Fusion protein of anti-TIGIT antibody and IL2 or variant and application thereof

Abstract

The present disclosure provides a fusion protein of an anti-TIGIT antibody and IL2, or a variant thereof, and an application thereof. Specifically, the present disclosure provides a fusion protein comprising: (a) a first polypeptide comprising an anti-TIGIT antibody or an antigen-binding fragment thereof; (b) a second polypeptide comprising interleukin-2 (IL-2) or a variant thereof having lymphocyte growth promoting activity wherein the second polypeptide is fused to the first polypeptide. The invention further provides application of the fusion protein in forward regulation of immune cell activity and / or improvement of immune response. And / or to treat cancer, an immunodeficient or inflammatory disease, or an infectious disease.

Description

Fusion protein of anti-TIGIT antibody and IL2 or its variant and its application Technical Field

[0001] This application belongs to the fields of biotechnology and medicine. Specifically, this application relates to antibody-interleukin fusion proteins, specifically comprising an antibody that specifically binds to TIGIT and interleukin-2 (IL-2) or various variants thereof. In addition, this application relates to polynucleotides encoding such fusion proteins, vectors and host cells expressing such fusion proteins. This application also relates to methods for producing and preparing such fusion proteins, as well as pharmaceutical compositions and treatment methods for treating diseases. Background Art

[0002] Interleukin-2 (IL2) is a T-cell growth factor induced by antigen stimulation. It can activate and promote the proliferation and differentiation of T cells, maintaining the growth and reproduction of various immune cells (such as B cells, natural killer cells, and macrophages). Therefore, IL2 plays an important role in the treatment of tumors and immunodeficiency diseases and is one of the first FDA-approved immunotherapy drugs for cancer.

[0003] In recent years, although wild-type IL2 has achieved remarkable results in the treatment of cancer, it has a short half-life in the body and may cause side effects such as fever, vomiting, diarrhea, dizziness and hypotension, prompting people to shift their research focus to new mutant IL2 proteins with stronger specificity and better therapeutic effects.

[0004] IL2 exerts its biological activity by binding to the IL2 receptor (IL2R) on the cell membrane. The IL2R receptor is a complex composed of an α chain (55kd), a β chain (75kd), and a γ chain (64kd). Only when the three chains are present at the same time can IL2 bind to IL2R with high affinity (Kd≈10 -11 M). Cells that express only the β and γ chains but lack the α chain are capable of signal transduction but can only bind to IL-2 with moderate affinity (Kd ≈ 10 -9 M). Cells expressing only the α chain bind to IL2 with low affinity (Kd≈10 -8 M) and is unable to perform cell signal transduction. The γ subunit does not bind to IL2 alone. High-affinity IL2Rs are mainly present in activated T cells, B lymphocytes, and NK cells. Most quiescent NK cells and macrophages express medium-affinity IL2Rs, while low-affinity IL2Rs are expressed in quiescent T cells (GAFFEN SL et al., Cytokine, 2004, 28(3): 109-123).

[0005] Studies have found that mutations in the IL2 protein can enhance its affinity for IL2Rβ. Compared with wild-type IL2, this novel IL2 mutant protein can better induce the proliferation of cytotoxic T cells and reduce the expansion of Treg cells, thereby improving anti-tumor effects (Levin et al., Nature 484:529 (2012)).

[0006] T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT, also known as WUCAM, Vstm3 or VSIG9) is a checkpoint molecule mainly expressed on the surface of immune cells such as NK cells, T cells, and Treg cells. It has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail and is a typical inhibitory receptor protein (Yu et al., Nat. Immunol. 10: 48-57, 2009).

[0007] TIGIT can inhibit the body's immune response through various mechanisms. By targeting the signaling pathway molecules of the T cell antigen receptor (TCR), TIGIT directly blocks the activation, proliferation, and acquisition of effector functions of naive T cells (Tn), and can inhibit the cell proliferation and inflammatory cytokine production of CD4+ T cells (Fourcade J et al., JCI Insight, 2018, 3(14): e121157). TIGIT can indirectly inhibit T cells by regulating the production of cytokines by dendritic cells (Yu et al., Nat. Immunol. 10: 48-57, 2009). TIGIT can also enhance the stability of Tregs and their inhibitory function on the proliferation of T cells that produce IFN-γ (Fourcade J et al., JCI Insight, 2018, 3(14): e121157).

[0008] In the tumor environment, TIGIT is highly expressed on the surfaces of NK cells and effector T cells associated with tumor infiltration or migration, while its ligand CD155 is highly expressed on the surface of tumor cells. Tumor cells directly act on NK cells and effector T cells through the binding of CD155 / TIGIT, inhibiting their activity (Li et al., J. Biol. Chem. 289:17647-17657, 2014).

[0009] TIGIT is a promising new immunotherapy target for enhancing immune responses and preventing and / or treating tumors, infections, or infectious diseases. CD155-blocking antibodies targeting TIGIT, particularly anti-human TIGIT antibodies, can unleash the tumor-killing activity of immune effector cells and are expected to achieve promising anti-tumor therapeutic effects.

[0010] Summary of the Invention

[0011] The present application provides a CD155 blocking antibody targeting TIGIT and its corresponding fusion protein.

[0012] In a first aspect of the present application, an anti-TIGIT antibody or an antigen-binding fragment thereof is provided.

[0013] In some embodiments, the anti-TIGIT antibody is a monoclonal antibody.

[0014] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is selected from the group consisting of:

[0015] (a) The anti-TIGIT antibody or antigen-binding fragment thereof can competitively bind to TIGIT with CD155 and its binding affinity to human TIGIT is EC 50 0.01 to 20 nM; and / or

[0016] (b) the anti-TIGIT antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions (VH CDRs) 1 to 3 and light chain complementary determining regions (VL CDRs) 1 to 3 having the following sequences: VH CDR1 having the following sequence: SEQ ID NO: 16; VH CDR2 having the following sequence: SEQ ID NO: 17; VH CDR3 having the following sequence: SEQ ID NO: 18; VL CDR1 having the following sequence: SEQ ID NO: 20; VL CDR2 having the following sequence: SEQ ID NO: 21; and VL CDR3 having the following sequence: SEQ ID NO: 22; and / or

[0017] (c) the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH having the following sequence: SEQ ID NO: 15; and / or a light chain variable region VL having the following sequence: SEQ ID NO: 19; and / or

[0018] (d) The anti-TIGIT antibody or antigen-binding fragment thereof comprises a VH and VL combination of the following sequences: SEQ ID NO: VH of SEQ ID NO: 15 and VL of SEQ ID NO: 19.

[0019] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises an antibody IgG1gamma constant region (e.g., an IgG1gamma constant region with accession number UniProtKB-P01857). In some embodiments, the constant region has a feature selected from the following group: (a) the constant region does not contain a mutation; (b) the constant region comprises one or more mutations that reduce antibody-mediated ADCC and CDC activity, such as amino acid mutations D265A and N297G, and / or L234A and L235A; (c) the constant region comprises a pair of heavy chain constant regions (CH), one of which comprises a mutation that introduces a knob structure (e.g., mutations S354C and / or T366W), and the other comprises a mutation that introduces a hole structure. (d) the constant region comprises a pair of CHs, one of which comprises an amino acid mutation that introduces a positive charge (e.g., mutations E356K and H435R), and the other CH comprises an amino acid mutation that introduces a negative charge (e.g., K439E), and wherein a stable dimer is formed by charge interaction, wherein the positions of the above mutations are according to the EU numbering method.

[0020] In some aspects, a fusion protein is provided, comprising: (a) a first polypeptide comprising an anti-TIGIT antibody or an antigen-binding fragment thereof; (b) a second polypeptide comprising interleukin-2 (IL-2) or a variant thereof having lymphocyte growth promoting activity, wherein the second polypeptide is fused to the first polypeptide.

[0021] In some embodiments, the first polypeptide comprises an anti-TIGIT antibody or antigen-binding fragment thereof, or a sequence having at least 95% sequence identity thereto, selected from the group consisting of:

[0022] (a) The anti-TIGIT antibody or antigen-binding fragment thereof can competitively bind to TIGIT with CD155 and its binding affinity to human TIGIT is EC 50 0.01 to 20 nM; and / or

[0023] (b) the anti-TIGIT antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions (VH CDRs) 1 to 3 and light chain complementary determining regions (VL CDRs) 1 to 3 having the following sequences: VH CDR1 having the following sequence: SEQ ID NO: 16; VH CDR2 having the following sequence: SEQ ID NO: 17; VH CDR3 having the following sequence: SEQ ID NO: 18; VL CDR1 having the following sequence: SEQ ID NO: 20; VL CDR2 having the following sequence: SEQ ID NO: 21; and VL CDR3 having the following sequence: SEQ ID NO: 22; and / or

[0024] (c) The anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH having the following sequence: SEQ ID NO: 15; and / or a light chain variable region VL having the following sequence: SEQ ID NO: 19.

[0025] In some embodiments, the first polypeptide comprises an antibody IgG1 gamma constant region (e.g., an IgG1 gamma constant region with accession number UniProtKB-P01857), wherein the constant region has a feature selected from the group consisting of:

[0026] (a) the constant region does not contain a mutation;

[0027] (b) the constant region comprises one or more mutations that reduce antibody-mediated ADCC and CDC activity, such as amino acid mutations D265A and N297G, or L234A and L235A;

[0028] (c) the constant region comprises a pair of heavy chain constant regions (CH), wherein one CH comprises a mutation introducing a knob structure (e.g., mutation S354C and / or T366W), and the other CH comprises a mutation introducing a hole structure (e.g., Y349C, T366S, L368A and / or Y407V), and wherein the knob structure matches the hole structure to form a stable dimer;

[0029] (d) the constant region comprises a pair of CHs, wherein one CH comprises an amino acid mutation that introduces a positive charge (e.g., mutations E356K and H435R), and the other CH comprises an amino acid mutation that introduces a negative charge (e.g., K439E), and wherein a stable dimer is formed by charge interaction,

[0030] The positions of the above mutations are based on the EU numbering system.

[0031] In some embodiments, the second polypeptide has one or more characteristics selected from the group consisting of:

[0032] (a) the second polypeptide further comprises a signal peptide;

[0033] (b) the IL2 is wild-type IL2 or a functional fragment thereof, such as that derived from humans, primates, or rodents;

[0034] (c) Compared with wild-type IL2, the binding affinity of the IL2 variant to the IL-2Rβ subunit is enhanced or weakened and / or the binding affinity of the IL2 variant to the IL-2Rα subunit is reduced.

[0035] In some embodiments, the IL2 variant comprises one or more mutations selected from the group consisting of R38L, L80F, R81D, L85V, I86V, I92F, and F42A, for example, a combination of L80F, R81D, L85V, I86V, and I92F and / or F42A, or R38L and F42A, wherein the positions of the mutations are according to EU numbering.

