CN115010810A - anti-CTLA-4 antibodies and uses thereof - Google Patents

anti-CTLA-4 antibodies and uses thereof Download PDF

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CN115010810A
CN115010810A CN202210210779.4A CN202210210779A CN115010810A CN 115010810 A CN115010810 A CN 115010810A CN 202210210779 A CN202210210779 A CN 202210210779A CN 115010810 A CN115010810 A CN 115010810A
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amino acid
acid sequence
antibody
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肖扬
李雪
周新然
赵立文
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Nanjing Sanhome Pharmaceutical Co Ltd
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Nanjing Sanhome Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention relates to the technical field of antibody medicaments, in particular to an anti-CTLA-4 antibody or an antigen binding fragment thereof, an anti-CTLA-4/anti-PD-1 antibody or an antigen binding fragment thereof, a pharmaceutical composition containing the anti-CTLA-4 antibody or the antigen binding fragment thereof or the anti-CTLA-4/anti-PD-1 antibody or the antigen binding fragment thereof, and applications of the antibodies and the antigen binding fragment thereof. The anti-CTLA-4 antibody and the anti-CTLA-4/anti-PD-1 antibody have obvious anti-tumor activity and can be applied to preparing anti-tumor medicaments.

Description

anti-CTLA-4 antibodies and uses thereof
Technical Field
The invention relates to the technical field of antibody medicaments, in particular to an anti-CTLA-4 antibody or an antigen binding fragment thereof, a double antibody comprising the anti-CTLA-4 antibody or the antigen binding fragment thereof and an anti-PD-1 antibody or the antigen binding fragment thereof, and application of the antibodies and the double antibodies.
Background
T lymphocytes play a key role in the adaptive immune response to antigens. Naive T cells require two signals for complete activation. The first signal is antigen-specific and is provided by the interaction of the T Cell Receptor (TCR) with the MHC/peptide complex on an Antigen Presenting Cell (APC). The second signal is a costimulatory signal, provided by the interaction between a receptor on the T cell and its ligand on the APC.
The costimulatory pathway between CD28 on T cells and B7-1(CD80) and B7-2(CD86) on APCs is the most critical pathway involved in T cell regulation. Following T cell activation, the negative regulatory receptor factor CTLA-4 (cytotoxic T lymphocyte-associated protein 4, also known as CD152) on T cells is upregulated. CTLA-4 is structurally homologous to CD28, but binds more tightly to the B7-1 and B7-2 ligands. CTLA-4 suppresses immune responses in two major ways, it competes with CD28 for B7-1 and B7-2 ligands to block co-stimulation, and it also signals in a negative way to inhibit T cell activation.
Blockade of CTLA-4 has been reported to increase T cell responses in vitro and in vivo, potentiate anti-tumor immunity, and enhance induced autoimmune diseases. Antibodies against CTLA-4 are described as immune-stimulatory modulators in a number of disease conditions, for example, in the treatment or prevention of viral and bacterial infections and for the treatment of cancer. Iplilimumab is a human anti-human CTLA-4 antibody that blocks CTLA-4 binding to B7-1 and B7-2 expressed on APC, thereby blocking negative downregulation of the immune response elicited by these molecular interactions.
Although Ipilimumab is already marketed for some cancer treatments, there is still a need for alternative anti-CTLA-4 antibodies.
In addition, PD-1 is also a key immune checkpoint receptor, which is expressed by activated T and B cells, mediating immunosuppression. PD-1 regulates T cell activation by binding its ligands programmed death ligand 1(PD-L1) and programmed death ligand 2 (PD-L2). PD-1 ligand is expressed on antigen presenting cells and in a variety of human cancers, and has been shown to down-regulate T cell activation and cytokine secretion when PD-1 ligand binds to PD-1.
The two checkpoints, CTLA-4 and PD-1, may be complementary immune checkpoints with different pathways of action. Therefore, research and development of double-resistant drugs capable of simultaneously acting on the two checkpoints also has significant clinical significance and wide market space.
Disclosure of Invention
In one aspect, the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising complementarity determining region 1(H1CDR1) of the heavy chain variable region, complementarity determining region 2(H1CDR2) of the heavy chain variable region and/or complementarity determining region 3(H1CDR3) of the heavy chain variable region and/or a light chain variable region comprising complementarity determining region 1(L1CDR1) of the light chain variable region, complementarity determining region 2(L1CDR2) of the light chain variable region and/or complementarity determining region 3(L1CDR3) of the light chain variable region.
In some embodiments, the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H1CDR1, H1CDR2, and H1CDR3 selected from the group consisting of:
(a1) amino acid sequences as shown in SEQ ID NO 1, 2 and 3;
(a2) amino acid sequences as shown in SEQ ID NO 1, 2 and 4; and
(a3) an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID Nos. 1, 2 and 3 or SEQ ID Nos. 1, 2 and 4; and
(2) the light chain variable region comprises an L1CDR1, an L1CDR2, and an L1CDR3 selected from the group consisting of:
(a4) amino acid sequences as shown in SEQ ID NO 5, 6 and 7; and
(a5) amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NO 5, 6 and 7.
In a specific embodiment, the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the H1CDR1, H1CDR2 and H1CDR3 of the heavy chain variable region are SEQ ID NOs 1, 2 and 3, respectively, and the L1CDR1, L1CDR2 and L1CDR3 of the light chain variable region are SEQ ID NOs 5, 6 and 7, respectively.
In a specific embodiment, the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the H1CDR1, H1CDR2, and H1CDR3 of the heavy chain variable region are SEQ ID NOs 1, 2, and 4, respectively, and the L1CDR1, L1CDR2, and L1CDR3 of the light chain variable region are SEQ ID NOs 5, 6, and 7, respectively.
In some embodiments, the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
(1) The amino acid sequence of the heavy chain variable region is selected from:
(b1) the amino acid sequence shown as SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30;
(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) an amino acid sequence shown as SEQ ID NO 27, 31, 32, 33, 34 and 35;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
In some specific embodiments, the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 26, the amino acid sequence of SEQ ID No. 26 obtained by substituting, deleting or adding one or more amino acids and having functional identity with SEQ ID No. 26 or having at least 85% sequence identity with SEQ ID No. 26, and the amino acid sequence of the light chain variable region is SEQ ID No. 27, the amino acid sequence of SEQ ID No. 27 obtained by substituting, deleting or adding one or more amino acids and having functional identity with SEQ ID No. 27 or having at least 85% sequence identity with SEQ ID No. 27.
In some specific embodiments, the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 28, the amino acid sequence of SEQ ID No. 28 that is obtained by substituting, deleting, or adding one or more amino acids and that is functionally identical to SEQ ID No. 28 or has at least 85% sequence identity to SEQ ID No. 28, and the amino acid sequence of the light chain variable region is SEQ ID No. 27, the amino acid sequence of SEQ ID No. 27 that is obtained by substituting, deleting, or adding one or more amino acids and that is functionally identical to SEQ ID No. 27 or has at least 85% sequence identity to SEQ ID No. 27.
In some embodiments, the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
(1) The amino acid sequence of the heavy chain variable region is selected from:
(b1) the amino acid sequences shown as SEQ ID NO. 29 and SEQ ID NO. 30;
(b2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1) and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) 31, 32, 33, 34 and 35;
(b5) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4) and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
In some specific embodiments, the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 29, the amino acid sequence of SEQ ID No. 29 that is obtained by substituting, deleting, or adding one or more amino acids and that is functionally identical to SEQ ID No. 29 or has at least 85% sequence identity to SEQ ID No. 29, and the amino acid sequence of the light chain variable region is SEQ ID No. 32, the amino acid sequence of SEQ ID No. 32 that is obtained by substituting, deleting, or adding one or more amino acids and that is functionally identical to SEQ ID No. 32 or has at least 85% sequence identity to SEQ ID No. 32.
In some specific embodiments, the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 30, the amino acid sequence of SEQ ID No. 30 that is obtained by substituting, deleting, or adding one or more amino acids and that is functionally identical to SEQ ID No. 30 or has at least 85% sequence identity to SEQ ID No. 30, and the amino acid sequence of the light chain variable region is SEQ ID No. 32, the amino acid sequence of SEQ ID No. 32 that is obtained by substituting, deleting, or adding one or more amino acids and that is functionally identical to SEQ ID No. 32 or has at least 85% sequence identity to SEQ ID No. 32.
In some particular embodiments, the anti-CTLA-4 antibody according to the invention is a murine antibody that further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and a light chain constant region of murine kappa, lambda chains or variants thereof.
In some preferred embodiments, the anti-CTLA-4 murine antibody according to the invention further comprises heavy chain constant regions of murine IgG1, IgG2a, IgG2b, IgG2c or variants thereof, and light chain constant regions of murine kappa chains or variants thereof.
In a preferred embodiment of the present invention, the antibody heavy chain of the anti-CTLA-4 chimeric antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or mutated sequences thereof, preferably a human IgG1 or IgG2 heavy chain constant region, or an IgG4 constant region that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity following amino acid mutation.
In some embodiments, the invention provides an anti-CTLA-4 humanized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or variants thereof and the light chain comprises a light chain constant region of human kappa, lambda chain, or variants thereof.
In some preferred embodiments, the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1 or IgG2 or variants thereof, and a light chain constant region of human kappa chain or variants thereof.
In some embodiments, the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or f (ab) 2 。
In another aspect, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region comprising complementarity determining region 1(H2CDR1) of the heavy chain variable region, complementarity determining region 2(H2CDR2) of the heavy chain variable region and/or complementarity determining region 3(H2CDR3) of the heavy chain variable region and/or a light chain variable region comprising complementarity determining region 1(L2CDR1) of the light chain variable region, complementarity determining region 2(L2CDR2) of the light chain variable region and/or complementarity determining region 3(L2CDR3) of the light chain variable region.
In some embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H2CDR1, H2CDR2, and H2CDR3 selected from the group consisting of:
(a1) amino acid sequences as shown in SEQ ID NO 8, 9 and 10; and
(a2) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a 1); and
(2) the light chain variable region comprises an L2CDR1, an L2CDR2, and an L2CDR3 selected from the group consisting of:
(a3) amino acid sequences as shown in SEQ ID NO 11, 12 and 13; and
(a4) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a 3).
In a specific embodiment, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, which has a heavy chain variable region in which the H2CDR1, H2CDR2, and H2CDR3 are SEQ ID NOs 8, 9, and 10, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 8, 9, and 10, and a light chain variable region in which the L2CDR1, L2CDR2, and L2CDR3 are SEQ ID NOs 11, 12, and 13, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 11, 12, and 13.
In some specific embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof according to the present invention is a monoclonal antibody or antigen-binding fragment thereof.
In some specific embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
In some specific embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein
(1) The amino acid sequence of the heavy chain variable region is selected from:
(b1) an amino acid sequence shown as SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50;
(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) an amino acid sequence shown as SEQ ID NO. 44, SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
In some specific embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 43, the amino acid sequence of SEQ ID No. 43 obtained by substituting, deleting or adding one or more amino acids and having functional identity with SEQ ID No. 43 or having at least 85% sequence identity with SEQ ID No. 43, and the amino acid sequence of the light chain variable region is SEQ ID No. 44, the amino acid sequence of SEQ ID No. 44 obtained by substituting, deleting or adding one or more amino acids and having functional identity with SEQ ID No. 44 or having at least 85% sequence identity with SEQ ID No. 44.
In some specific embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein
(1) The amino acid sequence of the heavy chain variable region is selected from:
(b1) an amino acid sequence shown as SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50;
(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
In some specific embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 46, the amino acid sequence of SEQ ID No. 46 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 46 or has at least 85% sequence identity to SEQ ID No. 46, and the amino acid sequence of the light chain variable region is SEQ ID No. 51, the amino acid sequence of SEQ ID No. 51 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 51 or has at least 85% sequence identity to SEQ ID No. 51.
In some specific embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 46, the amino acid sequence of SEQ ID No. 46 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 46 or has at least 85% sequence identity to SEQ ID No. 46, and the amino acid sequence of the light chain variable region is SEQ ID No. 52, the amino acid sequence of SEQ ID No. 52 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 52 or has at least 85% sequence identity to SEQ ID No. 52.
In some specific embodiments, the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO 46, the amino acid sequence of SEQ ID NO 46 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID NO 46 or has at least 85% sequence identity to SEQ ID NO 46, and the amino acid sequence of the light chain variable region is SEQ ID NO 53, the amino acid sequence of SEQ ID NO 53 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID NO 53 or has at least 85% sequence identity to SEQ ID NO 53.
In some specific embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region has the amino acid sequence of SEQ ID No. 46, wherein SEQ ID No. 46 has been substituted, deleted or added with one or more amino acids and has a functional identity to SEQ ID No. 46 or has at least 85% sequence identity to SEQ ID No. 46 and the amino acid sequences of H2CDR1, H2CDR2 and H2CDR3 are as set forth in SEQ ID nos. 8, 9 and 10, and the light chain variable region has the amino acid sequence of SEQ ID No. 52, wherein SEQ ID No. 52 has been substituted, deleted or added with one or more amino acids and has a functional identity to SEQ ID No. 52 or has at least 85% sequence identity to SEQ ID No. 52 and the amino acid sequences of L2CDR1, L2CDR2 and L2CDR3 are as set forth in SEQ ID No. 11, 12 and 13.
In some specific embodiments, the anti-PD-1 antibody according to the invention is a murine antibody, which further contains a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and a light chain constant region of murine kappa chain or variants thereof.
In some preferred embodiments, the anti-PD-1 murine antibody according to the present invention further contains heavy chain constant regions of murine IgG1, IgG2a, IgG2b, IgG2c or variants thereof, and light chain constant regions of murine kappa chains or variants thereof.
In a preferred embodiment of the present invention, the antibody light chain of the anti-PD-1 chimeric antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine kappa, lambda chain or mutated sequence thereof. The antibody heavy chain of the anti-PD-1 chimeric antibody or the antigen-binding fragment thereof further comprises a heavy G chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or mutated sequences thereof, preferably comprises a human IgG1, IgG2a, IgG2b, IgG2c heavy chain constant region, or an IgG4 constant region which is mutated by amino acids to significantly reduce ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity.
In some specific embodiments, the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, or a variant thereof, and a light chain constant region of human kappa, lambda chain, or a variant thereof. In some preferred embodiments, the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, or a variant thereof, and a light chain constant region of human kappa chain or a variant thereof.
In some embodiments, the invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or f (ab) 2.
Another aspect of the invention provides an isolated nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof according to the invention.
In some specific embodiments, an isolated nucleic acid according to the invention comprises a nucleotide sequence encoding a heavy chain variable region amino acid sequence as set forth in SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, or SEQ ID NO 50; and the nucleotide sequence of the amino acid sequence of the light chain variable region is shown as SEQ ID NO 44, SEQ ID NO 51, SEQ ID NO 52 and SEQ ID NO 53.
In a specific embodiment, an isolated nucleic acid according to the invention comprises a nucleotide sequence encoding heavy chain variable region SEQ ID NO 43; and a nucleotide sequence encoding the light chain variable region of SEQ ID NO. 44.
In a specific embodiment, an isolated nucleic acid according to the invention comprises a nucleotide sequence encoding heavy chain variable region SEQ ID NO 46; and a nucleotide sequence encoding the light chain variable region SEQ ID NO 52.
Another aspect of the invention provides an expression vector expressing the anti-PD-1 antibody or antigen-binding fragment thereof of the invention. An expression vector according to the invention comprises an isolated nucleic acid molecule of the invention.
Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
In some embodiments, the host cell according to the invention is selected from the group consisting of prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably E.coli. In another preferred embodiment, the host cell is a mammalian cell.
Another aspect of the invention provides a method of making an anti-PD-1 antibody or antigen-binding fragment thereof of the invention, comprising the steps of expressing the antibody in said host cell and isolating said antibody from the host cell.
Another aspect of the invention provides a pharmaceutical composition comprising an anti-PD-1 humanized antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides pharmaceutical compositions comprising an anti-PD-1 humanized antibody or antigen-binding fragment thereof of the present invention, and further comprising other active components, such as other antibodies, targeted drugs, and the like. In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is an aqueous buffered solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of a liposome.
The anti-PD-1 humanized antibody or an antigen-binding fragment thereof of the present invention may be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes. The formulations may be administered by any route, for example by infusion or bolus injection, by a route of absorption through epithelial or cutaneous mucosa (e.g. oral mucosa or rectum, etc.). Administration may be systemic or local. The formulations may be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulation.
Another aspect of the invention provides a method of inhibiting PD-1 activity, comprising administering to an individual in need thereof an anti-PD-1 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.
Another aspect of the present invention provides a method for detecting or assaying human PD-1, the method comprising the step of using the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
Another aspect of the invention provides a reagent for detecting or assaying human PD-1, which comprises an anti-PD-1 antibody or an antigen-binding fragment thereof of the present invention.
Another aspect of the present invention provides a method of treating a disease associated with PD-1, the method comprising administering to a subject a pharmaceutically effective amount of a PD-1 antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the above, or an isolated nucleic acid molecule of the above. Another aspect of the invention provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for a disease associated with PD-1. In some embodiments, the medicament for a disease associated with PD-1 is for treating a T cell dysfunctional disorder, such as a tumor, an immune disease, or an infectious disorder. In some embodiments, the tumor is non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, merkel cell carcinoma, adrenocortical carcinoma, hepatohepatocellular carcinoma, pancreatic ductal adenocarcinoma, pheochromocytoma, ganglioneuroma, endometrial carcinoma, and ovarian plasmacyclocarcinoma, and the like. In some embodiments, the immune disease is arthritis, inflammatory bowel disease, psoriasis. In some embodiments, the infectious disease is a chronic viral infection.
Another aspect of the present invention provides an anti-CTLA-4/anti-PD-1 antibody, comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises complementarity determining region 1 of the heavy chain variable region (H1CDR1), complementarity determining region 2 of the heavy chain variable region (H1CDR2), and/or complementarity determining region 3 of the heavy chain variable region (H1CDR3), and the light chain variable region comprises complementarity determining region 1 of the light chain variable region (L1CDR1), complementarity determining region 2 of the light chain variable region (L1CDR2), and/or complementarity determining region 3 of the light chain variable region (L1CDR 3); and the anti-PD-1 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to PD-1.
In some embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H1CDR1, H1CDR2, and H1CDR3 selected from the group consisting of:
(a1) amino acid sequences as shown in SEQ ID NO 1, 2 and 3;
(a2) amino acid sequences as shown in SEQ ID NO 1, 2 and 4; and
(a3) an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NO 1, 2 and 3 or SEQ ID NO 1, 2 and 4; and
(2) the light chain variable region comprises L1CDR1, L1CDR2, and L1CDR3 selected from the group consisting of:
(a4) amino acid sequences as shown in SEQ ID NO 5, 6 and 7; and
(a5) amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NO 5, 6 and 7.
