WO2020007368A1 - 低adcc/cdc功能性单抗及其制备方法与应用 - Google Patents

低adcc/cdc功能性单抗及其制备方法与应用 Download PDF

Info

Publication number
WO2020007368A1
WO2020007368A1 PCT/CN2019/094928 CN2019094928W WO2020007368A1 WO 2020007368 A1 WO2020007368 A1 WO 2020007368A1 CN 2019094928 W CN2019094928 W CN 2019094928W WO 2020007368 A1 WO2020007368 A1 WO 2020007368A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
monoclonal antibody
amino acid
acid sequence
adcc
Prior art date
Application number
PCT/CN2019/094928
Other languages
English (en)
French (fr)
Inventor
孙乐
李茂华
任文林
Original Assignee
北京天成新脉生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京天成新脉生物技术有限公司 filed Critical 北京天成新脉生物技术有限公司
Priority to JP2021521872A priority Critical patent/JP7193628B2/ja
Priority to US17/258,097 priority patent/US20210380700A1/en
Priority to EP19831301.7A priority patent/EP3825333A4/en
Publication of WO2020007368A1 publication Critical patent/WO2020007368A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Definitions

  • the invention relates to the preparation and application of therapeutic antibodies, mainly to the application of a low ADCC / CDC functional monoclonal antibody, and a method for reducing the ADCC / CDC and other functions of the antibody.
  • Antibody-dependent cell-mediated cytotoxicity refers to NK cells, macrophages, and neutrophils expressing IgG and Fc receptors.
  • the Fc segment of the IgG antibody binds while killing these target cells.
  • IgG antibodies can mediate the ADCC effect of these cells, and NK cells are the main cells that can exert ADCC effect.
  • NK cells are the main cells that can exert ADCC effect.
  • antibodies can only specifically bind to corresponding epitopes on target cells, and effector cells such as NK cells can kill any target cells that have been bound to the antibodies, so the antibodies and target cells The antigen binding is specific, and the killing effect of NK cells on target cells is non-specific.
  • CDC refers to the formation of MAC on the surface of target cells after the complement system is activated, causing target cells to lyse. This effect is called complement-dependent cytotoxicity. Complement can cause the lysis of a variety of bacteria and other pathogenic cells, and is an important defense mechanism for the body to resist infection by pathogens. Especially important for preventing the infection of Gram-negative bacteria. In some cases, the complement system can cause body tissue or cell damage and participate in the pathogenesis of hypersensitivity and autoimmune diseases.
  • the application of antibody drugs is not limited to infectious diseases and tumors.
  • the main method currently adopted is the amino acid N (297) at the glycosylation site of the weight chain.
  • Deglycosylation mutation that changes to A or changes antibody heavy chain to IgG2 / 4 subtype.
  • the deglycosylation of the heavy chain of the antibody resulted in too high a polymer content in the product, and the immunogenicity of the drug was significantly enhanced.
  • the incidence of anti-drug antibodies (ADA) was high after the patient was given multiple doses.
  • Antibodies of the IgG2 subtype are still capable of triggering monocyte-mediated ADCC and macrophage-mediated ADCP through binding to Fc ⁇ RIIa.
  • the antibody heavy chain is changed to IgG4 subtype, the antibody drug heavy chain of IgG4 subtype will automatically exchange with other human IgG4 heavy chains in the blood, forming half-molecules and bispecific functional monovalent antibodies.
  • the half-life of IgG2 / 4 antibody was significantly shorter than that of IgG1.
  • PD-1 is mainly expressed in activated T cells and B cells. Its function is to suppress the activation of immune cells. This is a normal homeostatic mechanism of the immune system because excessive T / B cell activation can cause autoimmune diseases. So PD-1 is a talisman for our body. However, the tumor microenvironment will induce infiltrating T cells to highly express PD-1 molecules, and tumor cells will highly express PD-1 ligands PD-L1 and PD-L2, resulting in the continued activation of the PD-1 pathway in the tumor microenvironment. Cell function is inhibited and tumor cells cannot be killed.
  • Antibodies to PD-1 and PD-L1 can block this pathway and partially restore the function of T cells, enabling these cells to continue to kill tumor cells.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the antibody drug has undergone deglycosylation mutations in the weight chain, resulting in excessive levels of multimers in the product.
  • the drug immunogenicity was significantly enhanced, and the incidence of anti-drug antibody (ADA) was as high as 41.5% after multiple administrations.
