptide or polynucleotide that does not exist in nature, as a non-limiting example, by combining polynucleotides or polypeptides that do not normally occur together.

[0079] The term "cross-reactivity" refers to the ability of the antibodies described herein to bind to antigens from different species. For example, the human CD3 antibodies described herein may also bind to CD3 from other species (e.g., cynomolgus CD3). Cross-reactivity can be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or binding to cells that physiologically express an antigen or otherwise interacting with cell functions that physiologically express an antigen. Examples of assays known in the art for determining binding affinity include surface plasmon resonance (e.g., Biacore) or similar techniques (e.g., Octet).

[0080] The technical solution of the present application is illustrated by the following non-limiting examples.

[0081] The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those in Sambrook et al. in Molecular cloning: A Laboratory Manual (Molecular cloning-A Laboratory manual. 3rd edition. 2001), or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise specified, the reagents and materials involved in the text are all commercially available, or can be prepared by those skilled in the art according to common knowledge.

[0082] Example 1 Preparation of HBsAg / CD3 bispecific antibody

[0083] Humanization of anti-CD3 antibodies:

[0084] The CDR sequence of antibody SP-34 was analyzed using the kabat system, and the results showed that the amino acid sequences of HCDR1-3 of SP-34 were shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the VH sequence of SP-34 antibody (Murine CD3 VH) was shown in SEQ ID NO: 17. The amino acid sequences of LCDR1-3 of antibody SP-34 were shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the VL sequence of SP-34 antibody (Murine CD3 VL) was shown in SEQ ID NO: 18.

[0085] The degree of humanization of antibody SP-34 was improved by chimerizing the framework regions of the heavy and light chain variable regions of human immunoglobulins.

[0086] By comparing the amino acid sequence of the heavy chain variable region of the mouse antibody SP-34 with the amino acid sequence of the known human germline immunoglobulin heavy chain, it was confirmed that the heavy chain variable region of the antibody SP-34 has 79% and 74% homology with the human germline IGHV3-73*01 and IGHV3-23*05, respectively, and can serve as a chimeric acceptor for the SP-34 heavy chain CDR.

[0087] By comparing the amino acid sequence of the light chain variable region of the mouse antibody SP-34 with the amino acid sequence of the known human immunoglobulin light chain, it was confirmed that the light chain variable region of the antibody SP-34 has 60.0% and 57% homology with the human germline IGLV7-43*01 and IGLV8-61*01, respectively, and can serve as a chimeric acceptor for the SP-34 light chain CDR.

[0088] In order to improve the affinity of the SP-34 antibody, some key amino acids in the chimeric heavy chain and light chain framework regions can be backmutated. SEQ ID NO: 19 (Humanized CD3 mAb VH1), SEQ ID NO: 20 (Humanized CD3mAbVH2), SEQ ID NO: 21 (Humanized CD3 mAb VH3), SEQ ID NO: 22 (Humanized CD3 mAb VH4), SEQ ID NO: 23 (Humanized CD3 mAb VH5), SEQ ID NO: 24 (Humanized CD3 mAb VH6) are the amino acid sequences of the heavy chain variable region of humanized SP-34; SEQ ID NO: 25 (Humanized CD3 mAb VL1), SEQ ID NO: 26 (Humanized CD3 mAb VL2), SEQ ID NO: 27 (Humanized CD3 mAb VL3), SEQ ID NO: 28 (Humanized CD3 mAb VL4), SEQ ID NO: 29 (Humanized CD3 mAb VL5) are the amino acid sequences of the light chain variable region of humanized SP-34.

[0089] Construction and production of expression vector for HBsAg / CD3 bispecific antibody:

[0090] Genewiz was commissioned to synthesize the DNA sequence encoding the HBsAg / CD3 bispecific antibody (the amino acid sequence encoded by it is shown in Table 1), and subcloned into the pcDNA3.1 vector through the restriction enzyme sites XbaI and NheI, respectively.