In some embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, or an antigen-binding fragment thereof, has:
the H1CDR1, H1CDR2 and H1CDR3 are heavy chain variable regions of SEQ ID NOs 1, 2 and 3 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2 and 3, respectively, and the L1CDR1, L1CDR2 and L1CDR3 are light chain variable regions of SEQ ID NOs 5, 6 and 7 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6 and 7, respectively; or
The H1CDR1, H1CDR2 and H1CDR3 are heavy chain variable regions of SEQ ID NOs 1, 2 and 4 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2 and 4, respectively, and the L1CDR1, L1CDR2 and L1CDR3 are light chain variable regions of SEQ ID NOs 5, 6 and 7 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6 and 7, respectively.
In some embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, or an antigen-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(b1) the amino acid sequence shown as SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30;
(b2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1) and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) an amino acid sequence shown as SEQ ID NO 27, 31, 32, 33, 34 and 35;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
In some embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
the amino acid sequence of the heavy chain variable region is SEQ ID NO 26, the amino acid sequence of the SEQ ID NO 26 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 26 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 26, the amino acid sequences of the H1CDR1, the H1CDR2 and the H1CDR3 are the amino acid sequences shown in SEQ ID NO 1, 2 and 3, the amino acid sequence of the light chain variable region is SEQ ID NO 27, the amino acid sequence of the SEQ ID NO 27 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 27 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 27, and the amino acid sequences of the L1CDR1, the L1CDR2 and the L1CDR3 are the amino acid sequences shown in SEQ ID NO 5, 6 and 7; or
The amino acid sequence of the heavy chain variable region is SEQ ID NO 28, the amino acid sequence of the SEQ ID NO 28 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 28 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 28, the H1CDR1, the H1CDR2 and the H1CDR3 are the amino acid sequences shown in SEQ ID NO 1, 2 and 4, the amino acid sequence of the light chain variable region is SEQ ID NO 27, the amino acid sequence of the SEQ ID NO 27 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 27 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 27, and the amino acid sequences of the L1CDR1, the L1CDR2 and the L1CDR3 are the amino acid sequences shown in SEQ ID NO 5, 6 and 7.
In some embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, or an antigen-binding fragment thereof, is a humanized antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(c1) an amino acid sequence shown as SEQ ID NO. 29 or SEQ ID NO. 30;
(c2) (c1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (c1), and has the same or similar function with the amino acid sequence shown in (c 1); and
(c3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (c 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(c4) 31, 32, 33, 34 and 35;
(c5) (c4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (c4), and has the same or similar function with the amino acid sequence shown in (c 4); and
(c6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (c 4).
In some embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, or an antigen-binding fragment thereof, is a humanized antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
The amino acid sequence of the heavy chain variable region is SEQ ID NO:29, the amino acid sequence of the SEQ ID NO:29 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:29 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO:29, the amino acid sequence of the light chain variable region is SEQ ID NO:32, the amino acid sequence of the SEQ ID NO:32 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:32 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO: 32; or
The amino acid sequence of the heavy chain variable region is SEQ ID NO 30, the amino acid sequence of the SEQ ID NO 30 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 30 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 30, the amino acid sequence of the light chain variable region is SEQ ID NO 32, and the amino acid sequence of the SEQ ID NO 32 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 32 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 32.
In some preferred embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody, comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or an antigen-binding fragment thereof is as defined in the above embodiments; and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining region 1(H2CDR1) of the heavy chain variable region, complementarity determining region 2(H2CDR2) of the heavy chain variable region and/or complementarity determining region 3(H2CDR3) of the heavy chain variable region and/or a light chain variable region comprising complementarity determining region 1(L2CDR1) of the light chain variable region, complementarity determining region 2(L2CDR2) of the light chain variable region and/or complementarity determining region 3(L2CDR3) of the light chain variable region.
Further preferably, in some embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or an antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H2CDR1, H2CDR2, and H2CDR3 selected from the group consisting of:
(A1) amino acid sequences as shown in SEQ ID NO 8, 9 and 10;
(A2) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a 1); and
(2) the light chain variable region comprises an L2CDR1, an L2CDR2, and an L2CDR3 selected from the group consisting of:
(A3) amino acid sequences as shown in SEQ ID NO 11, 12 and 13;
(A4) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (A3).
In some specific embodiments, the invention provides an anti-CTLA-4/anti-PD-1 antibody, comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein:
the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H1CDR1, H1CDR2, and H1CDR3 selected from the group consisting of:
(a1) amino acid sequences as shown in SEQ ID NO 1, 2 and 3;
(a2) amino acid sequences as shown in SEQ ID NO 1, 2 and 4; and
(a3) an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NO 1, 2 and 3 or SEQ ID NO 1, 2 and 4; and
(2) the light chain variable region comprises an L1CDR1, an L1CDR2, and an L1CDR3 selected from the group consisting of:
(a4) amino acid sequences as shown in SEQ ID NO 5, 6 and 7; and
(a5) an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6, and 7; and
the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H2CDR1, H2CDR2, and H2CDR3 selected from the group consisting of:
(A1) amino acid sequences as shown in SEQ ID NO 8, 9 and 10;
(A2) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a 1); and
(2) the light chain variable region comprises an L2CDR1, an L2CDR2, and an L2CDR3 selected from the group consisting of:
(A3) amino acid sequences as shown in SEQ ID NO 11, 12 and 13;
(A4) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (A3).
In a specific embodiment, the invention provides an anti-CTLA-4/anti-PD-1 antibody, comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region of the H1CDR1, H1CDR2, and H1CDR3 of SEQ ID NOs 1, 2, and 3, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2, and 3, and a light chain variable region of the L1CDR1, L1CDR2, and L1CDR3 of SEQ ID NOs 5, 6, and 7, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6, and 7; and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region in which the H2CDR1, H2CDR2, and H2CDR3 are SEQ ID NOs 8, 9, and 10, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 8, 9, and 10, and a light chain variable region in which the L2CDR1, L2CDR2, and L2CDR3 are SEQ ID NOs 11, 12, and 13, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 11, 12, and 13.
In a specific embodiment, the invention provides an anti-CTLA-4/anti-PD-1 antibody, comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region of the H1CDR1, H1CDR2, and H1CDR3 of SEQ ID NOs 1, 2, and 4, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2, and 4, and a light chain variable region of the L1CDR1, L1CDR2, and L1CDR3 of SEQ ID NOs 5, 6, and 7, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6, and 7; and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NOs 8, 9 and 10 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 8, 9 and 10, respectively, for the H2CDR1, H2CDR2 and H2CDR3, and a light chain variable region of SEQ ID NOs 11, 12 and 13 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 11, 12 and 13, respectively, for the L2CDR1, L2CDR2 and L2CDR 3.
In some specific embodiments, the anti-CTLA-4/anti-PD-1 antibody, the anti-CTLA-4 antibody or antigen-binding fragment thereof, and the anti-PD-1 antibody or antigen-binding fragment thereof, according to the present invention, are each independently a murine, chimeric, humanized, or fully human antibody.
In some specific embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(b1) the amino acid sequence shown as SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30;
(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) an amino acid sequence shown as SEQ ID NO 27, 31, 32, 33, 34, 35;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4); and
the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(B1) an amino acid sequence shown as SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50;
(B2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (B1) and has the same or similar function with the amino acid sequence shown in (B1); and
(B3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (B1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(B4) an amino acid sequence shown as SEQ ID NO. 44, SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53;
(B5) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (B4) and has the same or similar function with the amino acid sequence shown in (B4); and
(B6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (B4).
In some specific embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO 30, the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof, which is obtained by substituting, deleting or adding one or more amino acids to the SEQ ID NO 30, is functionally identical to SEQ ID NO 30 or has at least 85% sequence identity to SEQ ID NO 30, and the amino acid sequence of the light chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO 32, the amino acid sequence of the light chain variable region of the SEQ ID NO 32, which is obtained by substituting, deleting or adding one or more amino acids to the SEQ ID NO 32, is functionally identical to SEQ ID NO 32 or has at least 85% to SEQ ID NO 32 Amino acid sequences of sequence identity.
In some specific embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO:30, the amino acid sequence of SEQ ID NO:30, which is obtained by substituting, deleting, or adding one or more amino acids and is functionally identical to SEQ ID NO:30, or the amino acid sequence of which has at least 85% sequence identity to SEQ ID NO:30, and the amino acid sequences of H1CDR1, H1CDR2, and H1CDR3, which are as shown in SEQ ID NOs: 1, 2, and 4, and the amino acid sequence of the light chain variable region is SEQ ID NO:32, the amino acid sequence of SEQ ID NO:32, which is obtained by substituting, deleting, or adding one or more amino acids and is functionally identical to SEQ ID NO:32 or the amino acid sequence of which has the amino acid sequence of SEQ ID NO:32 Has at least 85% sequence identity and the L1CDR1, L1CDR2 and L1CDR3 have the amino acid sequences shown in SEQ ID NO 5, 6 and 7.
In some particular embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, wherein the anti-CTLA-4 antibody or anti-PD-1 antibody may be a murine antibody further comprising a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and a light chain constant region of murine kappa chain or variants thereof.
In some preferred embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, wherein the anti-CTLA-4 murine antibody further comprises a heavy chain constant region of murine IgG1 or IgG2, or a variant thereof, and a light chain constant region of murine kappa chain, or a variant thereof.
In some specific embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 antibody, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(c1) an amino acid sequence shown as SEQ ID NO. 30;
(c2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (c1) and has the same or similar functions with the amino acid sequence shown in (c 1); and
(c3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (c 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(c4) an amino acid sequence as shown in SEQ ID NO. 32;
(c5) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (c4) and has the same or similar function with the amino acid sequence shown in (c 4); and
(c6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (c 4); and
the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(C1) an amino acid sequence shown as SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50;
(C2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (C1) and has the same or similar functions with the amino acid sequence shown in (C1); and
(C3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (C1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(C4) amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53;
(C5) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (C4) and has the same or similar functions with the amino acid sequence shown in (C4); and
(C6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (C4).
In some specific embodiments, the present invention provides anti-CTLA-4/anti-PD-1 antibodies including an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO:30, the amino acid sequence of SEQ ID NO:30 obtained by substituting, deleting or adding one or more amino acids and having a functional identity to SEQ ID NO:30 or the amino acid sequence of SEQ ID NO:30 having at least 85% sequence identity and the H1CDR1, H1CDR2 and H1CDR3 are the amino acid sequences shown in SEQ ID NO:1, 2 and 4, and the amino acid sequence of the light chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO:32, SEQ ID NO:32 substituted with SEQ ID NO:32, An amino acid sequence which is obtained by deleting or adding one or more amino acids and has the function same as SEQ ID NO. 32 or an amino acid sequence which has at least 85 percent of sequence identity with SEQ ID NO. 32 and the L1CDR1, L1CDR2 and L1CDR3 are shown as SEQ ID NO. 5, 6 and 7; and the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO. 46, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 46 and having the same function as the amino acid sequence shown in SEQ ID NO. 46 or having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 46, and the H2CDR1, H2CDR2 and H2CDR3 are the amino acid sequences shown in SEQ ID NO. 8, 9 and 10, the amino acid sequence of the light chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO. 52, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 52 and having the same function as the amino acid sequence shown in SEQ ID NO. 52 or having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 52, and the L2CDR1, L2CDR2 and L2CDR3 are the amino acid sequence shown in SEQ ID NO. 11, SEQ ID NO. 46, 12 and 13.
In some specific embodiments, the present invention provides anti-CTLA-4/anti-PD-1 antibodies, which include an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO 28, the amino acid sequence of SEQ ID NO 28, which is obtained by substituting, deleting or adding one or more amino acids, is functionally identical to SEQ ID NO 28, or has at least 85% sequence identity to SEQ ID NO 28, and the amino acid sequences of H1CDR1, H1CDR2 and H1CDR3 are as shown in SEQ ID NO 1, 2 and 4, and the amino acid sequence of the light chain variable region of the anti-CTLA-4 antibody or the antigen-binding fragment thereof is SEQ ID NO 27, or the anti-PD-1 antibody or the antigen-binding fragment thereof, An amino acid sequence which is obtained by deleting or adding one or more amino acids and has the same function with SEQ ID NO. 27 or an amino acid sequence which has at least 85 percent of sequence identity with SEQ ID NO. 27 and the L1CDR1, L1CDR2 and L1CDR3 are shown as SEQ ID NO. 5, 6 and 7; and the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO. 46, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 46 and having the same function as the amino acid sequence shown in SEQ ID NO. 46 or having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 46, and the H2CDR1, H2CDR2 and H2CDR3 are the amino acid sequences shown in SEQ ID NO. 8, 9 and 10, the amino acid sequence of the light chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO. 52, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 52 and having the same function as the amino acid sequence shown in SEQ ID NO. 52 or having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 52, and the L2CDR1, L2CDR2 and L2CDR3 are the amino acid sequence shown in SEQ ID NO. 11, SEQ ID NO. 46, 12 and 13.
In some embodiments, the invention provides an anti-CTLA-4/anti-PD-1 humanized antibody wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or variants thereof and the light chain comprises a light chain constant region of human kappa, lambda or variants thereof.
In a preferred embodiment of the invention, the murine anti-CTLA-4/anti-PD-1 antibody may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, and/or further comprise a heavy chain constant region of a murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variant thereof.
In a preferred embodiment of the invention, the anti-CTLA-4/anti-PD-1 antibody according to the invention, the antibody light chain of the anti-CTLA-4 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine kappa, lambda chain or mutated sequences thereof. The heavy chain of the anti-CTLA-4 antibody or the antigen-binding fragment thereof further comprises heavy chain constant regions of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or mutated sequences thereof, and preferably comprises human IgG1, IgG2 and IgG4 heavy chain constant regions.
In some specific embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and a light chain constant region of human kappa, lambda chains or variants thereof. In some preferred embodiments, the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG4, or a variant thereof, and a light chain constant region of human kappa chain or a variant thereof.
In a preferred embodiment of the invention, the anti-CTLA-4/anti-PD-1 antibody according to the invention, the antibody heavy chain of the anti-PD-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutated sequence thereof, preferably a heavy chain constant region comprising human IgG or a mutated sequence thereof; the antibody light chain of the anti-PD-1 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine kappa, lambda chain or mutated sequences thereof.
In some specific embodiments, the anti-CTLA-4/anti-PD-1 antibody according to the invention, or antigen-binding fragment thereof, further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, and a light chain constant region of human kappa, lambda chains, or a variant thereof. In some preferred embodiments, the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG4 or a variant thereof, and a light chain constant region of human kappa chain or a variant thereof.
In some embodiments, the invention provides anti-CTLA-4/anti-PD-1 antibodies, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof are Fab, Fv, sFv, or f (ab), respectively 2 . In a specific embodiment, the anti-CTLA-4/anti-PD-1 antibody provided by the invention is scF (ab) 2 。
Preferably, the anti-CTLA-4/anti-PD-1 antibody in the above embodiments of the invention is an anti-CTLA-4/anti-PD-1 bispecific antibody. In some embodiments, the bispecific antibody is a human antibody or a humanized antibody. In some embodiments, one of the binding specificities is for CTLA-4 and the other binding specificity is for any other antigen. In some embodiments, one of the binding specificities is for CTLA-4 and the other binding specificity is for PD-1. In some embodiments, the bispecific antibody can bind two different epitopes of CTLA-4. The bispecific antibodies can also be used to localize cytotoxic agents to CTLA-4-expressing cells. These antibodies possess a CTLA-4 binding arm and a cytotoxic agent binding arm, such as saporin, anti-interferon-alpha, vinca alkaloids, ricin A chain, methotrexate or radioactive congenersA site hapten. Bispecific antibodies of the invention can be prepared as full length antibodies or antibody fragments (e.g., F (ab') 2 Bispecific antibodies).
Methods of making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule is usually carried out by an affinity chromatography step, which is rather cumbersome and results in low yields of product. Similar methods are disclosed in WO93/08829 and Traveckereral, EMBO J.10:3655 (1991).
According to a different approach, antibody variable regions with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant region sequences. In some embodiments, the fusion is to an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, a heavy chain constant region (CH1) comprising the site necessary for binding to a light chain is present in at least a portion of the fusion. The DNA encoding the immunoglobulin heavy chain fusion fragment and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used for construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in the same ratio leads to high yields or when the ratio is of no particular significance.
In one embodiment of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation route, it was found that the asymmetric structure facilitates the separation of the desired bispecific substance from the unwanted immunoglobulin chain composition. This process is disclosed in WO 94/04690. For further information on the generation of bispecific antibodies see, e.g., Sureshetal, Methods in Enzymology 121:210 (1986).
According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The interface comprises at least a portion of the antibody constant region CH3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as dimers.
Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one heteroconjugate antibody may be conjugated to avidin and another heteroconjugate antibody may be conjugated to biotin. Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.
Bispecific antibodies of the invention can be generated from antibody fragments. For example, bispecific antibodies can be prepared using chemical ligation techniques. Brennanetal, Science 229:81(1985) describes the proteolytic cleavage of intact antibodies to yield F (ab') 2 A method for fragmenting. These fragments are cleaved in the presence of the dithiol complexing agent sodium arsenite (to stabilize adjacent dithiols and prevent intermolecular disulfide formation). The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody.
Fab' -SH fragments can be recovered directly from E.coli and these fragments can be chemically coupled to form bispecific antibodies. A fully humanized bispecific antibody F (ab') is described by Shalaby et al, J.exp.Med.175:217-225(1992) 2 And (4) generation of molecules. Each Fab' fragment was secreted separately from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies.
In some embodiments, bispecific antibody fragments of the invention can be generated and isolated directly from recombinant cell cultures. For example, bispecific antibodies can be generated using leucine zippers (Kostelnyetal, J.Immunol.148(5): 1547-. Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are cleaved at the hinge region to form monomers, and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Diabody technology provides additional mechanisms for making bispecific antibody fragments. The bispecific antibody fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on the other fragment, thereby forming two antigen binding sites. In another embodiment, bispecific antibody fragments may be constructed by using single chain fv (sFv) dimers.
Multivalent antibodies having more than two valencies are contemplated by the invention, e.g., trispecific antibodies can be made. Multivalent antibodies can be internalized (and/or catabolized) faster than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies of the invention can be multivalent antibodies having three or more antigen binding sites (e.g., tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. In some embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In some embodiments, the multivalent antibody comprises four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable regions. The multivalent antibody of the invention may further comprise at least two (e.g., four) light chain variable region polypeptides. A multivalent antibody of the invention can comprise, for example, about two to about eight light chain variable region polypeptides. The light chain variable region polypeptide of the invention comprises a light chain variable region, and optionally further comprises a CL domain.
In bispecific antibodies comprising a CTLA-4 targeting moiety and a PD-1 targeting moiety, one of the CTLA-4 targeting moiety and the PD-1 targeting moiety can be a full length antibody, and the other can be an antigen-binding fragment (e.g., scFv) comprising a heavy chain CDR, a light chain CDR, or a combination thereof. A full-length antibody targeting one of the CTLA-4 and PD-1 proteins and an antigen-binding fragment targeting the other protein can be chemically linked (e.g., covalently linked), either directly or through a linking peptide. An antigen-binding fragment (e.g., an scFv) can be linked, either directly or through a linking peptide, to the N-terminus of a full-length antibody (e.g., the N-terminus of a light chain or heavy chain of a full-length antibody), the C-terminus of a full-length antibody (e.g., the C-terminus of a heavy chain (or Fc or CH3 domain) of a full-length antibody), or both.