  • the U.S. Food and Drug Administration authorized the accelerated approval of Merck's Pembrolizumab (trade name: Keytruda) for the market for the treatment of advanced or unresectable melanoma that no longer responds.
  • the United States Food and Drug Administration approved Keytruda for the treatment of solid tumors with microsatellite instability, becoming the first antibody drug to treat a variety of cancers.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the antibody drug uses an IgG4 subtype that has a weak affinity for the Fc ⁇ R receptor and lacks the ability to activate complement.
  • IgG4 subtype antibodies have Fab arm replacement, there is a chance of forming half-antibodies or bispecific antibodies, increasing the potential risk of drug use, and the production process of IgG4 subtype antibodies is not as mature as IgG1 subtype antibodies (mainly manifested in structural stability) Properties, physical and chemical properties, anti-aggregation ability, expression, etc.).
  • the constant region of the antibody can be modified without affecting the affinity and specificity of the antibody, the ADCC / CDC function of the antibody can be reduced, and the antibody drug can be prevented from forming a large number of multimers, reducing the immunity of the antibody drug.
  • Primitiveness on the one hand, can improve the safety of monoclonal antibodies; on the other hand, it can increase the half-life of the drug, which can also improve the efficacy while reducing the dosage.
  • CTLA-4 also known as CD152, is a transmembrane protein encoded by the CTLA-4 gene. It is expressed on activated CD4 + and CD8 + T cells and has a high degree of homology with the co-stimulatory molecule receptor (CD28) on the surface of T cells. Sex. CTLA-4 and CD28 are members of the immunoglobulin superfamily, and both bind to the same ligands CD86 (B7-2) and CD80 (B7-1). The key to CTLA-4's immune regulation is to control CD4 + FoxP3-, CD8 + T cells and regulatory T cells (Treg). CTLA-4 can stop the response of activated T cells and mediate the suppressive function of Treg.
  • CTLA-4 inhibits T cell responses mainly through two ways: one is to compete with CD28 to bind B7 or recruit phosphatase to the intracellular domain portion of CTLA-4 to reduce TCR (T cell cell receptor). ) And CD28 signals. The other is to reduce the expression level of CD80 and CD86 in antigen presenting cells (APC) or remove them from APC through transocytosis, which reduces CD28's participation in T cell activation.
  • APC antigen presenting cells
  • CTLA-4 also mediates dendritic cells binding to CD80 / CD86 and induces the expression of tryptophan degrading enzyme IDO, which leads to the inhibition of TCR.
  • CTLA-4 antibodies bind to CTLA-4 to reduce Treg and activate TCR.
  • indications for the approval of PD-1 and PD-L1 antibodies include advanced melanoma, head and neck squamous cell carcinoma, non-small cell lung cancer (NSCLC), urothelial carcinoma, Hodgkin lymphoma, Merkel cell carcinoma ( Merkel's cell carcinoma) and microsatellite unstable (MSI-H) solid tumors, while the indications for CTLA-4 antibody ipilimumab are limited to advanced melanoma and high-risk relapsed melanoma.
  • ipilimumab is less effective in treating advanced melanoma and has a higher incidence of immune-related side effects. This brings risks and inconveniences to the use of clinical patients, which greatly limits the market share and sales share of the antibody drug.
  • the main advancement direction of antibodies against CTLA4 is more effective combination therapy against multiple targets.
  • the combination of Ipilimumab and Nivolumab anti-PD-1 antibody
  • the combined treatment of CTLA4 antibody with many star antibody drugs (PD-1 antibody Nivolumab, PD-L1 antibody Atezolizumab, etc.) has effectively increased its market share.
  • the CTLA4 antibody Ipilimumab uses the IgG1 subtype, which has a strong ADCC / CDC function. To a certain extent, this strong ADCC effect will cause side effects induced by immunotherapy. This is mainly through the FcR-mediated specific elimination of tumors. Produced by Treg cells in the microenvironment. Therefore, low ADCC / CDC effect antibodies will effectively reduce the chance of ADCC causing corresponding side effects.
  • Ipilimumab for BMS is anti-CTLA-4 human IgG1 at a dose of 3 mg / kg. It is administered intravenously for a total of four doses every 3 weeks. Yipi antibody has very strong side effects. When taken alone, 10-20% of patients have severe adverse reactions or even die because of excessive activation of T cells and reproduction. The antitumor effect of combination with O drug increased greatly, however, more than half of the patients had severe side effects of grade 3-4. Permanently discontinue medication for serious adverse reactions.