[0036] In some embodiments, the first polypeptide and the second polypeptide are connected by a linker, for example, the linker is a glycine linker, such as Gn, or a glycine / serine linker, such as (GS) n 、(GGS) n 、(GGGS) n 、(GGGGS) n or (GGGGGS) n wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0037] In some embodiments, the second polypeptide is linked to the N-terminus and / or C-terminus of the antibody heavy chain in the first polypeptide.

[0038] In some aspects of the present application, an isolated nucleic acid molecule or a construct or vector comprising the nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the fusion protein described in the present disclosure.

[0039] In some embodiments, the nucleic acid molecule, construct or vector comprises a nucleotide sequence selected from SEQ ID NO: 1, 2, 4, 5, 29, 31 or 33, or a sequence having more than 90% homology to the nucleotide sequence and having the same biological activity.

[0040] In some aspects of the present application, a cell is provided, comprising the fusion protein, nucleic acid molecule, construct or vector of the present application.

[0041] In some aspects of the present application, a composition is provided, comprising the fusion protein, nucleic acid molecule, construct, vector, or cell of the present application; and a carrier.

[0042] In some aspects of the present application, provided is the use of the fusion protein, nucleic acid molecule, construct or vector, cell or composition of the present application in the preparation of a drug for immunotherapy.

[0043] In some embodiments, the drug is used to positively regulate immune cell activity and / or enhance immune response. In some embodiments, the drug is used to treat cancer, immunodeficiency disease or inflammatory disease, or infectious disease.

[0044] In some embodiments, the cancer is selected from bladder cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, squamous cell carcinoma, skin cancer, central nervous system tumors, lymphoma, leukemia, sarcoma, virus-related cancers, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin or non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or head and neck cancer, and any combination thereof.

[0045] In some embodiments, the inflammatory disease or autoimmune disease is selected from type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft-versus-host disease, psoriasis, alopecia areata, HCV-induced vasculitis, Sjögren's syndrome, pemphigus, ankylosing spondylitis, Behçet's disease, Wegener's granulomatosis, autoimmune hepatitis, sclerosing cholangitis, Guillain-Strauss syndrome, and macrophage activation syndrome.

[0046] In some embodiments, the infectious disease is selected from the group consisting of infections caused by pathogenic viruses, bacteria, fungi, and parasites.

[0047] In some aspects of the present application, a method for producing the fusion protein described in the present application is provided, the method comprising: culturing the cells of the present application under conditions suitable for expressing the fusion protein; and recovering the fusion protein.

[0048] Those skilled in the art may arbitrarily combine the aforementioned technical solutions and technical features without departing from the inventive concept and protection scope of this application. Other aspects of this application will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The present application will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the implementation scheme of the present application and are not intended to limit the scope of the present application.

[0050] Figure 1: Schematic diagram of the structure of an exemplary anti-TIGIT antibody-IL2 fusion protein of the present application:

[0051] Figure 1A: TIGIT antibody-IL2 fusion protein linked to the N-terminus of the antibody, such as KD5201J and control protein-2;

[0052] Figure 1B: TIGIT antibody-IL2 fusion proteins formed by linking to the C-terminus of the antibody, such as KD5201E (i.e., HC1-IL2mut / LC1 (ART-Ig)), KD5201F (i.e., HC1-IL2wt / LC1 (ART-Ig)), KD5201K and control protein-1 (i.e., IgG1HC-IL2mut / LC (ART-Ig)).

[0053] Figure 2: Flow cytometric analysis of the binding of anti-human TIGIT monoclonal antibodies to membrane TIGIT.

[0054] Figure 3: ELISA study showing the competitive binding of anti-human TIGIT monoclonal antibodies and CD155 to recombinant human TIGIT.

[0055] Figure 4 shows the promoting effect of anti-human TIGIT monoclonal antibodies on IFNγ production by PHA-activated PBMCs.

[0056] Figure 5: Shows the effect of anti-human TIGIT humanized monoclonal antibody in inhibiting mouse tumor growth in vivo:

[0057] Figure 5A: Relationship between tumor volume and time for each mouse in each group;

[0058] FIG5B : Tumor weight of each mouse in each group at the end of the experiment (D17).

[0059] Figure 6: Identification of the prepared anti-TIGIT antibody-IL2 fusion protein by SDS-PAGE method:

[0060] Figure 6A: SDS-PAGE identification results of KD5201E, KD5201F and control protein-1;

[0061] FIG6B : SDS-PAGE identification results of KD5201J, KD5201K and control protein-2.

[0062] Figure 7: SEC-HPLC identification of the prepared anti-TIGIT antibody-IL2 fusion protein:

[0063] Figure 7A: SEC-HPLC profile of KD5201F;

[0064] Figure 7B: SEC-HPLC profile of KD5201E;

[0065] FIG7C : SEC-HPLC profile of control protein-1.

[0066] Figure 8: BLI study of the binding affinity of anti-TIGIT antibody-IL2 fusion protein to recombinant IL2Rα and IL2Rβ subunits:

[0067] Figure 8A: Binding of KD5201F to IL2Rα;

[0068] Figure 8B: Binding of KD5201F to IL2Rβ;

[0069] Figure 8C: Binding of KD5201E to IL2Rα;

[0070] FIG8D : Binding of KD5201E to IL2Rβ.

[0071] Figure 9: Flow cytometric study of the binding affinity of anti-TIGIT antibody HC-IL2 fusion protein to membrane IL2Rαγ and βγ subunits:

[0072] Figure 9A: 293F cells were co-transfected with IL2Rα and γ expression plasmids or IL2Rβ and IL2Rγ expression plasmids, and IL2Rαγ dimers or IL2Rβγ dimers were expressed on the surface of 293F cells;

[0073] Figure 9B: Binding of KD5201F and KD5201E and control antibody control protein-1 to 293F cells expressing IL2Rαγ dimer and IL2Rβγ dimer;

[0074] Figure 9C: Binding study of KD5201F, KD5201K, and KD5201J to 293F cells expressing IL-2Rαγ dimer;

[0075] FIG9D : Binding study of KD5201F, KD5201K and KD5201J to 293F cells expressing IL-2Rαβγ trimer.

[0076] Figure 10: Study on the in vitro stimulation of CTLL-2 lymphocyte growth by TIGIT antibody-IL2 fusion protein.

[0077] Figure 11: ELISA study of TIGIT antibody-IL2 fusion protein binding to TIGIT in vitro

[0078] Figure 12: Flow cytometry study of TIGIT antibody-IL2 fusion protein binding to membrane TIGIT in vitro:

[0079] Figure 12A: Binding of KD5201K and KD5201J to membrane TIGIT;

[0080] FIG12B : Binding of KD5201K and KD5201F to membrane TIGIT.

[0081] Figure 13: Study on the stimulation of NK92 cell growth by TIGIT antibody-IL2 fusion protein:

[0082] FIG13A : Detection of TIGIT expression in NK92 cells using flow cytometry;

[0083] Figure 13B: Study on the effect of TIGIT antibody-IL2 fusion protein binding to TIGIT on stimulating NK92 cell growth.

[0084] Figure 14: Study on in vitro activation of KN92 cells by TIGIT antibody-IL2 fusion protein.

[0085] Figure 15: Study on the effect of fusion protein on the growth of CD4+ and CD8+ lymphocytes in vivo:

[0086] FIG15A : Effect of fusion protein on CD4 + T lymphocyte growth in vivo;

[0087] FIG15B : Effects of fusion proteins on CD8+ T lymphocyte growth in vivo.

[0088] Figure 16: Study on the inhibition of tumor growth by fusion protein in vivo:

[0089] FIG16A : Effect of fusion protein on tumor volume in vivo;

[0090] FIG16B : Effect of fusion proteins on tumor weight in vivo.

[0091] In the above figures, * indicates p < 0.05, ** indicates p < 0.01; KD5201E indicates HC1-IL2mut / LC1 (ART-Ig), and KD5201F = HC1-IL2wt / LC1 (ART-Ig). DETAILED DESCRIPTION

[0092] The present disclosure provides a fusion protein of an anti-TIGIT antibody and IL2 or a variant thereof, the fusion protein comprising an anti-TIGIT antibody and an IL2 molecule, or a variant of one or both of them. Compared with the wild-type IL2 molecule, the mutant interleukin-2 molecule can enhance the affinity for IL2Rβ, reduce the affinity for IL2Rα while enhancing the affinity for IL2Rβ, or reduce the affinity for IL2Rβ, and reduce the affinity for IL2Rα while reducing the affinity for IL2Rβ. On the other hand, the TIGIT antibody-IL2 fusion protein can delay the half-life of IL2 and has better biological activity than IL2 applied alone; its TIGIT antibody portion guides the fusion protein to target the tumor environment, forming a high concentration area around the tumor cells, thereby reducing the side effects caused by IL2. The TIGIT antibody can also release the tumor-killing activity of immune effector cells, synergistically kill tumor cells, and achieve a stronger tumor-suppressing effect.

[0093] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the examples may be combined. All features disclosed herein may be used in any combination, and each feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.

[0094] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0095] Fusion protein (peptide)

[0096] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising an anti-TIGIT antibody polypeptide and an IL2 polypeptide, wherein the components of the fusion protein are connected to each other directly or through a linker via a peptide bond. For the sake of clarity, the individual peptide chains of the antibody components of the fusion protein can be non-covalently linked, for example, through a disulfide bond. "Fusion" means that the components are connected by peptide bonds directly or via one or more peptide linkers. As used herein, the term "element" or "component" refers to an amino acid sequence that constitutes a part of a fusion protein. The term "unit" or "monomer" refers to the basic fragment of the element that constitutes the function.

[0097] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0098] "Antibody fragments" refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to the antigen that binds to the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies.

[0099] The term "monoclonal antibody (mAb)" as used herein refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody.

[0100] Monoclonal antibodies and fragments thereof can be produced by various methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by the hybridoma method (first proposed by Kohler et al., Nature, 256:495 (1975)) or by recombinant DNA methods (U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0101] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0102] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0103] The anti-TIGIT antibodies in the fusion protein disclosed herein include mouse antibodies, chimeric antibodies, and humanized antibodies. The antibody includes a heavy chain variable region (VH), the heavy chain variable region includes three complementary determining regions (CDRs), and the antibody includes a light chain variable region (VL), the light chain variable region includes three CDRs. In some embodiments, non-human monoclonal antibodies can be humanized by the following approaches: (1) homologous replacement, using human FRs with greater homology to the non-human counterparts for replacement; (2) surface remodeling, remodeling the amino acid residues on the surface of non-human CDRs and FRs to make them similar to the outline or FR pattern of human antibody CDRs; (3) compensatory changes, reshaping key position amino acid residues to compensate for CDR transplantation; (4) positioning conservation, humanized monoclonal antibodies are humanized using FR conservative sequences as templates, but retaining key amino acid residues in the variable regions of non-human monoclonal antibodies.

