WO2019062642A1

WO2019062642A1 - Double targeting fusion protein targeting pd-1 or pd-l1 and targeting vegf family and use thereof

Info

Publication number: WO2019062642A1
Application number: PCT/CN2018/106741
Authority: WO
Inventors: 胡品良; 邹敬; 洪伟东; 何芸; 白洁; 宋凌云; 杨文第
Original assignee: 北京比洋生物技术有限公司
Priority date: 2017-09-29
Filing date: 2018-09-20
Publication date: 2019-04-04
Also published as: CN109575140A; CN109575140B

Abstract

A double targeting fusion protein targeting PD-1 or PD-L1 and targeting VEGF family. Said double targeting fusion protein comprises (i) an anti-PD-1 antibody or an anti-PD-L1 antibody, and (ii) a VID effectively linked at the C-terminus of each of two heavy chains of said anti-PD-1 antibody or anti-PD-L1 antibody. Provided are a polynucleotide encoding the double targeting fusion protein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or the vector, and use of the double targeting fusion protein in the treatment, prevention and/or diagnosis of diseases associated with PD-1 activity, PD-L1 activity and VEGF family activity in an individual.

Description

Dual-targeted fusion protein targeting PD-1 or PD-L1 and targeting VEGF family and use thereof

Technical field

The present invention generally relates to the field of medical biotechnology. In particular, the present invention relates to targeting programmed death-1 (PD-1) or programmed death-1 ligand (PD-L1) and targeting blood vessels a dual targeting fusion protein of the Vascular Endothelial Cell Growth Factor (VEGF) family, a polynucleotide encoding the dual targeting fusion protein, a vector comprising the polynucleotide, comprising the Host cells of a polynucleotide or vector, and the use of the dual targeting fusion protein in treating, preventing and/or diagnosing a disease associated with PD-1 or PD-L1 activity and VEGF family activity in an individual.

Background technique

Immune checkpoint is a type of inhibitory signaling molecule present in the immune system that prevents tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and is involved in maintaining tolerance to autoantigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264). The study found that one of the reasons why tumor cells can escape the immune system and lose control of proliferation is to use the inhibitory signaling pathway of the immune checkpoint, thereby inhibiting the activity of T lymphocytes, so that T lymphocytes can not effectively exert the killing effect on tumors. (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146).

Programmed death protein-1 (PD-1) is an important immunological checkpoint protein and is currently an important target for tumor immunotherapy. PD-1 was first discovered in 1992, and its gene cloning and expression indicated that PD-1 activation can induce programmed cell death of T cells. PD-1 protein was found on activated T cells, B cells and myeloid cells. PD-1 is also inducibly expressed in macrophages, dendritic cells, and monocytes. There is no PD-1 expression on the resting lymphocyte surface.

PD-1 is a 55kDa type I transmembrane protein with a cytoplasmic region containing an immunoreceptor tyrosine inhibitory motif and homology to CD28 and CTLA-4. Two cell surface glycoprotein ligands of PD-1 have been identified, Programmed Death Protein Ligand 1 (PD-L1) and Programmed Death Protein Ligand 2 (PD-L2). Ligand expression of PD-1 has been found on many cancer cells, including human lung cancer, ovarian cancer, colon cancer, and various myeloma. In addition, PD-1 ligands are highly expressed on the surface of various epithelial cancers, hematological cancers, and other malignant tumor cells. In tumor patients, the expression of PD-1 ligands such as PD-L1 is often associated with poor prognosis of cancer (Iwai Y et al, Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD -L1 blockade, PNAS, 2002, 99(19): 12293-7).

The binding of PD-1 to the ligand of PD-1 plays an important role in regulating T lymphocyte activity and maintaining peripheral immune tolerance. Binding of PD-1 to the ligand of PD-1 leads to T cell apoptosis, immune non-response, T cell "depletion" and secretion of IL-10. Therefore, PD-1 functions to limit T cell activation, inhibit T cell proliferation, and increase tolerance to antigen. Upregulation of PD-1 expression on activated lymphocytes can lead to inhibition of acquired or innate immune responses, resulting in tumor infiltrating lymphocytes (including T lymphocytes), although they have tumor antigen specificity, Since the ligand of PD-1 on tumor cells binds to PD-1 on tumor infiltrating lymphocytes to produce a signal that inhibits the activation of tumor infiltrating lymphocytes, tumor cells can escape the killing of tumor cells by the immune system.

Studies have shown that these tumor-infiltrating lymphocytes expressing PD-1 are dysfunctional lymphocytes whose biological function can be restored by blocking the binding of PD-1 to the ligand of PD-1. At present, antibodies that inhibit the binding of PD-1 to PD-1 mainly include anti-PD-1 monoclonal antibodies and anti-PD-L1 monoclonal antibodies, but also products for PD-L2.

Currently, the more mature anti-PD-1 antibodies are the Nivolumab of BMS, and the Pembrolizumab of Merck. Nawu monoclonal antibody (trade name)

) is a fully humanized IgG4 antibody molecule, pemizumab (trade name)

) is a humanized IgG4 antibody molecule. The anti-PD-1 monoclonal antibody binds to PD-1 on T lymphocytes and inhibits the binding of PD-1 to its ligands PD-L1 and PD-L2, thereby promoting T lymphocyte activation, proliferation and immunity. Activated cytokines such as IL-2, and relieve the inhibition of PD-1 on immune surveillance of T lymphocytes with anti-tumor activity. The indications for NAV monoclonal antibodies currently approved by the US Food and Drug Administration include: melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, etc.; indications for pemimab include: head and neck cancer, non- Small cell lung cancer, melanoma, etc. Regarding the anti-PD-L1 antibody, atezolizumab developed by Roche, avelumab developed by Merck KGaA and Pfizer, and durvalumab developed by AstraZeneca also showed therapeutic effects on tumors.

Although anti-PD-1 antibodies and anti-PD-L1 antibodies have therapeutic effects on tumors, their average therapeutic efficiency is only about 20%, and the five-year survival rate of lung cancer is only 16%. A significant proportion of cancer patients still have no response to treatment with anti-PD-1 antibodies and anti-PD-L1 antibodies. Therefore, how to improve the effectiveness of cancer treatment is still a difficult problem that needs to be solved in the field of cancer treatment.

On the other hand, tumor angiogenesis is also an important cause of rapid tumor growth (Ferrara N and Alitalo K, Clinical applications of angiogenic growth factors and their inhibitors, Nat Med., 1999; 5(12): 1359-64) . On the surface and deep of the tumor, blood vessels of varying thickness can be seen everywhere, and nutrients and oxygen of life are transported to the tumor tissue through these blood vessels. Tumor angiogenesis is a fairly complex process that is regulated positively and negatively by multiple factors. Among these various factors, the vascular endothelial growth factor family is one of the most potent positive regulators, which plays a role in stimulating neovascularization. Vascular endothelial growth factor and vascular endothelial growth inhibitory factor exist in normal tissues at the same time and remain relatively balanced. This balance allows human blood vessels to be normally formed and differentiated. However, during tumor growth, the number of VEGF family molecules increases sharply, and the imbalance between regulation and angiogenesis inhibitors, thereby greatly promoting the proliferation and migration of vascular endothelial cells, improving vascular permeability, and inhibiting tumors. Apoptosis provides a good microenvironment for tumor growth and metastasis.

The VEGF family contains six closely related polypeptides, each of which is a highly conserved homodimeric glycoprotein, 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. The expression of VEGF-A (including its splices such as VEGF ₁₆₅ ) is associated with microvessel density in some solid tumors, and the concentration of VEGF-A in tissues is associated with the prognosis of solid tumors such as breast, lung, prostate and colon cancers. . The biological activity of each VEGF family member is mediated by one or more of the cell surface VEGF receptor (VEGFR) family, including VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR). Flk-1), VEGFR3 (also known as Flt-4), etc., wherein VEGFR1 and VEGFR2 are closely related to angiogenesis, and VEGF-C/D/VEGFR3 is closely related to lymphangiogenesis. The main biological functions of the VEGF family include: (1) selective promotion of mitosis of vascular endothelial cells, stimulation of endothelial cell proliferation and promotion of angiogenesis; (2) improvement of permeability of blood vessels, especially microvessels, and extravasation of 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 to secrete collagenase and vascular endothelial cells. Plasminogen, in order to degrade the vascular basement membrane, at the same time, the newly formed microvascular basement membrane inside the tumor tissue is imperfect, this property makes the tumor easy to enter the blood circulation; (4) Other effects: VEGF family can induce gaps in the epithelial cells and open Window phenomenon can activate cytoplasmic vesicles and organelles of epithelial cells; VEGF family directly stimulates endothelial cells to release proteolytic enzymes, degrades matrix, releases more VEGF family molecules, accelerates tumor development, and extracellular protease activates extracellular Matrix binding and release of the VEGF family; VEGF family makes plasma proteins (including fiber) by increasing vascular permeability Release of retinoic acid, forming a cellulose network, providing a good matrix for tumor growth, development and metastasis; (5) VEGF family inhibits the body's immune response and promotes invasion 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).

In the VEGF family, the 40% amino acid sequence of placental growth factor (PLGF) is homologous to VEGF-A and participates in the formation of neovascular and collateral vessels under pathological conditions. The biological function of PLGF is activated by specific binding to its receptor VEGFR1/Flt-1. VEGFR1/Flt-1 has strong biological activity, and its ligand can mediate the interaction between endothelial cells and stromal cells, as well as the differentiation and maturation of endothelial cells. PLGF can promote trophoblast proliferation and differentiation in early pregnancy, induce endothelial cell proliferation and migration, resist endothelial cell apoptosis, increase vascular permeability, enhance the biological activity of low concentration VEGF, and participate in various tumor blood vessels. An important pro-angiogenic factor produced. Excessive PLGF expression leads to an increase in tumor growth and survival of blood vessels. PLGF was observed to be expressed in all vascular-rich tumors in primary tumors, whereas tumors with few vascularities only partially expressed PLGF. Therefore, PLGF can be used to explain the mechanism of tumor neovascularization, and inhibition of tumor growth can be achieved by inhibiting the biological activity of PLGF.

