WO2020143720A1 - 阻断血管内皮细胞生长且活化t细胞的多靶向融合蛋白和包含其的药物组合物 - Google Patents

阻断血管内皮细胞生长且活化t细胞的多靶向融合蛋白和包含其的药物组合物 Download PDF

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WO2020143720A1
WO2020143720A1 PCT/CN2020/071213 CN2020071213W WO2020143720A1 WO 2020143720 A1 WO2020143720 A1 WO 2020143720A1 CN 2020071213 W CN2020071213 W CN 2020071213W WO 2020143720 A1 WO2020143720 A1 WO 2020143720A1
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antibody
fusion protein
domain
vegfr
seq
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PCT/CN2020/071213
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French (fr)
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胡品良
邹敬
洪伟东
何芸
白洁
宋凌云
杨文第
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北京比洋生物技术有限公司
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Priority to JP2021538762A priority Critical patent/JP2022517920A/ja
Priority to US17/421,874 priority patent/US20220106389A1/en
Priority to EP20738951.1A priority patent/EP3909986A4/en
Publication of WO2020143720A1 publication Critical patent/WO2020143720A1/zh

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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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    • 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
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention generally relates to the field of medical biotechnology.
  • the present invention relates to a multi-target fusion protein comprising (i) vascular endothelial cell growth inhibitory domain; (ii) immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD)
  • the multi-target fusion protein and pharmaceutical composition of the present invention can treat or prevent cancerous diseases in individuals.
  • the tumor microenvironment is a complex environment on which tumor cells depend for survival and development. It consists of cellular components and non-cellular components. Among them, cellular components include tumor cells themselves, immune cells, endothelial cells, etc.; non-cellular components include cytokines, chemokines, etc. . With the study of tumors, people realize that the generation, growth and metastasis of tumors are regulated by the tumor microenvironment. The tumor microenvironment determines whether tumor cells can grow preferentially.
  • tumor cells express PD-L1, thereby utilizing the inhibitory signaling pathway of immune checkpoints (ie, PD-1/PD-L1 inhibitory Signaling pathway) to inhibit T lymphocyte activity, where PD-L1 is not expressed in normal human tissues.
  • T cell receptor T cell receptor
  • MHC major histocompatibility complex
  • PD-1 and CD28 co-localize on the T lymphocyte membrane, and the target of PD-1 mediated immunosuppression is mainly CD28, not TCR.
  • PD-1 rapidly recruits Shp2 phospholipase after binding to PD-L1, Shp2 phospholipase preferentially dephosphorylates CD28, which is stronger than dephosphorylation of TCR, thereby inhibiting T cell function by inactivating CD28 signaling (Hui E. et al., T cell costimulatory receptor, CD28 is a primary target for PD-1-mediated inhibition, Science, 2017, 355(6332): 1428-1433).
  • CTLA-4 expressed on the surface of T cells has a high degree of homology with the costimulatory molecule CD28 on the surface of T cells, and they have the same ligands CD86 (B7-2) or CD80 (B7-1).
  • the combination of CTLA-4 and B7 molecules usually inhibits the activation of T cells, so blocking the immune checkpoint B7/CTLA-4 pathway can enhance tumor-specific T cell activation.
  • tumor cells also release angiogenic factors, such as vascular endothelial growth factor (Vascular Endothelial Cell Growth Factor (VEGF)), resulting in a surge in the number of VEGF.
  • VEGF vascular endothelial growth factor
  • VEGFR cell surface receptor
  • Anti-PD-1 antibody drugs such as Bristol-Myers Squibb (BMS)'s Nivolumab and Merck's Pembrolizumab; Bristol-Myers Squibb (BMS)'s anti-PD-1 antibody CTLA-4 antibody Ipilimumab (trade name Yervoy); Genentech's human-mouse chimeric anti-VEGF antibody Bevacizumab (trade name Avastin); Sanofi-aventis and Regeneron Aflibercept and the like developed as VEGF-Trap.
  • BMS Bristol-Myers Squibb
  • BMS Bristol-Myers Squibb
  • CTLA-4 antibody Ipilimumab trade name Yervoy
  • Genentech's human-mouse chimeric anti-VEGF antibody Bevacizumab trade name Avastin
  • Sanofi-aventis and Regeneron Aflibercept and the like developed as VEGF-Trap.
  • multi-targeted fusion proteins can simultaneously specifically target multiple signaling pathways involved in tumorigenesis and development, there is a multi-targeted fusion that can improve the tumor microenvironment by blocking the growth of vascular endothelial cells and activating T cells Protein, and the need to combine the multi-target fusion protein with other anti-cancer drugs.
  • the inventors have developed a group of multi-targeted fusion proteins that block the growth of vascular endothelial cells and activate T cells through vigorous research, which include (i) vascular endothelial growth inhibitory domain; (ii) immunoglobulin Fc domain ; And (iii) CD80 extracellular domain (ECD).
  • the multi-target fusion protein can improve the tumor microenvironment and improve the effect of tumor immunotherapy from two aspects, one of which is to improve the tumor microenvironment through the CD80 extracellular domain (ECD) of the multi-target fusion protein )
  • ECD extracellular domain
  • the CD80 extracellular domain binds to PD-L1 as an immune checkpoint to alleviate the PD-1/PD-L1 inhibitory signaling pathway , "Brake" the immune system; through the CD80 extracellular domain binds CTLA-4 and exerts the same function as the anti-CTLA-4 antibody ipilimumab to inhibit regulatory T cells (Treg); through the CD80 extracellular domain
  • the inhibitor domain blocks the VECD80 extracellular domain (EC
  • the multi-target fusion protein of the present invention comprises (i) an antigen-binding fragment derived from an anti-VEGF antibody and/or an anti-VEGFR antibody and/or a VEGFR extracellular receptor functional region; (ii) an immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD).
  • the antigen-binding fragment derived from the anti-VEGF antibody contained in the multi-target fusion protein may be derived from any antigen-binding fragment of the anti-VEGF antibody, as long as it can bind to VEGF and thereby block or inhibit the binding of VEGF to its receptor VEGFR Antibody.
  • the anti-VEGF antibodies include anti-VEGF antibodies known in the art and anti-VEGF antibodies developed in the future.
  • the antigen-binding fragment of the anti-VEGF antibody is Fab, Fab′, F(ab′) 2 , Fv, single-chain Fv of the anti-VEGF antibody; preferably, the antigen-binding fragment of the anti-VEGF antibody Contains all 6 heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence selected from SEQ ID NO: 1/2 and 3/4, or with all 6 heavy chain CDRs and one or more of the light chain CDRs have a sequence of one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions); more preferably, the The antigen-binding fragment of the anti-VEGF antibody comprises the paired heavy chain variable region sequence/light chain variable region sequence selected from SEQ ID NOs: 1/2 and 3/4, or the paired heavy chain variable region sequence / Light chain variable region sequence having a sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the antigen-binding fragment derived from the anti-VEGFR antibody contained in the multi-target fusion protein may be derived from any antigen-binding fragment of the anti-VEGFR antibody, as long as it can bind to VEGFR and thereby block or inhibit the binding of VEGF to its receptor VEGFR Antibody.
  • the anti-VEGFR antibodies include anti-VEGFR antibodies known in the art and anti-VEGFR antibodies developed in the future.
  • the antigen-binding fragment of the anti-VEGFR antibody is Fab, Fab', F(ab') 2 , Fv, single-chain Fv of the anti-VEGFR antibody; preferably, the antigen-binding fragment of the anti-VEGFR antibody All 6 heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 5/6, or with all 6 heavy chain CDRs One or more CDRs in the light chain CDR have a sequence of one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions); more preferably, the anti-VEGFR antibody is antigen-binding
  • the fragment comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 5/6, or has at least 90% of the paired heavy chain variable region sequence/light chain variable region sequence, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequences with
  • the VEGFR extracellular receptor functional region contained in the multi-target fusion protein may be any VEGFR extracellular receptor functional region, as long as it is a VEGFR cell capable of binding VEGF and thereby blocking or inhibiting the binding of VEGF to its receptor VEGFR
  • the external receptor functional area is sufficient.
  • the VEGFR extracellular receptor functional area comprises VEGFR1 immunoglobulin-like domain 2 and VEGFR2 immunoglobulin-like domain 3; or the VEGFR extracellular receptor functional area comprises VEGFR1 immunoglobulin-like domain Domain 2 and immunoglobulin-like domain 3 of VEGFR2 and immunoglobulin-like domain 4 of VEGFR2; or the VEGFR extracellular receptor functional region contains immunoglobulin-like domain 2 of VEGFR1; more preferably, The VEGFR extracellular receptor functional region has any one selected from the amino acid sequence shown in SEQ ID NO: 7-9 or at least 90%, 91%, 92%, and the amino acid sequence shown in SEQ ID NO: 7-9. Amino acid sequences with 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the (ii) immunoglobulin Fc domain contained in the multi-target fusion protein may be any immunoglobulin Fc domain, in particular, the (ii) is a human immunoglobulin Fc domain.
  • the immunoglobulin Fc domain is the Fc domain of an IgG class antibody, in particular the Fc domain of an IgG 1 subclass, IgG 2 subclass, IgG 4 subclass antibody.
  • the immunoglobulin Fc domain contained in the multi-target fusion protein of the present invention is the Fc domain of an IgG 1 subclass antibody, particularly the Fc structure of a human IgG 1 subclass antibody area.
  • the immunoglobulin Fc domain contained in the multi-target fusion protein of the present invention is the Fc domain of an IgG 4 subclass antibody, in particular the Fc structure of a human IgG 4 subclass antibody area.
  • the (ii) immunoglobulin Fc domain in the multi-target fusion protein of the present invention comprises the Fc domain of the amino acid sequence shown in SEQ ID NO: 10, 11, or 12, or comprises the amino acid sequence of SEQ ID NO: Fc domain having an amino acid sequence shown in 10, 11 or 12 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity .
  • CD80 (ECD) contained in the multi-target fusion protein is part of the extracellular domain of CD80.
  • the CD80 ECD comprises the CD80 immunoglobulin V (IgV) region (CD80-IgV).
  • the CD80 ECD comprises CD80 immunoglobulin V and C regions (CD80-IgVIgC).
  • the CD80 ECD is a human CD80 ECD, preferably the CD80 ECD comprises human CD80 IgV.
  • the CD80-IgV has the amino acid sequence shown in SEQ ID NO: 13, or at least 90%, 91%, 92%, 93%, 94% with the amino acid sequence of SEQ ID NO: 13 , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences.
  • the CD80-IgVIgC has the amino acid sequence shown in SEQ ID NO: 14, or at least 90%, 91%, 92%, 93%, 94%, the amino acid sequence of SEQ ID NO: 14; Amino acid sequences of 95%, 96%, 97%, 98%, 99% or more identity.
  • the multi-target fusion protein further comprises a peptide linker between (i), (ii) and/or (iii); preferably, the peptide linker comprises one or more amino acids, More preferably it contains at least 5 amino acids and most preferably it contains a peptide linker selected from SEQ ID NO: 20-46.
  • the multi-target fusion protein is in the order of (i), (ii), and (iii) from N-terminus to C-terminus; (iii), (i), and (ii); or ( The order of iii), (ii) and (i) is effectively connected.
  • the multi-target fusion protein of the invention comprises
  • CD80 ECD an immunoglobulin Fc domain in the form of a dimer operatively linked at the C-terminus of CD80 ECD; and a derivative derived from an operative link at the C-terminus of the immunoglobulin Fc domain in the form of the dimer Anti-VEGF antibodies and/or antigen-binding fragments of anti-VEGFR antibodies;
  • CD80 ECD an immunoglobulin Fc domain in the form of a dimer operatively linked at the C-terminus of CD80 ECD; and VEGFR cells operatively linked at the C-terminus of the immunoglobulin Fc domain in the form of the dimer External receptor functional area;
  • the antibody is an IgG class antibody, in particular an IgG 1 subclass, IgG 2 subclass, IgG 4 subclass antibody, more particularly an IgG 4 subclass antibody; also preferably, the IgG 4 subclass antibody
  • the antibody comprises an amino acid substitution at position S228 in the Fc domain, more preferably the amino acid substitution S228P; further preferably, the light chain type of the antibody is kappa or lambda, preferably kappa;
  • the full-length anti-VEGF antibody is Bevacizumab
  • the full-length anti-VEGFR antibody is Ramucirumab
  • the multi-target fusion protein of the invention is selected from
  • a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 80 and the second subunit of the fusion protein of SEQ ID NO: 82;
  • a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 84 and the second subunit of the fusion protein of SEQ ID NO: 86;
  • a fusion protein comprising the fusion protein subunit of SEQ ID NO: 88.
  • the present invention also provides a polynucleotide encoding the multi-target fusion protein of the present invention, a vector comprising the polynucleotide encoding the multi-target fusion protein of the present invention, preferably an expression vector, and most preferably glutamine with a double expression cassette Amide synthase expression vector.
  • the present invention provides a host cell comprising the polynucleotide or vector of the present invention.
  • the host cell is a CHO, HEK293, or NSO cell.
  • the present invention also provides a method for producing the multi-target fusion protein of the present invention, which includes step (i) culturing the host cell of the present invention under conditions suitable for expressing the multi-target fusion protein of the present invention, and (ii ) Recovery of the multi-target fusion protein of the present invention.
  • the present invention also provides a pharmaceutical composition in which the multi-target fusion protein of the present invention is combined with an anti-PD-1 antibody.
  • the multi-target fusion protein of the present invention shows good synergy when used in combination with an anti-PD-1 antibody, thereby being able to better achieve the purpose of inhibiting tumor growth.
  • the anti-PD-1 antibody may be any anti-PD-1 antibody, as long as it can inhibit or reduce the binding of PD-1 to its ligand, including the prior art Known anti-PD-1 antibodies and anti-PD-1 antibodies developed in the future.
  • the anti-PD-1 antibody comprises SEQ ID NO: 47/48, 49/50, 51/52, 53/54, 55/56, 57/58, 59/60, 61/ All six heavy chain CDRs and light chains contained in the paired heavy chain variable region sequence/light chain variable region sequence of 62, 63/64, 65/66, 67/68, 69/70 and 71/72 CDR, or a sequence with one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions) from one or more of the six heavy chain CDRs and light chain CDRs ;
  • the anti-PD-1 antibody comprises selected from SEQ ID NO: 47/48, 49/50, 51/52, 53/54, 55/56, 57/58, 59/60, 61/62 , 63/64, 65/66, 67/68, 69/70 and 71/72 paired heavy chain variable region sequence/light chain variable region sequence, or the paired heavy chain variable region
  • the present invention provides the use of the multi-target fusion protein and pharmaceutical composition of the present invention for preparing a medicament for treating or preventing cancerous diseases (eg, solid tumors and soft tissue tumors) in an individual, preferably , Cancerous diseases are melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), kidney cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer , Pancreatic cancer, prostate cancer, head and neck cancer, stomach cancer, hematological malignancies (eg, lymphoma); in particular, the disease is colon cancer or triple negative breast cancer; preferably, wherein the individual is a mammal , More preferably human.
  • cancerous diseases are melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), kidney cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer , Pancre
  • FIGS. 1A and B provide schematic diagrams of the multi-target fusion protein of the present invention, wherein FIG. 1A illustrates a multi-target fusion protein containing an antigen-binding fragment of an antibody, an immunoglobulin Fc domain, and CD80 ECD from the N-terminus to the C-terminus.
  • FIG. 1B illustrates a schematic diagram of a fusion protein comprising the CD80, ECD, immunoglobulin Fc domain, and VEGFR extracellular receptor functional region from the N-terminus to the C-terminus.
  • Figure 2 Shows the results of each protein of the invention prepared and purified in Example 2 by SDS-PAGE electrophoresis and staining with Coomassie blue in the presence of a reducing agent (5mM 1,4-dithiothreitol) .
  • Lane 1 Protein molecular weight standard marker
  • Lane 2 Fusion protein BY24.4
  • Lane 3 Fusion protein BY24.5
  • Lane 4 Fusion protein BY24.12
  • Lane 5 Fusion protein BY31.19
  • Lane 6 Antibody BY18 .1
  • Figure 3 shows the effect of the multi-target fusion protein of the present invention and its combination with anti-PD-1 antibody on IFN- ⁇ secretion in the mixed lymphocyte reaction (MLR) of Example 5.
  • MLR mixed lymphocyte reaction
  • Figure 4 shows the inhibitory effect of the multi-target fusion protein of the present invention and its combination with anti-PD-1 antibody on tumor growth in the animal model of Example 6.
  • the present invention provides a multi-target fusion protein that blocks the growth of vascular endothelial cells and activates T cells and a pharmaceutical composition containing the multi-target fusion protein.
  • the invention also provides a method for producing the multi-target fusion protein, and the use of the multi-target fusion protein or pharmaceutical composition in the treatment or prevention of cancerous diseases in an individual.
  • the term “comprising” or “including” means including the recited element, integer, or step, but does not exclude any other element, integer, or step.
  • PD-1/PD-L1 inhibitory signaling pathway refers to any intracellular signaling pathway triggered by the binding of PD-1 to PD-L1.
  • misalignment As used herein, "mitigation,” “interference,” “inhibition,” or “blocking” of the PD-1/PD-L1 inhibitory signaling pathway can be used interchangeably and refers to (i) interfering with PD-1 and PD-L1. Interactions; and/or (ii) lead to the inhibition of at least one biological function of the PD-1/PD-L1 signaling pathway.
  • the "relief”, “interference”, “inhibition” or “blocking" of the PD-1/PD-L1 signaling pathway caused by the specific binding of the multi-target fusion protein of the present invention to PD-L1 need not be a complete remission , Interfere, inhibit or block.
  • CD28/B7 signaling pathway can be used interchangeably in this article, which means (i) the signaling pathway that stimulates cell activation through the binding of CD28 and CD80 ; And/or (ii) a signaling pathway that stimulates cell activation by binding CD28 to CD86.
  • CD80 and CD86 are both transmembrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF) with highly similar structures, and are also collectively called B7 molecules.
  • the extracellular domain of CD80 and CD86 is composed of an immunoglobulin V (IgV) domain and an immunoglobulin C (IgC) domain.
  • the mature CD80 molecule consists of 254 amino acids, of which the extracellular region consists of 208 amino acids, the transmembrane region has 25 amino acids and the intracellular region has 21 amino acids.
  • the mature CD86 molecule is composed of 303 amino acids, of which the extracellular region is composed of 222 amino acids, the transmembrane region is 20 amino acids and the intracellular region is 61 amino acids.
