WO2020200210A1 - 抗pd-l1/vegf双功能抗体及其用途 - Google Patents

抗pd-l1/vegf双功能抗体及其用途 Download PDF

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WO2020200210A1
WO2020200210A1 PCT/CN2020/082535 CN2020082535W WO2020200210A1 WO 2020200210 A1 WO2020200210 A1 WO 2020200210A1 CN 2020082535 W CN2020082535 W CN 2020082535W WO 2020200210 A1 WO2020200210 A1 WO 2020200210A1
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antibody
vegf
present
bifunctional
seq
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PCT/CN2020/082535
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English (en)
French (fr)
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朱向阳
张凤雪
蔡明清
张雷
陈时
俞玲
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华博生物医药技术（上海）有限公司
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Priority to KR1020217015804A priority Critical patent/KR20210082219A/ko
Priority to AU2020255712A priority patent/AU2020255712B2/en
Priority to JP2021525632A priority patent/JP7283806B2/ja
Priority to US17/295,394 priority patent/US20220002418A1/en
Priority to EP20783433.4A priority patent/EP3954712A4/en
Publication of WO2020200210A1 publication Critical patent/WO2020200210A1/zh

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    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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Definitions

  • the present invention belongs to the field of tumor immunology, and specifically relates to anti-PD-L1/VEGF bifunctional antibodies and uses thereof.
  • Tumours can be divided into two categories: benign tumors and malignant tumors according to the cell characteristics of new organisms and the degree of harm to the body.
  • malignant tumor diseases are the major diseases that endanger human health in today's society, and their lethality ranks second.
  • Common tumors include liver cancer, lung cancer, stomach cancer, breast cancer, bladder cancer, etc.
  • Asia accounted for nearly half Of the 9.6 million cancer deaths, Asia accounts for nearly 70%.
  • immunotherapy refers to a treatment method that refers to a low or hyperactive immune state of the body, artificially enhancing or inhibiting the body's immune function to achieve the purpose of curing diseases.
  • immunotherapy methods which are suitable for the treatment of many diseases.
  • Tumor immunotherapy aims to activate the human immune system, relying on autoimmune function to kill cancer cells and tumor tissues, thereby controlling and eliminating tumors.
  • the target of immunotherapy is not tumor cells and tissues, but the body's own immune system. Including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy and small molecule inhibitors.
  • monoclonal antibody immune checkpoint inhibitors include monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy and small molecule inhibitors.
  • the good news of tumor immunotherapy has continued. At present, it has shown strong anti-tumor activity in the treatment of solid tumors such as melanoma, non-small cell lung cancer, kidney cancer and prostate cancer.
  • Immunotherapy drugs have been approved by the US FDA (Food and Drug Administration, FDA) for clinical application.
  • bifunctional antibodies are the direction of antibody drug research and development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control. Therefore, the development of bifunctional antibodies has been difficult. .
  • the purpose of the present invention is to provide an anti-tumor double antibody with stable structure, good specificity and easy preparation.
  • the first aspect of the present invention provides a bifunctional antibody, the bifunctional antibody comprising:
  • the anti-PD-L1 antibody or element and the anti-VEGF antibody or element are connected by a connecting peptide.
  • the connecting peptide includes an antibody constant region sequence.
  • the anti-VEGF antibody or element is connected to a region of the anti-PD-L1 antibody selected from the following group: heavy chain variable region, heavy chain constant region, light chain variable region, Or a combination.
  • the anti-VEGF antibody or element is connected to the beginning of the heavy chain variable region of the anti-PD-L1 antibody.
  • the anti-VEGF antibody or element is connected to the end of the heavy chain constant region of the anti-PD-L1 antibody.
  • the anti-PD-L1 antibody or element is connected to a region of the anti-VEGF antibody selected from the group consisting of heavy chain variable region, heavy chain constant region, light chain variable region, Or a combination.
  • the anti-PD-L1 antibody or element is connected to the beginning of the heavy chain variable region of the anti-VEGF antibody.
  • the anti-PD-L1 antibody or element is connected to the end of the heavy chain constant region of the anti-VEGF antibody.
  • the element includes the extracellular region of a ligand, receptor or protein.
  • the anti-PD-L1 antibody is selected from the group consisting of nanobodies, single-chain antibodies, and double-chain antibodies.
  • the anti-PD-L1 antibody is selected from the following group: animal-derived antibodies (such as murine antibodies), chimeric antibodies, and humanized antibodies.
  • the humanized antibody includes a fully humanized antibody.
  • the anti-PD-L1 element includes the receptor of PD-L1 (such as PD-1) or the extracellular region of a protein.
  • the anti-VEGF antibody is selected from the group consisting of nanobodies, single-chain antibodies, and double-chain antibodies.
  • the anti-VEGF antibody is selected from the group consisting of animal-derived antibodies (such as murine antibodies), chimeric antibodies, and humanized antibodies.
  • the anti-VEGF element includes a VEGF receptor (such as VEGFR) or the extracellular region of a protein.
  • the anti-VEGF antibody or element is in a monovalent form or a multivalent form (such as a bivalent form).
  • the number of the anti-VEGF antibody or element is 1-6, preferably 1-4.
  • the bifunctional antibody is a homodimer.
  • the bifunctional antibody has the structure shown in formula I from N-terminus to C-terminus:
  • Each D is independently an anti-VEGF antibody or element, and at least one D is an anti-VEGF antibody or element;
  • L1, L2, L3, L4, L5, L6 are each independently a key or joint element
  • VL stands for the light chain variable region of the anti-PD-L1 antibody
  • CL stands for the light chain constant region of the anti-PD-L1 antibody
  • VH stands for the variable region of the heavy chain of the anti-PD-L1 antibody
  • CH stands for the heavy chain constant region of the anti-PD-L1 antibody
  • ⁇ represents a disulfide bond or a covalent bond
  • the bifunctional antibody has the activity of simultaneously binding PD-L1 and binding VEGF.
  • D in Formula I are each independently an element that is absent or anti-VEGF, and at least one D is an anti-VEGF element.
  • the joint elements may be the same or different.
  • the L1, L2, L3, L4, L5, or L6 are each independently selected from GS, GGGGS (SEQ ID NO.: 14), GGGGSGGGS (SEQ ID NO.: 15), GGSGGSGGSGGSGGS (SEQ ID NO.: 16).
  • the heavy chain variable region (VH) of the anti-PD-L1 antibody includes the following three complementarity determining region CDRs:
  • the light chain variable region (VL) of the anti-PD-L1 antibody includes the following three complementarity determining region CDRs:
  • amino acid sequence is CDR2’ of GIS.
  • the heavy chain variable region (VH) of the anti-PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO.: 1 or 8.
  • the light chain variable region (VL) of the anti-PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO.: 2 or 9.
  • the anti-VEGF element includes the second extramembrane D2 (VEGFR1D2) of vascular endothelial cell growth factor receptor 1 (VEGFR1).
  • the anti-VEGF element has an amino acid sequence as shown in SEQ ID NO.:10.
  • the diabody is a diabody.
  • the bifunctional antibody has a heavy chain (H chain) and a light chain (L chain).
  • the H chain of the bifunctional antibody has an amino acid sequence as shown in SEQ ID NO.: 11 or SEQ ID NO.: 12.
  • the L chain of the bifunctional antibody has an amino acid sequence as shown in SEQ ID NO.:13.
  • the antibody is in the form of a drug conjugate.
  • the bifunctional antibody further contains (preferably coupled with) a detectable label, a targeting label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the bifunctional antibody is conjugated with a tumor targeting marker conjugate.
  • the bifunctional antibody further includes an active fragment and/or derivative of the bifunctional antibody, wherein the active fragment and/or the derivative retains 70% of the bifunctional antibody. -100% (such as 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) anti-PD-L1 activity and 70-100% Anti-VEGF activity.
  • the derivative of the antibody has at least 85% sequence identity with the antibody of the present invention.
  • the derivative of the antibody is a sequence of the antibody of the present invention that retains at least 85% identity after one or several amino acid deletions, insertions and/or substitutions.
  • the derivative of the antibody has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99% sequence identity.
  • substitutions are conservative substitutions.
  • the bifunctional antibody has a structure shown in formula Ia or Ib from N-terminus to C-terminus:
  • D is an anti-VEGF element
  • L1 is no or joint element
  • VL stands for the light chain variable region of the anti-PD-L1 antibody
  • CL stands for the light chain constant region of the anti-PD-L1 antibody
  • VH stands for the variable region of the heavy chain of the anti-PD-L1 antibody
  • CH stands for the heavy chain constant region of the anti-PD-L1 antibody
  • the bifunctional antibody has the activity of simultaneously binding PD-L1 and binding VEGF.
  • the VH includes CDR1 shown in SEQ ID NO.:3, CDR2 shown in SEQ ID NO.:4, and CDR3 shown in SEQ ID NO.:5.
  • the VL includes CDR1' shown in SEQ ID NO.: 6, CDR2' whose amino acid sequence is GIS, and CDR3' shown in SEQ ID NO.: 7.
  • the heavy chain variable region (VH) of the anti-PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO.: 1 or 8.
  • the light chain variable region (VL) of the anti-PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO.: 2 or 9.
  • the second aspect of the present invention provides an isolated polynucleotide encoding the bifunctional antibody according to the first aspect of the present invention.
  • the polynucleotide has a polynucleotide encoding the L chain of the bifunctional antibody.
  • the polynucleotide has a polynucleotide encoding the H chain of the bifunctional antibody.
  • the ratio of the polynucleotide encoding the L chain to the polynucleotide encoding the H chain is 1:1.
  • the third aspect of the present invention provides a vector containing the polynucleotide of the second aspect of the present invention.
  • the vector contains all the polynucleotides in the second aspect of the present invention.
  • the vectors respectively contain the polynucleotides in the polynucleotides of the second aspect of the present invention.
  • the vector is an expression vector.
  • the vector includes a plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • the fourth aspect of the present invention provides a genetically engineered host cell, the host cell contains the vector according to the third aspect of the present invention or the genome integrates the polynucleotide according to the second aspect of the present invention .
  • the fifth aspect of the present invention provides a method for preparing the antibody of the first aspect of the present invention, including the steps:
  • step (ii) Purifying and/or separating the mixture obtained in step (i) to obtain the bifunctional antibody according to the first aspect of the present invention.
  • the purification can be purified and separated by a protein A affinity column to obtain the target antibody.
  • the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
  • the sixth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition containing:
  • the pharmaceutical composition also contains an additional anti-tumor agent.
  • the pharmaceutical composition is in unit dosage form.
  • the anti-tumor agent comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, levatinib, or pembrolizumab.
  • the anti-tumor agent and the bifunctional antibody can be separately present in a separate package, or the anti-tumor agent can be coupled with the bifunctional antibody.
  • the dosage form of the pharmaceutical composition includes a gastrointestinal administration dosage form or a parenteral administration dosage form.
  • the parenteral administration dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
  • an immunoconjugate comprising:
  • a coupling part selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
  • the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce Enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (for example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the antibody portion and the coupling portion are coupled through a chemical bond or linker.