In some embodiments, bispecific antibodies of the invention comprise full length anti-CTLA-4 antibodies, antigen-binding fragments of anti-PD-1 antibodies (e.g., scFab, scFv), and linking peptides therebetween. In other embodiments, bispecific antibodies of the invention comprise full length anti-PD-1 antibodies, antigen-binding fragments of anti-CTLA-4 antibodies (e.g., scFab, scFv), and linking peptides therebetween.
In some embodiments, the scFv comprised in the bispecific antibody of the present invention may comprise a heavy chain variable region and a light chain variable region in any order. For example, the scFv comprised in the bispecific antibody may comprise the heavy chain variable region and the light chain variable region and optionally a linker peptide therebetween in the N-terminal to C-terminal direction, or alternatively, the scFv comprised in the bispecific antibody of the present invention may comprise the light chain variable region and the heavy chain variable region and optionally a linker peptide therebetween in the N-terminal to C-terminal direction.
In some embodiments, the linking peptide can include, for example, Gly, Asn, and/or Ser residues, and can also include neutral amino acids, such as Thr and/or Ala. Amino acid sequences suitable for use in the linker peptide may be those known in the relevant art. Meanwhile, the length of the linker peptide can be variously determined within such a limit that the function of the fusion protein is not affected. For example, a connecting peptide may be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the linker peptide may be represented by (GmSl) n (m, l, and n are independently integers from about 1 to about 10, particularly from about 2 to about 5).
In another embodiment, the PD-1 targeting moiety and the CTLA-4 targeting moiety can both be full length antibodies or antigen binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof.
In another embodiment, the bispecific antibody may be in the form of a heterodimer comprising a first arm comprising a pair of heavy and light chains targeting one of CTLA-4 and PD-1 and a second arm comprising a pair of heavy and light chains targeting the other.
In one embodiment, the full-length antibody may be in the form of a full-length immunoglobulin (e.g., IgG, IgM, IgA, IgE, or IgD, e.g., human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen-binding fragment may be selected from the group consisting of Fab, Fab ', F (ab') 2 Fd, Fv, scFv, scFab, single-chain antibody, sdFv, etc. For example, the full length antibody may be in the form of a full length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen binding fragment may be an scFv.
For example, the antibodies described herein may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, drug, toxin, or label). In some specific embodiments, the anti-CTLA-4/anti-PD-1 bispecific antibody according to the invention, the anti-CTLA-4 antibody or antigen-binding fragment thereof is of the structure (VH) -linked peptide- (VL) and the anti-PD-1 antibody or antigen-binding fragment thereof is of the structure (VL-CL) -linked peptide- (VH). In some specific embodiments, the linker peptide is in the form of (GGGGS) n, wherein n is 1 to 12, preferably 3 to 10, more preferably 3 to 8, e.g., 3, 4, 5, 6, 7, 8 GGGGS repeats. In some specific embodiments, the anti-CTLA-4/anti-PD-1 bispecific antibody according to the invention consists of two peptide chains, wherein:
the structure of peptide chain 1is shown in FIG. 1;
the structure of peptide chain 2 is shown in FIG. 2;
wherein:
the VH of the anti-PD-1 antibody is the heavy chain variable region of the anti-PD-1 antibody;
linker peptide 1, linker peptide 2 are each a flexible linker peptide of a repeating sequence of the form (GGGGS) n, wherein n is 1-10;
the VH of the anti-CTLA-4 antibody is the heavy chain variable region of the anti-CTLA-4 antibody;
the anti-CTLA-4 antibody VL is a light chain variable region of the anti-CTLA-4 antibody;
the anti-PD-1 antibody VL-CL is the light chain of the anti-PD-1 antibody.
In some specific embodiments, the anti-CTLA-4/anti-PD-1 bispecific antibody according to the invention consists of two peptide chains, wherein the amino acid sequence of peptide chain 1is SEQ ID NO:55 and the amino acid sequence of peptide chain 2 is SEQ ID NO: 54. In other specific embodiments, the anti-CTLA-4/anti-PD-1 bispecific antibody according to the invention consists of two peptide chains, wherein the amino acid sequence of peptide chain 1is SEQ ID NO:56 and the amino acid sequence of peptide chain 2 is SEQ ID NO: 54. Another aspect of the invention provides an isolated nucleic acid. In some embodiments, the isolated nucleic acid according to the invention encodes an anti-CTLA-4/anti-PD-1 antibody of the invention. In some embodiments, an isolated nucleic acid according to the invention encodes an anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention. In other embodiments, an isolated nucleic acid according to the invention encodes an anti-PD-1 antibody of the invention, or an antigen-binding fragment thereof.
In a specific embodiment, an isolated nucleic acid according to the invention comprises a nucleotide sequence encoding the heavy chain variable region of an anti-CTLA-4 antibody or antigen-binding fragment thereof as set forth in SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30 and a nucleotide sequence encoding the light chain variable region of an anti-CTLA-4 antibody or antigen-binding fragment thereof as set forth in SEQ ID NO 27, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35. In another specific embodiment, an isolated nucleic acid according to the invention comprises a nucleotide sequence encoding the heavy chain variable region of an anti-PD-1 antibody or antigen-binding fragment thereof as set forth in SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50 and a nucleotide sequence encoding the light chain variable region of an anti-PD-1 antibody or antigen-binding fragment thereof as set forth in SEQ ID NO 44, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53.
Another aspect of the invention provides an expression vector. In some embodiments, the expression vector of the invention expresses an anti-CTLA-4/anti-PD-1 bispecific antibody of the invention. In some embodiments, the expression vector of the invention expresses an anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention. In other embodiments, the expression vector of the invention expresses an anti-PD-1 antibody or antigen-binding fragment thereof of the invention. In some embodiments, the expression vector of the invention, the vector expressing the anti-CTLA-4 antibody or antigen-binding fragment thereof and the vector expressing the anti-PD-1 antibody or antigen-binding fragment thereof of the invention are heterologous expression vectors. An expression vector according to the invention comprises an isolated nucleic acid molecule of the invention.
Another aspect of the invention provides a Chimeric Antigen Receptor (CAR) fusion protein comprising an anti-CTLA-4 antibody or antigen-binding fragment thereof and/or an anti-PD-1 antibody or antigen-binding fragment thereof of the invention. In some embodiments, the chimeric antigen receptor fusion protein comprises an anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention, which is a single chain variable fragment (scFv) of a VH and VL against a CTLA-4 antigen. In other embodiments, the chimeric antigen receptor fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof of the invention, which is a single chain variable fragment (scFv) directed against the VH and VL of the PD-1 antigen. In other embodiments, the chimeric antigen receptor fusion protein comprises a first single-chain variable fragment (scFv) for VH and VL of the CTLA-4 antigen and a second single-chain variable fragment (scFv) for VH and VL of the PD-1 antigen. The first scFv against the VH and VL of the CTLA-4 antigen has the H1CDR1, H1CDR2, and H1CDR3 of the heavy chain variable region and the L1CDR1, L1CDR2, and L1CDR3 of the light chain variable region described in the above embodiments. The second scFv against VH and VL of PD-1 antigen has H2CDR1, H2CDR2 and H2CDR3 of the heavy chain variable region and L2CDR1, L2CDR2 and L2CDR3 of the light chain variable region described in the above embodiments.
In some specific embodiments, the present invention provides an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof which is a scFv-structured bispecific antibody or antigen-binding fragment thereof comprising a CTLA-4 binding moiety at the C-terminus and a PD-1 binding moiety at the N-terminus, wherein
(1) The C-terminal CTLA-4 binding part amino acid sequence is selected from:
(b1) the amino acid sequences shown as SEQ ID NO. 55 and SEQ ID NO. 56;
(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the amino acid sequence of the N-terminal PD-1 binding moiety is selected from the group consisting of:
(b4) an amino acid sequence as shown in SEQ ID NO. 54;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
In some specific embodiments, the invention provides a bispecific antibody against CTLA-4 and PD-1 or an antigen-binding fragment thereof, wherein the N-terminal PD-1 binding moiety amino acid sequence is SEQ ID No. 54, the amino acid sequence of SEQ ID No. 54 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 54 or has at least 85% sequence identity with SEQ ID No. 54, and the C-terminal CTLA-4 binding moiety amino acid sequence is SEQ ID No. 55, the amino acid sequence of SEQ ID No. 55 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 55 or has at least 85% sequence identity with SEQ ID No. 55.
In some specific embodiments, the invention provides a bispecific antibody against CTLA-4 and PD-1 or an antigen-binding fragment thereof, wherein the N-terminal PD-1 binding moiety amino acid sequence is SEQ ID No. 54, the amino acid sequence of SEQ ID No. 54 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 54 or has at least 85% sequence identity with SEQ ID No. 54, and the C-terminal CTLA-4 binding moiety amino acid sequence is SEQ ID No. 56, the amino acid sequence of SEQ ID No. 56 that is obtained by substituting, deleting or adding one or more amino acids and that is functionally identical to SEQ ID No. 56 or has at least 85% sequence identity with SEQ ID No. 56.
In some specific embodiments, the anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, and a light chain constant region of human kappa, lambda chains, or a variant thereof. In some preferred embodiments, the anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a light chain constant region of human kappa chain or a variant thereof.
In some embodiments, the invention provides an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or f (ab) 2.
Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
In some embodiments, the host cell according to the invention is selected from the group consisting of prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably E.coli. In another preferred embodiment, the host cell is a mammalian cell.
Another aspect of the invention provides a method of making an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the invention comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
Another aspect of the invention provides a pharmaceutical composition comprising an anti-CTLA-4/anti-PD-1 bispecific humanized antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides pharmaceutical compositions comprising the anti-CTLA-4/anti-PD-1 bispecific humanized antibodies or antigen-binding fragments thereof of the present invention, and further comprising other active components, such as other antibodies, targeted drugs, and the like. In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is an aqueous buffered solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of a liposome.
The anti-CTLA-4/anti-PD-1 bispecific humanized antibody or an antigen-binding fragment thereof of the present invention may be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical formulation suitable for oral or parenteral administration. Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes. The formulations may be administered by any route, for example by infusion or bolus injection, by a route of absorption through epithelial or cutaneous mucosa (e.g. oral mucosa or rectum, etc.). Administration may be systemic or local. The formulations may be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulation.
Another aspect of the invention provides a method of inhibiting CTLA-4 and/or PD-1 activity, comprising administering to an individual in need thereof an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.
Another aspect of the invention provides the use of an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for inhibiting CTLA-4 and/or PD-1 activity. In some embodiments, the medicament that inhibits CTLA-4 and/or PD-1 activity is for treating leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma, and/or melanoma. In some embodiments, the present invention provides the use of the above-described anti-B7-H3 antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of a tumor, preferably, the tumor is selected from the group consisting of leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma, and melanoma.
The anti-CTLA-4/anti-PD-1 bispecific antibody or the antigen binding fragment thereof provided by the invention has higher affinity and stability, obvious anti-tumor effect and low toxicity, can be applied to preparation of medicaments for treating various tumor diseases, and has wide market prospect.
Definition of
Unless defined otherwise, the meanings of scientific and technical terms used herein are those commonly understood by those of skill in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligo-or polynucleotide chemistry and hybridization described herein are those well known and commonly employed in the art. For recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection), standard techniques are used. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions or methods commonly used in the art or described herein. The foregoing techniques and methods are generally used as described in various comprehensive and more specific documents that are well known in the art and that are cited and discussed in this specification. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual) (2 nd edition, Cold Spring Harbor Laboratory Press, N.Y. (1989)). Nomenclature used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry, and laboratory methods and techniques described herein are those well known and commonly used in the art.
In the present invention, the term "at least 80% sequence identity" refers to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In the present invention, the term "at least 85% sequence identity" refers to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some preferred embodiments, the sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and percent identity determination between two sequences can be performed by the BLASTN/BLASTP algorithm on the National Center For Biotechnology institute website.
In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in a three-dimensional space in positions relative to each other to form an antigen-binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs". The assignment of amino acids to each domain is defined according to Kabat "sequences of proteins of immunological interest" (national institutes of health, Bessesda, Md., 1987 and 1991) or Chothia and Lesk, J.mol.biol.196: 901-.
The "antibody" of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody may be a whole antibody and any antigen binding fragment or single chain thereof. An "antibody" of the present invention includes any protein or peptide containing at least a portion of an Ig molecule having biological activity for binding an antigen. Examples of "antibodies" of the invention include, but are not limited to, CDRs of a heavy or light chain or a ligand-binding portion thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework regions, or any portion thereof.
The "antigen binding fragment" of the present invention includes Fab fragment, Fab 'fragment, F (ab') 2 fragment having antigen binding activity, Fv fragment and scFv fragment binding to human CTLA-4 or PD-1. The Fv fragment contains the variable regions of the antibody heavy and light chains, but lacks the constant region, and has the smallest antibody fragment with the entire antigen-binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined into a single polypeptide chain using different linkers, known as single chain antibodies or single chain fv (scFv). The anti-CTLA-4 or anti-PD-1 antibodies of the invention can be single chain variable fragments (scFv) derived from a single chain polypeptide of an antibody, which retains the ability to bind antigen. Examples of scfvs include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods for making scFv are well known to those skilled in the art.
An antibody according to the present invention refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e. a molecule comprising an antigen binding site that specifically binds to (is immunologically reactive with) an antigen. "specific binding" refers to an antibody that reacts with one or more antigenic determinants of an antigen without reacting with other polypeptides or with very low affinity (Kd)>10 -6 ) Binding to other polypeptides. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab 'and F (ab') 2 fragments, Fv, scFv and Fab expression libraries. Monoclonal antibodies (mabs) are antibodies derived from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma techniques, recombinant techniques, phage display techniques, and synthetic techniques such as CDR grafting or other known techniques.
The murine antibody of the invention is a monoclonal antibody to human CTLA-4 prepared according to the knowledge and skill in the art. The preparation is carried out by injecting a test subject with CTLA-4 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.
The chimeric antibody is formed by fusing a variable region of a murine antibody with a constant region of a human antibody, and can reduce an immune response reaction induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into human carrier, and finally expressing chimeric antibody molecule in eukaryotic industrial system or prokaryotic industrial system.
The "humanized antibody" of the present invention is also referred to as a CDR-grafted antibody, and is an antibody produced by grafting mouse CDR sequences into a human antibody variable region Framework (FR). Such variable region framework sequences may be obtained from public DNA databases or published references, for example from ImmunoGeneTiCs (IMGT) website http:// imgt. cities. fr or from J.Immunoglobu, 2001ISBN 012441351.
A "bispecific antibody" as described herein refers to a monoclonal antibody having binding specificity for at least two different antigens.
The "peptide linker" according to the present invention may be those comprising any of 1 to 10, particularly 2 to 50 amino acids, and may comprise any kind of amino acid without any limitation.
Drawings
FIG. 1is a schematic diagram of the structure of peptide chain 1 of an anti-CTLA-4/anti-PD-1 bispecific antibody.
Figure 2 is a schematic structural diagram of peptide chain 2 of the anti-CTLA-4/anti-PD-1 bispecific antibody.
Figure 3 is the results of experiments (FACS) showing that anti-CTLA-4 humanized antibody blocks CTLA-4 binding to CD80 activity, wherein the abscissa is antibody concentration (nM) and the ordinate is fluorescence intensity.
Figure 4 is the results of experiments (FACS) showing that anti-CTLA-4 humanized antibody blocks CTLA-4 binding to CD86 activity, wherein the abscissa is antibody concentration (nM) and the ordinate is fluorescence intensity.
FIG. 5 is a schematic structural diagram of the bispecific anti-CTLA-4/PD-1 antibody of ScFv structure of the present invention.
Figure 6 is the results of a bispecific antibody binding assay (ELISA) with CTLA-4, where the abscissa is antibody concentration (nM) and the ordinate is absorbance at OD 450.
FIG. 7 is the results of an Experiment (ELISA) for binding of bispecific antibody to PD-1, where the abscissa is the antibody concentration (nM) and the ordinate is the absorbance at OD 450.
FIG. 8 shows the results of the binding experiments (FACS) of bispecific antibody to cell surface CTLA-4, wherein the abscissa is the antibody concentration (nM) and the ordinate is the fluorescence intensity.
FIG. 9 shows the results of the bispecific antibody binding experiment (FACS) to cell surface PD-1, wherein the abscissa is the antibody concentration (nM) and the ordinate is the fluorescence intensity.
Figure 10 is the results of experiments (FACS) with bispecific antibodies blocking CTLA-4 binding to CD80 activity, with antibody concentration (nM) on the abscissa and fluorescence intensity on the ordinate.
Figure 11 is the results of experiments (FACS) with bispecific antibodies blocking CTLA-4 binding to CD86 activity, with antibody concentration (nM) on the abscissa and fluorescence intensity on the ordinate.
FIG. 12 is the results of an experiment (FACS) in which bispecific antibody blocks PD-1 binding to PD-L1 activity, with antibody concentration (nM) on the abscissa and fluorescence intensity on the ordinate.
FIG. 13 shows the experimental results of CD80 blocking activity (FACS) of bispecific antibody on bistable transfected cell lines, where the abscissa is antibody concentration (nM) and the ordinate is fluorescence intensity.
FIG. 14 shows the experimental results of CD86 blocking activity (FACS) of bispecific antibody on bistable transfected cell lines, where the abscissa is antibody concentration (nM) and the ordinate is fluorescence intensity.
FIG. 15 shows the experimental results of PD-1 blocking activity (FACS) of bispecific antibody on bistable transfected cell lines, wherein the abscissa is the antibody concentration (nM) and the ordinate is the fluorescence intensity.
Detailed Description
The following representative examples are intended to better illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures not specified in the following examples are generally carried out under conventional conditions such as antibody technical laboratory manual, molecular cloning manual, etc. in cold spring harbor, or under conditions recommended by manufacturers of raw materials or commercial products. The materials and reagents used in the examples were all commercially available unless otherwise specified.
Example 1: preparation of CTLA-4 antigenic protein
1. Construction of expression vectors for antigenic proteins
Synthesizing a gene segment for coding the full length of the extracellular region of the human CTLA-4 protein, wherein the amino acid sequence design is shown as SEQ ID NO. 14. The nucleotide sequence of the expression plasmid is cloned to a eukaryotic expression plasmid pTargetT to obtain an expression plasmid pTargetT-hCTLA-4.
Synthesizing the gene segment of coding the full length of the extracellular region of the monkey source CTLA-4 protein, and designing the amino acid sequence as shown in SEQ ID NO. 15. The nucleotide sequence of the expression plasmid is cloned to a eukaryotic expression plasmid pTargetT to obtain the expression plasmid pTargetT-cynoCTLA-4.