  • CD80 also known as B7.1
  • B7.1 is expressed on the surface of T cells, B cells, dendritic cells and monocytes, and binds CD28, PD-L1 and CTLA-4 with lower affinity through its immunoglobulin V (IgV) region ,
  • IgV immunoglobulin V
  • CD80 and CD28 is 4 ⁇ M
  • the binding affinity of CD80 and PD-L1 is ⁇ 1.7 ⁇ M
  • the binding affinity of CD80 and CTLA-4 is 0.2 ⁇ M
  • CD86 binds CD28 and CTLA-4, but not PD-L1.
  • Soluble CD80 (eg, CD80-Fc) can produce continuous activation of T lymphocytes through the CD28/B7 costimulatory pathway and stimulate the production of interferon.
  • CD80-Fc maintains T lymphocytes to produce interferon in vitro, which is even more effective than anti-PD-1 antibody or anti-PD-L1 antibody.
  • CD80-Fc soluble CD80
  • anti-PD-L1 antibodies Ostrand-Rosenberg S et al., Novel Strategies for inhibiting PD-1 pathway-mediated immune suppression
  • CD80-Fc can inhibit PD-1/PD-L1 pathway-mediated immunosuppression by binding to PD-L1, and deliver costimulatory signals to T cells activated by the CD28/B7 costimulatory pathway, thereby enhancing T lymphocyte activation.
  • CD80-Fc can alleviate the immunosuppressive effect of PD-1/PD-L1 pathway and activate tumor immunoreactive T cells.
  • soluble CD86 for example, CD86-Fc
  • CD86-Fc can also activate CD28, and even produce a 3-5 fold activation effect of CD80-Fc, but because CD86 does not bind PD-L1, the final CD80-Fc activation of T lymphocytes is strong
  • CD86-Fc Haile, ST, et al., Soluble, CD80, restores, T, cell, activation, and overcomes, tumor, programmed, death,ligand, 1-mediated immunity, depression, J. Immunol., September 1, 2013; 191(5): 2829-36
  • CD80-Fc has the following effects: (i) When CD80-Fc is used alone, its tumor suppressing effect is better than that of PD-L1 antibody (AACR ANNUAL MEETING, April 14-18, 2018, Illinois, USA (Chicago, California); (ii) CD80-Fc promotes lymphocytes to infiltrate tumor tissues, and the effect is better than PD-L1 antibody (Horn LA et al., Soluble CD80ProteinDelaysTumor Growth and PromotesTumor-InfiltratingLymphocytes, CancerImmunolRes.
  • CD80-Fc When CD80-Fc is used alone, it has a better tumor suppressing effect than PD-1/PD-L1 pathway inhibitors, and is compatible with PD-1 antibodies There is synergy when combined. Five Prime even believes that CD80-Fc is superior to T cell agonists such as GITRL, OX40L and 4-1BBL. Seeing the good immunotherapy effect of CD80-Fc, Five's Prime Therapeutics, Inc.'s CD80-Fc project FPT155 plans to conduct clinical trials in the near future.
  • B7/CTLA-4 pathway and “B7/CTLA-4 signaling pathway” are used interchangeably and refer to (i) signaling pathway caused by the binding of CD80 to CTLA-4; and/or ( ii) Signaling pathway caused by CD86 binding to CTLA-4.
  • VEGF/VEGFR pathway and "VEGF/VEGFR signaling pathway” are used interchangeably herein, and refer to binding to one or more of the cell surface receptor VEGFR family through one or more of the VEGF family Mediated signaling pathway.
  • the VEGF family contains six closely related polypeptides, which are highly conserved homodimeric glycoproteins, with six subtypes: VEGF-A, -B, -C, -D, -E, and placental growth factor ( placental growth factor (PLGF)), with molecular weights ranging from 35 to 44 kDa.
  • VEGF-A (including its splices such as VEGF 165 ) is related to the microvessel density of some solid tumors, and the concentration of VEGF-A in the tissue is related to the prognosis of solid tumors such as breast cancer, lung cancer, prostate cancer and colon cancer. .
  • each VEGF family member is mediated by one or more of the cell surface VEGF receptor (VEGFR) family, which includes VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR , Flk-1), VEGFR3 (also known as Flt-4), etc., where VEGFR1, VEGFR2 are closely related to angiogenesis, and VEGF-C/D/VEGFR3 is closely related to lymphangiogenesis.
  • VEGFR1 also known as Flt-1
  • VEGFR2 also known as KDR , Flk-1
  • VEGFR3 also known as Flt-4
  • the main biological functions of the VEGF family include: (1) Selectively promote vascular endothelial cell mitosis, stimulate endothelial cell proliferation and promote blood vessel formation; (2) Improve the permeability of blood vessels, especially microvessels, and deposit extravasated plasma macromolecules In the extravascular matrix, it provides nutrition for the growth of tumor cells and the establishment of a new capillary network; (3) promotes the proliferation and metastasis of tumors, which depend on the VEGF family for vascular endothelial cells to secrete collagenase and Plasminogen is used to degrade the vascular basement membrane. At the same time, the newly formed microvascular basement membrane inside the tumor tissue is imperfect.
  • VEGF can be used as an immunosuppressive molecule to suppress the body's immunity Response to promote infiltration and metastasis of malignant tumors (Lapeyre-Prost A et al., Immunomodulatory Activity of VEGF in Cancer, Int Rev Cell Mol Biol., 2017; 330:295-342); (5) Other effects: VEGF family can induce Epithelial cells have gaps and open windows, which can activate cytoplasmic vesicles and organelles of the epithelial cells; the VEGF family directly stimulates endothelial cells to release proteolytic enzymes, degrade the matrix, release more VEGF family molecules, accelerate tumor development, extracellular Proteases can also activate the binding of extracellular matrix and the release of VEGF family; VEGF family releases plasma proteins (including fibrinogen) by increasing vascular permeability to form a cellulose network, which provides good for tumor growth, development and metastasis Matrix; VEGF family promote
  • Bevacizumab brand name Avastin developed by Genentech is a recombinant human-mouse chimeric anti-VEGF antibody, which can prevent the activation of VEGFR by blocking the binding of VEGF-A to VEGFR. This plays an anti-angiogenic role. Bevacizumab is currently used for the treatment of metastatic colorectal cancer, lung cancer, breast cancer, pancreatic cancer, kidney cancer, etc.
  • Aflibercept developed by Sanofi-aventis and Regeneron is a kind of VEGF-Trap, which is obtained by fusing the second extracellular domain of VEGFR1 and the third extracellular domain of VEGFR2 with human IgG1 constant region
  • a kind of fusion protein can play an anti-tumor effect on some tumor patients by inhibiting angiogenesis.
  • the term "specific binding” means selective to the binding of an antigen or a molecule of interest and can be distinguished from unwanted or non-specific interactions.
  • the specific binding may be by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technique (analysis on BIAcore instrument) (Liljeblad et al., Analysis of ofagalacto- IgG in rhumatoid arthritis using surface plasmon resonance, Glyco J., 2000, 17, 323-329).
  • vascular endothelial growth inhibitory domain refers to the part of the vascular endothelial growth inhibitor that blocks VEGF/VEGFR signaling, and the part is a region that functions to inhibit vascular endothelial growth.
  • the vascular endothelial growth inhibitory domain may be, for example, one or more variable domains of anti-VEGF antibodies (also called antibody variable regions), one or more variable domains of anti-VEGFR antibodies, or VEGFR extracellularly. Provided by the body ribbon.
  • Treg regulatory T cells
  • the term "regulatory T cells (Treg)” represents a specific subset of T lymphocytes that are essential for maintaining self-tolerance.
  • the Treg cells with repressor function can be distinguished from other T lymphocytes by expressing the transcription factor FOXP3 and other cell markers such as CD127 low , CTLA-4 + , LAP, CD39 + , PD-1 + , GARP, etc. .
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k off and k on, respectively ).
  • K D dissociation constant
  • association rate constant k off and k on, respectively .
  • Affinity can be measured by common methods known in the art. A specific method for measuring affinity is surface plasmon resonance (SPR).
  • antibody is used in the broadest sense herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), as long as they exhibit the desired antigen binding activity .
  • the antibody may be a complete antibody of any type and subtype (eg, IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2) (eg, having two full-length light chains and two full-length heavy chains) chain).
  • whole antibody full length antibody
  • complete antibody complete antibody
  • whole antibody is used interchangeably herein to refer to an antibody that has a structure that is substantially similar to the structure of a natural antibody.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation, which normally determines the class of antibody to which it belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation.
  • ⁇ light chain and ⁇ light chain refer to the two main antibody light chain types.
  • Bispecific antibodies are artificial hybrid antibodies that have two different heavy/light chain pairs and have two different binding sites. Bispecific antibodies can be prepared by a variety of methods including fusion of hybridomas or ligation of Fab' fragments.
  • antigen-binding fragment of an antibody is a portion or segment of an antibody or antibody chain that has fewer amino acid residues than an intact or complete antibody or antibody chain, and is capable of binding antigen or binding to an intact antibody (ie, derived from an antigen-binding fragment Intact antibodies) compete for antigen binding.
  • Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to Fab, Fab', F(ab') 2 , Fv, and single-chain Fv.
  • the Fab fragment is a monovalent fragment composed of VL , VH , CL and CH1 domains.
  • Fab fragments can be obtained by papain digestion of complete antibodies.
  • the complete antibody is digested under the disulfide bond of the hinge region by pepsin to produce F(ab′) 2 , which is a dimer of Fab′ and is a bivalent fragment.
  • F(ab') 2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, thus converting the F(ab') 2 dimer to Fab' monomer.
  • Fab' monomers are basically Fab fragments with hinge regions (for more detailed descriptions of other antibody fragments, see: Fundamental Immunology, edited by WE Paul, Raven Press, NY (1993)).
  • the Fv fragment consisting of V L and V H domains of a single arm of an antibody composition.
  • the two domains V L and V H of the Fv fragment are encoded by independent genes, using recombinant methods, they can be connected by a synthetic linker that enables the two domains to be produced as a single protein chain.
  • the V L and V H regions of the single protein chain pair to form a single chain Fv.
  • the antibody fragment can be obtained by a chemical method, a recombinant DNA method, or a protease digestion method.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • an IgG-like immunoglobulin is a heterotetrameric glycoprotein composed of two light chains and two heavy chains bonded by disulfide bonds of about 150,000 daltons. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 And CH3), also known as the heavy chain constant region.
  • VH variable region
  • CH1 And CH3 constant domains
  • each immunoglobulin light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light chain (CL)
  • VL variable region
  • CL constant light chain
  • the heavy chains of immunoglobulins can be assigned to one of five categories, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG) or ⁇ (IgM), some of which can be further divided into sub Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ), and ⁇ 2 (IgA 2 ).
  • the light chain of an immunoglobulin can be divided into one of two types based on the amino acid sequence of its constant domain, called ⁇ and ⁇ .
  • An immunoglobulin is basically composed of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.
  • Fc domain or "Fc region” is used herein to define the C-terminal region of the immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the natural immunoglobulin "Fc domain” contains two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA, and IgD class antibodies; or The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) from the two heavy chains of IgM and IgE class antibodies.
  • the numbering of amino acid residues in the Fc region or the heavy chain constant region is based on, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health, Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also called the EU index) is used for numbering.
  • Human immunoglobulin is an immunoglobulin that has an amino acid sequence corresponding to an immunoglobulin produced by a human or human cell or from a non-human using a human immunoglobulin library or other sequence encoding human immunoglobulin Source derivation.
  • the “percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in this specification and, if necessary, after introducing gaps to achieve the maximum percentage of sequence identity, regardless of any When conservative substitutions are part of sequence identity, the percentage of amino acid residues in the candidate sequence that are the same as the amino acid residues of the specific amino acid sequence shown in this specification.
  • operably linked means that the specified components are in a relationship that allows them to function in the intended manner.
  • N-terminal refers to the last amino acid at the N-terminal
  • C-terminal refers to the last amino acid at the C-terminal
  • fusion refers to the direct connection of two or more components by peptide bonds or effective connection via one or more peptide linkers.
  • host cell refers to a cell into which a foreign polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom.
  • the host cell is any type of cell system that can be used to produce the multi-target fusion protein of the present invention.
  • the host cell includes a cultured cell, and also includes a transgenic animal, a transgenic plant, or a cell within a cultured plant tissue or animal tissue.
  • mammals include, but are not limited to domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, human and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large animals) mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, human and non-human primates such as monkeys
  • rabbits eg, mice and large animals
  • rodents eg, mice and large animals
  • treatment refers to a clinical intervention intended to alter the natural course of the disease in the individual being treated. Desirable therapeutic effects include but are not limited to preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and relieving or improving the prognosis.
  • the multi-target fusion protein or pharmaceutical composition of the invention is used to delay disease progression or to slow the progression of the disease.
  • anti-tumor effect refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduced tumor volume, reduced tumor cell number, reduced tumor cell proliferation, or reduced tumor cell survival.
  • tumor tumor cell proliferation
  • tumor cell survival a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduced tumor volume, reduced tumor cell number, reduced tumor cell proliferation, or reduced tumor cell survival.
  • tumor cancer
  • the present invention provides a novel multi-target fusion protein comprising (i) vascular endothelial growth inhibitory domain; (ii) immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD).
  • the (i), (ii) and/or (iii) are optionally operably linked by a peptide linker.
  • the multi-target fusion protein of the present invention is a heterotetrameric glycoprotein composed of two first subunits of fusion proteins and two second subunits of fusion proteins that are disulfide bonded.
  • the multi-target fusion protein of the invention is a homodimeric or heterodimeric glycoprotein bonded by disulfide bonds.
  • the multi-target fusion protein of the present invention blocks the growth of vascular endothelial cells and activates T lymphocytes.
  • the multi-target fusion protein can block the growth of vascular endothelial cells by blocking the VEGF/VEGFR pathway, on the other hand, it can inhibit the PD-1/PD-L1 inhibitory signaling pathway by activating the CD28/B7 costimulatory pathway, B7/CTLA-4 signaling pathway inhibits the function of regulatory T cells (Treg) to activate T lymphocytes, thereby improving the tumor microenvironment and improving the effect of tumor immunotherapy.
  • the multi-target fusion protein of the present invention binds to VEGF or VEGFR with a dissociation constant (K D ) of 10 -8 M or less, for example, 10 -9 M to 10 -12 M; and CD28, PD-L1 and CTLA-4 specifically bind.
  • K D dissociation constant
  • the "vascular endothelial growth inhibitory domain" in the multi-target fusion protein of the present invention can specifically bind to VEGF and/or VEGFR, including but not limited to antigen-binding fragments derived from anti-VEGF antibodies and/or anti-VEGFR antibodies and/or VEGFR extracellular receptor functional area.
  • the antigen-binding fragments derived from the anti-VEGF antibody and/or anti-VEGFR antibody contained in the multi-target fusion protein of the present invention enable the multi-target fusion protein of the present invention to have high affinity, for example, 10-8 M D or less, preferably with a K D of 10 -9 M to 10 -12 M, specifically binds to VEGF and/or VEGFR, and thereby blocks the signaling pathway mediated by the binding of VEGF to its receptor VEGFR.
  • the antigen-binding fragment of the multi-target fusion protein of the present invention comprises the heavy chain variable region (VH) and/or light chain variable region of the anti-VEGF antibody and/or anti-VEGFR antibody in Table 1.
  • VL amino acid sequence is substantially the same sequence, for example, with the paired heavy chain variable region sequence / light chain variable region sequence shown in Table 1 has at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or more sequences with sequence identity.
  • the antigen-binding fragment of the multi-target fusion protein of the present invention comprises a pair of heavy chain variable region sequence/light chain variable selected from SEQ ID NO: 1/2, 3/4, and 5/6 All six heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the region sequence.
  • CDRs heavy chain complementarity determining regions
  • Methods and techniques for identifying CDRs in the amino acid sequences of heavy chain variable regions and light chain variable regions are known in the art and can be used to identify specific heavy chain variable regions and/or light chains disclosed herein CDR in the amino acid sequence of the variable region.
  • Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat definition method, Chothia definition method, and AbM definition method.
  • the anti-VEGF antibody or anti-VEGFR antibody as the source of the antigen-binding fragment in the multi-target fusion protein of the present invention can be classified into a ⁇ type or a ⁇ type based on the amino acid sequence of the light chain constant region, preferably a ⁇ type.
  • amino acid sequence of the light chain constant region of the anti-VEGF antibody or anti-VEGFR antibody are provided in Table 2 below.
  • the amino acid sequence of the anti-VEGF antibody or anti-VEGFR antibody derived from the antigen-binding fragment in the multi-target fusion protein of the present invention based on the constant region of its heavy chain is preferably an IgG class antibody, particularly an IgG 1 subclass and an IgG 2 subclass , IgG 4 subclass antibodies, more particularly IgG 4 subclass antibodies.
  • the IgG 4 subclass antibody contains an amino acid substitution that prevents arm-exchange at position S228 in the Fc region, in particular the amino acid substitution S228P.
  • amino acid sequence of the antibody heavy chain constant region are provided in Table 3 below.
  • the VEGFR extracellular receptor functional region contained in the multi-target fusion protein of the present invention is a part of the extracellular domain of VEGFR or a combination thereof.
  • the VEGFR receptor is a tyrosine kinase receptor located on the cell surface, and its extracellular region is composed of seven immunoglobulin (Ig)-like domains.
  • human VEGFR1 contains seven Ig-like domains numbered 1, 2, 3, 4, 5, 6, and 7, Ig-like domain 1 is at the N-terminus of the extracellular domain, and Ig-like domain 7 is extracellular C-terminal of the domain. Unless otherwise indicated herein, Ig-like domains are numbered sequentially from the N-terminus to the C-terminus of the VEGFR protein.
  • the VEGFR extracellular receptor functional region comprises at least one Ig-like domain of one or more VEGFRs selected from VEGFR1, VEGFR2, and VEGFR3. In some aspects, the VEGFR extracellular receptor functional region comprises at least 1, 2, 3, 4, 5, 6 but not more than 7 Ig-like domains of VEGFR. In another aspect, the VEGFR extracellular receptor functional region comprises 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 Ig-like domains of VEGFR.
  • VEGFR extracellular receptor functional region comprising at least one Ig-like domain of two or more VEGFRs.
  • the VEGFR extracellular receptor functional region comprises at least one Ig-like domain from two or more VEGFRs selected from VEGFR1, VEGFR2, and VEGFR3.
  • VEGFR extracellular receptor functional domains containing any combination of the seven Ig-like domains of each VEGFR.
  • the VEGFR extracellular receptor functional region may comprise Ig-like domain 2 of VEGFR1 (eg, human VEGFR1) and Ig-like domain 3 of VEGFR2 (eg, human VEGFR2).