Clinical studies have shown that the use of monoclonal antibodies or soluble VEGFR can block the binding of the VEGF family to its receptor, and block the transmission of the VEGF family signaling pathway is also one of the current methods for treating tumors. Bevacizumab (trade name: Avastin), developed by Genentech, is a recombinant human-mouse chimeric anti-VEGF antibody that blocks VEGFR from inactivation by blocking the binding of VEGF-A to VEGFR. This exerts an anti-angiogenic effect. Bevacizumab is currently used for first-line treatment of metastatic colorectal cancer, and may be used for the treatment of metastatic lung cancer, breast cancer, pancreatic cancer, and kidney cancer in the future. Bevacizumab is also one of the more successful antibody drugs developed. Aflibercept, developed by Sanofi-aventis and Regeneron, is a VEGF-Trap that fuses the second extracellular domain of VEGFR1 with the extracellular domain of VEGFR2 and the human IgG1 constant region. A fusion protein that exerts an anti-tumor effect on a subset of tumor patients by inhibiting angiogenesis.

Most tumor patients still do not respond to the currently available anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-VEGF antibodies, or VEGF-Trap alone.

In view of the importance of the immunological checkpoint proteins PD-1 and PD-L1 in regulating immune responses, and the role of the VEGF family in inhibiting tumor immunity and promoting tumor angiogenesis in the tumor microenvironment, there is still a need in the art for treatment of tumors. therapy. Preferably, such alternative therapies are capable of targeting both the immunosuppressive protein PD-1 or PD-L1 and the VEGF family of molecules having immunosuppressive and pro-angiogenic effects, resulting in activation of the immune system and tumor blood vessels. Regression, thereby showing efficacy in patients who do not respond to monotherapy targeting PD-1 or PD-L1 or who are not responding to a single treatment targeting the VEGF family. One approach to this type of alternative therapy is to co-administer two different biological products that target PD-1 or PD-L1 and target VEGF family molecules. Co-administration requires the injection of two separate products or a single injection of a combination of two different proteins. Although the two injections allowed flexibility in the amount of administration and administration time, it caused inconvenience and pain to the patient. In addition, although the combination preparation may provide some flexibility in the amount of administration, it is often difficult to find a formulation condition that allows the chemical and physical stability of the two proteins in solution because the molecular characteristics of the two proteins are different. In addition, co-administration and combination therapy of two different drugs may increase the additional cost to the patient and/or payer. Thus, there is still a need for alternative therapies for treating tumors, and preferably such alternative therapies comprise dual targeting fusion proteins that target PD-1 or PD-L1 and target the VEGF family.

The present invention provides a dual targeting novel fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, which is capable of inhibiting activation of the PD-1 pathway or the PD-L1 pathway and the VEGF family signaling pathway, And for treating, preventing, and/or diagnosing diseases associated with PD-1, PD-L1 activity, and VEGF family activity in an individual.

Summary of invention

The present invention discloses a novel dual targeting fusion protein targeting PD-1 or PD-L1 and targeting the VEGF family, a polynucleotide encoding the dual targeting fusion protein, and a vector comprising the polynucleotide Use of a host cell comprising the polynucleotide or vector, and the dual targeting fusion protein for treating, preventing and/or diagnosing a disease associated with PD-1, PD-L1 activity and VEGF family activity in an individual .

Thus, in one aspect, the invention provides a dual targeting fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, which inhibits binding of PD-1 to its ligand or inhibits PD -L1 binds to its receptor and inhibits the signaling pathway of the VEGF family, comprising (i) an anti-PD-1 antibody or an anti-PD-L1 antibody; and (ii) with said anti-PD-1 antibody or anti-PD- The L1 antibody is operably linked to at least two VEGF family familiy inhibiting domains (hereinafter abbreviated as VID).

In one embodiment, the dual targeting fusion protein of the invention comprises (i) an anti-PD-1 antibody or an anti-PD-L1 antibody; and (ii) two of said anti-PD-1 antibodies or anti-PD-L1 antibodies a VID operably linked to the C-terminus of each heavy chain in the heavy chain, optionally, said (i) and said (ii) being operably linked by a peptide linker, whereby two identical or different VIDs are each Their N-terminal amino acids are fused to the C-terminal amino acid of one of the anti-PD-1 antibodies or one of the heavy chains of the anti-PD-L1 antibody, optionally via a peptide linker, preferably, the VID comprises a VEGF family Part of the extracellular domain of the body.

The anti-PD-1 antibody contained in the dual targeting fusion protein may be any anti-PD-1 antibody as long as it is capable of inhibiting or reducing the binding of PD-1 to its ligand, including those known in the art. Anti-PD-1 antibody and anti-PD-1 antibody developed in the future. In one embodiment, the anti-PD-1 antibody comprises an antibody selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/ All heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequences of 16, 17/18, 19/20, 21/22, 23/24, and 120/121 Preferably, the anti-PD-1 antibody comprises SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, Paired heavy chain variable region sequence/light chain variable region sequences of 17/18, 19/20, 21/22, 23/24, and 120/121, or with the paired heavy chain variable region sequences/ a light chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; more preferably, The anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region of an anti-PD-1 antibody selected from the group consisting of nabumab, pidilizumab, and pemimumab, in particular, the anti-PD-1 antibody Selected from navumab, pidilizumab and pemizumab.

The anti-PD-L1 antibody contained in the dual targeting fusion protein may be any anti-PD-L1 antibody as long as it is capable of inhibiting or reducing PD-L1 binding to its receptor (for example, with PD-1 or CD80 (B7-1) The antibodies may be combined with either, including anti-PD-L1 antibodies known in the art and anti-PD-L1 antibodies developed in the future. In one embodiment, the anti-PD-L1 antibody in the fusion protein of the invention comprises a pair of heavy chain variable region sequences/light chain variable regions selected from the group consisting of SEQ ID NOs: 25/26, 27/28, and 29/30 All heavy chain CDRs and light chain CDRs contained in the sequence. Preferably, the anti-PD-L1 antibody comprises a pair of heavy chain variable region sequence/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 25/26, 27/28 and 29/30, or The heavy chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity More preferably, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab and durvalumab.

In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody is an IgG class antibody, in particular an IgG ₁ subclass, an IgG ₂ subclass, an IgG ₄ subclass antibody. In a preferred embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody comprised in the fusion protein of the invention is an IgG ₄ subclass antibody, in particular a human IgG ₄ subclass antibody. In one embodiment, the IgG ₄ subclass antibody comprises an amino acid substitution at position S228 (EU numbering) in the Fc region, in particular amino acid substitution S228P. The heavy chain constant region amino acid sequence of an exemplary IgG ₁ subclass anti-PD-1 antibody is shown in SEQ ID NO:33. The heavy chain constant region amino acid sequence of an exemplary IgG ₂ subclass anti-PD-1 antibody is shown in SEQ ID NO:34. The heavy chain constant region amino acid sequence of an exemplary IgG ₄ subclass anti-PD-1 antibody is shown in SEQ ID NO:35.

In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody comprises a variable region and a constant region of a full antibody. The antibody light chain constant region type in the dual targeting fusion protein of the present invention may be a kappa type or a lambda type, preferably a kappa type. The kappa type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO:31. The lambda-type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO:32.

The VID contained in the dual targeting fusion protein comprises a portion of the extracellular domain of the receptor of the VEGF family. In one embodiment, the VID comprises immunoglobulin (Ig)-like domain 2 (Domain 2, abbreviated as D2) of VEGFR1 and Ig-like domain 3 (Domain 3, abbreviated as D3) of VEGFR2. In a specific embodiment, the VEGFR1-D2/VEGFR2-D3 has the amino acid sequence set forth in SEQ ID NO: 63 or at least 90%, 91%, 92%, 93 of the amino acid sequence of SEQ ID NO: 63 Amino acid sequence of %, 94%, 95%, 96%, 97%, 98%, 99% or more identity. In one embodiment, the VID comprises VEGFR1-D2 and Ig-like domain 4 (Domain 4, abbreviated D4) of VEGFR2-D3 and VEGFR2. In a specific embodiment, the VEGFR1-D2/VEGFR2-D3-D4 has the amino acid sequence set forth in SEQ ID NO: 64 or at least 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 64 Amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. In one embodiment, the VID comprises VEGFR1-D2. In a specific embodiment, the VEGFR1-D2 has the amino acid sequence set forth in SEQ ID NO: 65 or at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 65 Amino acid sequence of 95%, 96%, 97%, 98%, 99% or more identity.

In one embodiment, the peptide linker joining the VID at the C-terminus of the heavy chain of the anti-PD-1 antibody or anti-PD-L1 antibody comprises one or more amino acids, preferably comprising a SEQ ID NO: 36 -62 peptide linker.

In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 73 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 75, hereinafter referred to as For the fusion protein BY24.3. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 77 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 79, hereinafter referred to as For the fusion protein BY24.7. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 81 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 83, hereinafter referred to as For the fusion protein BY24.4. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 85 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 87, hereinafter referred to as For the fusion protein BY24.5. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 89 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 91, hereinafter referred to as For the fusion protein BY24.6. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 93 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 95, hereinafter referred to as For the fusion protein BY24.8. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 97 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 99, hereinafter referred to as For the fusion protein BY24.9. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 101 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 103, hereinafter referred to as For the fusion protein BY24.10. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 105 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 107, hereinafter referred to as For the fusion protein BY24.11. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 109 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 111, hereinafter referred to as For the fusion protein BY24.12. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 113 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 115, hereinafter referred to as For the fusion protein BY24.13. In a specific embodiment, the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 117 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 119, hereinafter referred to as For the fusion protein BY24.14.