[0091] The BsAb2-5 series of bispecific antibodies are anti-HBsAg antibody C8 scFv (SEQ ID NO: 10) fused to the N-terminus of the heavy chain of SP-34 antibody (bispecific antibody number BsAb2-5) or humanized SP-34 antibody (bispecific antibody number BsAb2-5-001 to BsAb-2-5-030) through a linker; the BsAb2-6 series of bispecific antibodies are anti-HBsAg antibody C8 scFv (SEQ ID NO: 10) fused to the C-terminus of the heavy chain of SP-34 antibody (bispecific antibody number BsAb2-6) or humanized SP-34 antibody (bispecific antibody number BsAb2-6-001 to BsAb2-6-030) through a linker. The light and heavy chain sequences corresponding to each bispecific antibody and the combination of the VH and VL humanized sequences of the anti-CD3 antibody part are shown in Table 1. For example: BsAb2-5-001 bispecific antibody, its heavy chain is arranged from N-terminus to C-terminus as: C8 scFv-VH1-CH1-Fc, and the light chain is VL1-CL. Finally, two identical heavy chains and two identical light chains form homodimers through disulfide bonds; among them, C8 scFv (SEQ ID NO: 10) is a single-chain antibody against HBsAg; VH1 and VL1 are the humanized heavy chain variable region variant 1 and light chain variable region variant 1 of the murine antibody SP-34, respectively. Another example: BsAb2-6-012 bispecific antibody, its heavy chain is arranged from N-terminus to C-terminus as follows: VH3-CH1-Fc-C8 scFv, and the light chain is VL2-CL, wherein C8 scFv (SEQ ID NO: 10) is a single-chain antibody against HBsAg; VH3 and VL2 are respectively the humanized heavy chain variable region variant 3 and light chain variable region variant 2 of the murine antibody SP-34.

[0092] Table 1 The light and heavy chain sequences corresponding to the bispecific antibodies and the humanized VH and VL combinations of anti-CD3 antibodies

[0093]

[0094]

[0095] Expi293 cell expression system:

[0096] The Expi293 cell expression system (Invitrogen) expresses bispecific antibodies by transiently co-transfecting plasmids encoding antibody light and heavy chains (usually, heavy chains and heavy chain variants are mixed with light chains and light chain variants in a 1:1 ratio). Briefly, a mixture of plasmids encoding antibody light and heavy chains and Expi293 fectin (Invitrogen, #A14635CN) is transfected into Expi293 cells suspended in a shaking Erlenmeyer flask or stirred fermenter. 100 mL of 1x10 6cells / mLExpi293 cells were inoculated in 1L shaking Erlenmeyer flasks (Corning) and cultured at 125 rpm and 8% CO2. The next day, the density of Expi293 cells reached 2.5x10 6 cells / mL, and the viability is >95%, prepare 10mL Opti-MEM (Invitrogen) and add 100μg total plasmid DNA (1μg / mL, 50-μg heavy chain plasmid and 50μg light chain plasmid), which is solution A; prepare another 10mL Opti-MEM, and add 260μL Expi293 fectin (2.6μL / mL), which is solution B; after incubating the mixture of solution A and solution B at room temperature for 20 minutes, slowly add Expi293 cell culture medium. According to the instructions, enhancers 1 and 2 were added on the second day after transfection. After 5 days of suspension culture, the supernatant containing secreted antibodies was collected, and the antibodies were either directly purified from the supernatant or the supernatant was frozen and stored.

[0097] purification:

[0098] Purify the target protein from the filtered cell culture supernatant according to the standard procedure of affinity chromatography column. In brief, BsAb-2-5 and BsAb-2-6 were purified by protein A affinity chromatography (equilibrium buffer: 20mM sodium citrate, 20mM sodium phosphate, pH 7.5; elution buffer: 20mM sodium citrate, pH 3.0). After obtaining the target protein at pH 3.0, the pH of the sample solution was immediately adjusted to 7.0. Protein multi-aggregation was removed by size exclusion chromatography (Superdex200, GE Healthcare) with PBS (pH 7.2) or 20mM histidine, 50mM NaCl (pH 5.5) as the mobile phase to improve the purity of the antibody. If necessary, the samples can be combined and concentrated using Millipore Amicon Μltra (30MW) ultrafiltration tubes to obtain antibody standards, which can be aliquoted and stored at -20°C or -80°C. Some samples can also be provided for protein characterization analysis such as SDS-PAGE, SEC-HPLC or mass spectrometry.

[0099] Protein determination:

[0100] The molar extinction coefficient was calculated based on the antibody amino acid sequence (Pace et al., Protein Science, 1995, 4, 2411-1423), and the concentration of the antibody standard was measured at a wavelength of 280 nm.

