CTLA4 / TIGIT BINDING PROTEIN AND ITS PHARMACEUTICAL APPLICATIONS

Abstract

Provided are a CTLA4 / TIGIT binding protein and the pharmaceutical use thereof. Specifically, the present invention relates to an anti-CTLA4 / TIGIT antibody and a method for treating cancers and the pharmaceutical use thereof.

Description

CTLA4 / TIGIT binding protein and its medical use

[0001] This application claims priority to Chinese patent application CN202311110063.8 filed on August 31, 2023. Technical Field

[0002] The present disclosure relates to CTLA4 and TIGIT binding proteins, such as anti-CTLA4 / TIGIT antibodies, and their use in treating cancer. Background Art

[0003] During the anti-tumor immune response, T cell activation requires two signals. The first signal is antigen-specific and is initiated by the T cell receptor (TCR) complex recognizing the antigen peptide-MHC complex on the surface of the APC cell. The second signal is a co-stimulatory signal, which requires the participation of co-stimulatory molecules (such as CD28). When CD28 binds to the ligand CD80 / CD86, it further activates T cells, promotes T cell maturation and proliferation, and then initiates an immune response (Nat Rev Immunol. 2013 Apr; 13(4): 227-42.). Regulatory T cells (also known as Treg cells) are an important T cell subset with immunosuppressive effects. Treg cells exert their immunosuppressive function mainly through intercellular contact inhibition (such as expressing inhibitory receptors that interact with ligands on the surface of target cells) and non-contact inhibition (such as secreting various immunosuppressive cytokines such as IL-10) (Nat Immunol. 2019 Feb; 20(2): 218-231).

[0004] CTLA4 protein (cytotoxic T lymphocyte associated antigen-4, also known as CD152) is an immune checkpoint molecule mainly expressed on the surface of activated T cells and Treg cells. It competes with CD28 for binding to ligands CD80 and CD86, and CTLA4 has a higher affinity, exerting an immunosuppressive effect.

[0005] The approved CTLA4 monoclonal antibodies include ipilimumab and tremelimumab. These two antibodies themselves can cause characteristic CTLA4 toxicities in clinical practice, including dermatitis, diarrhea, and enteritis (Oncogene. 2015 Oct; 34(43): 5411-7.; J Transl Med. 2012 Nov 21; 10: 236.), suggesting that CTLA4 toxicity comes from two aspects: one is ADCC-mediated clearance of peripheral Treg cells, and the other is the inhibition of CTLA4 signals in peripheral blood, which disrupts the peripheral immune balance and produces immunotoxicity. Therefore, the development of CTLA4 antibodies that reduce side effects without reducing efficacy has important clinical application value.

[0006] TIGIT protein (T cell immunoreceptor with Ig and ITIM domains, also known as VSIG9) is an immune checkpoint molecule primarily expressed on activated T cells. TIGIT's ligands include CD155 and CD112. CD155 has a high affinity for TIGIT, while CD112 has a weaker affinity for TIGIT. At the same time, CD155 and CD112 are ligands for the co-stimulatory molecule CD226. TIGIT competes with CD226 for binding to CD155 and CD112 on the surface of tumor or APC cells, exerting an immunosuppressive effect (Trends Immunol. 2017 Jan; 38(1): 20-28.). TIGIT antibodies have a good safety profile in clinical practice (Biomedicines. 2021 Sep; 9(9): 1277.).

[0007] The present disclosure provides a novel anti-CTLA4 / TIGIT antibody that, through structural design, can selectively target Treg cells that co-express the target protein in tumors through an avidity effect, and has excellent tumor growth inhibition and killing effects, good drugability, and high potential clinical safety.

[0008] Summary of the Invention

[0009] The present disclosure provides CTLA4 / TIGIT binding proteins and their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and methods and related pharmaceutical uses for treating cancer, eliminating Treg cells, or enhancing T cells.

[0010] CTLA4 / TIGIT binding protein

[0011] The present disclosure provides a CTLA4 / TIGIT binding protein, which comprises a first binding domain that specifically binds to CTLA4 and a second binding domain that specifically binds to TIGIT, and can specifically bind to CTLA4 and TIGIT simultaneously or separately.

[0012] Regarding the first binding domain that specifically binds to CTLA4:

[0013] In some embodiments, the first binding domain that specifically binds to CTLA4 in the CTLA4 / TIGIT binding protein comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein: the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 14.

[0014] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0015] In some embodiments, the first binding domain that specifically binds to CTLA4 in the CTLA4 / TIGIT binding protein comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 1, 2, and 3, respectively, and the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0016] In some embodiments, the first binding domain that specifically binds to CTLA4 in the CTLA4 / TIGIT binding protein comprises VH1 and VL1, wherein: the VH1 comprises a HCDR having 1, 2, 3, 4 or 5 amino acid mutations compared with any of the aforementioned HCDRs; and / or the VL1 comprises a LCDR having 1, 2, 3, 4 or 5 amino acid mutations compared with any of the aforementioned LCDRs.

[0017] In some specific embodiments, the above-mentioned amino acid mutation is an amino acid replacement, substitution, modification, deletion and / or addition (e.g., conservative substitution of amino acids), and the mutation does not affect or substantially does not affect the function of the first binding domain that specifically binds to CTLA4.

[0018] In some embodiments, in the first binding domain of the CTLA4 / TIGIT binding protein that specifically binds to CTLA4:

[0019] The VH1 comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80%, at least 90% (as a non-limiting example, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) sequence identity thereto; and / or,

[0020] The VL1 comprises an amino acid sequence as shown in SEQ ID NO: 14 or having at least 80%, at least 90% (as a non-limiting example, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) sequence identity thereto.

[0021] In some embodiments, the first binding domain that specifically binds to CTLA4 in the CTLA4 / TIGIT binding protein comprises the anti-CTLA4 antibody in tremelimumab, for example, comprises the Fab region or Fv region of tremelimumab.

[0022] In some embodiments, the first binding domain that specifically binds to CTLA4 in the CTLA4 / TIGIT binding protein comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 27, 28 and 29, respectively, and the VL1 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 30, 31 and 32, respectively.

[0023] Regarding the second binding domain that specifically binds to TIGIT:

[0024] In some embodiments, the second binding domain that specifically binds to TIGIT in the CTLA4 / TIGIT binding protein comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein: the VH2 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 15, and the VL2 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 16.

[0025] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0026] In some embodiments, the second binding domain that specifically binds to TIGIT in the CTLA4 / TIGIT binding protein comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively.

[0027] In some embodiments, the second binding domain that specifically binds to TIGIT in the CTLA4 / TIGIT binding protein comprises VH2 and VL2, wherein: the VH2 comprises a HCDR having 1, 2, 3, 4 or 5 amino acid mutations compared with any of the aforementioned HCDRs; and / or, the VL2 comprises a LCDR having 1, 2, 3, 4 or 5 amino acid mutations compared with any of the aforementioned LCDRs.

[0028] In some specific embodiments, the above-mentioned amino acid mutation is an amino acid replacement, substitution, modification, deletion and / or addition (such as conservative substitution of amino acids), and the mutation does not affect or basically does not affect the function of the second binding domain that specifically binds to TIGIT.

[0029] In some embodiments, the second binding domain of the CTLA4 / TIGIT binding protein specifically binds to TIGIT, wherein:

[0030] The VH2 comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 80%, at least 90% (as a non-limiting example, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) sequence identity thereto; and / or,

[0031] The VL2 comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 80%, at least 90% (as a non-limiting example, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) sequence identity thereto.

[0032] In some embodiments, the second binding domain that specifically binds to TIGIT in the CTLA4 / TIGIT binding protein comprises an anti-TIGIT antibody in tiragolumab, domvanalimab, vibostolimab, or ociperlimab, for example, comprises the Fab region or Fv region of tiragolumab, domvanalimab, vibostolimab, or ociperlimab.

[0033] In some embodiments, the CTLA4 / TIGIT binding protein further comprises a human immunoglobulin Fc region. In some specific embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4, such as the Fc region of human IgG1.

[0034] In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule. In some embodiments, the Fc region helps to extend the in vivo half-life of the binding protein.

[0035] In some embodiments, the Fc comprises an amino acid mutation at one or more positions selected from 239, 332, and 330 (IgG1, EU index numbering) (or equivalent positions in other IgG isotypes). In some specific embodiments, the Fc has 239D / 332E / 330L, or 239D / 332E mutations; illustratively, S239D / I332E / A330L, or S239D / I332E.

[0036] In some embodiments, the Fc comprises a mutation selected from the group consisting of:

[0037] 1) 236 bits; for example, 236A; illustratively, G236A;

[0038] 2) at least one of 298, 333, and 334; for example, 298A / 333A / 334A; illustratively, S298A / E333A / K334A;

[0039] 3) at least one of 235, 243, 292, 300, 305, 396; for example, 243L / 292P / 300L / 305I / 396L, or 235V / 243L / 292P / 300L / 396L; illustratively, F243L / R292P / Y300L / V305I / P396L, or L235V / F243L / R292P / Y300L / P396L;

[0040] 4) 234Y / 235Q / 236W / 239M / 268D / 270E / 298A on one heavy chain and 270E / 326D / 330M / 334E on the other heavy chain; illustratively, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A on one heavy chain and D270E / K326D / A330M / K334E on the other heavy chain.

