WO2023143547A1

WO2023143547A1 - Anti-cd28 antibody and use thereof

Info

Publication number: WO2023143547A1
Application number: PCT/CN2023/073643
Authority: WO
Inventors: 黄俊杰; 徐振前; 张慧; 吴精博; 汪志炜; 陈俊有; 张少榆; 黄贤明; 李胜峰
Original assignee: 百奥泰生物制药股份有限公司
Priority date: 2022-01-30
Filing date: 2023-01-29
Publication date: 2023-08-03
Also published as: CN116514974A

Abstract

Disclosed are an anti-CD28 antibody or an antigen-binding fragment, and use thereof in treating or preventing a tumor, an autoimmune disease, or an inflammatory disease. The anti-CD28 antibody or the antigen-binding fragment comprises a heavy chain variable region represented by a sequence set forth in any one of SEQ ID NOs: 1-6, and a light chain variable region represented by a sequence set forth in SEQ ID NO: 7.

Description

Anti-CD28 antibody and its application

technical field

The present invention belongs to the field of immunology. Specifically, the present invention relates to an anti-CD28 antibody and its application.

technical background

CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes and plays an important role in the activation of T cells. It binds to B7 molecules on APCs (antigen presenting cells), mediates co-stimulation of T cells and promotes their survival, proliferation and cytokine production. The complete activation of naive T cells usually requires three signals, the first signal is the combination of antigen and antigen receptor, the second signal is co-stimulatory signal, and the third signal is cytokine signal. As a co-stimulatory molecule, CD28 is involved in the reception of the second signal.

When the specific pMHC (peptide-major histocompatibility complex) on the surface of DC (dendritic cells) combines multiple copies of a TCR on the surface of naive Th cells (helper T cells) or Tc cells (cytotoxic T cells) (T cell receptor) will transmit the first signal. The combination of TCR and pMHC will lead to the phosphorylation of ITAM on the CD3 chain immediately adjacent to TCR by Lck kinase. Other intracellular signal transduction-related enzymes are then recruited here, making contacts with the CD4 or CD8 co-receptors and the cytoplasmic tail of the CD3 chain. Together, these enzymes mediate an enzymatic cascade of chemical reactions that lead to the activation of multiple other enzymes. When this activation cascade occurs across multiple TCRs, T cells receive the first signal (Mak, Saunders et al. 2013, Primer to the immune response, Newnes.).

In most cases, the combination of TCR and pMHC is not sufficient to fully activate a naive Th cell or Tc cell, and a second signal in the form of co-stimulatory signaling is required. For Th cells, after receiving the first signal, the expression of CD28, an important co-stimulatory molecule on the surface of Th cells, is up-regulated. However, in order for CD28 to transmit the second signal to the Th cell nucleus, it must combine with the ligand B7 molecule on the surface of DC presenting activated pMHC. The activation of T cells is the basis for the continued proliferation and differentiation of T cells, and the complete activation of T cells requires the joint participation of the first signal and the second signal. T cells are recognized by the TCR, which is the first signal for T cell activation. TCR and CD3 molecule form a TCR-CD3 complex, and the function of CD3 molecule is to transduce the activation signal generated by TCR recognition antigen. The second signal (or co-stimulatory signal) is generated by the interaction between co-stimulatory molecules on the surface of APC or target cells and corresponding co-stimulatory molecules on the surface of T cells, and CD28 is an extremely important co-stimulatory molecule in T cell activation.

The first CD28 super agonistic antibody Theralizumab (TGN1412, TAB08) was designed for the treatment of autoimmune-related diseases (Attarwala 2010, J Young Pharm 2(3):332-336.), and began to be used for the first time in 2006. clinical trial. Theralizumab is currently used in Phase II clinical trials for the treatment of Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, and psoriasis.

The CD28 target lies more in the development of antagonist drugs. For example, FR-104 (JNJ-3133) of OSE Immunotherapeutics is a PEGylated monovalent anti-CD28 Fab antibody, which is used for the treatment of rheumatoid arthritis, organ transplant rejection, and postoperative complications. Bristol-Myers Squibb's Lulizumab is also an antagonist antibody against the CD28 domain and is used to treat systemic lupus erythematosus and Sjogren-Larsson syndrome. Other CD28-related drugs, such as oncolytic virus products, gene therapy products, and fusion protein products, are also in the clinical research stage for indications such as tumors, transplant complications, and autoimmune diseases.

Contents of the invention

The present invention relates to anti-CD28 antibody and its application. Specifically, the present invention relates to the following:

1. An anti-CD28 antibody or antigen-binding fragment, wherein the anti-CD28 antibody or antigen-binding fragment comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof:

(1) The sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10,

The sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11,

The sequence of HCDR3 comprises or consists of any one of the sequences shown in SEQ ID NO:12-17,

The sequence of LCDR1 comprises the sequence shown in SEQ ID NO:18 or consists of it,

The sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19,

The sequence of LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20;

(2) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:1, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 12 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it;

(3) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:2, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 11, the sequence of HCDR3 comprises Contains the sequence shown in SEQ ID NO:13 or consists of it, the sequence of LCDR1 contains the sequence shown in SEQ ID NO:18 or consists of it, the sequence of LCDR2 contains the sequence shown in SEQ ID NO:19 or consists of it , the sequence of LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20;

(4) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:3, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 14 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it;

(5) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:4, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 15 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it;

(6) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:5, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 16 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises or consisting of the sequence shown in SEQ ID NO: 20; or

(7) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:6, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 17 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, LCDR3 The sequence comprises or consists of the sequence shown in SEQ ID NO:20,

wherein said variant has 3, 2 or 1 amino acid difference (preferably conservative amino acid substitutions) or at least 80%, 85%, 90%, 91%, 92%, 93%, respectively, from said corresponding CDR sequence, respectively. 94%, 95%, 96%, 97%, 98%, 99% identity and the variants retain binding affinity to CD28.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment comprises one or more of HCDR1, HCDR2, and HCDR3 described above. In some embodiments, the anti-CD28 antibody or antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 described above. In some embodiments, the anti-CD28 antibody or antigen-binding fragment further comprises one or more of LCDR1, LCDR2, and LCDR3 described above.