In a specific embodiment, the fusion protein comprises (i) an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab, and durvalumab, and (ii) each of the two heavy chains of the anti-PD-L1 antibody A VID molecule operably linked to the C-terminus of the strand.

In one embodiment, the fusion protein specifically targets PD-1 or PD-L1 and VEGF family molecules, inhibiting signaling mediated by PD-1 or PD-L1 and VEGF family molecules. The fusion protein of the present invention binds not only PD-1 or PD-L1 with high affinity at the N-terminus, but also binds a plurality of VEGF factors with high affinity at the C-terminus. The structure of the fusion protein designed by the invention fully ensures the suitable physical spatial distance of the fusion protein to the two types of targets, and the fusion protein of this structure is specific to a molecule of PD-1 or PD-L1 and VEGF family molecules. Sexual binding does not affect the specific binding of the fusion protein to another molecule in the PD-1 or PD-L1 and VEGF family molecules.

The invention further provides a polynucleotide encoding a fusion protein of the invention, a vector comprising a polynucleotide encoding a fusion protein of the invention, preferably an expression vector, most preferably a glutamine synthetase expression vector having a double expression cassette. In another aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. The invention also provides a method for producing a fusion protein of the invention comprising the steps of (i) cultivating a host cell of the invention under conditions suitable for expression of a fusion protein of the invention, and (ii) recovering the fusion protein of the invention .

In one aspect, the invention provides a diagnostic kit and pharmaceutical composition comprising a fusion protein of the invention. Further, there is provided the use of a fusion protein, diagnostic kit or pharmaceutical composition of the invention for the treatment, prevention and/or diagnosis of a disease associated with PD-1 or PD-L1 activity and VEGF family activity, in particular For the treatment, prevention and/or diagnosis of cancerous diseases (eg solid tumors and soft tissue tumors), most particularly for the treatment, prevention and/or diagnosis of melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal tract Interstitial tumors (GIST), renal 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).

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention, which are described in detail below, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the presently preferred embodiments are shown. However, it is understood that the invention is not limited to the precise arrangements and means of the embodiments shown.

Figure 1: Schematic diagram showing the structure of a dual targeting fusion protein of the present invention targeting PD-1 or PD-L1 and targeting the VEGF family.

Figure 2: shows the results of the fusion protein of the present invention prepared and purified in Example 2 by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol). Lane 1 in Figure 2A: Protein molecular weight standard marker; Lane 2: Fusion protein BY24.3; Lane 3: Fusion protein BY24.4; Lane 4: Fusion protein BY24.5; Lane 5: Fusion protein BY24.6; Lane 6: fusion protein BY24.8; lane 7: fusion protein BY24.9; lane 8: fusion protein BY24.10; lane 9: fusion protein BY24.11; lane 1 in Figure 2B: protein molecular weight standard marker; lane 2 : fusion protein BY24.12; lane 3: fusion protein BY24.13; lane 4: fusion protein BY24.14; lane 5: antibody BY18.1; lane 6: protein 301-8; lane 7: fusion protein BY24.7.

Figure 3: shows the effect of the fusion protein BY24.3, antibody BY18.1 and protein 301-8 of the present invention on the body weight of experimental animals.

Figure 4: A schematic diagram showing the in vivo antitumor effect of the fusion protein BY24.3 of the present invention and the antibodies BY18.1 and 301-8.

Detailed description of the invention

The present invention provides fusion proteins and pharmaceutical compositions that block the PD-1 pathway or the PD-L1 pathway and the VEGF family signaling pathway at the immune checkpoint. The invention also provides methods for producing the fusion protein, and the use of the fusion protein in treating, preventing, and/or diagnosing a disease associated with PD-1 or PD-L1 activity and VEGF family activity in an individual.

Unless otherwise defined below, the terms in this specification are used as commonly used in the art.

I. Definition

The term "about" when used in connection with a numerical value is meant to encompass a numerical value within the range of the lower limit of 5% less than the specified numerical value and the upper limit of 5% greater than the specified numerical value.

The term "comprising" or "including", as used herein, is intended to include the recited elements, integers or steps, but does not exclude any other elements, integers or steps.

"PD-1 pathway" refers to any intracellular signaling pathway initiated by binding to PD-1, including but not limited to intracellular signaling pathways triggered by PD-1 binding to PD-L1, or PD-1 and The intracellular signaling pathway triggered by PD-L2 binding, or the intracellular signaling pathway triggered by the binding of PD-1 to both PD-L1 and PD-L2.

"PD-L1 pathway" refers to any intracellular signaling pathway initiated by binding to PD-L1, including but not limited to, intracellular signaling pathways triggered by PD-L1 binding to PD-1, or PD-L1 and Intracellular signaling pathway triggered by binding of CD80 (B7-1), or intracellular signaling pathway triggered by binding of PD-L1 to both PD-1 and CD80 (B7-1).

As used herein, the term "specifically binds" means selective for binding of an antigen or molecule of interest and may be distinguished from unwanted or non-specific interactions. The specific binding can be by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Analysis of agalacto- IgG in rheumatoid arthritis using surface plasmon resonance, Glyco J., 2000, 17, 323-329).

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. Was affinity molecule X for its partner Y can generally dissociation constant (K _D) is represented by the solution, the dissociation constant is the ratio of the dissociation rate constant and association rate constant (k _off, respectively, and k _on) of. Affinity can be measured by common methods known in the art. One specific method for measuring affinity is surface plasmon resonance (SPR).

The term "antibody" is used herein in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), so long as they exhibit the desired antigen binding activity. . The antibody may be an intact antibody molecule or a functional fragment of an intact antibody molecule including, but not limited to, for example, Fab, F(ab') ₂ . The constant region of an antibody can be altered (eg, mutated) to modify antibody properties (eg, to increase or decrease one or more of the following properties: antibody glycosylation, number of cysteine residues, effector cell function, or complement function) .

The terms "full antibody," "full length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure that is substantially similar in structure to the native antibody.

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule, which normally determines the class to which the antibody belongs.

The term "antibody light chain" refers to the lesser of the two types of polypeptide chains present in an antibody molecule. The kappa light chain and the lambda light chain refer to two major antibody light chain isoforms.

The "percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in the present specification and, if necessary, the vacancy is introduced to achieve the maximum percent sequence identity, and no consideration is given. The percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of the particular amino acid sequence shown in this specification when the conservative substitution is part of the sequence identity.

The term "operatively linked" means that the specified components are in a relationship that allows them to function in the intended manner.

A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein that promotes secretion of the protein out of the cell. The mature form of the extracellular protein has no signal sequence that is cleaved during the secretory process.

The term "N-terminus" refers to the last amino acid at the N-terminus, and the term "C-terminus" refers to the last amino acid at the C-terminus.

The term "fusion" refers to the direct attachment of two or more components by peptide bonds or by one or more peptide linkers.

The term "fusion protein" as used herein, refers to a fusion polypeptide molecule comprising an antibody light chain subunit and an antibody heavy chain-VID fusion subunit, wherein the antibody light chain subunit is the smaller of the polypeptide chains present in the fusion protein, The antibody heavy chain-VID fusion subunit is the larger of the polypeptide chains present in the fusion protein.

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. A host cell is any type of cellular system that can be used to produce a fusion protein of the invention. Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cultured plant tissues or cells within animal tissues.

The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse). In particular, the individual is a human.

The term "treatment" refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis. In some embodiments, the fusion proteins of the invention are used to delay disease progression or to slow the progression of the disease.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and liquid tumors.

II. Fusion protein

The present invention provides a dual targeting fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, comprising (i) an anti-PD-1 antibody or an anti-PD-L1 antibody; and (ii) and the anti-antibody The PD-1 antibody or the anti-PD-L1 antibody is operably linked to at least two VIDs, wherein the two components of the fusion protein are linked to each other directly or via a peptide linker. In addition, the individual peptide chains of the component (i) anti-PD-1 antibody or anti-PD-L1 antibody in the fusion protein may be linked, for example, by a disulfide bond.

In some embodiments, a fusion protein of the invention is a heterotetrameric glycoprotein consisting of two disulfide-bonded two antibody light chain subunits and two antibody heavy chain-VID fusion subunits. From the N-terminus to the C-terminus, each antibody heavy chain-VID fusion subunit has an antibody heavy chain followed by a VID wherein the antibody heavy chain and VID are linked directly by peptide bonds or by one or more peptide linkers.

The fusion proteins of the invention block the PD-1 pathway or the PD-L1 pathway at the immunological checkpoint and inhibit the VEGF family signaling pathway. The immunological checkpoint PD-1 pathway blocked by this fusion protein is the signaling pathway mediated by PD-1 binding to its ligand. The PD-L1 pathway blocked by this fusion protein is a signaling pathway mediated by the binding of PD-L1 to its receptor. The fusion protein inhibits the VEGF family signaling pathway by signaling mediated by the binding of VEGF-A, -B, -C, -D, -E, and PLGF to receptors of the VEGF family (eg, VEGFR1, VEGFR2, and VEGFR3) way.

In some embodiments, the fusion protein of the invention binds to PD-1 or PD-L1 at a dissociation constant (K _D ) of 10 ^-8 M or less, for example, 10 ^-9 M to 10 ^-12 M; The VEGF family specifically binds with a dissociation constant (K _D ) of 10 ^-8 M or less, for example, 10 ^-9 M to 10 ^-12 M.

-Anti-PD-1 antibody or anti-PD-L1 antibody

The anti-PD-1 antibody or anti-PD-L1 antibody contained in the fusion protein of the present invention is a heterotetrameric glycoprotein composed of two light-chain-bonded two light chains and two heavy chains.