[0104] This article can use not only complete monoclonal antibodies, but also its immunologically active antibody fragments (antigen-binding fragments), such as Fab, Fab′, F(ab′)2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGACH2, small antibody, F(ab′)3, tetrabodies, triabodies, bispecific antibodies, single-domain antibodies, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc, etc.

[0105] As used herein, the term "sequence identity" or "% identity" refers to the percentage of identical residues (e.g., amino acids or nucleic acids) between a candidate sequence and a reference sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. For example, as used herein, "at least 70% sequence identity" means that the sequence identity between the candidate sequence and the reference sequence is greater than 70%, such as 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any numerical value therein.

[0106] The term "Fc region" herein is used to define the C-terminal region of at least a portion of the constant region in an antibody heavy chain. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises IgG CH2 and IgG CH3 domains, which may be native sequences or contain mutations. For example, "knob-in-hole" (KiH) technology (Merchant et al (1998) Nat Biotech 16, 677-681) or electrostatic manipulation-based interaction technology (ART-Ig, as described in PCT / JP2006 / 306803) can be used to maximize the production of heterodimeric antibodies. Simply put, KiH technology involves introducing mutations into the tightly bound Fc region of the two heavy chains. One heavy chain introduces mutations that can form a knob (e.g., S354C and T366W, EU numbering), while the other heavy chain introduces mutations that can form a hole (e.g., Y349C, T366S, L368A, Y407V, EU numbering), thereby generating a more structurally stable heterodimeric antibody. ART-Ig technology simply involves introducing mutations that alter positive and negative charges into the tightly bound Fc region of the two heavy chains. One heavy chain introduces mutations that create positively charged amino acids (ART-Ig-P) (e.g., E356K and H435R, EU numbering), while the other heavy chain introduces mutations that create negatively charged amino acids (ART-Ig-N) (e.g., K439E, EU numbering), thereby generating a more structurally stable heterodimeric antibody. In this context, IL2 can be fused to either of the two heavy chains. For example, IL2 can be fused to the antibody heavy chain HC (hole) or HC (ART-Ig-P).

[0107] The anti-TIGIT antibody in the fusion protein herein can be obtained by methods known in the art or can be a TIGIT antibody known or obtained in the art. The anti-TIGIT antibody that can be used in the fusion protein herein can include one or more characteristics selected from the following group:

[0108] (i) specifically binds to human TIGIT in vivo or in vitro, for example, its binding affinity to human TIGIT is determined by ELISA 50 0.001nM~100nM, 0.01nM~50nM, 0.1nM~10nM;

[0109] (ii) competitively inhibiting the binding of CD155 to TIGIT. For example, the IC50 for specifically blocking the binding of CD155 to human TIGIT as determined by ELISA is 0.005 μg / ml to 1 μg / ml, 0.01 μg / ml to 0.9 μg / ml;

[0110] (iii) binds to human or primate TIGIT but not mouse TIGIT;

[0111] (iv) positively regulating the activity of immune cells (e.g., T cells and NK cells), such as increasing the production of pro-immune response cytokines (e.g., IFNγ) by human peripheral blood mononuclear cells (PBMCs) (e.g., lymphocytes, T cells), and reducing or reversing NK cell exhaustion;

[0112] (v) reducing or eliminating TIGIT-expressing cells, such as Treg cells, to reduce immunosuppression;

[0113] (vi) enhancing immune response; and

[0114] (vii) having preventive and / or therapeutic effects against tumors, infections or infectious diseases.

[0115] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof in the fusion protein comprises heavy chain complementary determining regions (VH CDR) 1 to 3 and light chain complementary determining regions (VL CDR) 1 to 3 having the following sequences or sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto: VH CDR1 having the following sequence: SEQ ID NO: 16; VH CDR2 having the following sequence: SEQ ID NO: 17; VH CDR3 having the following sequence: SEQ ID NO: 18; VL CDR1 having the following sequence: SEQ ID NO: 20; VL CDR2 having the following sequence: SEQ ID NO: 21; and VL CDR3 having the following sequence: SEQ ID NO: 22.

[0116] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof in the fusion protein comprises an anti-TIGIT antibody or antigen-binding fragment thereof having VH CDRs 1 to 3 with amino acid sequences as described below: SEQ ID NOs: 16, 17, and 18, respectively; and VL CDRs 1 to 3 with amino acid sequences as described below: SEQ ID NOs: 20, 21, and 22, respectively; or a sequence thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises the following VH and VL combination: VH of SEQ ID NO: 15 and VL of SEQ ID NO: 19; or a sequence thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0117] "Native IL2," also referred to as "wild-type IL2" (wtIL2), refers to naturally occurring IL2, as opposed to "modified IL2," also referred to as "mutant IL2" (mutIL2), which is mutated or modified relative to naturally occurring IL2, e.g., to alter one or more properties of native IL2, such as stability, activity, etc. Modified IL2 molecules can, for example, comprise modifications in the amino acid sequence, such as amino acid substitutions, deletions, or insertions.

[0118] In some embodiments, the mutant IL-2 molecule differs in the region of IL-2 that binds to the IL-2Rα subunit and the region of IL-2 that binds to the IL-2Rβ subunit. Introducing amino acid mutations in these two regions of IL-2 can alter the affinity of IL-2 for binding to the IL-2Rα subunit and the IL-2Rβ subunit. For example, the affinity of IL-2 mutants (L80F, R81D, L85V, I86V, I92F) for binding to the IL-2Rβ subunit is enhanced (Levin et al. (2012) Nature 484, 529-533), the affinity of IL-2 mutant (R38L) for binding to the IL-2Rβ subunit is weakened (Sauve et al. (1991) Proc Natl Acad Sci 88, 4636-4640), and the affinity of IL-2 mutant (F42A) for binding to the IL-2Rα subunit is weakened (Helen et al. (1995) Journal of Molecular Biology 247, 979-994). The IL-2 mutant gene (L80F, R81D, L85V, I86V, I92F) can be artificially synthesized, and then an amino acid mutation (F42A) can be introduced into the gene sequence using a gene point mutagenesis method to construct an IL-2mut gene (IL-2mut-1) encoding a weakened binding affinity to the IL-2Rα subunit and an enhanced binding affinity to the IL-2Rβ subunit, or an IL-2mut gene (IL-2mut-2) encoding a weakened binding to the IL-2Rα subunit and a weakened binding to the IL-2Rβ subunit can be constructed by introducing mutations (R38L, F42A) through artificial synthesis.

[0119] As described in the Background section, the structure and function of IL2 are well-researched and understood in the art. IL2 polypeptides known in the art may be used in this application, as well as natural variants and fragments thereof (e.g., splice variants or allelic variants), and non-natural variants having IL2 activity.

[0120] The wild-type human IL2 polypeptide used herein may comprise amino acids 21-153 (accession number UniProtKB-P60568) of the amino acid sequence of SEQ ID NO: 23 (human IL2), or be encoded by a nucleic acid molecule comprising SEQ ID NO: 24, or may be a homologous sequence having the same or similar activity as these proteins (for example, homologous sequences can be obtained using databases or alignment software known in the art).

[0121] The mutant IL2 polypeptides herein can be selected from: (a) polypeptides having one or more additional amino acid residues in two stretches of the aforementioned wild-type IL2 amino acid sequence; or (b) proteins or polypeptides derived from (a) that have substituted, deleted, or added one or more amino acids within the wild-type IL2 sequence and possess wild-type IL2 activity. For example, the mutant IL2 polypeptides herein may comprise the aforementioned mutations that reduce binding affinity for the IL-2Rα subunit and / or enhance binding affinity for the IL-2Rβ subunit; or, the mutant IL2 polypeptides herein may comprise the aforementioned mutations that reduce binding affinity for the IL-2Rα subunit and / or reduce binding affinity for the IL-2Rβ subunit. In some embodiments, the sequence of the mutant IL2 polypeptide may be as shown in SEQ ID NO:25 or encoded by a nucleic acid molecule comprising SEQ ID NO:26; or as shown in SEQ ID NO:35 or encoded by a nucleic acid molecule comprising SEQ ID NO:28.

[0122] The polypeptide elements and polypeptide elements in the fusion proteins disclosed herein are preferably encoded by human genes or homologous genes or family genes or are humanized. Variants of the proteins or polypeptides disclosed herein include (but are not limited to): deletion, insertion, and / or substitution of one or more (generally 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or similar properties generally does not alter the function of the protein or polypeptide. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein or polypeptide. For example, a fusion protein may or may not include an initial methionine residue and still possess the desired viral infection prevention and treatment activity.

[0123] Variants of polypeptides include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, induced mutants, and proteins encoded by sequences that hybridize to their protein-coding sequences under conditions of high or low stringency. Depending on the host used in the recombinant production protocol, the proteins or polypeptides of the present invention may be glycosylated or non-glycosylated.

[0124] In the fusion protein of the present invention, the components (anti-TIGIT antibody and IL2 molecule) are genetically fused to each other. The fusion protein can be designed so that its components are fused directly to each other or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods known in the art, and the efficacy can be tested. Additional sequences are also included to include cleavage sites to separate the various components of the fusion protein (if desired), such as endopeptidase recognition sequences.

[0125] The polypeptide elements or peptide units in the fusion protein herein can be connected by a linker. In the present application, a flexible linker is preferably used to allow interaction between the polypeptide elements or peptide units. The linker that can be used in the fusion protein of the present application can contain 2 to 300 amino acid residues, such as 5 to 100, 10 to 50, or 15 to 3 amino acid residues. An exemplary linker can be a glycine linker, such as (G) n or glycine / serine linkers, such as (GS) n 、(GGS) n 、(GGGS) n 、(GGGGS) n or (GGGGGS) n wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0126] The fusion protein herein may further comprise a signal peptide, such as an amino acid sequence having the function of directing secretion, localization and / or delivery of the fusion protein, which is generally 5-50 amino acids in length. In some embodiments, the signal peptide may be selected from, for example, tPA2 signal peptide, TFF2 signal peptide, IL-2 signal peptide, bPRL signal peptide, CD33 protein signal peptide, and the like.

[0127] The fusion protein herein may further comprise a label, for example, a label for purification, detection, or localization, such as a fluorescent label, a non-radioactive nuclide label, a biotin label, a phosphorylation modification label, or a peptide tag.

[0128] Encoding nucleic acid molecules, vectors, and host cells

[0129] Fusion proteins of the present invention can be obtained, for example, by solid phase peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant generation. For recombinant generation, one or more polynucleotides encoding the fusion protein (fragment) are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Conventional procedures can be used to easily separate and sequence such polynucleotides.

[0130] In some embodiments, a vector (preferably an expression vector) containing one or more fusion proteins or their components encoding nucleic acid molecules is provided. Methods known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the fusion protein (or its fragment) and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination.