  • the VEGFR extracellular receptor functional region may comprise Ig-like domains 1-3 of VEGFR1 (eg, human VEGFR1), Ig-like domains 2-3, VEGFR2 (eg, human) of VEGFR1 (eg, human VEGFR1) VEGFR2) Ig-like domain 1-3, VEGFR1 (eg human VEGFR1) Ig-like domain 2 and VEGFR2 (eg human VEGFR2) Ig-like domain 3-4, or VEGFR1 (eg human VEGFR1) Ig-like structure Domain 2 and Ig-like domain 3 of VEGFR3 (eg, human VEGFR3).
  • VEGFR1 eg, human VEGFR1
  • Ig-like domains 2-3 eg-like domains 2-3
  • Ig-like domains and other Ig-like domains that can be used as part of the VEGFR extracellular receptor functional area are described in more detail in U.S. Patent No. 7531173; Yu, DC, etc., Soluble, Vascular, Endothelial, Growth Factor, Decoy Receptor, FP3, Exerts, Potent Antiangiogenic Effects , Mol. Ther., 2012, 20(3): 938-947 and Holash, J. et al., VEGF-Trap: a VEGF blocker with potent antitumor effects, PNAS, 2002, 99(17): 11393-11398, all literature The overall introduction is used here as a reference.
  • the VEGFR extracellular receptor functional region has any one selected from the amino acid sequence shown in SEQ ID NO: 7-9 in Table 4 or at least 90 from the amino acid sequence shown in SEQ ID NO: 7-9 Amino acid sequences of %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the VEGFR extracellular receptor functional region in the multi-target fusion protein of the present invention can interact with VEGF with high affinity, for example, with a K D of 10 -8 M or less, preferably 10 -9 M to 10 -12 M.
  • the family binds specifically, and thereby inhibits the binding of the VEGF family to VEGFR on the cell surface and subsequent signaling.
  • the "immunoglobulin Fc domain" in the multi-target fusion protein of the present invention contains all the amino acid residues of the naturally occurring immunoglobulin Fc domain or a part of the amino acid residues of the naturally occurring immunoglobulin Fc domain.
  • the immunoglobulin Fc domain provides advantageous pharmacokinetic properties for the multi-target fusion protein of the invention, including but not limited to long serum half-life.
  • the immunoglobulin Fc domain also makes it possible to purify the multi-target fusion protein of the present invention by, for example, protein A affinity chromatography.
  • Immunoglobulin Fc domains are usually dimeric molecules.
  • the immunoglobulin Fc domain may be produced by papain digestion or trypsin digestion of intact (full-length) immunoglobulin or may be recombinantly produced, which comprises a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the IgG Fc region comprises IgG CH2 domain and IgG CH3 domain.
  • the immunoglobulin Fc domain has the amino acid sequence shown in SEQ ID NO: 10-12 in Table 5 or has at least 90%, 91%, 92% of the amino acid sequence shown in SEQ ID NO: 10-12 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences.
  • the IgG Fc region may also contain a peptide sequence obtained by performing additional sequence modification on SEQ ID NO: 10-12, for example, SEQ ID NO: 10-12
  • an amino acid substitution that prevents arm-exchange from occurring particularly amino acid substitution S228P, is included in the IgG Fc region at position S228.
  • the IgG4 Fc region comprises the amino acid substitution S228P.
  • the "extracellular domain (CDD) of CD80" in the multi-target fusion protein of the present invention contains all the amino acid residues of naturally occurring CD80 ECD or a part of the amino acid residues of naturally occurring CD80 ECD.
  • the CD80ECD comprises CD80 IgV.
  • the CD80ECD comprises human CD80 IgV.
  • the CD80ECD has the amino acid sequence shown in SEQ ID NO: 13 or 14 in Table 6.
  • CD80ECD can also contain peptide sequences obtained by additional sequence modification of SEQ ID NO: 13 and 14, for example, in SEQ ID NO: 13 and 14.
  • the modified peptide will retain the activity or function associated with the unmodified peptide.
  • the modified peptide usually has an amino acid sequence that is substantially homologous to the amino acid sequence of the unmodified sequence.
  • the amino acid sequence shown in SEQ ID NO: 13 or 14 has at least 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • vascular endothelial growth inhibitory domain vascular endothelial growth inhibitory domain
  • immunoglobulin Fc domain an immunoglobulin Fc domain
  • a "peptide linker" to which CD80 and ECD are optionally operatively linked are one or Peptides with multiple amino acids, generally about 2-20 amino acids. Peptide linkers are known in the art or described herein.
  • the peptide linker comprises at least 5 amino acids, preferably selected from AKTTPKLEEGEFSEAR (SEQ ID NO: 20); AKTTPKLEEGEFSEARV (SEQ ID NO: 21); AKTTPKLGG (SEQ ID NO: 22); SAKTTPKLGG ( SEQ ID NO: 23); SAKTTP (SEQ ID NO: 24); RADAAP (SEQ ID NO: 25); RADAAPTVS (SEQ ID NO: 26); RADAAAAGGPGS (SEQ ID NO: 27); RADAAAA (SEQ ID NO: 28) ); SAKTTPKLEEGEFSEARV (SEQ ID NO: 29); ADAAP (SEQ ID NO: 30); DAAPTVSIFPP (SEQ ID NO: 31); TVAAP (SEQ ID NO: 32); TVAAPSVFIFPP (SEQ ID NO: 33); QPKAAP (SEQ ID NO: 34); QPKAAPSVTLFPP (SEQ ID NO: 35); AKTTPP (SEQ ID NO: 36); A
  • multi-target fusion proteins comprising (i) vascular endothelial growth inhibitory domain; (ii) immunoglobulin Fc domain; and (iii) CD80 ECD in any order, including but not limited to multi-target fusion proteins From the N terminal to the C terminal in the order of (i), (ii) and (iii); the order of (iii), (i) and (ii); or the order of (iii), (ii) and (i) is valid connection.
  • the multi-target fusion protein comprises a full-length anti-VEGF antibody, a full-length anti-VEGFR antibody, or a full-length anti-VEGF and VEGFR bispecific antibody from N-terminus to C-terminus; and A CD80 ECD is effectively connected to the C-terminal of each heavy chain in the heavy chain.
  • the multi-target fusion protein comprises a full-length anti-VEGF antibody, a full-length anti-VEGFR antibody, or a full-length anti-VEGF and VEGFR bispecific antibody; each of the two heavy chains of the antibody One CD80 ECD operatively linked to the N-terminus of a heavy chain; and one CD80 ECD operatively linked to the N-terminus of each of the two light chains of the antibody.
  • the multi-target fusion protein comprises CD80 ECD from the N-terminus to the C-terminus; an immunoglobulin Fc domain in the form of a dimer operably linked at the C-terminus of CD80 ECD; and The C-terminus of the immunoglobulin Fc domain in the form of a polymer is operatively linked to an antigen-binding fragment derived from an anti-VEGF antibody and/or anti-VEGFR antibody.
  • the multi-target fusion protein comprises CD80 ECD from the N-terminus to the C-terminus; an immunoglobulin Fc domain in the form of a dimer operably linked at the C-terminus of CD80 ECD; and The C-terminus of the immunoglobulin Fc domain in the form of a polymer is effectively linked to the VEGFR extracellular receptor functional domain.
  • the multi-target fusion protein of the present invention can be obtained, for example, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production.
  • the polynucleotide encoding each subunit of the multi-target fusion protein is isolated and inserted into one or more vectors for further cloning and/or expression in the host cell.
  • the polynucleotide can be easily isolated and sequenced.
  • a vector comprising one or more polynucleotides of the present invention, preferably an expression vector.
  • the expression vector can be constructed using methods well known to those skilled in the art.
  • Expression vectors include but are not limited to viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • a glutamine synthetase high-efficiency expression vector with dual expression cassettes is used.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a suitable host cell for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.
  • a host cell comprising one or more polynucleotides of the invention.
  • host cells comprising the expression vector of the present invention are provided.
  • the term "host cell” refers to any kind of cellular system that can be engineered to produce the multi-target fusion protein of the present invention.
  • Host cells suitable for replicating and supporting the expression of the multi-target fusion protein of the present invention are well known in the art. As required, such cells can be transfected or transduced with a specific expression vector, and a large number of vector-containing cells can be cultivated for inoculation of large-scale fermenters to obtain a sufficient amount of the multi-target fusion protein of the present invention for clinical applications.
  • Suitable host cells include prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • prokaryotic microorganisms such as E. coli
  • eukaryotic microorganisms such as filamentous fungi or yeast
  • various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • a mammalian cell line suitable for suspension culture can be used.
  • Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2 ), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, Sp2/0 and so on.
  • the host cell is a CHO, HEK293 or NSO cell.
  • a method of producing a multi-target fusion protein of the invention comprising culturing a host cell as provided herein under conditions suitable for expression of the multi-target fusion protein,
  • the host cell contains a polynucleotide encoding the multi-target fusion protein, and the multi-target fusion protein is recovered from the host cell (or host cell culture medium).
  • the multi-target fusion protein prepared as described herein can be purified by known existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art.
  • the purity of the multi-target fusion protein of the present invention can be determined by any of a variety of well-known analysis methods including gel electrophoresis, high-performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the multi-target fusion proteins provided herein can be identified, screened, or characterized by various assays known in the art.
  • PD-1 is an immunosuppressive protein with two ligands, PD-L1 and PD-L2. It is known that the interaction between PD-1 and PD-L1 results in, for example, a reduction of tumor-infiltrating lymphocytes and/or immune escape of cancer cells. Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; when the interaction of PD-1 with PD-L2 is also blocked, the effect is additive (Iwai Y. etc. People, Involvement of PD-L1 tumors cells in the theescape from host immune system and tumor immunotherapy by PD-L1 blockade, Proc. Nat'l. Acad. Sci.
  • the present invention has developed a pharmaceutical composition for combination therapy, which comprises the multi-target fusion protein of the present invention and an anti-PD-1 antibody.
  • the pharmaceutical composition for combination therapy described herein can provide superior beneficial effects, such as enhanced anti-cancer effects, Reduced toxicity and/or reduced side effects.
  • the multi-target fusion protein and/or anti-PD-1 antibody of the present invention in a pharmaceutical composition can be administered at a lower dose or shorter administration time than is necessary to achieve the same therapeutic effect compared to monotherapy administration. Therefore, the present invention also discloses the use of pharmaceutical compositions for combination therapy to treat cancer. The effectiveness of the aforementioned pharmaceutical composition can be tested in cell models and animal models known in the art.
  • the anti-PD-1 antibody included in the combination therapy may be any anti-PD-1 antibody, as long as it can inhibit or reduce the binding of PD-1 to its ligand, including anti-PD-1 known in the art 1 Antibodies and anti-PD-1 antibodies developed in the future. Anti-PD-1 antibodies can specifically bind to PD-1 with a high affinity, for example at a K D of 10 -8 M or less, preferably from 10 -9 M to 10 -12 M, and thereby block The signal transduction pathway mediated by the binding of PD-1 to ligands PD-L1 and/or PD-L2.
  • the anti-PD-1 antibody in the combination therapy of the present invention comprises a sequence substantially the same as the amino acid sequence shown in Table 7, for example, the paired heavy chain variable region sequence shown in Table 7/
  • the light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the anti-PD-1 antibody in the combination therapy of the present invention comprises SEQ ID NO: 47/48, 49/50, 51/52, 53/54, 55/56, 57/58, 59/ All heavy chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of 60, 61/62, 63/64, 65/66, 67/68, 69/70 and 71/72 Light chain CDR.
  • Methods and techniques for identifying CDRs in the amino acid sequences of heavy chain variable regions and light chain variable regions are known in the art and can be used to identify specific heavy chain variable regions and/or light chains disclosed herein CDR in the amino acid sequence of the variable region.
  • Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat definition, Chothia definition, and AbM definition. See, for example, Kabat, Sequences of Proteins, Immunological Interests, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., Standard conformations, for the immunological globulins, J. Mol. Biol. 273 -948 (1997); and Martin AC and others, Modeling antibody hypervariable loops: a combined algorithm, Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989).
  • the anti-PD-1 antibody in the combination therapy of the present invention is selected from nivolumab (Nivolumab), pidilizumab, and pembrolizumab.
  • the pharmaceutical composition of the present invention may contain a "therapeutically effective amount” or “prophylactically effective amount” of the multi-target fusion protein of the present invention and an anti-PD-1 antibody.
  • “Therapeutically effective amount” refers to an amount that is effective to achieve the desired therapeutic result at the required dose for the required period of time.
  • the therapeutically effective amount can be varied according to various factors such as disease state, individual's age, sex, and weight.
  • a therapeutically effective amount is any amount whose toxic or harmful effects are less than the beneficial effects of treatment.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more Preferably at least about 80%.
  • a measurable parameter eg, tumor growth rate
  • the ability of the pharmaceutical composition of the present invention to inhibit measurable parameters can be evaluated in an animal model system that predicts efficacy in human tumors.
  • prophylactically effective amount refers to an amount that is effective to achieve the desired preventive result at the required dose for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • the multi-target fusion protein and anti-PD-1 antibody treatment regimen of the combination therapy of the present invention can cooperate with each other, the pharmaceutical composition of the present invention is Tumor immune escape is beneficial.
  • Co-administration of the pharmaceutical composition comprising the multi-target fusion protein and the anti-PD-1 antibody of the present invention can be carried out by separately administering the multi-target fusion protein or the anti-PD-1 antibody, or the single-target multi-target fusion protein Combined preparation with anti-PD-1 antibody.
  • the administration of the multi-target fusion protein and anti-PD-1 antibody in the combination therapy of the present invention allows flexibility in the amount and schedule of administration.
  • the multi-target fusion proteins and pharmaceutical compositions disclosed herein have therapeutic and prophylactic uses for cancer.
  • the multi-target fusion protein and its composition with an anti-PD-1 antibody can be administered to cultured cells in vitro or ex vivo or to a subject, for example, a human subject, to treat and/or prevent multiple A cancerous disease.
  • the present invention relates to the use of a multi-target fusion protein or a pharmaceutical combination of the present invention to treat or prevent a disease that requires blocking the growth of vascular endothelial cells and activating T cells in a subject, thereby inhibiting or reducing related Diseases such as the growth or appearance, metastasis, or recurrence of cancerous tumors.
  • Multi-target fusion proteins can be used alone to inhibit the growth of cancerous tumors or prevent their appearance.
  • the multi-target fusion protein can be administered in combination with other cancer therapeutic/prophylactic agents (eg, anti-PD-1 antibodies). When the multi-target fusion protein of the present invention and anti-PD-1 antibody are administered, this combination may be administered in any order or simultaneously.
  • the present invention provides a method of inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of the multi-target fusion protein or pharmaceutical composition described herein .
  • the present invention provides a method of preventing the appearance or metastasis or recurrence of tumor cells in a subject, the method comprising administering to the subject a prophylactically effective amount of the multi-target fusion protein described herein or Pharmaceutical composition.
  • cancers treated and/or prevented with multi-target fusion proteins or pharmaceutical compositions include but are not limited to solid tumors, hematological cancers (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma) ) And metastatic lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, such as sarcomas and cancers of multiple organ systems, such as invasion of the lung, breast, ovary, lymphoid, gastrointestinal (eg, colon), anus, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (e.g., brain, nerve, or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated Metastatic or locally recurrent nasopharyngeal carcinoma) and those of the pancreas, as well as adenocarcinomas, including malignant tumors, such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer.
  • the cancer can be early, middle, or advanced or metastatic cancer.
  • the cancer is selected from melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC ), ovarian cancer, pancreatic cancer, prostate cancer, head and neck tumors, gastric cancer, hematological malignancies (eg, lymphoma).
  • Example 1 Construction of a glutamine synthetase high-efficiency expression vector containing the gene of interest
  • nucleotide sequence of the light chain (BY18.1L) of anti-PD1 antibody BY18.1 SEQ ID NO: 73:
  • Anti-PD1 antibody BY18.1 light chain (BY18.1L) amino acid sequence (SEQ ID NO: 74):
  • the nucleotide sequence of the heavy chain (BY18.1H) of anti-PD1 antibody BY18.1 (SEQ ID NO: 75):
  • Amino acid sequence of heavy chain (BY18.1H) of anti-PD1 antibody BY18.1 (SEQ ID NO: 76):
  • METDTLLLWVLLLWVPGSTG is the signal peptide sequence.
  • the BY18.1L encoding nucleotides were double digested with XhoI-EcoRI, and the glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.) was used with XhoI -EcoRI double digestion, and then ligase the XhoI-EcoRI double digested BY18.1L coding nucleotide into the XhoI-EcoRI double digested glutamine synthetase efficient expression vector with double expression cassette, Obtained a high-efficiency expression vector for glutamine synthetase with dual expression cassettes into which BY18.1L coding nucleotides have been introduced; then, the BY18.1H
  • the BY18.1L-encoding nucleotide can also be linked into a glutamine synthetase high-efficiency expression vector with dual expression cassettes into which the BY18.1H-encoding nucleotide has been introduced to express and obtain the antibody BY18.1.
  • CD80 extracellular domain in Table 1 and the IgG4Fc sequence in Table 6 it was optimized to be a nucleotide sequence suitable for expression in Chinese hamster ovary cancer cells (CHO), and entrusted to Shanghai Jierui Bioengineering Co., Ltd. for synthesis as follows SEQ ID NO: 77 polynucleotide sequence.
  • the CD80-Fc fusion protein produced after the expression of the nucleotide sequence is also referred to herein as fusion protein BY31.19.
  • Nucleotide sequence of fusion protein BY31.19 (CD80-Fc, IgG4) (SEQ ID NO: 77)
  • Amino acid sequence of fusion protein BY31.19 (CD80-Fc, IgG4) (SEQ ID NO: 78)
  • the amino acid sequence " METDTLLLWVLLLWVPGSTG” is a signal peptide.
  • the BY31.19 encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) From Beijing Biyang Biotechnology Co., Ltd.).
  • the recombinant vector was sequenced and verified to be correct for CD80-Fc fusion protein expression.
  • the expressed CD80-Fc fusion protein was named fusion protein BY31.19.
  • the sequence of the heavy chain variable region and the light chain variable region of the anti-VEGF antibody in Table 2 Based on the sequence of the CD80 extracellular domain in Table 1, the sequence of the heavy chain variable region and the light chain variable region of the anti-VEGF antibody in Table 2, the sequence of the heavy chain constant region of the antibody in Table 3, and the light chain of the antibody in Table 4
  • the sequence of the constant region and the peptide linker sequence of SEQ ID NO: 20-46 are optimized to the nucleotide sequence suitable for expression in Chinese hamster ovary cancer cells (CHO), and Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the following SEQ ID NO: polynucleotide sequences shown in 79 and 81.