In one embodiment, from the N-terminus to the C-terminus, the heavy chain of each anti-PD-1 antibody or anti-PD-L1 antibody has a variable region (VH), also referred to as a variable heavy chain domain or heavy chain. The variable domains are followed by three constant domains (CH1, CH2 and CH3), also referred to as heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, the light chain of each anti-PD-1 antibody or anti-PD-L1 antibody has a variable region (VL), also known as a variable light chain domain or a light chain variable domain. , followed by a constant light chain (CL) domain, also known as the light chain constant region. The anti-PD-1 antibody or anti-PD-L1 antibody consists essentially of two Fab molecules and one Fc domain joined by a hinge region of an anti-PD-1 antibody or an anti-PD-L1 antibody.

The anti-PD-1 antibody or the anti-PD-L1 antibody contained in the fusion protein of the present invention can have a high affinity, for example, a K _{D of} 10 ^-8 M or less, preferably 10 ^-9 M to 10 ^-12 M, Binding to PD-1 or PD-L1, respectively, and thereby blocking the signaling pathway mediated by PD-1 binding to ligand PD-L1/PD-L2 or blocking PD-L1 and receptor PD- 1/CD80 (B7-1) binds to the mediated signaling pathway.

Examples of the paired heavy chain variable region (VH) and light chain variable region (VL) of the anti-PD-1 antibody contained in the fusion protein of the present invention are provided herein below in Table 1A. In addition, examples of the paired heavy chain variable region (VH) and light chain variable region (VL) of the anti-PD-L1 antibody contained in the fusion protein of the present invention are provided herein below in Table 1B. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody in the fusion protein of the invention comprises a sequence substantially identical to the amino acid sequence set forth in Table 1A or Table 1B, respectively, for example, with Table 1A or Table The paired heavy chain variable region sequence/light chain variable region sequence shown in 1B has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or more sequences of sequence identity.

Table 1A. Examples of heavy chain variable region and light chain variable region sequences of anti-PD-1 antibodies in fusion proteins

Table 1B. Examples of heavy chain variable region and light chain variable region sequences of anti-PD-L1 antibodies in fusion proteins

In one embodiment, the anti-PD-1 antibody in the fusion protein of the invention comprises an antibody selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/ All heavy chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequences of 15, 15/16, 17/18, 19/20, 21/22, 23/24, and 120/121 With light chain CDRs. In one embodiment, the anti-PD-L1 antibody in the fusion protein of the invention comprises a pair of heavy chain variable region sequences/light chain variable regions selected from the group consisting of SEQ ID NOs: 25/26, 27/28, and 29/30 All heavy chain CDRs and light chain CDRs contained in the sequence. 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 particular heavy chain variable regions and/or light chains disclosed herein. The CDRs 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 of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins., J. Mol. Biol. 273:927 -948 (1997); and Martin AC et al, Modeling antibody hypervariable loops: a combined algorithm, Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).

The anti-PD-1 antibody or the anti-PD-L1 antibody in the fusion protein of the present invention may be classified into a kappa type or a lambda type based on the amino acid sequence of the light chain constant region thereof, and is preferably a kappa type.

An example of the amino acid sequence of the anti-PD-1 antibody light chain constant region in the fusion protein of the invention is provided herein below in Table 2.

Table 2. Examples of anti-PD-1 antibody light chain constant region sequences in fusion proteins

The amino acid sequence of the anti-PD-1 antibody or the anti-PD-L1 antibody in the fusion protein of the present invention based on the heavy chain constant region thereof is preferably an IgG class antibody, particularly an IgG ₁ subclass, an IgG ₂ subclass, an IgG ₄ subclass. Antibodies, more particularly IgG ₄ subclass antibodies. Preferably, the IgG ₄ subclass anti-PD-1 antibody or anti-PD-L1 antibody comprises an amino acid substitution preventing the occurrence of arm-exchange at the S228 position in the Fc region, in particular amino acid substitution S228P.

Examples of the amino acid sequences of the heavy chain constant regions of the anti-PD-1 antibodies in the fusion proteins of the invention are provided in Table 3 below.

Table 3. Examples of anti-PD-1 antibody heavy chain constant region sequences in fusion proteins

- inhibition of the domain of the VEGF family (VID)

The "inhibiting VEGF family domain (VID)" in a fusion protein of the invention comprises a portion of the extracellular domain of VEGFR. The VEGFR receptor is a tyrosine kinase receptor located on the cell surface, and its extracellular domain is composed of seven immunoglobulin (Ig)-like domains. For example, human VEGFR1 comprises seven Ig-like domains numbered 1, 2, 3, 4, 5, 6, and 7, with Ig-like domain 1 at the N-terminus of the extracellular domain and Ig-like domain 7 outside the extracellular domain. The C end 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. In some embodiments, the VID comprises at least one Ig-like domain of one or more VEGFRs selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3. In some aspects, the VID comprises at least 1, 2, 3, 4, 5, 6, but no more than 7 Ig-like domains of the VEGFR. In another aspect, the VID 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.

VIDs comprising at least one Ig-like domain of two or more VEGFRs are also contemplated herein. In some embodiments, the VID comprises at least one Ig-like domain from two or more VEGFRs selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3. The VID of any combination of seven Ig-like domains comprising each VEGFR is contemplated herein. For example, a VID can comprise an Ig-like domain 2 of VEGFRl (eg, human VEGFRl) and an Ig-like domain 3 of VEGFR2 (eg, human VEGFR2). In another embodiment, the VID may comprise Ig-like domains 1-3 of VEGFR1 (eg, human VEGFR1), Ig-like domains 2-3 of VEGFR1 (eg, human VEGFR1), Ig-like structures of VEGFR2 (eg, human VEGFR2) Domains 1-3, Ig-like domain 2 of VEGFR1 (eg, human VEGFR1) and Ig-like domain 3-4 of VEGFR2 (eg, human VEGFR2), or Ig-like domain 2 and VEGFR3 of VEGFR1 (eg, human VEGFR1) (eg, Ig-like domain 3 of human VEGFR3). A more detailed description of these Ig-like domains and other Ig-like domains that can be used as part of the VID can be found in U.S. Patent No. 5,751,173; Yu DC et al., 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 of which are hereby incorporated by reference in their entirety. reference. In some embodiments, the VID has any one of the amino acid sequences selected from SEQ ID NOs: 63-65 in Table 4 or at least 90%, 91%, 92 from the amino acid sequence set forth in SEQ ID NOs: 63-65. Amino acid sequence of %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

Table 4 Examples of VID amino acid sequences in fusion proteins

The VID contained in the fusion protein of the present invention is capable of specifically binding to the VEGF family with high affinity, for example, a K _{D of} 10 ^-8 M or less, preferably 10 ^-9 M to 10 ^-12 M, and thereby Binding of the VEGF family to cell surface VEGFR and subsequent signaling are inhibited.

-peptide linker

The "peptide linker" optionally contained in the heavy chain C-terminus and the VID N-terminus of the anti-PD-1 antibody or anti-PD-L1 antibody in the fusion protein of the present invention is a peptide of one or more amino acids, generally about 2-20 amino acids. . Peptide linkers are known in the art or described herein.

In some embodiments, the peptide linker comprises at least 5 amino acids, preferably comprising selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 36); AKTTPKLEEGEFSEARV (SEQ ID NO: 37); AKTTPKLGG (SEQ ID NO: 38); SAKTTPKLGG ( SEQ ID NO: 39); SAKTTP (SEQ ID NO: 40); RADAAP (SEQ ID NO: 41); RADAAPTVS (SEQ ID NO: 42); RADAAAAGGPGS (SEQ ID NO: 43); RADAAAA (SEQ ID NO: 44) SAKTTPKLEEGEFSEARV (SEQ ID NO: 45); ADAAP (SEQ ID NO: 46); DAAPTVSIFPP (SEQ ID NO: 47); TVAAP (SEQ ID NO: 48); TVAAPSVFIFPP (SEQ ID NO: 49); QPKAAP (SEQ) ID NO: 50); QPKAAPSVTLFPP (SEQ ID NO: 51); AKTTPP (SEQ ID NO: 52); AKTTPPSVTPLAP (SEQ ID NO: 53); AKTTAP (SEQ ID NO: 54); AKTTAPSVYPLAP (SEQ ID NO: 55) ASTKGP (SEQ ID NO: 56); ASTKGPSVFPLAP (SEQ ID NO: 57); GGGGSGGGGSGGGGS (SEQ ID NO: 58); GENKVEYAPALMALS (SEQ ID NO: 59); GPAKELTPLKEAKVS (SEQ ID NO: 60); GHEAAAVMQVQYPAS (SEQ ID NO: 61); and a peptide linker of GGGGSGGGGSGGGGSA (SEQ ID NO: 62).

III. Production and purification of the dual targeting fusion protein of the invention

The dual targeting fusion proteins of the invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, a polynucleotide encoding an antibody light chain subunit of the dual targeting fusion protein and/or a polynucleotide encoding an antibody heavy chain-VID fusion subunit of the dual targeting fusion protein is isolated and inserted One or more vectors for further cloning and/or expression in a host cell. The polynucleotide can be easily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.

Expression vectors can be constructed using methods well known to those of skill in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC). In a preferred embodiment, a glutamine synthetase high expression vector having a dual expression cassette is used.

Once an expression vector comprising one or more polynucleotides of the invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.

In one embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In some embodiments, a host cell comprising an expression vector of the invention is provided. As used herein, the term "host cell" refers to any type of cellular system that can be engineered to produce a dual targeting fusion protein of the invention. Host cells suitable for replicating and supporting the expression of the dual targeting fusion proteins of the invention are well known in the art. Such cells can be transfected or transduced with a particular expression vector, as desired, and a large number of cells containing the vector can be grown for inoculating large scale fermenters to obtain a sufficient amount of the dual targeting fusion protein of the invention for clinical use. 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. Mammalian cell lines 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 (293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), Africa Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2) ), CHO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2/0. For a review of mammalian host cell lines suitable for protein production see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Standard techniques for expressing foreign genes in these host cell systems are known in the art. In one embodiment, a method of producing a dual targeting fusion protein of the invention, wherein the method comprises culturing a host cell as provided herein under conditions suitable for expression of the dual targeting fusion protein, The host cell comprises a polynucleotide encoding the dual targeting fusion protein and the dual targeting fusion protein is recovered from a host cell (or host cell culture medium).

