TECHNICAL FIELD

The invention relates to antibodies, in particular to a monoclonal antibody that specifically binds to human 4-1BB.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is NBSH-2020005p-seql.txt. The text file is 15.9 KB; was created on Apr. 9, 2020; and is being submitted via EFS-Web with the filing of the specification.

DESCRIPTION OF RELATED ART

Activation of T lymphocytes requires simulation of dual signals. The first signal is an antigen recognition signal provided by specific binding of antigen recognition receptors (TCR) on surfaces of T cells and MHC molecule-antigen peptide, and the second signal is a costimulatory signal (Chambers C A, et al. 1999. Curr Opin Cell Biol, 11(2):203-210) provided by binding of costimulatory molecules on surfaces of antigen presenting cells (APC) and corresponding receptors on the surfaces of the T cells. Only with combined action of the dual signals can the T cells be activated efficiently and proliferate to further play corresponding biological functions. In the absence of the costimulatory signal, the T cells are incapable or non-responsive (Seo S K, et al. 2003. J Immunol, 171(2): 576-583).

CD28 is the first costimulatory molecule discovered. The CD28/B7 synergistic stimulation signal plays a major role in the early stage of activation of the T cells, promotes its proliferation and maintains short-term survival (Boulougouris G, et al. 1998. J Immunol, 161(8): 3919-3924); 4-1BB/4-1BBL is a key costimulatory signal independent of the CD28/B7. Different from the CD28/B7 which plays the major role in the early stage of the activation of the T cells, the synergistic stimulation signal produced by the 4-1BB/4-1BBL acts mainly in the late response, can cooperate with the CD28 to further activate the T cells, especially is essential for maintaining the survival and effect functions of CD8+ T cells.

4-1BB (CD137; TNFRSF9) is a member of the tumor necrosis factor receptor superfamily and is a type I transmembrane glycoprotein expressed as a monomer or dimer on the surface of an activated T cell, mainly a CD8+ T cell and also expressed in a CD4+ T cell, a NK cell, a CD4+CD25+ regulatory T cell, and the like.

Early studies have shown that the 4-1BB molecule is an activated costimulatory molecule that not only provides costimulatory signals to activate the T cells, thereby activating and proliferate the T cells, secreting cytokines and enhancing cytotoxic activity thereof, but also mediates reverse costimulatory signal, induces activation and proliferation of the APC and secretion of cytokines (Vinay D S, et al. 2006. J Mol Med (Berl), 84(9): 726-736), In vitro experiments have shown that a 4-1BB signal can play an immunomodulatory role in enhancing cell anti-infection and inhibiting tumor growth by promoting proliferation of the CD8+T cells and the CD4+T cells and secretion of cytokines. In animal tumor models, the use of the 4-1BB monoclonal antibody or the 4-1BBL gene into tumor cells can effectively induce cell-mediated anti-tumor immune response and promote tumor regression. In the study of the number and function of CD8+T after infection with 4-1BB/4-1BBL-deficient mouse virus, it is found that 4-1BB/4-1BBL interaction can enhance the killing function of virus-specific CD8 T cells and further can promote the survival and proliferation of memory T cells and control viral infection (Fuse S, et al. 2007. J Immunol, 178(8): 5227-5236). Subsequent studies have found that 4-1BB molecules have costimulatory activity and inhibitory effects in immune regulation. When the agonistic 4-1BB antibody is applied to an autoimmune disease model, the expression of IFN-γ, IDO and TGF-beta is up-regulated, and CD11+CD8+ regulates the proliferation of T cells in a large number, thereby inhibiting the proliferation and function of CD4+T cells, inhibiting the development of autoimmune diseases (Kim Y H, et al. 2009. J Leukoc Biol, 85(5):817-825). Activation of the 4-1BB pathway not only promotes the infiltration of the T cells into a transplanted organ, shortens the survival time of the transplanted organ and exacerbates the host's resistance to graft rejection, but also promotes CD4+ and CD8+ T cell-mediated graft-versus-host responses. Thus, to block the 4-1BB pathway between donor T cells and the receptor can reduces the host's resistance to graft rejection.

Therefore, it is possible to regulate the immune function of lymphocytes by interfering with the action of the 4-1BB signaling pathway, that is, by activating or blocking the 4-1BB/4-1BBL signaling pathway by antibodies against human 4-1BB, so that purposes of treatment or prevention of diseases such as cancers, autoimmune diseases, viral infections or graft-versus-host reactions can be achieved.