[0041] In some embodiments, mutations are introduced into the Fc region that have the effect of enhancing antibody ADCC.

[0042] In some embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2), and a mutation is introduced that allows the two subunits (Fc1, Fc2) of the Fc region to pair to form a dimer, or a mutation that reduces homodimerization.

[0043] In some embodiments, the Fc region comprises a knob-into-hole mutation that promotes the association of the first subunit and the second subunit. For example, within the CH3 / CH3 interface, one, two, or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1; and one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1.

[0044] In some embodiments, the Fc1 has one or more amino acid substitutions at positions selected from 354, 356, 358, and 366, and the Fc2 has one or more amino acid substitutions at positions selected from 349, 356, 358, 366, 368, and 407. In some specific embodiments, the Fc1 contains a mutation at position 366, and the Fc2 contains a mutation at positions 366, 368, and 407, or any combination thereof. In some specific embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349. In some specific embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349, 366, 368, and 407.

[0045] In some embodiments, the Fc1 comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 comprises one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some specific embodiments, the Fc1 comprises a 366W mutation, and the Fc2 comprises a mutation selected from 366S, 368A, and 407V, or any combination thereof. In some specific embodiments, the Fc1 comprises a 354C or 356C mutation, and the Fc2 comprises a 349C mutation. Alternatively, in some specific embodiments, the Fc1 comprises 354C / 366W mutations, and the Fc2 comprises 349C / 366S / 368A / 407V mutations.

[0046] In some embodiments, the sequence of the Fc1 is shown in SEQ ID NO: 25, and the sequence of the Fc2 is shown in SEQ ID NO: 26.

[0047] In some embodiments, in the CTLA4 / TIGIT binding protein, the mispairing between the light and heavy chains is reduced by mutating the amino acid size and charge of the amino acids at the interface of the heavy chain CH1 and the light chain CL. For example, Roche exchanged the CH1 and CL domains and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187–11192(2011)); MedImmune introduced disulfide bonds by mutating the heavy chain F126C and the light chain S121C (Mazor et al., mAbs, 7(2), pp.377–389(2015)); Amgen further modified the CH1-CL region with electrostatic interactions (Liu et al., Journal of Biological Chemistry, 290(12), pp.7535–7562(2015)); and Lilly (Lewis et al., Nature Biotechnology, 32(2), pp.191–198 (2014)) and Genentech (Dillon et al., mAbs, 9(2), pp.213–230 (2017)) introduced mutations in both the variable and constant domains. WuXi AppTec replaced the constant region of an antibody with that of a TCR, as described in CN109535257A (incorporated herein by reference).

[0048] In some embodiments, the CTLA4 / TIGIT binding protein contains a linker.

[0049] In some embodiments, the linker is such as (G m S n ) h or (GGNGT) h (SEQ ID NO: 44) or (YGNGT) h (SEQ ID NO: 45) or (EPKSS) h (SEQ ID NO: 46), wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0050] In some embodiments, the linker is (G x S)y A linker, wherein x is selected from an integer of 1-5 (e.g., 1, 2, 3, 4, or 5), and y is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the linker is selected from G4S (SEQ ID NO: 47), GS, GAP, (G4S)2 (SEQ ID NO: 48), (G4S)3 (SEQ ID NO: 49), (G4S)4 (SEQ ID NO: 50), (G4S)5 (SEQ ID NO: 51), and ASGS (SEQ ID NO: 52).

[0051] In some embodiments, the CTLA4 / TIGIT binding protein contains only one first binding domain that specifically binds to CTLA4, and at least one second binding domain that specifically binds to TIGIT.

[0052] In some specific embodiments, the CTLA4 / TIGIT binding protein contains only one first binding domain that specifically binds to CTLA4, and only one second binding domain that specifically binds to TIGIT.

[0053] In some embodiments, in the CTLA4 / TIGIT binding protein, the first binding domain that specifically binds to CTLA4 is a Fab, and the second binding domain that specifically binds to TIGIT is a replaced Fab comprising a titin-T chain and an obscurin-O chain linked to a variable region, i.e., the original CH1 and CL of the Fab are replaced by the titin-T chain and the obscurin-O chain. In some embodiments, the VH2 of the second binding domain is fused directly or via a linker to the N-terminus of the titin-T chain, and the VL2 of the second binding domain is fused directly or via a linker to the N-terminus of the obscurin-O chain; or, the VH2 of the second binding domain is fused directly or via a linker to the N-terminus of the obscurin-O chain, and the VL2 of the second binding domain is fused directly or via a linker to the N-terminus of the titin-T chain.

[0054] In some embodiments, in the CTLA4 / TIGIT binding protein, the second binding domain that specifically binds to TIGIT is a Fab, and the first binding domain that specifically binds to CTLA4 is a replaced Fab, wherein the replaced Fab comprises a titin-T chain and an obscurin-O chain linked to a variable region, i.e., the original CH1 and CL of the Fab are replaced by the titin-T chain and the obscurin-O chain. In some embodiments, the VH1 of the first binding domain is fused directly or via a linker to the N-terminus of the titin-T chain, and the VL1 of the first binding domain is fused directly or via a linker to the N-terminus of the obscurin-O chain; or, the VH1 of the first binding domain is fused directly or via a linker to the N-terminus of the obscurin-O chain, and the VL1 of the first binding domain is fused directly or via a linker to the N-terminus of the titin-T chain.

[0055] The replacement technology of the Titin-T chain and the Obscurin-O chain is described in detail in WO2021139758A1, which is incorporated herein by reference in its entirety.

[0056] In some embodiments, the CTLA4 / TIGIT binding protein comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein

[0057] The first heavy chain, from N-terminus to C-terminus, is composed of: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region],

[0058] The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 2]-[Titin-T chain],

[0059] The second heavy chain, from N-terminus to C-terminus, consists of: [VH2]-[CH1]-[second subunit of the Fc region], and

[0060] The second light chain, from N-terminus to C-terminus, is: [VL2]-[CL]; or,

[0061] The first heavy chain, from N-terminus to C-terminus, consists of: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region],

[0062] The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 2]-[Titin-T chain],

[0063] The second heavy chain, from N-terminus to C-terminus, consists of: [VH1]-[CH1]-[second subunit of the Fc region], and

[0064] The second light chain, from N-terminus to C-terminus, is: [VL1]-[CL];

[0065] in,

[0066] - represents a peptide bond, and the linker 1, linker 2 and linker 3 may be the same or different, and may exist independently or not.

[0067] In some embodiments, the Obscurin-O chain is as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto, and the Titin-T chain is as set forth in SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereto.

[0068] In some embodiments, Linker 1 and Linker 2 are represented by GGGGS (SEQ ID NO: 35), and Linker 3 is absent.

[0069] In some embodiments, the CTLA4 / TIGIT binding protein comprises the following polypeptide chain combination:

[0070] a first heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 21 or having at least 90% sequence identity thereto,

[0071] a first light chain comprising an amino acid sequence as set forth in SEQ ID NO: 22 or having at least 90% sequence identity thereto,

[0072] a second heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 90% sequence identity thereto, and / or

[0073] A second light chain comprising an amino acid sequence as set forth in SEQ ID NO: 24, or at least 90% sequence identity thereto.

[0074] In some embodiments, a CTLA4 / TIGIT binding protein is provided that comprises a combination of polypeptide chains of SEQ ID NOs: 21-24.

[0075] In some embodiments, the CTLA4 / TIGIT binding protein contains two first binding domains that specifically bind to CTLA4, and two second binding domains that specifically bind to TIGIT.

[0076] In some embodiments, a CTLA4 / TIGIT binding protein is provided that comprises a combination of polypeptide chains of SEQ ID NOs: 33-35.

[0077] In some embodiments, the aforementioned CTLA4 / TIGIT binding protein has at least one of the following properties:

[0078] a) specifically binds to human CTLA4 or an epitope thereof;

[0079] b) specifically binds to human TIGIT or an epitope thereof;

[0080] c) The binding activity to cells co-expressing CTLA4 and TIGIT is better than that to cells expressing CTLA4 alone; for example, the EC 50 Ratio ≥ 2; illustratively, EC 50 Ratio ≥ 2, EC 50 Ratio ≥ 3, EC 50 Ratio ≥ 4, EC 50 Ratio ≥ 5, EC 50 Ratio ≥ 6, EC 50 Ratio ≥7, EC 50 Ratio ≥8, EC 50 Ratio ≥ 9, EC 50 Ratio ≥ 10, EC 50 Ratio ≥ 20, EC 50 ratio ≥30 or higher;

[0081] d) In the presence of both CTLA4 and TIGIT, the blocking activity against CTLA4-CD80 is better than that in the presence of only CTLA4; for example, the IC 50 Ratio ≥ 2, IC 50 Ratio ≥ 3, IC 50 Ratio ≥ 4, IC 50 Ratio ≥ 5, IC 50 Ratio ≥ 6, IC 50 Ratio ≥7, IC 50 Ratio ≥8, IC 50 Ratio ≥9, IC 50 Ratio ≥10, IC 50 Ratio ≥ 20, IC 50 ratio ≥30 or higher;

[0082] e) Elimination or suppression of Treg cells, for example, intratumoral Treg cells;

[0083] f) activating T cells and / or promoting T cell proliferation; for example, activating CD8+ T cells in tumors;

[0084] g) inhibiting tumor growth, or treating or alleviating cancer.