The dual targeting fusion protein prepared as described herein can be purified by known prior art 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, hydrophilicity, and the like, and these will be apparent to those skilled in the art.

The purity of the dual targeting fusion proteins of the invention can be determined by any of a variety of well known analytical methods, including gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activities of the dual targeting fusion proteins provided herein can be identified, screened or characterized by a variety of assays known in the art.

IV. Pharmaceutical Compositions and Kits

In another aspect, the invention provides compositions, for example, pharmaceutical compositions comprising a dual targeting fusion protein as described herein formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

The compositions of the invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. A common preferred composition is in the form of an injectable solution or an infusible solution. A preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the dual targeting fusion protein is administered by intravenous infusion or injection. In another preferred embodiment, the dual targeting fusion protein is administered by intramuscular, intraperitoneal or subcutaneous injection.

The phrases "parenteral administration" and "parenteral administration" as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.

Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes or lyophilized forms. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the dual-targeted fusion protein) in the required amount in a suitable solvent, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing base dispersion medium and other ingredients. A coating agent such as lecithin or the like can be used. In the case of dispersions, the proper fluidity of the solution can be maintained by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions of the compositions which delay the absorption, such as the monostearate and gelatin.

In certain embodiments, the dual targeting fusion proteins of the invention can be administered orally, for example, orally with an inert diluent or an edible carrier. The dual targeting fusion proteins of the invention may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets or incorporated directly into the subject's diet. For oral therapeutic administration, the compound can be incorporated with excipients and in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, glutinous rice papers It is used in the form of a wafer or the like. In order to administer a dual targeting fusion protein of the invention by a parenteral administration method, it may be desirable to coat or co-administer the dual targeting fusion protein with a material that prevents its inactivation. Therapeutic compositions can also be administered using medical devices known in the art.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a dual targeting fusion protein of the invention. "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required. The therapeutically effective amount can vary depending on various factors such as the disease state, the age, sex, and weight of the individual. A therapeutically effective amount is any amount that is toxic or detrimental to a therapeutically beneficial effect. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%. The ability of the dual targeting fusion proteins of the invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in an animal model system that predicts efficacy in human tumors.

By "prophylactically effective amount" is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered to the subject prior to the earlier stage of the disease or at an earlier stage of the disease.

Kits comprising the dual targeting fusion proteins described herein are also within the scope of the invention. The kit may contain one or more additional elements including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

V. Use of dual targeting fusion proteins

The dual targeting fusion proteins disclosed herein have diagnostic and therapeutic and prophylactic uses in vitro and in vivo. For example, these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, eg, a human subject, to treat, prevent, and/or diagnose a variety of PD-1 activity, PD-L1 activity, and A disease associated with VEGF family activity, such as cancer.

In one aspect, the invention provides for the detection of a PD-1 or PD-L1 and VEGF family molecule in a biological sample, such as a serum, semen or urine or a tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion) in vitro or in vivo. Diagnostic method. The diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with a dual targeting fusion protein as described herein or administering the dual targeting to a subject under conditions that allow interaction to occur The fusion protein and (ii) detect the formation of a complex between the dual targeting fusion protein and the sample (and optionally, a control sample). Formation of the complex indicates the presence of PD-1 or PD-L1 and VEGF family molecules, and may indicate the suitability or need for the treatment and/or prevention described herein.

In some embodiments, the PD-1 or PD-L1 and VEGF family molecules are detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval. Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR-technology (eg, RT-PCR) or in vivo imaging techniques. Generally, dual targeting fusion proteins used in in vivo and in vitro assays are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugates. Suitable detectable materials include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (eg, nuclear magnetic resonance) materials, and radioactive materials.

In some embodiments, the level and/or distribution of PD-1 or PD-L1 and VEGF family molecules is determined in vivo, eg, in a non-invasive manner (eg, by using suitable imaging techniques (eg, positron emission tomography) Detection (PET) scan) detection of a detectable marker of a dual targeting fusion protein of the invention. In one embodiment, for example, by detecting a PET reagent (eg, ¹⁸ F-fluorodeoxyglucose (FDG)) in a detectable manner Labeled dual-targeted fusion proteins of the invention, assay the level and/or distribution of PD-1 or PD-L1 and VEGF family molecules in vivo.

In one embodiment, the invention provides a diagnostic kit comprising the dual targeting fusion protein described herein and instructions for use.

In another aspect, the invention relates to the use of a dual targeting fusion protein for the treatment or prevention of a disease in need of enhancing an immune response and reducing angiogenesis in a subject, thereby inhibiting or reducing the growth or appearance of a related disease such as a cancerous tumor. , metastasis or recurrence. A dual targeting fusion protein can be used alone to inhibit or prevent the growth of cancerous tumors. Alternatively, the dual targeting fusion protein can be administered in combination with other cancer therapeutic/preventive agents. When the dual targeting fusion protein of the invention is administered in combination with one or more other drugs, the combination can be administered in any order or simultaneously.

Accordingly, in one embodiment, the invention provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a dual targeting fusion protein described herein. In another embodiment, the 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 a dual targeting fusion protein described herein.

In some embodiments, cancers treated and/or prevented with a dual targeting fusion protein include, but are not limited to, solid tumors, hematological cancer (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and metastatic Lesion. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems, such as invasive lungs, breasts, ovaries, lymphoid, gastrointestinal (eg, colon), anal, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (eg, brain, nerve or glial cells), head and neck, skin (eg, melanoma), nasopharynx (eg, 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 bowel cancer, and esophageal cancer. Cancer can be in early, intermediate or advanced stages or metastatic cancer.

In some embodiments, the cancer is selected from the group consisting of 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 cancer, stomach cancer, hematological malignancies (eg, lymphoma).

The following examples are described to aid in the understanding of the invention. The examples are not intended to be construed as limiting the scope of the invention in any way.

Example

Example 1. Construction of a high-efficiency expression vector for glutamine synthetase containing a gene of interest

(1) Synthesis of the coding nucleotide of anti-PD-1 antibody BY18.1 as a control and construction of expression vector

According to the amino acid sequence data of Navuzumab, number 9623 in the International Nonproprietary Name (INN) database, the following nucleotide sequences suitable for expression in Chinese hamster ovarian cancer cells (CHO) were optimized and commissioned by Shanghai Jierui Biotechnology Co., Ltd. Engineering Co., Ltd. synthesized the nucleotide sequence. The anti-PD-1 antibody produced after expression of the nucleotide sequence is referred to herein as antibody BY18.1.

The light chain (BY18.1L) nucleotide sequence of the anti-PD-1 antibody BY18.1 is shown in SEQ ID NO: 66; the light chain (BY18.1L) amino acid sequence of the anti-PD-1 antibody BY18.1 is SEQ ID NO: 67.

The heavy chain (BY18.1H) nucleotide sequence of the anti-PD-1 antibody BY18.1 is shown in SEQ ID NO: 68; the heavy chain (BY18.1H) amino acid sequence of the anti-PD-1 antibody BY18.1 is SEQ ID NO: 69.

In the amino acid sequence, "METDTLLLWVLLLWVPGSTG" is a signal peptide sequence.

Shanghai Jierui Bioengineering Co., Ltd. synthesized the above BY18.1L coding nucleotide sequence and BY18.1H coding nucleotide sequence. The BY18.1L coding nucleotide was digested with XhoI-EcoRI, and the glutamine synthetase high expression vector with double expression cassette (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.) was used for XhoI. -EcoRI double-digestion, and ligated the BY18.1L-encoding nucleotide double-digested with XhoI-EcoRI into a high-efficiency expression vector of glutamine synthetase with double expression cassette double-digested by XhoI-EcoRI. A high expression vector for glutamine synthetase having a double expression cassette into which a BY18.1L coding nucleotide has been introduced is obtained; then, the BY18.1H coding nucleotide is digested with XbaI-SalI, respectively, and BY18 has been introduced. .1L coding nucleotides of glutamine synthetase high expression vector with double expression cassette were digested with XbaI-SalI, and then ligated into the BY18.1H coding nucleotide double-digested by XbaI-SalI A glutamine synthetase high-efficiency expression vector with a double expression cassette into which a BY18.1L coding nucleotide has been introduced by XbaI-SalI digestion, thereby obtaining a nucleotide encoding BY18.1L and BY18.1H a glutamine synthetase having a double expression cassette encoding a nucleotide Expression vectors, expression correctly verified by sequencing, to obtain an anti-PD-1 antibody BY18.1.

Alternatively, the BY18.1L coding nucleotide can also be ligated into a glutamine synthetase high expression vector having a double expression cassette into which a BY18.1H coding nucleotide has been introduced, and the antibody BY18.1 is expressed and obtained.

(2) Synthesis and expression vector construction of protein 301-8 encoding nucleotide as a control

Based on the amino acid sequence data of abelibercept number 8739 in the International Nonproprietary Name (INN) database, optimized for the following nucleotide sequences suitable for expression in Chinese hamster ovarian cancer cells (CHO), and commissioned in Shanghai The nucleotide sequence was synthesized by Jerry Bioengineering Co., Ltd. The protein product produced after expression of the nucleotide sequence is referred to herein as protein 301-8.

The nucleotide sequence encoding the protein 301-8 is shown in SEQ ID NO: 70; the amino acid sequence of protein 301-8 is shown in SEQ ID NO: 71.

The nucleotide sequence encoding the protein 301-8 was digested with XhoI-EcoRI, and the glutamine synthetase high expression vector with double expression cassette was obtained (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd. The double-digested XhoI-EcoRI was used to ligate the XhoI-EcoRI double-digested protein 301-8-encoding nucleotide to the XhoI-EcoRI double-digested glutamine synthetase with double expression cassette. The vector was efficiently expressed, and a glutamine synthetase high-efficiency expression vector having a double expression cassette into which the nucleotide sequence encoding the protein 301-8 was introduced was obtained. After being verified by sequencing, it was used for expression, and the expressed protein was named as protein 301-8. The amino acid sequence of protein 301-8 is identical to the amino acid sequence of abecept disclosed in the prior art.