  • the anti-VEGF antibody-CD80 fusion protein produced after the expression of the nucleotide sequence is expressed herein as fusion protein BY24.4.
  • the amino acid sequence " METDTLLLWVLLLWVPGSTG” is a signal peptide.
  • the BY24.4L encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) (Beijing Biyang Biotechnology Co., Ltd.); by XbaI-SalI double digestion to clone BY24.4H encoding nucleotides to BY24.4L encoding nucleotides with a double expression cassette glutamine synthetase with high efficiency Expression vector; or vice versa.
  • the recombinant vector was sequenced and verified to be correct for expression.
  • the expressed anti-VEGF antibody-CD80 fusion protein was named fusion protein BY24.4.
  • sequence of the CD80 extracellular domain in Table 1 the sequence of the heavy chain variable region and the light chain variable region of the anti-VEGFR antibody in Table 2, the sequence of the heavy chain constant region of the antibody in Table 3, and the light chain of the antibody in Table 4
  • the sequence of the constant region and the peptide linker sequence of SEQ ID NO: 20-46 are optimized to be suitable for nucleotide sequence expression in Chinese hamster ovary cancer cells (CHO), and Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the following SEQ ID NO: Polynucleotide sequences shown in 83 and 85.
  • the anti-VEGFR antibody-CD80 fusion protein produced after the expression of the nucleotide sequence is expressed herein as fusion protein BY24.5.
  • the amino acid sequence " METDTLLLWVLLLWVPGSTG” is a signal peptide.
  • the BY24.5L encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) (Beijing Biyang Biotechnology Co., Ltd.); then cloned the BY24.5H encoding nucleotide by XbaI-SalI double digestion into the glutamine synthetase with the double expression cassette linked to the BY24.5L encoding nucleotide. Expression vector; or vice versa. The recombinant vector was sequenced and verified to be correct for expression.
  • the expressed anti-VEGFR antibody-CD80 fusion protein was named fusion protein BY24.5.
  • CD80 extracellular domain in Table 1 the sequence of IgG4Fc in Table 6, and the sequence of VEGFR functional region in Table 5 (VEGFR1-D2/VEGFR2-D3), it is optimized to be suitable for nuclear expression in Chinese hamster ovary cancer cells (CHO) Nucleotide sequence, and entrusted Shanghai Jierui Biological Engineering Co., Ltd. to synthesize the polynucleotide sequence shown in SEQ ID NO: 87
  • the CD80-Fc-VEGFR fusion protein produced after the expression of the nucleotide sequence is also referred to herein as fusion protein BY24.12.
  • Nucleotide sequence of fusion protein BY24.12 (CD80-Fc-VEGFR) (SEQ ID NO: 87)
  • the BY24.12 encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) From Beijing Biyang Biotechnology Co., Ltd.).
  • the recombinant vector was sequenced and verified to be correct for protein expression.
  • the expressed fusion protein was named fusion protein BY24.12.
  • each of the recombinant expression vector plasmid DNA containing the target gene prepared in Example 1 was 250ug and polyethylenimine (PEI) (Sigma, catalog number: 408727) 500ug was added 1ml of serum-free CD 293 culture solution was mixed well. After standing at room temperature for 8 minutes, the PEI/DNA suspension was added dropwise to a shake flask containing 100ml of cell suspension. Mix gently and place in 5% CO 2 at 37°C for shaking (120 rpm). After 5 days, the culture supernatant was collected.
  • PEI polyethylenimine
  • the target protein present in the culture supernatant collected in Example 2(1) above was purified using a HiTrap MabSelect SuRe 1ml column (GE Healthcare Products Life Catalog Number: 11-0034-93) equilibrated with pH 7.4 PBS solution.
  • the HiTrap MabSelect SuRe 1ml column was equilibrated with 10 column bed volumes of PBS solution at pH 7.4 at a flow rate of 0.5ml/min; the culture supernatant collected in Example 2(1) above was filtered with a 0.45 ⁇ m filter Then, load the sample onto a HiTrap MabSelect SuRe 1ml column equilibrated with pH 7.4 PBS solution; after loading the supernatant, first wash the column with PBS solution pH 7.4 at a flow rate of 0.5ml/min to wash 5-10 column bed volumes, and It was then eluted with 100 mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min. The elution peaks were collected, and the target proteins BY18.1, BY31.19, BY24.4, BY24.5, and BY24.12 existed in the elution peaks, respectively.
  • Recombinant human CD28 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 50103-M08H), recombined human PD-L1 (Beijing Bepsey Biotechnology Co., Ltd., catalog number: PD1-H5229) and recombined human CTLA-4 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11159-H08H) diluted to 0.5 ⁇ g/ml, 0.25 ⁇ g/ml, and 1.0 ⁇ g/ml and coated with 96-well ELISA plates (Corning Corporation, article number: 42592).
  • the fusion proteins BY24.4, BY24.5, BY24.12, and BY31.19 purified in the above Example 2(2) were diluted to 2000 ⁇ g/ml, and then serially diluted 3 times, a total of 16 gradients were diluted for each The concentration gradient was used for double well detection. 50 ⁇ l of the diluted sample was added to the above-mentioned 96-well plate coated with recombinant human CD28, recombinant human CTLA-4 or recombinant human PD-L1, and incubated at 37°C for 2 hours.
  • ELISA results show that the multi-target fusion proteins of the present invention BY24.4, BY24.5, BY24.12 and BY31.19 as positive controls can bind recombinant human PD-L1, recombinant human CD28 and CTLA-4.
  • Each fusion protein has the strongest binding ability to CTLA-4, followed by PD-L1, and the weakest to CD28.
  • Example 4 Using Biacore T100 to determine the affinity of the target protein of the present invention for the target
  • the anti-IgG antibody (GE Healthcare, Life Sciences, catalog number: BR-1008-39) was covalently fixed on the CM5 chip by amide coupling.
  • EDC N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • each target protein of the present invention prepared in Example 2 was diluted to 5 ⁇ g/ml, and the dilution was injected at a flow rate of 10 ⁇ L/min for 2 minutes.
  • Each target protein of the present invention prepared in Example 2 passed through its own Fc region. Covalently captured on the CM5 chip surface. The resulting complex is stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration.
  • antigen PD-1 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 10377-H08H
  • VEGF 165 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 11066-HNAH
  • mouse VEGF164 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 50159-MNAB
  • VEGFR2 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 10012-H02H1
  • concentration gradients 7nM, 22nm, 66nM, 200nM , 600nM.
  • Binding was measured by injecting each concentration at a flow rate of 30 ⁇ l/min for 180 seconds and a dissociation time of 600 seconds. The surface was regenerated by washing with 3M MgCl 2 solution at a flow rate of 10 ⁇ L/min for 30 seconds. Data analysis was performed using BIA evaluation software (BIAevaluation 4.1 software from GE Healthcare Biosciences AB, Sweden) to obtain the affinity data shown in Table 10 below.
  • the KD(M) of the fusion proteins BY24.4, BY24.5, BY24.12 and their respective targets are lower than 10 -9 M, indicating that they have high affinity with their respective targets, especially the fusion protein BY24.12 can bind Both mouse and human VEGF-A bind with high affinity and the difference is not very large.
  • Example 5 Effect of the target protein of the present invention and its composition on IFN- ⁇ secretion in mixed lymphocyte reaction (MLR)
  • CD4 + T lymphocytes and dendritic cells were purchased from Beijing Shihe Biotechnology Co., Ltd.
  • the CD4 + T lymphocytes and dendritic cells (DC) were derived from different healthy persons.
  • CD4 + T lymphocytes and dendritic cells (DC) were plated in 96-well cell culture plates at 1 ⁇ 10 5 cells/well and 1 ⁇ 10 4 cells/well, respectively.
  • the experiment was divided into 7 groups, namely BY18.1 group (1.0 ⁇ g/ml), BY24.4 group (1.14 ⁇ g/ml), BY24.4 (1.14 ⁇ g/ml) + BY18.1 (1.0 ⁇ g/ml) group , BY24.5 (1.14 ⁇ g/ml) group, BY24.5 (1.14 ⁇ g/ml) + BY18.1 (1.0 ⁇ g/ml) group, BY24.12 (0.84 ⁇ g/ml) group and BY24.12 (0.84 ⁇ g) /ml)+BY18.1 (1.0 ⁇ g/ml) group.
  • test results are as follows: compared with the antibody BY18.1 group (2221.8 ⁇ 364.5pg/ml), the fusion protein BY24.4 group alone (924.1 ⁇ 221.9pg/ml), BY24.5 group (760.1 ⁇ 286.8pg/ml) And BY24.12 group (793.4 ⁇ 139.2pg/ml) IFN- ⁇ secretion were significantly lower than antibody BY18.1 group (P ⁇ 0.01); but fusion proteins BY24.4, BY24.5 and BY24.12 were added respectively After BY18.1, IFN- ⁇ secretion was significantly increased.
  • the IFN- ⁇ secretion of each group was: BY24.4+BY18.1 group (3494.2 ⁇ 364.5pg/ml), BY24.5+BY18.1 group (3523.8 ⁇ 465.1pg/ml) and BY24.12+BY18.1 group (3801.8 ⁇ 702.2pg/ml).
  • the fusion proteins BY24.4, BY24.5, and BY24.12 combined with BY18.1 can cause a significant increase in IFN- ⁇ secretion (P ⁇ 0.01), and indicate that the antibody BY18.1 (PD-1 antibody )
  • the combination with the multi-target fusion proteins BY24.4, BY24.5 and BY24.12 of the present invention has a clear synergistic effect on the secretion of IFN- ⁇ .
  • Example 6 Synergy of the multi-target fusion protein of the present invention and PD-1 antibody
  • the main purpose of this example is to explore the synergistic effect of the multi-target fusion protein of the present invention and the PD-1 antibody in anti-tumor in vivo, so the dosage of the PD-1 antibody and each fusion protein is a low dose because of the high The dose may have a good tumor suppression effect, and no synergy between the PD-1 antibody and each fusion protein was observed.
  • mouse colon cancer cells CT26 (ATCC) in RPMI-1640 medium were inoculated subcutaneously on the right anterior rib of 6-week-old female BALB/c mice, 100 ⁇ l/mouse, inoculation volume was 1 ⁇ 10 6 Cells/mouse.
  • the time of inoculation of mouse-derived colon cancer cell CT26 was set to day 0.