  • the eighth aspect of the present invention provides the use of the bifunctional antibody according to the first aspect of the present invention or the immunoconjugate according to the seventh aspect of the present invention for preparing (a) detection reagents or kits And/or (b) preparing a pharmaceutical composition for preventing and/or treating cancer or tumor.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the tumor is selected from the group consisting of ovarian cancer, colon cancer, rectal cancer, melanoma (such as metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant Hematological diseases, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor and osteosarcoma.
  • melanoma such as metastatic malignant melanoma
  • Examples of other cancers that can be treated with the method of the present invention include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, intrauterine cancer Membrane cancer, vaginal cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis Cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer , Kidney cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumor
  • the tumor is rectal cancer, non-small cell lung cancer, melanoma, bladder cancer, or a combination thereof.
  • the tumor is a tumor that highly expresses PD-L1 and/or VEGF.
  • the drug or preparation is used to prepare a drug or preparation for preventing and/or treating diseases related to PD-L1 and/or VEGF (positive expression).
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is used to diagnose PD-L1 and/or VEGF related diseases.
  • the detection reagent or kit is used to detect PD-L1 and/or VEGF protein in a sample.
  • the detection reagent is a detection chip.
  • the ninth aspect of the present invention provides a CAR construct.
  • the antigen binding region of the CAR construct includes a binding region that specifically binds to PD-L1 and a binding region that specifically binds to VEGF, and the specific The binding region that sexually binds to PD-L1 has a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region includes CDR1 shown in SEQ ID NO.: 3, CDR2 shown in SEQ ID NO.: 4, and CDR3 shown in SEQ ID NO.: 5;
  • the VL includes CDR1' shown in SEQ ID NO.: 6, CDR2' whose amino acid sequence is GIS, and CDR3' shown in SEQ ID NO.: 7.
  • the binding region that specifically binds to VEGF includes the second extramembranous region D2 (VEGFR1D2) of vascular endothelial cell growth factor receptor 1 (VEGFR1).
  • the binding region that specifically binds to VEGF has an amino acid sequence as shown in SEQ ID NO.:10.
  • the present invention also provides a nucleic acid sequence encoding the CAR construct.
  • the present invention also provides a vector containing the nucleic acid sequence encoding the CAR construct.
  • the tenth aspect of the present invention provides a recombinant immune cell that expresses an exogenous CAR construct as described in the ninth aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells, T cells, NKT cells, or a combination thereof.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • the present invention also provides a method for treating tumors, comprising the steps of: administering to a subject in need a safe and effective amount of the bifunctional antibody according to the first aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention, or The immunoconjugate according to the seventh aspect of the present invention, or the immune cell according to the tenth aspect of the present invention, or a combination thereof.
  • Figure 1 shows the structure map of bifunctional antibodies 900387 and 900388.
  • Figure 2 shows the SDS-PAGE images of the bifunctional antibodies 900387 and 900388. Among them, lane 1: 900387 oxidized or reduced type; lane 2: 900388 oxidized or reduced type.
  • Figure 3 shows the sensor map of the non-specific adsorption detection of bispecific antibodies and non-target molecules.
  • Figure 4 shows the reactor culture results, including cell density (A), viability (B), pH (C), lactate metabolism (D), expression (E), and purity (F).
  • 223 Interval feeding, cooling to 33°C; 225: interval feeding, cooling to 31°C; feeding every day, cooling to 31°C.
  • Figure 5 shows the tumor volume (A) and relative tumor volume change trend (B) of each group of animals. Note: The figure shows the mean ⁇ SEM of the tumor volume of each group of animals, b.i.w. twice a week; i.v. intravenous injection.
  • Figure 6 shows the tumor weight (g) of each group on D32 days. Note: The figure shown is the mean ⁇ SEM of animal body weight in each group, b.i.w. twice a week; i.v. intravenous injection.
  • the inventors unexpectedly obtained a bifunctional antibody, which is composed of an anti-PD-L1 antibody and an anti-VEGF nanobody in series.
  • the bifunctional antibody of the present invention is a homodimer.
  • the bifunctional antibody of the present invention can simultaneously bind to PD-L1 and VEGF, thereby exerting a therapeutic effect on PD-L1 positive and/or VEGF tumor cells (especially malignant tumor cells). Therefore, the present invention Bifunctional antibodies can be developed as an anti-tumor drug with superior curative effect. On this basis, the inventor completed the present invention.
  • administering refers to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids.
  • administering can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells.
  • administering also mean treatment by a reagent, diagnostic, binding composition, or by another cell in vitro and ex vivo.
  • Treatment when applied to humans, animals or research subjects, refers to treatment, preventive or preventive measures, research and diagnosis; including anti-human PD-L1 antibodies and humans or animals, subjects, cells, tissues , Physiological compartment or physiological fluid contact.
  • treatment refers to the administration of an internal or external therapeutic agent, including any one of the anti-human PD-L1 antibodies and compositions of the present invention, to a patient who has one or more disease symptoms and is known
  • the therapeutic agent has a therapeutic effect on these symptoms.
  • the patient is administered in an amount (therapeutically effective amount) of a therapeutic agent effective to alleviate one or more disease symptoms.
  • the term “optional” or “optionally” means that the event or situation described later can occur but does not have to occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may have but does not have to be, and it can be 1, 2, or 3.
  • sequence identity in the present invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions.
  • sequence identity between the sequence described in the present invention and its identical sequence may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.
  • antibody is also called “immunoglobulin”, which can be a natural or conventional antibody, in which two heavy chains are connected to each other by disulfide bonds and each heavy chain and light chain are connected by disulfide bonds.
  • light chains There are two types of light chains, ⁇ (l) and ⁇ (k).
  • the light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, the variable region of the heavy chain (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH).
  • the variable regions of the light chain (VL) and heavy chain (VH) both determine the binding recognition and specificity of the antigen.
  • the constant domain (CL) of the light chain and the constant domain (CH) of the heavy chain confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the immunoglobulin Fab fragment and consists of the variable parts of one light chain and one heavy chain.
  • the specificity of an antibody depends on the structural complementarity between the antibody binding site and the epitope.
  • the antibody binding site is composed of residues mainly derived from hypervariable regions or complementarity determining regions (CDR). Occasionally, residues from non-hypervariable or framework regions (FR) affect the overall domain structure and thus the binding site.
  • Complementarity determining region or CDR refers to an amino acid sequence that collectively defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site.
  • the light chain and the heavy chain of an immunoglobulin each have three CDRs, which are referred to as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, and CDR3-H.
  • Conventional antibody antigen binding sites therefore include six CDRs, including a set of CDRs from each of the heavy and light chain v regions.
  • single domain antibody and “nanobody” have the same meaning, referring to cloning the variable region of the heavy chain of an antibody, and constructing a single domain antibody consisting of only one heavy chain variable region, which has complete functions The smallest antigen-binding fragment.
  • the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • variable means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure .
  • the CDRs in each chain are closely joined together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). Constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • FR framework region
  • the light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
  • human framework region is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .
  • monoclonal antibody refers to an antibody molecule with a single amino acid composition against a specific antigen, and should not be understood as requiring the production of the antibody by any specific method.
  • Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be recombinant, that is, produced by protein engineering.
  • the term "antigen" or "target antigen” refers to a molecule or part of a molecule that can be bound by an antibody or antibody-like binding protein.
  • the term further refers to a molecule or part of a molecule that can be used in animals to produce antibodies that can bind to an epitope of the antigen.
  • the target antigen can have one or more epitopes.
  • the antibody-like binding protein can compete with a complete antibody that recognizes the target antigen.
  • affinity is theoretically defined by the balanced association between the intact antibody and the antigen.
  • the affinity of the double antibody of the present invention can be evaluated or determined by KD value (dissociation constant) (or other measurement methods), such as Bio-layer Interferometry (BLI), which is measured and determined by FortebioRed96 instrument.
  • KD value dissociation constant
  • BLI Bio-layer Interferometry
  • linker refers to the insertion of an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold to exchange one or more amino acid residues of the immunoglobulin with dual variable regions. base.
  • the linker of the present invention refers to the linker L1, L2, L3 and L4, wherein the L1 and L4 linkers connect two anti-VEGF antibodies, and L2 connects the dual anti-heavy chain anti-VEGF antibody dimer of the present invention and the anti-PD-L1 antibody
  • the light chain variable regions VL and L3 connect the heavy chain constant region CH of the double antibody light chain anti-VEGF antibody dimer of the present invention and the anti-PD-L1 antibody.
  • linkers include monoglycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements that need to be implemented in the linker.
  • a preferred joint is as follows:
  • L1, L2, L3, L4, L5, or L6 are each independently selected from GS, GGGGS (SEQ ID NO.: 14), GGGGSGGGS (SEQ ID NO.: 15), GGSGGSGGSGGSGGS (SEQ ID NO.: 16).
  • PD-1 Programmed cell death protein-1
  • PD-1 is a negative costimulatory molecule discovered in recent years and belongs to the CD28 immunoglobulin superfamily. PD-1 is commonly expressed in activated T cells, B cells and myeloid cells. It has two natural ligands, namely programmed death ligand-1 (PD-L1) and PD-L2, both It belongs to the B7 superfamily and is expressed in antigen presenting cells, and PD-L1 is also expressed in various tissues. Among them, PD-L1 is an important negative immunoregulatory factor of PD-1, also known as B7-H1.
  • PD-1 co-inhibitory signal of T cell activation, inhibits T cell activation and proliferation, and plays Similar to the negative regulation of CTLA-4, it can induce T cell apoptosis.
  • the tumor microenvironment can also protect tumor cells from the destruction of immune cells, so that tumor cells cannot be recognized and immune escape phenomenon occurs.
  • the tumor microenvironment continuously expresses PD-L1, which makes the immune function of tumor patients extremely decreased.
  • MPDL3280A anti-PD-L1 monoclonal antibody
  • Avelumab anti-PD-L1 monoclonal antibody
  • the sequence of the anti-PD-L1 antibody of the present invention can be an antibody that is known or prepared by a conventional method or developed through screening.
  • the anti-PD-L1 antibody of the present invention is obtained after screening, and the heavy chain variable region includes CDR1 shown in SEQ ID NO.: 3, CDR2 shown in SEQ ID NO.: 4 and SEQ ID NO. : CDR3 shown in: 5;
  • the VL includes CDR1' shown in SEQ ID NO.: 6, CDR2' whose amino acid sequence is GIS, and CDR3' shown in SEQ ID NO.: 7.
  • Those skilled in the art can also modify or transform the anti-PD-L1 antibody of the present invention through techniques well known in the art, such as adding, deleting and/or substituting one or several amino acid residues to further increase the anti-PD-L1 affinity or Structural stability, and the modified or modified results can be obtained by conventional measurement methods.
  • the heavy chain variable region (VH) of the anti-PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO.: 1 or 8 (the underlined are the marked variable heavy chain regions). Region CDR1, CDR2, CDR3 amino acid sequence).
  • the light chain variable region (VL) of the anti-PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO.: 2 or 9 (the underline is annotated and the light chain can be The amino acid sequence of the variable regions CDR1, CDR2, CDR3).
  • the anti-PD-L1 antibody dimer of the present invention can be obtained by expressing HEK293 cells or CHO cells.
  • the anti-PD-L1 antibody of the present invention binds to mammalian PD-L1, preferably human PD-L1.
  • the binding affinity of the anti-PD-L1 antibody of the present invention to PD-L1 is 9.40E-10M, preferably not less than 5E-09M.