The amino acid sequences of the fused extracellular region of the human CTLA-4 protein and the hIgG1-Fc or His label are shown as SEQ ID NO:16 and SEQ ID NO:17, the amino acid sequences are subjected to codon optimization, nucleotide sequences of CTLA-4-hFc and CTLA-4-His with labels are synthesized, and the nucleotide sequences are respectively cloned to a eukaryotic expression plasmid pHR to obtain expression plasmids pHR-CTLA-4-hFc and pHR-CTLA-4-His.
The amino acid sequences of the fused extracellular domain of the human CTLA-4 protein and the mIgG1-Fc label are shown in SEQ ID NO. 18. After codon optimization is carried out on the sequence of the human CTLA-4 protein extracellular region, a nucleotide sequence of CTLA-4-mFc with a label is synthesized, and the nucleotide sequence is cloned to a eukaryotic expression plasmid pHR to obtain the expression plasmid pHR-CTLA-4-mFc.
The amino acid sequence of the fused extracellular region of the monkey-derived CTLA-4 protein and the hIgG1-Fc tag is shown as SEQ ID NO:19, the nucleotide sequence of the cynoCTLA-4-hFc with the tag is synthesized after codon optimization of the amino acid sequence, and the nucleotide sequence is cloned to a eukaryotic expression plasmid pHR to obtain the expression plasmid pHR-cynoCTLA-4-hIgG 1-Fc.
Synthesizing a gene segment for coding the full length of the extracellular region of the humanized CD80 protein, wherein the amino acid sequence design is shown as SEQ ID NO. 20. The nucleotide sequence thereof was cloned into a eukaryotic expression plasmid pTargetT to obtain an expression plasmid pTargetT-CD80 thereof.
Synthesizing a gene segment for coding the full length of the extracellular region of the humanized CD86 protein, wherein the amino acid sequence design is shown as SEQ ID NO. 21. The nucleotide sequence thereof was cloned into a eukaryotic expression plasmid pTargetT to obtain an expression plasmid pTargetT-CD 86.
The amino acid sequence of the fused extracellular domain of the humanized CD80 protein and the mIgG1-Fc tag is shown as SEQ ID NO. 22, and the nucleotide sequence of the CD80-mFc with the tag is synthesized after the codon optimization of the amino acid sequence. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CD 80-mFc.
The amino acid sequences of the fused extracellular domain of the humanized CD86 protein and the mIgG1-Fc tag are shown in SEQ ID NO. 23, and the nucleotide sequence of the CD86-mFc with the tag is synthesized after the codon optimization of the amino acid sequences. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CD 86-mFc.
The amino acid sequence of the fused human CD80 protein extracellular region and hIgG1-Fc label is shown as SEQ ID NO:24, the amino acid sequence is subjected to codon optimization to synthesize a CD80-hFc nucleotide sequence with a label, and the CD80-hFc nucleotide sequence is cloned to a eukaryotic expression plasmid pHR to obtain an expression plasmid pHR-CD 80-hFc.
The amino acid sequence of the fused human CD86 protein extracellular region and hIgG1-Fc label is shown as SEQ ID NO. 25, the amino acid sequence is subjected to codon optimization to synthesize a CD86-hFc nucleotide sequence with a label, and the CD86-hFc nucleotide sequence is cloned to a eukaryotic expression plasmid pHR to obtain an expression plasmid pHR-CD 86-hFc.
2. Expression and purification of antigenic proteins
(1) Construction of stably transfected cell lines expressing antigenic proteins
The eukaryotic expression plasmid pTargeT-hCTLA-4 was transfected into CHO-K1 cells (Shanghai cell of Chinese academy of sciences) by electrotransformation under a voltage of 160V and a square pulse of 15msecInstitute of physics), 5% CO at 37 deg.C 2 Culturing in an incubator with concentration. After 24h, pressure culture was carried out using a medium containing 1000. mu.g/ml G418(Gibco, # 10131-027). The positive rate of the transfected pool was determined by flow cytometry 16 days after transfection, and the cells with higher positive rate were plated (according to 1X 10) 6 Cell density at cell/ml, 100. mu.l/well, 96-well plate, cells were incubated with Iplilimumab antibody (trade name Yervoy, available from Bristol-Myers Squibb Company) and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibodies, mean values at a wavelength of 585nm were measured by flow cytometry (ACEABIO, Novocyte 2060R), and data analysis was performed using GraphPad generation. The positive cell strain is subcloned, and a cloned CHO-K1 cell strain is selected, and the cell strain expresses CTLA-4 molecules at a high level and is named as CHO-K1-CTLA-4.
Gene fragments encoding human CTLA-4 protein (SEQ ID NO:14) and full-length extracellular region (SEQ ID NO:41) of human PD1 protein were synthesized separately. Then, the plasmid is cloned to a eukaryotic plasmid pTargetT to obtain an expression plasmid pTargetT-hPD-1-hCTLA-4 thereof. pTargeT-hPD1-hCTLA-4 was transfected into CHO-K1 cells (Shanghai cell biology institute, Chinese academy of sciences) by electrotransformation at 160V voltage and 15msec square pulses, and the cells were incubated at 37 ℃ and 5% CO 2 Culturing in an incubator with concentration. After 24h, pressure culture was carried out using a medium containing 1000. mu.g/ml G418(Gibco, #10131-027) and 500. mu.g/ml hygromycin B. The positive rate of the transfected pool was determined by flow cytometry 16 days after transfection, and the cells with higher positive rate were plated (according to 1X 10) 6 Cell density of individual/ml, 100. mu.l/well, 96-well plate) were plated, cells were incubated with Iplilimumab antibody and Nivolumab antibody (purchased from Bristol-Myers Squibb Company) and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, mean values at a wavelength of 585nm were detected by flow cytometry (ACEABIO, Novocyte 2060R), and data analysis was performed using GraphPad generation. And subcloning the positive cell strain, and selecting a cloned CHO-K1 cell strain, wherein the cell strain expresses PD-1 and CTLA-4 molecules at a high level and is named as CHO-K1-PD 1-CTLA-4.
(2) Expression of tag antigen protein
In a 1L cell culture flask, the inoculation density is0.5 x 10 6 The cells/mL 293E cells (from ATCC) were inoculated with fresh, preheated FreeStyle293 expression medium to a total volume of 250mL at 37 ℃ with 8% CO 2 Humidified CO 2 The culture was carried out overnight in an incubator. 8.5mL FreeStyle293 expression culture medium is taken, 500 mu l of PEI solution of 1mg/mL is added, the mixture is mixed evenly, 250 mu g of plasmid to be transfected is added into 8.5mL FreeStyle293 expression culture medium and mixed evenly, wherein the labeled antigen protein plasmids pHR-CTLA-4-hFc, pHR-CTLA-4-His, pHR-cynoCTLA-4-hFc, pHR-CD80-hFc, pHR-CD86-hFc, pHR-CD80-mFc and pHR-CD86-mFc are transfected respectively. . Adding the mixed solution of PEI and FreeStyle293 expression medium into the plasmid, mixing well, adding into cell culture, standing at 37 deg.C and 8% CO 2 Humidified CO 2 Culturing in an incubator. Cells were fed on days 1 and 3 after cell transfection, and 2.5ml of glutamine (mother liquor concentration of 200mM) and 5ml of glucose (mother liquor concentration of 180g/L) were added to each flask. When the cell viability is reduced to 65% -75%, cell supernatants are collected. The cell culture was centrifuged at 1500rpm for 5min, and the supernatant was collected, and then centrifuged at 8000rpm for 20min, and the supernatant was collected.
(3) Affinity chromatography column purification
The protein was purified by using an affinity column using AKTA (GE, AKTA pure-150) according to its properties.
Example 2: preparation of anti-CTLA-4 monoclonal antibody
1. Preparation of hybridoma monoclonal
(1) Animal immunization
The hCTLA-4 antigen protein with different labels and adjuvant are used together to immunize experimental animals, wherein the experimental animals comprise Balb/c strain mice and SD rats. The animals were immunized with 50. mu.g of antigen for the first immunization and 25. mu.g of antigen for the later immunization. The immunological adjuvant may be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Beijing Boolong immuno-technologies, Inc.). Emulsifying antigen by adopting Freund's adjuvant, dropwise adding hTLA-4 antigen protein samples with different labels into adjuvant solution, and fully mixing while dropwise adding and vortexing, wherein the adjuvant is prepared by using a dosage reference instruction. Mixing well to form water-in-oil emulsion, and immunizing mouse. Adopting Quick Antibody-Mouse5W as an adjuvant, mixing hTLA-4 antigen protein samples with different labels and the Quick Antibody-Mouse5W according to the volume ratio of 1:1, and immunizing SD rats in an intramuscular injection mode after uniformly mixing. The immunization protocol is shown in table 1.
TABLE 1 animal immunization protocol
Figure BDA0003533158350000371
I.m. intramuscular injection; s.c. subcutaneous injection; i.p. intraperitoneal injection.
(2) Hybridoma fusion
Acquisition and preparation of splenocytes: mice/rats after booster immunizations were sacrificed and soaked in 75% alcohol. The spleen is dissected and taken out, ground by a grinding rod and filtered by a cell screen to prepare single cell suspension. The spleen cell suspension was centrifuged at 2000rpm for 5min and the supernatant was discarded. Adding 2mL of erythrocyte lysate, lysing erythrocytes at room temperature for 2min, adding PBS to 20mL, centrifuging at 1500rpm for 7min, discarding supernatant, and counting viable cells after resuspension. Sp2/0 cells were collected from the flask, centrifuged at 1000rpm for 5min and the supernatant discarded, resuspended and then counted for viable cells. According to the ratio of splenocytes: sp2/0 cells were mixed at a ratio of 1:1, centrifuged at 1500rpm for 7min and the supernatant discarded. Cells were resuspended in 20mL of electrotransfer buffer and centrifuged at 1500rpm for 7 min. Discard the supernatant and repeat once. Resuspending the cells with appropriate amount of electrotransfer buffer to ensure cell concentration of 2X 10 7 About one cell/mL. The cell suspension was added to a 9mL electrofusion tank for fusion. After the fusion, the cell suspension was transferred to 15mL of RPMI 1640 complete medium containing 20% FBS and left at room temperature for 20 min. The fused cells were resuspended in RPMI 1640 medium containing 1 XHAT, 1 XBIOYC 3, 20% FBS. The cell suspension was added to several 96-well cell culture plates at 100. mu.l/well to ensure a cell count of approximately 4X 10 per well 4 Each cell/well was cultured in a 37 ℃ cell incubator. After 5 days, the cells were supplemented with 100. mu.L/well of RPMI 1640 complete medium (20% FBS, 1 XHAT, 1 XBIOYC-3).
(3) Hybridoma and subclone screening
After one week of fusion, cell culture supernatants of the hybridoma female clones were selected, hybridoma female clones binding human hCTLA-4 protein and cynomolgus monkey CTLA-4 protein were screened by ELISA, and female clones capable of binding CHO-K1-CTLA-4 stably transfected cell lines were further screened by flow cytometry.
And (3) carrying out subcloning on the positive parent clone by using a limiting dilution method, culturing for one week, detecting the binding activity of the subclone supernatant and hCTLA-4 molecules by using ELISA, and screening a monoclonal cell strain secreting an anti-hCTLA-4 antibody. A plurality of better monoclonal cell strains are obtained. One of the anti-hCTLA-4 monoclonal antibodies was labeled as SHS 010-C92.
2. Preparation of monoclonal antibodies
Determining the monoclonal antibody parent clone strain according to the activity analysis result of the subclone supernatant, and carrying out amplification culture on the monoclonal antibody parent clone strain. The culture condition is 1640 culture medium containing 10% fetal calf serum, 1x NAEE, 1x sodium pyruvate and 1% streptomycin double antibody, when the cell confluence is more than 80%, the cells are subcultured and expanded, when the cells are cultured to about 50ml, the supernatant is collected, and the antibody is purified. The obtained antibody was confirmed to have good purity by SDS-PAGE gel electrophoresis.
3. Sequencing of monoclonal antibodies
The subcloned positive hybridoma cells were expanded, and a suitable amount of cells were used to extract total RNA according to the instructions of RNeasy Plus Mini Kit (Qiagen, 74134) Kit, and first strand cDNA was synthesized using Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription Kit.
Designing a specific primer (the 5' end contains a homologous arm sequence for homologous recombination with a eukaryotic expression vector) according to the mouse antibody subtype variable region, and carrying out PCR amplification on an antibody variable region gene by taking cDNA as a template so as to respectively obtain gene fragments of a mouse antibody light chain variable region and a mouse antibody heavy chain variable region; primers were designed (reference: 1. like Krebber, Susanne Bornhauser, journal Burmester et al. Reliable locking of functional antisense variable domains from hybrids and phosphor cell primers system. journal of Immunological Methods 1997,201: 35-55; 2.Simon
Figure BDA0003533158350000381
Antibody variable-region sequencing as a method for hybrid cell-line authentication,2008,78: 1071-. The sequencing results of SHS010-C92 are shown in Table 2.
TABLE 2 anti-CTLA-4 murine monoclonal antibody sequences
Antibodies Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence
SHS010-C92 SEQ ID NO:26 SEQ ID NO:27
CDR sequences of VH of antibody SHS 010-C92: the sequences of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:1, 2 and 3, and the CDR sequences of VL: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 5, 6 and 7 respectively.
Example 3: construction of anti-CTLA-4 chimeric antibodies
Respectively co-transforming the purified mouse antibody light chain and heavy chain variable region gene fragments and linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant regions into escherichia coli DH5 alpha competent cells, uniformly coating the mixed solution on the surface of an agar plate containing corresponding antibiotics, and respectively picking a plurality of single colonies for DNA sequencing after overnight culture in a constant-temperature incubator at 37 ℃; the chimeric antibody with correct sequencing was labeled as SHS 010-C92-CHI.
The chimeric antibody heavy-light chain plasmid is cotransfected with HEK293E cells, expression and purification are carried out to obtain the antibody, and then purity detection, activity analysis and affinity detection are carried out.
The chimeric antibody SHS010-C92-CHI is subjected to gene mutation by using a site-directed mutagenesis method to screen a better antibody. The G at the 24 th position of the CDR of the heavy chain of the SHS010-C92-CHI is mutated into E, the stability of the antibody is improved, and the marker is SHS 010-C92-G24E-CHI. The chimeric antibody sequencing results are shown in table 3.
TABLE 3 anti-CTLA-4 chimeric antibody sequences
Chimeric antibodies Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence
SHS010-C92-CHI SEQ ID NO:26 SEQ ID NO:27
SHS010-C92-CHI-G24E SEQ ID NO:28 SEQ ID NO:27
CDR sequences of the VH of antibody SHS 010-C92-CHI: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO:1, 2 and 3, and the CDR sequences of VL: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 5, 6 and 7 respectively. CDR sequences of the VH of antibody SHS 010-C92-CHI-G24E: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO:1, 2 and 4, and the CDR sequences of VL: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 5, 6 and 7 respectively.
Example 4: construction and production of anti-CTLA-4 humanized antibody
According to the immune activity analysis, a plurality of chimeric antibodies with good activity are selected for humanized antibody modification.
Firstly, comparing with a mouse antibody sequence in an immune gene database (IMGT) to confirm a murine germline of the variable region of the SHS010-C92-CHI antibody, and after homologous comparison, ensuring that the FR region of the heavy chain variable region sequence of the SHS010-C92-CHI antibody is most similar to a mouse antibody germline gene IGHV3-48 x 01; the FR sequence of the antibody light chain variable region most closely resembles the mouse antibody IGKV3-11 x 01. Using the frame region sequence FR1-FR3 of the SHS010-C92-CHI antibody as a template, searching a full-human frame with similar 3D structure but lower immunogenicity in a human frame region library to replace the FR1-FR3 sequence of the SHS010-C92-CHI, performing 3D modeling on the full-length heavy chain/light chain sequence, performing structural alignment analysis on the full-length heavy chain/light chain sequence and the heavy chain/light chain sequence of the original antibody, comprehensively considering antigenicity and 3D structural similarity, and finally selecting 2 humanized heavy chain variable regions (see SEQ ID NO:29 and 30) and 5 humanized light chain variable regions (see SEQ ID NO:31, 32, 33, 34 and 35) of the SHS010-C92-CHI for further optimization. The non-CDR sequence of the humanized antibody of SHS010-C92-CHI reaches over 95 percent of humanization.
Reverse transcribing the designed amino acid sequences of the light chain and the heavy chain variable region of the humanized antibody into corresponding nucleotide sequences, generating oligonucleotide fragments containing complementary sequences between adjacent fragments, annealing the oligonucleotide fragments through Overlap PCR, connecting, and amplifying complete nucleotide fragments of the light chain and the heavy chain variable region by using a specific primer (the 5' end contains a homologous arm sequence for homologous recombination with a eukaryotic expression vector); the purified light chain variable region nucleotide fragment and a linearized eukaryotic expression plasmid (pHR-hK) containing a human K light chain constant region are co-transformed into an escherichia coli DH5 alpha competent cell, the purified heavy chain variable region nucleotide fragment and a eukaryotic expression plasmid (pHR-IgG4) containing a human IgG4 heavy chain constant region are co-transformed into an escherichia coli DH5 alpha competent cell, the competent cells of the transformation plasmids are respectively and uniformly coated on the surfaces of agar plates containing corresponding antibiotics, and after overnight culture in a constant-temperature incubator at 37 ℃, a plurality of single colonies are respectively picked for DNA sequencing.
Inoculating the positive clones with correct sequencing into a2 XYT liquid culture medium containing corresponding antibiotics, performing shake culture at 37 ℃ for more than 12 hours, collecting thalli, performing plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids, and detecting the concentration and purity of the plasmids by using a nucleic acid quantitative analyzer.
The plasmid is transfected into HEK293E cells, and a large amount of antibodies are obtained through expression and purification, and purity detection, activity analysis and affinity detection are carried out.
The humanized antibody with good purity, activity and affinity was selected and labeled huC92-11, and its sequence is shown in Table 4.
TABLE 4 anti-CTLA-4 humanized antibody sequences
Figure BDA0003533158350000401
CDR sequences of VH of antibody HuC 92-11: the sequences of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:1, 2 and 4, and the CDR sequences of VL: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 5, 6 and 7 respectively.
Example 5: determination of binding Activity of anti-CTLA-4 antibody with human CTLA-4 (ELISA)
The binding activity of the antibodies was analyzed by ELISA. Human CTLA-4-His protein (1. mu.g/well, prepared in examples 1 and 2) was coated on a 96-well plate and incubated overnight at 4 ℃. Washed 3 times with 1xPBST and then blocked with 5% skim milk at 37 ℃ for 2 h. After 3 washes with 1xPBST, the anti-CTLA-4 antibody of the present invention was added as a primary antibody to the microplate starting at 10. mu.g/mL, with 5-fold gradient dilution, at 8 concentrations of 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, and 0.128ng/mL, respectively, and incubated at 37 ℃ for 2h with a control antibody of Iplilimumab (available from Bristol-Myers Squibb Company); after washing 5 times with 1 xPBT, the secondary antibody was incubated for 1h at 37 ℃ using Anti-Human IgG HRP (Jackson, 109-. After washing 5 times with 1xPBST, a color developing solution TMB was added, and after completion, the OD450 value was read with a microplate reader (thermo, Multiskan FC). Generation of EC Using GraphPad 50 。
The experimental results show thatEC of humanized anti-CTLA-4 antibody HuC92-11 of the invention 50 0.01nM, equivalent to the control antibody Iplilimumab (EC) 50 : 0.01nM) and has better binding ability with human CTLA-4.