(3) Synthesis of nucleotides encoding the double-targeted fusion protein of PD-1 and VEGF family and construction of expression vector

According to the heavy chain variable region and light chain variable region sequences of the anti-PD-1 antibody in Table 1A, the light chain constant region sequence of the antibody in Table 2, the heavy chain constant region sequence of the antibody in Table 3, and the VID in Table 4 The sequence, and the peptide linker sequence of SEQ ID NO: 36-62, were optimized to be suitable for nucleotide sequences expressed in Chinese hamster ovarian cancer cells (CHO), and Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize SEQ ID NO: The polynucleotide sequence shown by the even number in 72-118.

The light chain subunit (BY24.3L) nucleotide sequence of the fusion protein BY24.3 (κ, IgG4) is shown in SEQ ID NO: 72; the light chain subunit of the fusion protein BY24.3 (κ, IgG4) (BY24) .3L) The amino acid sequence is set forth in SEQ ID NO:73. The heavy chain-VID fusion subunit (BY24.3H) nucleotide sequence of the fusion protein BY24.3 (κ, IgG4) is shown in SEQ ID NO: 74; the heavy chain of the fusion protein BY24.3 (κ, IgG4) The VID fusion subunit (BY24.3H) amino acid sequence is set forth in SEQ ID NO:75.

The light chain subunit (BY24.7L) nucleotide sequence of the fusion protein BY24.7 (κ, IgG2) is shown in SEQ ID NO: 76; the light chain subunit of the fusion protein BY24.7 (κ, IgG2) (BY24) .7L) The amino acid sequence is set forth in SEQ ID NO:77. The heavy chain-VID fusion subunit (BY24.7H) nucleotide sequence of the fusion protein BY24.7 (κ, IgG2) is shown in SEQ ID NO: 78; the heavy chain of the fusion protein BY24.7 (κ, IgG2) The VID fusion subunit (BY24.7H) amino acid sequence is set forth in SEQ ID NO:79.

The light chain subunit (BY24.4L) nucleotide sequence of the fusion protein BY24.4 (κ, IgG4) is shown in SEQ ID NO: 80; the light chain subunit of the fusion protein BY24.4 (κ, IgG4) (BY24) .4L) The amino acid sequence is set forth in SEQ ID NO:81. The heavy chain-VID fusion subunit (BY24.4H) nucleotide sequence of the fusion protein BY24.4 (κ, IgG4) is shown in SEQ ID NO: 82; the heavy chain of the fusion protein BY24.4 (κ, IgG4) The VID fusion subunit (BY24.4H) amino acid sequence is set forth in SEQ ID NO:83.

The light chain subunit (BY24.5L) nucleotide sequence of the fusion protein BY24.5 (κ, IgG4) is shown in SEQ ID NO: 84; the light chain subunit of the fusion protein BY24.5 (κ, IgG4) (BY24) .5L) The amino acid sequence is set forth in SEQ ID NO:85. The heavy chain-VID fusion subunit (BY24.5H) nucleotide sequence of the fusion protein BY24.5 (κ, IgG4) is shown in SEQ ID NO: 86; the heavy chain of the fusion protein BY24.5 (κ, IgG4) The VID fusion subunit (BY24.5H) amino acid sequence is set forth in SEQ ID NO:87.

The light chain subunit (BY24.6L) nucleotide sequence of the fusion protein BY24.6 (κ, IgG2) is shown in SEQ ID NO: 88; the light chain subunit of the fusion protein BY24.6 (κ, IgG2) (BY24) .6L) The amino acid sequence is set forth in SEQ ID NO:89. The heavy chain-VID fusion subunit (BY24.6H) nucleotide sequence of the fusion protein BY24.6 (κ, IgG2) is shown in SEQ ID NO: 90; the heavy chain of the fusion protein BY24.6 (κ, IgG2) The VID fusion subunit (BY24.6H) amino acid sequence is set forth in SEQ ID NO:91.

The light chain subunit (BY24.8L) nucleotide sequence of the fusion protein BY24.8 (κ, IgG4) is shown in SEQ ID NO: 92; the light chain subunit of the fusion protein BY24.8 (κ, IgG4) (BY24) .8L) The amino acid sequence is set forth in SEQ ID NO:93. The heavy chain-VID fusion subunit (BY24.8H) nucleotide sequence of the fusion protein BY24.8 (κ, IgG4) is shown in SEQ ID NO: 94; the heavy chain of the fusion protein BY24.8 (κ, IgG4) The VID fusion subunit (BY24.8H) amino acid sequence is set forth in SEQ ID NO:95.

The light chain subunit (BY24.9L) nucleotide sequence of the fusion protein BY24.9 (κ, IgG4) is shown in SEQ ID NO: 96; the light chain subunit of the fusion protein BY24.9 (κ, IgG4) (BY24) .9L) The amino acid sequence is set forth in SEQ ID NO:97. The heavy chain-VID fusion subunit (BY24.9H) nucleotide sequence of the fusion protein BY24.9 (κ, IgG4) is shown in SEQ ID NO: 98; the heavy chain of the fusion protein BY24.9 (κ, IgG4) The VID fusion subunit (BY24.9H) amino acid sequence is set forth in SEQ ID NO:99.

The light chain subunit (BY24.10L) nucleotide sequence of the fusion protein BY24.10 (κ, IgG4) is shown in SEQ ID NO: 100; the light chain subunit of the fusion protein BY24.10 (κ, IgG4) (BY24) .10L) The amino acid sequence is set forth in SEQ ID NO:101. The heavy chain-VID fusion subunit (BY24.10H) nucleotide sequence of the fusion protein BY24.10 (κ, IgG4) is shown in SEQ ID NO: 102; the heavy chain of the fusion protein BY24.10 (κ, IgG4) The VID fusion subunit (BY24.10H) amino acid sequence is set forth in SEQ ID NO:103.

The light chain subunit (BY24.11L) nucleotide sequence of the fusion protein BY24.11 (κ, IgG4) is shown in SEQ ID NO: 104; the light chain subunit of the fusion protein BY24.11 (κ, IgG4) (BY24) The .11L) amino acid sequence is set forth in SEQ ID NO:105. The heavy chain-VID fusion subunit (BY24.11H) nucleotide sequence of the fusion protein BY24.11 (κ, IgG4) is represented by SEQ ID NO: 106; the heavy chain of the fusion protein BY24.11 (κ, IgG4) The VID fusion subunit (BY24.11H) amino acid sequence is set forth in SEQ ID NO:107.

The light chain subunit (BY24.12L) nucleotide sequence of the fusion protein BY24.12 (κ, IgG4) is shown in SEQ ID NO: 108; the light chain subunit of the fusion protein BY24.12 (κ, IgG4) (BY24) The .12L) amino acid sequence is set forth in SEQ ID NO:109. The heavy chain-VID fusion subunit (BY24.12H) nucleotide sequence of the fusion protein BY24.12 (κ, IgG4) is shown in SEQ ID NO: 110; the heavy chain of the fusion protein BY24.12 (κ, IgG4) The VID fusion subunit (BY24.12H) amino acid sequence is set forth in SEQ ID NO:111.

The nucleotide sequence of the light chain subunit (BY24.13L) of the fusion protein BY24.13 (κ, IgG4) is shown in SEQ ID NO: 112; the light chain subunit of the fusion protein BY24.13 (κ, IgG4) (BY24) .13L) The amino acid sequence is set forth in SEQ ID NO:113. The heavy chain-VID fusion subunit (BY24.13H) nucleotide sequence of the fusion protein BY24.13 (κ, IgG4) is shown in SEQ ID NO: 114; the heavy chain of the fusion protein BY24.13 (κ, IgG4) The VID fusion subunit (BY24.13H) amino acid sequence is set forth in SEQ ID NO:115.

The light chain subunit (BY24.14L) nucleotide sequence of the fusion protein BY24.14 (κ, IgG4) is shown in SEQ ID NO: 116; the light chain subunit of the fusion protein BY24.14 (κ, IgG4) (BY24) The .14L) amino acid sequence is set forth in SEQ ID NO:117. The heavy chain-VID fusion subunit (BY24.14H) nucleotide sequence of the fusion protein BY24.14 (κ, IgG4) is shown in SEQ ID NO: 118; the heavy chain of the fusion protein BY24.14 (κ, IgG4) The VID fusion subunit (BY24.14H) amino acid sequence is set forth in SEQ ID NO:119.

In the amino acid sequence of the fusion protein, "METDTLLLWVLLLWVPGSTG" is a signal peptide.

Using the same method as in the above Example 1 (1), BY24.3L, BY24.4L, LBY24.5L, BY24.6L, BY24.7L, BY24.8L, BY24.9L, BY24 were respectively digested by XhoI-EcoRI. .10L, BY24.11L, BY24.12L, BY24.13L and BY24.14L coding nucleotides are ligated to a high expression vector for glutamine synthetase with a double expression cassette (patent authorization number: CN104195173B, obtained from Beijing Biyang Bio Technology Co., Ltd.); BY24.3H, BY24.4H, LBY24.5H, BY24.6H, BY24.7H, BY24.8H, BY24.9H, BY24.10H, BY24.11H, by XbaI-SalI double digestion The BY24.12H, BY24.13H and BY24.14H coding nucleotides were respectively cloned into a glutamine synthetase high-efficiency expression vector with a double expression cassette to which the corresponding fusion protein light chain subunit coding nucleotide has been ligated; or vice versa Also. The recombinant vector was sequenced and verified for expression. The expressed dual targeting fusion proteins were named as fusion protein BY24.3, BY24.4, BY24.5, BY24.6, BY24.7, BY24.8, BY24.9, BY24.10, BY24.11, BY24. .12, BY24.13 and BY24.14.