[0131] The expression vector can be a part of a plasmid, a virus or a nucleic acid fragment. The expression vector comprises an expression cassette, wherein the polynucleotide (i.e., coding region) encoding the fusion protein (fragment) is cloned in an operable association with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a part of a nucleic acid that is composed of codons that are translated into amino acids. Two or more coding regions can be present in a single polynucleotide construct (e.g., on a single vector), or in a separate polynucleotide construct, such as on a separate (different) vector. In addition, any vector can contain a single coding region, or can comprise two or more coding regions, such as the vector of the present invention can encode one or more polypeptides, which are separated into final proteins via proteolytic cleavage after translation or co-translation. In addition, the vector, polynucleotide or nucleic acid of the present invention can encode a heterologous coding region, which is fused or unfused with the polynucleotide encoding the fusion protein (fragment) of the present invention or its variant or derivative. Heterologous coding regions include but are not limited to specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. A gene product, such as a polypeptide, is operably associated when the coding region for the gene product is associated with one or more regulatory sequences in such a way that expression of the gene product is placed under the influence or control of the regulatory sequences.

[0132] The polynucleotides and nucleic acid coding regions of the present invention can be combined with other coding regions encoding secretion or signal peptides, which guide the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if it is desired to secrete the fusion protein, the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the fusion protein of the present invention or its fragment. Those of ordinary skill in the art know that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cut off from the translated polypeptide to generate a secretory or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide is used, such as an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of the sequence that retains the ability to guide the secretion of the polypeptide to which it is operably associated. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0133] In some embodiments, a host cell comprising one or more polynucleotides or vectors of the present invention is provided. As used herein, the term "host cell" refers to any cell system that can be engineered to generate a fusion protein of the present invention or a fragment thereof. Host cells suitable for replication and supporting expression of fusion proteins are well known in the art. Where appropriate, such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be cultured for inoculation of large-scale fermenters to obtain sufficient amounts of fusion protein for clinical applications.

[0134] Suitable host cells include prokaryotic microorganisms such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. Examples of useful mammalian host cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), macaque kidney cells (MDCK), bovine rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells, MRC5 cells and FS4 cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0.

[0135] Host cells include not only cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0136] In one embodiment, a method for producing a fusion protein according to the present invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the fusion protein under conditions suitable for expression of the fusion protein, and recovering the fusion protein from the host cell (or host cell culture medium).

[0137] The recombinant fusion protein of the present application can be expressed or secreted into the cell extracellularly, intracellularly or on the cell membrane. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0138] Compositions or pharmaceutical products

[0139] Also provided herein is a product comprising an effective amount of the fusion protein herein, a vector comprising a fusion protein encoding molecule, a host cell or a composition, and a pharmaceutically or physiologically acceptable carrier. As used herein, the term "active substance" refers to the fusion protein herein, its encoding nucleic acid molecule, a construct or vector comprising the nucleic acid molecule, a host cell or the aforementioned composition.

[0140] In a preferred embodiment, the products herein can be used for applications in positively regulating immune cell activity and / or enhancing immune responses; and / or, treating cancer, immunodeficiency diseases or inflammatory diseases, or infectious diseases. As used herein, the terms "containing" or "comprising" include "comprising", "consisting essentially of", and "consisting of". As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0141] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers that are not essential active ingredients themselves and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A comprehensive discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0142] Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, glycerol, and ethanol. In addition, these carriers may also contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, etc. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous medium, wherein the pH is generally about 5 to 8, preferably about 6 to 8.

[0143] As used herein, the term "unit dosage form" means that for the convenience of administration, the composition of the present invention is prepared into the dosage form required for single administration, including but not limited to various solid doses (such as tablets, lyophilized powders), liquid doses (such as solutions), capsules, and sustained-release agents.

[0144] In another preferred embodiment of the present invention, the composition is in unit dosage form or multiple dosage form, and the content of the active substance is 0.01 to 2000 mg / dose, preferably 0.1 to 1500 mg / dose, and more preferably 1 to 1000 mg / dose. In another preferred embodiment of the present invention, 1 to 6 doses of the composition of the present invention are administered daily, preferably 1 to 3 doses; most preferably, the daily dose is 1 dose.

[0145] The pharmaceutical composition of the present invention can be prepared into various dosage forms as needed, and the dosage that is beneficial to the patient can be determined by the physician based on factors such as the patient type, age, weight and general disease condition, and the mode of administration. The form of the pharmaceutical composition of the present invention can be suitable for the desired mode of administration, such as intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrasplenic, intrarenal, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, local perfusion directly bathing the target cells, via a catheter, via lavage, as an emulsion, as a liquid composition (e.g., liposomes), or by other methods or any combination thereof.

[0146] To enhance efficacy, the active substances or products of the present invention may be used in combination with one another, or with other drugs and treatments, for example, for the prevention and treatment of cancer. For example, if the fusion protein of the present invention is used to prevent and / or treat cancer, other drugs or treatments clinically used for cancer treatment may be used simultaneously or sequentially. Such other drugs or treatments include, but are not limited to, chemotherapy drugs, radiotherapy, surgery, etc.

[0147] Cancers that can be treated with the fusion proteins herein include, but are not limited to, bladder cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, squamous cell carcinoma, skin cancer, central nervous system tumors, lymphoma, leukemia, sarcoma, virus-related cancers, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin or non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or head and neck cancer, and any combination thereof.

[0148] Inflammatory diseases or autoimmune diseases that can be treated with the fusion proteins herein are selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft-versus-host disease, psoriasis, alopecia areata, HCV-induced vasculitis, Sjögren's syndrome, pemphigus, ankylosing spondylitis, Behçet's disease, Wegener's granulomatosis, autoimmune hepatitis, sclerosing cholangitis, Guillain-Strauss syndrome, and macrophage activation syndrome.

[0149] The infectious diseases that can be treated with the fusion protein of the present invention are selected from the group consisting of infections caused by pathogenic viruses, bacteria, fungi, and parasites.

[0150] Example

[0151] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art may make appropriate modifications and variations to the present application, and these modifications and variations are all within the scope of the present application.

[0152] For experimental procedures in the following examples where specific conditions are not specified, conventional methods in the art may be employed, for example, as described in Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, New York, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and are also available from commercial companies.

[0153] Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0154] Example 1. Acquisition and performance evaluation of anti-TIGIT monoclonal antibodies and their coding sequences, and construction of anti-TIGIT antibody-IL2 fusion protein genes

[0155] 1.1 Obtaining anti-TIGIT monoclonal antibodies and their coding sequences

[0156] The anti-TIGIT antibodies are multiple anti-TIGIT humanized monoclonal antibodies invented by the applicant. The antibody preparation method and amino acid sequence can be found in the Chinese invention patent application (CN202111105544.0, "Monoclonal antibodies targeting TIGIT", application date September 22, 2021).

[0157] Specifically, according to conventional methods, Balb / c mice (6-8 weeks old, weighing approximately 18 g, female, purchased from the Experimental Animal Management Department of the National Institute of Family Planning) were immunized intraperitoneally and subcutaneously with recombinant human TIGIT (Human TIGIT His, Sino Biological, 10917-H08H Accession #NP 776160.2.Met1-Phe138, with a polyhistidine tag expressed at the C-terminus). Splenocytes were then harvested from the immunized mice. The splenocytes were fused with mouse myeloma cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCM18). After fusion, positive clones were screened by ELISA using TIGIT-Fc (Human TIGIT (Fc Tag): Sino Biological, 10917-H02H). Multiple clones were screened, among which clone number 7103-07 possessed an IgG1 heavy chain and a kappa light chain.

[0158] The obtained cell line was cultured and the cells were expanded. The supernatant was collected and the fusion protein was purified from the supernatant using a Protein-A affinity chromatography column. The gel was run for analysis, and 2 μg of reduced and non-reduced samples were loaded into each lane. The voltage was 150 V for 1 hour. The molecular weight of the non-reduced antibody was about 150 KD, and the molecular weight of the reduced antibody was about 50 KD and 25 KD, respectively, which was consistent with the molecular weight characteristics of the antibody. The binding of the obtained monoclonal antibody to the recombinant human TIGIT antigen (Human TIGIT (His), Sino Biological, 10917-H08H) was detected by ELISA. The results showed that the binding affinity of the 7103-07 monoclonal antibody to the TIGIT antigen EC 50 is: 0.075nM.

[0159] The total RNA of the hybridoma cells was extracted and reverse transcribed. The obtained cDNA was used as a template to amplify the VH and VL genes of the hybridoma antibody and sequenced to obtain the VH and VL sequences of the TIGIT mouse monoclonal antibody. Before humanizing the antibody, we first constructed a recombinant human-mouse chimeric antibody expression plasmid and constructed the V region of the hybridoma antibody into a chimeric antibody expression vector (pcDNA3.1), that is, the V region sequence was connected to an expression vector with a constant region of human IgG1 or human λ / κ, and the human-mouse chimeric antibody was transiently expressed and purified in HEK293F cells. Then, based on the mouse parent antibody sequence numbered 7103-07, a humanized antibody sequence was designed. The specific steps are as follows: First, use Discovery Studio and Antibody Modeling uses homology modeling methods to construct a three-dimensional molecular model of the variable region. Next, by comparing the existing antibody structures in the database, the variable region and CDR of the parent antibody are structurally simulated. At the same time, cDNA-derived human germline sequences with high homology to the murine parent antibody VH and VL are selected for comparison. The heavy chain VH selects IGHV1 with the highest homology as the humanized design template and designs the sequence. The light chain VL selects IGKV1 as the humanized design template and designs the sequence. A humanized anti-TIGIT monoclonal antibody with the following heavy chain and light chain sequence combination is obtained:

[0160] Table 1. Combinations of different light and heavy chains of humanized antibodies and their corresponding numbers

[0161] Table 2. P03479 heavy and light chain sequences

[0162] Based on the humanized design results, the sequence was constructed into the pcDNA3.4 vector. Plasmids were obtained after PCR, enzyme digestion, ligation, transformation, identification, sequencing, alignment, and extraction. Based on the humanized expression profile, the plasmids were expressed in ExpiCHO-S cells using the ExpiCHO-S Expression System (Thermo Fisher, A29133) for 7 days as required. Expression levels were measured by HPLC on day 6 of expression. Finally, the antibody protein was purified by Protein A affinity chromatography.

[0163] To determine whether humanized TIGIT monoclonal antibodies can bind to recombinant human TIGIT protein, ELISA test was performed. The results showed that the anti-human TIGIT humanized monoclonal antibody P03479 could bind to recombinant human TIGIT protein with an EC of 0.09 nM. 50 Binds to human TIGIT protein.

[0164] 1.2 Biofilm layer interferometry (BLI) to study the binding activity of antibodies to TIGIT

[0165] The dissociation constant (Kd) of the binding of chimeric TIGIT monoclonal antibody 7103-07 (hFc) and humanized TIGIT monoclonal antibody to recombinant human TIGIT was detected using a GATOR (ProbeLife) instrument and HFC (LOT#2007065 Tray 2), Human antibody Capture probe. The monoclonal antibody was diluted to 30nM in binding buffer (Q buffer [PBS (10mM pH7.4) + 0.02% Tween 20 + 0.2% BSA]. The recombinant human TIGIT protein was diluted 2-fold in Q buffer to different concentrations, ranging from 40nM to 10nM. The kinetic association assay was initiated by placing the antibody capture sensor in the above serially diluted antigen solution.