  • the anti-PD-L1 antibody of the present invention is a humanized antibody.
  • Vascular endothelial growth factor (vascular endothelial growth factor), also called VEGF.
  • VEGF protein was successfully purified and identified by scientists from two biotech companies in the United States in 1989, and its gene sequence was cloned and determined, proving that VPF and VEGF are the same protein encoded by the same gene.
  • VEGF has six isoforms: VEGF-A, -B, -C, -D, and -E; its molecular weight ranges from 35 to 44kDa, and each isoform is specifically related to three "vascular endothelial growth Specific combinations of factor receptors" (VEGFR-1, -2, and -3) are combined.
  • VEGFR-1, -2, and -3 vascular endothelial growth Specific combinations of factor receptors
  • VEGF is a highly conserved homodimeric glycoprotein. Two single chains with a molecular weight of 24kDa each form a dimer with disulfide bonds. The monomers decomposed by VEGF are inactive, and the removal of N2 glycosyl has no effect on biological effects, but may play a role in cell secretion. Due to the different shearing methods of mRNA, at least 5 protein forms such as VEGF121, VEGF145, VEGF165, VEGF185, and VEGF206 are produced respectively. Among them, VEGF121, VEGF145 and VEGF165 are secreted soluble proteins that can directly act on vascular endothelial cells and promote vascular endothelial cells.
  • VEGFR1D2 is selected as the anti-VEGF element.
  • VEGFR1D2 of the present invention is connected to two identical VEGFR1D2 are connected by a linker to appear as a dimer.
  • Bispecific Antibody is an unnatural antibody that can target two different antigens or proteins at the same time, block two different signal pathways, and stimulate a specific immune response.
  • the role of and bifunctionality in tumor immunotherapy is becoming more and more important, and it has become a research hotspot in antibody engineering treatment of tumors in the world today.
  • bispecific antibodies mainly mediate the killing of tumors by immune cells in tumor immunotherapy; combine dual targets, block dual signaling pathways, and exert unique or overlapping functions, which can effectively prevent drug resistance; Strong specificity, targeting and reduced off-target toxicity; effective reduction of treatment costs and other advantages (taken from the antibody circle), so the use of bispecific antibody drugs can reduce the chance of tumor cell escape, eliminate tumor cells, and improve efficacy.
  • Bispecific antibodies can be prepared by two-hybridoma cells, chemical coupling, recombinant genes, etc., among which recombinant gene technology has strong flexibility in binding sites and yield. According to incomplete statistics, there are currently more than 60 bispecific antibodies. According to their characteristics and structural differences, the structure of bispecific antibodies mainly includes bispecific antibodies containing Fc fragments (IgG-like bispecific antibodies with Fc Mediated effector function) and bispecific antibody without Fc fragment (non-IgG-like bispecific antibody, which functions through antigen binding capacity, has the advantages of small molecular weight and low immunogenicity).
  • Blinatumomab is a bispecific antibody for CD19 and CD3.
  • Blincyto is the first bispecific antibody approved by the US FDA.
  • bispecific antibodies As used herein, the terms “bispecific antibodies”, “bifunctional antibodies”, “antibodies of the present invention”, “biantibodies of the present invention”, “biantibodies”, and “bifunctional fusion antibodies” are used interchangeably and refer to simultaneous binding to PD -Anti-PD-L1/VEGF bispecific antibody to L1 and VEGF.
  • the bifunctional antibody includes:
  • the bifunctional antibody has the structure shown in formula I from N-terminus to C-terminus:
  • Each D is independently an anti-VEGF antibody or element, and at least one D is an anti-VEGF antibody or element;
  • L1, L2, L3, L4, L5, L6 are each independently a key or joint element
  • VL stands for the light chain variable region of the anti-PD-L1 antibody
  • CL stands for the light chain constant region of the anti-PD-L1 antibody
  • VH stands for the variable region of the heavy chain of the anti-PD-L1 antibody
  • CH stands for the heavy chain constant region of the anti-PD-L1 antibody
  • ⁇ represents a disulfide bond or a covalent bond
  • the bifunctional antibody has the activity of simultaneously binding PD-L1 and binding VEGF.
  • the double antibody of the present invention is formed by the fusion of PD-L1 antibody and VEGR1D2, and has two pairs of symmetrical peptide chains.
  • Each pair of peptide chains contains a light chain L chain and a heavy chain H chain. All peptides The chains are all connected by disulfide bonds, and any pair of peptide chains has the L chain and H chain structures shown in formula Ia or Ib from N to C terminal:
  • D is an anti-VEGF element (VEGR1D2)
  • L1 is no or joint element
  • VL stands for the light chain variable region of the anti-PD-L1 antibody
  • CL stands for the light chain constant region of the anti-PD-L1 antibody
  • VH stands for the variable region of the heavy chain of the anti-PD-L1 antibody
  • CH stands for the heavy chain constant region of the anti-PD-L1 antibody
  • the bifunctional antibody has the activity of simultaneously binding PD-L1 and binding VEGF.
  • a preferred H chain is shown in SEQ ID NO.: 11 or SEQ ID NO.: 12, and a preferred L chain is shown in SEQ ID NO.: 13.
  • the double antibodies of the present invention include not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies. As used herein, the terms “fragment”, “derivative” and “analog” refer to polypeptides that substantially maintain the same biological function or activity as the antibody of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with substitution groups in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or with Fusion protein formed by 6His tag).
  • these fragments, derivatives and analogs are within the scope well known to those skilled in the art.
  • the double antibody of the present invention refers to an antibody that has anti-PD-L1 and anti-VEGF activities and includes two of the above-mentioned structures of formula I.
  • the term also includes variant forms of the antibody having the same function as the double antibody of the present invention, including the two above-mentioned structures of formula I.
  • These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and the addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the dual antibodies of the present invention.
  • the variant forms of the double antibody include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention are included in the variant forms of the double antibody.
  • “conservative variants of the double antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, compared with the amino acid sequence of the double antibody of the present invention. Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by amino acid substitutions.
  • the present invention also provides polynucleotide molecules encoding the aforementioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • nucleic acid (and nucleic acid combination) of the present invention can be used to produce the recombinant antibody of the present invention in a suitable expression system.
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • One feasible method is to use artificial synthesis to synthesize relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned suitable DNA sequence and a suitable promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the expression level of the bispecific antibody can reach 3.9g/L, the purity is above 97%, and the lactic acid can be metabolized well during the culture process.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • the double antibody of the present invention can be used alone, or can be combined or coupled with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nano magnetic particles; 8. Tumor therapeutic agents (for example, cisplatin) or any form of anti-tumor drugs.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned bispecific antibody or active fragment or fusion protein of the present invention, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavity injection.
  • routes including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavity injection.
  • the pharmaceutical composition of the present invention can be directly used to bind PD-L1 protein molecules or PD-L1, and thus can be used to treat tumors.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned Nanobody (or conjugate) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptide of the present invention can also be used with
  • bispecific antibodies can be used alone, and the dosage regimen can be adjusted to obtain the best objective response. For example, single administration, or multiple administrations over a period of time, or the dose can be reduced or increased proportionally to the urgency of the treatment situation.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • the bifunctional antibody of the present invention can bind PD-L1 and VEGF at the same time, and keep the binding configuration of the bifunctional antibody and the binding target unchanged, and the molecule is stable.
  • the bispecific antibody HB0025 of the present invention can specifically bind to recombinant human PD-L1 and recombinant human VEGF165 (KD less than 10 -5 M), and has no non-specific electrostatic and hydrophobic binding with non-target molecules ; In the preliminary culture conditions, the expression of bispecific antibodies reached 3.9g/L, and the purity was above 97%.
  • the bifunctional antibody of the present invention has high binding affinity to the binding target, good blocking activity, and exhibits a certain dual-target synergistic effect, which can effectively kill tumor cells (especially tumors with high PD-L1 and VEGF expression), Thereby significantly reducing tumor volume and tumors, treating cancer, especially solid tumors.
  • the bispecific antibody of the present invention has higher binding activity to PD-L1 and/or VEGF.
  • the affinity of the bispecific antibody of the present invention to PD-L1 is about 1E-10 mol; the affinity to VEGF165 is about 1E-11 mol.
  • the preparation method of the present invention is simple and feasible.
  • the anti-VEGF/PD-L1 bispecific antibody applied by the present invention will have a good application prospect.
  • the anti-PD-L1 antibody of the present invention is a mouse monoclonal antibody obtained by immunizing mice with human PD-L1-His protein (heavy chain variable region and light chain variable region sequences are as SEQ ID NO.:1 and SEQ ID NO.: 2), and then humanized the humanized monoclonal antibody (900339).
  • the heavy chain sequence is shown in SEQ ID NO.:8, and the light chain sequence is shown in SEQ ID NO.:9.
  • the artificially synthesized VEGFR1D2 (sequence shown in SEQ ID NO.: 10) is connected to the 5'end or 3'end of the heavy chain expression vector by connecting Linker (GGSGGSGGSGGSGGS, SEQ ID NO.: 16), and the light chain vector (1 1) Co-transfected into CHO-S cells, cultured at 37°C, 5% CO 2 , 130 rpm/min for 7 days, and centrifuged to collect the supernatant. The supernatant was centrifuged at 4000 rpm for 10 min, and filtered with a 0.45 ⁇ m filter membrane to collect the filtrate. After the filtrate was purified by the Protein A affinity column, antibodies 900387 and 900388 were obtained.
  • the structure map is shown in Figure 1.
  • the purified protein was tested by SEC_UPLC, and the purity was greater than 98%, and was tested by SDS-PAGE.
  • the results of SDS-PAGE reduction or non-reduction electrophoresis detection are shown in Figure 2.
  • the bispecific antibodies 900387 and 900388 were prepared.
  • the protein number of 900387 is HB0025, and the relevant test results of the following experiments are all expressed as HB0025, and its heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO.: 11 and SEQ ID NO.: 13, respectively.
  • the SPR method was used to measure antibody-antigen binding kinetics and affinity.
  • the bispecific antibody HB0025 has a good affinity with recombinant human PD-L1; the bispecific antibody HB0025 has a good affinity with recombinant human VEGF165.
  • the SPR method was used to determine the non-specific adsorption effects of antibodies and non-target molecules.
  • Soybean pancreatic inhibitor 1-S type Sigma, T-2327
  • Method Put the Series S Sensor Chip CM5 chip at room temperature to equilibrate for 20-30 minutes, and load it into the Biacore 8K instrument.
  • the amino coupling kit was used to fix egg white lysozyme and soybean pancreatic inhibitor 1-S type to CM5 chip respectively.
  • the injection buffer is HBS-EP+1X, and 4 equilibrium cycles are set. Dilute the anti-lysozyme rabbit polyclonal antibody, anti-trypsin inhibitor antibody, and humanized monoclonal antibody to 1000 nM with equilibration buffer, set a flow rate of 5 ⁇ L/min, injection channels 1, 2 and 3, Flow Cell 1 and 2.
  • the binding time is 10min, and the dissociation time is 15min.
  • the regeneration flow rate is 50 ⁇ L/min, first 0.85% phosphoric acid is regenerated for 60s, and then 50mM sodium hydroxide is used for 30s.