Example 2. Expression and purification of fusion protein

(1) Transient expression of fusion protein

293F (purchased from Invitrogen, catalog number: 11625-019) cells were suspension cultured in serum-free CD 293 medium (purchased from Invitrogen, catalog number: 11913-019). The cell culture was centrifuged before transfection to obtain a cell pellet, and the cells were suspended in fresh serum-free CD 293 culture medium to adjust the cell concentration to 1 × 10 ⁶ cells/ml. The cell suspension was placed in a shake flask. Taking 100 ml of the cell suspension as an example, the recombinant expression vector 250 ug of the recombinant expression vector prepared in Example 1 and 500 ug of polyethylenimine (PEI) (Sigma, catalog number: 408727) were added to 1 ml of serum-free CD 293 medium. After mixing and allowing to stand at room temperature for 8 minutes, the PEI/DNA suspension was added dropwise to a shake flask in which 100 ml of the cell suspension was placed. Mix gently, place in a 5% CO ₂ shaker at 37 ° C (120 rpm). The culture supernatant was collected after 5 days.

According to the same method, transient expression produced antibody BY18.1 as a control and protein 301-8 as a control.

(2) Purification of expressed protein

The fusion protein present in the culture supernatant collected in the above Example 2 (1) was purified using a HiTrap MabSelect SuRe 1 ml column (GE Healthcare Life Sciences product, catalog number: 11-0034-93) equilibrated with a pH 7.4 PBS solution. Briefly, a HiTrap MabSelect SuRe 1 ml column was equilibrated with a pH 7.4 PBS solution at a volume of 10 bed volumes at a flow rate of 0.5 ml/min; the culture supernatant collected in the above Example 2 (1) was filtered through a 0.45 μm filter. Thereafter, the sample was loaded onto a HiTrap MabSelect SuRe 1 ml column equilibrated with a pH 7.4 PBS solution; after loading the supernatant, the column was first washed with a pH 7.4 PBS solution at a flow rate of 0.5 ml/min for 5-10 bed volumes, and This was followed by elution with 100 mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min. The elution peak was collected and the protein of interest was present in the elution peak.

The purity and molecular weight of the fusion protein were analyzed by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol). The result is shown in Figure 2. The predicted values of the molecular weight theory and the actual measured values are shown in Table 5. Because of the glycosylation of proteins in eukaryotic expression systems, the actual measured molecular weight is slightly higher than the theoretical prediction.

Table 5 Molecular weight of purified expressed protein

Example 3. Detection of binding of the fusion protein of the present invention to human PD-1 and recombinant human VEGF-A using an ELISA method

The antigen PD-1 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H) and antigen VEGF ₁₆₅ (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11066-HNAH) were diluted to 0.5 μg/ Ml and 0.02 μg/ml were coated with 96-well ELISA plates (purchased from Corning, Inc., Cat. No. 42592). The double-targeted fusion protein purified in the above Example 2 (2) was diluted to 5 μg/ml, and then subjected to 3-fold serial dilution, and a total of 9 gradients were diluted, and each concentration gradient was subjected to multi-well detection. The diluted sample was added to 50μl were PD-1 antigen VEGF ₁₆₅ antigen-coated 96-well plates or, for 2 hours 37 ℃. After washing 3 times, horseradish peroxidase-labeled goat anti-human secondary antibody (product of Beijing Zhongxiu Jinqiao Co., Ltd., product number: ZDR-5301) was added and incubated at 37 ° C for 1 hour. After washing 3 times, 3,3',5,5'-tetramethylbenzidine (TMB) substrate color developing solution (Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0050) was added at 50 μl/well. After 10 min, 2N H ₂ SO ₄ to terminate the color development. The absorbance OD value of each well was measured at 450 nm using an ELISA reader. The same ELISA procedure was performed on the antibody BY 18.1 as a control and the protein 301-8 as a control.

The ELISA results showed that the fusion proteins of the present invention were BY24.3, BY24.4, BY24.5, BY24.6, BY24.7, BY24.8, BY24.9, BY24, as well as the antibody BY18.1 as a control. 10. BY24.11, BY24.12, BY24.13, and BY24.14 can specifically bind to PD-1; similarly to protein 301-8 as a control, the fusion proteins of the present invention are BY24.3, BY24.4, LBY24.5, BY24.6, BY24.7, BY24.8, BY24.9, BY24.10, BY24.11, BY24.12, BY24.13, and BY24.14 also specifically bind to VEGF-A.

GraphPadPrism5 software application, the protein concentration of each fusion protein absorbance OD values were plotted and data fitted to generate specific binding fusion protein mediated half maximal effective concentration values ₅₀ EC. The results are shown in Table 6 below.

Table 6 Binding effect of the fusion protein of the present invention on PD-1 and VEGF-A

According to the results of Table 6, the novel fusion proteins constructed by the present invention are capable of binding PD-1 with high affinity, wherein the fusion proteins are BY24.4, BY24.5, BY24.7, BY24.8, BY24.10, BY24. 11. BY24.12 and BY24.14 bind to PD-1 with a greater affinity than the antibody BY18.1, even the fusion protein BY24.8, which is about 10-fold more affinity than the antibody BY18.1. The fusion protein BY24.3 and the antibody BY18.1 The affinity for PD-1 is basically the same; the novel fusion proteins constructed by the present invention are also capable of binding VEGF-A with high affinity, wherein the fusion protein BY24.3 shows high affinity binding to VEGF- similar to protein 301-8 as a control. A.

Example 4 Determination of the affinity of the fusion protein of the invention using Biacore T100

in

Surface plasmon resonance measurements were performed on a T100 instrument (GE Healthcare Biosciences AB, Sweden) at 25 °C.

First, an anti-IgG antibody (GE Healthcare Life Sciences, catalog number: BR-1008-39) was covalently immobilized on a CM5 chip by amide coupling. The CM5 chip was activated with 60 μl of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 60 μl of N-hydroxysuccinimide (NHS), followed by 5 μl of anti- IgG antibody plus 95 μl of dilution buffer HBST (0.1 M HEPES, 1.5 M NaCl, pH 7.4, plus 0.005% Tween 20) was filtered through a 0.2 um filter, and the anti-IgG antibody was covalently immobilized on the CM5 chip by amide coupling. On top, a capture system of approximately 9,000-14,000 resonance units (RU) is produced. The CM5 chip was blocked with 120 μl of ethanolamine.

Then, the fusion protein of the present invention prepared in Example 2, the antibody BY18.1 and the protein 301-8 were each diluted to 5 μg/ml, and the dilution was injected at a flow rate of 10 μL/min for 2 minutes to prepare 1600 RU of Example 2. The fusion protein of the present invention, antibody BY18.1 and protein 301-8 were non-covalently captured on the surface of the CM5 chip by the respective Fc regions. The resulting complex was stabilized by cross-linking with EDC/NHS to avoid baseline drift during measurement and regeneration.

The antigen will be combined with PD-1 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H), VEGF ₁₆₅ (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11066-HNAH), VEGF-B (Biovision product, catalog number: 4642-20) and PLGF-1 (Biovision product, catalog number: 4739-25) were formulated to the following concentration gradients: 7 nM, 22 nm, 66 nM, 200 nM, 600 nM. Binding was measured by injecting each concentration for 180 seconds at a flow rate of 30 μl/min with a dissociation time of 600 seconds. The surface was regenerated by washing with a 3 M MgCl ₂ solution at a flow rate of 10 μL/min for 30 seconds. Data analysis was performed using BIA evaluation software (BIAevaluation 4.1 software, from GE Healthcare Biosciences AB, Sweden), and the affinity data shown in Table 7 below was obtained.

Table 7 Binding of each protein to VEGF family molecule and PD-1

Note: ND: not detected

According to the data shown in Table 7, the fusion protein BY24.3 binds to VEGF-A, VEGF-B and PLGF-1 with high affinity, and the affinity reaches 9.59×10 ^-12 M, 1.23×10 ^-9 M and 1.82, respectively. ×10 ^-10 M, and the affinity (PDD) for PD-1 is substantially identical to the antibody BY18.1 as a control, and the fusion protein BY24.7 binds PD-1 with a greater affinity (KD) than the antibody BY18.1; fusion Protein BY24.3 showed high affinity binding to VEGF-A similar to protein 301-8 as a control; fusion protein BY24.3 also bound VEGF-B and PLGF-1 with high affinity.

This demonstrates that the dual targeting fusion proteins of the invention bind to PD-1 with similar or better affinity to anti-PD-1 antibodies and have excellent ability to bind to multiple VEGFs.

The results in Table 7 also indicate that the affinity results as determined by surface plasmon resonance (SPR) technique of Example 4 are highly consistent with the affinity results determined by ELISA of Example 3.

Example 5: Inhibition of tumor growth by a fusion protein of the invention in a humanized B-hPD-1 mouse model

5×10 ⁵ MC38 murine colon cancer cells (obtained from ATCC, USA) in 0.1 mL DMEM medium were inoculated into 6-week-old female B-hPD-1 humanized mice (obtained from Beijing 100) Ossetu Gene Biotechnology Co., Ltd., product number: B-CM-001) The right front flank is subcutaneous. The tumor grew up in the mouse. When the tumor volume reached about 108 mm ³ , the tumor-bearing mice were randomly divided into groups of 6 and 4 groups, respectively: PBS solvent control group, protein 301-8 group (3.3 mg/kg), fusion protein BY24.3 Group (6.4 mg/kg) and antibody BY18.1 group (5 mg/kg), the dosage of each administration group was based on the dose of antibody BY18.1, the protein 301-8 group, the fusion protein BY24.3 group and the antibody. The doses administered for the BY18.1 group are equivalent in molar amount. The time of the first administration was set to day 0. All groups were administered intraperitoneally (ip), once every three days, six times in a row, and the experiment was terminated three days after the last administration. Tumor volume and mouse body weight were measured twice a week, and mouse body weight and tumor volume were recorded. At the end of the experiment, the animals were euthanized, the tumor weighed stripping, pictures, calculate tumor growth inhibition rate (T umor G rowth I nhibition% ) and tumor weight inhibition rate (I nhibition R ate of T umor W eight%). The formula used to calculate TGI% is: [1 - (mean of tumor volume change in the drug-administered group / mean change in tumor volume of the PBS solvent control group)] x 100%, and the formula used to calculate IRTW% is: [1- (administration group) Tumor weight / PBS solvent control tumor weight)) x 100%. The experiment was carried out in Beijing Baiao Saitu Gene Biotechnology Co., Ltd.