BRIEF SUMMARY OF THE INVENTION

The technical problem to be solved by the invention is to provide an antibody which specifically binds to human 4-1BB and does not block the binding of h4-1BBL to h4-1BB. The agonistic 4-1BB monoclonal antibody can also activate the 4-1BB/4-1BBL signaling pathway, enhance the T cell-mediated immune response, is anti-tumor, anti-infective and plays a regulation role in autoimmune diseases.

In order to solve the above technical problems, the invention adopts the following technical solutions:

The object of the invention is to provide an agonistic 4-1BB monoclonal antibody or antigen-binding fragment thereof; the agonistic 4-1BB monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the heavy chain variable region comprises a CDR1 region having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 3; and the light chain variable region comprises a CDR1 region having an amino acid sequence as shown in SEQ ID NO: 4, a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 6.

According to one embodiment, the agonistic 4-1BB monoclonal antibody or the antigen binding fragment thereof comprises the heavy chain variable region as shown in SEQ ID NO: 7.

According to another embodiment, the agonistic 4-1BB monoclonal antibody or the antigen binding fragment thereof comprises the light chain variable region as shown in SEQ ID NO: 8.

According to preferred embodiment, the agonistic 4-1BB monoclonal antibody or the antigen binding fragment comprises the heavy chain variable region as shown in SEQ ID NO: 7 and the light chain variable region as shown in SEQ ID NO: 8.

According to some embodiments, the agonistic 4-1BB monoclonal antibody has one or more of the following properties:

(a) specifically binding to the human 4-1BB,

(b) activating the T cells,

(c) inhibiting tumor cell growth and

(d) treating cancers.

According to one specific embodiment, the agonistic 4-1BB monoclonal antibody comprises a heavy chain and a light chain.

In the invention, the agonistic 4-1BB monoclonal antibody is IgG, IgA, IgE, IgM or IgD, preferably IgG.

Furthermore, the agonistic 4-1BB monoclonal antibody is IgG1, IgG2, IgG3 or IgG4, preferably subclass IgG1.

Another object of the invention is to provide a humanized anti-human 4-1BB which is formed by humanized transformation of the agonistic 4-1BB monoclonal antibody.

According to a preferred embodiment, the humanized anti-human 4-1BB antibody comprises a light chain and a heavy chain; the sequence of the light chain is as shown in SEQ ID NO:9; and the sequence of the heavy chain is as shown in SEQ ID NO:10.

The third object of the invention is to provide a preparation method of the humanized anti-human 4-1BB antibody, in which, the agonistic 4-1BB monoclonal antibody is subjected to humanized transformation by adopting a way of template replacement.

The fourth object of the invention is to provide a derivative of the agonistic 4-1BB monoclonal antibody; the derivative of the agonistic 4-1BB monoclonal antibody is an antibody obtained after the amino acid sequence of the agonistic 4-1BB monoclonal antibody is modified or an antibody obtained after the amino acid sequence of the humanized anti-human 4-1BB antibody is modified.

The fifth object of the invention is to provide a nucleic acid encoding the agonistic 4-1BB monoclonal antibody, the antigen-binding fragment thereof or the humanized anti-human 4-1BB antibody.

The sixth object of the invention is to provide a host cell expressing the nucleic acid.

The seventh object of the invention is to provide a preparation method of the agonistic 4-1BB monoclonal antibody, the preparation method comprising the steps of

(a) immunizing mice by using antigens to prepare hybridoma cells;

(b) screening positive hybridoma cells;

(c) cloning the nucleic acid sequence of the light and heavy chain variable regions of the antibody, and ligating with the constant region of human IgG1 to construct a eukaryotic expression vector; and

(d) transfecting a host cell expression antibody, and purifying the antibody to obtain a monoclonal antibody against 4-1BB.

The eighth object of the invention is to provide application of the agonistic 4-1BB monoclonal antibody or antigen-binding fragment thereof in preparation of drugs for inhibiting tumor cell growth, inflammation and development of autoreactive diseases.

The ninth object of the invention is to provide a pharmaceutical composition, comprising the agonistic 4-1BB monoclonal antibody or the antigen-binding fragment thereof and a pharmaceutically acceptable vector.

Due to implementation of the above technical solutions, the invention, compared with the prior art, has the following advantages:

The invention provides a monoclonal antibody against h4-1BB or a humanized anti-human 4-1BB antibody which specifically binds to h4-1BB to further activate the T cells. The antibody of the invention has application potentials in treating of a variety of cancers by means of immunomodulatory effects.