  • Table 3 The results of the non-specific adsorption of bispecific antibody HB0025 and non-target molecules are shown in Table 3, and the sensor diagram of the detection of non-specific adsorption of bispecific antibody and non-target molecules is shown in Figure 3.
  • the binding signals of HB0025 with soybean pancreatic inhibitor 1-S and lysozyme are all less than 20, so it can be considered that the four samples have no non-specific electrostatic and hydrophobic binding. In turn, it can reduce the immunity to the human body after the antibody is injected into the human body, causing a series of immune diseases such as hemangioma.
  • the amino groups of egg white lysozyme and soybean pancreatic inhibitor can be determined After being coupled and fixed to the CM5 chip, the activity is normal and the activity remains good (if the combined signal is less than 20, it can be considered that the sample has no non-specific electrostatic and hydrophobic binding).
  • this CHO-K1 cell line can metabolize lactic acid very well; the cell growth status is good, and the survival rate is higher than 90% when harvested at 16 days; cooling down There is no significant difference in expression between 33°C and 31°C, about 3g/L; daily feeding can significantly increase cell density and expression, and the final expression is 3.9g/L; the purity of the whole culture process does not decrease, and it is maintained at about 97%.
  • hCD34+ humanized mice were inoculated with human lung adenocarcinoma HCC-827 cells in the right armpit. When the tumor grew to an average of about 100-150 mm 3 , 48 tumor-bearing mice were tested according to the hCD45+ ratio in the peripheral blood and the tumor volume.
  • the tumor volume of the PBS vehicle control group (G1) mice continued to increase (see Figure 5), indicating that the HCC827 lung cancer cell hCD34+ humanized mouse subcutaneously transplanted tumor model was successfully established.
  • G2 animals were given HB0023 1mg/kg, the tumor volume increased relatively slowly after administration.
  • the tumor volume and relative tumor volume were relatively reduced, but there was no statistical difference; at the end of the D32 test, compared with the vehicle Compared with the group, the tumor weight was relatively reduced (P>0.05, see Figure 5, Figure 6), and the tumor inhibition rate was 17.48%.
  • G6 animals were given HB002.1T 1mg/kg, the tumor volume increased slowly after administration.
  • the tumor volume and relative tumor volume were significantly reduced (P ⁇ 0.05, see Figure 5); at the end of the D32 test, Compared with the vehicle control group, the tumor weight was significantly reduced (P ⁇ 0.05, see Figure 6), and the tumor inhibition rate was 64.08%.
  • mice in G3, G4, and G5 groups were given HB0025 1, 3, and 10 mg/kg, respectively.
  • the tumor volume increased slowly, and the tumor volume and relative tumor volume were significantly lower than that of the vehicle control group (p ⁇ 0.05); at the end of the D32 test, Compared with the vehicle control group, the tumor weight was significantly reduced (P ⁇ 0.05, see Figure 6), and the tumor inhibition rates were 70.87%, 84.47%, and 85.44%, respectively, indicating that HB0025 can effectively inhibit hCD34+ humanized mouse HCC827 lung cancer subcutaneously
  • the effective tumor suppressor dose is 1 mg/kg.
  • the affinity of the bispecific antibody 900388 (heavy chain amino acid sequence and light chain amino acid sequence shown in SEQ ID NO.: 12 and SEQ ID NO.: 13) to the bispecific antibody 900388 prepared in Example 1 and non-target molecules
  • the non-specific adsorption effect and in vivo drug efficacy of HBV were tested.
  • the experimental method was the same as that in Example 2-4, and HB0025 was replaced with bispecific antibody 900388.
  • bispecific antibody 900388 has good affinity with recombinant human PD-L1 and recombinant human VEGF165, and has no non-specific electrostatic and hydrophobic binding. It can also treat tumors well and significantly reduce the volume of animal tumors. And tumor weight.
  • the anti-VEGF/PD-L1 bispecific antibody of the present invention can be expressed in CHO-K1 cells and can be further purified by affinity chromatography.
  • the resulting bispecific antibody can bind to PD-L1 positive cells and VEGF positive cells.
  • the antibody has good affinity, not only has good anti-VEGF biological activity, but also perfectly retains the biological activity of anti-PD-L1 antibody. Therefore, the anti-VEGF/PD-L1 bispecific antibody of the present invention will have a good application prospect.

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Abstract

本发明提供了一种抗PD-L1/VEGF双特异性抗体及其用途，具体地，本发明提供一种双功能抗体，包括：（a）抗PD-L1的抗体或元件；和（b）与所述抗PD-L1的抗体或元件相连接的抗VEGF的抗体或元件。本发明的双功能抗体可同时与VEGF及PD-L1结合，从而发挥对VEGF和PD-L1阳性的肿瘤细胞（尤其是恶性肿瘤细胞）的治疗作用。

Description

抗PD-L1/VEGF双功能抗体及其用途 技术领域
本发明属于肿瘤免疫学领域，具体地，涉及抗PD-L1/VEGF双功能抗体及其用途。
背景技术
肿瘤(tumour)根据新生物的细胞特性及对机体的危害性程度，可分为良性肿瘤和恶性肿瘤两大类；其中恶性肿瘤疾病是当今社会上危害人类健康的重大疾病，致死程度高居第二，常见肿瘤有肝癌、肺癌、胃癌、乳腺癌、膀胱癌等。据报道，2018年全球有大约1810万癌症新发病例和960万癌症死亡病例(剔除非黑色素瘤皮肤癌后分别为1700万和950万)，其中新增1810万癌症病例中，亚洲占据近一半，960万癌症死亡患者中，亚洲占近七成。
恶性肿瘤由于其个体差异，一般会对大部分患者进行综合治疗，即综合采用手术、化疗、放疗、免疫治疗、中医中药治疗、介入治疗、微波治疗等手段，以期较大幅度地提高治愈率，并改善患者的生活质量。其中免疫治疗(immunotherapy)是指针对机体低下或亢进的免疫状态，人为地增强或抑制机体的免疫功能以达到治疗疾病目的的治疗方法。免疫治疗的方法有很多，适用于多种疾病的治疗。肿瘤的免疫治疗旨在激活人体免疫***，依靠自身免疫机能杀灭癌细胞和肿瘤组织，从而控制与清除肿瘤的一种治疗方法。与以往的手术、化疗、放疗和靶向治疗不同的是，免疫治疗针对的靶标不是肿瘤细胞和组织，而是人体自身的免疫***。包括单克隆抗体类免疫检查点抑制剂、治疗性抗体、癌症疫苗、细胞治疗和小分子抑制剂等。近几年，肿瘤免疫治疗的好消息不断，目前已在多种肿瘤如黑色素瘤，非小细胞肺癌、肾癌和***癌等实体瘤的治疗中展示出了强大的抗肿瘤活性，多个肿瘤免疫治疗药物已经获得美国FDA(Food and Drug Administration,FDA)批准临床应用。
目前上市在售的抗体药物多为单克隆抗体，治疗性单克隆抗体已被用于治疗癌症、自身免疫病、炎症和其他疾病，多数是针对一个靶标的特异性。然而，病人接受单克隆抗体治疗可能产生耐药性或无应答。并且有些疾病在体内的影响因素是多方面的，包括不同的信号通路、不同的细胞因子和受体的调节机制等，单一靶点的免疫疗法似乎并不足以摧毁癌细胞。因此，需要通过组合不同的药物，或是使用多特异性抗体的多重靶向策略来实现。
双功能抗体虽然是抗体药物研发的方向，但面临诸多挑战，比如临床前评价模型、表达量低、稳定性差、工艺复杂、质控差异性大等问题，因此一直以来双功能抗体的研发困难重重。