In some embodiments, if the anti-CD28 antibody or antigen-binding fragment comprises one or more of the above-described LCDR1, LCDR2, and LCDR3, it further comprises one of the above-described HCDR1, HCDR2, and HCDR3 or more.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof:

The sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10,

The sequence of LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment, wherein the anti-CD28 antibody or antigen-binding fragment comprises the heavy chain variable region of the sequence shown in SEQ ID NO: 1 or a variant thereof.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment, wherein the anti-CD28 antibody or antigen-binding fragment comprises the light chain variable region of the sequence shown in SEQ ID NO: 7 or a variant thereof.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment, wherein the anti-CD28 antibody or antigen-binding fragment comprises the heavy chain variable region of the sequence shown in SEQ ID NO: 1 or a variant thereof and the sequence shown in SEQ ID NO: 7 The light chain variable regions of the indicated sequences or variants thereof.

In some embodiments, the variant has 3, 2 or 1 amino acid difference (preferably conservative amino acid substitutions) or at least 80%, 85%, 90%, 91%, 92% difference from the corresponding sequence, respectively. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, and the variants retain binding affinity to CD28.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment thereof, wherein the anti-CD28 antibody or Antigen-binding fragments comprise the following CDRs or variants thereof:

(1) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:1, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

The sequence of HCDR1 comprises the sequence shown in SEQ ID NO:10 or consists of it, the sequence of HCDR2 comprises the sequence shown in SEQ ID NO:11 or consists of it, the sequence of HCDR3 comprises the sequence shown in SEQ ID NO:12 or Consisting of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consisting of the sequence shown;

(2) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:2, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

The sequence of HCDR1 comprises the sequence shown in SEQ ID NO:10 or consists of it, the sequence of HCDR2 comprises the sequence shown in SEQ ID NO:11 or consists of it, the sequence of HCDR3 comprises the sequence shown in SEQ ID NO:13 or Consisting of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consisting of the sequence shown;

(3) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:3, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

The sequence of HCDR1 comprises the sequence shown in SEQ ID NO:10 or consists of it, the sequence of HCDR2 comprises the sequence shown in SEQ ID NO:11 or consists of it, the sequence of HCDR3 comprises the sequence shown in SEQ ID NO:14 or Consisting of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consisting of the sequence shown;

(4) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:4, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

The sequence of HCDR1 comprises the sequence shown in SEQ ID NO:10 or consists of it, the sequence of HCDR2 comprises the sequence shown in SEQ ID NO:11 or consists of it, the sequence of HCDR3 comprises the sequence shown in SEQ ID NO:15 or Consisting of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consisting of the sequence shown;

(5) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 5, SEQ ID LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in NO:7;

The sequence of HCDR1 comprises the sequence shown in SEQ ID NO:10 or consists of it, the sequence of HCDR2 comprises the sequence shown in SEQ ID NO:11 or consists of it, the sequence of HCDR3 comprises the sequence shown in SEQ ID NO:16 or Consisting of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consisting of the sequence shown; or

(6) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:6, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

The sequence of HCDR1 comprises the sequence shown in SEQ ID NO:10 or consists of it, the sequence of HCDR2 comprises the sequence shown in SEQ ID NO:11 or consists of it, the sequence of HCDR3 comprises the sequence shown in SEQ ID NO:17 or Consisting of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consisting of the sequence shown,

In some embodiments, the CDRs are according to the Kabat numbering system.

2. The anti-CD28 antibody or antigen-binding fragment according to item 1, wherein the anti-CD28 antibody comprises a sequence shown in any one of SEQ ID NO: 1-6 or a heavy chain variable region consisting of it or a variant thereof.

In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(1) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 1 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(2) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 2 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(3) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 3 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(4) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(5) A heavy chain variable region comprising or consisting of the sequence shown in SEQ ID NO:5, a light chain variable region comprising or consisting of the sequence shown in SEQ ID NO:7; or

(6) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 6 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it.

In some embodiments, the variant has 3, 2 or 1 amino acid difference (preferably conservative amino acid substitutions) or at least 80%, 85%, 90%, 91%, respectively, from the corresponding variable region, respectively. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity and the variants retain binding affinity to CD28.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment further comprises a light chain constant region and/or a heavy chain constant region.

In some embodiments, the light chain constant region of the antibody or antigen-binding fragment is a kappa or lambda chain constant region.

In some embodiments, the antibody or antigen-binding fragment is of an isotype of IgG, IgM, IgA, IgE, or IgD.

In some embodiments, the antibody or antigen-binding fragment is IgGl, IgG2, IgG3, or IgG4.

In some embodiments the antibody or antigen-binding fragment is an IgG1 antibody.

In some embodiments, the light chain constant region comprises or consists of the sequence shown in SEQ ID NO:8, and the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO:9.

In some embodiments, wherein the antibody or antigen-binding fragment is a monoclonal antibody.

In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

3. The anti-CD28 antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ₂ , F(ab) ₂ , Fd, Fv, Fab/ c, scFv, scFv multimer, disulfide bond-stabilized Fv (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), diabody, disulfide-stabilized diabody (ds-Diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody (sdAb), a nanobody, a domain antibody (dAb) or a bivalent domain antibody.