Example 6: determination of binding Activity of anti-CTLA-4 antibody with cynomolgus monkey CTLA-4 (ELISA)
The binding activity of the antibodies was analyzed by ELISA. Cynomolgus monkey CTLA-4-His (1. mu.g/well, prepared in examples 1 and 2) was coated on a 96-well microplate. The anti-CTLA-4 antibody is used as a primary antibody, the primary antibody is diluted from 10 mu g/mL by 5 times of gradient and added into an ELISA plate, the concentration is 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL and 0.128ng/mL respectively, the incubation is carried out at 37 ℃ for 2h, and the control antibody is Iplilimumab. The secondary antibody was treated with Anti-Human IgG HRP (Jackson, 109-. EC Generation Using GraphPad 50 The results are shown in Table 5.
TABLE 5 binding Activity of anti-CTLA-4 antibodies to cynomolgus monkey CTLA-4
Figure BDA0003533158350000411
The experimental result shows that the humanized anti-CTLA-4 antibody HuC92-11 has better binding capacity with cynomolgus monkey CTLA-4.
Example 7: determination of the blocking Activity of anti-CTLA-4 antibodies against CTLA-4 and its ligands (ELISA)
The blocking activity of the antibody on CTLA-4 and its ligand was analyzed by ELISA. Human CTLA-4-His protein (1. mu.g/well, prepared in examples 1 and 2) was coated on a 96-well plate and incubated overnight at 4 ℃. Washed 3 times with 1xPBST and then blocked with 5% skim milk at 37 ℃ for 2 h. After washing with 1xPBST for 3 times, preparing CD80-mFc with the concentration of 1 mug/mL and CD86-mFc ligand solution with the concentration of 5 mug/mL, taking the ligand solution as a diluent, taking the anti-CTLA-4 antibody as a primary antibody, starting from 25 mug/mL, diluting with 5 times of gradient, adding the antibody into an ELISA plate, and taking 8 concentrations as 25000ng/mL, 5000ng/mL and 1000 ng/L respectivelymL, 200ng/mL, 40ng/mL, 8ng/mL, 1.6ng/mL, 0.32ng/mL, incubation at 37 ℃ for 2h, control antibody is Iplilimumab; after washing 5 times with 1xPBST, the secondary antibody was incubated for 1h at 37 ℃ using Anti-mouse IgG HRP (Jackson, 109. sub. 035. sub. 003, 1: 5000). After washing 5 times with 1xPBST, a color developing solution TMB was added, and after completion, the OD450 value was read with a microplate reader (thermo, Multiskan FC). IC Generation Using GraphPad 50 The results are shown in tables 6 and 7.
TABLE 6 anti-CTLA-4 humanized antibodies block CTLA-4 binding to CD80
Figure BDA0003533158350000421
TABLE 7 anti-CTLA-4 humanized antibodies block binding of CTLA-4 to CD86
Figure BDA0003533158350000422
The results of the experiments show that the humanized anti-CTLA-4 antibody HuC92-11 of the present invention is capable of blocking CTLA-4 binding to its ligand, and the blocking activity is stronger than that of the control antibody Iplilimumab.
Example 8: determination of binding of anti-CTLA-4 antibodies to cell surface hCTLA-4 (FACS)
The binding activity of the antibody to CTLA-4 on the cell surface of CHO-K1-CTLA-4 was analyzed by FACS. CHO-K1-CTLA-4 cells were digested, resuspended in a solution of 2% FBS-PBS, and counted. The cells were plated at 1 × 10 per well 5 The anti-CTLA-4 antibody is used as a primary antibody, the primary antibody is added into the cell plate by gradient dilution from 20 mu g/mL, the concentration is 8, the concentration is 20000ng/mL, 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL and 0.64ng/mL respectively, the cell plate is incubated for 1h at the temperature of 4 ℃, and the control antibody is Iplilimumab; the secondary antibody was PE-Anti-Human IgG (Biolegend, Cat. No.409303, 1.25. mu.l/well), and the fluorescence intensity generated by binding of the antibody to the cell surface was measured by flow cytometry after washing, and the results are shown in Table 8.
TABLE 8 binding Activity of anti-CTLA-4 antibodies with cell surface hCTLA-4
Figure BDA0003533158350000431
The experimental result shows that the humanized anti-CTLA-4 antibody HuC92-11 has better binding capacity with cell surface CTLA-4.
Example 9: determination of the blocking Activity of anti-CTLA-4 antibodies against CTLA-4 and its ligands (FACS)
FACS analysis was performed to analyze the ability of the antibodies to block binding of ligands by CTLA-4 on the surface of CHO-K1-CTLA-4 cells. CHO-K1-CTLA-4 cells were digested, resuspended in a solution of 2% FBS-PBS, and counted. The cells were plated at 1 × 10 per well 5 The anti-CTLA-4 antibody is used as a primary antibody, the primary antibody is added into the cell plate by gradient dilution from 20 mu g/mL, the concentration of the primary antibody is 20000ng/mL, 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL and 0.64ng/mL, the diluted antibody is mixed with 1 mu g/mL CD80-mFc or 1 mu g/mL CD86-mFc 1:1, and then the cell plate is incubated for 1h at the temperature of 4 ℃, and the control antibody is Iplilimumab; the secondary antibody was washed with PE-Anti-mouse IgG (Biolegend, Cat. No.409303, 1.25. mu.l/well) and then the fluorescence intensity of the binding of the ligand to the cell surface was measured by flow cytometry, and the results are shown in FIGS. 3 and 4.
The experimental results show that the humanized anti-CTLA-4 antibody HuC92-11 of the present invention is capable of blocking binding of CTLA-4 to its ligand, and has stronger blocking activity than control antibody Iipilimumab.
Example 10: preparation of anti-PD-1 antibody-associated antigen protein
1. Construction of expression vectors for antigenic proteins
1) Construction of expression vector for PD-1 antigen protein
The amino acid sequence of the extracellular region of the human PD-1 protein and the amino acid sequence of a Tag of hIgG1-Fc or mIgG1-Fc or His Tag are designed, and the amino acid sequence design is respectively shown as SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38. After the amino acid sequence is subjected to codon optimization, PD-1-hFc, PD-1-mFc and PD-1-His of PD-1 protein extracellular region gene fragments with labels are synthesized and are respectively cloned into a eukaryotic expression plasmid pHR, and expression plasmids pHR-PD-1-hFc, pHR-PD-1-mFc and pHR-PD-1-His of the PD-1 protein extracellular region gene fragments are obtained.
2) Expression vector construction of PD-L1 ligand protein
The amino acid sequence of the extracellular region of the humanized PD-L1 protein and the amino acid sequence of a hIgG1-Fc or mIgG1-Fc label are designed, and the amino acid sequence design is respectively shown as SEQ ID NO 39 and SEQ ID NO 40. After the amino acid sequence is subjected to codon optimization, gene fragments PD-L1-hFc, PD-L1-mFc and PD-L1-His of the PD-L1 protein extracellular region with labels are synthesized and are respectively cloned into a eukaryotic expression plasmid pHR, and expression plasmids pHR-PD-L1-hFc and pHR-PD-L1-mFc are obtained.
2. Expression and purification of antigenic proteins
1) Construction of stably transfected cell line for antigen protein
Synthesizing a gene fragment for coding the full length of the PD-1 protein, designing an amino acid sequence as shown in SEQ ID NO:41, and cloning the gene fragment to a eukaryotic expression plasmid pTargeT to obtain an expression plasmid pTargeT-PD-1 thereof.
The eukaryotic expression plasmid pTargeT-PD-1 was transfected into CHO-K1 cells (Shanghai cell biology institute of Chinese academy of sciences) by electrotransformation at 160V voltage with a square pulse of 15msec, and cultured in an incubator at 37 ℃ and 5% CO 2. After 24h, the medium was pressure-cultured in DME/F12 complete medium containing 1000. mu.g/ml G418. After 16 days, the pool positive rate was measured by FACS, and the cell viability rate was calculated. Cells were incubated with PE anti-human PD-1 antibody (Biolegend, inc.,621607), mean and parent% values were read with a flow cytometer (ACEA, Novocyte), and data analysis was performed using GraphPad generation. Positive pool cells with a cell viability of 90% or more were sub-clonally plated (cell density of 0.5/well, 200. mu.l/well) at 37 ℃ in 5% CO 2 Cultured in an incubator. After the subclones were grown for 12 days, the cloned CHO-K1 cell line was selected, FACS screened for PD-1 high expression subclones, and designated hPD-1-CHO-K1.
2) Construction of stably transfected cell lines for ligand protein
Synthesizing a gene fragment for coding the full length of the PD-L1 protein, designing an amino acid sequence as shown in SEQ ID NO:42, and cloning the gene fragment to a eukaryotic expression plasmid pTargeT to obtain the expression plasmid pTargeT-PD-L1.
The eukaryotic expression plasmid pTargeT-PD-L1 was transfected into CHO-K1 cells (Shanghai cell biology institute of Chinese academy of sciences) at a voltage of 160V and a square pulse of 15msec by electrotransformation, and the cells were incubated at 37 ℃ and 5% CO 2 Cultured in an incubator. After 24h, the medium was pressure-cultured in DME/F12 complete medium containing 1000. mu.g/ml G418. After 16 days, the pool positive rate was measured by FACS, and the cell viability rate was calculated. Cells were incubated with PE anti-human PD-L1 antibody (nano Biological inc., 10084-R312-P), mean and parent% values were read with a flow cytometer (ACEA, Novocyte), and data analysis was performed using GraphPad generation. Positive pool cells with a cell viability of 90% or more were sub-clonally plated (cell density of 0.5/well, 200. mu.l/well) at 37 ℃ in 5% CO 2 Cultured in an incubator. After the subclones were grown for 12 days, the cloned CHO-K1 cell line was selected, FACS screened for PD-L1 high expression subclones, and designated hPD-L1-CHO-K1.
3) Expression of tag protein
The inoculation density in a 1L cell culture flask is 0.5X 10 6 293E cells per mL, fresh, pre-warmed FreeStyle293 expression medium to 250mL total volume after inoculation, 37 ℃, 8% CO 2 Humidified CO 2 The culture was carried out overnight in an incubator. Adding 1mg/mL PEI solution 500 mul into 8.5mL FreeStyle293 expression culture medium, mixing uniformly, adding 250 mul plasmid to be transfected into 8.5mL FreeStyle293 expression culture medium, mixing uniformly, wherein the labeled antigen protein plasmids pHR-PD-1-hFc, pHR-PD-1-mFc and pHR-PD-1-His are transfected respectively; the tag ligand protein plasmids pHR-PD-L1-hFc and pHR-PD-L1-mFc were transfected respectively. Adding the mixed solution of PEI and FreeStyle293 expression medium into the plasmid, mixing well, adding into cell culture, standing at 37 deg.C and 8% CO 2 Humidified CO 2 Culturing in an incubator. Cells were fed on days 1 and 3 after cell transfection, and 2.5ml of glutamine (mother liquor concentration of 200mM) and 5ml of glucose (mother liquor concentration of 180g/L) were added to each flask. When the cell viability is reduced to 65% -75%, collecting cell supernatant.The cell culture was centrifuged at 1500rpm for 5min and the supernatant was collected, and further centrifuged at 8000rpm for 20min and the supernatant was collected.
4) Affinity chromatography column purification
The protein was purified by using an affinity column using AKTA (GE, AKTA pure-150) according to its properties.
Example 11: preparation of anti-PD-1 monoclonal antibody
1. Preparation of hybridoma monoclonal
(1) Animal immunization
The experimental mice of C57 and SJL strains are immunized by a method of immunizing anti-PD-1 antigen proteins (PD-1-His, purchased from Sino Biological Inc., 10377-H08H), PD-1-hFc and PD-1-mFc) with different labels together with adjuvant, wherein 50 mu g of antigen is used for the first immunization, and 25 mu g of antigen is used for the later immunization; the SD strain experimental rat is immunized by adopting a method of co-immunizing anti-PD-1 antigen proteins with different labels and an adjuvant, wherein 100 mu g of antigen is used for the first time, and 50 mu g of antigen is used for the later period.
The immunoadjuvant may be Quick Antibody-Mouse5W (Beijing Boolong Immunol technologies, Inc.) or Titer Max (Sigma) interspersed with CpG (Kinsley Biotechnology limited Synthesis)/Alum (thermo) adjuvants. The PD-1 antigen protein samples with different labels are added into the adjuvant solution drop by drop and vortex to mix fully, and the adjuvant is carried out by using the dosage reference instruction. Mixing to form water-in-oil emulsion, and immunizing mouse and rat.
Cell lines expressing high levels of PD-1 molecules, such as hPD-1-CHO-K1, are also used to immunize rats to produce antibodies. Trypsinizing the hPD-1-CHO-K1 positive single cells obtained in example 1 under culture, centrifuging at 1000rpm for 5min, discarding the supernatant, resuspending the cell pellet in PBS, sampling and counting with a cell counter, centrifuging the remaining sample at 1000rpm for 5min, discarding the supernatant, resuspending the cell pellet in PBS, and adding an appropriate amount of PBS to obtain 1X10 8 Individual cells/ml of cell suspension. Experimental group mice were immunized 1X10 each 7 And (4) cells.
The immunization protocol is shown in tables 9 and 10:
TABLE 9 mouse immunization protocol
Figure BDA0003533158350000461
TABLE 10 rat immunization protocol
Figure BDA0003533158350000462
I.m. intramuscular injection; s.c. subcutaneous injection; i.p. intraperitoneal injection.
(2) Hybridoma fusion
Acquisition and preparation of splenocytes: the boosted mice/rats were sacrificed and soaked in 75% alcohol. Spleen is dissected and taken out, ground by a grinding rod and filtered by a cell screen to prepare single cell suspension. The spleen cell suspension was centrifuged at 2000rpm for 5min and the supernatant was discarded. Adding 2mL of erythrocyte lysate, lysing erythrocytes at room temperature for 2min, adding PBS to 20mL, centrifuging at 1500rpm for 7min, discarding supernatant, and counting viable cells after resuspension. Sp2/0 cells were collected from the flask, centrifuged at 1000rpm for 5min and the supernatant discarded, resuspended and then counted for viable cells. According to the ratio of splenocytes: sp2/0 cells were mixed at a ratio of 1.5:1, centrifuged at 1500rpm for 7min and the supernatant discarded. Cells were resuspended in 20mL of electrotransfer buffer and centrifuged at 1500rpm for 7 min. Discard the supernatant and repeat once. Resuspending the cells with appropriate amount of electrotransfer buffer to ensure cell concentration of 2X 10 7 About one cell/mL. The cell suspension was added to a 9mL electrofusion tank for fusion. After the fusion, the cell suspension was transferred to 15mL of H-SFM complete medium containing 20% FBS, and left at room temperature for 20 min. The fused cells were resuspended in H-SFM medium containing 1 XHAT, 1 XBIOYC-3, 2% FBS. The cell suspension was added to several 96-well cell culture plates at 100. mu.l/well to ensure a cell count of approximately 4X 10 per well 4 Each cell/well was cultured in a 37 ℃ cell incubator. After 5 days, the cells were supplemented with 100. mu.L/well of H-SFM complete medium (containing 2% FBS, 1 XHAT, 1 XBIOYC-3).
(3) Screening of hybridoma and subclone supernatants
Primary screening: one week after fusion, cell supernatants were taken and screened by ELISA for hybridoma supernatants that bind PD-1-His protein or cell surface PD-1, and PD-1-His was used to screen antibodies against PD-1 but not hFc, mFc. Hybridoma supernatants were then analyzed for the ability to block the interaction of PD-1 and PD-L1 by ELISA. The specific method comprises the following steps: PD-L1-mFc is coated on an enzyme label plate, a mixture of recombinant human protein PD-1-hFc and hybridoma supernatant is added for incubation for 2h, anti-human IgG Fc specific antibody (Jackson Immuno Research) marked by HRP is added for incubation for 1h, and the absorbance at 450nm is detected by an enzyme label instrument.
And (4) retesting: carrying out amplification culture on the screened hybridoma parent clones with the binding capacity and the blocking capacity, and carrying out retesting on the ELISA binding activity; screening hybridoma supernatants capable of binding to PD-1 on the surface of hPD-1-CHO-K1 cells by FACS; screening out hybridoma supernatant capable of combining cyno-PD-1-His protein by ELISA; hybridoma supernatants that crossed positively through three experiments were used as candidate positive clones.
And (3) carrying out subcloning on the positive cell strain by using a limiting dilution method, culturing for one week, detecting the binding activity of a subcloned supernatant and a PD-1 molecule and the activity of blocking the interaction of the PD-1 and the PD-L1 by using ELISA, and screening the double-positive cell strain. One of the anti-PD-1 monoclonal antibodies was labeled as SHS 006-P01.
2. Preparation of monoclonal antibodies
And (3) carrying out amplification culture on the monoclonal antibody parent clone strain. The culture condition is 1640 culture medium containing 10% fetal calf serum, 1 XNAEE, 1 Xsodium pyruvate and 1% streptomycin double antibody, when the cell confluence is more than 80%, the cells are subcultured and expanded, when the cells are cultured to about 50ml, the supernatant is collected, and the antibody is purified. The obtained antibody was confirmed to have good purity by SDS-PAGE gel electrophoresis.
3. Sequencing of monoclonal antibodies
The subcloned positive hybridoma cells were expanded, and a suitable amount of cells were used to extract total RNA according to the instructions of RNeasy Plus Mini Kit (Qiagen, 74134) Kit, and first strand cDNA was synthesized using Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription Kit.
Designing specific primer (5' end contains homologous arm sequence for homologous recombination with eukaryotic expression vector) according to rat antibody subtype variable region, and using cDNA as templateCarrying out PCR amplification on the antibody variable region gene so as to respectively obtain gene segments of the rat antibody light chain and heavy chain variable regions; primers were designed (reference: 1. like Krebber, Susanne Bornhauser, journal Burmester et al. Reliable cloning of functional anti-variant variants from a hybridoma and a spelen cell strains expression. journal of Immunological Methods 1997,201: 35-55; 2.Simon Korenmha
Figure BDA0003533158350000482
DNA sequencing was performed as a method for hybrid cell-line authentication,2008,78: 1071-. The sequencing results of SHS006-P01 are shown in Table 11.
TABLE 11 anti-PD-1 murine monoclonal antibody sequences
Figure BDA0003533158350000481
CDR sequences of VH of antibody SHS 006-P01: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 8, 9 and 10, and the CDR sequences of VL: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 11, 12 and 13 respectively.