  • the tumor-bearing mice were randomly divided into 6 mice in each group, for a total of 5 groups. Calculate the molar amount based on the molecular weight, and administer the fusion proteins BY31.19, BY24.12 and anti-mPD-1 antibody in equivalent molar amounts.
  • the grouping and administration doses are: vehicle (PBS) control group; fusion protein BY31.19 (1.6mg/kg) group; fusion protein BY24.12 (2.5mg/kg,) group; anti-mPD-1 ( 3.0mg/kg, purchased from BioXcell, clone number: RMP1-14, product number: BE0146) group; fusion protein BY24.12 (2.5mg/kg) + anti-mPD-1 (3mg/kg) group.
  • mice were euthanized, serum and tumors were collected, and the serum was stored at -80°C.
  • the tumors were weighed and photographed, and then fixed into formal samples by formalin fixation and paraffin embedding (FFPE).
  • FFPE formalin fixation and paraffin embedding
  • the tumor growth inhibition rates (TGI%) of BY31.19 group, BY24.12 group, PD-1 group, BY24.12+anti-mPD-1 group were 15%, 17%, 24%, and 47% (in terms of tumor volume) have different degrees of inhibition.
  • the immunosuppressive protein BY24.12 combined with PD-1 antibody has a significantly better tumor suppression effect than BY24.12 and PD-1 when used alone, so there is a synergistic effect between the two.

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Abstract

本发明提供了一种阻断血管内皮细胞生长且活化T细胞的多靶向融合蛋白，其包含(i)血管内皮生长抑制结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。本发明还提供了编码所述多靶向融合蛋白的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及包含所述多靶向融合蛋白和抗PD-1抗体的药物组合物。本发明的所述多靶向融合蛋白和药物组合物能够在个体中治疗或预防癌性疾病。

Description

阻断血管内皮细胞生长且活化T细胞的多靶向融合蛋白和包含其的药物组合物 技术领域
本发明总体上涉及医药生物技术领域。具体地，本发明涉及包含(i)血管内皮细胞生长抑制剂结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)的多靶向融合蛋白、编码所述多靶向融合蛋白的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及包含所述多靶向融合蛋白和抗PD-1抗体的药物组合物。本发明的所述多靶向融合蛋白和药物组合物能够在个体中治疗或预防癌性疾病。
背景技术
肿瘤微环境是肿瘤细胞赖以生存和发展的复杂环境，由细胞成分和非细胞成分组成，其中细胞成分包括肿瘤细胞本身、免疫细胞、内皮细胞等；非细胞成分包括细胞因子、趋化因子等。随着对肿瘤的研究，人们认识到了肿瘤的生成、生长、转移受到肿瘤微环境的调控。肿瘤微环境决定了肿瘤细胞是否得以优势生长。
在肿瘤微环境中，通常存在T淋巴细胞活性的抑制，导致T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S，Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.，Nat Rev Drug Discov，2013，12(2)：130-146)，例如，肿瘤细胞通过表达PD-L1，由此利用免疫检查点的抑制性信号通路(即，PD-1/PD-L1抑制性信号通路)来抑制T淋巴细胞活性，其中所述PD-L1在正常人体组织是不表达的。
以前认为PD-1与其配体PD-L1结合后，通过抑制T细胞受体(TCR)/主要组织相容性复合物(MHC)来实现对T淋巴细胞激活的抑制，但近来Hui等的研究表明，PD-1和CD28共定位于T淋巴细胞膜，PD-1介导的免疫抑制作用靶标主要为CD28，而非TCR，具体而言，PD-1结合PD-L1后快速募集Shp2磷脂酶，Shp2磷脂酶优先地使CD28去磷酸化，这强于对TCR的去磷酸化，从而通过失活CD28信号传导来抑制T细胞功能(Hui E.等人，T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition，Science，2017，355(6332)：1428-1433)。
Kamphorst AO等人证实了CD28共刺激信号的激活是T细胞“再激活”的重要条件之一(Kamphorst AO等人，Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent，Science，2017，355(6332)：1423-1427)。CD28/B7(即，T淋巴细胞表面的共刺激分子CD28与CD86(也称为B7-2)或CD80(也称为B7-1)结合的)共刺激途径的活化对于荷瘤小鼠和慢性病毒感染期间抗PD-1抗体的治疗效果至关重要，如果用抗B7抗体阻断B7分子与CD28的结合，则抗PD-1抗体对肿瘤的抑制作用显著降低。另有研究表明，可溶性CD80在小鼠肿瘤系统中具有体内治疗功效(Horn LA等人，Soluble CD80 Protein  Delays Tumor Growth and Promotes Tumor-Infiltratmg Lymphocytes，Cancer Immunol Res.2018；6(1)：59-68)。在肿瘤微环境中CD80和CD86低表达或不表达，这也是造成肿瘤免疫逃避的重要机制之一。
另外，在T细胞表面表达的CTLA-4和T细胞表面的共刺激分子CD28具有高度的同源性，它们拥有相同的配体CD86(B7-2)或CD80(B7-1)。CTLA-4与B7分子结合通常会抑制T细胞的活化，因此阻断免疫检查点B7/CTLA-4通路可以增强肿瘤特异性T细胞激活作用。
另一方面，在肿瘤微环境中，肿瘤细胞还通过释放促血管生成因子，例如血管内皮细胞生长因子(Vascular Endothelial Cell Growth Factor(VEGF))，导致VEGF数量激增，VEGF通过与其细胞表面受体VEGFR结合介导了血管内皮细胞的分裂增殖和迁移、提高了血管通透性、抑制肿瘤细胞凋亡，为肿瘤的生长和转移提供了良好的微环境。
目前，已批准上市了用来阻断PD-1/PD-L1抑制性信号通路、阻断B7/CTLA-4通路、或阻断VEGF/VEGFR通路的单靶向肿瘤治疗药物，它们分别例如为抗PD-1抗体药物，如百时美施贵宝(BMS)公司的纳武单抗(Nivolumab)和默克(Merck)公司的派姆单抗(Pembrolizumab)；百时美施贵宝(BMS)公司的抗CTLA-4抗体伊匹单抗(Ipilimumab，商品名为Yervoy)；基因泰克(Genentech)公司的人鼠嵌合抗VEGF抗体贝伐单抗(Bevacizumab，商品名Avastin)；Sanofi-aventis公司和Regeneron公司研制的作为VEGF-Trap的阿柏西普(aflibercept)等。但是，有相当一部分的肿瘤患者对单靶向肿瘤治疗无应答或产生耐药性，例如，目前抗PD-1和PD-L1抗体药物平均的治疗有效率仅为20％左右，肺癌的五年生存率仅16％。因此，在许多情况下，需要采用多靶向疗法来避免患者对单靶向肿瘤治疗无应答或产生耐药性的问题。
由于多靶向融合蛋白能够同时特异性靶向参与肿瘤发生和发展的多种信号传导通路，因此，存在研发能够通过阻断血管内皮细胞生长且活化T细胞来改善肿瘤微环境的多靶向融合蛋白、以及将所述多靶向融合蛋白与其他抗癌药联用的需要。
发明概述
本发明人通过锐意研究，开发了一组阻断血管内皮细胞生长且活化T细胞的多靶向融合蛋白，其包含(i)血管内皮生长抑制剂结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。所述多靶向融合蛋白能够从两个方面改善肿瘤微环境，并提高肿瘤免疫治疗效果，其中对肿瘤微环境的改善一方面是通过所述多靶向融合蛋白的CD80胞外结构域(ECD)与CD28、PD-L1和CTLA-4的特异性结合实现的，具体而言，通过CD80胞外结构域结合PD-L1这一免疫检查点，缓解PD-1/PD-L1抑制性信号通路，给免疫系统“松刹车”；通过CD80胞外结构域结合CTLA-4而发挥同抗CTLA-4抗体伊匹单抗同样的抑制调节性T细胞(Treg)的功能；通过CD80胞外结构域结合CD28来活化CD28/B7共刺激途径，并激活T淋巴细胞， 为淋巴细胞的激活“加油门”；对肿瘤微环境的改善另一方面是通过所述多靶向融合蛋白的血管内皮细胞生长抑制剂结构域阻断VEGF/VEGFR通路来抑制肿瘤新生血管的生成，使肿瘤组织血管正常化，从而更多的淋巴细胞侵润到肿瘤组织，以及解除VEGFs对免疫细胞的抑制作用。
在一个实施方案中，本发明的多靶向融合蛋白包含(i)衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段和/或VEGFR胞外受体功能区；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。
所述多靶向融合蛋白中包含的衍生自抗VEGF抗体的抗原结合片段可以衍生自任何抗VEGF抗体的抗原结合片段，只要是能够结合VEGF，并由此阻断或抑制VEGF与其受体VEGFR结合的抗体即可。所述抗VEGF抗体包括现有技术中已知的抗VEGF抗体和将来研发出的抗VEGF抗体。在一个实施方案中，所述抗VEGF抗体的抗原结合片段是抗VEGF抗体的Fab、Fab′、F(ab′) 2、Fv、单链Fv；优选地，所述抗VEGF抗体的抗原结合片段包含选自SEQ ID NO：1/2和3/4的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，或者与所述全部6个重链CDR与轻链CDR中的一个或多个CDR具有一个、两个、三个、四个、或五个氨基酸变化(例如，氨基酸置换或缺失)的序列；更优选地，所述抗VEGF抗体的抗原结合片段包含选自SEQ ID NO：1/2和3/4的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；最优选地，所述抗VEGF抗体的抗原结合片段是Bevacizumab或Ranibizumab的Fab。
所述多靶向融合蛋白中包含的衍生自抗VEGFR抗体的抗原结合片段可以衍生自任何抗VEGFR抗体的抗原结合片段，只要是能够结合VEGFR，并由此阻断或抑制VEGF与其受体VEGFR结合的抗体即可。所述抗VEGFR抗体包括现有技术中已知的抗VEGFR抗体和将来研发出的抗VEGFR抗体。在一个实施方案中，所述抗VEGFR抗体的抗原结合片段是抗VEGFR抗体的Fab、Fab′、F(ab′) 2、Fv、单链Fv；优选地，所述抗VEGFR抗体的抗原结合片段包含SEQ ID NO：5/6的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，或者与所述全部6个重链CDR与轻链CDR中的一个或多个CDR具有一个、两个、三个、四个、或五个氨基酸变化(例如，氨基酸置换或缺失)的序列；更优选地，所述抗VEGFR抗体的抗原结合片段包含SEQ ID NO：5/6的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；最优选地，所述抗VEGFR抗体的抗原结合片段是Ramucirumab的Fab。
所述多靶向融合蛋白中包含的VEGFR胞外受体功能区可以是任何VEGFR胞外受体功能区，只要是能够结合VEGF，并由此阻断或抑制VEGF与其受体VEGFR结合的VEGFR胞外 受体功能区即可。优选地，所述VEGFR胞外受体功能区包含VEGFR1的免疫球蛋白样结构域2和VEGFR2的免疫球蛋白样结构域3；或者所述VEGFR胞外受体功能区包含VEGFR1的免疫球蛋白样结构域2以及VEGFR2的免疫球蛋白样结构域3和VEGFR2的免疫球蛋白样结构域4；或者所述VEGFR胞外受体功能区包含VEGFR1的免疫球蛋白样结构域2；更优选地，所述VEGFR胞外受体功能区具有任一选自SEQ ID NO：7-9所示的氨基酸序列或与SEQ ID NO：7-9所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
所述多靶向融合蛋白中包含的(ii)免疫球蛋白Fc结构域可以是任何免疫球蛋白Fc结构域，特别地，所述(ii)是人免疫球蛋白Fc结构域。在一个实施方案中，所述免疫球蛋白Fc结构域是IgG类抗体的Fc结构域，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体的Fc结构域。在一个优选的实施方案中，包含于本发明多靶向融合蛋白中的所述免疫球蛋白Fc结构域是IgG 1亚类抗体的Fc结构域，特别地是人IgG 1亚类抗体的Fc结构域。在一个优选的实施方案中，包含于本发明多靶向融合蛋白中的所述免疫球蛋白Fc结构域是IgG 4亚类抗体的Fc结构域，特别地是人IgG 4亚类抗体的Fc结构域。在一些实施方案中，本发明多靶向融合蛋白中的(ii)免疫球蛋白Fc结构域包含SEQ ID NO：10、11或12所示氨基酸序列的Fc结构域，或者包含与SEQ ID NO：10、11或12所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc结构域。
所述多靶向融合蛋白中包含的(iii)CD80 ECD是CD80的胞外结构域的一部分。在一个实施方案中，所述CD80 ECD包含CD80免疫球蛋白V(IgV)区(CD80-IgV)。在一个实施方案中，所述CD80 ECD包含CD80免疫球蛋白V区和C区(CD80-IgVIgC)。在一个实施方案中，所述CD80 ECD是人CD80 ECD，优选地所述CD80 ECD包含人CD80 IgV。在一个具体实施方案中，所述CD80-IgV具有SEQ ID NO：13所示的氨基酸序列，或与SEQ ID NO：13的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。在一个实施方案中，所述CD80-IgVIgC具有SEQ ID NO：14所示的氨基酸序列，或与SEQ ID NO：14的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
在一个实施方案中，所述多靶向融合蛋白还包含所述(i)、(ii)和/或(iii)之间的肽接头；优选地，所述肽接头包含一个或多个氨基酸，更优选地包含至少5个氨基酸，最优选地包含选自SEQ ID NO：20-46的肽接头。
在一个实施方案中，所述多靶向融合蛋白从N端至C端以(i)、(ii)和(iii)的顺序；(iii)、(i)和(ii)的顺序；或者(iii)、(ii)和(i)的顺序有效连接。
在一些实施方案中，本发明的多靶向融合蛋白包含
(a)全长抗VEGF抗体、全长抗VEGFR抗体或者全长抗VEGF和VEGFR双特异性抗体；和在所述抗体的两条重链中的每一重链的C端有效连接的一个CD80 ECD；
(b)全长抗VEGF抗体、全长抗VEGFR抗体或者全长抗VEGF和VEGFR双特异性抗体；在所述抗体的两条重链中的每一重链的N端有效连接的一个CD80 ECD；和在所述抗体的两条轻链中的每一轻链的N端有效连接的一个CD80 ECD；
(c)CD80 ECD；在CD80 ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段；
或者
(d)CD80 ECD；在CD80 ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的VEGFR胞外受体功能区；
优选地，所述抗体是IgG类抗体，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体，更特别地是IgG 4亚类抗体；还优选地，所述IgG 4亚类抗体在Fc结构域中第S228位置处包含氨基酸置换，更优选地是氨基酸置换S228P；进一步优选地，所述抗体的轻链型别为κ型或λ型，优选地为κ型；
优选地，所述全长抗VEGF抗体是Bevacizumab，所述全长抗VEGFR抗体是Ramucirumab。
在一些实施方案中，本发明的多靶向融合蛋白选自
(1)包含SEQ ID NO：80的融合蛋白第一亚基和SEQ ID NO：82的融合蛋白第二亚基的融合蛋白；
(2)包含SEQ ID NO：84的融合蛋白第一亚基和SEQ ID NO：86的融合蛋白第二亚基的融合蛋白；
(3)包含SEQ ID NO：88的融合蛋白亚基的融合蛋白。
本发明还提供了编码本发明的多靶向融合蛋白的多核苷酸、包含编码本发明多靶向融合蛋白的多核苷酸的载体，优选地表达载体，最优选地具有双表达盒的谷氨酰胺合成酶表达载体。在另一方面，本发明提供了包含本发明多核苷酸或载体的宿主细胞。在一个实施方案中，所述宿主细胞是CHO、HEK293或NSO细胞。本发明也提供了一种用于产生本发明多靶向融合蛋白的方法，包括步骤(i)在适于表达本发明的多靶向融合蛋白的条件下培养本发明的宿主细胞，和(ii)回收本发明的多靶向融合蛋白。
在另一方面，本发明还提供了本发明的多靶向融合蛋白与抗PD-1抗体组合的药物组合物。本发明的多靶向融合蛋白与抗PD-1抗体联用时显示出了较好的协同性，由此能够更好地实现抑制肿瘤生长的目的。
当与本发明的多靶向融合蛋白组合使用时，抗PD-1抗体可以是任何抗PD-1抗体，只要是能够抑制或减少PD-1与其配体结合的抗体即可，包括现有技术中已知的抗PD-1抗体和将来研发出的抗PD-1抗体。在一些实施方案中，所述抗PD-1抗体包含选自SEQ ID NO：47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70和71/72的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻 链CDR，或者与所述全部6个重链CDR与轻链CDR中的一个或多个CDR具有一个、两个、三个、四个、或五个氨基酸变化(例如，氨基酸置换或缺失)的序列；进一步优选地，所述抗PD-1抗体包含选自SEQ ID NO：47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70和71/72的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；最优选地，所述抗PD-1抗体选自纳武单抗(Nivolumab)、pidilizumab和派姆单抗(Pembrolizumab)。
在又一个方面，本发明提供了本发明的多靶向融合蛋白、药物组合物的用途，用于制备在个体中治疗或预防癌性疾病(例如，实体瘤和软组织瘤)的药物，优选地，癌性疾病是黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)；特别地，所述疾病是结肠癌或三阴性乳腺癌；优选地，其中所述个体是哺乳动物，更优选地是人。
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入本文作为参考。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1A和B提供了本发明的多靶向融合蛋白的示意图，其中图1A例示了从N端至C端包含抗体的抗原结合片段、免疫球蛋白Fc结构域和CD80 ECD的多靶向融合蛋白的结构示意图；图1B例示了从N端至C端包含CD80 ECD、免疫球蛋白Fc结构域和VEGFR胞外受体功能区的融合蛋白的结构示意图。
图2：显示了实施例2中制备并纯化的本发明的各目的蛋白在还原剂(5mM 1，4-二硫苏糖醇)存在下通过SDS-PAGE电泳并用考马斯蓝染色后的结果。泳道1：蛋白分子量标准标志物；泳道2：融合蛋白BY24.4；泳道3：融合蛋白BY24.5；泳道4：融合蛋白BY24.12；泳道5：融合蛋白BY31.19；泳道6：抗体BY18.1
图3：显示了在实施例5的混合淋巴细胞反应(MLR)中本发明的多靶向融合蛋白及其与抗PD-1抗体组合后对IFN-γ分泌的影响。柱1：BY18.1组；柱2：BY24.4组；柱3：BY18.1+BY24.4组；柱4：BY24.5组；柱5：BY18.1+BY24.5组；柱6：BY24.12组；柱7：BY18.1+BY24.12 组。
图4：显示了在实施例6的动物模型中本发明的多靶向融合蛋白及其与抗PD-1抗体组合后对抗肿瘤生长的抑制作用。
发明详述
本发明提供了阻断血管内皮细胞生长且活化T细胞的多靶向融合蛋白和包含所述多靶向融合蛋白的药物组合物。本发明还提供了用于产生该多靶向融合蛋白的方法，以及该多靶向融合蛋白或药物组合物在个体中治疗或预防癌性疾病中的用途。
除非下文中另外定义，否则本说明书中的术语如本领域通常所用那样使用。
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
“PD-1/PD-L1抑制性信号通路”、“PD-1/PD-L1信号通路”、“PD-1/PD-L1信号传导途径”、“PD-1/PD-L1途径”在本文中可以互换使用，是指任何通过PD-1与PD-L1结合而引发的细胞内信号传导途径。
本文所用的“缓解”、“干扰”、“抑制”或“阻断”PD-1/PD-L1抑制性信号传导途径可以互换使用，是指(i)干扰PD-1和PD-L1之间的相互作用；和/或(ii)导致PD-1/PD-L1信号传导途径的至少一种生物学功能的抑制。由本发明的多靶向融合蛋白与PD-L1特异性结合后导致的“缓解”、“干扰”、“抑制”或“阻断”PD-1/PD-L1信号传导途径不需要是完全的缓解、干扰、抑制或阻断。
在本文中“CD28/B7信号传导途径”、“CD28/B7共刺激途径”、“CD28/B7通路”可以互换使用，是指(i)通过CD28与CD80结合而刺激细胞活化的信号传导途径；和/或(ii)通过CD28与CD86结合而刺激细胞活化的信号传导途径。