4. A polypeptide selected from the group consisting of:

(1) Polypeptides comprising the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or variants thereof, wherein the polypeptides are part of an anti-CD28 antibody specifically binding to CD28, the Said antibody also comprises the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(2) a polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody also comprises the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

(3) a polypeptide comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 13 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the The antibodies also contain the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(4) A polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or a variant thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody also comprises the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:13;

(5) A polypeptide comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 14 or variants thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody also comprises the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(6) A polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody should also comprise the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:14;

(7) A polypeptide comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 15 or variants thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody should also comprise the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(8) Polypeptides comprising the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 or variants thereof, wherein the polypeptides are part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody should also comprise the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:15;

(9) A polypeptide comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 16 or variants thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody should also comprise the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(10) A polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or a variant thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody should also comprise the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:16;

(11) A polypeptide comprising sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 17 or variants thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds to CD28, so Said antibody should also comprise the sequence shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(12) A polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or a variant thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds CD28, specifically Sexually binding to CD28, the antibody should also comprise the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:17;

(13) A polypeptide comprising the sequence shown in SEQ ID NO: 1 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody that specifically binds CD28, and the antibody further comprises SEQ ID NO :7 the sequence shown;

(14) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody that specifically binds CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 1;

(15) A polypeptide comprising the sequence shown in SEQ ID NO: 2 or a variant thereof, wherein the polypeptide specifically binds to CD28 as part of an anti-CD28 antibody that specifically binds to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 7;

(16) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide specifically binds to CD28 as part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 2;

(17) A polypeptide comprising the sequence shown in SEQ ID NO: 3 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 7;

(18) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 3;

(19) A polypeptide comprising the sequence shown in SEQ ID NO: 4 or a variant thereof, wherein the polypeptide is a part of an anti-CD28 antibody that specifically binds to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 7 sequence;

(20) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 4;

(21) A polypeptide comprising the sequence shown in SEQ ID NO: 5 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 7;

(22) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 5;

(23) A polypeptide comprising the sequence shown in SEQ ID NO: 6 or a variant thereof, wherein the polypeptide specifically binds CD28 as part of an anti-CD28 antibody that specifically binds CD28, and the antibody further comprises SEQ ID NO : the sequence shown in 7; or

(24) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide, as a part of an anti-CD28 antibody, specifically binds to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 6 sequence;

Wherein the variant sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of the corresponding sequence shown in the sequence number, Preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and retains binding affinity to CD28, or the corresponding sequence as indicated by the sequence number Than a sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retaining binding affinity to CD28.

In some embodiments, the polypeptide that is part of an anti-CD28 antibody is selected from the group consisting of:

(1) a polypeptide comprising the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or variants thereof;

(2) a polypeptide comprising the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:13 or variants thereof;

(3) a polypeptide comprising the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 14 or a variant thereof;

(4) a polypeptide comprising the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 15 or a variant thereof;

(5) a polypeptide comprising the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:16 or variants thereof;

(6) A polypeptide comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 17 or variants thereof.

In some embodiments, the anti-CD28 antibody further comprises the sequences shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof.

In some embodiments, the polypeptide that is part of an anti-CD28 antibody that specifically binds CD28 comprises the sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or variants thereof.

In some embodiments, the anti-CD28 antibody specifically binding to CD28 further comprises SEQ ID NO: 10, SEQ ID NO: 11 and a sequence selected from SEQ ID NO: 12-17 or a variant thereof.

(1) a polypeptide comprising the sequence shown in SEQ ID NO: 1 or a variant thereof;

(2) a polypeptide comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;

(3) a polypeptide comprising the sequence shown in SEQ ID NO: 3 or a variant thereof;

(4) polypeptide, it comprises the sequence shown in SEQ ID NO:4 or its variant;

(5) a polypeptide comprising the sequence shown in SEQ ID NO: 5 or a variant thereof; or

(6) A polypeptide comprising the sequence shown in SEQ ID NO: 6 or a variant thereof.

In some embodiments, the anti-CD28 antibody further comprises the sequence shown in SEQ ID NO: 7 or a variant thereof.

In some embodiments, the polypeptide that is part of an anti-CD28 antibody that specifically binds CD28 comprises the sequence shown in SEQ ID NO: 7 or a variant thereof.

In some embodiments, the anti-CD28 antibody further comprises a sequence selected from SEQ ID NO: 1-6 or variants thereof.

In some embodiments, the variant sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and retain binding affinity to CD28, or as indicated by the sequence number One or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the corresponding sequence and retain the binding affinity with CD28 sequence.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment or polypeptide.

5. Biomaterials, for

(1) a nucleic acid molecule encoding the anti-CD28 antibody or antigen-binding fragment thereof of any one of items 1-3, or the polypeptide of item 4;

(2) a vector comprising a nucleic acid molecule encoding the anti-CD28 antibody or antigen-binding fragment thereof described in any one of items 1-3, or the polypeptide described in item 4; or

(3) A host cell comprising a nucleic acid molecule encoding the anti-CD28 antibody or antigen-binding fragment thereof according to any one of items 1-3, or the polypeptide according to item 4.

In some embodiments, the host cell is selected from CHO cells, HEK cells (such as HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells or murine L cells.

The present invention also provides a method for preparing the anti-CD28 antibody or antigen-binding fragment of the present invention, comprising: culturing the above-mentioned host cells to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment thereof.

6. Conjugates, comprising the anti-CD28 antibody or antigen-binding fragment thereof and a coupling portion according to any one of items 1-3, wherein the coupling portion is a purification tag (such as a His tag), a detectable Markers, drugs, toxins, cytokines, enzymes, or combinations thereof.