  • Ipilimumab not only temporarily blocks the suppression of the immune system by the CTLA-4 signaling pathway, but also kills T-regulatory cells because of its own ADCC function, which relieves the patient's immune suppression regulation once and for all, leading to the immune system. Over activated. If ADCC / CDC can be modified for ipilimumab to obtain the neutralizing effect of the original drug and remove the ADCC toxic and side effects, it will greatly reduce the immunotoxicity related to immunotherapy and significantly reduce the serious side effects of grade 3-4. Incidence, more secure.
  • a first object of the present invention is to provide a method for reducing ADCC / CDC function of an antibody and its application.
  • the second object of the present invention is to provide a series of low ADCC / CDC, low multimer, and low immunogenic therapeutic antibodies including multiple monoclonal antibodies such as anti-PD-L1, anti-PD-1, and anti-CTLA-4. And its applications.
  • the present invention adopts the following technical solutions.
  • the invention provides a method for reducing or removing ADCC / CDC function of an IgG1 subtype antibody.
  • the method inserts a flexible amino acid sequence into the constant region of the heavy chain of the original IgG1 antibody.
  • the method inserts a flexible amino acid sequence between the CDR3 and CH2 regions of the heavy chain of the original IgG1 subtype antibody.
  • the insertion of a flexible amino acid sequence blocks the mechanical stress transmission generated after the antibody variable region binds the antigen, so that the constant region of the antibody heavy chain and the Fc receptor and / or complement binding site cannot be fully exposed and weakened.
  • the antibody binds to NK cells, macrophages, and neutrophils to killer cells expressing the IgG Fc receptor or to complement, and cannot or reduce the signal that induces ADCC and CDC.
  • the IgG1 antibody monoclonal antibody is PD-L1, PD-1, CTLA4, TNF- ⁇ , PCSK9, NGF, C5, A ⁇ , IL-6, IL-17A, IL23A, HGF , CMET, Notch1, CCL11, IL6R, IL-31R, IL-1B, IL-20, CD40, CD47, DKK1 / 2, TIGIT, 4-1BB, IGF-1R, LL4, PDGFR2, HER3, IGF1 / 2, RANKL , Sclerostin, GCGR, CGRP, ANGPTL3, IL-13, IL-4R, CSF-1R, TFPI, FCGRT, CD47 antibodies, such as Atezolizumab mAb, Pembrolizumab mAb, Ipilimumab mAb, Eculizumab mAb, evolocumab mAb, erenumab MAb, ful
  • the flexible amino acid sequence includes, but is not limited to, GGSGGS, GSGGSGG, GSGGSGGG, GGGGSGGG, GSGSG, GGSGG, GGS, GGSGS, and GGGS.
  • the flexible amino acid sequence insertion site includes, but is not limited to, GG (138/139), SS (177/178), SG (178/179), SS (184/185), SSS (191/192/193), LL (235/236), GG (237/238).
  • the invention also provides an application of a flexible amino acid sequence in improving antibody stability / reducing antibody immunogenicity / extending antibody half-life.
  • the flexible amino acid sequence is inserted into the constant region of the heavy chain of the original IgG1 antibody.
  • the flexible amino acid sequence is inserted between CDR3 and CH2 of the heavy chain of the original IgG1 antibody.
  • the invention also provides a class of low ADCC / CDC, low multimer, and low immunogenic therapeutic antibodies, including monoclonal antibodies against PD-L1.
  • the invention also provides a method for preparing low ADCC / CDC, low multimer, low immunogenic therapeutic antibody, including monoclonal antibody against PD-L1.
  • the invention is based on the antibody-induced ADCC / CDC action mechanism.
  • a flexible amino acid sequence into the constant region of the antibody heavy chain and cutting off the mechanical stress transmission generated after the antibody variable region binds to the antigen, the antibody drug heavy chain constant region and Fc are affected.
  • the body and / or complement complement site cannot be fully exposed, weakening the antibody drug binding to killer cells expressing IgG Fc receptors such as NK cells, macrophages and neutrophils, or to complement, which cannot or reduce the induction of ADCC and CDC and other signals.
  • a flexible amino acid sequence is inserted between the CDR1 and the CH2 region of the heavy chain of the IgG1 antibody based on mutation of amino acid A at position 298 of the Atezolizumab antibody drug of Genentech company to return to N to restore glycosylation.
  • the stress transmission in the variable region and the constant region of the antibody is passed, so as to reduce the ADCC / CDC function without significantly increasing the formation of multimers.