[0101] Example 2 Binding properties of HBsAg / CD3 bispecific antibodies

[0102] The binding properties of the HBsAg / CD3 bispecific antibody in Example 1 to HBsAg, human CD3ε and rhesus CD3ε recombinant protein were respectively detected by standard ELISA experiments. FIG. 2A to FIG. 2D Table 2 shows that all BsAb-2-5 and BsAb-2-6 series constructs have similar super strong binding ability to HBsAg recombinant protein.

[0103] Table 2 Binding ability of HBsAg / CD3 bispecific antibody to human CD3ε, rhesus monkey CD3ε and HBsAg recombinant protein

[0104]

[0105]

[0106] The binding properties of BsAb-2-5 and BsAb-2-6 series constructs to human CD3ε and rhesus CD3ε are shown in FIG. 3A to FIG. 3D , 4A to 4D As shown in Table 2, compared with other molecules, BsAb2-5-001, BsAb2-5-006, BsAb2-5-011, BsAb2-5-016, BsAb2-5-021, BsAb2-5-026, BsAb2-6-001, BsAb2-6-006, BsAb2-6-011, BsAb2-6-016, BsAb2-6-021, and BsAb2-6-026 molecules derived from the combination with the VL1 variant have significantly reduced binding ability to human and rhesus monkey CD3ε recombinant proteins. However, bispecific antibodies derived from the VL4 of BsAb2-5-004, BsAb2-5-009, BsAb2-5-014, BsAb5-019, BsAb2-5-024, BsAbB2-5-029, BsAb2-6-004, BsAb2-6-009, BsAb-2-6-014, BsAb2-6-019, BsAb2-6-024, and BsAb2-6-029 molecules showed no cross-binding to human CD3ε and rhesus CD3ε.

[0107] The binding properties of the HBsAg / CD3 bispecific antibody in Example 1 to CD3ε on the surface of the human leukemia cell line Jurkat and HBsAg on the surface of the human liver cancer cell line HepG2-LMS-660 were detected by flow cytometry. Specifically, the two cell lines were collected in FACS buffer (PBS containing 2% BSA), counted and adjusted to a cell density of 2x10 6cells / mL. The cell suspension was then dispensed into a U-shaped 96-well plate at a volume of 50 μL per well, and 50 μL of HBsAg / CD3 bispecific antibody or human IgG isotype diluted in gradient with FACS buffer was added as a negative control (concentration range of 300 nM to 0.017 nM). After incubation at room temperature for 60 minutes, the cells were collected by centrifugation (3 minutes, 300 x g) and washed twice with 150 μL / well FACS buffer. Then, 100 μL of 5000-fold diluted APC-labeled anti-human Fc secondary antibody (Jackson, #615-605-214) was added to resuspend the cells and incubated at room temperature for 30 minutes. Wash 3 times with 150 μL / well FACS, collect the cells by centrifugation, resuspend the cells with 100 μL FACS buffer, and detect on a flow cytometer (Beckman). Finally, FlowJo was used to process the data and GraphPad Prism was used for graph fitting.

[0108] FIG. 5A to FIG. 5B The binding properties of BsAb2-5 and BsAb2-6 series constructs to CD3ε on the surface of Jurkat cells are shown. All tested BsAb-2-6 series constructs can bind to CD3ε on the surface of Jurkat cells. Among them, the binding activity of BsAb-2-6, BsAb-2-6-017, BsAb-2-6-018 and BsAb-2-6-020 is optimal, while the binding properties of BsAb-2-6-001, BsAb-2-6-006, BsAb-2-6-011, BsAb-2-6-016, BsAb-2-6-021 and BsAb-2-6-026 derived from the combination of VL1 variants are significantly reduced. Similarly, compared with the parent bsAb2-5, bsAb2-5-006, bsAb2-5-011 and bsAb2-5-021 are almost not bound to hCD3ε. This result is consistent with the binding characteristics of hCD3ε recombinant protein in Table 2.

[0109] Figure 5C The binding properties of BsAb-2-5 and BsAb-2-6 constructs to HBsAg expressed on the surface of HepG2-LMS-660 cells are shown. All tested BsAb-2-5 and BsAb-2-6 constructs can bind to HBsAg on the surface of HepG2-LMS-660 cells with comparable binding abilities. This result indicates that the antibody binding properties at the HBsAg end are unchanged under the two different structures of BsAb-2-5 and BsAb-2-6.