In some embodiments, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, an organic Chromogenic substances, chemotherapeutic agents, biotoxins, polyethylene glycols, or enzymes.

7. A kit comprising the anti-CD28 antibody or antigen-binding fragment thereof according to any one of items 1-3, and the conjugate according to item 6.

In some embodiments, the kit also includes a second antibody that specifically recognizes the anti-CD28 antibody; optionally, the second antibody also includes a detectable label, such as a radioisotope, a fluorescent substance, a chemical Luminescent substances, colored substances or enzymes.

In some embodiments, the kit is used to detect the presence or level of CD28 in a sample.

8. A pharmaceutical composition comprising the anti-CD28 antibody or antigen-binding fragment thereof according to any one of items 1-3, and the conjugate according to item 6.

In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients.

In some embodiments, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

9. The anti-CD28 antibody or its antigen-binding fragment described in any one of items 1-3, and the conjugate described in item 6 are used in the treatment and/or prevention of diseases or in the preparation of medicaments for treatment and/or prevention of diseases use.

10. A kit comprising (1) the anti-CD28 antibody or antigen-binding fragment thereof according to any one of items 1-3, the conjugate described in item 6, and (2) antibodies against other antigens or their antigens A binding fragment, and/or a cytotoxic agent, and optionally, instructions for use.

11. A method for treating or preventing a disease, comprising administering a therapeutically effective amount of the anti-CD28 antibody or antigen-binding fragment thereof according to any one of items 1-3, or the conjugate described in item 6, to a subject.

In some embodiments, the disease is a tumor, an autoimmune disease, or an inflammatory disease, such as a disease selected from the group consisting of Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, Organ transplant rejection, postoperative complications, Sjogren-Larsson syndrome.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Terms involved in the present invention have conventional meanings understood by those skilled in the art. Where a term has two or more definitions as used and/or accepted in the art, the definitions of the term used herein are intended to include all meanings.

"Antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof. The term "antibody" therefore includes molecules containing Any protein or peptide that is at least part of a biologically active immunoglobulin molecule. Examples of antibodies include, but are not limited to, heavy chains, light chains, complementarity determining regions (CDRs) of ligand binding portions thereof, heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions (CH), light chain constant region (CL), framework region (FR) or any part thereof or at least a part of a binding protein. The CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3).

Those of ordinary skill in the art will understand that the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen. Given the known antibody heavy and light chain variable region sequences, there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, each application of the definition to the CDRs of an antibody or variant thereof will be within the scope of the terms as defined and used herein. Given the variable region amino acid sequence of the antibody, one of skill in the art can generally determine which residues are contained in a particular CDR, without reliance on any experimental data outside of the sequence itself.

The term "polypeptide" is intended to encompass the singular as well as the plural "polypeptides" and refers to a molecule composed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain" or any other term used to refer to a chain of two or more amino acids is included in the definition of "polypeptide", and "polypeptide" may be defined by in place of, or interchangeably with, any of the above terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.

"Amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine amino acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

In this field, substitutions with amino acids with similar or similar properties usually do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. For the purposes of comparing two or more amino acid sequences, the percentage "sequence homology" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be determined by using [section The number of amino acid residues in an amino acid sequence that is identical to the amino acid residue at the corresponding position in the second amino acid sequence] divided by [the total number of amino acid residues in the first amino acid sequence] and multiplied by [100%], where the second Every deletion, insertion, substitution or addition of an amino acid residue in an amino acid sequence - compared to the first amino acid sequence - is considered to be a difference in a single amino acid residue (position), i.e., as defined in the present invention "Amino Acid Differences". Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as NCBI Multiple Alignment (https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt. cgi?LINK_LOC=BlastHomeLink). Methods for determining the degree of sequence identity are described, for example, in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A. Some other techniques, computer algorithms and settings. Generally, the amino acid sequence having the greatest number of amino acid residues is considered the "first" amino acid sequence for the purposes of determining the percentage "sequence identity" between two amino acid sequences according to the calculation method set out above, And another amino acid sequence is considered a "second" amino acid sequence.

In addition, in determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which amino acid residues are replaced with similar chemical Another amino acid residue substitution in the structure and which has little or no substantial effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are known in the art, for example, from WO 04/037999, GB-A-3357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and can be based on WO 04/ 037999 and the relevant teachings of WO 98/49185 and other references cited therein to select such alternative (preferred) types and/or combinations.

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid, ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), side chains of β-branches (for example, threonine, valine, isoleucine amino acids) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, nonessential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or composition of side chain families.

Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between these two amino acids.

In some embodiments, the conservative substitution is preferably a substitution wherein one amino acid residue within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small Aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e ) Aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Ala by Gly or by Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly replaced by Ala or replaced by Pro; His replaced by Asn or replaced by Gln; Ile replaced by Leu or replaced by Val; Leu replaced by Ile or replaced by Val; Lys replaced by Arg, replaced by Gln or replaced by Glu; Met Substitution of Leu, substitution of Tyr or substitution of Ile; substitution of Phe by Met, substitution of Leu or substitution of Tyr; substitution of Ser by Thr; substitution of Thr by Ser; substitution of Trp by Tyr; substitution of Tyr by Trp; and/or substitution of Phe To Val, to Ile or to Leu.