The results are shown in Figures 3 and 4. In this experiment, the inhibitory effect of the fusion protein BY24.3 of the present invention and the antibody BY18.1, protein 301-8 as a drug control on the growth of subcutaneous xenografts of colon cancer of MC38 mice was observed.

Throughout the experiment, all animals were in good mental condition and no animals died. At the end of the experiment (day 21 after the first dose), the animals in each group weighed an average of about 19 g. Comparing the body of the fusion protein BY24.3 of the present invention with the antibody BY18.1 group and the protein 301-8 group as drug control, and the PBS solvent control group, there was no significant difference (P>0.05), indicating that the animal The fusion protein BY24.3 of the present invention was well tolerated (Fig. 3).

At the end of the experiment, the mean tumor volume ± standard error of the PBS solvent control group was 1386 ± 170 mm ³ , while the mean tumor volume ± standard error of the fusion protein BY24.3 and protein 301-8 groups were 452 ± 69, 1023 ± 256, respectively. They were 73.3% and 28.1%, respectively, and IRTW% were 74.6% and 25.7%, respectively. The fusion protein BY24.3 was significantly different from the tumor volume of the PBS solvent control group (P<0.05), indicating that the fusion protein BY24.3 has significant tumor suppressive effect. Protein 301-8 had a certain tumor suppressive effect, but there was no significant difference compared with the PBS solvent control group (P>0.05). As a control, the antibody BY18.1 had a mean tumor volume ± standard error of 739 ± 128, a TGI% of 50.6%, and an IRTW% of 46.0%, which was significantly different from the tumor volume of the PBS solvent control group (P < 0.05). The fusion protein BY24.3 showed excellent tumor inhibition, and there was a significant difference compared with the tumor inhibition effect of the antibody BY18.1 group and the protein 301-8 group as a drug control. The antitumor efficacy results of this experiment were further confirmed in the comparison of tumor weights in mice.

Table 8 Comparison of inhibitory effects on tumor growth in a humanized B-hPD-1 mouse model

组别Group	平均肿瘤体积±标准误Mean tumor volume ± standard error	肿瘤生长抑制率(TGI％)Tumor growth inhibition rate (TGI%)	肿瘤重量抑制率(IRTW％)Tumor weight inhibition rate (IRTW%)
溶剂对照组Solvent control	1386±170mm ³ 1386±170mm ³	－-	－-
蛋白301-8对照组Protein 301-8 control group	1023±256mm ³ 1023±256mm ³	28.1％28.1%	25.7％25.7%
抗体BY18.1对照组Antibody BY18.1 control group	739±128mm ³ 739±128mm ³	50.6％50.6%	46.0％46.0%
融合蛋白BY24.3组Fusion protein BY24.3 group	452±69mm ³ 452±69mm ³	73.3％73.3%	74.6％74.6%

While certain representative embodiments and details have been shown for the purposes of the present invention, it will be apparent to those skilled in the art In this regard, the scope of the invention is limited only by the following claims.

Claims

A dual targeting fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, which inhibits the binding of PD-1 to its ligand or inhibits the binding of PD-L1 to its receptor, and inhibits a signaling pathway of the VEGF family, which comprises

(i) an anti-PD-1 antibody or an anti-PD-L1 antibody;

(ii) at least two VEGF family-inhibiting domains (VIDs) operably linked to the anti-PD-1 antibody or the anti-PD-L1 antibody.
The dual targeting fusion protein of claim 1 comprising

(i) an anti-PD-1 antibody or an anti-PD-L1 antibody;

(ii) a VEGF family-inhibiting domain (VID) operably linked at the C-terminus of each of the two heavy chains of the anti-PD-1 antibody or the anti-PD-L1 antibody,

Thus, two identical or different VIDs are each operably linked at their N-terminal amino acids to the C-terminal amino acid of one of the anti-PD-1 antibodies or one of the heavy chains of the anti-PD-L1 antibody;

Preferably, said (i) and said (ii) are operably linked by a peptide linker; preferably said peptide linker comprises one or more amino acids, more preferably at least 5 amino acids, most preferably comprises a SEQ ID NO: peptide linker of 36-62.
The dual-targeting fusion protein according to claim 1 or 2, wherein the anti-PD-1 antibody or anti-PD-L1 antibody is an IgG class antibody, particularly an IgG 1 subclass, an IgG 2 subclass, an IgG 4 subunit. An antibody, more particularly an IgG 4 subclass antibody; preferably, the IgG 4 subclass antibody comprises an amino acid substitution at position S228 in the Fc region, more preferably amino acid substitution S228P.
The dual-targeting fusion protein according to any one of claims 1 to 3, wherein the light chain of the anti-PD-1 antibody or the anti-PD-L1 antibody is of a kappa type or a lambda type, preferably a kappa type.
The dual targeting fusion protein of any one of claims 1 to 4, wherein the VID comprises a portion of an extracellular domain of a receptor of the VEGF family, preferably, the VID comprises an immunoglobulin-like form of VEGFR1 Domain 2 and immunoglobulin-like domain 3 of VEGFR2; or the VID comprises immunoglobulin-like domain 2 of VEGFR1 and immunoglobulin-like domain 3 of VEGFR2 and immunoglobulin-like domain 4 of VEGFR2; The VID comprises an immunoglobulin-like domain 2 of VEGFR1; more preferably, the VID has any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 63-65 or SEQ ID NOS: 63-65 The amino acid sequence has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
The dual targeting fusion protein of any one of claims 1 to 5, wherein the anti-PD-1 antibody comprises a SEQ ID NO: 1/2, 3/4, 5/6, 7/8, Paired heavy chain variable region sequences/light chain variable 9/10, 11/12, 13/14, 15/16, 17/18, 19/20, 21/22, 23/24, and 120/121 All of the heavy chain CDRs and light chain CDRs contained in the region sequence, preferably, the anti-PD-1 antibody comprises a plurality selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/ Paired heavy chain variable region sequence/light chain variable region sequences of 10, 11/12, 13/14, 15/16, 17/18, 19/20, 21/22, 23/24, and 120/121 Or with the paired heavy chain variable region sequence/light chain variable region sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 More preferably, the anti-PD-1 antibody comprises a heavy chain of an anti-PD-1 antibody selected from the group consisting of Nivolumab, pidilizumab, and Pembrolizumab. a variable region and a light chain variable region, in particular, the anti-PD-1 antibody is selected from the group consisting of nabumab, pidilizumab, and pemizumab;

Wherein the anti-PD-L1 antibody comprises all heavy chain CDRs contained in a pair of heavy chain variable region sequence/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 25/26, 27/28 and 29/30 And the light chain CDR, preferably, the anti-PD-L1 antibody comprises a pair of heavy chain variable region sequence/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 25/26, 27/28 and 29/30, Or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy chain variable region sequence/light chain variable region sequence More or more sequence identity; more preferably, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab and durvalumab.
A dual targeting fusion protein according to any one of claims 1 to 6 which is selected from

(1) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 73 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 75;

(2) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 77 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 79;

(3) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 81 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 83;

(4) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 85 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 87;

(5) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 89 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 91;

(6) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 93 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 95;

(7) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 97 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 99;

(8) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 101 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 103;

(9) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 105 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 107;

(10) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 109 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 111;

(11) a dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 113 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 115;

(12) A dual targeting fusion protein comprising the anti-PD-1 antibody light chain subunit of SEQ ID NO: 117 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 119.
A polynucleotide encoding the dual targeting fusion protein of any of claims 1-7.
A vector, preferably an expression vector, most preferably a glutamine synthetase expression vector having a double expression cassette comprising the polynucleotide of claim 8.
A host cell comprising the polynucleotide of claim 8 or the vector of claim 9.
A method for producing the dual targeting fusion protein of any one of claims 1-7, the method comprising the step of (i) cultivating the claim 10 under conditions suitable for expression of the dual targeting fusion protein Said host cell, and (ii) recovering said dual targeting fusion protein.
A pharmaceutical composition comprising the dual targeting fusion protein of any of claims 1-7 and a pharmaceutically acceptable carrier.
Use of the dual-targeted fusion protein of any of claims 1-7 and the pharmaceutical composition of claim 12 for the preparation or treatment of PD-1 activity, PD-L1 activity and A drug for a disease associated with VEGF family activity, preferably, the disease is a cancerous disease (for example, solid tumor and soft tissue tumor), more preferably melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal tract Quality 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, lymph Preferably, the individual is a mammal, more preferably a human.
A diagnostic kit comprising the fusion protein of any of claims 1-7 and optionally a label or reagent for coupling.
The diagnostic kit according to claim 14, comprising the fusion protein of any one of claims 1 to 7 which is labeled with a positron emission tomography detectable label, preferably the label is 18 F-fluorodeoxyglucose.
Use of the diagnostic kit of claim 14 or 15 for the preparation of a medicament for diagnosing a disease associated with PD-1 activity, PD-L1 activity and CD28 activity in an individual, preferably the disease is a cancerous disease (eg, solid tumors and soft tissue tumors), more preferably 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 cancer, gastric cancer, hematological malignancies (eg, lymphoma); preferably, wherein the individual is a mammal, more preferably a human .