[0085] In some embodiments, the aforementioned CTLA4 / TIGIT binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0086] In some embodiments, the CTLA4 / TIGIT binding protein of the present disclosure is an anti-CTLA4 / TIGIT antibody, such as an anti-CTLA4 / TIGIT bispecific antibody or an anti-CTLA4 / TIGIT multispecific antibody.

[0087] The antibodies include antigen-binding fragments, including but not limited to Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. In some specific embodiments, the antigen-binding fragment includes Fab, Fv, sFv, Fab', F(ab')2.

[0088] In some embodiments, anti-CTLA4 / TIGIT antibodies are provided that compete with the aforementioned CTLA4 / TIGIT binding proteins of the present disclosure for binding to CTLA4 and / or TIGIT, or compete for binding to the same epitope of CTLA4 and / or TIGIT.

[0089] In some embodiments, anti-CTLA4 / TIGIT antibodies are provided that block the binding of the aforementioned CTLA4 / TIGIT binding proteins of the present disclosure to CTLA4 and / or TIGIT.

[0090] In some embodiments, a protein or molecule is provided, comprising any of the aforementioned CTLA4 / TIGIT binding proteins disclosed herein. For example, the protein or molecule is a conjugate, and the conjugate may comprise any detectable label.

[0091] The CTLA4 / TIGIT binding proteins (e.g., anti-CTLA4 / TIGIT antibodies) provided by the present disclosure can improve the binding ability to cells co-expressing CTLA4 and TIGIT through the avidity effect, thereby enhancing the blocking activity against CTLA4-CD80.

[0092] The CTLA4 / TIGIT binding protein provided by the present invention selectively eliminates Treg cells in tumors, causes an increase in CD8+ T cells in tumors, reduces toxicity, improves anti-tumor activity, has an excellent dosing window and drugability, and provides a solution for the clinical treatment of tumors.

[0093] Polynucleotides and vectors

[0094] The present disclosure provides polynucleotides encoding the CTLA4 / TIGIT binding proteins provided by the present disclosure.

[0095] In some embodiments, the polynucleotide may be RNA, DNA, or cDNA.According to some embodiments of the present disclosure, the polynucleotide of the present disclosure is a substantially isolated nucleic acid.

[0096] The nucleic acid of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector may be, in particular, an expression vector, i.e., a vector that provides for expression of the CTLA4 / TIGIT binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host. Regulatory elements and other elements useful or necessary for the expression of the CTLA4 / TIGIT binding protein of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0097] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0098] host cells

[0099] The present disclosure provides recombinant host cells that express or are capable of expressing one or more CTLA4 / TIGIT binding proteins of the present disclosure, and / or contain a nucleic acid or vector of the present disclosure.

[0100] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0101] Exemplary bacterial cells include, for example, cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0102] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0103] Illustratively, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0104] The present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0105] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0106] Production or preparation method

[0107] The present disclosure provides a method for preparing any of the CTLA4 / TIGIT binding proteins of the present disclosure. In some embodiments, the method comprises the following steps:

[0108] - culturing the host cell of the present disclosure under conditions suitable for expression of the antibody; and

[0109] - recovering the target protein expressed by the host cell from the culture; and

[0110] - Optionally, further purification and / or modification of the protein of interest of the present disclosure is included.

[0111] The CTLA4 / TIGIT binding proteins of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0112] Methods and reagents for recombinantly producing polypeptides, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, isolation and purification techniques suitable for producing target proteins, such as binding proteins or antibodies, are well known to those skilled in the art. Methods for producing and purifying antibodies are well known in the art and can be found in the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). The engineered antibodies disclosed herein can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors to produce antibodies. The culture medium containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and multimers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be frozen immediately, such as at -70°C, or freeze-dried.

[0113] However, the CTLA4 / TIGIT binding proteins of the present disclosure can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0114] Composition

[0115] The present disclosure provides a composition comprising any one or any combination of the following: any CTLA4 / TIGIT binding protein provided by the present disclosure, a polynucleotide encoding a CTLA4 / TIGIT binding protein, and a vector.

[0116] In some embodiments, the pharmaceutical composition contains an amount of the CTLA4 / TIGIT binding protein, encoding polynucleotide, or vector as described above that is effective for treating, alleviating, or preventing cancer.

[0117] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0118] In some embodiments, the unit dosage of the pharmaceutical composition may contain 0.01 to 99 weight % of CTLA4 / TIGIT binding protein; or the amount of CTLA4 / TIGIT binding protein contained in a unit dose of the pharmaceutical composition is 0.1-2000 mg; in some embodiments, 1-1000 mg.

[0119] In some embodiments, an article or product is provided comprising the aforementioned CTLA4 / TIGIT binding protein, polynucleotide, and / or vector. Optionally, the article comprises a container and a label. The container, for example, a bottle, syringe, or test tube, holds a composition effective for treating a condition. The label on or associated with the container indicates that the composition is used to treat the selected condition. The composition contains the aforementioned CTLA4 / TIGIT binding protein, polynucleotide, and / or vector.

[0120] Kits and Tests

[0121] The present disclosure provides a kit comprising the aforementioned CTLA4 / TIGIT binding proteins, polynucleotides, vectors, and compositions. The present disclosure also provides methods, systems, or devices for detecting CTLA4 or TIGIT in vivo or in vitro, comprising treating a sample with the aforementioned binding proteins, polynucleotides, vectors, and compositions of the present disclosure.

[0122] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0123] (1) contacting a sample with a CTLA4 / TIGIT binding protein, polynucleotide, vector or composition disclosed herein;

[0124] (2) detecting a complex formed between the aforementioned binding protein, polynucleotide, carrier or composition and the sample; and / or

[0125] (3) contacting a reference sample (e.g., a control sample) with the binding protein and polynucleotide; and

[0126] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample or subject compared to the control sample or subject indicates the presence of CTLA4 and TIGIT in the sample.

[0127] In other embodiments, the in vivo detection method, system or device may include:

[0128] (1) administering the aforementioned binding protein, polynucleotide or vector to a subject; and

[0129] (2) Detecting the formation of a complex among the binding protein, polynucleotide, carrier, and subject.

[0130] Detection can include determining the location or time of complex formation. The aforementioned binding protein and nucleic acid are labeled with a detectable substance, and the detection of the substance (e.g., CTLA4, TIGIT) of the protein or nucleic acid that can be bound is achieved by detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. The formation of complexes between binding proteins, nucleic acids, and CTLA4 or TIGIT can be detected by measuring substances that are bound or not bound to CTLA4 or TIGIT or by visualizing them. Conventional detection assays can be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry. For detection purposes, the binding proteins and nucleic acids of the present disclosure can be labeled with fluorophore chromophores. In some embodiments, diagnostic reagents comprising the above-mentioned nucleic acids and binding proteins are also provided, as well as related diagnostic uses.

[0131] In some embodiments, a kit is also provided, comprising the aforementioned binding protein, polynucleotide, and instructions for diagnostic use. The kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the binding protein may be formulated as a pharmaceutical composition.

[0132] Methods for treating diseases and pharmaceutical uses

[0133] The present disclosure provides uses and methods of CTLA4 / TIGIT binding proteins, encoding polynucleotides, vectors or pharmaceutical compositions in preventing, treating or alleviating diseases or conditions.

[0134] In some embodiments, the present disclosure provides at least one of the following uses of the CTLA4 / TIGIT binding protein, or encoding polynucleotide, vector, or pharmaceutical composition:

[0135] 1) Use in the preparation of drugs for preventing, treating or alleviating diseases or conditions;

[0136] 2) for preventing, treating or alleviating a disease or condition;

[0137] 3) Used to activate T cells;

[0138] 4) Use in the preparation of drugs for activating T cells;

[0139] 5) Used to eliminate or inhibit Treg cells;

[0140] 6) Use in the preparation of drugs for eliminating or inhibiting Treg cells;

[0141] 7) Used to selectively kill CTLA4 and TIGIT double-positive cells;

[0142] 8) Application in the preparation of drugs for selectively killing CTLA4 and TIGIT double-positive cells;

[0143] 9) Used to reduce peripheral toxicity, improve side effects and enhance safety.

[0144] In some embodiments, the present disclosure provides a method for preventing, treating, or ameliorating a disease or condition, comprising administering to a patient or subject an effective amount of the CTLA4 / TIGIT binding protein or encoding polynucleotide, vector, or pharmaceutical composition of the present disclosure for preventing, treating, or ameliorating the disease or condition.

[0145] In some embodiments, the disease or disorder is a tumor or cancer. In some specific embodiments, the cancer is colorectal cancer, non-small cell lung cancer, or breast cancer.

[0146] In some embodiments, the CTLA4 / TIGIT binding proteins, or encoding polynucleotides, vectors, and pharmaceutical compositions of the present disclosure can be administered by any suitable method known in the art, and administration can be systemic or local.