The term "antibody fragment" or "antigen-binding fragment" includes, but is not limited to, F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, Fd, Fab/c, complementarity determining region (CDR) fragments, Single-chain Fvs (scFv), disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), diabody, disulfide-stabilized Diabodies (ds-Diabody), scFv multimers (such as scFv dimers, scFv trimers), multispecific antibodies formed from a part of antibodies containing one or more CDRs, nanobodies, single domains antibody (sdAb), domain antibody (dAb), bivalent domain antibody, or any other antibody that binds to an antigen but does not contain a complete antibody structure body fragment. Regardless of structure, an antigen-binding fragment includes any polypeptide or polypeptide complex capable of binding to the same antigen as the parent antibody or parent antibody fragment. Mao C S et al. "Disulfide stabilized Fv Fragments (dsFv): a New Type of Engineering Antibody Fragments". Progress in Biochemistry and Biophysics, 1998, 25(6):525-526 introduced the structure of dsFv. Holt et al. "Domain antibodies: proteins for therapy" Trends in Biotechnology (2003): Vol.21, No.11:484-490 reviewed antigen-binding fragments called "domain antibodies" or dAbs, which contain only the VH of the antibody or VL domains and thus smaller than eg Fab and scFv. dAbs are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. The Fab/c fragment contains the Fc and Fab determinants, wherein the "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain. The term "antibody fragment" includes aptamers, aptamer spiegelmers and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that, like an antibody, binds to a specific antigen to form a complex. Typically, antibody fragments have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.

The antibodies, antigen-binding fragments disclosed herein include derivatives that are modified, that is, modified by covalent linkage of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent linkage does not prevent the antibody or antigen-binding fragment from binding to the epitope combined. Antibodies or antigen-binding fragments can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. wait. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

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

In the present invention, the CD28 antigen can be from mammals, such as humans, rats, mice, and monkeys.

Antibodies described herein can be derived from any animal, including birds and mammals. Preferably, the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin.

The term "isolated" used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, antibodies refers to the isolated components of the cell's natural environment, such as DNA or RNA. One or more of the isolated molecules. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include fragments of nucleic acid that do not occur in nature, and do not exist in nature. "Isolated" is also used to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptide is intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. will usually be prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values The range between any two values of , including the endpoints, or any value therein.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide which is said to "encode" a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation may result in the polypeptide and/or fragments thereof.

"Treatment" means therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, slow down, ameliorate or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, amelioration, remission, alleviation or disappearance of disease state (whether partial or total), prolongation and Expected survival without treatment, etc. Those in need of treatment include patients already with a condition or disorder, susceptible to the condition or disorder, or in need of prevention of the condition or disorder, who can or are expected to benefit from administration of an antibody, antigen-binding fragment or pharmaceutical composition disclosed herein For patients who would benefit from testing, diagnostic procedures and/or treatment.

The effective dose and regimen for treating a particular patient will depend on various factors, including the particular antibody, antigen-binding fragment or derivative used, the patient's age and weight, general health, sex and diet, and the time of administration, Frequency of excretion, drug combination, and severity of the particular condition being treated. These factors are in the judgment of the medical caregiver, who is within the purview of those of ordinary skill in the art. The dosage used can be determined by principles of pharmacology and pharmacokinetics well known in the art. In some embodiments, the antibody of the present invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight each time. In some embodiments, administration is weekly, or monthly.

"Pharmaceutically acceptable" means a material listed in a Pharmacopoeia for use in medicine for animals, especially for humans. Furthermore, a "pharmaceutically acceptable carrier and/or excipient" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, Ethylene Glycol, Water, Ethanol wait. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with suitable amounts of carriers and/or excipients to provide a form suitable for administration to the patient. The formulation should be suitable for the mode of administration. The parent formulation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection to human body according to conventional procedures. Compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredients are presented alone or in combination in unit dosage form, eg, as a dry lyophilized powder or water-free concentrate, in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is administered by infusion, the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection can be used so that the active ingredient can be mixed before administration.

Antibodies or antigen-binding fragments of the invention include neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions derived from, for example, hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and with anions derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, Salts formed by cations of amines, 2-ethylaminoethanol, histidine, procaine, etc. The antibodies described herein can be neutral, ie, have substantially no net charge, eg, the antibody is electrically neutral when the pH of the antibody-containing composition is the isoelectric point of the antibody. The antibodies described herein may exist in a positive ion form, eg, when the pH is below the isoelectric point, the antibody molecule exhibits an overall positive charge. The antibodies described herein may exist in the form of negative ions, such as when the pH is above the isoelectric point, the antibody molecule as a whole exhibits a negative charge.

"About" refers to the usual error range for the corresponding value readily known to those skilled in the relevant art. In some embodiments, reference to "about" herein refers to the described numerical value and the range of ±10%, ±5% or ±1%.

"EC ₅₀ " means the half-maximal effect concentration (concentration for 50% of maximal effect) refers to the concentration that can cause 50% of the maximal effect.

Description of the drawings:

Figure 1. Binding curves of different anti-CD28 antibodies to human CD28.

Figure 2. Binding curves of different anti-CD28 antibodies to rat CD28.

Figure 3. Binding curves of different anti-CD28 antibodies to mouse CD28.

Figure 4. Functional test of anti-CD28 antibody in solution state.

Figure 5. Test of the ability of anti-CD28 antibody to activate T cells with the participation of anti-CD3 antibody, in which αCD3 in the figure is mouse anti-human CD3 antibody.

Antibody preparation

Various methods for preparing antibodies are known in the art, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, phage display library and other methods for preparing antibodies.

Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, and the like can be selected, constructed, and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, which in its entirety includes The supplementary content is incorporated by reference in its entirety.

In some embodiments, the DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein in a conventional manner, placed into an expression vector, and then transfected into a host cell, and cultured in a medium to produce the transfected host cell. Monoclonal antibodies. In some embodiments, an antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence. High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and other cellular promoters such as muscle Kinetin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc. Commonly used mammalian host cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells.

In some embodiments, the inserted gene fragment needs to contain selection markers, common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation. Transfect the constructed plasmid into host cells without the above genes, and undergo selective culture Medium culture, the successfully transfected cells grow in large numbers and produce the desired target protein. The obtained antibody can be separated or purified by conventional technical means, such as protein A-sepharose, ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography and the like.