“CD80”和“CD86”均为跨膜糖蛋白，属结构高度相似的免疫球蛋白超家族(IgSF)成员，亦统称B7分子。CD80和CD86胞外区有一个免疫球蛋白V(IgV)区和免疫球蛋白C(IgC)区构成。成熟CD80分子由254个氨基酸组成，其中胞外区由208个氨基酸、跨膜区25个氨基酸和胞内区21个氨基酸。类似地，成熟CD86分子由303个氨基酸组成，其中胞外区由222个氨基酸、跨膜区20个氨基酸和胞内区61个氨基酸。CD80，亦称B7.1，表达于T细胞、B细胞、树突细胞和单核细胞的表面，通过其免疫球蛋白V(IgV)区以较低亲和力结合CD28、PD-L1和CTLA-4，其中CD80与CD28的结合亲和力为4μM；CD80与PD-L1的结合亲和力为～1.7μM；CD80与CTLA-4的结合亲和力为0.2μM(Butte MJ等人，Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses，Immunity， 2007年7月；27(1)：111-122)。CD86结合CD28和CTLA-4，但不结合PD-L1。
可溶性CD80(例如，CD80-Fc)能够通过CD28/B7共刺激途径对T淋巴细胞产生持续的激活作用，并刺激产生干扰素。实验表明，CD80-Fc在体外维持T淋巴细胞产生干扰素，甚至比抗PD-1抗体或抗PD-L1抗体更有效。在体内抑制肿瘤生长方面，可溶性CD80(例如，CD80-Fc)的抑瘤效果比抗PD-L1抗体更有效(Ostrand-Rosenberg S等人，Novel strategies for inhibiting PD-1 pathway-mediated immune suppression while simultaneously delivering activating signals to tumor-reactive T cells，Cancer Immunol Immunother.，2015年10月；64(10)：1287-93)。CD80-Fc能通过结合PD-L1来抑制PD-1/PD-L1途径介导的免疫抑制，并向通过CD28/B7共刺激途径激活的T细胞递送共刺激信号，从而增强T淋巴细胞活化。总之，CD80-Fc可以缓解PD-1/PD-L1途径的免疫抑制作用，同时激活肿瘤免疫反应性T细胞。虽然可溶性CD86(例如，CD86-Fc)也可激活CD28，甚至可产生CD80-Fc的3-5倍激活效应，但由于CD86不结合PD-L1，最终CD80-Fc对T淋巴细胞的激活作用强于CD86-Fc(Haile ST等人，Soluble CD80 restores T cell activation and overcomes tumor cell programmed death ligand 1-mediated immune suppression.，J Immunol.，2013年9月1日；191(5)：2829-36)。根据已有的研究表明CD80-Fc具有如下作用：(i)CD80-Fc单独使用时，抑制肿瘤的效果好于PD-L1抗体(AACR ANNUAL MEETING，2018年4月14-18日，美国，伊利诺伊州，芝加哥)；(ii)CD80-Fc促进淋巴细胞侵润肿瘤组织，且效果好于PD-L1抗体(Horn LA等人，Soluble CD80 Protein Delays Tumor Growth and Promotes Tumor-Infiltrating Lymphocytes，Cancer Immunol Res.，2018年1月；6(1)：59-68)；(iii)CD80-Fc单独使用时，抑制肿瘤的效果好于PD-1/PD-L1途径的抑制剂，并且与PD-1抗体联用时有协同作用。Five Prime公司甚至认为CD80-Fc优于GITRL、OX40L和4-1BBL等T细胞激动剂。由于看到了CD80-Fc良好的免疫治疗效果，Five Prime Therapeutics，Inc.的CD80-Fc项目FPT155计划在最近开展临床试验。
在本文中“B7/CTLA-4通路”、“B7/CTLA-4信号传导途径”可以互换使用，是指(i)通过CD80与CTLA-4结合而引起的信号传导途径；和/或(ii)通过CD86与CTLA-4结合而引起的信号传导途径。
在本文中“VEGF/VEGFR通路”、“VEGF/VEGFR信号传导途径”可以互换使用，是指通过VEGF家族中的一种或多种与细胞表面受体VEGFR家族中的一种或多种结合介导的信号传导途径。VEGF家族包含六种密切相关的多肽，分别是高度保守的同源二聚体糖蛋白，有六个亚型：VEGF-A、-B、-C、-D、-E、和胎盘生长因子(placental growth factor(PLGF))，分子量从35至44kDa不等。VEGF-A(包括其剪接物如VEGF 165)的表达与一些实体瘤的微血管密度具有相关性，并且组织中VEGF-A的浓度与乳腺癌、肺癌、前列腺癌和结肠癌等实体瘤的预后有关。每个VEGF家族成员的生物学活性通过细胞表面VEGF受体(VEGFR)家族中的一种或多种介导，所述VEGFR家族包括VEGFR1(也称为Flt-1)、VEGFR2(也称为KDR、Flk-1)、VEGFR3(也称为Flt-4)等，其中VEGFR1、VEGFR2与血管的生成关系密切，VEGF-C/D /VEGFR3则与淋巴管生成密切相关。VEGF家族的主要生物学功能包括：(1)选择性促进血管内皮细胞有丝分裂，刺激内皮细胞增殖并促进血管形成；(2)提高血管尤其是微小血管的通透性，使血浆大分子外渗沉积在血管外的基质中，为肿瘤细胞的生长和新生毛细血管网的建立提供营养；(3)促进肿瘤的增殖和转移，所述肿瘤的增殖和转移依赖VEGF家族使血管内皮细胞分泌胶原酶和纤溶酶原，借以降解血管基底膜，同时，肿瘤组织内部新形成的微血管基膜不完善，这种性质使肿瘤易于进入血循环；(4)VEGF可作为一种免疫抑制分子，抑制机体的免疫反应，促进恶性肿瘤的浸润与转移(Lapeyre-Prost A等人，Immunomodulatory Activity of VEGF in Cancer，Int Rev Cell Mol Biol.，2017；330：295-342)；(5)其他作用：VEGF家族可诱导上皮细胞出现间隙及开窗现象，可活化上皮细胞的胞质小泡及细胞器；VEGF家族直接刺激内皮细胞释放蛋白水解酶，降解基质，释放更多的VEGF家族分子，加速肿瘤的发展，细胞外蛋白酶又可激活细胞外基质的结合性和VEGF家族的释放；VEGF家族通过增加血管通透性使血浆蛋白(包括纤维蛋白原)释放，形成纤维素网络，为肿瘤生长、发展和转移提供了良好的基质；VEGF家族促进异常血管的生成，阻碍免疫细胞侵润等作用。
临床研究显示利用抗VEGF单克隆抗体、抗VEGFR单克隆抗体、或可溶性VEGFR能够阻断VEGF家族与其受体的结合，并阻碍VEGF家族信号通路的传导。基因泰克(Genentech)公司研发的贝伐单抗(Bevacizumab，商品名Avastin)是一种重组的人鼠嵌合抗VEGF抗体，可通过阻断VEGF-A与VEGFR的结合，使VEGFR无法活化，由此发挥抗血管生成的作用。贝伐单抗目前用于转移性结直肠癌、肺癌、乳癌、胰脏癌、肾脏癌等的治疗。Sanofi-aventis公司和Regeneron公司研制的阿柏西普(aflibercept)是一种VEGF-Trap，其是将VEGFR1胞外第2个结构域和VEGFR2胞外第3个结构域与人IgG1恒定区融合获得的一种融合蛋白，能通过抑制血管生成而对一部分肿瘤患者发挥抗肿瘤作用。
如本文所用，术语“特异性结合”意指对抗原或目的分子的结合具有选择性并且可以与不想要的或非特异的相互作用区别。所述特异性结合可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术，例如表面等离子体共振(SPR)技术(在BIAcore仪上分析)(Liljeblad等人，Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance，Glyco J.，2000，17，323-329)测量。
术语“血管内皮生长抑制剂结构域”指阻断VEGF/VEGFR信号传导的血管内皮生长抑制剂的部分，所述部分是发挥抑制血管内皮生长的区域。血管内皮生长抑制剂结构域可以由例如一个或多个抗VEGF抗体的可变结构域(也称作抗体可变区)、一个或多个抗VEGFR抗体的可变结构域、或VEGFR胞外受体功能区提供。
术语“调节性T细胞(Treg)”代表对维持自我耐受性至关重要的特定T淋巴细胞亚群。具有阻抑物功能的所述Treg细胞能通过细胞内表达转录因子FOXP3以及其他细胞标志物如CD127 低、CTLA-4 +、LAP、CD39 +、PD-1 +、GARP等与其他T淋巴细胞区分。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X 对其配偶物Y的亲和力可以通常由解离常数(K D)代表，解离常数是解离速率常数和缔合速率常数(分别是k off和k on)的比例。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是表面等离子体共振法(SPR)。
术语“抗体”在本文中以最广意义使用并且包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)，只要它们显示出所需的抗原结合活性即可。抗体可以是任何型和亚型(例如，IgM、IgD、IgG1、IgG2、IgG3、IgG4、IgE、IgA1和IgA2)的完整抗体(例如，具有两个全长的轻链和两个全长的重链)。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指一种抗体，所述抗体具有基本上与天然抗体结构相似的结构。
术语“抗体重链”指以其天然存在构象在抗体分子中存在的两种类型多肽链中的较大者，其在正常情况下决定抗体所属的类别。
术语“抗体轻链”指以其天然存在构象在抗体分子中存在的两种类型多肽链中的较小者。κ轻链和λ轻链指两个主要的抗体轻链型别。
“双特异性抗体”是具有两个不同的重链/轻链对且具有两个不同的结合部位的人工杂合抗体。可以通过多种方法，包括杂交瘤融合或Fab’片段的连接制备双特异抗体。
术语抗体的“抗原结合片段”是比完整或完全抗体或抗体链更少的氨基酸残基的抗体或抗体链的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv。所述Fab片段是一种由V L、V H、C L和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab′) 2，其为Fab’的二聚体，是双价片段。F(ab′) 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，因此将F(ab′) 2二聚体转化为Fab′单体。Fab′单体基本上是具有铰链区的Fab片段(其它抗体片段的更详细的描述请参见：基础免疫学(Fundamental Immunology)，W.E.Paul编辑，Raven Press，N.Y.(1993))。所述Fv片段由抗体单臂的V L和V H结构域组成。另外，虽然Fv片段的两个结构域V L和V H由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性接头连接，在所述单条蛋白链中V L区和V H区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗体片段。
术语“免疫球蛋白”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫球蛋白是由二硫键结合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端，每条免疫球蛋白重链具有一个可变区(VH)，也称作可变重链域或重链可变结构域，随后是三个恒定结构域(CH1、CH2和CH3)，也称作重链恒定区。类似地，从N端至C端，每条免疫球蛋白轻链具有一个可变区(VL)，也称作可变轻链域或轻链可变结构域，随后一个恒定轻链(CL)结构域，也称作轻链恒定区。免疫球蛋白的重链可以归属5个类别之一，称作α(IgA)、 δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，其中某些类别可以进一步划分成亚类，例如γ 1(IgG1)、γ 2(IgG2)、γ 3(IgG 3)、γ 4(IgG 4)、α 1(IgA 1)和α 2(IgA 2)。免疫球蛋白的轻链可以基于其恒定结构域的氨基酸序列而划分成两种型之一，称作κ和λ。免疫球蛋白基本上由借助免疫球蛋白铰链区连接的两个Fab分子和一个Fc结构域组成。
术语“Fc结构域”或“Fc区”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的两条重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如Kabat等人，Sequences of Proteins of Immunological Interes，第5版，Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述的EU编号体系(也称作EU索引)进行编号。
“人免疫球蛋白”是这样一种免疫球蛋白，其拥有对应于人或人细胞产生的免疫球蛋白的氨基酸序列或从利用人免疫球蛋白库或其他编码人免疫球蛋白的序列的非人来源衍生。
氨基酸序列的“同一性百分数(％)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。
术语“有效连接”意指指定的各组分处于一种允许它们以预期的方式起作用的关系。
术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
术语“融合”指将两个或多个组分由肽键直接连接或借助一个或多个肽接头有效连接。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明多靶向融合蛋白的任何类型的细胞系统。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体是人。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的多靶向融合蛋白或药物组合物用来延缓疾病发展或用来减慢疾病的进展。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体 积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。术语“肿瘤”、“癌症”和“癌性疾病”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
II.多靶向融合蛋白
本发明提供了一种新型的多靶向融合蛋白，其包含(i)血管内皮生长抑制剂结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。
在一些实施方案中，所述(i)、(ii)和/或(iii)任选地通过肽接头有效连接。
在一些实施方案中，本发明的多靶向融合蛋白是由二硫键键合的两个融合蛋白第一亚基和两个融合蛋白第二亚基组成的异四聚体糖蛋白。
在一些实施方案中，本发明的多靶向融合蛋白是由二硫键键合的同二聚体或异二聚体糖蛋白。
本发明的多靶向融合蛋白阻断血管内皮细胞生长且活化T淋巴细胞。该多靶向融合蛋白一方面能够通过阻断VEGF/VEGFR通路来阻断血管内皮细胞生长，另一方面能够通过活化CD28/B7共刺激途径、抑制PD-1/PD-L1抑制性信号通路、由B7/CTLA-4信号传导途径抑制调节性T细胞(Treg)功能来活化T淋巴细胞，从而改善肿瘤微环境，并提高肿瘤免疫治疗效果。
在一些实施方案中，本发明的多靶向融合蛋白以10 -8M或更小、例如以10 -9M至10 -12M的解离常数(K D)与VEGF或VEGFR结合；且与CD28、PD-L1和CTLA-4特异性结合。
以下对本发明多靶向融合蛋白的各组分分别进行描述。
-血管内皮生长抑制剂结构域
本发明多靶向融合蛋白中的“血管内皮生长抑制剂结构域”能够特异性结合VEGF和/或VEGFR，包括但不限于衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段和/或VEGFR胞外受体功能区。
在一个实施方案中，本发明多靶向融合蛋白中包含的衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段使得本发明的多靶向融合蛋白能够以高亲和力，例如以10 -8M或更小、优选地以10 -9M至10 -12M的K D与VEGF和/或VEGFR特异性结合，并由此阻断VEGF与其受体VEGFR结合所介导的信号传导途径。
本文在下表1中提供了本发明多靶向融合蛋白中包含的抗VEGF抗体或抗VEGFR抗体的抗原结合片段中成对重链可变区(VH)和轻链可变区(VL)的例子。在一些实施方案中，本发明多靶向融合蛋白中的抗原结合片段包含与表1中抗VEGF抗体和/或抗VEGFR抗体的重链可变区(VH)和/或轻链可变区(VL)的氨基酸序列基本上同一的序列，例如，与表1中所示的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
表1.抗VEGF抗体或抗VEGFR抗体的抗原结合片段中重链可变区和轻链可变区序列的例子
Figure PCTCN2020071213-appb-000001
在一个实施方案中，本发明多靶向融合蛋白中的抗原结合片段包含选自SEQ ID NO：1/2、3/4和5/6的成对重链可变区序列/轻链可变区序列中所含的全部6个重链互补决定区(CDR)与轻链CDR。用于鉴定重链可变区与轻链可变区的氨基酸序列中的CDR的方法及技术为本领域中已知的，且可用于鉴定本文公开的特定重链可变区及/或轻链可变区的氨基酸序列中的CDR。可用于鉴定CDR边界的示例性公知技术包括例如Kabat界定法、Chothia界定法以及AbM界定法。参见，例如Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health，Bethesda，Md.(1991)；Al-Lazikani等人，Standard conformations for the canonical structures of immunoglobulins.，J.Mol.Biol.273：927-948(1997)；以及Martin AC等人，Modeling antibody hypervariable loops：a combined algorithm，Proc.Natl.Acad.Sci.USA 86：9268-9272(1989)。
作为本发明多靶向融合蛋白中的抗原结合片段来源的抗VEGF抗体或抗VEGFR抗体可以基于其轻链恒定区的氨基酸序列而划分为κ型或λ型，优选为κ型。
本文在下表2中提供了抗VEGF抗体或抗VEGFR抗体轻链恒定区的氨基酸序列的例子。
表2.抗体的轻链恒定区序列的例子
Figure PCTCN2020071213-appb-000002
Figure PCTCN2020071213-appb-000003
作为本发明多靶向融合蛋白中的抗原结合片段来源的抗VEGF抗体或抗VEGFR抗体基于其重链恒定区的氨基酸序列优选地是IgG类抗体，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体，更特别地是IgG 4亚类抗体。优选地，所述IgG 4亚类抗体在Fc区中第S228位置处包含防止发生臂交换(arm-exchange)的氨基酸置换，特别地是氨基酸置换S228P。
本文在下表3中提供了抗体重链恒定区的氨基酸序列的例子。
表3.抗体的重链恒定区序列的例子
Figure PCTCN2020071213-appb-000004
在一个实施方案中，本发明多靶向融合蛋白中包含的VEGFR胞外受体功能区是VEGFR的胞外结构域的一部分或其组合。VEGFR受体是位于细胞表面的一种酪氨酸激酶受体，其胞外区由7个免疫球蛋白(Ig)样结构域组成。例如，人VEGFR1包含编号为1、2、3、4、5、6和7的七个Ig样结构域，Ig样结构域1在胞外结构域的N端，Ig样结构域7在胞外结构域 的C端。除非本文另外指出，否则Ig样结构域从VEGFR蛋白的N端至C端顺序编号。在一些实施方案中，VEGFR胞外受体功能区包含选自VEGFR1、VEGFR2和VEGFR3的一种或多种VEGFR的至少一个Ig样结构域。在一些方面，VEGFR胞外受体功能区包含VEGFR的至少1、2、3、4、5、6个但不超过7个Ig样结构域。在另一方面，VEGFR胞外受体功能区包含VEGFR的1至7、1至6、1至5、1至4、1至3或1至2个Ig样结构域。
本文还考虑了包含两种或多种VEGFR的至少一个Ig样结构域的VEGFR胞外受体功能区。在一些实施方案中，VEGFR胞外受体功能区包含来自两种或多种选自VEGFR1、VEGFR2和VEGFR3的VEGFR的至少一个Ig样结构域。本文考虑了包含每种VEGFR的七个Ig样结构域的任意组合的VEGFR胞外受体功能区。例如，VEGFR胞外受体功能区可以包含VEGFR1(例如人VEGFR1)的Ig样结构域2和VEGFR2(例如人VEGFR2)的Ig样结构域3。在另一实施方案中，VEGFR胞外受体功能区可以包含VEGFR1(例如人VEGFR1)的Ig样结构域1-3、VEGFR1(例如人VEGFR1)的Ig样结构域2-3、VEGFR2(例如人VEGFR2)的Ig样结构域1-3、VEGFR1(例如人VEGFR1)的Ig样结构域2和VEGFR2(例如人VEGFR2)的Ig样结构域3-4，或VEGFR1(例如人VEGFR1)的Ig样结构域2和VEGFR3(例如人VEGFR3)的Ig样结构域3。这些Ig样结构域和其他可以用作VEGFR胞外受体功能区的部分的Ig样结构域的更详细描述见美国专利号7531173；Yu DC等，Soluble vascular endothelial growth factor decoy receptor FP3 exerts potent antiangiogenic effects，Mol.Ther.，2012，20(3)：938-947和Holash，J.等，VEGF-Trap：a VEGF blocker with potent antitumor effects，PNAS，2002，99(17)：11393-11398，全部文献在此以其整体引入作为参考。在一些实施方案中，VEGFR胞外受体功能区具有任一选自表4中SEQ ID NO：7-9所示的氨基酸序列或与SEQ ID NO：7-9所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
表4多靶向融合蛋白中的VEGFR胞外受体功能区氨基酸序列的例子
Figure PCTCN2020071213-appb-000005
Figure PCTCN2020071213-appb-000006
本发明多靶向融合蛋白中的VEGFR胞外受体功能区能够以高的亲和力，例如以10 -8M或更小、优选地以10 -9M至10 -12M的K D，与VEGF家族特异性结合，并由此抑制VEGF家族与细胞表面VEGFR的结合和随后的信号传导。
-免疫球蛋白Fc结构域
本发明多靶向融合蛋白中的“免疫球蛋白Fc结构域”包含天然存在的免疫球蛋白Fc结构域的全部氨基酸残基或包含天然存在的免疫球蛋白Fc结构域的一部分氨基酸残基。免疫球蛋白Fc结构域对本发明的多靶向融合蛋白提供有利的药代动力学特性，包括但不限于长血清半寿期。另外，免疫球蛋白Fc结构域还使得通过例如蛋白A亲和层析纯化本发明的多靶向融合蛋白成为可能。
免疫球蛋白Fc结构域通常是二聚体分子。可以通过木瓜蛋白酶消化或胰蛋白酶消化完整(全长)免疫球蛋白来产生或可以重组产生免疫球蛋白Fc结构域，其包含CH2结构域、CH3结构域和可选的CH4结构域。