[0147] In some embodiments, the dosage regimen can be adjusted to obtain the optimal intended response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the therapeutic situation.

[0148] The ordinal numbers "first", "second", "third", "1", "2", "3", etc. (such as "first subunit", "Fc2", "third chain", "linker 2") in this disclosure are only used to distinguish different features, elements, components or steps, and are not intended to limit the quantity, order or level. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figures 1A to 1D show the expression of CTLA4 and TIGIT in tumor patient samples from single-cell sequencing results analyzed by bioinformatics. Figure 1A shows the co-expression ratio of CTLA4 and TIGIT on tumor Tregs in breast cancer (BRCA), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC); Figure 1B shows the expression levels of CTLA4 and TIGIT on tumor Tregs in BRCA, CRC, and NSCLC; Figure 1C shows the co-expression ratio of CTLA4 and TIGIT in various immune cells; and Figure 1D shows the co-expression ratio of CTLA4 and TIGIT on tumor Tregs in patients with CRC or NSCLC.

[0150] Figure 2 is a flow cytometry analysis of the expression levels of CTLA4, TIGIT, or co-expression of CTLA4, TIGIT, or both on Tregs within the tumor in the CTLA4-TIGIT dual humanized mouse MC38 transplant tumor model.

[0151] Figures 3A to 3C show flow cytometry analysis of the binding activity of anti-CTLA4 / TIGIT antibodies to cell surface CTLA4 and TIGIT antigens. Figures 3A and 3B show binding to CHOK1-CTLA4 cells, and Figure 3C shows binding to CHOK1-TIGIT cells.

[0152] Figures 4A and 4B show flow cytometry analysis of the blocking activity of anti-CTLA4 / TIGIT antibodies against CTLA4-CD80 and TIGIT-CD155. Figure 4A shows the blocking of CTLA4-CD80 cell binding, and Figure 4B shows the blocking of TIGIT-CD155 cell binding.

[0153] Figures 5A and 5B show flow cytometry analysis of the binding activity of anti-CTLA4 / TIGIT antibodies to HEK293-CTLA4 single-positive cells and HEK293-CTLA4 / TIGIT double-positive cells. Figure 5A shows the binding activity of Ipi, and Figure 5B shows the binding activity of As-3.

[0154] Figure 6 shows the ELISA assay for the blocking activity of anti-CTLA4 / TIGIT antibodies against CTLA4-CD80.

[0155] Figures 7A and 7B show the ADCC activity of anti-CTLA4 / TIGIT antibodies against HEK293-CTLA4 single-positive cells and HEK293-CTLA4 / TIGIT double-positive cells detected by LDH. Figure 7A shows the ADCC activity of Ipi, and Figure 7B shows the ADCC activity of As-3.

[0156] FIG8 shows the ADCC killing activity of NK cells against iTreg mediated by anti-CTLA4 / TIGIT antibodies detected by LDH.

[0157] Figure 9 shows the CDC killing activity mediated by anti-CTLA4 / TIGIT antibodies detected by CTG.

[0158] Figures 10A and 10B are in vivo validations of the inhibitory effect of anti-CTLA4 / TIGIT antibodies on MC38 xenograft tumors in CTLA4-TIGIT dual human mice.

[0159] Figure 11 is an in vivo verification of the effects of anti-CTLA4 / TIGIT antibodies on intratumoral and peripheral Treg and CD8+T cells in the CTLA4-TIGIT dual human mouse MC38 transplant tumor model.

[0160] Figures 12A and 12B: Exemplary structures of anti-CTLA4 / TIGIT antibodies. DETAILED DESCRIPTION

[0161] Definition of terms

[0162] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0163] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0164] "CTLA4" or "CTLA4" or "CTLA4 protein" or "CTLA4 polypeptide" may optionally include any such protein or variant, conjugate, or fragment thereof, including but not limited to known or wild-type CTLA4 as described herein, as well as any naturally occurring splice variants, amino acid variants, or isoforms. The complete CTLA4 sequence can be found at GenBank Accession No. NP_005205.

[0165] "TIGIT" or "TIGIT protein" or "TIGIT polypeptide" may optionally include any such protein or variant, conjugate, or fragment thereof, including but not limited to known or wild-type TIGIT as described herein, as well as any naturally occurring splice variant, amino acid variant, or isoform. The complete TIGIT sequence can be found at GenBank Accession No. NP_776160.

[0166] The term "functional variant" includes but is not limited to homologs, fragments, truncations, mutants, modifications, etc. of the wild-type protein. The functional variant of the protein has improved, reduced or maintained protein activity compared to the wild-type protein.

[0167] The "binding protein" and "binding molecule" of the present disclosure cover any protein, polypeptide or any molecule comprising the protein or polypeptide that can specifically bind to an antigen (such as CTLA4 or TIGIT) or a fragment or epitope thereof, including but not limited to antibodies as defined in the present disclosure.

[0168] Throughout this disclosure, "polypeptide," "peptide," or "protein" are used interchangeably to refer to a polymer of amino acid residues, or an aggregate of multiple polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptide sequences are typically described with the left-hand end of the polypeptide sequence being the amino terminus (N-terminus, N-terminus) and the right-hand end of the polypeptide sequence being the carboxyl terminus (C-terminus, C-terminus).

[0169] "Antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, bispecific or multispecific antibodies (e.g., trispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, antigen-binding domains), as long as they exhibit the desired antigen-binding activity. Antibodies of the present disclosure include recombinant antibody forms.

[0170] Antibodies, also known as immunoglobulins, are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of immunoglobulins (Ig) heavy chains vary, resulting in different antigenicity. This classification leads to the classification of immunoglobulins into five classes, or isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same Ig class, the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds are further divided into subclasses. For example, IgG is divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on their constant regions. Each of the five Ig classes can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly, forming the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, forming the constant region (C region). The variable region consists of three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0171] "Antigen-binding fragment" or "antigen-binding domain" encompasses Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0172] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0173] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0174] Table 1. Relationships between CDR numbering systems

[0175] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0176] Although in specific embodiments, one numbering system (such as Kabat) is used to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent technical solutions.

[0177] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antigen-binding site of an antibody molecule and a corresponding antigen epitope). The binding affinity between two molecules can be determined by determining the dissociation equilibrium constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of the complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and FACS, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore. TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D K value and non-target interaction D The value is evaluated.

[0178] Avidity refers to the strength of binding between the antigen-binding site of an intact antibody molecule and several corresponding antigenic epitopes. For example, an anti-CTLA4 / TIGIT antibody binds to both CTLA4 and TIGIT antigens.

[0179] Typically, "specific binding" refers to binding of a binding molecule (binding protein) to an epitope on an antigen. The CTLA4 / TIGIT binding proteins of the present disclosure will bind to an epitope as measured in a Biacore or KinExA or Fortibio assay at a specific binding affinity of ≤10 -7 M, preferably ≤10 -8 The dissociation equilibrium constant (K D ) binds to the antigen (i.e. CTLA4 or TIGIT) or its epitope. -4 M's K D Values ​​are generally considered to indicate nonspecific binding. Specific binding of a binding molecule to an antigen or epitope can be determined by any suitable means known in the art, including, for example, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry sorting (FACS) as described herein.

[0180] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0181] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two nucleic acid sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0182] "Obscurin-O chain" or "O chain" refers to a peptide segment of the obscurin protein containing the obscurin Ig-like 1 domain, or a functional variant thereof, that is 87-117 amino acids in length. The obscurin-O chain is capable of forming a complex with the titin Ig-like 152 domain through intermolecular interactions. The functional obscurin-O chain variants are characterized by partial amino acid mutations in the wild-type O chain, but still contain a peptide segment capable of forming a complex with the titin Ig-like 152 domain through intermolecular interactions.

[0183] "Titin-T chain" or "T chain" refers to a 78-118 amino acid peptide segment of the titin protein containing the titin Ig-like 152 domain, or a functional variant thereof. The titin-T chain is capable of binding to the obscurin Ig-like 1 or obscurin Ig-like 1 domain through intermolecular interactions to form a complex. The functional T chain variant is a variant of the wild-type T chain with partial amino acid mutations, but still contains a peptide segment that binds to the obscurin Ig-like 1 or obscurin Ig-like 1 domain through intermolecular interactions to form a complex.

[0184] Obscurin-O chain or titin-T chain can be used to replace the CH1 or CL domain of an antibody without affecting the binding of the antibody VH and VL and the binding of the antibody to the antigen epitope.

[0185] "Nucleic acid" or "polynucleotide" are used interchangeably herein to refer to any DNA or RNA molecule that is single-stranded or double-stranded and, in the case of single-stranded molecules, its complementary sequence, preferably double-stranded DNA.

[0186] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable vehicle" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents.

[0187] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0188] "Proliferative disease" refers to a condition associated with some degree of abnormal cell proliferation. In one embodiment, a proliferative condition refers to cancer. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0189] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0190] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0191] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0192] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to."