因此，本领域迫切开发一种特异性佳、疗效好且易于制备的抗肿瘤双抗。
发明内容
本发明的目的在于提供一种结构稳定、特异性佳且易于制备的抗肿瘤双抗。
本发明的第一方面，提供了一种双功能抗体，所述双功能抗体包括：
(a)抗PD-L1的抗体或元件；和
(b)与所述抗PD-L1的抗体或元件相连接的抗VEGF的抗体或元件。
在另一优选例中，所述抗PD-L1的抗体或元件和所述抗VEGF的抗体或元件通过连接肽相连。
在另一优选例中，所述连接肽包括抗体恒定区序列。
在另一优选例中，所述抗VEGF的抗体或元件连接到所述抗PD-L1的抗体的选自下组的区域：重链可变区、重链恒定区、轻链可变区、或其组合。
在另一优选例中，所述抗VEGF的抗体或元件连接到所述抗PD-L1的抗体的重链可变区的起始端。
在另一优选例中，所述抗VEGF的抗体或元件连接到所述抗PD-L1的抗体的重链恒定区的末端。
在另一优选例中，所述抗PD-L1的抗体或元件连接到所述抗VEGF的抗体的选自下组的区域：重链可变区、重链恒定区、轻链可变区、或其组合。
在另一优选例中，所述抗PD-L1的抗体或元件连接到所述抗VEGF的抗体的重链可变区的起始端。
在另一优选例中，所述抗PD-L1的抗体或元件连接到所述抗VEGF的抗体的重链恒定区的末端。
在另一优选例中，所述的元件包括配体、受体或蛋白的胞外区。
在另一优选例中，所述的抗PD-L1的抗体选自下组：纳米抗体、单链抗体、双链抗体。
在另一优选例中，所述的抗PD-L1的抗体选自下组：动物源抗体(如鼠源抗体)、嵌合抗体、人源化抗体。
在另一优选例中，所述的人源化抗体包括全人源化抗体。
在另一优选例中，所述的抗PD-L1的元件包括PD-L1的受体(如PD-1)或蛋白的胞外区。
在另一优选例中，所述的抗VEGF的抗体选自下组：纳米抗体、单链抗体、双链抗体。
在另一优选例中，所述的抗VEGF的抗体选自下组：动物源抗体(如鼠源抗体)、嵌合抗体、人源化抗体。
在另一优选例中，所述的抗VEGF的元件包括VEGF的受体(如VEGFR)或蛋白的胞外区。
在另一优选例中，所述的抗VEGF的抗体或元件是单价形式的或多价形式的(如二价形式)。
在另一优选例中，所述双功能抗体中，所述的抗VEGF的抗体或元件的数量为1-6，较佳地，1-4。
在另一优选例中，所述双功能抗体为同源二聚体。
在另一优选例中，所述双功能抗体从N端到C端具有式I所示的结构：
Figure PCTCN2020082535-appb-000001
其中，
D各自独立地为无或抗VEGF的抗体或元件，且至少一个D为抗VEGF的抗体或元件；
L1、L2、L3、L4、L5、L6各自独立地为键或接头元件；
VL代表抗PD-L1抗体的轻链可变区；
CL代表抗PD-L1抗体的轻链恒定区；
VH代表抗PD-L1抗体的重链可变区；
CH代表抗PD-L1抗体的重链恒定区；
“～”代表二硫键或共价键；
“-”代表肽键；
其中，所述双功能抗体具有同时结合PD-L1以及结合VEGF的活性。
在另一优选例中，式I中的D均各自独立地为无或抗VEGF的元件，且至少一个D为抗VEGF的元件。
在另一优选例中，所述接头元件可以相同或不相同。
在另一优选例中，所述L1、L2、L3、L4、L5或L6各自独立地选自GS、GGGGS(SEQ ID NO.:14)、GGGGSGGGS(SEQ ID NO.:15)、GGSGGSGGSGGSGGS(SEQ ID NO.:16)。
在另一优选例中，所述的抗PD-L1抗体的重链可变区(VH)包括以下三个互补决定区CDR：
SEQ ID NO.:3所示的CDR1，
SEQ ID NO.:4所示的CDR2，和
SEQ ID NO.:5所示的CDR3；和/或
所述的抗PD-L1抗体的轻链可变区(VL)包括以下三个互补决定区CDR：
SEQ ID NO.:6所示的CDR1’，
氨基酸序列为GIS的CDR2’，和
SEQ ID NO.:7所示的CDR3’。
在另一优选例中，所述的抗PD-L1抗体的重链可变区(VH)具有如SEQ ID NO.:1或8所示的氨基酸序列。
在另一优选例中，所述的抗PD-L1抗体的轻链可变区(VL)具有如SEQ ID NO.:2或9所示的氨基酸序列。
在另一优选例中，所述的抗VEGF的元件包括血管内皮细胞生长因子受体1(VEGFR1)的第二膜外区D2(VEGFR1D2)。
在另一优选例中，所述的抗VEGF的元件具有如SEQ ID NO.:10所示的氨基酸序列。
在另一优选例中，所述的双功能抗体为双链抗体。
在另一优选例中，所述的双功能抗体具有重链(H链)和轻链(L链)。
在另一优选例中，所述双功能抗体的H链具有如SEQ ID NO.:11或SEQ ID NO.:12所示的氨基酸序列。
在另一优选例中，所述双功能抗体的L链具有如SEQ ID NO.:13所示的氨基酸序列。
在另一优选例中，所述的抗体为药物偶联物形式。
在另一优选例中，所述双功能抗体还含有(优选偶联有)可检测标记物、靶向标记、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述双功能抗体偶联有肿瘤靶向标记偶联物。
在另一优选例中，所述双功能抗体还包括所述双功能抗体的活性片段和/或衍生物，其中，所述活性片段和/或所述衍生物保留了所述双功能抗体的70-100％(如70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、100％)的抗PD-L1活性和70-100％的抗VEGF活性。
在另一优选例中，所述抗体的衍生物具有与本发明抗体至少85％的序列同一性。
在另一优选例中，所述抗体的衍生物是本发明抗体经过一个或几个氨基酸缺失、***和/或取代后并保持至少85％的同一性的序列。
在另一优选例中，所述抗体的衍生物具有与本发明抗体至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性。
在另一优选例中，所述的取代为保守性取代。
在另一优选例中，所述双功能抗体从N端到C端具有式Ia或Ib所示的结构：
Figure PCTCN2020082535-appb-000002
其中，
D为抗VEGF的元件；
L1为无或接头元件；
VL代表抗PD-L1抗体的轻链可变区；
CL代表抗PD-L1抗体的轻链恒定区；
VH代表抗PD-L1抗体的重链可变区；
CH代表抗PD-L1抗体的重链恒定区；
“～”代表二硫键；
“-”代表肽键；
其中，所述双功能抗体具有同时结合PD-L1以及结合VEGF的活性。
在另一优选例中，所述VH包括SEQ ID NO.:3所示的CDR1、SEQ ID NO.:4所示的CDR2和SEQ ID NO.:5所示的CDR3。
在另一优选例中，所述VL包括SEQ ID NO.:6所示的CDR1’、氨基酸序列为GIS的CDR2’和SEQ ID NO.:7所示的CDR3’。
在另一优选例中，所述的抗PD-L1抗体的重链可变区(VH)具有如SEQ ID NO.:1或8所示的氨基酸序列。
在另一优选例中，所述的抗PD-L1抗体的轻链可变区(VL)具有如SEQ ID NO.:2或9所示的氨基酸序列。
本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码本发明的第一方面所述的双功能抗体。
在另一优选例中，所述多核苷酸具有编码所述双功能抗体L链的多核苷酸。
在另一优选例中，所述多核苷酸具有编码所述双功能抗体H链的多核苷酸。
在另一优选例中，所述多核苷酸中，编码L链的多核苷酸和编码H链的多核苷酸的比例为1:1。
本发明的第三方面，提供了一种载体，所述载体含有本发明的第二方面所述的多核苷酸。
在另一优选例中，所述载体同时含有本发明第二方面所述多核苷酸中的所有多核苷酸。
在另一优选例中，所述载体分别含有本发明第二方面所述多核苷酸中的多核苷酸。
在另一优选例中，所述载体为表达载体。
在另一优选例中，所述载体包括质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第四方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有本发明的第三方面所述的载体或基因组中整合有本发明的第二方面所述的多核苷酸。
本发明的第五方面，提供了一种制备本发明的第一方面所述抗体的方法，包括步骤：
(i)在合适的条件下，培养本发明的第四方面所述的宿主细胞，获得含有本发明的第一方面所述的双功能抗体的混合物；和
(ii)对步骤(i)中得到的混合物进行纯化和/或分离，从而获得本发明的第一方面所述的双功能抗体。
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。
本发明的第六方面，提供了一种药物组合物，所述药物组合物含有：
(I)如本发明的第一方面所述的双功能抗体；和
(II)药学上可接受的载体。
在另一优选例中，所述药物组合物中还含有额外的抗肿瘤剂。
在另一优选例中，所述药物组合物为单元剂型。
在另一优选例中，所述抗肿瘤剂包含紫杉醇、多柔比星、环磷酰胺、阿西替尼、乐伐替尼、或派姆单抗。
在另一优选例中，所述的抗肿瘤剂可以与所述双功能抗体单独存在于独立的包装内，或所述抗肿瘤剂可以与所述双功能抗体偶联。
在另一优选例中，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型。
在另一优选例中，所述的胃肠外给药剂型包括静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射、或腔内注射。
本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物包括：
(a)如本发明的第一方面所述的双功能抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述偶联物部分选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
本发明的第八方面，提供了如本发明的第一方面所述双功能抗体或如本发明的第七方面所述的免疫偶联物的用途，用于制备(a)检测试剂或试剂盒；和/或(b)制备预防和/或治疗癌症或肿瘤的药物组合物。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述肿瘤选自下组：卵巢癌、结肠癌、直肠癌、黑色素瘤(例如转移的恶性黑色素瘤)、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、***、子宫体瘤和骨肉瘤。可以用本发明的方法治疗的其他癌症的例子包括:骨癌、膜腺癌、皮肤癌、***癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、***癌、***癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌***癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、***癌、慢性或急性白血病，包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾孟癌、中枢神经***(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波因肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
在另一优选例中，所述肿瘤为直肠癌、非小细胞性肺癌、黑色素瘤、膀胱癌、或其组合。
在另一优选例中，所述肿瘤为高表达PD-L1和/或VEGF的肿瘤。
在另一优选例中，所述药物或制剂用于制备预防和/或治疗与PD-L1和/或VEGF(表达阳性的)相关的疾病的药物或制剂。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的检测试剂或试剂盒用于诊断PD-L1和/或VEGF相关疾病。
在另一优选例中，所述检测试剂或试剂盒用于检测样品中PD-L1和/或VEGF蛋白。
在另一优选例中，所述的检测试剂为检测片。
本发明的第九方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区域包括特异性结合于PD-L1的结合区和特异性结合于VEGF的结合区，并且所述特异性结合于PD-L1的结合区具有重链可变区和轻链可变区，其中
所述重链可变区包括SEQ ID NO.:3所示的CDR1、SEQ ID NO.:4所示的CDR2和SEQ ID NO.:5所示的CDR3；
所述VL包括SEQ ID NO.:6所示的CDR1’、氨基酸序列为GIS的CDR2’和SEQ ID NO.:7所示的CDR3’。
在另一优选例中，所述特异性结合于VEGF的结合区包括血管内皮细胞生长因子受体1(VEGFR1)的第二膜外区D2(VEGFR1D2)。
在另一优选例中，所述特异性结合于VEGF的结合区具有如SEQ ID NO.:10所示的氨基酸序列。
本发明还提供了编码所述CAR构建物的核酸序列。
本发明还提供了含有所述编码所述CAR构建物的核酸序列的载体。
本发明的第十方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明第九方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞、NKT细胞、或其组合。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明还提供了一种***的方法，包括步骤：向需要的对象施用安全有效量的本发明第一方面所述的双功能抗体、或本发明第六方面所述的药物组合物、或本发明第七方面所述的免疫偶联物、或本发明第十方面所述的免疫细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了双功能抗体900387和900388结构图谱。