在一个实施方案中，IgG Fc区包含IgG CH2结构域和IgG CH3结构域。优选地，免疫球蛋白Fc结构域具有表5中SEQ ID NO：10-12所示的氨基酸序列或者具有与SEQ ID NO：10-12所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
表5多靶向融合蛋白中的免疫球蛋白Fc结构域氨基酸序列的例子
Figure PCTCN2020071213-appb-000007
Figure PCTCN2020071213-appb-000008
除了如SEQ ID NO：10-12所定义的序列之外，IgG Fc区还可以包含对SEQ ID NO：10-12进行额外的序列修饰后获得的肽序列，例如对SEQ ID NO：10-12中的氨基酸残基进行一个或多个氨基酸替换、缺失或衍生后获得的肽序列。在一个实施方案中，在IgG Fc区中第S228位置处包含防止发生臂交换(arm-exchange)的氨基酸置换，特别地是氨基酸置换S228P。在一个实施方案中，IgG4 Fc区包含氨基酸置换S228P。
-CD80的胞外结构域(ECD)
本发明多靶向融合蛋白中的“CD80的胞外结构域(ECD)”包含天然存在的CD80ECD的全部氨基酸残基或包含天然存在的CD80ECD的一部分氨基酸残基。在一些实施方案中，所述CD80ECD包含CD80 IgV，优选地，所述CD80 ECD包含人CD80 IgV，更优选地，所述CD80ECD具有表6中SEQ ID NO：13或14所示的氨基酸序列。
表6多靶向融合蛋白中的CD80ECD氨基酸序列的例子
Figure PCTCN2020071213-appb-000009
除了如SEQ ID NO：13和14所定义的序列之外，CD80ECD还可以包含对SEQ ID NO：13和14进行额外的序列修饰后获得的肽序列，例如对SEQ ID NO：13和14中的氨基酸残基进行一个或多个保守性替换、缺失或衍生后获得的肽序列，只要具有与未修饰的肽基本上相同的活性或功能即可。经修饰的肽将保留与未修饰肽相关的活性或功能。经修饰的肽通常具有与未修饰序列的氨基酸序列基本上同源的氨基酸序列，例如，与SEQ ID NO：13或14所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
-肽接头
本发明的多靶向融合蛋白中将(i)血管内皮生长抑制剂结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80 ECD任选地有效连接的“肽接头”是一个或多个氨基酸、一般约2-20个氨基酸的肽。本领域已知或本文中描述了肽接头。
在一些实施方案中，所述肽接头包含至少5个氨基酸，优选地包含选自 AKTTPKLEEGEFSEAR(SEQ ID NO：20)；AKTTPKLEEGEFSEARV(SEQ ID NO：21)；AKTTPKLGG(SEQ ID NO：22)；SAKTTPKLGG(SEQ ID NO：23)；SAKTTP(SEQ ID NO：24)；RADAAP(SEQ ID NO：25)；RADAAPTVS(SEQ ID NO：26)；RADAAAAGGPGS(SEQ ID NO：27)；RADAAAA(SEQ ID NO：28)；SAKTTPKLEEGEFSEARV(SEQ ID NO：29)；ADAAP(SEQ ID NO：30)；DAAPTVSIFPP(SEQ ID NO：31)；TVAAP(SEQ ID NO：32)；TVAAPSVFIFPP(SEQ ID NO：33)；QPKAAP(SEQ ID NO：34)；QPKAAPSVTLFPP(SEQ ID NO：35)；AKTTPP(SEQ ID NO：36)；AKTTPPSVTPLAP(SEQ ID NO：37)；AKTTAP(SEQ ID NO：38)；AKTTAPSVYPLAP(SEQ ID NO：39)；ASTKGP(SEQ ID NO：40)；ASTKGPSVFPLAP(SEQ ID NO：41)；GGGGSGGGGSGGGGS(SEQ ID NO：42)；GENKVEYAPALMALS(SEQ ID NO：43)；GPAKELTPLKEAKVS(SEQ ID NO：44)；GHEAAAVMQVQYPAS(SEQ ID NO：45)；GGGGSGGGGSGGGGSA(SEQ ID NO：46)的肽接头。
-多靶向融合蛋白
本文提供了按任意顺序包含(i)血管内皮生长抑制剂结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80 ECD的多靶向融合蛋白，包括但不限于多靶向融合蛋白从N端至C端以(i)、(ii)和(iii)的顺序；(iii)、(i)和(ii)的顺序；或者(iii)、(ii)和(i)的顺序有效连接。
在一个实施方案中，所述多靶向融合蛋白从N端至C端包含全长抗VEGF抗体、全长抗VEGFR抗体或者全长抗VEGF和VEGFR双特异性抗体；和在所述抗体的两条重链中的每一重链的C端有效连接的一个CD80 ECD。
在另一个实施方案中，所述多靶向融合蛋白包含全长抗VEGF抗体、全长抗VEGFR抗体或者全长抗VEGF和VEGFR双特异性抗体；在所述抗体的两条重链中的每一重链的N端有效连接的一个CD80 ECD；和在所述抗体的两条轻链中的每一轻链的N端有效连接的一个CD80 ECD。
在又一个实施方案中，所述多靶向融合蛋白从N端至C端包含CD80 ECD；在CD80 ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段。
在又一个实施方案中，所述多靶向融合蛋白从N端至C端包含CD80 ECD；在CD80 ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的VEGFR胞外受体功能区。
III.本发明的多靶向融合蛋白的生产和纯化
本发明的多靶向融合蛋白可以例如通过固态肽合成(例如Merrifield固相合成)或重组生产获得。为了重组生产，将编码所述多靶向融合蛋白的各亚基的多核苷酸分离并插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。使用常规方法，可以轻易地分离所述多核苷酸并将其测序。在一个实施方案中，提供了包含本发明的一种或多种多核苷酸的载体，优 选地表达载体。
可以使用本领域技术人员熟知的方法来构建表达载体。表达载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个优选的实施方案中，使用了具有双表达盒的谷氨酰胺合成酶高效表达载体。
一旦已经制备了用于表达的包含本发明的一种或多种多核苷酸的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。
在一个实施方案中，提供了包含一种或多种本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。如本文所用，术语“宿主细胞”指可以工程化以产生本发明的多靶向融合蛋白的任何种类的细胞系统。适于复制和支持本发明的多靶向融合蛋白表达的宿主细胞是本领域熟知的。根据需要，这类细胞可以用特定表达载体转染或转导，并且可以培育大量含有载体的细胞用于接种大规模发酵器以获得足够量的本发明多靶向融合蛋白用于临床应用。合适的宿主细胞包括原核微生物，如大肠杆菌，真核微生物如丝状真菌或酵母，或各种真核细胞，如中国仓鼠卵巢细胞(CHO)、昆虫细胞等。可以使用适于悬浮培养的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK 293或293F细胞)、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、CHO细胞、NSO细胞、骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO、HEK293或NSO细胞。
本领域已知在这些宿主细胞系统中表达外源基因的标准技术。在一个实施方案中，提供了产生本发明的多靶向融合蛋白的方法，其中所述方法包括在适于表达所述多靶向融合蛋白的条件下培养如本文中提供的宿主细胞，所述宿主细胞包含编码所述多靶向融合蛋白的多核苷酸，并且从宿主细胞(或宿主细胞培养基)回收所述多靶向融合蛋白。
如本文所述制备的多靶向融合蛋白可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于如净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。
可以通过多种熟知分析方法中的任一种方法确定本发明的多靶向融合蛋白的纯度，所述熟知分析方法包括凝胶电泳、高效液相色谱等。可以通过本领域已知的多种测定法，鉴定、筛选或表征本文提供的多靶向融合蛋白的物理/化学特性和/或生物学活性。
IV.联合疗法
PD-1是一种免疫抑制蛋白，其有两种配体，分别为PD-L1和PD-L2。已知PD-1和PD-L1之间的相互作用导致例如肿瘤浸润淋巴细胞的减少和/或癌细胞的免疫逃避。通过抑制PD-1与PD-L1或PD-L2的局部相互作用可以逆转免疫抑制；当PD-1与PD-L2的相互作用也被阻断时，该效果是相加的(Iwai Y.等人，Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade，Proc.Nat′l.Acad.Sci.USA，2002，99：12293-7)。鉴于免疫检查点PD-1的信号传导在调节免疫应答中的重要性，本发明开发了用于联合疗法的药物组合物，其包含本发明的多靶向融合蛋白和抗PD-1抗体。
与使用本发明多靶向融合蛋白的单一疗法或抗PD-1抗体的单一疗法相比，本文所述的用于联合疗法的药物组合物可以提供优越的有益效果，例如增强的抗癌效果、降低的毒性和/或减少的副作用。例如，药物组合物中的本发明多靶向融合蛋白和/或抗PD-1抗体可以以比单一疗法施用相比达到相同的治疗效果所需的更低剂量或更短的施用时间施用。因此，本发明还公开了使用用于联合疗法的药物组合物来治疗癌症。可以在本领域已知的细胞模型和动物模型中检测前述药物组合物的有效性。
所述联合疗法中包含的抗PD-1抗体可以是任何抗PD-1抗体，只要是能够抑制或减少PD-1与其配体结合的抗体即可，包括现有技术中已知的抗PD-1抗体和将来研发出的抗PD-1抗体。抗PD-1抗体能够以高的亲和力，例如以10 -8M或更小、优选地以10 -9M至10 -12M的K D，与PD-1特异性结合，并由此阻断PD-1与配体PD-L1和/或PD-L2结合所介导的信号传导途径径。
本文在下表7中提供了本发明联合疗法中包含的抗PD-1抗体的成对重链可变区(VH)和轻链可变区(VL)的例子。在一些实施方案中，本发明联合疗法中的抗PD-1抗体包含与表7中所示的氨基酸序列基本上同一的序列，例如，与表7所示的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
表7.联合疗法中的抗PD-1抗体的重链可变区和轻链可变区序列的例子
Figure PCTCN2020071213-appb-000010
Figure PCTCN2020071213-appb-000011
Figure PCTCN2020071213-appb-000012
在一个实施方案中，本发明联合疗法中的抗PD-1抗体包含选自SEQ ID NO：47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70和71/72的成对重链可变区序列/轻链可变区序列中所含的全部重链CDR与轻链CDR。用于鉴定重链可变区与轻链可变区的氨基酸序列中的CDR的方法及技术为本领域中已知的，且可用于鉴定本文公开的特定重链可变区及/或轻链可变区的氨基酸序列中的CDR。可用于鉴定 CDR边界的示例性公知技术包括例如Kabat界定法、Chothia界定法以及AbM界定法。参见，例如Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health，Bethesda，Md.(1991)；Al-Lazikani等人，Standard conformations for the canonical structures of immunoglobulins.，J.Mol.Biol.273：927-948(1997)；以及Martin AC等人，Modeling antibody hypervariable loops：a combined algorithm，Proc.Natl.Acad.Sci.USA 86：9268-9272(1989)。
在一个实施方案中，本发明联合疗法中的抗PD-1抗体选自纳武单抗(Nivolumab)、pidilizumab和派姆单抗(Pembrolizumab)。
本发明的药物组合物可以包含“治疗有效量”或“预防有效量”的本发明所述多靶向融合蛋白和抗PD-1抗体。“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。可以根据多种因素如疾病状态、个体的年龄、性别和重量等变动治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的受试者，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约60％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的药物组合物抑制可度量参数(例如，肿瘤体积)的能力。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量小于治疗有效量。
由于在肿瘤免疫中，如在实施例中所证实的那样，本发明联合疗法中的多靶向融合蛋白和抗PD-1抗体的治疗方案能够相互协同，因此，本发明的药物组合物对于防止肿瘤的免疫逃逸是有利的。共施用本发明的包含多靶向融合蛋白和抗PD-1抗体的药物组合物可以通过分别独立地施用多靶向融合蛋白或抗PD-1抗体实施，也可以单次施用多靶向融合蛋白和抗PD-1抗体的联合制剂。本发明联合疗法中的多靶向融合蛋白和抗PD-1抗体的施用允许给药量和时程的灵活性。
V.多靶向融合蛋白和药物组合物的用途
本文公开的多靶向融合蛋白和药物组合物具有癌症的治疗性和预防性用途。例如，可以将多靶向融合蛋白以及其与抗PD-1抗体的组合物施用至体外或离体的培养细胞或施用至受试者，例如，人类受试者，以治疗和/或预防多种癌性疾病。
在一个方面，本发明涉及使用多靶向融合蛋白或本发明的药物组合物体内用来治疗或预防需要在受试者中阻断血管内皮细胞生长且活化T细胞的疾病，从而抑制或减少相关疾病如癌性肿瘤的生长或出现、转移或复发。可以单独使用多靶向融合蛋白以抑制癌性肿瘤的生长或者预防其出现。备选地，多靶向融合蛋白可以与其他癌症治疗剂/预防剂(例如，抗PD-1抗体)组合施用。当本发明的多靶向融合蛋白与抗PD-1抗体施用时，这种组合可以按任何顺序施用或者同时施用。
因此，在一个实施方案中，本发明提供一种抑制受试者中肿瘤细胞生长的方法，所述方 法包括向受试者施用治疗有效量的本文所述的多靶向融合蛋白或药物组合物。在另一个实施方案中，本发明提供一种防止受试者中肿瘤细胞出现或者转移或者复发的方法，所述方法包括向受试者施用预防有效量的本文所述的多靶向融合蛋白或药物组合物。
在一些实施方案中，用多靶向融合蛋白或药物组合物治疗和/或预防的癌包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓瘤)及转移性病灶。在一个实施方案中，癌是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器官系统的肉瘤和癌，如侵袭肺、乳房、卵巢、淋巴样、胃肠道的(例如，结肠)、肛门、生殖器和生殖泌尿道(例如，肾、膀胱上皮、膀胱细胞、前列腺)、咽、CNS(例如，脑、神经的或神经胶质细胞)、头和颈、皮肤(例如，黑素瘤)、鼻咽(例如，分化或未分化的转移性或局部复发性鼻咽癌)和胰的那些癌、以及腺癌，包括恶性肿瘤，如结肠癌、直肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌和食道癌。癌症可以处于早期、中期或晚期或是转移性癌。
在一些实施方案中，癌选自黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1、包含目的基因的谷氨酰胺合成酶高效表达载体的构建
(1)抗PD1抗体BY18.1的编码核苷酸的合成及表达载体的构建
根据International Nonproprietary Name(INN)数据库中编号为9623的纳武单抗的氨基酸序列数据，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的下述核苷酸序列，并委托上海捷瑞生物工程有限公司合成该核苷酸序列。所述核苷酸序列表达后产生的抗PD1抗体在本文中表示为抗体BY18.1。
抗PD1抗体BY18.1的轻链(BY18.1L)核苷酸序列(SEQ ID NO：73)：
Figure PCTCN2020071213-appb-000013
Figure PCTCN2020071213-appb-000014
抗PD1抗体BY18.1的轻链(BY18.1L)氨基酸序列(SEQ ID NO：74)：
Figure PCTCN2020071213-appb-000015
抗PD1抗体BY18.1的重链(BY18.1H)核苷酸序列(SEQ ID NO：75)：
Figure PCTCN2020071213-appb-000016
Figure PCTCN2020071213-appb-000017
抗PD1抗体BY18.1的重链(BY18.1H)氨基酸序列(SEQ ID NO：76)：
Figure PCTCN2020071213-appb-000018
其中带下划线部分“ METDTLLLWVLLLWVPGSTG”为信号肽序列。
上海捷瑞生物工程有限公司合成了上述BY18.1L编码核苷酸序列和BY18.1H编码核苷酸序列。分别将BY18.1L编码核苷酸用XhoI-EcoRI双酶切，将具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比洋生物技术有限公司)用XhoI-EcoRI双酶切，再通过连接酶将经XhoI-EcoRI双酶切的BY18.1L编码核苷酸连接入经XhoI-EcoRI双酶切的具有双表达盒的谷氨酰胺合成酶高效表达载体，获得已导入了BY18.1L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体；然后，分别将BY18.1H编码核苷酸用XbaI-SalI双酶切，将已导入了BY18.1L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体用XbaI-SalI双酶切，再通过连接酶将经XbaI-SalI双酶切的BY18.1H编码核苷酸连接入经XbaI-SalI双酶切的已导入了BY18.1L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，由此获得了已导入BY18.1L编码核苷酸和BY18.1H编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，经测序验证正确后表达，获得抗PD1抗体BY18.1。
备选地，也可以将BY18.1L编码核苷酸连接入已导入了BY18.1H编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，表达并获得抗体BY18.1。
(2)包含CD80胞外结构域和免疫球蛋白Fc区的融合蛋白的编码核苷酸的合成及表达载体的构建
根据表1中CD80胞外结构域的序列、以及表6中IgG4Fc序列，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的核苷酸序列，并委托上海捷瑞生物工程有限公司合成如下SEQ ID NO：77的多核苷酸序列。所述核苷酸序列表达后产生的CD80-Fc融合蛋白在本文中也表示为融合蛋白BY31.19。
融合蛋白BY31.19(CD80-Fc，IgG4)的核苷酸序列(SEQ ID NO：77)
Figure PCTCN2020071213-appb-000019
Figure PCTCN2020071213-appb-000020
融合蛋白BY31.19(CD80-Fc，IgG4)的氨基酸序列(SEQ ID NO：78)
Figure PCTCN2020071213-appb-000021
其中氨基酸序列“ METDTLLLWVLLLWVPGSTG”为信号肽。
使用上述实施例1(1)相同的方法，通过XhoI-EcoRI双酶切将BY31.19编码核苷酸连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比洋生物技术有限公司)。将重组载体测序验证正确后用于CD80-Fc融合蛋白表达。所表达的CD80-Fc融合蛋白命名为融合蛋白BY31.19。
(3)包含抗VEGF抗体与CD80胞外结构域的融合蛋白BY24.4的编码核苷酸的合成及表达载体的构建
根据表1中CD80胞外结构域的序列、表2中抗VEGF抗体的重链可变区和轻链可变区序列、表3中抗体的重链恒定区序列、表4中抗体的轻链恒定区序列、以及SEQ ID NO：20 -46的肽接头序列，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的核苷酸序列，并委托上海捷瑞生物工程有限公司合成如下SEQ ID NO：79、81所示的多核苷酸序列。所述核苷酸序列表达后产生的抗VEGF抗体-CD80融合蛋白在本文中表示为融合蛋白BY24.4。
融合蛋白BY24.4(κ，IgG4)的第一亚基(BY24.4L)核苷酸序列(SEQ ID NO：79)：
Figure PCTCN2020071213-appb-000022
融合蛋白BY24.4(κ，IgG4)的第一亚基(BY24.4L)氨基酸序列(SEQ ID NO：80)：
Figure PCTCN2020071213-appb-000023
融合蛋白BY24.4(κ，IgG4)的第二亚基(BY24.4H)核苷酸序列(SEQ ID NO：81)：
Figure PCTCN2020071213-appb-000024
Figure PCTCN2020071213-appb-000025
融合蛋白BY24.4(κ，IgG4)的第二亚基(BY24.4H)氨基酸序列(SEQ ID NO：82)：
Figure PCTCN2020071213-appb-000026
其中氨基酸序列“ METDTLLLWVLLLWVPGSTG”为信号肽。
使用上述实施例1(1)相同的方法，通过XhoI-EcoRI双酶切将BY24.4L编码核苷酸连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比 洋生物技术有限公司)；再通过XbaI-SalI双酶切将BY24.4H编码核苷酸克隆至已连接了BY24.4L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体；或者反之亦然。将重组载体测序验证正确后用于表达。所表达的抗VEGF抗体-CD80融合蛋白命名为融合蛋白BY24.4。
(4)包含抗VEGFR抗体与CD80胞外结构域的融合蛋白BY24.5的编码核苷酸的合成及表达载体的构建