Example 12: construction of anti-PD-1 chimeric antibody
Respectively co-transforming the purified mouse anti-light chain and heavy chain variable region gene fragments and linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant regions into escherichia coli DH5 alpha competent cells, uniformly coating the mixed solution on the surface of an agar plate containing corresponding antibiotics, and respectively picking a plurality of single colonies for DNA sequencing after overnight culture in a constant-temperature incubator at 37 ℃; the chimeric antibody with correct sequencing was labeled as SHS006-P01 CHI.
The heavy chain variable region and the light chain variable region of the chimeric antibody SHS006-P01CHI are identical to those of the murine antibody SHS 006-P01.
Inoculating the positive clone with correct sequencing into a2 XYT liquid culture medium containing corresponding antibiotics, carrying out shake culture at 37 ℃ for more than 12 hours, then collecting thalli and carrying out plasmid extraction, thereby obtaining chimeric antibody light chain and heavy chain expression plasmids, and detecting the concentration and purity of the plasmids by using a nucleic acid quantitative analyzer.
The chimeric antibody is transfected into HEK293E cells, and a large amount of antibody is obtained through expression and purification, and purity detection, activity analysis and affinity detection are carried out.
Example 13: construction and production of anti-PD-1 humanized antibody
According to the results of activity analysis, affinity and the like, a plurality of chimeric antibodies with good activity are selected for humanized antibody modification.
Firstly, comparing with a rat antibody sequence in an immune gene database (IMGT) to confirm the murine germline of the variable region of the SHS006-P01CHI antibody, and after homologous comparison, ensuring that the FR region of the heavy chain variable region sequence of the SHS006-P01CHI antibody is most similar to the rat antibody germline gene IGHV2-26 x 01; the FR sequence of the antibody light chain variable region most closely resembles the rat antibody IGKV2-28 x 01. Using the frame region sequence FR1-FR3 of the SHS006-P01CHI antibody as a template, searching a full-human frame with similar 3D structure but low immunogenicity in a human frame region library to replace the FR1-FR3 sequence of the SHS006-P01CHI, carrying out 3D modeling on the full-length heavy chain/light chain sequence, carrying out structural alignment analysis on the full-length heavy chain/light chain sequence and the original antibody heavy chain/light chain sequence, comprehensively considering antigenicity and 3D structural similarity, and carrying out back mutation on an amino acid site which plays a key role in stabilizing the antibody structure in structural simulation into a murine amino acid residue. Finally, 6 humanized heavy chain variable regions (see SEQ ID NOS: 45, 46, 47, 48, 49, 50) and 3 humanized light chain variable regions (see SEQ ID NOS: 51, 52, 53) of the SHS006-P01CHI were selected for further optimization. The sequence of the non-CDR region of the SHS006-P01CHI humanized antibody reaches over 95 percent of humanization.
Reverse transcribing the designed amino acid sequences of the light chain and the heavy chain variable region of the humanized antibody into corresponding nucleotide sequences, generating oligonucleotide fragments containing complementary sequences between adjacent fragments, annealing the oligonucleotide fragments through Overlap PCR, connecting, and amplifying complete nucleotide fragments of the light chain and the heavy chain variable region by using a specific primer (the 5' end contains a homologous arm sequence for homologous recombination with a eukaryotic expression vector); the purified light chain variable region nucleotide fragment and a linearized eukaryotic expression plasmid containing an IgG4 light chain constant region are co-transformed into an escherichia coli DH5 alpha competent cell, the purified heavy chain variable region nucleotide fragment and a eukaryotic expression plasmid containing an IgG4 heavy chain constant region with S228P/L235E mutation are co-transformed into an escherichia coli DH5 alpha competent cell, the competent cells of the transformation plasmid are respectively and uniformly coated on the surfaces of agar plates containing corresponding antibiotics, and a plurality of single colonies are respectively picked for DNA sequencing after overnight culture in a constant-temperature incubator at 37 ℃.
And (3) inoculating the positive clone with correct sequencing into a2 XYT liquid culture medium containing corresponding antibiotics, carrying out shake culture at 37 ℃ for more than 12 hours, then collecting thalli and carrying out plasmid extraction, thereby obtaining humanized antibody light chain and heavy chain expression plasmids, and detecting the concentration and purity of the plasmids by using a nucleic acid quantitative analyzer.
The plasmid is transfected into HEK293E cells, and a large amount of antibodies are obtained through expression and purification, and purity detection, activity analysis and affinity detection are carried out.
Selecting humanized antibodies with good purity, activity and affinity, wherein one of the humanized antibodies is marked as SHS006-HuP01-22 (abbreviated as HuP01-22) and the sequences are shown in Table 12.
TABLE 12 anti-PD-1 humanized antibody sequences
Figure BDA0003533158350000501
CDR sequences of the VH of antibody SHS006-HuP 01-22: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NO 8, 9 and 10, and the CDR sequences of VL: the sequences of CDR1, CDR2 and CDR3 are SEQ ID NOS: 11, 12 and 13, respectively.
Example 14: determination of binding Activity of anti-PD-1 antibody to monkey PD-1 (ELISA)
The binding activity of the antibodies was analyzed using Protein based ELISA. Cynomolgus monkey PD-1-His (0.1. mu.g/well, SB, Cat. No.90311-C08H) was coated on a 96-well plate and incubated overnight at 4 ℃. The anti-PD-1 antibody of the invention is used as a primary antibody, and is added into an enzyme label plate by 5-fold gradient dilution from 10 mu g/mL, and the total number of the primary antibody is 8The concentration of the mixture was 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, and 0.13ng/mL, respectively, and the mixture was incubated at 37 ℃ for 1.5 hours. The secondary antibody was treated with Anti-Human IgG HRP (Jackson, 109-. EC Generation Using GraphPad 50 。
EC of humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention 50 Is 0.088nM, with better binding ability to cynomolgus PD-1.
Example 15: determination of binding Activity of anti-PD-1 antibody to human PD-1 (ELISA)
The binding activity of the antibodies was analyzed by ELISA. Human PD-1-His protein (2. mu.g/ml, produced by oneself) was coated onto a 96-well microplate and incubated overnight at 4 ℃. After 3 washes with 1XPBST, they were blocked overnight at 4 ℃ with 5% skimmed milk. After washing 3 times by 1XPBST, the anti-PD-1 antibody of the invention is used as a primary antibody, and is diluted and added to an enzyme label plate by 5 times of gradient from 1 mug/mL, and the concentrations are respectively 1000ng/mL, 200ng/mL, 40ng/mL, 8ng/mL, 1.6ng/mL, 0.32ng/mL, 0.064ng/mL and 0ng/mL for 8 concentrations, and the antibody is Nivolumab at 37 ℃; after 3 washes with 1XPBST, the secondary antibody was incubated with Anti-Human IgG HRP (Jackson, 109-. After washing 5 times with 1XPBST, a color developing solution TMB was added, and after completion, the OD450 value was read by a microplate reader (thermo, Multiskan FC). Generation of EC Using GraphPad 50 。
EC in binding of humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention to human PD-1 50 Is 0.18nM, and has better binding ability to human PD-1.
Example 16: determination of binding Activity of anti-PD-1 antibody to cell surface PD-1 (FACS)
The binding activity of the antibody was analyzed by FACS. hPD-1-CHO-K1 cells at 1X10 per well 5 The anti-PD-1 antibody of the invention is added to the cell plate in a gradient dilution manner starting from 3.3. mu.g/mL as a primary antibody, and the concentrations are 3333ng/mL, 1111ng/mL, 370ng/mL, 123ng/mL, 41ng/mL, 13.7ng/mL, 4.57ng/mL and 1.52ng/mL respectively, and the concentration is 8The body is Nivolumab, and incubation is carried out for 1h at 37 ℃; washing the Cell stabilizing buffer for 3 times; the secondary antibody was incubated with PE anti-human IgG Fc (Biolegend, 409304, 0.8. mu.l/well) at 4 ℃ for 30min in the absence of light; after washing the Cell stabilizing buffer 3 times, the mean value at a wavelength of 585nm was measured by a flow cytometer (ACEABIO, Novocyte). Generation of EC Using GraphPad 50 。
EC in binding of humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention to cell surface PD-1 50 The molecular weight of the polypeptide is 0.11nM, and the polypeptide has better binding capacity with cell surface PD-1.
Example 17: determination of affinity of anti-PD-1 antibody to human PD-1 protein
The affinity of the humanized anti-PD-1 antibody prepared in the above example for binding to the antigen PD-1-His (Nano Biological, 10377-H08H) was determined using Fortebio Octet. The humanized anti-PD-1 antibody was diluted to a concentration of 10. mu.g/ml with SD buffer (PBS + 0.02% Tween20+ 0.1% BSA), the antigen PD-1-His was diluted with SD buffer at a 5-fold concentration gradient to a concentration of 5. mu.g/ml, 1. mu.g/ml, 0.2. mu.g/ml, 0. mu.g/ml, and the affinity was determined by using AHC sensor immobilized antibody according to the protocol of Fortebio Ocet RED96, with the specific parameters and experimental results shown in Table 13.
TABLE 13 determination of the affinity of anti-PD-1 antibodies to human PD-1 protein
Figure BDA0003533158350000511
Figure BDA0003533158350000521
The experimental result shows that the humanized anti-PD-1 antibody SHS006-HuP01-22 has better binding affinity with human PD-1 protein.
Example 18: detection of anti-PD-1 antibodies blocking the binding of PD-1 to PD-L1 (FACS)
The ability of the anti-PD-1 antibodies of the invention to block the binding of PD-1 to cell surface PD-L1 was examined using FACS. hPD-L1-CHO-K1 positive cell line as PD-L1 provider, anti-PD diluted in gradientThe binding ability of PD-1-hFc to PD-L1-CHO-K1 was observed in the presence of-1 antibody. The change in PD-1-hFc was monitored using PE coat anti-human IgG (Biolegend, 405307, 0.8. mu.l/well). Flow cytometer (ACEABIO, Novocyte) read mean value at 585nm wavelength and IC generation using GraphPad 50 。
Experimental results show that the humanized anti-PD-1 antibody SHS006-HuP01-22 of the invention blocks EC of human PD-1 binding to PD-L1 50 Is 0.77nM, and has better blocking ability.
Example 19: in vitro drug efficacy testing of anti-PD-1 antibodies
The in vitro efficacy of the anti-PD-1 antibody is detected by adopting a method of stimulating PBMC twice by SEB.
Resuscitating the PBMC according to the required cell amount, adding the PBMC into 8-9ml of IMDM complete culture medium, centrifuging at 1200rpm for 10min, and discarding the supernatant; resuspending with a proper amount of culture medium, counting with a hemocytometer, adding into a 6-well plate, simultaneously adding SEB solution with a final concentration of 100ng/ml, and incubating for 48 h; after 48h, centrifuging at 1200rpm for 10min, discarding the supernatant, washing 1-2 times by using IMDM complete culture medium, resuspending by using an appropriate amount of culture medium, counting by using a hemocytometer, resuspending to 1M/mL, and adding 100 mu L/hole into a 96-hole plate; nivolumab and Isotype were prepared in IMDM complete medium at 4 fold concentration (i.e., 40. mu.g/mL) at 50. mu.L/well, labeled, and vortexed; adding the antibody solution into the corresponding hole, adding 50 mu L/hole culture medium into a control group, placing a 96-hole plate in an incubator at 37 ℃, and incubating cells and the antibody for 1 h; after 1h, preparing SEB solution by using IMDM complete culture medium according to the dosage of 50 mu L/hole and the concentration of 4 times (400ng/ml), and adding the SEB solution into the corresponding hole; and placing the 96-well plate at 37 ℃, incubating in a 5% CO2 incubator for 72h, centrifuging to remove the supernatant, collecting 150 mu L of cell-free supernatant, diluting according to a certain proportion, and detecting the concentrations of IFN-gamma and IL-2 in the supernatant according to the specification of an hIFN-gamma (R & D system Cat: DY285B) and hIL-2(R & D system Cat: DY202) ELISA detection kit.
The experimental result shows that the SHS006-HuP01-22 has better IFN-gamma release promoting and IL-2 release promoting capabilities.
The present invention also detects the release of the chimeric antibody in vitro cytokine IL-2 using the luciferase reporter gene method (NFAT), and the results are shown in Table 14.
TABLE 14 in vitro cytokine Release from anti-PD-1 chimeric antibodies
Figure BDA0003533158350000531
The results show that SHS006-P01CHI is able to block PD-1 binding to PD-L1 well, leading to a downstream NFAT signaling response.
Example 20: construction of anti-CTLA-4/anti-PD-1 bispecific antibody expression vector and protein expression purification
The anti-PD-1 antibody used in the invention is a mouse monoclonal antibody SHS006-P01 (heavy chain variable region: SEQ ID NO:43, light chain variable region: SEQ ID NO: 44) obtained by screening after immunizing a mouse with human PD-1-his protein, and a humanized monoclonal antibody SHS006-HuP01-22 (heavy chain variable region: SEQ ID NO:46, light chain variable region: SEQ ID NO: 52) obtained by screening after humanization.
The anti-CTLA-4 antibody is a mouse monoclonal antibody obtained by screening a human CTLA-4-his protein immunized mouse, then a chimeric antibody SHS010-C92-CHI-G24E (the heavy chain variable region sequence is SEQ ID NO:28, and the light chain variable region sequence is SEQ ID NO:27) is obtained, and a humanized monoclonal antibody HuC92-11 (the heavy chain variable region sequence is SEQ ID NO:30, and the light chain variable region sequence is SEQ ID NO:32) is obtained by screening after the antibody is humanized.
The invention utilizes the variable region sequences of the humanized antibodies of the anti-PD-1 and the anti-CTLA-4 to construct the anti-CTLA-4/anti-PD-1 bispecific antibody. This bispecific antibody is a bispecific antibody comprising the symmetric structure of an scFv, as shown in figure 5. Specifically, the bispecific antibody of the present invention is prepared by linking the scFv of an anti-CTLA-4 antibody to the C-terminus of the entire anti-PD-1 antibody heavy chain via a flexible linker peptide comprising GGGGS repeats.
The bispecific antibody sequence of the invention has a schematic structure:
the structure of peptide chain 1is shown in FIG. 1;
the structure of peptide chain 2 is shown in fig. 2;
wherein:
anti-PD-1 antibody VH: is a humanized anti-PD-1 monoclonal antibody SHS006-HuP01-22 heavy chain variable region;
linker peptide 1: a flexible linker peptide which is a repeat sequence of 3 GGGGS;
anti-CTLA-4 antibody VH: is a humanized anti-CTLA-4 monoclonal antibody HuC92-11 heavy chain variable region;
linker peptide 2: a flexible linker peptide which is a repeat of 4 GGGGS;
anti-CTLA-4 antibody VL: is a humanized anti-CTLA-4 monoclonal antibody HuC92-11 light chain variable region;
anti-PD-1 antibody VL-CL: is a humanized anti-PD-1 monoclonal antibody SHS006-HuP01-22 light chain;
according to the above symmetrical structure, the variable region sequences of anti-PD-1 antibody SHS006-HuP01-22 and anti-CTLA-4 antibody SHS010-HuC92-11 were used to construct bispecific antibody ScFv- [ P01-22-C92-11], the amino acid sequence of peptide chain 1is shown as SEQ ID NO:55, and the amino acid sequence of peptide chain 2 is shown as SEQ ID NO: 54. In addition, bispecific antibody ScFv- [ P01-22-C92-12] is constructed, the amino acid sequence of the peptide chain 1is shown as SEQ ID NO:56, and the amino acid sequence of the peptide chain 2 is shown as SEQ ID NO: 54. The bispecific antibody ScFv- [ P01-22-C92-11] and ScFv- [ P01-22-C92-12] peptide chain 1 comprises CDR sequences: the sequences of H1CDR1, H1CDR2, H1CDR3, L1CDR1, L1CDR2, L1CDR3, H2CDR1, H2CDR2 and H2CDR3 are SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 and 10 respectively, and the peptide chain 2 comprises CDR sequences: the sequences of L2CDR1, L2CDR2 and L2CDR3 are SEQ ID NOS 11, 12 and 13 respectively.
Inoculating the positive clones of the constructed and sequenced double-antibody peptide chain 1 and peptide chain 2 into a2 XYT liquid culture medium containing corresponding antibiotics, carrying out shake culture at 37 ℃ for more than 12 hours, then collecting thalli and carrying out plasmid extraction, thereby obtaining humanized antibody light chain and heavy chain expression plasmids, and detecting the concentration and purity of the plasmids by using a nucleic acid quantitative analyzer.
The plasmid is transfected into HEK293E cells, and a large amount of antibodies are obtained through expression and purification, and purity detection, activity analysis and affinity detection are carried out. The bispecific antibody with better purity, activity and affinity was selected and labeled as BsAB0192-1 (i.e., antibody ScFv- [ P01-22-C92-11).
Example 21 binding Activity assay (ELISA) with human CTLA-4 and PD-1
The binding activity of the antibodies was analyzed by ELISA. Human CTLA-4-His protein (1. mu.g/well, prepared in examples 1 and 2) and PD-1-His protein (1. mu.g/well, available from Beijing Yiqiao Shen Biotechnology, cat # 50124-M08H) were coated onto 96-well microtiter plates and incubated overnight at 4 ℃. Washed 3 times with 1xPBST and then blocked with 5% skim milk at 37 ℃ for 2 h. After washing 3 times by using 1xPBST, the bispecific antibody provided by the invention is used as a primary antibody, and is diluted and added into an enzyme label plate from 10 mu g/mL in a 5-fold gradient manner, the concentrations of the bispecific antibody are 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL and 0.128ng/mL respectively, the bispecific antibody is incubated for 2 hours at 37 ℃, and the control antibodies are Iplilimumab and Nivolumab respectively; after washing 5 times with 1xPBST, the secondary antibody was incubated for 1h at 37 ℃ using Anti-Human IgG HRP (Jackson, 109-. After washing 5 times with 1xPBST, a color developing solution TMB was added, and after completion, the OD450 value was read with a microplate reader (thermo, Multiskan FC). Generation of EC Using GraphPad 50 The results are shown in FIGS. 6 and 7.
The experimental result shows that the bispecific antibody BsAB0192-1 has better binding capacity with the human CTLA-4 and the human PD-1.
Example 22 determination of the binding of antibodies to cell surface hTLA-4 and hPD-1 (FACS)
FACS was used to analyze the binding ability of the antibodies to CTLA-4 on the surface of CHO-K1-CTLA-4 and PD-1 on the surface of CHO-K1-PD-1. CHO-K1-CTLA-4 cells and CHO-K1-PD-1 cells were digested, resuspended in a solution of 2% FBS-PBS, and counted. The cells were plated at 1 × 10 per well 5 The bispecific antibody provided by the invention is used as a primary antibody, and is added into the cell plate by gradient dilution from 20 mu g/mL to total 8 concentrations which are 20000ng/mL, 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL and 0.64ng/mL respectively, the cell plate is incubated for 1h at 4 ℃, and the control antibodies are respectively illimumab and Nivolumab; the secondary antibody was PE-Anti-Human IgG (Biolegend, Cat. No.409303, 1.25. mu.l/well), washed, and then the fluorescence intensity of the antibody bound to the cell surface was measured by flow cytometry, and the secondary antibody was usedGraphPad Generation EC 50 The results are shown in FIGS. 8 and 9.