图2显示了双功能抗体900387和900388的SDS-PAGE图。其中，泳道1:900387氧化型或还原型；泳道2:900388氧化型或还原型。
图3显示了双特异性抗体与非靶分子非特异吸附检测传感图。
图4显示了反应器培养结果，包括细胞密度(A)、活率(B)、pH(C)、乳酸代谢(D)、表达量(E)、纯度(F)。223：间隔补料，降温至33℃；225：间隔补料，降温至31℃；每天补料，降温至31℃。
图5显示了各组动物肿瘤体积(A)和相对肿瘤体积变化趋势(B)。注:图中所示为各组动物肿瘤体积平均值±SEM，b.i.w.一周两次；i.v.静脉注射。
图6显示了D32天各组肿瘤重量(g)。注:图中所示为各组动物体重平均值±SEM，b.i.w.一周两次；i.v.静脉注射。
具体实施方式
本发明人通过广泛而深入的研究，意外地获得一种双功能抗体，由抗PD-L1的抗体和抗VEGF纳米抗体串联而成。优选地，本发明的双功能抗体为同源二聚体。体外实验证实，本发明的双功能抗体可同时与PD-L1及VEGF结合，从而发挥对PD-L1阳性和/或VEGF的肿瘤细胞(尤其是恶性肿瘤细胞)的治疗作用，因此，本发明的双功能抗体可以被开发为一种疗效优越的抗肿瘤药物。在此基础上，本发明人完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括抗人PD-L1抗体与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的任何一种抗人PD-L1抗体及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
本发明所述的“序列同一性”表示当具有适当的替换、***或缺失等突变的情况下最佳比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其具有同一性的序列之间的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,100％。
通常，“抗体”也称为“免疫球蛋白“其可以是天然或常规的抗体，其中两条重链通过二硫键彼此连接且每条重链与轻链通过二硫键连接。存在两种类型的轻链，λ(l)和κ(k)。存在五种主要的重链种类(或同型)，其决定抗体分子的功能活性：IgM、IgD、IgG、IgA和IgE。每种链包含不同的序列结构域。轻链包括两个结构域或区，可变结构域(VL)和恒定结构域(CL)。重链包括四个结构域，重链可变区(VH)和三个恒定区(CH1、CH2和CH3，统称为CH)。轻链(VL)和重链(VH)的可变区都决定对抗原的结合识别和特异性。轻链的恒定结构域(CL)和重链的恒定区(CH)赋予重要的生物性质如抗体链结合、分泌、经胎盘的移动性、补体结合和与Fc受体(FcR)的结合。Fv片段是免疫球蛋白Fab片段的N-末端部分且由一条轻链和一条重链的可变部分组成。抗体的特异性取决于抗体结合位点和抗原决定区间的结构互补。抗体结合位点由主要来自高度可变区或互补决定区(CDR)的残基组成。偶尔，来自非高度可变或框架区(FR)的残基影响整体结构域结构且进而影响结合位点。互补决定区或CDR指共同限定结合亲和力和天然免疫球蛋白结合位点天然Fv区的特异性的氨基酸序列。免疫球蛋白的轻链和重链各具有三个CDR，分另称为CDR1-L、CDR2-L、 CDR3-L和CDR1-H、CDR2-H、CDR3-H。常规抗体抗原结合位点因此包括六个CDR，包含来自每个重链和轻链v区的CDR集合。
如本文所用，术语“单域抗体”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β-折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“框架区”(FR)指***CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“单克隆抗体”或“mAb”指针对特定抗原的具有单一氨基酸组成的抗体分子，且不应理解为需要通过任何特定方法产生该抗体。单克隆抗体可由B细胞或杂交瘤的单一克隆产生，但还可为重组的，即通过蛋白工程产生。
如本文所用，术语“抗原”或“靶抗原”指能够由抗体或抗体样结合蛋白所结合的分子或分子的部分。该术语进一步指能够用于动物以产生能够与该抗原的表位结合的抗体的分子或分子的部分。靶抗原可具有一个或多个表位。对于每种由抗体或由抗体样结合蛋白识别的靶抗原，抗体样结合蛋白能够与识别靶抗原的完整抗体竞争。
如本文所用，术语“亲和力”理论上通过完整抗体和抗原间的平衡缔合来定义。本发明双抗的亲和力可以通过KD值(解离常数)(或其它测定方式)进行评估或测定，例如生物膜层干涉技术(Bio-layer interferometry BLI)，使用FortebioRed96仪器测量确定。
如本文所用，术语“接头”是指***免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。本发明所 述的接头是指接头L1、L2、L3和L4，其中L1和L4接头连接两个抗VEGF抗体，L2连接本发明双抗重链抗VEGF抗体二聚体与抗PD-L1抗体的轻链可变区VL，L3连接本发明双抗轻链抗VEGF抗体二聚体与抗PD-L1抗体的重链恒定区CH。
合适的接头实例包括单甘氨酸(Gly)、或丝氨酸(Ser)残基，接头中氨基酸残基的标识和序列可随着接头中需要实现的次级结构要素的类型而变化。一种优选的接头如下：
L1、L2、L3、L4、L5或L6各自独立地选自GS、GGGGS(SEQ ID NO.:14)、GGGGSGGGS(SEQ ID NO.:15)、GGSGGSGGSGGSGGS(SEQ ID NO.:16)。
抗PD-L1抗体
细胞程序性死亡受体-1(programmed cell death protein，PD-1)是近年来发现的一种负性共刺激分子,为CD28免疫球蛋白超家族。PD-1普遍表达于活化的T细胞，B细胞及髓系细胞，其有两个天然配体，即程序性死亡配体-1(programmed death ligand 1，PD-L1)和PD-L2，均属于B7超家族，在抗原递呈细胞中均有表达，PD-L1在多种组织也有表达。其中PD-L1是PD-1的一种重要负性免疫调节因子，又称B7-H1，其与PD-1的结合介导T细胞活化的共抑制信号，抑制T细胞活化和增殖，起到类似于CTLA-4的负调节作用，可诱导T细胞的凋亡。而且有研究报道表明肿瘤微环境也可以保护肿瘤细胞免受免疫细胞的破坏，使得肿瘤细胞不能被识别，发生免疫逃逸现象。而且肿瘤微环境可持续性表达PD-L1，使得肿瘤患者的免疫功能极度下降。
华裔科学家陈列平实验室首先发现PD-L1在肿瘤组织高表达，而且调节肿瘤浸润CD8T细胞的功能。因此，以PD-1/PD-L1为靶点的免疫调节对抗肿瘤有重要的意义。近年来，已有多种Anti-PD-1/PD-L1抗体在肿瘤免疫治疗的临床研究迅速开展。目前Pembrolizumab和Nivolumab已被FDA批准用于晚期黑色素瘤，最近Nivolumab也已被美国FDA批准用于晚期鳞状非小细胞肺癌的治疗。另外，MPDL3280A(anti-PD-L1单抗)，Avelumab(anti-PD-L1单抗)等也已进入多个晚期临床研究中，覆盖非小细胞癌，黑色素瘤，膀胱癌等多个瘤种。
本发明抗PD-L1抗体的序列可以采用已知的或用常规方法制备的或经筛选开发的抗体。优选地，本发明抗PD-L1抗体是经过筛选得到的，所述重链可变区包括SEQ ID NO.:3所示的CDR1、SEQ ID NO.:4所示的CDR2和SEQ ID NO.:5所示的CDR3；所述VL包括SEQ ID NO.:6所示的CDR1’、氨基酸序列为GIS的CDR2’和SEQ ID NO.:7所示的CDR3’。
Figure PCTCN2020082535-appb-000003
本领域技术人员也可以通过本领域熟知的技术对本发明抗PD-L1抗体进行修饰或改造，例如添加、缺失和/或取代一个或几个氨基酸残基，从而进一步增加抗PD-L1的亲和力或结构稳定性，并通过常规的测定方法获得修饰或改造后的结果。
在另一优选例中，所述的抗PD-L1抗体的重链可变区(VH)具有如SEQ ID NO.:1或8所示的氨基酸序列(下划线为标注的依次为重链可变区CDR1，CDR2，CDR3的氨基酸序列)。
Figure PCTCN2020082535-appb-000004
在另一优选例中，所述的抗PD-L1抗体的轻链可变区(VL)具有如SEQ ID NO.:2或9所示的氨基酸序列(下划线为标注的依次为轻链链可变区CDR1，CDR2，CDR3的氨基酸序列)。
Figure PCTCN2020082535-appb-000005
本发明抗PD-L1抗体二聚体可由HEK293细胞或CHO细胞表达获得。
本发明抗PD-L1抗体与哺乳动物PD-L1结合，优选为人PD-L1结合。
本发明抗PD-L1抗体与PD-L1的结合亲和力9.40E-10M，优选不低于5E-09M。
在另一优选例中，本发明抗PD-L1抗体为人源化抗体。
抗VEGF抗体
血管内皮生长因子(vascular endothelial growth factor)，又叫VEGF。VEGF蛋白是于1989年由美国的两间生物科技公司的科学家分别成功纯化与鉴定，并克隆与测定了其基因序列，证明VPF与VEGF是同一基因编码的同一蛋白。VEGF有六个等型(isoforms)：VEGF-A,-B,-C,-D,及-E；其分子量从35至44kDa不等，每个等型特异性地与三个“血管内皮生长因子受体”(VEGFR-1,-2,及-3)的特定组合相结合。但研究表明个这几种受体与VEGF家族分子的亲和力不同，其中VEGFR1与VEGF家族的亲和力相对较高。
VEGF是高度保守的同源二聚体糖蛋白。二条分子量各为24kDa的单链以二硫键组成二聚体。VEGF分解的单体无活性，去除N2糖基对生物效应无影响，但可能在细胞分泌中起作用。由于mRNA不同的剪切方式，产生出分别VEGF121、VEGF145、VEGF165、VEGF185、VEGF206等至少5种蛋白形式，其中VEGF121、VEGF145、VEGF165是分泌型可溶性蛋白，能直接作用于血管内皮细胞促进血管内皮细胞增殖，增加血管通透性。1990年，美国哈佛大学Folkman博士提出著名的Folkman理论，即肿瘤组织生长，必须依靠新生血管生成来提供足够的氧气和营养物质来维持，因此被认为是VEGF临床应用的基础。参照专利CN103319610，发明人发现在VEGFR1第二膜外区D2(Domain2)增加侧翼序列，具有很强的VEGF结合活性，由此开发出一类重组融合蛋白类药物，例如VEGFR1D2-Fc。
在本发明的一个优选例中，选择VEGFR1D2作为抗VEGF的元件。
Figure PCTCN2020082535-appb-000006
优选地，本发明VEGFR1D2无论连接于抗PD-L1抗体的哪一端，均由接头连接两个相同的VEGFR1D2从而以二聚体形式出现。
双功能抗体(双特异性抗体)
双特异性抗体(Bispecific Antibody，bsAb)是一种非天然抗体，它能同时靶向两种不同的抗原或蛋白，阻断两种不同的信号通路，激发具有特异性的免疫反应，其特异性和双功能性在肿瘤免疫治疗中的作用越来越重要，已成为当今世界抗体工程***方面的研究热点。研究表明，双特异性抗体在肿瘤免疫治疗方面主要有介导免疫细胞对肿瘤的杀伤；结合双靶点，阻断双信号通路，发挥独特的或重叠的功能，可以有效防止耐药性；具有强特异性、靶向性和降低脱靶毒性；有效降低治疗成本等优势(摘自抗体圈)，因此采用双特异性抗体药物可以降低肿瘤细胞逃逸几率，清除肿瘤细胞，提高疗效。
双特异性抗体可通过双杂交瘤细胞，化学偶联，重组基因等手段制备，其中重组基因技术在结合位点以及产量等方面灵活性强。据不完全统计，目前已有60多种双特异性抗体，根据其特点以及结构差异性，双特异性抗体结构主要有含Fc片段的双特异性抗体(IgG-like双特异性抗体，具有Fc介导的效应功能)和不含Fc片段的双特异性抗体(non-IgG-like双特异性抗体，通过抗原结合力发挥作用，具有分子量小、免疫原性低等优势)两种结构。2014年12月03日美国FDA审批安进公司研发的双特异性抗体Blincyto(Blinatumomab)上市，用于急性淋巴细胞白血病的治疗。Blinatumomab为CD19、CD3双特异性抗体，Blincyto(Blinatumomab)是美国FDA审批的第一个双特性抗体。
如本文所用，术语“双特异性抗体”、“双功能抗体”“本发明抗体”、“本发明双抗”“双抗”、“双功能融合抗体”可互换使用，是指同时结合PD-L1和VEGF的抗PD-L1/VEGF双特异性抗体。
在本发明中，所述双功能抗体包括：
(a)抗PD-L1的抗体或元件；和
(b)与所述抗PD-L1的抗体或元件相连接的抗VEGF的抗体或元件。
在一优选实施方式中，所述双功能抗体从N端到C端具有式I所示的结构：
Figure PCTCN2020082535-appb-000007
其中，
D各自独立地为无或抗VEGF的抗体或元件，且至少一个D为抗VEGF的抗体或元件；
L1、L2、L3、L4、L5、L6各自独立地为键或接头元件；
VL代表抗PD-L1抗体的轻链可变区；
CL代表抗PD-L1抗体的轻链恒定区；
VH代表抗PD-L1抗体的重链可变区；
CH代表抗PD-L1抗体的重链恒定区；
“～”代表二硫键或共价键；
“-”代表肽键；