[0166] Table 3 shows the BLI test results. The binding affinity of the humanized antibody #P03479 of 7103-07 to TIGIT is weaker than that of its parent human-mouse chimeric antibody 7103-07 (hFc) to TIGIT. Their Kd are 8.43x 10 -9 M and 2.96x 10 -9 M.

[0167] Table 3. Summary of binding kinetic constants of TIGIT mAbs to recombinant human TIGIT

[0168] 1.3 Flow cytometry to study the binding of human-mouse chimeric and humanized monoclonal antibodies to TIGIT protein on the cell membrane

[0169] A CHO-K cell line stably expressing full-length human TIGIT (CHO-K-TIGIT) was constructed as follows: a plasmid containing the full-length human TIGIT cDNA (NM_173799.2) (purchased from Sino Biological) was used to clone the cDNA into the mammalian cell expression vector pcDNA3.1 (Invitrogen) containing the rat glutamine synthetase gene. The constructed plasmid was then transfected into CHO-K cells using electroporation (Bio-Rad, Gene Pulser Xcell). The transfected cells were cultured in OptiCHO medium (Invitrogen) for 24-48 hours, after which the medium was replaced with selection medium containing OptiCHO, 5 μg / ml recombinant human insulin, and 10 μM aminosulfoxide methionine (MSX). Cells were cultured at 37°C in an incubator with 8% CO2. Three weeks later, the screened cultured cells were subjected to flow cytometry sorting (FACS) using anti-TIGIT antibodies to obtain a CHO-K monoclonal cell line expressing human TIGIT on the cell membrane.

[0170] The flow cytometric method for studying TIGIT antibody binding to cell membrane TIGIT is briefly described as follows: First, prepare FACS buffer (1× PBS + 0.5% BSA). Take the desired number of CHO-K-TIGIT cells (approximately 1×105 to 5×105 cells per sample). Dilute TIGIT mAbs (7103-07 (hFc), P03479) and tiragolumab (Roche, currently in Phase III clinical trials; used as a positive control) at a specific ratio in FACS buffer and resuspend the cells in a volume of approximately 100 μL. Incubate at 4°C for 30 min. Wash the cells once with FACS buffer and resuspend them in FITC-anti-human IgG (Abcam: 6854) diluted in FACS buffer. Incubate at 4°C for 30 min. Finally, wash the cells with FACS buffer, resuspend them in 200 μL of FACS buffer, and analyze them.

[0171] Figure 2 shows the results of flow cytometry studies. As shown in the figure, TIGIT humanized monoclonal antibody P03479 binds well to CHO-TIGIT and binds to EC on the CHO-TIGIT cell membrane. 50 was 0.144 μg / mL (0.963 nM), compared to the EC of the parent (7103-07 (hFc)). 50 0.201 μg / mL (1.347 nM) and the EC of tislelizumab 50 0.574 μg / mL (3.844 nM), the affinity of P03479 is higher.

[0172] 1.4 Inhibition of recombinant human TIGIT-CD155 binding by chimeric and humanized TIGIT monoclonal antibodies

[0173] To determine whether human-mouse chimeric and humanized TIGIT monoclonal antibodies can inhibit the binding of CD155 to TIGIT, in vitro testing was performed using the ELISA method. Tiragolumab was used as a positive control. The antigen TIGIT (Human TIGIT (His), Sino Biological, 10917-H08H) was diluted to 1 μg / mL with a NaHCO3 solution at pH = 9.6, and 50 μL per well was added to a 96-well ELISA plate and refrigerated at 4°C overnight. The next day, after washing twice with PBS, the plates were blocked with 3% BSA, 100 μL per well, incubated at 37°C for 1.5 hours, and washed 4 times with PBST. Equal volumes of graded diluted TIGIT monoclonal antibody and CD155-mFC (6 μg / mL) (Human CD155 (Fc Tag): Sino Biological, 10109-H02H) were mixed and added to the blocked and washed ELISA plate, incubated at 37°C for 2 h, and then washed four times with PBST. Then, a certain proportion of diluted secondary antibody HRP anti-mFC (Jackson Immuno Research, 115-035-164) was added and incubated at 37°C for 1.5 h. Finally, after washing four times with PBST, TMB colorimetric solution was added, and the plate was incubated at 37°C for 10-15 min. The plate was read on a microplate reader (wavelength 450 nm and 655 nm).

[0174] Figure 3 shows the results of the ELISA study. As shown in the figure, all TIGIT monoclonal antibodies can specifically block the binding of TIGIT to CD155. The inhibitory activity IC of humanized mAb P03479 is 50 The blocking ability was 0.107 μg / mL (0.714 nM), and the IC of human-mouse chimeric monoclonal antibody (7103-07 (hFc)) was similar to that of human-mouse chimeric monoclonal antibody (7103-07 (hFc)). 50 The IC value of 0.154 μg / mL (1.033 nM) is comparable to or better than that of tislelizumab. 50 The value was 0.378 μg / mL (2.534 nM).

[0175] 1.5 In vitro enhancement of INFγ production by human PBMCs

[0176] Upon activation, T lymphocytes secrete the cytokine IFNγ. To test the function of TIGIT monoclonal antibodies as positive regulators of T cell activation, we conducted the following experiments to demonstrate the enhanced IFNγ production in PBMCs induced by TIGIT signaling blockade. The method is briefly described as follows: frozen human peripheral blood mononuclear cells (PBMCs) were thawed and adjusted to a cell density of 5 × 10⁶ cells / mL. The TIGIT antibody to be tested was diluted to a concentration of 20 μg / mL and PHA to a concentration of 6 μg / mL in RPMI 1640 medium with 10% FBS. 50 μL of PHA dilution and 50 μL of antibody dilution were added to a 96-well plate. Finally, 100 μL of the cell suspension was added to the 96-well plate, gently mixed, and incubated at 37°C in a 5% CO₂ incubator for 4 days. After 4 days, 100 μL of the supernatant was aspirated and assayed for IFNγ levels.

[0177] Figure 4 shows the test results. As shown, the humanized TIGIT monoclonal antibody P03479 promoted IFNγ release from PHA-activated PBMCs. The IFNγ release in the presence of humanized TIGIT antibody P03479 was 81% higher than that in the control group (no humanized TIGIT monoclonal antibody), and the effect was significantly superior to the 16% release of tisleliumab.

[0178] 1.6 Functional study of inhibiting tumor growth in mice in vivo

[0179] Since the antibodies of the present invention do not bind to mouse TIGIT, we used human TIGIT knock-in mice (C57BL / 6) to study the in vivo anti-tumor effect of the antibodies. Human TIGIT knock-in mice were raised in an SPF-level environment. In this example, the mouse tumor model was MC38 colon cancer. The method is briefly described as follows: Mouse colon cancer MC38 tumor cells were obtained from Shanghai South Model Organisms Technology Co., Ltd. 24 7-9 week old human TIGIT knock-in C57 / B6 mice were divided into 3 groups (4 males and 4 females per group) and 1×10 MC38 cells were injected subcutaneously into the mice's armpits. 6 On the day of inoculation (D0), mice in each group were given PBS (control group), recombinant human IgG1 10 mg / kg (control group), and TIGIT humanized antibody P03479 10 mg / kg by intraperitoneal injection. The drug was administered twice a week for a total of 4 times. The tumor volume (i.e., long diameter × short diameter) was measured at each administration. 2 The mice were weighed. The experiment was terminated on D17. The mice were sacrificed by cervical dislocation, and the tumors were removed and weighed.

[0180] The results are shown in Figure 5. Figure 5A shows the relationship between tumor volume and time for each mouse in each group, and Figure 5B shows the tumor weight for each mouse in each group at the end of the experiment (D17). The experimental results showed that the humanized TIGIT antibody P03479 effectively inhibited the growth of mouse tumors (Figures 5A and 5B). At the end of the experiment (D17), the average tumor weight of mice in the P03479 group was significantly lower than that in the hIgG1 group (p < 0.05).

[0181] 2. Antibody Modification

[0182] To reduce antibody-mediated ADCC and CDC activity, amino acid mutations D265A and N297G (DANG) (EU numbering) or L234A and L235 (LALA) (EU numbering) were introduced into the Fc region of the antibody to reduce antibody binding to Fc gamma receptors (FcγRs) and C1q (Lund et al (1996) J. Immunol. 157, 4963-4969; Tao and Morrison, (1989) J. Immunol. 143, 25952-601). The modified antibody genes were obtained by artificial synthesis (Jin Weizhi, China; the same below).

[0183] 3. Wild-type IL2 and mutant IL2

[0184] The full-length wild-type human IL2 (WT) gene (including the signal peptide sequence, SEQ ID NO: 23) was artificially synthesized. The regions of IL2 that bind to the IL2Rα subunit differ from those that bind to the IL2Rβ subunit. Introducing amino acid mutations in these two regions of IL2 can alter the binding affinity of IL2 to the IL2Rα and IL2Rβ subunits. IL2 mutants (L80F, R81D, L85V, I86V, I92F) have enhanced binding affinity to the IL2Rβ subunit (Levin et al. (2012) Nature 484, 529-533), while the IL2 mutant (F42A) has decreased binding affinity to the IL2Rα subunit. An IL2 mutant gene (L80F, R81D, L85V, I86V, I92F) was artificially synthesized, and then an amino acid mutation (F42A) was introduced into the gene sequence using gene point mutagenesis to construct the IL2mut-1 gene (SEQ ID NO: 26) encoding an IL-2Rα subunit with reduced binding affinity and an enhanced binding affinity for the IL2Rβ subunit. An IL-2mut-2 gene (SEQ ID NO: 28) encoding an IL-2Rα subunit with reduced binding affinity and an IL-2Rβ subunit was constructed by artificially introducing mutations (R38L, F42A).

[0185] IL2mut is characterized by the following mutations:

[0186] a) Mut-1 (F42A / L80F, R81D, L85V, I86V, I92F)—reduces IL2 / IL2Rα interaction and enhances IL2 / IL2Rβ interaction;

[0187] b) Mut-2(R38L / F42A)—reduces IL2 / IL2Rα and IL2 / IL2Rβ interactions.

[0188] 4. Construction of fusion protein expression vector

[0189] IL2 or its mutants are linked to peptide GSGGGGS (SEQ ID NO: 27) or (G4S) n Fusion to the N-terminus or C-terminus of one of the heavy chains of the antibody. To achieve fusion of only one of the two heavy chains of the antibody to IL2, we use the "knob-in-hole" (KiH) technology (Merchant et al (1998) Nat Biotech 16, 677-681) or the electrostatic manipulation interaction-based technology (ART-Ig) (PCT / JP2006 / 306803) to maximize the production of heterodimeric antibodies.