[0110] Example 3 HBsAg / CD3 bispecific antibody induces T cell activation

[0111] The NFAT luciferase reporter gene Jurkat engineered cells purchased from BPS Bioscience integrate the gene that stably expresses firefly luciferase under the control of the NFAT response element into the genome of Jurkat cells, which can be used to evaluate the HBsAg / CD3 bispecific antibody-induced T cell activation and non-specific activation of the NFAT signaling pathway. Specifically, NFAT-Luc Jurkat cells were revived in RPMI-1640 medium containing 10% fetal bovine serum and 1 mg / mL G418. For high-throughput screening, 384-well plates with white transparent bottoms pre-coated with 2 μg / mL HBsAg (standing at 37°C for 2 hours) were used to collect cells and adjust the density to 2x10 6 cells / mL, 20 μL / well of cells were inoculated into 384-well plates, and 10 μL of RPMI-1640 medium was added with 3-fold dilution of HBsAg / CD3 bispecific antibody (concentration range of 27nM to 0.032nM). In order to strictly screen HBsAg cross-linked HBsAg / CD3 bispecific antibodies, an HBsAg uncoated control group was set up, that is, 27nMHBsAg / CD3 and NFAT-Luc Jurkat were mixed and cultured in HBsAg uncoated wells as negative controls. After the above cells were incubated at 37°C and 5% CO2 for 6 hours, 15 μL of luciferase substrate OneGlo (Promega # E6120) was added to each well. After incubation for 5 minutes, the chemiluminescence value was read on the TECAN INFINITE MPLEX microplate reader, and the data was processed using GraphPad Prism.

[0112] FIG. 6A to FIG. 6B The experimental results showed that HBsAg / CD3 bispecific antibodies can induce T cell activation only in the presence of HBsAg antigen. Among them, BsAb-2-6-017 has the strongest ability to activate T cells, and BsAb-2-6-001, BsAb-2-6-006, BsAb-2-6-011, BsAb-2-6-016, BsAb-2-6-021, and BsAb-2-6-026 derived from the combination with VL1 variants have significantly reduced their ability to activate T cells, and their EC50 values ​​differ by 5-10 times. The T cell activation ability of BsAb-2-5-017 is comparable to that of BsAb-2-6-017; however, under the same structure, BsAb-2-5-001, BsAb-2-5-006, BsAb-2-5-011, BsAb-2-5-016, BsAb-2-5-021 and BsAb-2-5-026 failed to induce T cell activation.

[0113] Example 4 HBsAg / CD3 bispecific antibody induces human T cell proliferation

[0114] The ability of the HBsAg / CD3 bispecific antibody in Example 1 to induce the proliferation of human primary T cells under HBsAg cross-linking was detected by flow cytometry. Specifically, 2 μg / mL HBsAg recombinant protein was pre-coated on a U-bottom 96-well plate (standing at 37°C for 2 hours); at the same time, human peripheral blood mononuclear cells were quickly thawed in a 37°C water bath and plated at 1×10 6 Resuspend in pre-warmed RPMI-1640 medium at a density of 2x10 cells / mL and stain with 1 μM CFSE for 5 minutes at room temperature in the dark. Add an equal volume of RPMI-1640 complete medium to terminate the staining reaction and wash once to remove free CFSE. Collect and resuspend CSFE-labeled PBMC cells in complete RPMI-1640 medium and adjust the density to 2x10 6 cells / mL. 50 μL of cell suspension and 50 μL of 5-fold diluted HBsAg / CD3 bispecific antibody in RPMI-1640 medium were added to 96-well plates pre-coated with HBsAg (concentration ranged from 50 nM to 0.016 nM). In order to strictly screen HBsAg-crosslinked HBsAg / CD3 bispecific antibodies, an HBsAg uncoated control group was added, i.e., PBMC cells containing 50 nM HBsAg / CD3 and CFSE-labeled were mixed and cultured in HBsAg uncoated wells as negative controls. After the above cells were cultured at 37°C and 5% CO2 for 3 days, they were washed 3 times with 150 μL / well FACS, the cells were collected by centrifugation (5 minutes, 300 x g), and 100 μL FACS buffer was added to resuspend the cells and detected on a flow cytometer (Beckman). Finally, FlowJo software was used to analyze the total cells. The percentage of total T cell proliferation level was determined by all CFSE dilution peaks.