其中，所述双功能抗体具有同时结合PD-L1以及结合VEGF的活性。
在一优选实施方式中，本发明双抗由PD-L1抗体以及VEGR1D2融合而成，且具有相互对称的两对肽链，每对肽链含有轻链L链和重链H链，所有的肽链均由二硫键相连，其 中任一一对肽链从N端到C端具有式Ia或Ib中所示的L链和H链结构：
Figure PCTCN2020082535-appb-000008
其中，
D为抗VEGF的元件(VEGR1D2)；
L1为无或接头元件；
VL代表抗PD-L1抗体的轻链可变区；
CL代表抗PD-L1抗体的轻链恒定区；
VH代表抗PD-L1抗体的重链可变区；
CH代表抗PD-L1抗体的重链恒定区；
“～”代表二硫键；
“-”代表肽键；
其中，所述双功能抗体具有同时结合PD-L1以及结合VEGF的活性。
式Ia或式Ib中，一种优选的H链如SEQ ID NO.:11或SEQ ID NO.:12所示，一种优选地L链如SEQ ID NO.:13所示。
H链：(VEGR1D2在重链可变区的氨基酸序列)
Figure PCTCN2020082535-appb-000009
H链：(VEGR1D2在重链恒定区的氨基酸序列)
Figure PCTCN2020082535-appb-000010
L链：
Figure PCTCN2020082535-appb-000011
而两条如式Ia或式Ib结构式所示的序列通过H链的二硫键相连，从而形成对称的双功能抗体结构。
本发明双抗不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明双抗指具有抗PD-L1以及抗VEGF活性的、包括两条上述式I结构的抗体。该术语还包括具有与本发明双抗相同功能的、包括两条上述式I结构的抗体的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明双抗的活性片段和活性衍生物。
该双抗的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
在本发明中，“本发明双抗的保守性变异体”指与本发明双抗的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
编码核酸和表达载体
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明的核酸(以及核酸组合)可用于在合适的表达***产生本发明的重组抗体。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在早期培养条件中，双特异性抗体的表达量可达3.9g/L，纯度均在97％以上，且在培养过程中可以很好地代谢乳酸。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的双抗可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.肿瘤治疗剂(例如，顺铂)或任何形式的抗肿瘤药物等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有本发明上述的双特异性抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。
本发明的药物组合物可直接用于结合PD-L1蛋白分子或PD-L1，因而可用于***。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
在本发明中，可单独使用双特异性抗体，通过调整给药方案以获得最佳目的反应。例如，单次给药，或在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
1.本发明双功能抗体可以同时结合PD-L1以及VEGF，且保持双功能抗体与结合靶标的结合构型不变，分子稳定。
2.本发明的双特异性抗体HB0025能够很好地特异性与重组人PD-L1、重组人VEGF165结合(KD小于10 -5M)，并且与非靶分子无非特异性的静电和疏水结合作用；在初步的培养条件中，双特异性抗体表达量达到3.9g/L，纯度均在97％以上。
3.本发明双功能抗体与结合靶标的结合亲和力高，阻断活性好，并表现一定的双靶点协同效应，能够有效地杀伤肿瘤细胞(尤其是高表达PD-L1和VEGF的肿瘤)，从而显著减小肿瘤体积和肿瘤，治疗癌症，特别是实体瘤。
4.相比单独的抗PD-L1抗体(如HB0023)和抗VEGF融合蛋白(如HB002.1T)，本发明双特异性抗体与PD-L1和/或VEGF的结合活性更高。具体地，本发明双特异性抗体与PD-L1的亲和力约为1E-10mol；与VEGF165的亲和力约为1E-11mol。
5.本发明制备方法简便可行。本发明申请的抗VEGF/PD-L1双特异性抗体将具有良好的应用前景。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，实施例中所用的材料或试剂均为市售产品。
实施例1 双功能抗体900387和900388表达载体构建及蛋白的表达纯化
本发明的抗PD-L1抗体是通过用人PD-L1-His蛋白免疫小鼠后筛选得到小鼠单克隆抗 体(重链可变区和轻链可变区序列分别如SEQ ID NO.:1和SEQ ID NO.:2所示)，再将其人源化得到的人源化单克隆抗体(900339)。其中重链序列如SEQ ID NO.:8所示，轻链序列如SEQ ID NO.:9所示。人工合成的VEGFR1D2(序列如SEQ ID NO.:10所示)通过连接Linker(GGSGGSGGSGGSGGS,SEQ ID NO.:16)连接在重链表达载体的5’末端或3’末端，与轻链载体(1:1)共转染到CHO-S细胞中，37度，5％CO 2，130rpm/min培养7天后，离心收集上清。将上清4000rpm离心10min，并用0.45μm滤膜过滤，收集滤液。滤液经Protein A亲和柱纯化后，得到抗体900387和900388，其结构图谱见图1。纯化后蛋白经SEC_UPLC检测，纯度均大于98％，并进行SDS-PAGE检测，SDS-PAGE还原或非还原电泳检测结果见图2。
900339-VH SEQ ID NO：8(下划线为标注的依次为重链可变区CDR1，CDR2，CDR3的氨基酸序列)
Figure PCTCN2020082535-appb-000012
900339-VL SEQ ID NO：9(下划线为标注的依次为轻链链可变区CDR1，CDR2，CDR3的氨基酸序列)
Figure PCTCN2020082535-appb-000013
制备了双特异性抗体900387和900388。其中900387的蛋白号为HB0025，后面实验相关检测结果均以HB0025表述，其重链氨基酸序列和轻链氨基酸序列分别如SEQ ID NO.:11和SEQ ID NO.:13所示。
实施例2 双特异性抗体的亲和力检测
在本实施例中，使用SPR方法测定抗体-抗原结合动力学及亲和力。
材料与仪器：
重组人PD-L1，Sino Biological，10084-H08H
重组人VEGF165,Sino Biological，11066-HNAH
氨基偶联试剂盒，GE，BR-1000-50
HBS-EP(10X)，GE，BR-1006-69
人抗体捕获试剂盒(Human Antibody Captrue Kit)，GE，BR-1008-39
S系列传感器芯片(Series S Sensor Chip CM5)，GE，BR-1005-30
BIACORE，GE，Biacore 8K
方法：按照人抗体捕获试剂盒氨基偶联法准备Anti-Human Capture-CM5芯片。将芯片置室温平衡20-30min，装入Biacore 8K仪器；用平衡缓冲液将瞬转蛋白稀释至实验工作浓度；抗原用平衡缓冲液稀释至50nM，再3倍稀释度7个浓度梯度，并设置2个零浓度(即平衡缓冲液)和一个重复浓度(一般为最低浓度重复)；按照抗体，抗原，再生的次序，循环往复对10个抗原浓度(2个零浓度，7个梯度浓度及1个重复浓度)进行实验分析，抗 原进样流速30μL/分钟，结合时间120秒，解离时间600秒；分析完成后，选用对应的分析程序分析数据，确认无明显reference binding，选用Kinetics，1:1binding modle，拟合数据，获得人鼠嵌合抗体和人源化抗体的动力学相关参数Ka，Kd和KD值。双特异性抗体HB0025与重组人PD-L1、重组人VEGF165的亲和力如表1与表2所示。
表1：双特异性抗体HB0025与重组人PD-L1亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(M)
HB0025 4.762E+05 4.018E-04 8.438E-10
表2：双特异性抗体HB0025与重组人VEGF165亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(M)
HB0025 9.496E+06 2.799E-04 2.947E-11
结果表明，双特异性抗体HB0025与重组人PD-L1有较好的亲和力；双特异性抗体HB0025与重组人VEGF165有较好的亲和力。
实施例3 双特异性抗体与非靶标分子的非特异性吸附效应
在本实施例中，使用SPR方法测定抗体与非靶标分子的非特异吸附效应。
材料与仪器：
蛋清溶菌酶，Sigma，L3790
大豆胰抑制剂1-S型，Sigma，T-2327
氨基偶联试剂盒，GE，BR-1000-50
HBS-EP(10X)，GE，BR-1006-69
抗溶菌酶的兔多克隆抗体，ABcam，Ab391
抗胰蛋白酶抑制剂抗体，LifeSpan Biosciences，LS-C76609
0.85％磷酸溶液，ProteOn，176-2260
50mM氢氧化物，ProteOn，176-2230
S系列传感器芯片CM5，GE，BR-1005-30
BIACORE，GE，Biacore 8K
方法：将Series S Sensor Chip CM5芯片置室温平衡20-30min，装入Biacore 8K仪器。采用氨基偶联试剂盒将蛋清溶菌酶和大豆胰抑制剂1-S型分别固定到CM5芯片。进样缓冲液为HBS-EP+1X，设置4个平衡循环。用平衡缓冲液将抗溶菌酶的兔多克隆抗体，抗胰蛋白酶抑制剂抗体，人源化单克隆抗体稀释至1000nM，设置流速5μL/min，进样通道1，2和3，Flow Cell 1和2。结合时间10min，解离时间15min。再生流速50μL/min，先0.85％磷酸再生60s，再用50mM氢氧化钠再生30s。双特异性抗体HB0025与非靶分子非特异吸附结果如表3所示，双特异性抗体与非靶分子非特异吸附检测传感图，如图3所示。
表3：双特异性抗体HB0025与非靶分子非特异吸附检测结果
Figure PCTCN2020082535-appb-000014
Figure PCTCN2020082535-appb-000015
注：一般认为扣除Buffer的影响后，响应值在20RU以下相互作用较弱，可以忽略；超过20RU即认为有明显的相互作用；100RU以上则是很强的相互作用。
从结果来看，扣除缓冲液的本底，HB0025与大豆胰抑制剂1-S型和溶菌酶结合的信号均小于20，因此可以认为四个样品均无非特异性的静电和疏水结合作用。进而可降低抗体注入人体后，对人体产生免疫，造成血管瘤等一系列免疫病。
采用SPR技术，通过抗溶菌酶的兔多克隆抗体和抗胰蛋白酶抑制剂抗体分别对蛋清溶菌酶和大豆胰抑制剂1-S型的质控结果，可以确定蛋清溶菌酶和大豆胰抑制剂氨基偶联固到CM5芯片后活性正常，活性也保持良好(如结合的信号均小于20，可以认为样品无非特异性的静电和疏水结合作用)。
实施例4 双特异性抗体的体内药效检测
4.1双特异性抗体制备
采用BalanA培养基，添加一定的补料，进行培养，如图4所示，此CHO-K1细胞株可以很好地代谢乳酸；细胞生长状态好，16天收获时活率高于90％；降温至33℃和31℃，表达无明显差异，约3g/L；每天补料可以明显提高细胞密度和表达量，最终表达3.9g/L；整个培养过程纯度无下降，维持在97％左右。
4.2体内药效实验设计以及结果
HB0025的体内药效。hCD34+人源化小鼠右侧腋下接种人肺腺癌HCC-827细胞，当肿瘤生长到平均约100-150mm 3左右时，48只荷瘤小鼠根据外周血中hCD45+的比例和肿瘤体积被随机分成6组，每组8只动物，分别经尾静脉给予G1组PBS、G2组HB0023(抗PD-L1单克隆抗体，实施例1中的人源化单克隆抗体900339)1mg/kg、G3组HB0025 1mg/kg、G4组HB0025 3mg/kg、G5组HB0025 10mg/kg、G6组HB002.1T(抗VEGF融合蛋白，专利号CN103319610B)1mg/kg，2次/周，给药9次。2次/周测量肿瘤体积和小鼠体重。实验过程中当某组中动物的平均肿瘤体积超过2000mm 3或实验结束时，对动物进行安乐死处理，称瘤重。
实验期间，PBS溶媒对照组(G1)小鼠的肿瘤体积持续增长(见图5)，表明成功建立了HCC827肺癌细胞hCD34+人源化小鼠皮下移植瘤模型。G2动物给予HB0023 1mg/kg，给药后瘤体积增长相对缓慢，与同期溶媒对照组相比，瘤体积和相对瘤体积相对减小，但未见统计学差异；D32试验结束时，与溶媒对照组相比，肿瘤重量相对降低(P>0.05，见图5，图6)，肿瘤抑制率为17.48％。G6动物给予HB002.1T 1mg/kg，给药后瘤体积增长缓慢，与同期溶媒对照组相比，瘤体积和相对瘤体积明显减小(P<0.05，见图5)；D32试验结束时，与溶媒对照组相比，肿瘤重量明显降低(P<0.05，见图6)，肿瘤抑制率为64.08％。