[0193] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0194] Example

[0195] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0196] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0197] Example 1. Analysis of CTLA4 and TIGIT expression in tumors

[0198] 1. Single-cell sequencing analysis of CTLA4 and TIGIT co-expression in tumor patient samples

[0199] In this example, the inventors used bioinformatics to analyze the results of single-cell sequencing of patient samples from a variety of high-incidence cancers in the TISCH database, including breast cancer (BRCA), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). The data came from a public data set included in the database. The data were normalized by TPM (the unit of expression was Log(TPM / 10+1)), the cell clustering algorithm used the Louvain algorithm, and the dimensionality reduction and visualization analysis used the UMAP algorithm. The inventors analyzed the co-expression ratio and expression level of TIGIT and CTLA4 on different immune cells in the tumor, as well as the difference in co-expression ratios between intratumoral Treg and peripheral Treg.

[0200] The results are shown in Figures 1A to 1D. CTLA4 and TIGIT are highly co-expressed on intratumoral Tregs in a variety of highly prevalent cancers (Figure 1A). Their expression levels on intratumoral Tregs are comparable across a variety of highly prevalent cancers (Figure 1B). Among intratumoral immune cells, CTLA4 and TIGIT have the highest co-expression ratio on Tregs compared to other immune cells (Figure 1C). The co-expression ratio of CTLA4 and TIGIT on intratumoral Tregs is significantly higher than that on peripheral Tregs (Figure 1D). The above results from human tumor samples demonstrate that CTLA4 and TIGIT are selectively highly expressed and co-expressed on intratumoral Tregs.

[0201] 2. Analysis of CTLA4 and TIGIT Co-expression in the MC38 Colon Cancer Model of the CTLA4 / TIGIT Dual Humanized Mouse

[0202] Experimental methods: CTLA4 / TIGIT double humanized mice (purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were adaptively bred for 7 days. MC38 cells (purchased from Sunran Shanghai Biotechnology Co., Ltd.) were inoculated at 5×10 5 The cells / 0.1 mL / mouse were inoculated subcutaneously on the right side of double humanized mice. The weight and tumor volume of the mice were measured twice a week. Five mice with tumors of 300-500 mm were selected. 3 The mice were euthanized, the tumors were removed and photographed, and anticoagulated blood and tumors were collected for flow cytometry detection (flow panel: Live / dead, mCD45, mCD3, mCD4, mCD25, mFoxp3, hCTLA4, hTIGIT). The data were statistically analyzed (Mean±SEM), and p<0.05 was considered to be significantly different.

[0203] The results are shown in Figure 2. TIGIT expression on tumor Tregs was significantly higher than that on peripheral blood Tregs; CTLA4 expression on tumor Tregs was significantly higher than that on peripheral blood Tregs; and the co-expression ratio of CTLA4 and TIGIT on tumor Tregs was significantly higher than that on peripheral blood Tregs. The above results from double-humanized mice also showed that CTLA4 and TIGIT were significantly highly expressed and co-expressed on Tregs in tumor tissue.

[0204] Example 2. Design and preparation of anti-CTLA4 / TIGIT antibodies

[0205] In this example, anti-CTLA4 / TIGIT antibodies As-3 and S-15 were designed.

[0206] The As-3 molecule (Figure 12A) contains two heavy chains and two light chains. The CDRs used by the CTLA4 end are shown in SEQ ID NOs: 1-6, and the heavy chain variable region (VH) and light chain variable region (VL) are shown in SEQ ID NOs: 13-14; the CDRs used by the TIGIT end are shown in SEQ ID NOs: 7-12, and the heavy chain variable region (VH) and light chain variable region (VL) are shown in SEQ ID NOs: 15-16.

[0207] To prevent and reduce mispairing of heavy and light chains, Obscurin-O and Titin-T are shown in SEQ ID NOs: 17-18, and CH1 and CK are shown in SEQ ID NOs: 19-20.

[0208] To prevent and reduce heavy chain mispairing, Fc1 and Fc2 with KIH mutations were used, and DLE mutations were used to enhance ADCC. The sequences are shown in SEQ ID NOs: 25-26.

[0209] The heavy chain sequence of As-3 is shown in SEQ ID NOs: 21 and 23, and the light chain sequence is shown in SEQ ID NOs: 22 and 24.

[0210] The S-15 molecule (as shown in Figure 12B) is a symmetrical bispecific antibody containing one heavy chain and two light chains. The CDRs used on the CTLA4 side are shown in SEQ ID NOs:27-32; the CDRs used on the TIGIT side are shown in SEQ ID NOs:7-12. To prevent and reduce mispairing of the heavy and light chains, the Obscurin-O and Titin-T used are shown in SEQ ID NOs:17-18, and the CH1 and Cκ are shown in SEQ ID NOs:19-20. The heavy chain sequence of S-15 is shown in SEQ ID NO:33, the TIGIT light chain sequence is shown in SEQ ID NO:34, and the CTLA4 light chain sequence is shown in SEQ ID NO:35.

[0211] Table 2. CDRs of anti-CTLA4 / TIGIT antibodies (Kabat numbering convention)

[0212] >As-3 VH1

[0213] >As-3 VL1

[0214] >As-3 VH2

[0215] >As-3 VL2

[0216] >Obscurin-O

[0217] >Titin-T

[0218] >CH1

[0219] >Cκ

[0220] >As-3 H1

[0221] >As-3 L1

[0222] >As-3 H2

[0223] >As-3 L2

[0224] >IgG1 Fc1(S354C,T366W,S239D / A330L / I332E)

[0225] >IgG1 Fc2 (Y349C, T366S, L368A, Y407V, S239D / A330L / I332E)

[0226] >S-15 H1(S239D / A330L / I332E)

[0227] >S-15 L1

[0228] >S-15 L2

[0229] In the above sequence, the underlined straight line is the CDR, the underlined curved line is the linker, and the italicized part is the Fc region.

[0230] The polynucleotide sequence encoding the antibody was cloned into the pTT5 vector and then transfected into CHOK1 cells. The target antibodies As-3 and S-15 were obtained by conventional expression, purification and detection.

[0231] The control antibody sequences used in this disclosure are as follows:

[0232] >Anti-CTLA4 antibody Ipi heavy chain

[0233] >Anti-CTLA4 antibody Ipi light chain

[0234] >Anti-TIGIT antibody S2 heavy chain

[0235] >Anti-TIGIT antibody S2 light chain

[0236] >Anti-CTLA4 antibody Tre heavy chain

[0237] Anti-CTLA4 antibody Tre light chain

[0238] >Anti-TIGIT antibody RG6058 heavy chain

[0239] >Anti-TIGIT antibody RG6058 light chain

[0240] Example 3. Detection of Anti-CTLA4 / TIGIT Antibody Binding Affinity to Antigen

[0241] 1. Biacore detection

[0242] The affinity kinetics of the interaction between anti-CTLA4 / TIGIT antibodies and human TIGIT and human CTLA4 antigens were determined using a Biacore biomacromolecule interaction instrument using surface plasmon resonance (SPR) technology.

[0243] Experimental methods:

[0244] Affinity kinetic analysis of the interaction between anti-CTLA4 / TIGIT antibodies and human CTLA4 (Acro Biosystems, Cat#CT4-H5229) and human TIGIT (Acro Biosystems, Cat#TIT-H52H5) antigens was performed using a Protein A biosensor chip (GE Healthcare, Cat#29127556). The test antibodies were prepared at a certain concentration in HBS-EP+ buffer solution as ligands and captured in the Fc2 (Flow cell 2, Fc2) channel of the chip at a flow rate of 10 μL / min and a capture time of 30 s. CTLA4 and TIGIT were prepared at specific concentrations in HBS-EP+ buffer and injected into the chip's reference channel Fc2 (Flow cell 1, Fc1) and the detection channel Fc2 in HBS-EP+ buffer. The analytes were serially diluted two-fold over a total of seven concentration points at a flow rate of 30 μL / min, with an association time of 120 seconds and a dissociation time of 1200 seconds. Finally, the cells were regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, Cat# BR-1003-54). Regeneration conditions were: a regeneration time of 30 seconds and a flow rate of 30 μL / min.

[0245] Affinity kinetics analysis of the interaction between human CTLA4 and human TIGIT antigens and anti-CTLA4 / TIGIT antibodies was performed using a CM5 chip. Anti-histidine antibodies were covalently coupled to the Fc1 and Fc2 channels of a CM5 biosensor chip (GE Healthcare, Cat#29149603) according to the instructions for the His Capture Kit (Cytiva, Cat#29234602). Human CTLA4 or human TIGIT antigens were captured on the Fc2 (Fc2) channel of the chip at a flow rate of 10 μL / min and a capture time of 30 s. Monovalent anti-CTLA4 / TIGIT antibodies were prepared as analytes in HBS-EP+ buffer and serially diluted two-fold to a total of seven concentration points. The flow rate was 30 μL / min, the association time was 100 s, and the dissociation time was 1800 s. Finally, 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was used for regeneration under the following conditions: regeneration time 30 s, flow rate 30 μL / min.

[0246] Data statistics and analysis: Biacore 8K Control Software was used to collect and save SPR signals, and then Biacore 8K Evaluation software was used for data processing.

[0247] Experimental results: The affinity dissociation constants of AS-3 for human CTLA4 or TIGIT antigens are shown in Table 3-1 (1:1 binding). The results show that AS-3 has similar one-arm affinities to the anti-CTLA4 antibody Ipi and the anti-TIGIT antibody S2.