根据表1中CD80胞外结构域的序列、表2中抗VEGFR抗体的重链可变区和轻链可变区序列、表3中抗体的重链恒定区序列、表4中抗体的轻链恒定区序列、以及SEQ ID NO：20-46的肽接头序列，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的核苷酸序列，并委托上海捷瑞生物工程有限公司合成如下SEQ ID NO：83、85所示的多核苷酸序列。所述核苷酸序列表达后产生的抗VEGFR抗体-CD80融合蛋白在本文中表示为融合蛋白BY24.5。
融合蛋白BY24.5(κ，IgG4)的第一亚基(BY24.5L)核苷酸序列(SEQ ID NO：83)：
Figure PCTCN2020071213-appb-000027
融合蛋白BY24.5(κ，IgG4)的第一亚基(BY24.5L)氨基酸序列(SEQ ID NO：84)：
Figure PCTCN2020071213-appb-000028
融合蛋白BY24.5(κ，IgG4)的第二亚基(BY24.5H)核苷酸序列(SEQ ID NO：85)：
Figure PCTCN2020071213-appb-000029
Figure PCTCN2020071213-appb-000030
融合蛋白BY24.5(κ，IgG4)的第二亚基(BY24.5H)氨基酸序列(SEQ ID NO：86)：
Figure PCTCN2020071213-appb-000031
Figure PCTCN2020071213-appb-000032
其中氨基酸序列“ METDTLLLWVLLLWVPGSTG”为信号肽。
使用上述实施例1(1)相同的方法，通过XhoI-EcoRI双酶切将BY24.5L编码核苷酸连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比洋生物技术有限公司)；再通过XbaI-SalI双酶切将BY24.5H编码核苷酸克隆至已连接了BY24.5L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体；或者反之亦然。将重组载体测序验证正确后用于表达。所表达的抗VEGFR抗体-CD80融合蛋白命名为融合蛋白BY24.5。
(5)包含CD80胞外结构域、免疫球蛋白Fc区和VEGFR胞外受体功能区的融合蛋白的编码核苷酸的合成及表达载体的构建
根据表1中CD80胞外结构域的序列、表6IgG4Fc序列、和表5中VEGFR功能区序列(VEGFR1-D2/VEGFR2-D3)，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的核苷酸序列，并委托上海捷瑞生物工程有限公司合成如下SEQ ID NO：87所示的多核苷酸序列。所述核苷酸序列表达后产生的CD80-Fc-VEGFR融合蛋白在本文中也表示为融合蛋白BY24.12。
融合蛋白BY24.12(CD80-Fc-VEGFR)的核苷酸序列(SEQ ID NO：87)
Figure PCTCN2020071213-appb-000033
Figure PCTCN2020071213-appb-000034
融合蛋白BY24.12(CD80-Fc-VEGFR)的氨基酸序列(SEQ ID NO：88)
Figure PCTCN2020071213-appb-000035
使用上述实施例1(1)相同的方法，通过XhoI-EcoRI双酶切将BY24.12编码核苷酸连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比洋生物技术有限公司)。将重组载体测序验证正确后用于蛋白表达。所表达的融合蛋白命名为融合蛋白BY24.12。
实施例2、目的蛋白的表达和纯化
(1)目的蛋白的瞬时表达
将293F(购自Invitrogen公司，目录号：11625-019)细胞悬浮培养于无血清CD 293培养液(购自Invitrogen公司，目录号：11913-019)中。转染前离心细胞培养物，获得细胞沉淀，用新鲜的无血清CD 293培养液悬浮细胞，将细胞浓度调整为1×10 6个细胞/ml。将细胞悬浮液置于摇瓶中。以100ml细胞悬浮液为例，分别将实施例1制备的包含目的基因的每一种重组表达载体质粒各DNA 250ug和聚乙烯亚胺(polyethylenimine(PEI))(Sigma，目录号：408727)500ug加入1ml无血清CD 293培养液中混匀，室温静置8分钟后，将PEI/DNA混悬液逐滴加入放置有100ml细胞悬浮液的摇瓶中。轻轻混匀，置于5％CO 2、37℃摇床培养(120转/分钟)。5天后收集培养上清。
(2)表达蛋白的纯化
用pH 7.4 PBS溶液平衡的HiTrap MabSelect SuRe 1ml柱(GE Healthcare Life Sciences产品，目录号：11-0034-93)纯化上述实施例2(1)收集的培养上清中存在的目的蛋白。简而言之，用pH 7.4的PBS溶液以10个柱床体积平衡HiTrap MabSelect SuRe 1ml柱，流速为0.5ml/分钟；将上述实施例2(1)收集的培养上清用0.45μm滤膜过滤后，载样至用pH 7.4 PBS溶液平衡的HiTrap MabSelect SuRe 1ml柱；装载上清液后，将该柱首先用pH 7.4的PBS溶液以流速0.5ml/分钟洗涤5-10个柱床体积，并随后用100mM柠檬酸缓冲液(pH 4.0)以流速0.5ml/分钟洗脱。收集洗脱峰，目的蛋白BY18.1、BY31.19、BY24.4、BY24.5、BY24.12分别存在于洗脱峰中。
在还原剂(5mM 1，4-二硫苏糖醇)存在下通过SDS-PAGE电泳并用考马斯蓝染色，分别分析各目的蛋白BY18.1、BY31.19、BY24.4、BY24.5、BY24.12的纯度和分子量。结果如下表8所示，其中给出了分子量理论预测值和实际测定值。因真核表达系统中存在对蛋白质的糖基化作用，故分子量实际测定值略高于理论预测值。
表8经纯化的表达蛋白的分子量大小
Figure PCTCN2020071213-appb-000036
实施例3、使用ELISA方法检测特异性结合作用
分别将重组人CD28(北京义翘神州生物技术有限公司产品，目录号：50103-M08H)、重组人PD-L1(北京百普赛斯生物科技有限公司，目录号：PD1-H5229)和重组人CTLA-4(北京义翘神州生物技术有限公司产品，目录号：11159-H08H)稀释至0.5μg/ml、0.25μg/ml和1.0μg/ml并包被96孔ELISA板(Corning公司，货号：42592)。将上述实施例2(2)纯化的融合蛋白BY24.4、BY24.5、BY24.12、和BY31.19稀释至2000μg/ml，然后进行3倍系列稀释，共稀释16个梯度，对每个浓度梯度进行复孔检测。将稀释样品50μl分别加入上述经重组人CD28、重组人CTLA-4或重组人PD-L1包被的96孔板中，37℃孵育2小时。洗涤3次后，加入辣根过氧化物酶标记的山羊抗人二级抗体(北京中衫金桥公司产品，产品号：ZDR-5301)，37℃孵育1小时。洗涤3次后，加入3，3′，5，5′-四甲基联苯胺(TMB)底物显色液(北京康为世纪生物科技有限公司，产品号：CW0050)50μl/孔。10分钟后，加入2N的H 2SO 4终止显色。使用ELISA读数仪在450nm处测定每孔的吸光度OD值。
应用GraphPadPrism5软件，将融合蛋白BY24.4、BY24.5、BY24.12、和BY31.19的蛋白质浓度对吸光度OD值作图，并且拟合数据以产生融合蛋白介导的特异性结合作用的半数最大有效浓度EC50值。结果如下表9所示。
表9各融合蛋白对人PD-L1、人CD28和人CTLA-4的结合作用
Figure PCTCN2020071213-appb-000037
ELISA结果显示，本发明的多靶向融合蛋白BY24.4、BY24.5、BY24.12和作为阳性对照的BY31.19均能结合重组人PD-L1、重组人CD28和CTLA-4。各融合蛋白对CTLA-4的结合能力最强，PD-L1次之，结合CD28最弱。
实施例4、使用Biacore T100测定本发明的目的蛋白对靶标的亲和力
在
Figure PCTCN2020071213-appb-000038
T100仪器(GE Healthcare Biosciences AB，瑞典)上于25℃进行表面等离子体共振测量。
首先，通过酰胺偶联将抗IgG抗体(GE Healthcare Life Sciences，目录号：BR-1008-39)共价固定在CM5芯片上。使用60μl N-乙基-N′-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和60μl N-羟基琥珀酰亚胺(NHS)活化CM5芯片，然后将5μl抗IgG抗体加95μl稀释缓冲液HBST(0.1M HEPES，1.5M NaCl，pH7.4，加0.005％吐温20)经0.2um滤膜过滤后， 通过酰胺偶联将抗IgG抗体共价固定在CM5芯片上，产生约9000-14000共振单位(RU)的捕获系统。使用120μl乙醇胺封闭CM5芯片。
然后，将实施例2制备的本发明的各目的蛋白分别稀释为5μg/ml，以流速10μL/分钟注射该稀释液2分钟，实施例2制备的本发明的各目的蛋白通过各自的Fc区非共价地捕获到CM5芯片表面上。通过与EDC/NHS交联来稳定所得的复合物，以避免在测量和再生期间的基线漂移。
分别将结合抗原PD-1(北京义翘神州生物技术有限公司产品，目录号：10377-H08H)、VEGF 165(北京义翘神州生物技术有限公司产品，目录号：11066-HNAH)、鼠VEGF164(北京义翘神州生物技术有限公司产品，目录号：50159-MNAB)和VEGFR2(北京义翘神州生物技术有限公司产品，目录号：10012-H02H1)配制为如下浓度梯度：7nM、22nm、66nM、200nM、600nM。通过以流速30μl/分钟注射每个浓度180秒，解离时间600秒，测量结合。通过用3M MgCl 2溶液以流速10μL/分钟洗涤30秒使表面再生。使用BIA评价软件(BIAevaluation 4.1 software，来自GE Healthcare Biosciences AB，瑞典)进行数据分析，获得下表10所示的亲和力数据。
表10各目的蛋白质与靶标的结合
Figure PCTCN2020071213-appb-000039
融合蛋白BY24.4、BY24.5、BY24.12与各自靶标的KD(M)均低于10 -9M，说明它们与各自靶标均有有较高亲和力，尤其是融合蛋白BY24.12可以结合鼠和人VEGF-A，二者均以高亲和力结合并且差异不是很大。
实施例5、混合淋巴细胞反应(MLR)中本发明的目的蛋白及其组合物对IFN-γ分泌的影响
自北京时合生物科技有限公司购得CD4 +T淋巴细胞和树突状细胞(DC)，所述CD4 +T淋巴细胞和树突状细胞(DC)来源于不同的健康人。将CD4 +T淋巴细胞和树突状细胞(DC)分别按1×10 5个细胞/孔和1×10 4个细胞/孔铺种在96孔细胞培养板中。实验分为7组，分别为BY18.1组(1.0μg/ml)、BY24.4组(1.14μg/ml)、BY24.4(1.14μg/ml)+BY18.1(1.0μg/ml) 组、BY24.5(1.14μg/ml)组、BY24.5(1.14μg/ml)+BY18.1(1.0μg/ml)组、BY24.12(0.84μg/ml)组和BY24.12(0.84μg/ml)+BY18.1(1.0μg/ml)组。每组3个复孔，按要求加入抗体BY18.1、融合蛋白BY24.4、BY24.5、BY24.12、或抗体BY18.1与各融合蛋白BY24.4、BY24.5、BY24.12的组合至所示的终浓度。最后补加含10％胎牛血清的1640培养基，使终体积为200μl。37℃，5％CO 2培养。
培养5天后，这7个实验组的成团细胞均较多，并有相当部分的梭状和贴壁细胞出现。取培养上清，用依科赛生物科技有限公司IFN-γ试剂盒(货号：EH008-96 ELISA)检测各组IFN-γ表达水平。
检测结果如下：与抗体BY18.1组(2221.8±364.5pg/ml)相比较，单独的融合蛋白BY24.4组(924.1±221.9pg/ml)、BY24.5组(760.1±286.8pg/ml)和BY24.12组(793.4±139.2pg/ml)的IFN-γ分泌均显著地低于抗体BY18.1组(P＜0.01)；但融合蛋白BY24.4、BY24.5和BY24.12分别加入BY18.1后，IFN-γ分泌均明显增高，各组的IFN-γ分泌分别为：BY24.4+BY18.1组(3494.2±364.5pg/ml)、BY24.5+BY18.1组(3523.8±465.1pg/ml)和BY24.12+BY18.1组(3801.8±702.2pg/ml)。由此可见，融合蛋白BY24.4、BY24.5和BY24.12分别与BY18.1组合能够导致IFN-γ的分泌显著增加(P＜0.01)，并且表明了抗体BY18.1(PD-1抗体)与本发明的多靶向融合蛋白BY24.4、BY24.5和BY24.12的组合对IFN-γ的分泌有明显协同作用。
实施例6、本发明的多靶向融合蛋白与PD-1抗体的协同作用
本实施例的主要目的是探讨本发明的多靶向融合蛋白与PD-1抗体在体内抗肿瘤方面的协同作用，因此PD-1抗体和各融合蛋白的剂量均采用的是低剂量，因为高剂量有可能对肿瘤抑制效果较好而观察不到PD-1抗体和各融合蛋白相互间的协同作用。
将RPMI-1640培养基中的小鼠源结肠癌细胞CT26(ATCC)接种于约18g，6周龄雌性BALB/c小鼠右侧前肋部皮下，100μl/小鼠，接种量为1x10 6个细胞/小鼠。将小鼠源结肠癌细胞CT26接种的时间设定为第0天。当平均肿瘤体积达到93mm 3左右时，将荷瘤小鼠随机分组，每组6只小鼠，共5组。根据分子量大小计算摩尔量，以等同的摩尔量施用融合蛋白BY31.19、BY24.12和抗mPD-1抗体。具体而言，分组和施用剂量分别为：溶媒(PBS)对照组；融合蛋白BY31.19(1.6mg/kg)组；融合蛋白BY24.12(2.5mg/kg，)组；抗mPD-1(3.0mg/kg，购自BioXcell，克隆号：RMP1-14，产品号：BE0146)组；融合蛋白BY24.12(2.5mg/kg)+抗mPD-1(3mg/kg)组。一周给药2次。每周对肿瘤体积进行3次测量，在进行肿瘤体积测量的同时，称量小鼠体重，记录小鼠体重和肿瘤体积的变化与给药时间的关系。实验结束时，将小鼠安乐死，采集血清和肿瘤，血清冻存至-80℃保存，肿瘤在称重拍照后经福尔马林固定和石蜡包埋(FFPE)制备成组织样本备用。对各数据进行统计学分析并计算各治疗组与溶媒对照组的相对肿瘤体积比值(T/C)％(即：(给药组肿瘤体积变化的均值/PBS溶媒对照组肿瘤体积变化的均值)x 100％)和肿瘤生长抑制率( Tumor  Growth  Inhibition％)，为(1-T/C)％。
在实验结束时，BY31.19组、BY24.12组、PD-1组、BY24.12+抗mPD-1组的肿瘤生长抑制率(TGI％)分别为15％、17％、24％、和47％(以瘤体积计)，均有不同程度的抑制作用。免疫融合蛋白BY24.12与PD-1抗体联合使用时对肿瘤的抑制作用显著好于BY24.12和PD-1单独使用时的肿瘤抑制作用，因此二者之间存在协同作用。
各组动物在给药后体重均持续增加，总体状况良好，未见明显异常，各组均无明显停药或动物死亡。

Claims (14)

  1. 一种阻断血管内皮细胞生长且活化T细胞的多靶向融合蛋白，其包含(i)血管内皮生长抑制剂结构域；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。
  2. 根据权利要求1所述的多靶向融合蛋白，其中所述(i)衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段和/或VEGFR胞外受体功能区；
    优选地，所述抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段是抗VEGF抗体和/或抗VEGFR抗体的Fab、Fab′、F(ab′) 2、Fv、单链Fv；更优选地，所述抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段包含选自SEQ ID NO：1/2、3/4和5/6的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，或者与所述全部6个重链CDR与轻链CDR中的一个或多个CDR具有一个、两个、三个、四个、或五个氨基酸变化(例如，氨基酸置换或缺失)的序列；进一步优选地，所述抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段包含选自SEQ ID NO：1/2、3/4和5/6的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；最优选地，所述抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段是Bevacizumab、Ranibizumab或Ramucirumab的Fab；
    优选地，VEGFR胞外受体功能区包含VEGFR1的免疫球蛋白样结构域2和VEGFR2的免疫球蛋白样结构域3；或者所述VEGFR胞外受体功能区包含VEGFR1的免疫球蛋白样结构域2以及VEGFR2的免疫球蛋白样结构域3和VEGFR2的免疫球蛋白样结构域4；或者所述VEGFR胞外受体功能区包含VEGFR1的免疫球蛋白样结构域2；更优选地，所述VEGFR胞外受体功能区具有任一选自SEQ ID NO：7-9所示的氨基酸序列或与SEQ ID NO：7-9所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
  3. 根据权利要求1或2所述的多靶向融合蛋白，其中所述(ii)是人免疫球蛋白Fc结构域；优选地，所述(ii)是人IgG1、IgG2、IgG3或IgG4的Fc结构域；更优选地，所述(ii)包含SEQ ID NO：10、11或12所示氨基酸序列的Fc结构域，或者包含与SEQ ID NO：10、11或12所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc结构域。
  4. 根据权利要求1-3中任一项所述的多靶向融合蛋白，其中所述(iii)包含人CD80 ECD；优选地，所述(iii)包含人CD80IgV或人CD80IgVIgC；更优选地，所述(iii)具有SEQ ID NO：13或14所示的氨基酸序列或与SEQ ID NO：13或14所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的多靶向融合蛋白，其还包含所述(i)、(ii)和/或(iii)之间的肽接头；优选地，所述肽接头包含一个或多个氨基酸，更优选地包含至少5个氨基酸， 最优选地包含选自SEQ ID NO：20-46的肽接头。
  6. 根据权利要求1-5中任一项所述的多靶向融合蛋白，其中所述融合蛋白从N端至C端以(i)、(ii)和(iii)的顺序；(iii)、(i)和(ii)的顺序；或者(iii)、(ii)和(i)的顺序有效连接。
  7. 根据权利要求6所述的多靶向融合蛋白，其包含
    (a)全长抗VEGF抗体、全长抗VEGFR抗体或者全长抗VEGF和VEGFR双特异性抗体；和在所述抗体的两条重链中的每一重链的C端有效连接的一个CD80 ECD；
    (b)全长抗VEGF抗体、全长抗VEGFR抗体或者全长抗VEGF和VEGFR双特异性抗体；在所述抗体的两条重链中的每一重链的N端有效连接的一个CD80 ECD；和在所述抗体的两条轻链中的每一轻链的N端有效连接的一个CD80 ECD；
    (c)CD80 ECD；在CD80 ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗VEGF抗体和/或抗VEGFR抗体的抗原结合片段；
    或者
    (d)CD80 ECD；在CD80 ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的VEGFR胞外受体功能区；
    优选地，所述抗体是IgG类抗体，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体，更特别地是IgG 4亚类抗体；还优选地，所述IgG 4亚类抗体在Fc结构域中第S228位置处包含氨基酸置换，更优选地是氨基酸置换S228P；进一步优选地，所述抗体的轻链型别为κ型或λ型，优选地为κ型；
    优选地，所述全长抗VEGF抗体是Bevacizumab，所述全长抗VEGFR抗体是Ramucirumab。
  8. 根据权利要求1-7中任一项所述的多靶向融合蛋白，其选自
    (1)包含SEQ ID NO：80的融合蛋白第一亚基和SEQ ID NO：82的融合蛋白第二亚基的融合蛋白；
    (2)包含SEQ ID NO：84的融合蛋白第一亚基和SEQ ID NO：86的融合蛋白第二亚基的融合蛋白；
    (3)包含SEQ ID NO：88的融合蛋白亚基的融合蛋白。
  9. 多核苷酸，其编码权利要求1-8中任一项所述的多靶向融合蛋白。
  10. 载体，优选地表达载体，最优选地具有双表达盒的谷氨酰胺合成酶表达载体，所述载体包含权利要求9所述的多核苷酸。
  11. 宿主细胞，其包含权利要求9所述的多核苷酸或权利要求10所述的载体，优选地，所述宿主细胞是CHO、HEK293或NSO细胞。
  12. 用于产生权利要求1-8中任一项所述的多靶向融合蛋白的方法，所述方法包括步骤(i)在适于表达所述多靶向融合蛋白的条件下培养权利要求11所述的宿主细胞，和(ii)回收所述融蛋白。
  13. 药物组合物，其包含权利要求1-8中任一项所述的多靶向融合蛋白和抗PD-1抗体，优选地，所述抗PD-1抗体包含选自SEQ ID NO：47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70和71/72的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，或者与所述全部6个重链CDR与轻链CDR中的一个或多个CDR具有一个、两个、三个、四个、或五个氨基酸变化(例如，氨基酸置换或缺失)的序列；进一步优选地，所述抗PD-1抗体包含选自SEQ ID NO：47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70和71/72的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；最优选地，所述抗PD-1抗体选自纳武单抗(Nivolumab)、pidilizumab和派姆单抗(Pembrolizumab)。
  14. 权利要求1-8中任一项所述的多靶向融合蛋白、权利要求13所述的药物组合物的用途，用于制备在个体中治疗或预防癌性疾病(例如，实体瘤和软组织瘤)的药物，优选地，癌性疾病是黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)；特别地，所述疾病是结肠癌或三阴性乳腺癌；优选地，其中所述个体是哺乳动物，更优选地是人。
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CN111423512B (zh) 2024-01-05
US20220106389A1 (en) 2022-04-07

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