[0115] Fig. 7A Figures 7C to 7C illustrate that HBsAg / CD3 bispecific antibodies induce T cell proliferation in the presence of HBsAg antigen. Among them, BsAb-2-6-017 has the strongest ability to stimulate T cell proliferation, and the ability of BsAb-2-6-001, BsAb-2-6-006, BsAb-2-6-011, BsAb-2-6-016, BsAb-2-6-021, and BsAb-2-6-026 derived from the combination with the VL1 variant to induce T cell proliferation is significantly reduced. Similarly, the ability of BsAb-2-5 to induce T cell proliferation is comparable to that of BsAb-2-6-017; while under the same structure, BsAb-2-5-001, BsAb-2-5-006, BsAb2-5-011, and BsAb-2-5-021 cannot induce T cell proliferation.

[0116] Example 5 HBsAg / CD3 bispecific antibody induces cytokine secretion in human T cells

[0117] The ability of the HBsAg / CD3 bispecific antibody in inducing human primary T cells to secrete cytokines under HBsAg cross-linking in Example 1 was analyzed. Specifically, 2 μg / mL HBsAg recombinant protein was pre-coated on a U-bottom 96-well plate (standing at 37°C for 2 hours); at the same time, human peripheral blood mononuclear cells (PBMCs) were quickly thawed in a 37°C water bath and plated at 1x10 6 Cells were transferred to pre-warmed RPMI-1640 medium at a density of cells / mL. 100 μL of PBMC cell suspension and 10 μL of RPMI-1640 medium with 5-fold dilution of HBsAg / CD3 bispecific antibody were added to 2 μg / mL HBsAg-coated 96-well plates (concentration range of 50 nM to 0.016 nM). In order to test whether HBsAg / CD3 bispecific antibody can non-specifically induce cytokine secretion without HBsAg cross-linking, an HBsAg uncoated control group was added, that is, 50 nM of HBsAg / CD3 and primary PBMC cells were mixed and cultured in HBsAg uncoated wells as negative controls. After the above cells were cultured at 37°C and 5% CO2 for 3 days, the supernatant was collected by centrifugation (20 minutes, 3000xg) and transferred to a new 96-well plate for CBA analysis according to the manufacturer's instructions (BD). The production of IFN-γ, TNF-α, IL-2, IL-4, IL-6 and IL-10 by human Th1 / Th2 cells was measured and the data were read out using GraphPad Prism.

[0118] Figure 8 A to Figure 8 F shows that HBsAg / CD3 bispecific antibodies successfully induced T cell activation in the presence of HBsAg antigen, and then secreted Th1 / Th2 cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, TNF-α). Among them, BsAb-2-6-017 has the strongest ability to induce T cells to secrete cytokines, while BsAb-2-6-001, BsAb-2-6-011, and BsAb-2-6-016 derived from the combination with VL1 variants have weaker ability to induce T cells to secrete cytokines. Under the same conditions, BsAb-2-5-001 failed to induce T cells to secrete related cytokines.

[0119] Example 6 HBsAg / CD3 bispecific antibody induces human T cell cytotoxicity

[0120] The cytotoxicity of the HBsAg / CD3 bispecific antibody in Example 1 was tested for redirecting T cells to lyse HBsAg-positive HepAD38 cells under HBsAg crosslinking. Specifically, the cells were diluted with Collagen (PBS 1:30) and then

[0121] The E-Plate 96 culture plate was placed at 37°C for 30 min. The RTCA was turned on to set the detection program. 50 μL of M10 complete cell culture medium was added to each well of the pre-coated E-Plate 96 culture plate to determine the baseline. HepAD38 target cells in the logarithmic growth phase were collected by 0.25% trypsin digestion, and the cells were resuspended in M10 complete culture medium and the cell concentration was adjusted to 1×10 5 / mL, 100μL / well, 1×10 4 Target cells were added to E-Plate 96 cells / well and allowed to stand for 5 minutes. The cell growth curve was continuously detected by RTCA for 16 hours. The next day, total human peripheral blood T cells induced by 400 IU / mL human IL2 for 7 days were collected as effector cells and the cell density was adjusted to 3×10 5 / mL; HBsAg / CD3 bispecific antibody was diluted 3 times with M10 complete medium (concentration range 300nM~1.23nM); 50μL effector T cells (effect cell: target cell is 3:1) and 50μL gradient diluted HBsAg / CD3 bispecific antibody were mixed with HepAD38 target cells. In order to detect the non-specific killing of HepAD38 cells by effector T cells, a group of effector T cells and HepAD38 cells but without antibody was added as a negative control. After RTCA was used to continuously detect the cell growth curve for 72 hours, the RTCA detection program was terminated, the file was saved, and the cell continuous growth curve (Plot) was exported.