[0248] The affinity dissociation constants for the interaction between human CTLA4 or human TIGIT antigens and anti-CTLA4 / TIGIT antibodies are shown in Table 3-2 (1:1 binding). The results showed that due to its single-arm design, AS-3 had reduced binding ability to CTLA4 or TIGIT antigens compared to the two-armed anti-CTLA4 antibody Ipi and the two-armed anti-TIGIT antibody S2.

[0249] Table 3-1. Binding affinity of anti-CTLA4 / TIGIT antibodies to antigens CTLA4 and TIGIT

[0250] Table 3-2. Binding affinity of anti-CTLA4 / TIGIT antibodies to antigens CTLA4 and TIGIT

[0251] 2. FACS detection

[0252] FACS assay was used to detect the binding activity of anti-CTLA4 / TIGIT antibodies to human CTLA4 and human TIGIT proteins expressed on the cell surface.

[0253] Experimental Methods: CHOK1-CTLA4 cells were obtained from CHOK1 cells stably expressing human CTLA4 protein, and CHOK1-TIGIT cells were obtained from CHOK1 cells stably expressing human TIGIT protein. The cell culture medium used was F12K (Gibco, Cat#21127-022) containing 10% inactivated fetal bovine serum (Gibco, Cat#10091-148). The buffer was sterile PBS (containing 2% fetal bovine serum). CHOK1-CTLA4 or CHOK1-TIGIT cells were washed twice with the buffer and 1×10 cells were plated per well. 5 Cells were seeded in 96-well U-bottom plates and serially diluted samples were added. The cells were incubated at 4°C for 1 hour and washed twice with assay buffer. Alexa Fluor 647-conjugated goat anti-human (IgG, Fcγ fragment specific) antibody (Jackson ImmunoResearch, Cat# 109-605-098) was then added and incubated at 4°C for 30 minutes. After washing twice with assay buffer, fluorescence signals were read by flow cytometry. The results are shown in Figures 3A to 3C and Tables 3-3, 3-4, and 3-5.

[0254] Experimental Results: As-3 has binding ability to the CTLA4 protein on the surface of CHOK1-CTLA4 cells. Due to its single-arm design, its affinity for CTLA4 is weaker than that of Ipi and Tre. As-3 has good binding ability to the TIGIT protein on the surface of CHOK1-TIGIT cells, comparable to the anti-TIGIT antibodies S2 and RG6058. S-15's binding ability to CTLA4 on the surface of CHOK1-CTLA4 cells is comparable to that of Tre, and its binding ability to the TIGIT protein on the surface of CHOK1-TIGIT cells is comparable to that of the anti-TIGIT antibody S2. hIgG1 is an isotype control antibody.

[0255] Table 3-3. Results of As-3 binding to CHOK1-CTLA4 cells

[0256] Table 3-4. Results of S-15 binding to CHOK1-CTLA4 cells

[0257] Table 3-5. Results of anti-CTLA4 / TIGIT antibody binding to CHOK1-TIGIT cells

[0258] NA indicates that EC cannot be obtained by fitting 50 or IC 50 .

[0259] Example 4. Detection of Anti-CTLA4 / TIGIT Antibody Blocking Activity of Ligand Binding to Antigen on Cells

[0260] In this example, FACS experiments were used to detect whether anti-CTLA4 / TIGIT antibodies blocked the binding of CTLA4 expressed on the cell surface to CD80, and whether they blocked the binding activity of TIGIT expressed on the cell surface to CD155.

[0261] Experimental Methods: CHOK1-CTLA4 cells were derived from CHOK1 cells stably expressing human CTLA4 protein, CHOK1-TIGIT cells were derived from CHOK1 cells stably expressing human TIGIT protein, CHOK1-CD80 cells were derived from CHOK1 cells stably expressing human CD80 protein, and CHOK1-CD155 cells were derived from CHOK1 cells stably expressing human CD155 protein. The cell culture medium used was F12K (Gibco, Cat#21127-022) supplemented with 10% inactivated fetal bovine serum (Gibco, Cat#10091-148). The buffer used was sterile PBS (containing 2% fetal bovine serum). Wash CHOK1-CTLA4, CHOK1-TIGIT, CHOK1-CD80, and CHOK1-CD155 cells twice with buffer, label the antigen-expressing cells and ligand-expressing cells with CFSE (Invitrogen, Cat#C34554) and Far Red (Invitrogen, Cat#C34564), respectively. After labeling, wash away the excess fluorescent dye with experimental buffer. 5 antigen-expressing cells and 1×10 5 Cells expressing the corresponding ligands were seeded in a 96-well U-bottom plate, and the test samples were added with gradient dilutions. After incubation at 37°C for 2 hours, the fluorescence signal value was read using a flow cytometer.

[0262] Experimental Results: See Figures 4A and 4B, Tables 4-1 and 4-2. FACS analysis showed that As-3 blocked the binding of CHOK1-CTLA4 and CHOK1-CD80 cells. Due to its single-arm design, the blocking effect was weaker than that of Ipi and Tre. As-3 also had a good blocking effect on the binding of CHOK1-TIGIT and CHOK1-CD155 cells, comparable to the anti-TIGIT antibodies S2 and RG6058. The blocking effect of S-15 on the binding of CHOK1-CTLA4 and CHOK1-CD80 cells was comparable to that of Tre. The blocking effect of S-15 on the binding of CHOK1-TIGIT and CHOK1-CD155 cells was also comparable to that of the anti-TIGIT antibody S2. Ipilimumab and Tremelimumab induce characteristic CTLA4 toxicity in clinical practice, so the inventors selected As-3, which has weaker blocking activity, for further validation.

[0263] Table 4-1. Results of anti-CTLA4 / TIGIT antibodies blocking CTLA4-CD80 binding

[0264] Table 4-2. Results of anti-CTLA4 / TIGIT antibodies blocking TIGIT-CD155 binding

[0265] NA indicates that EC cannot be obtained by fitting 50 or IC 50 .

[0266] Example 5. Detection of the binding ability and avidity effect of anti-CTLA4 / TIGIT antibodies on cells co-expressing CTLA4 and TIGIT

[0267] 1. Use FACS experiments to detect the binding activity of anti-CTLA4 / TIGIT antibodies to single-positive cells expressing human CTLA4 and double-positive cells co-expressing CTLA4 and TIGIT.

[0268] Experimental Methods: HEK293-CTLA4 cells were obtained from HEK293 cells stably expressing human CTLA4 protein, and HEK293-CTLA4 / TIGIT cells were obtained from HEK293-CTLA4 cells stably expressing human TIGIT protein. The cell culture medium was DMEM (Gibco, Cat#11995-065) containing 10% inactivated fetal bovine serum (Gibco, Cat#10091-148). HEK293-CTLA4 or HEK293-CTLA4 / TIGIT cells were washed twice with buffer (PBS containing 2% fetal bovine serum) and 1×10 cells were plated per well. 5Cells were seeded in 96-well U-bottom plates and serially diluted samples were added. The cells were incubated at 4°C for 1 hour and washed twice with assay buffer. Alexa Fluor 647-labeled goat anti-human (IgG, Fcγ fragment specific) antibody (Jackson ImmunoResearch, Cat#109-605-098) was then added and incubated at 4°C for 30 minutes. After washing twice with assay buffer, fluorescence signals were read by flow cytometry.

[0269] Experimental results: See Figures 5A and 5B, Table 5. As-3 showed superior binding activity to double-positive cells compared to HEK293-CTLA4 single-positive cells, while Ipi showed comparable binding activity to double-positive and single-positive cells. This suggests that As-3 enhances its binding to cells co-expressing CTLA4 and TIGIT through an avidity effect.

[0270] Table 5. Binding activity of anti-CTLA4 / TIGIT antibodies to HEK293-CTLA4 single positive cells and HEK293-CTLA4 / TIGIT double positive cells

[0271] 2. Use ELISA to detect the ability of anti-CTLA4 / TIGIT antibodies to block CTLA4-CD80 in the presence of TIGIT protein.

[0272] Experimental methods: Two groups were set up for comparison. In the first group, CTLA4 protein (Acro Biosystems, Cat#CT4-H5229) and an irrelevant protein were simultaneously coated in a 96-well plate (Costar, Cat#3590). In the second group, CTLA4 protein and TIGIT protein (Acro Biosystems, Cat#TIT-H52H5) were simultaneously coated in a 96-well plate (Costar, Cat#3590). The concentrations were kept the same as those in the first group. Both groups were incubated overnight at 4°C, washed three times, and then 3% BSA was added. After incubation at room temperature for 1 hour, the plate was washed three times. A constant concentration of biotin-labeled CD80 protein (AMSBIO, Cat#71114-1) and serially diluted test antibodies were added. The plate was incubated at room temperature for 2 hours, washed three times, and SA-HRP (Jackson ImmunoResearch, Cat#016-030-084) was added and incubated at room temperature for 1 hour, followed by washing six times. The OD450 value was read using EnVision using a colorimetric solution.