Detailed ways

The present invention is specifically described below by way of examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, the third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, but they were conventional products that could be purchased from the market.

Example 1: Preparation and affinity determination of anti-CD28 antibody

1. Expression of anti-CD28 antibody

The nucleic acid encoding the variable region of the antibody described in Table 1 is transferred into an expression vector containing the human Ig kappa light chain constant region framework (SEQ ID NO: 8) and the IgG1 heavy chain constant region framework (SEQ ID NO: 9) , and expressed antibodies by transient transfection of HEK293F host cells.

Table 1. List of anti-CD28 antibody heavy chain variable region sequences and light chain variable region sequences (wherein the CDR sequences are underlined)

Human Ig kappa light chain constant region sequence (SEQ ID NO: 8):

Human IgG1 heavy chain constant region sequence (SEQ ID NO: 9):

2. Affinity determination of anti-CD28 antibody and human CD28

Under the condition of 2-8°C, coat human CD28 protein (Sino Biological, product number: 11524-HCCH) at a final concentration of 2 μg/mL overnight, and add 100 μl per well into the ELISA plate. Discard the coating solution the next day and block with 5% skimmed milk ELISA plate, 250 μl per well, incubated in a 37°C incubator for 2 hours. After washing the ELISA plate twice with PBST buffer (prepared by PBS phosphate buffered saline + Tween 20), 100 μl of different anti-CD28 antibodies were added respectively. The control antibody was a mouse anti-human CD28 antibody (BD Biosciences, CAT#555725). The initial concentration of anti-CD28 antibody was 10 μg/mL, diluted 3 times, and the secondary antibody was HRP-coupled goat anti-human IgG (Jackson, catalog number: 109-035-098). ELISA tests showed that these 6 antibodies were different from human CD28 degree of integration (see Figure 1 and Table 2).

Table 2. Binding tests of different anti-CD28 monoclonal antibodies to human CD28

The human CD28 protein was coupled with biotin, and the affinity of different concentrations of anti-CD28 monoclonal antibodies to the human CD28 antigen was detected on a Fortebio instrument using an SA sensor. The affinity values of the six antibodies are shown in Table 3.

Table 3. Affinity detection of different anti-CD28 monoclonal antibodies and human CD28

Example 2: Research on the binding activity of CD28 monoclonal antibody to CD28 protein of different species

Dilute the CD28 monoclonal antibody with PBS as the diluent to a final concentration of 1 μg/mL, add 100 μl per well to the ELISA plate, and coat overnight at 2-8°C. The next day, discard the coating solution and block the ELISA plate with 5% skimmed milk, 250 μl per well, and incubate in a 37°C incubator for 2 hours. After washing the ELISA plate twice with PBST buffer, add rat CD28 protein (Sino Biological, Cat. The concentration is 3 μg/mL, 3-fold dilution, 100 μl per well, and then incubated at 37°C for 1 hour. After washing with PBST buffer for 3 times, HRP-labeled anti-his secondary antibody (1:5000 dilution, Genscript, catalog number: A00612) was added, and then incubated at 37°C for 1 hour. After washing 5 times with PBST buffer, add TMB to develop color for about 15-20 minutes, add 0.1M H ₂ SO ₄ to stop color development, and read 450 on a microplate reader Absorbance value at nm. The fitting results with four parameter equations are shown in Fig. 2 and Fig. 3.

Since the sequence of the extracellular region of the human CD28 protein is 100% identical to that of the cynomolgus monkey CD28 protein, it can be seen from Figure 1 in Example 1 that except for the strong binding of P64-2D6 to human or cynomolgus monkey CD28, the other five CD28 The antibody has relatively weak binding ability to human or cynomolgus monkey CD28. Figure 2 shows that except for P64-2D6 and P64-2D8 having strong affinity to rat CD28 protein, the other four antibodies have relatively weak affinity to rat CD28. It can be seen from Figure 3 that, except for P64-2D6 and P64-2D8 having strong affinity to mouse CD28 protein, the other four antibodies have relatively weak affinity to mouse CD28.

Example 3: Functional detection of anti-CD28 antibody

The genetically engineered Jurkat T cell system was used to detect the ability of CD28 monoclonal antibody to activate T cells. This cellular system consists of a luciferase reporter gene driven by expression of the NFAT-responsive element (NFAT-RE) or NF-κb-responsive element or IL-2 promoter. Jurkat cells themselves contain TCR/CD3 receptors, and when cells containing NFAT-RE response elements or NF-κb response element-driven luciferase reporter cells are combined with appropriate TCR/CD3 ligands or anti-TCR/CD3 antibodies, TCR will Transduces intracellular signals leading to NFAT-RE or NF-κb-mediated luminescence. Similarly, when TCR/CD3 effector cells (IL-2) are co-engaged with anti-TCR/CD3 and anti-CD28 stimulators, receptor-mediated signaling leads to IL-2 promoter-mediated luminescence.