[0190] Simply put, KiH technology introduces mutations in the tightly bound regions of the Fc of the two heavy chains. One heavy chain introduces the mutations S354C and T366W (EU numbering method) that can form a knob, and the other heavy chain introduces the mutations Y349C, T366S, L368A, and Y407V (EU numbering method) that can form a hole, thereby producing a more stable heterodimeric antibody structure.

[0191] Simply put, ART-Ig technology involves introducing positive and negative charge mutations in the tightly bound Fc regions of the two heavy chains. One heavy chain incorporates the positively charged amino acid mutations E356K and H435R (EU numbering), while the other heavy chain incorporates the negatively charged amino acid mutation K439E (EU numbering). This creates a more stable heterodimeric antibody structure. IL2 can be fused to either heavy chain. In the present invention, IL2 is fused to either the HC (hole) or HC (ART-Ig-P) of the antibody heavy chain.

[0192] The anti-TIGIT antibody heavy chain and IL2 genes were amplified separately using PCR. The IL2 gene was then linked to the N-terminus or C-terminus of the antibody heavy chain using overlap extension PCR to construct the IL2-HC or TIGIT HC-IL2 genes (see the schematic diagrams in Figures 1A and 1B, respectively). HC (also referred to herein as HC1) represents the heavy chain of the TIGIT humanized antibody P03479.

[0193] Table 4. Nucleic acid molecules fused to the N-terminus of the TIGIT antibody heavy chain and corresponding amino acid sequence numbers

[0194] Table 5. Nucleic acid molecules and corresponding amino acid sequence numbers of fusion protein antibody heavy and light chains

[0195] Table 6. Nucleic acid molecules fused to the C-terminus of the TIGIT antibody heavy chain and corresponding amino acid sequence numbers

[0196] In addition, the IgG1 used as a control sample for the TIGIT antibody-IL2 fusion protein of the present invention can be any human or humanized anti-human protein antibody. The IgG1 used in the present invention is a humanized anti-TNFR2 antibody and a PD-1 antibody pembrolizumab:

[0197] Table 7. Control molecule nucleic acid molecules and corresponding amino acid sequence numbers

[0198] Table 8. IL-2 wild-type (wt) and variant (mut) nucleic acid sequence and amino acid sequence number

[0199] The gene constructed above was cloned into the mammalian cell expression vector pKD2.4, which contains a CMV promoter and a BGH-polyA signal sequence for transcription termination, and the cloning sites are NotI and XbaI.

[0200] Example 2. Preparation of anti-TIGIT antibody-IL2 fusion protein

[0201] The aforementioned expression plasmids were co-transfected into mammalian human 293F cells in appropriate combinations to transiently express various anti-TIGIT antibody-IL2 fusion proteins (Sinobio transient expression kit), or to construct stably expressing CHO cell lines. The transfected cells or stably expressing cells were cultured in serum-free medium, and the antibody-IL2 fusion proteins were then purified from the culture supernatant using a protein A affinity chromatography column. The purified antibody fusion proteins were characterized by SDS-PAGE and analyzed by SEC-HPLC.

[0202] Table 9. TIGIT antibody-IL2 fusion proteins prepared using KiH technology

[0203] Table 10. TIGIT antibody-IL2 fusion proteins prepared using ART-Ig technology

[0204] Table 11. Control antibody-IL2mut fusion proteins prepared using ART-Ig technology are as follows:

[0205] In the above table, HC (or HC1) and LC (or LC1) represent the heavy chain and light chain of the humanized antibody P03479, respectively. The antibody of control sample-1 is a humanized anti-TNFR2 antibody, and the antibody of control sample-2 is PD-1 pembrolizumab.

[0206] SDS-PAGE identification of these proteins is shown in Figure 6. Figure 6A shows the SDS-PAGE identification results of KD5201E, KD5201F, and control protein-1. Lanes 1, 4, and 6 are electrophoresis of KD5201E, KD5201F, and control protein-1 under non-reducing conditions, respectively, while lanes 2, 5, and 7 are electrophoresis under reducing conditions. Lane 3 is electrophoresis of human IgG1 under reducing conditions. Electrophoresis of the three fusion proteins under reducing conditions showed three bands: the smallest molecular weight band is the normal light chain, with a molecular weight of approximately 24 kDa; the largest molecular weight band is the heavy chain fused to IL2 at the C-terminus, with a molecular weight of approximately 65 kDa; and the middle band is the other heavy chain of the heterodimeric antibody, with a molecular weight of approximately 49 kDa.

[0207] Figure 6B shows the SDS-PAGE analysis of KD5201J, KD5201K, and control protein-2. Lanes 3 and 7 represent electrophoresis of KD5201J and KD5201K under non-reducing conditions, respectively, while lanes 4 and 8 represent electrophoresis under reducing conditions. Lanes 1 and 5 represent electrophoresis of human IgG1 under non-reducing conditions, while lanes 2 and 6 represent electrophoresis of human IgG1 under reducing conditions. One of the heavy chains of KD5201J does not contain a TIGIT-binding domain Fab, but rather IL-2mut-2 linked to the N-terminus of the hinge region of IgG4 Fc. Therefore, its molecular weight is smaller than that of the other heavy chain containing the TIGIT-binding domain Fab (Figure 6B, lane 4). The complete KD5201J molecule is also smaller than a normal IgG1 (Figure 6B, lane 3). The molecular structure of KD5201K is similar to that of KD5201E and KD5201F, and the SDS-PAGE results were the same as theirs ( FIG. 6B , lanes 7 and 8).

[0208] The structure of the SEC-HPLC analysis is shown in Figure 7. The spectra of KD5201F, KD5201E, and control fusion protein-1 are shown in Figures 7A, 7B, and 7C, respectively. The results showed that the obtained antibody-IL2 fusion protein was a dimer in the SEC-HPLC analysis, with no multimers.

[0209] The above results indicate that the desired antibody-IL2 fusion protein was obtained through the preparation.

[0210] Example 3. Affinity detection of fusion protein binding to recombinant IL2Rα subunit and IL2Rβ subunit in vitro

[0211] The binding affinity of the purified anti-TIGIT antibody-IL2 fusion protein to recombinant IL2Rα subunit and IL2β subunit (Sinobio) was detected by biomembrane interferometry (BLI).

[0212] FIG8 shows the BLI detection results of the binding affinities of KD5201F (containing IL2WT) and KD5201E (containing IL2mut-1) to the IL2Rα subunit and IL2β subunit, respectively.

[0213] The results showed that KD5201F (containing IL2WT) had a strong affinity for the IL2Rα subunit, with a binding constant KD of approximately 3.0 nM (Figure 8A), while its affinity for the IL2Rβ subunit was weak (almost undetectable) (Figure 8B). In contrast to KD5201F, KD5201E (containing IL2mut-1) had a weaker affinity for the IL2Rα subunit (Figure 8C) but a stronger affinity for the IL2Rβ subunit, with a binding constant KD of approximately 8.2 nM (Figure 8D).

[0214] The BLI results were consistent with our expectations, showing that KD5201F had a strong affinity for the IL2Rα subunit and a weak affinity for the IL2Rβ subunit. KD5201E, in contrast to KD5201F, had a strong affinity for IL2Rβ but a weakened binding affinity for the IL2Rα subunit.

[0215] Example 4. Affinity detection of fusion protein binding to IL2Rα subunit and IL2Rβ subunit on cell membrane in vitro

[0216] Flow cytometry was used to detect the binding affinity of anti-TIGIT antibody-IL2 fusion protein to IL2Rα subunit and IL2Rβ subunit on the cell membrane.

[0217] First, we co-transfected 293F cells with human IL2Rα, IL2Rβ, and IL2Rγ expression plasmids (Sinobio) to transiently express the following IL2R complexes: IL2Rαβγ (trimer), IL2Rαγ (dimer), and IL2Rβγ (dimer). Antibodies against IL2Rα, IL2Rβ, and IL2Rγ (source) were used to detect the expression of the corresponding IL2R subunits on the cell membrane. Flow cytometry revealed that IL2Rαγ and IL2Rβγ were expressed on the 293F cell membrane (Figure 9A).

[0218] We then used flow cytometry to detect the activity of KD5201F, KD5201E, and control protein-1 in binding to the above-mentioned two 293F cells expressing IL2R complexes (αγ and βγ). Figure 9B shows the results of flow cytometry. The results showed that KD5201F (IL2WT) had a strong affinity for IL2Rαγ, but a very weak binding ability to the IL2Rβγ subunit. In contrast to KD5201F, KD5201E and control protein-1 (both containing IL2mut-1) had a weak affinity for IL2Rαγ, but a stronger binding ability to IL2Rβγ. These results are consistent with the results of Example 3.

[0219] We then examined the relationship between the binding of KD5201F, KD5201J, and KD5201K to 293F cells expressing IL-2Rαγ and IL-2Rαβγ and protein concentration (Figures 9C and 9D). Figure 9C shows that KD5201F has a high binding activity to membrane IL-2Rαγ (EC 50 Figure 9D shows that KD5201F has a high binding activity to membrane IL-2Rαβγ (EC 50 About 0.77 μg / mL), while KD5201J and KD5201K did not bind or had very weak binding activity to membrane IL-2Rαβγ.

[0220] We used flow cytometry to examine the binding activity of KD5201F (IL2WT), KD5201E (IL2mut-1), a control protein (IL2mut-1), KD5201J (IL2mut-2), and KD5201K (IL2mut-2) to IL-2Rαβγ, IL2Rαγ, and dimeric IL2Rβγ on the cell membrane. The results were consistent with our expectations and those from BLI analysis, showing that KD5201F, containing IL2WT, had strong affinity for the IL2Rαγ subunit and weak affinity for the IL2Rβγ subunit. The antibody-IL2mut-1 fusion protein (including KD5201E and control protein-1) had strong binding affinity for IL2Rβγ, while its binding to IL2Rαγ was significantly weakened or even eliminated. The binding activity of antibody-IL2mut-2 fusion proteins (including KD5201J and KD5201K) to both IL-2Rα and IL-2Rβ was weakened.

[0221] Example 5. Study on the biological function of IL2 fusion protein in stimulating CTLL-2 cell proliferation in vitro

[0222] IL2 is a key growth factor for lymphocyte growth and can stimulate the proliferation of various lymphocytes, including the mouse T lymphocyte CTLL-2. Human IL2 binds to mouse IL2R with an affinity comparable to that of human IL2R. We used CTLL-2 cells to test the biological activity of the TIGIT antibody-IL2 fusion protein of the present invention in stimulating lymphocyte growth.

[0223] CTLL-2 (China Food and Drug Administration) was cultured in RPMI1640 medium (Gibco) containing 10% FBS (fetal bovine serum) (Gibco) and recombinant human (rh) IL-2 (Beijing Shuanglu Pharmaceutical). On the day before the experiment, 1×10 4 Cells were plated in 0.2 mL of culture medium (10% FBS, without rhIL2) and serially diluted anti-TIGIT antibody-IL2 fusion proteins to be tested, HC1-IL2wt / LC1 (ART-Ig) and HC1-IL2mut / LC1 (ART-Ig), as well as a control antibody IgG1-HC-IL2mut / LC (ART-Ig) fusion protein, were added to each well. After two days of culture, cell growth was assessed using a CCK-8 cell growth assay kit.