The experimental result shows that the bispecific antibody BsAB0192-1 has better binding capacity with cell surface CTLA-4 and PD-1.
Example 23 measurement of blocking Activity of antibodies against CTLA-4 and its ligands (FACS)
FACS analysis was used to analyze the ability of the antibodies to block binding of CTLA-4 to the ligand on the surface of CHO-K1-CTLA-4. CHO-K1-CTLA-4 cells were digested, resuspended in a solution of 2% FBS-PBS, and counted. The cells were plated at 1 × 10 per well 5 The bispecific antibody provided by the invention is used as a primary antibody, the primary antibody is added into the cell plate by gradient dilution from 20 mu g/mL, the concentration is 20000ng/mL, 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL and 0.64ng/mL, the diluted antibody is mixed with 1 mu g/mL CD80-mFc or 1 mu g/mL CD86-mFc 1:1, and then the cell plate is incubated for 1h at the temperature of 4 ℃, and the control antibody is Iplilimumab; the secondary antibody was washed with PE-Anti-mouse IgG (Biolegend, Cat. No.409303, 1.25. mu.l/well) and then the fluorescence intensity produced by binding of the ligand to the cell surface was measured by flow cytometry, and the results are shown in FIGS. 10 and 11.
The experimental result shows that the bispecific antibody BsAB0192-1 can better block the combination of CTLA-4 on the cell surface and the ligands CD80 and CD86 thereof.
Example 24 measurement of blocking Activity of antibodies against PD-1 and its ligands (FACS)
FACS was used to analyze the ability of the antibody to block the binding of PD-1 to the ligand on the surface of CHO-K1-PD-1. After digestion of CHO-K1-PD-1 cells, they were resuspended in a solution of 2% FBS-PBS and counted. The cells were plated at 1 × 10 per well 5 The bispecific antibody provided by the invention is used as a primary antibody, and is added into the cell plate by gradient dilution from 10 mu g/mL to total 8 concentrations of 10000ng/mL, 3333.33ng/mL, 1111.11ng/mL, 370.37ng/mL, 123.45ng/mL, 41.15ng/mL, 13.71ng/mL and 4.57ng/mL respectively, after the diluted antibody is mixed with 10 mu g/mL PD-L1-mFc 1:1, the mixture is incubated for 1h at 4 ℃, and the control antibody is Nivolumab; the secondary antibody used PE-Anti-mouse IgG (Biolegend, Cat)No.409303, 1.25 μ l/well), and the fluorescence intensity generated by binding of the ligand to the cell surface was measured using a flow cytometer after washing, and the results are shown in fig. 12.
The experimental result shows that the bispecific antibody BsAB0192-1 can better block the combination of PD-1 on the cell surface and the ligand PD-L1 thereof.
Example 25 blocking Activity of antibodies on cells co-expressing CTLA-4 and PD-1 (FACS)
The ability of bispecific antibodies to block binding of CTLA-4 to its ligand was determined using CHO-K1 cells co-expressing CTLA-4 and PD-1. After digestion of CHO-K1-PD-1-CTLA-4 cells, the cells were resuspended in a solution of 2% FBS-PBS and counted. The cells were plated at 1 × 10 per well 5 The bispecific antibody provided by the invention is used as a primary antibody, the primary antibody is added into the cell plate by gradient dilution from 20 mu g/mL, the concentration is 20000ng/mL, 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL and 0.64ng/mL, the diluted antibody is mixed with 1 mu g/mL CD80-mFc or 1 mu g/mL CD86-mFc 1:1, and then the cell plate is incubated for 1h at the temperature of 4 ℃, and the control antibody is Iplilimumab; the secondary antibody was washed with PE-Anti-mouse IgG (Biolegend, Cat. No.409303, 1.25. mu.l/well) and then the fluorescence intensity of the binding of the ligand to the cell surface was measured by flow cytometry, and the results are shown in FIGS. 13 and 14.
The ability of bispecific antibodies to block binding of PD-1 to its ligand was determined using CHO-K1 cells co-expressing CTLA-4 and PD-1. After CHO-K1-PD-1-CTLA-4 cells were digested, the cells were treated at 1X10 per well 5 The cell plate is paved in a way of individual cells, the bispecific antibody of the invention is used as a primary antibody, the primary antibody is added into the cell plate by gradient dilution, the total concentration is 8, the concentration is 10000ng/mL, 3333.33ng/mL, 1111.11ng/mL, 370.37ng/mL, 123.45ng/mL, 41.15ng/mL, 13.71ng/mL and 4.57ng/mL respectively, after the diluted antibody is mixed with 10 μ g/mL PD-L1-mFc 1:1, the mixture is incubated for 1h at the temperature of 4 ℃, and the contrast antibody is Nivolumab; the secondary antibody was washed with PE-Anti-mouse IgG (Biolegend, Cat. No.409303, 1.25. mu.l/well) and then the fluorescence intensity produced by binding of the ligand to the cell surface was measured by flow cytometry, and the results are shown in FIG. 15.
The experimental result shows that the bispecific antibody BsAB0192-1 can better block the binding capacity of CTLA-4 and PD-1 and ligands thereof on bistable transfected cell strains, and the blocking activity is superior to that of corresponding monoclonal antibodies HuC92-11 and HuP01-22, and is superior to that of control antibodies Iplilimumab and Nivolumab.
Example 26 bispecific antibody affinity assay with human CTLA-4 and human PD-1
The affinity of the obtained bispecific antibody binding to the antigens CTLA-4-His and PD-1-His was determined by Fortebio Octet. The antibody was diluted to 10. mu.g/ml using SD buffer PBS + 0.02% Tween20+ 0.1% BSA, and then immobilized using AHC sensor, and antigens (CTLA-4-His, PD-1-His) were prepared at three different concentrations of 1.5. mu.g/ml, 0.3. mu.g/ml, and 0.06. mu.g/ml using SD buffer, and the control antibodies were Iplilimumab and Nivolumab, and affinity measurements were performed according to the protocol of fortio Octet RED96, and the specific parameters and experimental results are shown in Table 15.
TABLE 15 measurement results of the affinity of the antibody for CTLA-4 and PD-1
Figure BDA0003533158350000571
The experimental results show that the bispecific antibodies of the invention bind to human CTLA-4 and human PD-1 proteins with higher affinity than the control antibodies.
Example 27 MC38 antitumor experiment in transplantation tumor model
1. Experimental Material
(1) Test cells and animals
MC38 cells were purchased from the American Type Culture Collection (ATCC);
B6-hPD 1/hTLA-4 mice, female, 6-8 weeks old, weighing 18-20 g, purchased from Nanjing Collection drug Kangji Biotech;
(2) test and reference substances
Control Iplilimumab, purchased from Beijing Yi Qiao Shen Biotech (cat # 68052-H001), was used as a positive control; a control Nivolumab, purchased from Bristol-Myers Squibb Company, was used as a positive control; before the test, the PD-1 antibody SHS006-P01-22 of the present invention was formulated with PBS to 1 mg/mL.
2. Experimental methods
MC38 transplantation tumor model
Inoculating human tumor cell MC38 to the right side of mouse, wherein the number of inoculated cells is 5 × 10 6 A/only. When the tumor grows to the average volume of 140mm 3 Grouping is started, the tail vein administration is carried out 2 times per week, the tumor diameter is measured twice per week by using a vernier caliper, and the tumor volume is calculated according to the formula: v is 0.5a × b 2 And a and b represent the major and minor diameters of the tumor, respectively, and the relative tumor proliferation rate (T/C) was calculated. Tumor volume measured for 24 days following dosing was recorded and tumor volume growth curves were plotted using GraphPad Prism, with the results shown in table 16.
TABLE 16 MC38 results of antitumor test in transplanted tumor model
Figure BDA0003533158350000581
Experimental results show that the anti-PD-1 antibody can better inhibit tumor growth.
Example 28 human lymphoma Raji-PDL1 transplantation tumor model anti-tumor test
1. Experimental Material
(1) Test cells and animals
Raji human lymphoma cells were purchased from Beijing Bai Biotech, Inc. The Raji-PDL1 cell line was constructed by a conventional method.
NOD-Scid mice, female, 5-8 weeks old, weighing 18-20 g, purchased from Baiosai Tou, GenBank Biotechnology Ltd;
(2) test and reference substances
The Nivolumab antibody was purchased from Baishi Mei Shi Guibao, and the ipilimumab was purchased from Beijing Yi Qiao Shenzhou science and technology, Inc.
Before the experiment, the anti-CTLA-4/PD-1 double-antibody BsAB0192-1 is prepared into 2.66mg/mL by PBS, the Nivolumab and the ipilimumab are prepared into 2mg/mL by PBS, and the mixture is stored at 4 ℃.
(3) Experimental methods
RMPI1640 medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin was used at 37 ℃ with 5% CO 2 Culturing human lymphoma cells Raji-PDL1 in the incubator. When the saturation degree of the cells is 80% -90%, collecting the cells, counting and inoculating. The right rear edge of the mice was inoculated with 50. mu.l of PBS containing 5X 106 cells in a final volume of 100. mu.l mixed with 50. mu.l of Matrigel, at a cell count of 5X 106/mouse. When the tumor grows to reach the volume of 100mm 3 Grouping is started, the abdominal cavity is administrated 3 times a week, the tumor diameter is measured by a vernier caliper three times a week, the tumor volume is calculated, and the calculation formula of the tumor volume is as follows: v is 0.5a × b 2 And a and b represent the major and minor diameters of the tumor, respectively. The tumor suppressor therapeutic effect of the antibody was evaluated relative to the tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C% ═ TRTV/CRTV × 100% (TRTV: treatment RTV; CRTV: negative control RTV). RTV-V21/V0, where V0 is the tumor volume measured at the time of group administration (i.e., d0) and V25 is the tumor volume measured 25 days after administration. Tumor volumes of the last Day (Day 25) of dosing and vehicle groups were analyzed using T-test and GraphPad Prism. The results are shown in Table 17.
TABLE 17 results of antitumor test of human lymphoma Raji-PDL1 transplanted tumor model
Figure BDA0003533158350000591
The four groups, Nivolumab 20mg/kg, Ipiumumab 20mg/kg, Nivolumab and Ipiumumab 10mg/kg +10mg/kg, and BsAB 0192-126.6 mg/kg, had the same molarity. Experimental results show that the double-antibody BsAB0192-1 has a better effect of inhibiting tumor growth under the same molar concentration, and the effect is better than that of the combined administration of Nivolumab, Iplimumab and Nivolumab and Iplimumab.
Although the present invention has been described in detail above, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not to be limited by the above detailed description but is only limited by the claims.
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Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val
100 105 110
Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile
115 120 125
Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly
130 135 140
Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser
145 150 155 160
Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe
165 170 175
Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys
180 185 190
Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu
195 200 205
Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn Lys
210 215 220
Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr
225 230 235 240
Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
245 250 255
Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val
260 265 270
His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser
275 280 285
Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys
290 295 300
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu
305 310 315 320
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
325 330 335
Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
340 345 350
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
355 360 365
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
370 375 380
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
385 390 395 400
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
405 410 415
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
420 425 430
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
435 440 445
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
450 455 460
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
465 470 475 480
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
485 490 495
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
500 505 510
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
515 520 525
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
530 535 540
Ser Phe Ser Arg Thr Pro Gly Lys
545 550
<210> 19
<211> 455
<212> PRT
<213> Artificial
<220>
<223> CTLA-4-hIgG1-Fc fusion protein
<400> 19
Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Arg Leu Asn Leu Ala
1 5 10 15
Thr Arg Thr Arg Pro Tyr Thr Leu Leu Phe Ser Leu Leu Phe Ile Pro
20 25 30
Val Phe Ser Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala
35 40 45
Asn Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly
50 55 60
Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln
65 70 75 80
Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr
85 90 95
Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val
100 105 110
Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile
115 120 125
Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Met Gly Ile Gly
130 135 140
Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser
145 150 155 160
Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe
165 170 175
Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys
180 185 190
Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu
195 200 205
Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn Glu
210 215 220
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 20
<211> 288
<212> PRT
<213> Artificial
<220>
<223> CD80 protein extracellular domain
<400> 20
Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr
1 5 10 15
Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys
20 25 30
Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu
35 40 45
Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile
50 55 60
Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp
65 70 75 80
Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr
85 90 95
Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly
100 105 110
Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg
115 120 125
Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr
130 135 140
Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile
145 150 155 160
Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu
165 170 175
Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp
180 185 190
Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met
195 200 205
Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg
210 215 220
Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro
225 230 235 240
Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly
245 250 255
Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg
260 265 270
Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val
275 280 285
<210> 21
<211> 329
<212> PRT
<213> Artificial
<220>
<223> CD86 protein extracellular domain
<400> 21
Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met
1 5 10 15
Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe
20 25 30
Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln
35 40 45
Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val
50 55 60
Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser
65 70 75 80
Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg
85 90 95
Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile
100 105 110
His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser
115 120 125
Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile
130 135 140
Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile
145 150 155 160
His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys
165 170 175
Asn Ser Thr Ile Glu Tyr Asp Gly Val Met Gln Lys Ser Gln Asp Asn
180 185 190
Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro
195 200 205
Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys
210 215 220
Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln
225 230 235 240
Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val
245 250 255
Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys
260 265 270
Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu
275 280 285
Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro
290 295 300
Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser
305 310 315 320
Ser Cys Asp Lys Ser Asp Thr Cys Phe
325
<210> 22
<211> 617
<212> PRT
<213> Artificial
<220>
<223> CD80-mIgG1-Fc fusion protein
<400> 22
Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr
1 5 10 15
Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys
20 25 30
Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu
35 40 45
Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile
50 55 60
Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp
65 70 75 80
Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr
85 90 95
Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly
100 105 110
Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg
115 120 125
Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr
130 135 140
Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile
145 150 155 160
Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu
165 170 175
Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp
180 185 190
Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met
195 200 205
Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg
210 215 220
Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro
225 230 235 240
Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly
245 250 255
Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg
260 265 270
Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val
275 280 285
Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp
290 295 300
Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe
305 310 315 320
Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly
325 330 335
Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser
340 345 350
Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr
355 360 365
Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
370 375 380
Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro
385 390 395 400
Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
405 410 415
Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
420 425 430
Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
435 440 445
Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
450 455 460
Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His
465 470 475 480
Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
485 490 495
Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser
500 505 510
Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met
515 520 525
Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro
530 535 540
Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn
545 550 555 560
Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
565 570 575
Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
580 585 590
Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr
595 600 605
Lys Ser Phe Ser Arg Thr Pro Gly Lys
610 615
<210> 23
<211> 658
<212> PRT
<213> Artificial
<220>
<223> CD86-mIgG1-Fc fusion protein
<400> 23
Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met
1 5 10 15
Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe
20 25 30
Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln
35 40 45
Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val
50 55 60
Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser
65 70 75 80
Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg
85 90 95
Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile
100 105 110
His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser
115 120 125
Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile
130 135 140
Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile
145 150 155 160
His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys
165 170 175
Asn Ser Thr Ile Glu Tyr Asp Gly Val Met Gln Lys Ser Gln Asp Asn
180 185 190
Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro
195 200 205
Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys
210 215 220
Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln
225 230 235 240
Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val
245 250 255
Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys
260 265 270
Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu
275 280 285
Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro
290 295 300
Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser
305 310 315 320
Ser Cys Asp Lys Ser Asp Thr Cys Phe Lys Thr Thr Ala Pro Ser Val
325 330 335
Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr
340 345 350
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr
355 360 365
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
370 375 380
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser
385 390 395 400
Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala
405 410 415
Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile
420 425 430
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly
435 440 445
Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile
450 455 460
Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp
465 470 475 480
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
485 490 495
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg
500 505 510
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
515 520 525
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
530 535 540
Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
545 550 555 560
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
565 570 575
Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp
580 585 590
Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
595 600 605
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu
610 615 620
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His
625 630 635 640
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro
645 650 655
Gly Lys
<210> 24
<211> 520
<212> PRT
<213> Artificial
<220>
<223> CD80-hIgG1-Fc fusion protein
<400> 24
Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr
1 5 10 15
Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys
20 25 30
Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu
35 40 45
Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile
50 55 60
Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp
65 70 75 80
Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr
85 90 95
Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly
100 105 110
Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg
115 120 125
Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr
130 135 140
Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile
145 150 155 160
Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu
165 170 175
Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp
180 185 190
Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met
195 200 205
Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg
210 215 220
Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro
225 230 235 240
Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly
245 250 255
Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg
260 265 270
Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val
275 280 285
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
290 295 300
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
305 310 315 320
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
325 330 335
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
340 345 350
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
355 360 365
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
370 375 380
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
385 390 395 400
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
405 410 415
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
420 425 430
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
435 440 445
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
450 455 460
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
465 470 475 480
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
485 490 495
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
500 505 510
Ser Leu Ser Leu Ser Pro Gly Lys
515 520
<210> 25
<211> 561
<212> PRT
<213> Artificial
<220>
<223> CD86-hIgG1-Fc fusion protein
<400> 25
Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met
1 5 10 15
Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe
20 25 30
Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln
35 40 45
Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val
50 55 60
Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser
65 70 75 80
Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg
85 90 95
Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile
100 105 110
His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser
115 120 125
Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile
130 135 140
Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile
145 150 155 160
His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys
165 170 175
Asn Ser Thr Ile Glu Tyr Asp Gly Val Met Gln Lys Ser Gln Asp Asn
180 185 190
Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro
195 200 205
Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys
210 215 220
Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln
225 230 235 240
Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val
245 250 255
Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys
260 265 270
Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu
275 280 285
Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro
290 295 300
Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser
305 310 315 320
Ser Cys Asp Lys Ser Asp Thr Cys Phe Glu Pro Lys Ser Ser Asp Lys
325 330 335
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
340 345 350
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
355 360 365
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
370 375 380
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
385 390 395 400
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
405 410 415
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
420 425 430
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
435 440 445
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
450 455 460
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
465 470 475 480
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
485 490 495
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
500 505 510
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
515 520 525
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
530 535 540
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
545 550 555 560
Lys
<210> 26
<211> 118
<212> PRT
<213> mouse source (Mus musculus)
<220>
<223> CTLA-4 heavy chain variable region
<400> 26
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Tyr Ser Gly Thr Tyr Ser Tyr His Ser Asp Thr Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Glu Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Arg Asp Tyr Asp Asn Gly Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 27
<211> 107
<212> PRT
<213> mouse source (Mus musculus)
<220>
<223> CTLA-4 light chain variable region
<400> 27
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Val Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Val Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Lys Arg Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 28
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CTLA-4 heavy chain variable region
<400> 28
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Tyr Ser Gly Thr Tyr Ser Tyr His Ser Asp Thr Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Glu Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Arg Asp Tyr Asp Asn Glu Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 29
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 heavy chain variable region
<400> 29
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Tyr Ser Gly Thr Tyr Ser Tyr His Ser Asp Thr Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Asp Asn Gly Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 30
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 heavy chain variable region
<400> 30
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Tyr Ser Gly Thr Tyr Ser Tyr His Ser Asp Thr Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Arg Asp Tyr Asp Asn Glu Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 31
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 light chain variable region
<400> 31
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Val Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Lys Arg Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 32
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 light chain variable region
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Val Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Lys Arg Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 33
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 light chain variable region
<400> 33
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Val Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Val Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Lys Arg Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 34
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 light chain variable region