[0273] Experimental Results: See Figure 6 and Table 6. ELISA results showed that when coated with CTLA4 and an unrelated protein, As-3's blocking activity against CTLA4-CD80 was weaker than that of Ipi. However, when coated with both CTLA4 and TIGIT, the blocking activity of As-3 and Ipi was comparable. This suggests that in the presence of both CTLA4 and TIGIT, As-3 enhances its blocking activity against CTLA4-CD80 through an avidity effect, thereby attenuating the immunosuppressive effects of CTLA-4.

[0274] Table 6. Blocking activity of anti-CTLA4 / TIGIT antibodies against CTLA4-CD80

[0275] Example 6. Anti-CTLA4 / TIGIT Antibody-Mediated ADCC Killing and Detection of ADCC's Ability to Clear Tregs

[0276] 1. The lactate dehydrogenase (LDH) assay was used to evaluate the ADCC killing activity of anti-CTLA4 / TIGIT antibodies mediated by NK cells against single-positive cells expressing CTLA4 and double-positive cells co-expressing CTLA4 and TIGIT.

[0277] Experimental Methods: Cryopreserved PBMCs were purchased from Shanghai Saili Biotechnology Co., Ltd. (hereinafter referred to as the same) and cultured in RPMI1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum (Gibco, Cat#10091-148) after thawing and incubated at 37°C overnight. The next day, single-positive and double-positive cells were collected and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum at a density of 2 × 10 5 cells / mL, and then 50 μL / well was seeded into a 96-well plate, and 50 μL of the serially diluted antibody to be tested was added. PBMCs were collected and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum, and the density was adjusted to 2×10 6 Cells / mL were seeded in the above experimental plates at 100 μL / well and incubated in a 37°C, 5% CO2 incubator for 4 hours. The cell culture plates were removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. The supernatant was then purified by CytoTox Non-Radioactive Cytotoxicity Assay Kit was used to detect LDH levels.

[0278] Experimental Results: See Figures 7A and 7B, Table 7. As-3-mediated cytotoxicity against HEK293-CTLA4 / TIGIT double-positive cells was superior to that against HEK293-CTLA4 single-positive cells. Ipi-mediated cytotoxicity against double-positive and single-positive cells was comparable, indicating that As-3 enhanced its selective cytotoxicity against cells co-expressing CTLA4 and TIGIT.

[0279] Table 7. Results of ADCC killing activity of NK cells mediated by anti-CTLA4 / TIGIT antibodies against HEK293-CTLA4 single positive cells and HEK293-CTLA4 / TIGIT double positive cells

[0280] NA indicates that EC cannot be obtained by fitting 50 or IC 50 .

[0281] 2. Use lactate dehydrogenase (LDH) detection to evaluate the ADCC killing activity of NK cells against Treg cells mediated by anti-CTLA4 / TIGIT antibodies.

[0282] Experimental method: First, Treg cells were induced according to EasySep TM Human CD4 + CD4 T Cell Enrichment Kit (STEMCELL, CAT#19052) was used to isolate CD4 T cells from fresh PBMCs. + CD4 T cells were cultured using RPMI 1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum (Gibco, Cat#10091-148). + The T cell density was adjusted to 2.5 × 10 6 / mL, a total volume of 20mL, added with loaded Anti-Biotin MACSiBead Particles (Miltenyi Biotec, Cat#130-091-441) (Bead:cell=1:1), human IL-2 (PEPROTECH, Cat#200-02-50UG) (400U / mL), rapamycin (SELLECK, Cat#S1039) (100ng / mL), TGFβ (PEPROTECH, Cat#100-21) (20ng / mL), atRA (Sigma, Cat#R2625-100MG) (100nM), and cultured at 37°C, 5% CO2 for 3 days. The cell density was adjusted to 1.2-2.5×10 6 / mL, supplemented with 20U / mL human IL-2, and cultured for 3 days at 37 degrees and 5% CO2. Anti-Biotin MACSiBead Particles were removed according to the T Cell Activation / Expansion Kit (Miltenyi Biotec, Cat#130-091-441) and used Regulatory CD4 + CD25 + T Cell Kit (life, Cat#11363D) was used to separate and purify CD4 + CD25 + T cells were lysed and beads were removed to obtain beads-free induced Treg (iTreg) cells. The lactate dehydrogenase (LDH) assay was used to evaluate the ADCC killing activity of antibody-mediated NK cells against iTreg. After thawing, frozen PBMCs were cultured in RPMI1640 (Gibco, Cat#10491A-01) culture medium containing 10% inactivated fetal bovine serum (Gibco, Cat#10091-148) and incubated at 37°C overnight. The next day, iTregs were collected and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum and the density was adjusted to 1×10 5 cells / mL, and then 50 μL / well was seeded into a 96-well plate, and 50 μL of the serially diluted antibody to be tested was added. PBMCs were collected and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum, and the density was adjusted to 5×10 6 Cells / mL were seeded in the above experimental plates at 100 μL / well and incubated in a 37°C, 5% CO2 incubator for 4 hours. The cell culture plates were removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. The supernatant was then purified by CytoTox Non-Radioactive Cytotoxicity Assay Kit was used to detect LDH levels.

[0283] Experimental results: See Figure 8 and Table 8. ADCC assay results showed that As-3 mediated ADCC cytotoxicity against iTreg was stronger than that of Ipi.

[0284] Table 8. Results of ADCC killing activity of NK cells against iTreg cells mediated by anti-CTLA4 / TIGIT antibodies

[0285] NA indicates that EC cannot be obtained by fitting 50 or IC 50 .

[0286] Example 7. Detection of CDC effect mediated by anti-CTLA4 / TIGIT antibodies

[0287] The CTG assay was used to assess the CDC effector activity mediated by anti-CTLA4 / TIGIT antibodies.

[0288] Experimental Methods: CHOK1-CTLA4 cells were obtained by stably transfecting CHOK1 cells expressing human CTLA4 protein. The cell culture medium was F12K (Gibco, Cat#21127-022) containing 10% inactivated fetal bovine serum (Gibco, Cat#10091-148). CHOK1-CTLA4 cells were harvested and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum at a density of 2×10 5 Cells were plated at 50 μL / well in a 96-well plate. 40 μL of serially diluted test antibody was added, followed by 10 μL of complement (QUIDEL, Cat#A113). The mixture was incubated overnight at 37°C in a 5% CO2 incubator. The cell culture plate was removed, and cells were lysed by adding Triton X-ray to the positive control wells. 50 μL of CTG (Promega, Cat#PR-G7573) was then added to each well. The plates were incubated at room temperature for 10 minutes, and fluorescence was read using Envision. Results are shown in Figure 9 and Table 9.

[0289] CDC test results showed that the CDC effect of CTLA4 / TIGIT antibodies was significantly weaker than that of Ipi. This suggests that anti-CTLA4 / TIGIT antibodies can reduce the CDC effect through DLE mutation, which may improve the clinical side effects of CTLA4 antibodies (such as hypophysitis) and enhance safety.

[0290] Table 9. Results of CDC effects mediated by anti-CTLA4 / TIGIT antibodies

[0291] NA indicates that EC cannot be obtained by fitting 50 or IC 50 .

[0292] Example 8. Evaluation of the anti-tumor efficacy of anti-CTLA4 / TIGIT antibodies in a CTLA4 / TIGIT dual humanized mouse model

[0293] The calculation method used in this embodiment is:

[0294] Tumor volume V = 1 / 2 × a × b 2 , where a and b represent length and width respectively.

[0295] TGITV (%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0296] (Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; Vi: mean tumor volume of the solvent control group on day i of administration, V0: mean tumor volume of the solvent control group on day 0 of administration)

[0297] CR% (complete tumor regression ratio) = complete tumor regression (tumor volume < 50 mm for at least 2 consecutive measurements) 3 ) of mice / number of mice enrolled.

[0298] Data Statistics and Analysis: Raw data from measurements and observations must be recorded. Analyses and processing are performed based on the raw data, with results presented as mean ± SEM. Tumor volume should also be statistically analyzed, with a p < 0.05 considered significant. Both statistical and biological significance should be considered when analyzing the results.

[0299] 1. Efficacy testing in the CTLA4 / TIGIT dual-humanized mouse MC38 colon cancer xenograft model

[0300] Experimental methods: CTLA4 / TIGIT dual humanized mice (Animal Center of Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were bred for 7 days. MC38 cells were cultured at 5×10 5 The 0.1 mL / mouse was inoculated subcutaneously on the right side of the mouse, and the average tumor volume reached 95 mm 3 Thirty mice were randomly divided into five groups, with six mice in each group, namely G1: 1×PBS, G2: Ipi (0.3 mg / kg), G3: Ipi (1 mg / kg), G4: As-3 (0.3 mg / kg), G5: As-3 (1 mg / kg). Drug administration began on the day of grouping (Day 0). The route of administration was intraperitoneal injection. The drug was administered twice a week for a total of six times. The body weight and tumor volume of the mice were measured twice a week during the administration and observation period. At the end of the experiment, the animals were euthanized, the tumors were removed and photographed, and the tumor growth inhibition rate (TGI) was calculated. TV ).

[0301] The results are shown in Figure 10A and Table 10. The results showed that As-3 exhibited significant antitumor activity in a dose-dependent manner, and its antitumor activity was comparable to that of Ipi, without causing significant weight changes in mice (Figure 10B).