[0224] The experimental results are shown in Figure 10. Figure 10A shows that KD5201E containing IL2mut-1 stimulates the growth of CTLL-2 cells. 50 The bioactive EC of KD5201F containing IL2wt was 15.3 ng / mL.50 There was no significant difference between the two groups (11.1 ng / mL), indicating that IL2mut-1 had a good activity in stimulating lymphocyte growth in vitro.

[0225] Figure 10B shows the comparison of the growth stimulating activity of KD5201F with KD5201J and KD5201K containing IL2mut-2 in CTLL-2 cells. 50 The activity of stimulating CTLL-2 cell growth was much lower than that of KD5201F (EC 50 This result is consistent with their IL-2R binding activity (Example 4).

[0226] Example 6. Study on the binding of TIGIT antibody-IL2 fusion protein to TIGIT in vitro

[0227] The activity of the TIGIT antibody-IL2 fusion protein of the present invention in binding to TIGIT in vitro was detected by ELISA. The method is as follows: the plate was coated with 50 μl of NaHCO3 (pH 9.6) buffer containing 1 μL / mL recombinant human TIGIT-His at 4°C overnight. The next day, after washing twice with PBS, it was blocked with 3% BSA, 100 μL per well, incubated at 37°C for 1.5h, and washed 4 times with PBST. The TIGIT antibody-IL2 fusion protein was gradiently diluted with binding solution (TBST containing 1% BSA), added to the blocked and washed ELISA plate (50 μl per well), and incubated at 37°C for 2h. After washing four times with PBST, a diluted secondary antibody (alkaline phosphatase-labeled anti-hFc, Jackson Immuno Research) was added to each well and incubated at 37°C for 1.5 h. Finally, after washing four times with PBST, PNPP colorimetric solution was added and incubated at 37°C for 10-15 min. The wells were then read using a microplate reader (wavelengths of 405 nm and 490 nm). The humanized TIGIT monoclonal antibody P03479 was used as a positive control.

[0228] The experimental results are shown in Figure 11. The experimental results show that KD5201K binds to TIGIT with high affinity, and its binding activity is similar to that of the control antibody P03479. 50 The affinity of KD5201J to TIGIT is very weak, and the EC 50 The serum titer of KD5201J was 531 ng / mL, which was probably because KD5201J had only one TIGIT-binding domain Fab, while KD5201K and P03479 each had two TIGIT-binding domain Fabs.

[0229] Example 7. Study on the binding of fusion protein to TIGIT on cell membrane

[0230] Flow cytometry was used to detect the binding activity of the anti-TIGIT antibody-IL2 fusion protein to TIGIT on the cell membrane. First, a Jurkat cell line stably expressing human TIGIT was constructed. The full-length human TIGIT gene (Sinobio) was amplified by PCR and cloned into the mammalian cell expression vector pcDNA3.1 (Invitrogen) using the cloning enzymes Not I and Xba I. The TIGIT expression plasmid was transfected into Jurkat cells to construct a Jurkat cell line stably expressing TIGIT (TIGIT-Jurkat).

[0231] The flow cytometric method for studying the binding of TIGIT antibody-IL2 fusion protein to cell membrane TIGIT is briefly described as follows: First, prepare FACS buffer (1× PBS + 0.5% BSA). Take the desired number of TIGIT-Jurkat cells (approximately 1×105 to 5×105 cells per sample). Dilute the TIGIT antibody-IL2 fusion protein at a specific ratio in FACS buffer and incubate with TIGIT-Jurkat cells in a volume of approximately 100 μL for 30 minutes at 4°C. Wash the cells once with FACS buffer and resuspend them in FITC-anti-human IgG (Abcam: 6854) diluted in FACS buffer. Incubate at 4°C for 30 minutes. Finally, wash the cells with FACS buffer and resuspend them in 200 μL of FACS buffer before analysis. The parental TIGIT antibody P03479 was used as a control.

[0232] The results of flow cytometry are shown in Figure 12. Figure 12A shows that KD5201K binds to membrane TIGIT with high affinity, EC 50 The affinity of P03479 to membrane TIGIT was 85.5 ng / mL, slightly lower than that of P03479 (EC 50 The affinity of KD5201J to membrane TIGIT is very weak, EC 50 This result is consistent with the results of in vitro binding to recombinant TIGIT (Example 6).

[0233] Figure 12B shows the binding affinity of KD5201F and KD5201K to membrane TIGIT, and their binding activity EC 50 The concentrations of the two antibodies were 62.8 ng / mL and 31.2 ng / mL, respectively, which were comparable to the activity of the parental TIGIT antibody P03479 in binding to membrane TIGIT.

[0234] Example 8. Study on in vitro stimulation of NK92 cell growth by TIGIT antibody-IL2 fusion protein

[0235] NK92 cells are human malignant non-Hodgkin's lymphoma cells that rely on IL-2 for growth. NK92 cells express TIGIT ( Figure 13A ). We used NK92 cells to study the stimulatory effect of the TIGIT antibody-IL2 fusion protein of the present invention on NK92 cell growth, examining whether this stimulatory effect on NK92 cell growth depends on the interaction between the TIGIT antibody-IL2 fusion protein and TIGIT on NK92 cells.

[0236] NK92 cells were cultured in medium containing recombinant human IL-2. Prior to the experiment, cells were washed once and incubated in medium without IL-2 for 2 hours. Serial dilutions of a TIGIT antibody-IL2 fusion protein or / and a TIGIT antibody (P03479) at a final concentration of 10 μg / mL were then added. P03479 blocks the binding of the TIGIT-IL2 fusion protein to TIGIT on the NK92 cell membrane. After 48 hours of culture, cell count was determined.

[0237] The experimental results are shown in Figure 13. Figure 13A shows that NK92 cells express TIGIT. Figure 13B shows that KD5201K (both IL-2Rα and β subunit binding activities are weakened) stimulates the growth of NK92 cells. 50 The EC of KD5201K to stimulate NK92 cell growth was 0.86 ng / mL in the presence of P03479 (blocking TIGIT on NK92 cells). 50 The activity of KD5201F (containing IL-2WT) in stimulating NK92 cell growth in the absence or presence of P03479 was 4.6 ng / mL, which was much lower than that in the absence of P03479. This indicates that KD5201K stimulates the growth of NK92 cells by binding to TIGIT on the cell membrane. There was no significant difference in the activity of KD5201F (containing IL-2WT) in stimulating NK92 cell growth in the absence or presence of P03479. 50 The results are as follows: 0.94 ng / mL and 0.67 ng / mL respectively. KD5201F containing IL-2WT can bind to IL-2R on the NK92 cell membrane with high affinity. The binding of KD5201F to TIGIT on NK92 cells does not significantly enhance the binding of KD5201F to NK92 cells. Therefore, KD5201F stimulates the growth of NK92 cells independently of TIGIT. KD5201K containing IL2mut-2 has a very weak affinity for IL-2R. Therefore, when it cannot bind to TIGIT on NK92 cells (P03479 exists to block TIGIT on NK92 cells), the activity of KD5201K (EC 50 =4.6 ng / mL) was much lower than the activity of KD5201F (EC 50However, after KD5201K binds to TIGIT on NK92 cells, it significantly enhances the growth of NK92 cells stimulated by KD5201K, and the stimulatory activity is comparable to that of KD5201F (0.86 ng / mL vs 0.94 ng / mL), indicating that the growth of NK92 cells stimulated by KD5201K depends on the binding of KD5201K to TIGIT on the cell membrane.

[0238] Example 9. Study on in vitro activation of NK92 by TIGIT antibody-IL2 fusion protein

[0239] IL-2 can activate NK92 cells in vitro and increase the expression of CD107A molecules on the cell surface. We used an in vitro activation experiment of NK92 cells to study the effect of the TIGIT antibody-IL2 fusion of the present invention on NK92 cell activation.

[0240] NK92 cells were cultured in medium containing recombinant human IL-2. Before the experiment began, the cells were washed once and cultured overnight in medium without IL-2. Serial dilutions of the TIGIT antibody-IL2 fusion protein were then added. After 48 hours of culture, the cells were harvested and fluorescently labeled anti-CD107a antibodies were added. The fluorescence intensity of the cells was then measured by flow cytometry.

[0241] The experimental results showed (Figure 14) that KD5201J (containing IL2mut-2) can enhance the expression of CD107a in NK92 cells and activate active EC 50 The concentration of control protein-2, which also contains IL2mut-2, is about 0.7 ng / mL. However, since it is not a TIGIT antibody, it cannot enhance the activity of IL2 by binding to TIGIT of NK92 cells. Therefore, the control protein-2 activates the active EC of NK92 cells. 50 This experimental result also shows that the activation of NK92 cells by TIGIT antibody-IL2mut-2 depends on its binding to TIGIT on the cell membrane.

[0242] Example 10. Study on the effect of fusion protein on the growth of CD4+ and CD8+ lymphocytes in vivo

[0243] We used human TIGIT knock-in mice to study the stimulatory effect of the anti-TIGIT-IL2 fusion protein of the present invention on the growth of T lymphocytes CD4+ and CD8+ in vivo.

[0244] Fifty-six 8-10 week old C57BL / 6 mice (Biocytogenarian, half male and half female) were divided into 7 groups (8 mice / group) and injected with different doses of the fusion protein KD5201F (i.e., HCl-IL2wt / LC1 (ART-Ig)) and KD5201E (i.e., HCl-IL2mut / LC1 (ART-Ig)) of the present invention or PBS (control group) through the tail vein. The doses of the fusion protein were 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg, respectively, and the injection volume was 0.2 mL. After 4 days, blood was collected from the eyes, and fluorescently labeled anti-mouse CD4 or CD8 antibodies were added. After incubation for 1 hour, the cells were washed three times with PBS, and the fluorescence intensity of each sample was detected by flow cytometry, and the content of CD4+ and CD8+ cells in the blood was calculated.

[0245] FIG15A shows that the CD4+ cell content in the blood of mice treated with the fusion protein was decreased compared with that of the control mice, but there was no difference between the effects of KD5201F and KD5201E on CD4+ cells.

[0246] Figure 15B shows that the number of CD8+ cells in the blood of mice treated with the fusion protein increased compared to control mice. A 0.1 mg / kg dose of KD5201F had no effect on CD8+ cell counts, and only at higher doses did it increase CD8+ cell counts. However, KD5201E increased CD8+ cell counts at low doses, with the increase being very significant at higher doses. The effects of KD5201E at doses of 0.1 mg / kg and 1 mg / kg on CD8+ cell counts were statistically significantly different from those of KD5201F at the same dose (p < 0.01).

[0247] Example 11. In vivo study of tumor growth inhibition

[0248] We used human TIGIT knock-in mice to study the function of the anti-TIGIT antibody-IL2 fusion protein of the present invention in inhibiting the growth of homologous tumors.