  • the low-immunogenic anti-PD-L1 monoclonal antibody of the present invention is modified by amino acids in the non-antigen binding region in the original Atezolizumab sequence to reduce its immunogenicity.
  • the original Atezolizumab sequence has variable heavy chain length and light chain.
  • the amino acid sequences of the regions are shown in SEQ ID Nos. 1, 2 respectively.
  • the low immunogenic anti-PD-L1 monoclonal antibody provided by the present invention has the amino acid sequence of its heavy chain as shown in SEQ ID NO. 3 or 4, and the amino acid sequence of its light chain as SEQ ID NO. 2.
  • the light chain of the anti-human PD-L1 monoclonal antibody provided by the present invention is shown as L0 (SEQ ID No. 2), the full length of the heavy chain has H-1 (SEQ ID No. 3), and H-2 (SEQ ID No. 4) Any one of these two base sequences.
  • the present invention screens out two monoclonal antibody sequences that both retain the original affinity for binding to human PD-L1 and can specifically block PD-
  • the combination of 1 and PD-L1 reduces the content of multimers, which can effectively reduce their immunogenicity.
  • the verification results of ADCC activity during the transformation process show that the two monoclonal antibody sequences obtained by the screening of the present invention The ADCC activity was consistent with the original Atezolizumab.
  • the two low-immunogenic anti-PD-L1 monoclonal antibodies obtained by the present invention are H-1L0 and H-2L0, respectively.
  • the biological material is an expression cassette, an expression vector, an engineered bacterium, or a cell.
  • the invention provides the application of the above two low-immunogenic anti-PD-L1 monoclonal antibodies in the preparation of a medicine for treating diseases.
  • the diseases are tumors, immune decline and so on.
  • the invention provides the application of the above-mentioned anti-PD-L1 monoclonal antibody in preparing a medicine for treating diseases with PD-L1 as a target.
  • the medicament is a medicament for anti-tumor and immune decline diseases.
  • the invention provides a medicine or a detection reagent containing the above-mentioned anti-PD-L1 monoclonal antibody.
  • the invention provides the application of the above-mentioned low ADCC / CDC functional anti-PD-L1 monoclonal antibody in the treatment of diseases that target PD-L1 or PD-1.
  • the present invention provides the application of the above-mentioned low ADCC / CDC functional anti-PD-L1 monoclonal antibody in killing tumor cells or treating immune-capable degenerative diseases.
  • the anti-PD-L1 monoclonal antibody of the present invention may be combined with a second therapeutic agent or a therapeutic form.
  • Anti-PD-L1 antibodies can be combined with cancer treatments including administration of CTLA-4 antibodies or CD47 antibodies.
  • Non-limiting examples of combinations include anti-PD-L1 antibodies in combination with anti-CTLA-4 antibodies for the treatment of melanoma or non-small cell lung cancer.
  • Treatment with the anti-PD-L1 antibody of the invention can be combined with chemotherapy.
  • Chemotherapy using cytotoxic agents will cause cancer cells to die, thereby increasing the release of tumor antigens.
  • the increased effectiveness of tumor antigens can lead to synergy with anti-PD-L1 treatment.
  • Treatment with the PD-L1 mAb of the present invention can be combined with surgery to remove cancer cells from a subject.
  • the anti-PD-L1 antibody of the present invention can be combined with a therapy capable of producing a synergistic effect with PD-L1 blockade, the therapy including a targeted drug for hormone elimination or inhibition of angiogenesis, or a targeting that has activity in tumor cells Protein-targeted drugs, all of which lead to increased tumor cell death and increased effectiveness of immunostimulatory tumor antigens.
  • the anti-PD-L1 monoclonal antibody of the present invention can be combined with another therapeutic antibody for treating cancer or infectious diseases.
  • Specific examples may be: a combination of an anti-PD-L1 antibody and an antibody that targets PD-1 or CTLA-4.
  • the invention provides a method for preparing the above-mentioned anti-PD-L1 monoclonal antibody, comprising the following steps:
  • the present invention uses Pymol software to analyze the structure of Atezolizumab of Genentech, find a relatively flexible region between the variable region and the constant region of the antibody, and insert a flexible amino acid sequence in the corresponding region to the corresponding sequence Perform gene synthesis, sequencing, and select the correct sequence for sequencing.
  • the present invention performs full gene synthesis on the corresponding modified sequence, performs sequencing on the synthesized gene, and selects the correct sequence for the next operation to change the light chain.