[0122] Fig. 9 The real-time curve of exemplary HBsAg / CD3 bispecific antibodies inducing T cells to kill HBsAg-positive liver cancer cells HepAD38 is shown. The results show that HBsAg / CD3 bispecific antibodies have a very significant killing effect on HepAD38 cells, and are dose-dependent. Among them, BsAb-2-6-017 has the best killing effect on HepAD38 at 33nM, and BsAb-2-6-011 and BsAb-2-6-016 also show good killing levels at a dose of 300nM; while BsAb2-6-001 and bsAb2-6-006 also show incomplete killing under the same conditions. However, BsAb2-5-001 failed to successfully induce the killing effect of effector T cells on HepAD38 cells under the same conditions.

[0123] Example 7 Surface plasmon resonance (SPR) characterization of HBsAg / CD3 bispecific antibody

[0124] Biacore T800 (GE Healthcare) was used to characterize the affinity constants of HBsAg / CD3 bispecific antibodies binding to human / cynomolgus monkey CD3ε. Specifically, the exemplary HBsAg / CD3 bispecific antibodies were fixed on a proteinA chip at a flow rate of 10 μL / min, with gradient diluted human CD3ε or cynomolgus CD3ε or HBsAg antigen as the mobile phase, the binding conditions were 10 μL / min x 180 seconds, the dissociation conditions were 10 μL / min x 400 seconds, and the regeneration conditions were pH1.5Gly, 30 μL / min x 30 seconds. All binding kinetic experiments were carried out at 25 ° C. The binding constant (Ko) and dissociation constant (Kd) were determined using the 1:1 kinetic binding model fitting of Biacore T200Evaluation software 3.2.

[0125] Fig.10 and Fig.11 The kinetic curves of binding of BsAb-2-5 and BsAb-2-6 constructs to human and cynomolgus monkey CD3 recombinant proteins are shown, respectively. Fig.12 The kinetic curves of binding of BsAb-2-5 and BsAb-2-6 constructs to HBsAg are shown.

[0126] As shown in Table 3, Fig.10 and Fig.12 As shown, the exemplary HBsAg / CD3 bispecific antibodies (BsAb-2-6-001, BsAb-2-6-006, BsAb-2-6-011, BsAb-2-6-016, BsAb-2-6-017 and BsAb-2-5-001) have a strong binding ability to HBsAg with a KD of 1E-11, but show a differentiated binding ability to human CD3ε with a KD of 1E-11. D The values ​​ranged from 1E-8 to 1E-10; all constructs showed good cross-reactivity to cynomolgus monkey CD3ε (e.g. Fig.11 ), suggesting that cynomolgus monkeys can be used as non-human primate models to conduct preclinical safety and toxicity studies of HBsAg / CD3 bispecific antibodies.

[0127] Table 3 Binding affinity of HBsAg / CD3 bispecific antibody to human CD3ε, cynomolgus monkey CD3ε and HBsAg

[0128] Example 8 Determination of the purity of HBsAg / CD3 bispecific antibodies by size exclusion chromatography

[0129] The purity of the bispecific antibody in Example 1 was tested by size exclusion chromatography. The HPLC system (1260 Infinity II model, Agilent Technologies) and XBridge SEC column (3.5 μm, 7.8X300

[0130]

[0131]

[0132] mm, Waters) for SEC-HPLC characterization. Specifically, mobile phase A consisted of 67mM Na2HPO4, 33mM NaH2PO4, 100mM Na2SO4 at pH 7.0. The flow rate was set to 0.5mL / min, 100% mobile phase A, column temperature 30°C, for 30 minutes. 100μg of protein was injected into the chromatographic column and detected at 280nm, 10Hz. Data processing was performed using CDS2 (Agilent).