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Val Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Val Lys Pro Gly Lys Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Lys Arg Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 35
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized CTLA-4 light chain variable region
<400> 35
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Val Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Val Lys Pro Gly Lys Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Lys Arg Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 36
<211> 525
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-1-hIgG1-Fc fusion protein
<400> 36
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
Ile Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys
290 295 300
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
305 310 315 320
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
325 330 335
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
340 345 350
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
355 360 365
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
370 375 380
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
385 390 395 400
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
405 410 415
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
420 425 430
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
435 440 445
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
450 455 460
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
465 470 475 480
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
485 490 495
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
500 505 510
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
515 520 525
<210> 37
<211> 760
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-1-mIgG1-Fc fusion protein
<400> 37
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
Phe Ser Pro Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys
290 295 300
Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe
305 310 315 320
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val
325 330 335
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
340 345 350
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
355 360 365
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
370 375 380
Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val
385 390 395 400
Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro
405 410 415
Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
420 425 430
Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp
435 440 445
Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr
450 455 460
Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser
465 470 475 480
Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu
485 490 495
Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His
500 505 510
His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys Phe Ser Pro Glu
515 520 525
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
530 535 540
Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
545 550 555 560
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
565 570 575
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
580 585 590
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
595 600 605
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
610 615 620
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
625 630 635 640
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
645 650 655
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
660 665 670
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
675 680 685
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
690 695 700
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
705 710 715 720
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
725 730 735
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
740 745 750
Ser Phe Ser Arg Thr Pro Gly Lys
755 760
<210> 38
<211> 294
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-1-his fusion protein
<400> 38
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
His His His His His His
290
<210> 39
<211> 527
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-L1-hIgG1-Fc fusion protein
<400> 39
Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu
1 5 10 15
Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu
35 40 45
Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val
50 55 60
Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn
65 70 75 80
Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu
115 120 125
Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140
Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro
145 150 155 160
Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly
165 170 175
Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val
180 185 190
Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys
195 200 205
Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile
210 215 220
Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp
225 230 235 240
Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val
245 250 255
Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys
260 265 270
Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu
275 280 285
Glu Thr Ile Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys Thr His
290 295 300
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
305 310 315 320
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
325 330 335
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
340 345 350
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
355 360 365
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
370 375 380
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
385 390 395 400
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
405 410 415
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
420 425 430
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
435 440 445
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
450 455 460
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
465 470 475 480
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
485 490 495
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
500 505 510
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
515 520 525
<210> 40
<211> 526
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-L1-mIgG1-Fc fusion protein
<400> 40
Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu
1 5 10 15
Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu
35 40 45
Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val
50 55 60
Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn
65 70 75 80
Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu
115 120 125
Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140
Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro
145 150 155 160
Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly
165 170 175
Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val
180 185 190
Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys
195 200 205
Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile
210 215 220
Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp
225 230 235 240
Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val
245 250 255
Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys
260 265 270
Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu
275 280 285
Glu Thr Phe Ser Pro Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro
290 295 300
Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe
305 310 315 320
Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro
325 330 335
Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
340 345 350
Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr
355 360 365
Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
370 375 380
Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
385 390 395 400
Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser
405 410 415
Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro
420 425 430
Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val
435 440 445
Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly
450 455 460
Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp
465 470 475 480
Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp
485 490 495
Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His
500 505 510
Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
515 520 525
<210> 41
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-1 protein
<400> 41
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 42
<211> 290
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PD-L1 protein
<400> 42
Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu
1 5 10 15
Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu
35 40 45
Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val
50 55 60
Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn
65 70 75 80
Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu
115 120 125
Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140
Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro
145 150 155 160
Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly
165 170 175
Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val
180 185 190
Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys
195 200 205
Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile
210 215 220
Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp
225 230 235 240
Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val
245 250 255
Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys
260 265 270
Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu
275 280 285
Glu Thr
290
<210> 43
<211> 117
<212> PRT
<213> rat source (Mus musculus)
<220>
<223> PD-1 heavy chain variable region
<400> 43
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Ile Lys Gln Thr Thr Gly Gln Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Pro Asp Asp Ser Ala Ala Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser
115
<210> 44
<211> 113
<212> PRT
<213> rat source (Mus musculus)
<220>
<223> PD-1 light chain variable region
<400> 44
Asp Ile Val Met Ala Gln Thr Pro Ser Ser Gln Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser
20 25 30
Glu Asn Lys Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Phe Gly Val
50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Thr Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Glu Gln
85 90 95
Tyr Tyr Asp Ile Leu Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 45
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 heavy chain variable region
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 46
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 heavy chain variable region
<400> 46
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 47
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 heavy chain variable region
<400> 47
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 48
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 heavy chain variable region
<400> 48
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 49
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 heavy chain variable region
<400> 49
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 50
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 heavy chain variable region
<400> 50
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 51
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 light chain variable region
<400> 51
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser
20 25 30
Glu Asn Lys Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Phe Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Glu Gln
85 90 95
Tyr Tyr Asp Ile Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 52
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 light chain variable region
<400> 52
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser
20 25 30
Glu Asn Lys Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Phe Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Glu Gln
85 90 95
Tyr Tyr Asp Ile Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 53
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Humanized PD-1 light chain variable region
<400> 53
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser
20 25 30
Glu Asn Lys Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Phe Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Glu Gln
85 90 95
Tyr Tyr Asp Ile Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 54
<211> 220
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Peptide chain 2 of CTLA-4/PD-1 bispecific antibody
<400> 54
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser
20 25 30
Glu Asn Lys Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Phe Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Glu Gln
85 90 95
Tyr Tyr Asp Ile Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 55
<211> 704
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Peptide chain 1 of CTLA-4/PD-1 bispecific antibody
<400> 55
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val
450 455 460
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser
465 470 475 480
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser Trp Val
485 490 495
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Glu Ile Ser Tyr
500 505 510
Ser Gly Thr Tyr Ser Tyr His Ser Asp Thr Val Thr Gly Arg Phe Thr
515 520 525
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser
530 535 540
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Arg Asp Tyr
545 550 555 560
Asp Asn Glu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
565 570 575
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
595 600 605
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln
610 615 620
Val Ile Thr Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
625 630 635 640
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
645 650 655
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
660 665 670
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
675 680 685
Gly Lys Arg Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
690 695 700
<210> 56
<211> 704
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Peptide chain 1 of CTLA-4/PD-1 bispecific antibody
<400> 56
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Trp Tyr
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Asn Thr Arg Ser Gly Asp Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Gly Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val
450 455 460
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser
465 470 475 480
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser Trp Val
485 490 495
Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ala Glu Ile Ser Tyr
500 505 510
Ser Gly Thr Tyr Ser Tyr His Ser Asp Thr Val Thr Gly Arg Phe Thr
515 520 525
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser
530 535 540
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Arg Asp Tyr
545 550 555 560
Asp Asn Glu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
565 570 575
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
595 600 605
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln
610 615 620
Val Ile Thr Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
625 630 635 640
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
645 650 655
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
660 665 670
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
675 680 685
Gly Lys Arg Pro Pro Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys
690 695 700

Claims (18)

1.An anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H1CDR1, H1CDR2, and H1CDR3 selected from the group consisting of:
(a1) 1, 2 and 3 as shown in SEQ ID NO;
(a2) amino acid sequences as shown in SEQ ID NO 1, 2 and 4; and
(a3) an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NO 1, 2 and 3 or SEQ ID NO 1, 2 and 4; and
(2) the light chain variable region comprises an L1CDR1, an L1CDR2, and an L1CDR3 selected from the group consisting of:
(a4) amino acid sequences as shown in SEQ ID NO 5, 6 and 7; and
(a5) amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NO 5, 6 and 7.
2. The anti-CTLA-4 antibody or antigen-binding fragment thereof of claim 1, having:
the H1CDR1, H1CDR2 and H1CDR3 are heavy chain variable regions of SEQ ID NOs 1, 2 and 3 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2 and 3, respectively, and the L1CDR1, L1CDR2 and L1CDR3 are light chain variable regions of SEQ ID NOs 5, 6 and 7 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6 and 7, respectively; or
The H1CDR1, H1CDR2 and H1CDR3 are heavy chain variable regions of SEQ ID NOs 1, 2 and 4 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2 and 4, respectively, and the L1CDR1, L1CDR2 and L1CDR3 are light chain variable regions of SEQ ID NOs 5, 6 and 7 or amino acid sequences having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6 and 7, respectively.
3. The anti-CTLA-4 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(b1) the amino acid sequence shown as SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30;
(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) an amino acid sequence shown as SEQ ID NO 27, 31, 32, 33, 34 and 35;
(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).
4. The anti-CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein
The amino acid sequence of the heavy chain variable region is SEQ ID NO 26, the amino acid sequence of the SEQ ID NO 26, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 26 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 26, the amino acid sequence of the light chain variable region is SEQ ID NO 27, the amino acid sequence of the SEQ ID NO 27, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 27 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 27;
the amino acid sequence of the heavy chain variable region is SEQ ID NO 28, the amino acid sequence of the SEQ ID NO 28, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 28 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 28, the amino acid sequence of the light chain variable region is SEQ ID NO 27, the amino acid sequence of the SEQ ID NO 27, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 27 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 27;
the amino acid sequence of the heavy chain variable region is SEQ ID NO:29, the amino acid sequence of the SEQ ID NO:29 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:29 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO:29, the amino acid sequence of the light chain variable region is SEQ ID NO:32, the amino acid sequence of the SEQ ID NO:32 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:32 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO: 32; or
The amino acid sequence of the heavy chain variable region is SEQ ID NO 30, the amino acid sequence of the SEQ ID NO 30 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 30 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 30, the amino acid sequence of the light chain variable region is SEQ ID NO 32, and the amino acid sequence of the SEQ ID NO 32 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 32 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 32.
5. An anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof, wherein:
the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H1CDR1, H1CDR2, and H1CDR3 selected from the group consisting of:
(a1) amino acid sequences as shown in SEQ ID NO 1, 2 and 3;
(a2) amino acid sequences as shown in SEQ ID NO 1, 2 and 4; and
(a3) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a1) or (a 2); and
(2) the light chain variable region comprises an L1CDR1, an L1CDR2, and an L1CDR3 selected from the group consisting of:
(a4) amino acid sequences as shown in SEQ ID NO 5, 6 and 7;
(a5) amino acid sequences as shown in SEQ ID NO 5, 6 and 7; and
(a6) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a4) or (a 5); and
the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the heavy chain variable region comprises a H2CDR1, H2CDR2, and H2CDR3 selected from the group consisting of:
(A1) amino acid sequences as shown in SEQ ID NO 8, 9 and 10;
(A2) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (a 1); and
(2) the light chain variable region comprises an L2CDR1, an L2CDR2, and an L2CDR3 selected from the group consisting of:
(A3) amino acid sequences as shown in SEQ ID NO 11, 12 and 13;
(A4) an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in (A3).
6. The anti-CTLA-4/anti-PD-1 antibody of claim 5, wherein:
the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the H1CDR1, H1CDR2, and H1CDR3 of SEQ ID NOs 1, 2, and 3, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2, and 3, and a light chain variable region of the L1CDR1, L1CDR2, and L1CDR3 of SEQ ID NOs 5, 6, and 7, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 5, 6, and 7; or
The anti-CTLA-4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region in which the H1CDR1, H1CDR2, and H1CDR3 are SEQ ID NOS: 1, 2, and 4, respectively, or have an amino acid sequence with at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOS: 1, 2, and 4, and a light chain variable region in which the L1CDR1, L1CDR2, and L1CDR3 are SEQ ID NOS: 5, 6, and 7, respectively, or have an amino acid sequence with at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOS: 5, 6, and 7.
7. The anti-CTLA-4/anti-PD-1 antibody of claim 5 or 6, wherein:
the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(b1) the amino acid sequences shown as SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30;
(b2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1) and has the same or similar functions with the amino acid sequence shown in (b 1); and
(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(b4) an amino acid sequence shown as SEQ ID NO 27, 31, 32, 33, 34 and 35;
(b5) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4) and has the same or similar function with the amino acid sequence shown in (b 4); and
(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4); and
the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
(1) the amino acid sequence of the heavy chain variable region is selected from:
(B1) an amino acid sequence shown as SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50;
(B2) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (B1) and has the same or similar function with the amino acid sequence shown in (B1); and
(B3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (B1); and
(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:
(B4) an amino acid sequence shown as SEQ ID NO. 44, SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53;
(B5) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (B4) and has the same or similar function with the amino acid sequence shown in (B4); and
(B6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (B4).
8. The anti-CTLA-4/anti-PD-1 antibody of any one of claims 5 to 7, wherein:
the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is SEQ ID NO:30, the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is obtained by substituting, deleting or adding one or more amino acids in the SEQ ID NO:30 and has at least 85% of sequence identity with the SEQ ID NO:30, the amino acid sequences of the H1CDR1, the H1CDR2 and the H1CDR3 are shown as SEQ ID NO:1, 2 and 4, the amino acid sequence of the light chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is shown as SEQ ID NO:32, the amino acid sequence of the SEQ ID NO:32 is obtained by substituting, deleting or adding one or more amino acids in the SEQ ID NO:32 and has at least 85% of sequence identity with the SEQ ID NO:32, and the amino acid sequences of the L1CDR1, L1CDR2 and L1CDR3 are shown as SEQ ID NO:5, 5, 6 and 7; or
The amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is SEQ ID NO 28, the amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is obtained by substituting, deleting or adding one or more amino acids in the SEQ ID NO 28 and has at least 85% of sequence identity with the SEQ ID NO 28, the H1CDR1, the H1CDR2 and the H1CDR3 are the amino acid sequences shown in SEQ ID NO 1, 2 and 4, the amino acid sequence of the light chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is SEQ ID NO 27, the amino acid sequence of the SEQ ID NO 27 is obtained by substituting, deleting or adding one or more amino acids in the light chain variable region of the anti-CTLA-4 antibody or the antigen binding fragment thereof is the amino acid sequence of the L1CDR1, L1CDR2 and L1CDR3 are the amino acid sequences shown in SEQ ID NO 5,5, 6 and 7.
9. The anti-CTLA-4/anti-PD-1 antibody of any one of claims 5 to 8, wherein:
the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO 43, the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the SEQ ID NO 43 and has the same function with the SEQ ID NO 43 or has at least 85% of sequence identity with the SEQ ID NO 43, the H2CDR1, the H2CDR2 and the H2CDR3 are the amino acid sequences shown in SEQ ID NO 8, 9 and 10, the amino acid sequence of the light chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO 44, the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the SEQ ID NO 44 and has the same function with the SEQ ID NO 44 or has at least 85% of sequence identity with the SEQ ID NO 44, the L2CDR1, L2CDR2 and L2CDR3 are the amino acid sequences shown in SEQ ID NO 11, 12 and 13; or
The amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO. 46, the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 46 and has the same function with the SEQ ID NO. 46 or has at least 85% of sequence identity with the SEQ ID NO. 46, the H2CDR1, the H2CDR2 and the H2CDR3 are the amino acid sequences shown in SEQ ID NO. 8, 9 and 10, the amino acid sequence of the light chain variable region of the anti-PD-1 antibody or the antigen binding fragment thereof is the amino acid sequence shown in SEQ ID NO. 52, the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 52 and has the same function with the SEQ ID NO. 52 or has at least 85% of sequence identity with the SEQ ID NO. 52, the L2CDR1, L2CDR2 and L2CDR3 are the amino acid sequences shown in SEQ ID NO. 11, SEQ ID NO. 11, 12 and 13.
10. The anti-CTLA-4/anti-PD-1 antibody of any one of claims 5-9, wherein the antibody is a bispecific antibody.
11. The anti-CTLA-4 antibody or antigen-binding fragment thereof of any of claims 1-4 or the anti-CTLA-4/anti-PD-1 antibody of any of claims 5-10, wherein the antibody is a humanized antibody or a fully human antibody.
12. An isolated nucleic acid encoding the anti-CTLA-4 antibody or antigen-binding fragment thereof of any of claims 1-4 or the anti-CTLA-4/anti-PD-1 antibody of any of claims 5-10.
13. The nucleic acid of claim 12, comprising:
(1) the heavy chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment thereof is encoded by the nucleotide sequence shown in SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30; and
(2) the light chain variable region of the anti-CTLA-4 antibody or antigen binding fragment thereof is encoded by the nucleotide sequence of SEQ ID NO 27, 31, 32, 33, 34, 35; and/or
(3) A nucleotide sequence encoding the heavy chain variable region of the anti-PD-1 antibody or an antigen-binding fragment thereof as shown in SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50; and
(4) the light chain variable region of the anti-PD-1 antibody or antigen binding fragment thereof is encoded by the nucleotide sequence of SEQ ID NO 44, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53.
14. An expression vector comprising the nucleic acid of claim 12 or 13.
15. A host cell transformed with the expression vector of claim 14, said host cell being selected from the group consisting of prokaryotic and eukaryotic cells, preferably mammalian cells.
16. A method of making the anti-CTLA-4 antibody or antigen-binding fragment thereof of any of claims 1-4 or the anti-CTLA-4/anti-PD-1 antibody of any of claims 5-10, comprising the steps of expressing an antibody in the host cell of claim 15, and isolating the antibody from the host cell.
17. A pharmaceutical composition comprising the anti-CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 1-4 or the anti-CTLA-4/anti-PD-1 antibody of any one of claims 5-10 and a pharmaceutically acceptable carrier.
18. Use of the anti-CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 1 to 4 or the anti-CTLA-4/anti-PD-1 antibody of any one of claims 5 to 10 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for inhibiting CTLA-4 and/or PD-1 activity, preferably for the treatment of a tumor.
CN202210210779.4A 2021-03-05 2022-03-04 anti-CTLA-4 antibodies and uses thereof Pending CN115010810A (en)

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CN104974253A (en) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 Anti-CTLA-4/PD-1 bispecific antibody as well as preparation method and application thereof
CN104987421A (en) * 2015-05-13 2015-10-21 北京比洋生物技术有限公司 Anti-CTLA-4 and PD-1 dual variable domain immunoglobulin
EP3389714A4 (en) * 2015-12-14 2019-11-13 MacroGenics, Inc. Bispecific molecules having immunoreactivity with pd-1 and ctla-4, and methods of use thereof
CN105754990A (en) * 2016-01-29 2016-07-13 深圳精准医疗科技有限公司 Preparation method and application of PD-1/CTLA-4 (programmed death-1/cytotoxic T lymphocyte antigen-4) bispecific antibody
CN116478289A (en) * 2017-05-19 2023-07-25 上海药明生物技术有限公司 Novel CTLA-4 monoclonal antibody
WO2019179391A1 (en) * 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel bispecific pd-1/ctla-4 antibody molecules
CN110343180B (en) * 2019-07-25 2021-03-30 北京免疫方舟医药科技有限公司 anti-CTLA-4 antibodies and uses thereof

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