  • the region-designed enzyme digestion site is Kpn I + BamH I
  • the heavy chain variable region-designed enzyme digestion site is KpnI + AgeI, which are respectively linked to the expression vector pJH16 vector and transformed into E. coli DH5 ⁇ to obtain the chimeric heavy and light chain.
  • Antibody expression vector Simultaneously sequence and compare the constructed antibodies;
  • the present invention selects a corresponding combination to construct a stable strain using electrotransfection, and at the same time uses MTX to screen the degree of antibody expression, and performs monoclonal screening on the stable strain after pressurization, and finally selects the antibody yield Higher stable strains for subsequent experiments;
  • the monoclonal antibody produced by the present invention has a lower immune response in mice than Atezolizumab.
  • the anti-PD-L1 antibody and Atezolizumab provided by the present invention are directed at the same site of PD-L1, but their immunogenicity and antibody conformation are different from Atezolizumab. Compared with Atezolizumab, it has significantly improved stability, greatly reduced immunogenicity, and animals. Prolonged drug half-life in vivo is expected to be a very ideal bio-targeted therapeutic antibody. By modifying the sequence of the Atezolizumab monoclonal antibody constant region, the present invention will significantly reduce the risk of the antibody drug producing immunogenicity in the patient.
  • the examples of the present invention show that the affinity of the modified Atezolizumab antibody of the present invention and PD-L1 is similar to that of the original Atezolizumab, and the ADA titer is significantly reduced, the half-life of the antibody drug is prolonged, and the therapeutic effect is improved.
  • the invention also provides an IgG1 subtype low ADCC / CDC functional monoclonal antibody against human PD-1 and application of the monoclonal antibody.
  • the invention is based on the antibody-induced ADCC / CDC action mechanism.
  • the antibody is changed from the IgG4 subtype to the IgG1 subtype, and a flexible segment is inserted between the IgG1 antibody heavy chain CDR3 and CH2 regions Amino acid sequence, the constant region of the monoclonal antibody heavy chain and the Fc receptor and / or complement binding site cannot be fully exposed, weakening the monoclonal antibody binding to NK cells, macrophages and neutrophils expressing IgG receptor killer cells Or combined with complement, can not or reduce the signal to induce ADCC and CDC, the original full-length amino acid sequence of the heavy chain of the original Pembrolizumab monoclonal antibody is shown in SEQ ID NO. 5, the full-length amino acid sequence of the light chain is shown in SEQ ID ID NO. 6 shown.
  • the full-length heavy chain of the IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody provided by the present invention contains the amino acid sequence shown in SEQ ID No. 7 or the amino acid sequence shown in SEQ ID No. 7 A variant whose full length of the light chain contains the amino acid sequence shown in SEQ ID No. 6 or a variant of the amino acid sequence described in SEQ ID No. 6.
  • the antibody heavy chain constant region or a variant thereof is IgG1.
  • the antibody of the present invention may include: inserting a flexible amino acid sequence between the CDR3 and CH2 regions of the heavy chain of the IgG1 antibody, blocking the stress transmission of the variable region and the constant region of the antibody after the antibody binds to the antigen, and reducing the ADCC / CDC function while reducing the antibody Difficulty in the development of subsequent production processes to improve the anti-polymerization, expression and stability of antibodies.
  • the IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody provided by the present invention has the light chain full-length amino acid shown as L0 (SEQ ID No. 6), and the full-length heavy chain amino acid sequence has H1 (SEQ ID No. 7).
  • the present invention screens for the H2L0 monoclonal antibody sequence. It not only maintains the original affinity of binding to human PD-1, but can specifically block the binding of PD-1 to PD-L1, and reduces the functional activity of ADCC and CDC. Because it is an IgG1 subtype monoclonal antibody, relative For the original Pembrolizumab monoclonal antibody as an IgG4 subtype, the production process of the monoclonal antibody of the present invention is mature, the difficulty is low, and the anti-polymerization, expression and stability of the antibody are improved.
  • the present invention provides an expression vector containing any of the IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibodies light and heavy chain genes.
  • a host bacteria, a host cell or an expression cassette containing the expression vector is also within the scope of the present invention.
  • the invention provides a method for increasing the activity of an immune cell, said method comprising administering a therapeutically effective amount of an antibody of the invention to a subject in need of treatment.
  • the method can be used for treating cancer, and can be used for treating immunocompromised diseases.
  • the invention also includes the further administration of a second therapeutic agent or form.
  • the invention also includes the use of the above-mentioned anti-PD-1 monoclonal antibody in the preparation of a medicament for improving immune function.