[0302] Table 10. Inhibitory effects of anti-CTLA4 / TIGIT antibodies on MC38 xenografts in CTLA4-TIGIT dual humanized mice

[0303] 2. Efficacy testing in the CTLA4 / TIGIT dual-humanized mouse MC38 colon cancer model

[0304] Experimental methods: CTLA4 / TIGIT dual humanized mice (Animal Center of Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were bred for 7 days. MC38 cells were cultured at 5×10 5 The average tumor volume in the right subcutaneous tissue of B-hCTLA4 / hTIGIT humanized mice was 95 mm 3 At 4 pm, mice were randomly divided into three groups according to tumor size, with three mice in each group. G1 received 1×PBS, G2 received Ipi (1 mg / kg), and G3 received As-3 (1 mg / kg). On Day 19 and Day 21, mice in Groups G1 to G3 were each administered once, for a total of two doses, via intraperitoneal injection. On Day 24, anticoagulated blood (anticoagulant EDTA-K2, 150 μL / mouse) and tumors were collected from mice in Groups G1 to G3 for flow cytometry analysis (flow cytometry panel: Live / dead, mCD45, mCD3, mCD4, mCD8, mCD25, mFoxp3). Body weight and tumor volume were measured twice weekly for all mice in the PD group during the dosing and observation period. At the end of the experiment, the animals were euthanized, and the tumors were removed and photographed.

[0305] Experimental results: See Figure 11 and Table 11. As-3 selectively eliminated intratumoral Tregs, with a stronger effect than Ipi (Figure 11B), but had no significant inhibitory effect on peripheral Tregs (Figure 11A). As-3 also induced an increase in intratumoral CD8+ T cells (Figure 11D), with a stronger effect than Ipi, but had no significant increase in peripheral CD8+ T cells (Figure 11C).

[0306] Table 11. Effects of anti-CTLA4 / TIGIT antibodies on Treg and CD8+ T cells in and around MC38 transplanted tumors in CTLA4-TIGIT dual humanized mice

Claims

1. CTLA4 / TIGIT-containing binding protein a first binding domain that specifically binds to CTLA4 and a second binding domain that specifically binds to TIGIT, wherein the first binding domain that specifically binds to CTLA4 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2 and HCDR3 within the amino acid sequence set forth under SEQ ID NO: 13, and VL1 comprises LCDR1, LCDR2 and LCDR3 within the amino acid sequence set forth under SEQ ID NO: 14, a second binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein VH2 comprises HCDR1, HCDR2 and HCDR3 within the amino acid sequence set forth under SEQ ID NO: 15, and VL2 comprises LCDR1, LCDR2 and LCDR3 within the amino acid sequence set forth under SEQ ID NO: 16, the CDRs are defined in accordance with the numbering system according to Kabat, IMGT, Chothia, AbM or Contact; Preferably, VH1 comprises HCDR1, HCDR2 and HCDR3 with the amino acid sequences set forth under SEQ ID NO: 1, 2 and 3, respectively, and VL1 comprises LCDR1, LCDR2 and LCDR3 with the amino acid sequences set forth under SEQ ID NO: 4, 5 and 6, respectively; Preferably, VH2 comprises HCDR1, HCDR2 and HCDR3 with the amino acid sequences set forth under SEQ ID NO: 7, 8 and 9, respectively, and VL2 comprises LCDR1, LCDR2 and LCDR3 with the amino acid sequences set forth under SEQ ID NO: 10, 11 and 12, respectively; more preferably, the CTLA4 / TIGIT binding protein is an anti-CTLA4 / TIGIT antibody.

2. The CTLA4 / TIGIT binding protein of claim 1, wherein in the first binding domain that specifically binds to CTLA4, VH1 comprises the amino acid sequence set forth under SEQ ID NO: 13, or an amino acid sequence characterized by at least 90% sequence identity thereto; and VL1 comprises the amino acid sequence set forth under SEQ ID NO: 14, or an amino acid sequence characterized by at least 90% sequence identity thereto.

3. The CTLA4 / TIGIT binding protein of claim 1 or 2, wherein in the second binding domain that specifically binds to TIGIT, VH2 comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity thereto; and VL2 comprises the amino acid sequence set forth under SEQ ID NO: 16, or an amino acid sequence characterized by at least 90% sequence identity thereto.

4. The CTLA4 / TIGIT binding protein of any one of claims 1-3, further comprising a human immunoglobulin Fc region; wherein preferably the Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4; more preferably, the Fc region is a human IgG1 Fc region, and optionally, the Fc region comprises at least one mutation represented by 239D, 330L and 332E; where the mutation is defined in accordance with the Eu numbering system.

5. The CTLA4 / TIGIT binding protein of claim 4, wherein the Fc region comprises a first subunit and a second subunit; wherein preferably the first subunit of the Fc region comprises a mutation that provides for the formation of a protrusion, and the second subunit of the Fc region comprises a mutation that provides for the formation of a cavity.

6. The CTLA4 / TIGIT binding protein of claim 5, wherein The Fc region comprises a first subunit comprising a mutation at position 366 and a second subunit comprising a mutation at a position selected from 366, 368 and 407, or any combination thereof; or The Fc region comprises a first subunit containing a mutation at position 354 or 356 and a second subunit containing a mutation at position 349; or the Fc region comprises a first subunit containing a mutation at position 354 or 356 and a second subunit containing mutations at positions 349, 366, 368 and 407; preferably The Fc region comprises a first subunit comprising the 366W mutation and a second subunit comprising a mutation selected from 366S, 368A and 407V, or any combination thereof; or The Fc region comprises a first subunit containing a 354C or 356C mutation and a second subunit containing a 349C mutation; or The Fc region contains a first subunit containing the 354C / 366W mutations and a second subunit containing the 349C / 366S / 368A / 407V mutations.

7. The CTLA4 / TIGIT binding protein of any one of claims 1-6, further comprising a linker, wherein preferably the linker is (G m S n ) h , or (G m Qn ), or (GGNGT) h , or (YGNGT) h , or (EPKSS) h , where each of m and n is independently selected from an integer ranging from 1 to 8, and h is independently selected from an integer ranging from 1 to 20.

8. The CTLA4 / TIGIT binding protein of any one of claims 5-7, comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein the first heavy chain from the N-terminus to the C-terminus sequentially contains [VH1]-[linker 1]-[obscurin-O chain]-[linker 3]-[first subunit of the Fc region], the first light chain from the N-terminus to the C-terminus sequentially contains [VL1]-[linker 2]-[titin-T chain], the second heavy chain from the N-terminus to the C-terminus sequentially contains [VH2]-[CH1]-[second subunit of the Fc region], and the second light chain from the N-terminus to the C-terminus sequentially contains [VL2]-[CL]; where is a peptide bond, the amino acid sequence of the obscurin-O chain is set forth under SEQ ID NO: 17, the amino acid sequence of the titin T chain is set forth under SEQ ID NO: 18, wherein linker 1, linker 2 and linker 3 may be the same or different and may be independently present or absent; preferably, the amino acid sequences of linker 1 and linker 2 are GGGGS, and linker 3 is absent.

9. The CTLA4 / TIGIT binding protein of any one of claims 1-8, comprising the following combination of polypeptide chains: a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90% sequence identity therewith, a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90% sequence identity therewith, a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90% sequence identity thereto, and a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90% sequence identity thereto.

10. A polynucleotide encoding a CTLA4 / TIGIT binding protein according to any one of claims 1-9.

11. A vector containing a polynucleotide according to claim 10.

12. A host cell containing a polynucleotide according to claim 10 or a vector according to claim 11.

13. A pharmaceutical composition containing a CTLA4 / TIGIT binding protein according to any one of claims 1 to 9, a polynucleotide according to claim 10, or a vector according to claim 11; wherein optionally the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or adjuvants.

14. A method for producing a CTLA4 / TIGIT binding protein, comprising culturing a host cell according to claim 12 and expressing the CTLA4 / TIGIT binding protein; wherein optionally the method further comprises the step of isolating and / or purifying the CTLA4 / TIGIT binding protein.

15. The use of a CTLA4 / TIGIT binding protein according to any one of claims 1-9, a polynucleotide according to claim 10, a vector according to claim 11, or a pharmaceutical composition according to claim 13, represented by any of the following variants (1)-(3): (1) for use in the treatment or amelioration of cancer or in the manufacture of a medicament for the treatment or amelioration of cancer; (2) for use in depleting or suppressing the activity of Treg cells or in the manufacture of a medicament for depleting or suppressing the activity of Treg cells; (3) for use in activating T cells or in the manufacture of a medicament for activating T cells; wherein preferably the Treg cells are intratumoral Treg cells; preferably the T cells are intratumoral T cells, more preferably intratumoral CD8+ T cells; wherein the cancer is preferably selected from colorectal cancer, non-small cell lung cancer and breast cancer.

16. A method for treating or reducing the severity of cancer manifestations, including stages administering to a subject a therapeutically effective amount of a CTLA4 / TIGIT binding protein according to any one of claims 1-9, a polynucleotide according to claim 10, a vector according to claim 11, or a pharmaceutical composition according to claim 13.