[0133] like Fig.13 The SEC-HPLC characterization shown shows that the HBsAg / CD3 bispecific antibody has a high purity, suggesting that such molecules have good pharmaceutical developability.

[0134] Example 9 In vitro neutralization activity of HBsAg / CD3 bispecific antibodies

[0135] The HBV-infected Huh7-NTCP cell model was used to analyze the in vitro neutralization efficacy of the HBsAg / CD3 bispecific antibody in Example 1. Specifically, Huh7-NTCP cells were resuspended in DMEM medium containing 10% FBS and 2.5% DMSO and the density was adjusted to 3.5×10 6 cells / mL. According to 100μL / well (1×10 4Huh7-NTCP was inoculated into a 96-well plate coated with collagen and cultured overnight. The next day, the HBV virus was diluted and incubated with 100, 10, 0nM (HBV virus alone as a system control) of BsAb2-6-001, BsAb2-6-011 or C8 mAb, and then added to Huh7-NTCP cells. After 24 hours of HBV infection, the HBV virus was removed, and Huh7-NTCP cells were cultured with fresh DMEM complete medium for 5 to 10 days. The HBeAg concentration in the supernatant was determined using an HBeAg enzyme-linked immunosorbent assay kit (the reading is the neutralization capacity).

[0136] Fig.14 The results showed that both BsAb2-6-001 and BsAb2-6-011 had strong virus neutralization activity at doses of 10 nM and 100 nM, corresponding to HBeAg values ​​as low as single-digit PEIU / mL, which was comparable to the neutralization potency of the parent C8 mAb. In contrast, the HBeAg value of the HBV virus group was approximately 60 PEIU / mL, indicating that the detection system was successfully infected with HBV virus.

[0137] Example 10 Pharmacodynamic study of HBsAg / CD3 bispecific antibody in mouse transplanted tumor model

[0138] The in vivo antitumor activity of the HBsAg / CD3 bispecific antibody produced in Example 1 was evaluated in immunodeficient mice inoculated with the HBS+HepAD38 cell line. In this model, HepAD38 cells (1E7 cells per mouse) were inoculated subcutaneously on the right back of NOG mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). One week later, the mice were reconstituted with 1.5E7 human T cells purified from healthy donors. Two weeks later, when the tumor size reached 100-200mm3 and the hCD45 ratio was >5%, the mice were grouped. Treatment started on day 0 with intravenous injection of bsAb2-6-011 at doses of 0.3mpk and 1mpk twice a week, with the PBS treatment group as a vehicle and the anti-RSV IgG treatment group as an isotype control. Tumor size and body weight were measured every 2-3 days. Tumor volume (mm3) was calculated based on caliper measurement as: [D*d2] / 2

[0139] The results are as follows Fig.15A As shown, bsAb2-6-011 effectively reduced HepAD38 tumor volume at doses of 0.3 mpk and 1 mpk, while tumor growth of isotype and vehicle control groups was not affected. On day 26 after treatment, the average tumor volume of mice treated with bsAb2-6-011 was significantly smaller than that of PBS and isotype control mice.

[0140] Fig. 15BThe percentage of human leukocytes in the blood of mice at the end point of the experiment is shown. The percentage of human leukocytes (hCD45+ cells) in the peripheral blood of mice in the PBS and isotype control groups was comparable, while a slight enhancement was observed in mice treated with 1mkp bsAb2-6-011, indicating T cell activation and expansion after treatment with the HBsAg / CD3 bispecific antibody. There was no significant change in the body weight of mice in all treatment groups (see Fig. 15C ).

[0141] Example 11 Pharmacokinetic study of HBsAg / CD3 bispecific antibody

[0142] The pharmacokinetic characteristics of the HBsAg / CD3 bispecific antibody were evaluated in cynomolgus monkeys. Specifically, three male monkeys and three female monkeys were randomly divided into three groups and received a single intravenous injection of BsAb2-6-011 at a dose of 0.1 mpk, 1 mpk, and 5 mpk, respectively. The total concentration of BsAb2-6-011 in plasma samples before and after administration at different time points within 7 days was determined by ELISA, and the data were analyzed.