  • the anti-PD-1 mAb of the present invention can be combined with a second therapeutic agent or therapeutic form.
  • Anti-PD-1 antibodies can be combined with cancer treatments including administration of recombinant cytokines or secretion of immune factors.
  • Non-limiting examples of combinations include the use of anti-PD-1 antibodies in combination with recombinant IL-2 or recombinant IFN2 for the treatment of melanoma or renal cell carcinoma.
  • Recombinant IL-2 increases T cell production in cancer patients.
  • Recombinant IFN2 not only inhibits the growth of cancer cells in treated patients, but also increases the expression of PD-1 inhibitory ligands on cancer cells, antigen presenting cells and other somatic cells.
  • the anti-PD-1 mAb of the present invention can be combined with other cytokines that are considered to be useful for treating cancer or infectious diseases.
  • the anti-PD-1 monoclonal antibody or antibody fragment of the present invention can be combined with a vaccine to prevent or treat cancer or infectious diseases.
  • an anti-PD-1 monoclonal antibody may be combined with a protein, peptide or DNA vaccine containing one or more antigens associated with the cancer or infection to be treated or a tree stimulated with said antigen Vaccine consisting of dendritic cells.
  • the invention provides the use of a vaccine against PD-1 and (weakened) cancer cells or whole viruses.
  • the invention provides a combination of anti-PD-1 therapy and a genetically engineered whole-cell cancer vaccine that secretes GM-CSF.
  • anti-PD-1 monoclonal antibodies of the invention can be combined with treatments that are considered to be the standard of care in cancer or infectious diseases.
  • the basic principle of the combination is that the concurrently enhanced immune activation by anti-PD-1 will induce or promote an initial clinical response to the standard of care treatment, induce a long-term clinical response, and long-term immune control of the disease.
  • Treatment with the anti-PD-1 antibodies of the invention can be combined with chemotherapy.
  • Chemotherapy using cytotoxic agents will cause cancer cells to die, thereby increasing the release of tumor antigens.
  • Such increased effectiveness of tumor antigens can lead to synergy with anti-PD-1 treatment.
  • Treatment with the PD-1 mAb of the present invention can be combined with surgery to remove cancer cells from a subject.
  • the anti-PD-1 antibody of the present invention can be combined with a therapy capable of producing a synergistic effect with PD-1 blockade, the therapy including a targeted drug for hormone elimination or inhibition of angiogenesis, or a targeting that is active in tumor cells Protein-targeted drugs, all of which lead to increased tumor cell death and increased effectiveness of immunostimulatory tumor antigens.
  • a therapy capable of producing a synergistic effect with PD-1 blockade the therapy including a targeted drug for hormone elimination or inhibition of angiogenesis, or a targeting that is active in tumor cells Protein-targeted drugs, all of which lead to increased tumor cell death and increased effectiveness of immunostimulatory tumor antigens.
  • an increase in T cell activation can lead to durable immune control of cancer.
  • the anti-PD-1 mAb of the present invention can be combined with another therapeutic antibody for treating cancer or infectious diseases.
  • Specific examples may include: a combination of an anti-PD-1 antibody and an antibody targeting Her2 / neu or an EGF receptor; a combination of an anti-PD-1 antibody and an anti-CTLA-4; an anti-PD-1 antibody or an antibody fragment and a targeting OX40 antibody combination.
  • the biological material is an expression cassette, an expression vector, an engineered bacterium, or a cell.
  • the invention provides the application of the IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody in the preparation of a medicine for treating diseases or a medicine for improving immune function.
  • the diseases are tumors, immune decline and so on.
  • the invention provides the application of the IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody in the preparation of a medicine for treating diseases with PD-L1 as a target.
  • the medicament is a medicament for anti-tumor and immune decline diseases.
  • the invention provides a medicine or a detection reagent containing the above IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody.
  • the invention provides the application of the above-mentioned IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody in the treatment of diseases that target PD-L1 or PD-1.
  • the invention provides the application of the IgG1 subtype low ADCC / CDC functional PD-1 monoclonal antibody in killing tumor cells or treating immune degenerative diseases.
  • the present invention uses Pymol software to analyze the structure of Pembrolizumab of Merck Company in the United States, replace the antibody from an IgG4 subtype to an IgG1 subtype, and insert a flexible amino acid sequence between the IgG1 antibody heavy chain CDR3 and the CH2 region. It can reduce the ADCC / CDC function and reduce the difficulty of subsequent production of antibodies, improve the anti-polymerization, expression and stability of antibodies, and avoid the formation of half-antibodies and bispecific antibodies caused by the heavy chain replacement characteristics of IgG4 subtypes.