[0249] Forty 8-10 week old C57BL / 6 mice (Biocytogenarian, half male and half female) were divided into five groups (8 mice / group) and injected with different doses of the fusion proteins KD5201F and KD5201E of the present invention or PBS (control group) from the tail vein. The doses of the fusion proteins were 0.1 mg / kg and 0.3 mg / kg, respectively, and the injection volume was 0.2 mL. Four days later (D4), the mice were subcutaneously inoculated with mouse colon cancer cells MC38, 1×106 cells / mouse. The second, third, and fourth doses of fusion protein or PBS were then administered on D14, D17, and D24, respectively. The experiment was terminated at D31, and the tumors were weighed.

[0250] The experimental results are shown in (Figure 16). Figure 16A shows the relationship between the average tumor volume of mice in each group and time, and Figure 16B shows the tumor weight of mice in each group at the end of the experiment (D31). The experimental results showed that KD5201E can significantly inhibit or delay the growth of MC38 tumors. Compared with the control group, 0.1 mg / kg and 0.3 mg / kg inhibited tumor growth by 27% and 70%, respectively (p < 0.01) (Figure 16B). Among them, the tumors of 3 mice in the 0.3 mg / kg dose group completely disappeared, and the effect of KD5201E in inhibiting tumor growth is proportional to the dose. KD5201F at a dose of 0.1 mg / kg can inhibit the growth of MC38 tumors, but the high dose (0.3 mg / kg) has almost no inhibitory function. The effect of KD5201F on tumor growth has no dose-effect relationship.

[0251] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and that such equivalents also fall within the scope of the claims appended hereto.

[0252] Appendix 1: Comparison of the sequence of this application with the sequence of PCT / CN2022 / 093346 (original):

[0253] *: SEQ ID NO in PCT / CN2022 / 093346 specification and sequence listing

[0254] Appendix 2: Comparison of the names of fusion proteins, antibodies, IL-2 or their fragments in this application with those in PCT / CN2022 / 093346

Claims

1. A fusion protein comprising: (a) a first polypeptide comprising an anti-TIGIT antibody or an antigen-binding fragment thereof; (b) a second polypeptide comprising interleukin-2 (IL-2) or a variant thereof having lymphocyte growth promoting activity, in, The second polypeptide is fused to the first polypeptide.

2. The fusion protein according to claim 1, wherein The first polypeptide comprises an anti-TIGIT antibody or antigen-binding fragment thereof, or a sequence having at least 95% sequence identity thereto, selected from the group consisting of: (a) The anti-TIGIT antibody or antigen-binding fragment thereof can compete with CD155 for binding to TIGIT and its binding affinity to human TIGIT is EC 50 0.01 to 20 nM; and / or (b) The anti-TIGIT antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (VH CDRs) 1 to 3 and light chain complementarity determining regions (VL CDRs) 1 to 3 selected from the group consisting of: VH CDR1 with the following sequence: SEQ ID NO: 16; VH CDR2 with the following sequence: SEQ ID NO: 17; VH CDR3 with the following sequence: SEQ ID NO: 18; VL CDR1 having the following sequence: SEQ ID NO: 20; VL CDR2 having the following sequence: SEQ ID NO: 21; and VL CDR3 of the following sequence: SEQ ID NO: 22; and / or (c) The anti-TIGIT antibody or antigen-binding fragment thereof comprises a VH and VL combination of the following sequences: VH of SEQ ID NO: 15 and VL of SEQ ID NO:

19.

3. The fusion protein according to claim 1, wherein The first polypeptide comprises an antibody IgG1gamma constant region (e.g., an IgG1gamma constant region with accession number UniProtKB-P01857), wherein the constant region has a feature selected from the group consisting of: (a) the constant region does not contain a mutation; (b) the constant region comprises one or more mutations that reduce antibody-mediated ADCC and CDC activity, such as amino acid mutations D265A and N297G, and / or L234A and L235A; (c) the constant region comprises a pair of heavy chain constant regions (CH), wherein one CH comprises a mutation introducing a knob structure (e.g., mutation S354C and / or T366W), and the other CH comprises a mutation introducing a hole structure (e.g., Y349C, T366S, L368A and / or Y407V), and wherein the knob structure matches the hole structure to form a stable dimer; (d) the constant region comprises a pair of CHs, wherein one CH comprises an amino acid mutation that introduces a positive charge (e.g., mutations E356K and H435R), and the other CH comprises an amino acid mutation that introduces a negative charge (e.g., K439E), and wherein a stable dimer is formed by charge interaction, The positions of the above mutations are based on the EU numbering system.

4. The fusion protein according to claim 1, wherein The second polypeptide has one or more characteristics selected from the group consisting of: (a) the second polypeptide further comprises a signal peptide; and / or (b) the IL2 is wild-type IL2 or a functional fragment thereof, such as that derived from humans, primates, or rodents; and / or (c) Compared with wild-type IL2, the binding affinity of the IL2 variant to the IL-2Rβ subunit is enhanced or weakened, and / or the binding affinity of the IL2 variant to the IL-2Rα subunit is reduced.

5. The fusion protein according to claim 1, wherein The IL2 variant comprises one or more mutations selected from the group consisting of L80F, R81D, L85V, I86V, I92F, F42A, R38L, for example comprising the combination of mutations L80F, R81D, L85V, I86V and I92F and / or mutation F42A, or R38L and F42A, wherein the positions of the above mutations are according to the EU numbering system.

6. The fusion protein according to claim 1, wherein The first polypeptide and the second polypeptide are connected by a linker, for example, the linker is a glycine linker, such as G n , or glycine / serine linkers such as (GS) n 、(GGS) n 、(GGGS) n 、(GGGGS) n or (GGGGGS) n wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and / or The second polypeptide is linked to the N-terminus and / or C-terminus of the antibody heavy chain in the first polypeptide.

7. The fusion protein according to claim 1, wherein The heavy chain of the first polypeptide is fused to the second polypeptide to form an amino acid sequence having SEQ ID NO: 34 or having at least 80% sequence identity thereto: and / or If present, the heavy chain not linked to the second polypeptide in the fusion protein has the amino acid sequence of SEQ ID NO: 32 or has at least 80% sequence identity thereto; and / or If present, the light chain in the fusion protein has the amino acid sequence of SEQ ID NO: 7 or has at least 80% sequence identity thereto.

8. The fusion protein according to claim 1, wherein The fusion protein comprises a sequence combination selected from the group consisting of: (a) SEQ ID NOs: 34, 32, and 7; (b) SEQ ID NOs: 12, 8, and 7; (c) SEQ ID NOs: 13, 9, and 7; and (d) SEQ ID NOs: 30, 8 and 7.

9. An isolated nucleic acid molecule or a construct or vector comprising said nucleic acid molecule, wherein: The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 8.

10. The nucleic acid molecule, construct or vector according to claim 9, comprising a nucleotide sequence selected from SEQ ID NO: 1, 2, 4, 5, 29, 31 or 33, or a sequence having more than 90% homology to the nucleotide sequence and having the same biological activity.

11. A cell comprising the fusion protein of any one of claims 1 to 8, or the nucleic acid molecule, construct or vector of claim 9 or 10.

12. A composition comprising the fusion protein of any one of claims 1 to 8, the nucleic acid molecule, construct or vector of claim 9 or 10, or the cell of claim 11; and a carrier.

13. Use of the fusion protein according to any one of claims 1 to 8, the nucleic acid molecule, construct or vector according to claim 9 or 10, the cell according to claim 11 or the composition according to claim 12 in the preparation of a medicament for immunotherapy.

14. The use according to claim 13, wherein: The drug is used to positively regulate immune cell activity and / or enhance immune response; and / or, the drug is used to treat cancer, immunodeficiency disease or inflammatory disease, or infectious disease.

15. The use according to claim 14, wherein: The cancer is selected from the group consisting of bladder cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, squamous cell carcinoma, skin cancer, melanoma, central nervous system tumors, sarcoma, virus-related cancers, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, liver cancer, salivary gland cancer, kidney cancer, basal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, nasopharyngeal cancer, malignant pleural mesothelioma, Hodgkin's or non-Hodgkin's lymphoma, myeloma, leukemia, myelodysplastic syndrome, and any combination thereof; The inflammatory disease, autoimmune disease or pathogen infection disease is selected from: type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft-versus-host disease, psoriasis, alopecia areata, atopic dermatitis, HCV-induced vasculitis, Sjögren's syndrome, pemphigus, ankylosing spondylitis, Behcet's disease, Wegener's granulomatosis, autoimmune hepatitis, sclerosing cholangitis, Guillain-Strauss syndrome and macrophage activation syndrome, autoimmune thyroiditis, autoimmune uveitis, aplastic anemia, HIV, viral hepatitis, HTLV-1 virus infection, lymphocytic choriomeningitis virus infection, parasitic infection (echocystis, schistosoma japonicum, Plasmodium).

16. A method for producing the fusion protein according to any one of claims 1 to 8, comprising: Cultivating the cell according to claim 11 under conditions suitable for expressing the fusion protein; and recovering the fusion protein.

17. An anti-TIGIT antibody or an antigen-binding fragment thereof, comprising: (a) heavy chain complementarity determining regions (VH CDRs) 1 to 3 and light chain complementarity determining regions (VL CDRs) 1 to 3 having the following sequences: VH CDR1 with the following sequence: SEQ ID NO: 16; VH CDR2 with the following sequence: SEQ ID NO: 17; VH CDR3 with the following sequence: SEQ ID NO: 18; VL CDR1 having the following sequence: SEQ ID NO: 20; VL CDR2 having the following sequence: SEQ ID NO: 21; and VL CDR3 of the following sequence: SEQ ID NO: 22; and / or (e) A VH and VL combination having the following sequences: VH of SEQ ID NO: 15 and VL of SEQ ID NO:

19.

18. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 17, further comprising an antibody IgG1gamma constant region (e.g., an IgG1gamma constant region with accession number UniProtKB-P01857), wherein the constant region has a feature selected from the group consisting of: (a) the constant region does not contain a mutation; (b) the constant region comprises one or more mutations that reduce antibody-mediated ADCC and CDC activity, such as amino acid mutations D265A and N297G, and / or L234A and L235A; (c) the constant region comprises a pair of heavy chain constant regions (CH), wherein one CH comprises a mutation introducing a knob structure (e.g., mutation S354C and / or T366W), and the other CH comprises a mutation introducing a hole structure (e.g., Y349C, T366S, L368A and / or Y407V), and wherein the knob structure matches the hole structure to form a stable dimer; (d) the constant region comprises a pair of CHs, wherein one CH comprises an amino acid mutation that introduces a positive charge (e.g., mutations E356K and H435R), and the other CH comprises an amino acid mutation that introduces a negative charge (e.g., K439E), and wherein a stable dimer is formed by charge interaction, in, The positions of the above mutations are according to EU numbering.

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