Resuspend the cells and dilute the antibody in RPMI 1640 medium (purchased from Gibco) containing 10% FBS (fetal bovine serum, purchased from Gibco). 200,000 IL-2-Jurkat cells/well were added to the corresponding wells of the 96-well plate, 50 μL per well. The final concentration of CD28 monoclonal antibody was 1 μg/mL, and the final concentration of positive control mouse anti-human CD28 antibody (BD Biosciences, CAT#555725) and negative control human IgG1 isotype control was also 1 μg/mL. The final concentration of anti-human IgG Fc specific (Sigma, CAT#I2136-BULK) was 2.5 μg/mL. The amount of diluted antibody in the corresponding well was 50 μL, and the final volume of each well was 100 μL. After adding the samples, incubate in a 37°C CO ₂ cell incubator for 6 hours, add 50 μL of luciferase substrate reaction solution, and then shake in a microplate reader for 10 seconds to detect relative light units (RLU). CD28 monoclonal antibody alone in solution state will not cause T cell activation, but after adding anti-human IgG Fc specific for cross-linking, antibodies P64-1B11, P64-1H3, P64-2D6, P64-1G12, P64- 2D5 and P64-2D8 can induce the activation of T cells (see Figure 4). As we all know, there is a certain amount of immunoglobulin (IgG) in the human body. If these immunoglobulins are cross-linked with CD28 antibody, it may prompt CD28 antibody to activate T cells, resulting in a large release of cytokines, resulting in some adverse reactions. reaction. It can be seen from Figure 4 that under the same conditions, compared with other monoclonal antibodies, P64-2D6 has a lower ability to activate T cells after adding anti-human IgG Fc specific for cross-linking, indicating that it is safer.

In another experiment, mouse anti-human CD3 antibody (BD Biosciences, CAT#555336) was coated overnight, The next day, the coating solution was discarded, and IL-2-Jurkat cells and CD28 monoclonal antibody were added. The amount of cells added and the concentration and amount of antibodies added were consistent with those shown in Figure 4. The Fab fragment of the anti-human CD3 antibody used in this example can specifically recognize the CD3ε chain in the TCR-CD3 complex, activate T cells, and initially activate T cells. But only with the participation of the first signal, CD3 antibody alone is not enough to activate T cells, but combined with P64-1B11 or P64-2D6 can strongly activate T cells, that is, with the joint participation of the second signal, it can make T cells T cells were further activated (see Figure 5).

Claims

An anti-CD28 antibody or antigen-binding fragment, wherein the anti-CD28 antibody or antigen-binding fragment comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof:

(1) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:1, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 12 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it;

(2) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:2, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 13 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it;

(3) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:3, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 14 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it;

(4) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:4, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 15 or be made up of it, the sequence of LCDR1 comprises the sequence shown in SEQ ID NO:18 The sequence or consists of it, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20;

(5) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:5, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 16 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises or consisting of the sequence shown in SEQ ID NO: 20; or

(6) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:6, LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7;

Preferably according to the Kabat numbering system, the sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, the sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, and the sequence of HCDR3 comprises SEQ ID NO: The sequence shown in 17 or consists of it, the sequence of LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, the sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, the sequence of LCDR3 comprises The sequence shown in SEQ ID NO:20 or consisting of it,

(7) The sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10,

The sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11,

The sequence of HCDR3 comprises or consists of any one of the sequences shown in SEQ ID NO:12-17,

The sequence of LCDR1 comprises the sequence shown in SEQ ID NO:18 or consists of it,

The sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19,

The sequence of LCDR3 comprises the sequence shown in SEQ ID NO:20 or consists of it,

wherein said variant has 3, 2 or 1 amino acid difference (preferably conservative amino acid substitutions) or at least 80%, 85%, 90%, 91%, 92%, 93%, respectively, from said corresponding CDR sequence, respectively. 94%, 95%, 96%, 97%, 98%, 99% identity and the variants retain binding affinity to CD28.
Anti-CD28 antibody or antigen-binding fragment comprising one or more of the following HCDR1, HCDR2, and HCDR3:

The sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10,

The sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11,

The sequence of HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NO: 12-17.
Anti-CD28 antibodies or antigen-binding fragments comprising the following HCDR1, HCDR2, and HCDR3:

The sequence of HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10,

The sequence of HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11,

The sequence of HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NO: 12-17.
The anti-CD28 antibody or antigen-binding fragment of claim 2 or 3, further comprising one or more of the following LCDR1, LCDR2, and LCDR3:

The sequence of LCDR1 comprises the sequence shown in SEQ ID NO:18 or consists of it,

The sequence of LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19,

The sequence of LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20.
The anti-CD28 antibody or antigen-binding fragment of any one of claims 1-4, wherein the anti-CD28 antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region selected from the group consisting of , or its variants:

(1) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 1 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(2) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 2 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(3) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 3 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(4) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 or consisting of it, and a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it;

(5) a heavy chain variable region comprising or consisting of the sequence set forth in SEQ ID NO:5, a light chain variable region comprising or consisting of the sequence set forth in SEQ ID NO:7; or

(6) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 6 or consisting of it, a light chain variable region comprising the sequence shown in SEQ ID NO: 7 or consisting of it,

wherein said variant has 3, 2 or 1 amino acid difference (preferably conservative amino acid substitutions) or at least 80%, 85%, 90%, 91%, 92%, 93% respectively from said corresponding variable region , 94%, 95%, 96%, 97%, 98%, 99% identity, and the variants retain binding affinity to CD28.
The anti-CD28 antibody or antigen-binding fragment of any one of claims 1-4, wherein the anti-CD28 antibody or antigen-binding fragment comprises a heavy chain variable region, and the heavy chain variable region comprises a sequence selected from SEQ ID NO: The sequence shown in any one of 1-6 or consists of it.
The anti-CD28 antibody or antigen-binding fragment of claim 6, wherein the anti-CD28 antibody or antigen-binding fragment further comprises a light chain variable region, and the light chain variable region comprises the sequence shown in SEQ ID NO: 7 or consists of it.
The anti-CD28 antibody or antigen-binding fragment according to any one of claims 1-7, wherein the antibody or antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , F(ab) 2 , Fd, Fv , Fab/c, complementary determining region fragments, scFv, scFv multimer, disulfide bond stable Fv (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), double chain antibody (Diabody), Disulfide-stabilized diabodies (ds-Diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, single domain antibodies (sdAbs), nanobodies, domain antibodies or bivalent domains Antibody.
The anti-CD28 antibody or antigen-binding fragment of any one of claims 1-8, wherein the anti-CD28 antibody or antigen-binding fragment comprises a heavy chain constant region, and the heavy chain constant region is of IgG1, IgG2, IgG3 or IgG4 Heavy chain constant region.
The anti-CD28 antibody or antigen-binding fragment of any one of claims 1-9, wherein the anti-CD28 antibody or antigen-binding fragment comprises a light chain constant region, and the light chain constant region is a kappa or lambda light chain constant region.
The anti-CD28 antibody or antigen-binding fragment according to any one of claims 1-10, wherein the anti-CD28 antibody or antigen-binding fragment comprises the light chain constant region shown in SEQ ID NO:8 and the light chain constant region shown in SEQ ID NO:9 heavy chain constant region.
A polypeptide specifically binding to CD28 selected from the group consisting of:

(1) Polypeptides comprising the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or variants thereof, wherein the polypeptides are part of an anti-CD28 antibody specifically binding to CD28, the Said antibody also comprises the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(2) a polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody also comprises the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

(3) Polypeptides comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 13 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody also comprises the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(4) Polypeptides comprising the sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 or variants thereof, wherein the polypeptides are part of an anti-CD28 antibody specifically binding to CD28, the Said antibody also comprises the sequence shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:13;

(5) a polypeptide comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 14 or variant, wherein said polypeptide is part of an anti-CD28 antibody specifically binding to CD28, said antibody further comprising the sequences shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20;

(6) Polypeptides comprising the sequences shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequence shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:14;

(7) Polypeptides comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 15 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequence shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(8) Polypeptides comprising the sequences shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequence shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:15;

(9) Polypeptides comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 16 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequence shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(10) A polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequence shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:16;

(11) A polypeptide comprising the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 17 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequence shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

(12) A polypeptide comprising the sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof, wherein the polypeptide is part of an anti-CD28 antibody specifically binding to CD28, the Said antibody should also comprise the sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:17;

(13) A polypeptide comprising the sequence shown in SEQ ID NO: 1 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 7 sequence;

(14) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 1 sequence;

(15) A polypeptide comprising the sequence shown in SEQ ID NO: 2 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 7 sequence;

(16) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide is used as a specific binding agent A part of an anti-CD28 antibody that incorporates CD28, said antibody also comprising the sequence shown in SEQ ID NO:2;

(17) A polypeptide comprising the sequence shown in SEQ ID NO:3 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO:7 sequence;

(18) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 3 sequence;

(19) A polypeptide comprising the sequence shown in SEQ ID NO: 4 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 7 sequence;

(20) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 4 sequence;

(21) A polypeptide comprising the sequence shown in SEQ ID NO: 5 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 7 sequence;

(22) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 5 sequence;

(23) A polypeptide comprising the sequence shown in SEQ ID NO: 6 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 7 sequence; or

(24) A polypeptide comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the polypeptide is used as a part of an anti-CD28 antibody specifically binding to CD28, and the antibody further comprises the sequence shown in SEQ ID NO: 6 sequence;

Wherein the variant sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of the corresponding sequence shown in the sequence number, Preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and retains binding affinity to CD28, or the corresponding sequence as indicated by the sequence number Than a sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retaining binding affinity to CD28.
biomaterials, for

(1) a nucleic acid molecule, which encodes the anti-CD28 antibody or antigen-binding fragment according to any one of claims 1-11, or the polypeptide according to claim 12;

(2) a vector comprising a nucleic acid molecule encoding the anti-CD28 antibody or antigen-binding fragment of any one of claims 1-11, or the polypeptide of claim 12; or

(3) A host cell comprising a nucleic acid molecule encoding the anti-CD28 antibody or antigen-binding fragment of any one of claims 1-11, or the polypeptide of claim 12.
A conjugate comprising the anti-CD28 antibody or antigen-binding fragment of any one of claims 1-11 and A coupling moiety, wherein the coupling moiety is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; or, the coupling moiety is a radioactive isotope, a fluorescent substances, chemiluminescent substances, colored substances, chemotherapeutic agents, biotoxins, polyethylene glycols or enzymes.
A kit comprising the anti-CD28 antibody or antigen-binding fragment according to any one of claims 1-11 and the conjugate according to claim 14; optionally, the kit further comprises a second antibody specific for Optionally, the second antibody further includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; optionally, the kit is used for Detecting the presence or level of CD28 in the sample; optionally, the kit further includes antibodies against other antigens or antigen-binding fragments thereof, and/or cytotoxic agents, and optionally, instructions for use.
A pharmaceutical composition comprising the anti-CD28 antibody or antigen-binding fragment of any one of claims 1-11, the conjugate of claim 14; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable or, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
The anti-CD28 antibody or antigen-binding fragment according to any one of claims 1-11, the conjugate according to claim 14, and the pharmaceutical composition according to claim 16 are used for treating and/or preventing diseases or preparing for treatment and/or use in medicines for the prevention of disease.
The use according to claim 17, the disease is a tumor, an autoimmune disease or an inflammatory disease, such as a disease selected from the group consisting of: Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, silver Psoriasis, organ transplant rejection, postoperative complications, Sjogren-Larsson syndrome.
A method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the anti-CD28 antibody or antigen-binding fragment thereof of any one of claims 1-11, the conjugate of claim 14, or the conjugate of claim 16 The pharmaceutical composition.
The method of claim 19, wherein the disease is a tumor, an autoimmune disease or an inflammatory disease, such as a disease selected from the group consisting of Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, psoriasis disease, organ transplant rejection, postoperative complications, Sjogren-Larsson syndrome.