  • the affinity of the monoclonal antibody of the present invention for binding to human PD-1 is similar to that of the original Pembrolizumab antibody, which can specifically block the binding of PD-1 to cell surface PD-L1, prolong the half-life of antibody drugs, improve the efficacy, and have excellent clinical Value.
  • the invention also provides an IgG1 subtype low ADCC / CDC functional monoclonal antibody against human CTLA4 and application of the monoclonal antibody.
  • the invention is based on the antibody-induced ADCC / CDC action mechanism.
  • a flexible amino acid sequence is inserted between the IgG1 subtype antibody Ipilimumab heavy chain CDR3 and the CH2 region, so that the monoclonal antibody heavy chain constant region Binding site with Fc receptor and / or complement cannot be fully exposed, weakening the monoclonal antibody binding to NK cells, macrophages and neutrophils expressing IgG Fc receptor killer cells or binding to complement, unable or reduced ADCC induction
  • the full-length amino acid sequence of the original Ipilimumab monoclonal antibody is shown in SEQ ID NO.8, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO.9.
  • the present invention utilizes Pymol software to analyze the structure of ipilimumab of Bristol-Myers Squibb (BMS) in the United States, and inserts a flexible amino acid sequence between the CDR3 and CH2 regions of the IgG1 antibody heavy chain to reduce ADCC / CDC function while reducing antibodies Side effects in treatment, improve anti-polymerization, expression and stability of antibodies.
  • the affinity of the monoclonal antibody of the present invention for binding to human CTLA4 is similar to that of the original ipilimumab antibody, which can specifically block the binding of CTLA4 to CD80 and CD86, reduce the side effects of antibody drug treatment, improve the efficacy of antibody drugs, and has excellent clinical application value.
  • the IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibody provided by the present invention has a full-length heavy chain containing the amino acid sequence shown in SEQ ID No. 10 and a full-length light chain containing SEQ ID No. 9 Amino acid sequence.
  • the antibody heavy chain constant region is of the IgG1 subtype.
  • the antibody of the present invention may include: inserting a flexible amino acid sequence between the CDR3 and CH2 regions of the heavy chain of the IgG1 antibody, blocking the stress transmission of the variable region and the constant region of the antibody after the antibody binds to the antigen, and reducing the ADCC / CDC function while reducing the antibody Difficulty in the development of subsequent production processes to improve the anti-polymerization, expression and stability of antibodies.
  • the IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibody provided by the present invention has a light chain full-length amino acid as shown in L0 (SEQ ID No. 9) and a heavy chain full-length amino acid sequence as shown in H1 (SEQ ID ID NO .10) sequence.
  • the present invention screens H1L0 monoclonal antibody sequences. It not only maintains the original affinity of binding to human CTLA4, can specifically block the binding of CTLA4 to its ligand, but also reduces ADCC and CDC functional activities.
  • the present invention provides an expression vector containing the light and heavy chain genes of any of the IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibodies.
  • a host bacteria, a host cell or an expression cassette containing the expression vector is also within the scope of the present invention.
  • the invention provides a method for increasing the activity of an immune cell, said method comprising administering a therapeutically effective amount of an antibody of the invention to a subject in need of treatment.
  • the method can be used for treating cancer, and can be used for treating immunocompromised diseases.
  • the invention also includes the further administration of a second therapeutic agent or form.
  • the invention also includes the use of the above-mentioned anti-CTLA4 monoclonal antibody in the preparation of a medicament for improving immune function.
  • the anti-CTLA4 monoclonal antibody of the present invention can be combined with a second therapeutic agent or therapeutic form.
  • Anti-CTLA4 antibodies can be combined with cancer treatments including the administration of PD-1 antibodies or PD-L1 antibodies.
  • Non-limiting examples of combinations include anti-CTLA4 antibodies in combination with anti-PD-1 antibodies for the treatment of melanoma or non-small cell lung cancer.
  • Treatment with the anti-CTLA4 antibodies of the invention can be combined with chemotherapy.
  • Chemotherapy using cytotoxic agents will cause cancer cells to die, thereby increasing the release of tumor antigens.
  • Such increased effectiveness of tumor antigens can lead to synergy with anti-CTLA4 treatment.
  • Treatment with the CTLA4 mAb of the present invention can be combined with surgery to remove cancer cells from a subject.