G3、G4、G5组动物分别给予HB0025 1、3、10mg/kg，给药后瘤体积增长缓慢，肿瘤 体积和相对肿瘤体积显著低于同期溶媒对照组(p<0.05)；D32试验结束时，与溶媒对照组相比，肿瘤重量明显减小(P<0.05，见图6)，肿瘤抑制率分别为70.87％、84.47％、85.44％，表明HB0025能有效抑制hCD34+人源化小鼠HCC827肺癌皮下移植瘤的生长，有效抑瘤剂量为1mg/kg。
与给予1mg/kg HB0023、HB002.1T单靶点抗体/融合蛋白相比，给予等剂量双特异性抗体HB0025的动物肿瘤体积、相对肿瘤体积、瘤重均相应减小(见图5、图6)，表现一定的双靶点协同效应。
实施例5
本实施例对实施例1中制备的双特异性抗体900388(重链氨基酸序列和轻链氨基酸序列分别如SEQ ID NO.:12和SEQ ID NO.:13所示)的亲和力、与非靶标分子的非特异性吸附效应和体内药效进行了检测，实验方法同实施例2-4，将其中的HB0025替换为双特异性抗体900388。
表4 双特异性抗体900388与PD-L1和VEGF165亲和力
抗原 抗体 ka(1/Ms) kd(1/s) KD(M) 动力学Chi 2(RU 2) tc
PD-L1 900388 6.332E+5 5.217E-4 8.240E-10 0.0876 4.464E+7
VEGF165 900388 1.030E+7 4.104E-4 3.984E-11 1.31 1.105E+8
结果发现，双特异性抗体900388与重组人PD-L1和重组人VEGF165均有较好的亲和力，并且无非特异性的静电和疏水结合作用，也能够很好地***，显著减小动物肿瘤体积和瘤重。
以上实施例表明，本发明的抗VEGF/PD-L1双特异性抗体可以在CHO-K1细胞中表达，能够进一步通过亲和层析纯化。所得到的双特异性抗体可以结合PD-L1阳性的细胞和VEGF阳性的细胞。此外，该抗体有较好的亲和力，既有良好的抗VEGF的生物学活性，又完美保留了抗PD-L1抗体的生物学活性。由此，本发明申请的抗VEGF/PD-L1双特异性抗体将具有良好的应用前景。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种双功能抗体，其特征在于，所述双功能抗体包括：
    (a)抗PD-L1的抗体或元件；和
    (b)与所述抗PD-L1的抗体或元件相连接的抗VEGF的抗体或元件。
  2. 如权利要求1所述的双功能抗体，其特征在于，所述双功能抗体从N端到C端具有式I所示的结构：
    Figure PCTCN2020082535-appb-100001
    其中，
    D各自独立地为无或抗VEGF的抗体或元件，且至少一个D为抗VEGF的抗体或元件；
    L1、L2、L3、L4、L5、L6各自独立地为键或接头元件；
    VL代表抗PD-L1抗体的轻链可变区；
    CL代表抗PD-L1抗体的轻链恒定区；
    VH代表抗PD-L1抗体的重链可变区；
    CH代表抗PD-L1抗体的重链恒定区；
    “～”代表二硫键或共价键；
    “-”代表肽键；
    其中，所述双功能抗体具有同时结合PD-L1以及结合VEGF的活性。
  3. 如权利要求1所述的双功能抗体，其特征在于，所述的抗PD-L1抗体的重链可变区(VH)包括以下三个互补决定区CDR：
    SEQ ID NO.:3所示的CDR1，
    SEQ ID NO.:4所示的CDR2，和
    SEQ ID NO.:5所示的CDR3；和/或
    所述的抗PD-L1抗体的轻链可变区(VL)包括以下三个互补决定区CDR：
    SEQ ID NO.:6所示的CDR1’，
    氨基酸序列为GIS的CDR2’，和
    SEQ ID NO.:7所示的CDR3’。
  4. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1所述的双功能抗体。
  5. 一种载体，其特征在于，所述载体含有权利要求4所述的多核苷酸。
  6. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有权利要求5所述的载体或基因组中整合有权利要求4所述的多核苷酸。
  7. 一种制备权利要求1所述抗体的方法，其特征在于，包括步骤：
    (i)在合适的条件下，培养权利要求6所述的宿主细胞，获得含有权利要求1所述的双功能抗体的混合物；和
    (ii)对步骤(i)中得到的混合物进行纯化和/或分离，从而获得权利要求1所述的双功能抗体。
  8. 一种药物组合物，其特征在于，所述药物组合物含有：
    (I)如权利要求1所述的双功能抗体；和
    (II)药学上可接受的载体。
  9. 一种免疫偶联物，其特征在于，所述免疫偶联物包括：
    (a)如权利要求1所述的双功能抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  10. 如权利要求1所述双功能抗体或如权利要求9所述的免疫偶联物的用途，其特征在于，用于制备(a)检测试剂或试剂盒；和/或(b)制备预防和/或治疗癌症或肿瘤的药物组合物。
PCT/CN2020/082535 2019-04-01 2020-03-31 抗pd-l1/vegf双功能抗体及其用途 WO2020200210A1 (zh)

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AU2020255712A AU2020255712B2 (en) 2019-04-01 2020-03-31 Anti-PD-L1/VEGF bifunctional antibody and use thereof
JP2021525632A JP7283806B2 (ja) 2019-04-01 2020-03-31 抗ｐｄ－ｌ１／ｖｅｇｆ二機能性抗体およびその用途
US17/295,394 US20220002418A1 (en) 2019-04-01 2020-03-31 Anti-pd-l1/vegf bifunctional antibody and use thereof
EP20783433.4A EP3954712A4 (en) 2019-04-01 2020-03-31 BIFUNCTIONAL ANTI-PD-L1/VEGF ANTIBODIES AND THEIR USE

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206843A1 (en) * 2021-03-31 2022-10-06 Wuxi Biologics (Shanghai) Co., Ltd. A bispecific anti-pd-l1/vegf antibody and uses thereof
WO2023087344A1 (zh) * 2021-11-22 2023-05-25 杭州翰思生物医药有限公司 重组抗体及其应用
CN116731188A (zh) * 2022-03-02 2023-09-12 三优生物医药(上海)有限公司 抗pd-l1和vegf双特异性抗体及其应用

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109929037B (zh) * 2019-04-01 2023-03-17 华博生物医药技术（上海）有限公司 针对程序性死亡配体的结合物及其应用
CN109942712B (zh) * 2019-04-01 2022-12-20 华博生物医药技术（上海）有限公司 抗pd-l1/vegf双功能抗体及其用途
JP2023511898A (ja) * 2020-01-21 2023-03-23 ウーシー・バイオロジクス・（シャンハイ）・カンパニー・リミテッド 二重特異性抗ｐｄ－ｌ１／ｖｅｇｆ抗体及びその使用
TWI813934B (zh) * 2020-01-21 2023-09-01 大陸商甫康（上海）健康科技有限責任公司 一種雙特異性抗pd-l1/vegf抗體及其用途
CN114106190A (zh) * 2020-08-31 2022-03-01 普米斯生物技术(珠海)有限公司 一种抗vegf/pd-l1双特异性抗体及其用途
CN114573701A (zh) * 2020-12-02 2022-06-03 上海华奥泰生物药业股份有限公司 抗PD-L1/TGF-β双功能抗体及其用途
CN114685674B (zh) * 2020-12-29 2023-09-29 瑞阳(苏州)生物科技有限公司 一种抗体融合蛋白及其应用
CN116917334A (zh) * 2021-02-22 2023-10-20 浙江道尔生物科技有限公司 一种具有抗癌活性的多结构域融合蛋白
CN117295769A (zh) * 2021-02-22 2023-12-26 浙江道尔生物科技有限公司 一种具有抗癌活性的双功能融合蛋白
WO2024032664A1 (zh) * 2022-08-09 2024-02-15 上海济煜医药科技有限公司 一种靶向pd-l1和vegf的抗体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103319610A (zh) 2013-07-05 2013-09-25 华博生物医药技术（上海）有限公司 新型重组融合蛋白及其制法和用途
WO2018035084A1 (en) * 2016-08-16 2018-02-22 Epimab Biotherapeutics, Inc. Monovalent asymmetric tandem fab bispecific antibodies
CN109942712A (zh) * 2019-04-01 2019-06-28 华博生物医药技术（上海）有限公司 抗pd-l1/vegf双功能抗体及其用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015000181A1 (zh) * 2013-07-05 2015-01-08 华博生物医药技术（上海）有限公司 新型重组融合蛋白及其制法和用途
CN105175545B (zh) * 2015-10-20 2019-01-25 安徽瀚海博兴生物技术有限公司 一种抗vegf-抗pd-1双功能抗体及其应用
CN106986939B (zh) * 2017-03-27 2019-06-07 顺昊细胞生物技术（天津）股份有限公司 抗pd-1和tem-8双特异性抗体及其应用
CN109096396B (zh) * 2017-06-20 2022-01-04 华兰生物工程股份有限公司 一种抗pd-l1人源化纳米抗体及其应用
CN109053895B (zh) * 2018-08-30 2020-06-09 中山康方生物医药有限公司 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途
US11407832B2 (en) * 2018-12-03 2022-08-09 Immuneonco Biopharmaceuticals (Shanghai) Inc. Recombinant protein targeting PD-L1 and VEGF

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103319610A (zh) 2013-07-05 2013-09-25 华博生物医药技术（上海）有限公司 新型重组融合蛋白及其制法和用途
WO2018035084A1 (en) * 2016-08-16 2018-02-22 Epimab Biotherapeutics, Inc. Monovalent asymmetric tandem fab bispecific antibodies
CN109942712A (zh) * 2019-04-01 2019-06-28 华博生物医药技术（上海）有限公司 抗pd-l1/vegf双功能抗体及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM, vol. 243, 1968, pages 3558
KABAT ET AL., NIH PUBL. NO. 91-3242, vol. I, 1991, pages 647 - 669
SAMBROOK. ET AL.: "in Molecule Clone: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3954712A4

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206843A1 (en) * 2021-03-31 2022-10-06 Wuxi Biologics (Shanghai) Co., Ltd. A bispecific anti-pd-l1/vegf antibody and uses thereof
WO2023087344A1 (zh) * 2021-11-22 2023-05-25 杭州翰思生物医药有限公司 重组抗体及其应用
CN116731188A (zh) * 2022-03-02 2023-09-12 三优生物医药(上海)有限公司 抗pd-l1和vegf双特异性抗体及其应用

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