[0143] like Fig.16A The pharmacokinetic characteristics shown in Figure 2 indicate that the initial distribution and clearance of BsAb2-6-011 in monkeys is rapid, with a half-life of ≈63 hours. In addition, no significant changes in body weight were observed in monkeys treated with 5 mpk BsAb2-6-011 (see Fig. 16B ).

Claims

1. A bispecific antibody comprising: (a) a first polypeptide chain, comprising a heavy chain of an anti-human CD3 antibody, wherein the heavy chain is fused to an anti-hepatitis B surface antigen antibody single-chain variable fragment (HBsAg scFv), and the hepatitis B surface antigen comprises a small hepatitis B virus surface antigen, a medium hepatitis B virus surface antigen, or a large hepatitis B virus surface antigen; and (b) a second polypeptide chain comprising a light chain of an anti-human CD3 antibody.

2. The bispecific antibody according to claim 1, characterized in that: The HBsAg scFv is fused to the N-terminus or C-terminus of the heavy chain of the anti-human CD3 antibody.

3. The bispecific antibody according to claim 2, characterized in that: The HBsAg scFv is fused to the N-terminus or C-terminus of the heavy chain of the anti-human CD3 antibody via a glycine-serine linker.

4. The bispecific antibody according to claim 3, characterized in that: The glycine-serine linker is selected from one or a combination of (GGGGS)n of SEQ ID NO:33, (GGGSG)n of SEQ ID NO:34, (GSGGG)n of SEQ ID NO:35, (GSGGGP)n of SEQ ID NO:36, (GSEPS)n of SEQ ID NO:37, (GGEGGGP)n of SEQ ID NO:38, (GGEGGGSEGGGS)n of SEQ ID NO:39, and (GGGSGGGG)n of SEQ ID NO:40, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

5. The bispecific antibody according to claim 1, characterized in that: The HBsAg scFv comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:1 to SEQ ID NO:6, respectively; and / or The CD3 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:11 to SEQ ID NO:16, respectively.

6. The bispecific antibody according to claim 5, characterized in that: The HBsAg scFv comprises a VH domain and a VL domain; the VH domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7; and the VL domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

8.

7. The bispecific antibody according to claim 6, characterized in that: The VH domain and VL domain of the HBsAg scFv are connected via a YOL linker; preferably, the YOL linker comprises the amino acid sequence shown in SEQ ID NO:

9.

8. The bispecific antibody according to claim 7, characterized in that: The HBsAg scFv comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

10.

9. The bispecific antibody according to claim 1, characterized in that: The anti-human CD3 antibody comprises a VH domain and a VL domain: the VH domain amino acid sequence comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; The VL domain amino acid sequence includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:

29.

10. The bispecific antibody according to claim 1, characterized in that: The bispecific antibody is of IgG1-κ subtype.

11. The bispecific antibody according to claim 1, characterized in that: The bispecific antibody further comprises an alanine at position 234 and an alanine at position 235 of the heavy chain, wherein the residue numbering is according to the EU index.

12. The bispecific antibody according to claim 1, characterized in that: The first polypeptide chain comprises SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52; and / or The second polypeptide chain includes SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:

57.

13. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bispecific antibody according to any one of claims 1 to 12.

14. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 13.

15. A cell, characterized in that The cell comprises the nucleic acid molecule of claim 13 or the vector of claim 14.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the bispecific antibody of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the expression vector of claim 14 or the cell of claim 15, and one or more pharmaceutically acceptable excipients, diluents or carriers.

17. Use of the bispecific antibody of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the expression vector of claim 14, the cell of claim 15 or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating a disease caused by HBV infection.

18. The use according to claim 17, characterized in that The conditions caused by HBV infection include hepatitis, liver fibrosis, liver cirrhosis or liver cancer.

19. A CD3 antibody or an antigen-binding fragment thereof, characterized in that: The CD3 antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain: the VH domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24; and / or The VL domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:

29.

20. The CD3 antibody or antigen-binding fragment thereof according to claim 19, wherein: The amino acid sequence of the VH domain is shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24; and the amino acid sequence of the VL domain is shown in SEQ ID NO:

25.

21. A bispecific antigen-binding molecule, characterized in that The bispecific antigen-binding molecule comprises the CD3 antibody or antigen-binding fragment thereof according to claim 19.

22. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CD3 antibody or antigen-binding fragment thereof according to claim 19 or 20.

23. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bispecific antigen-binding molecule of claim 21.

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