  • the anti-CTLA4 antibody of the present invention can be combined with a therapy capable of producing a synergistic effect with CTLA4 blockade, which therapy includes a targeted drug for hormone elimination or inhibition of angiogenesis, or a target for a protein having activity in tumor cells All of these drugs lead to increased tumor cell death and increased effectiveness of immunostimulatory tumor antigens.
  • a therapy capable of producing a synergistic effect with CTLA4 blockade which therapy includes a targeted drug for hormone elimination or inhibition of angiogenesis, or a target for a protein having activity in tumor cells All of these drugs lead to increased tumor cell death and increased effectiveness of immunostimulatory tumor antigens.
  • an increase in T cell activation can lead to durable immune control of cancer.
  • the anti-CTLA4 monoclonal antibodies of the present invention can be combined with another therapeutic antibody for treating cancer or infectious diseases.
  • Specific examples may be a combination of an anti-CTLA4 antibody and an antibody that targets PD-1 or PD-L1.
  • the biological material is an expression cassette, an expression vector, an engineered bacterium, or a cell.
  • the invention provides the application of the above-mentioned IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibody in the preparation of a medicine for treating a disease or a medicine for improving immune function.
  • the diseases are tumors, immune degeneration and the like.
  • the invention provides the application of the above-mentioned IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibody in the preparation of a medicine for treating diseases with CTLA4 as a target.
  • the medicine is an antitumor medicine and an immune degenerative medicine.
  • the present invention provides a combined application of the above-mentioned IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibody in the treatment of diseases that target PD-L1 or PD-1.
  • the invention provides the application of the IgG1 subtype low ADCC / CDC functional CTLA4 monoclonal antibody in killing tumor cells or treating immune degenerative diseases.
  • the method for reducing ADCC / CDC function of an antibody of the present invention can reduce ADCC / CDC function without significantly increasing multimer formation.
  • the modified monoclonal antibody and the original monoclonal antibody aimed at the same site and removed ADCC function, but the antibody conformation was different, the stability was significantly improved, the immunogenicity was significantly reduced, and the half-life was extended. Effectively avoid the side effects caused by ADCC / CDC function, improve the curative effect, and have excellent clinical application value.
  • Figure 1 Sequence alignment results of the modified PD-L1 monoclonal antibody (H-1L0) of the present invention and the original Atezolizumab antibody heavy chain.
  • Figure 2 Affinity evaluation results of the modified PD-L1 monoclonal antibody of the present invention and the original Atezolizumab antibody.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the absorbance at 450 nm.
  • FIG. 3 ADCC activity experiments of two PD-L1 monoclonal antibodies (H-1L0, H-2L0) and original Atezolizumab antibody after the transformation of the present invention, the ordinate is the percentage of Raji-PDL1 cell lysis, the abscissa is the sample concentration, and IMM25 is ADCC Positive antibody was used as positive control, and original drug Atezolizumab was used as negative control for ADCC experiment.
  • Fig. 4 Experimental results of PD-L1 monoclonal antibodies inhibiting PD-L1 and PD-1 active cells after the modification of the present invention.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the mean unit of luciferase activity.
  • Figure 5 ADA evaluation comparison between the modified PD-L1 monoclonal antibody and the original Atezolizumab antibody after the modification of the present invention.
  • the abscissa is the serum dilution ratio, and the ordinate is the absorbance at 450nm.
  • FIGS 6A and 6B are HPLC-SEC results of the original Atezolizumab antibody and the modified PD-L1 monoclonal antibody of the present invention, respectively.
  • FIG. 7A and FIG. 7B are glycotype analysis of the PD-L1 monoclonal antibody modified by the present invention.
  • FIG. 7A is the result of the glycoform analysis mass spectrum of the original research Atezolizumab
  • FIG. 7B is the result of the glycoform analysis mass spectrum of the modified PD-L1 monoclonal antibody.
  • FIG. 8 shows the results of thermal stability experiments of the PD-L1 monoclonal antibody and the original Atezolizumab antibody modified by the present invention.
  • the abscissa is the concentration (M) of the antibody
  • the ordinate is the absorbance at 450 nm.
  • Figure 9 Sequence alignment results of the modified PD-1 monoclonal antibody of the present invention and the original heavy chain of the original Pembrolizumab antibody.
  • Figure 10 Affinity evaluation results of the modified PD-1 monoclonal antibody of the present invention and the original Pembrolizumab antibody.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the absorbance at 450 nm.
  • FIG 11 ADCC activity experiment of the modified PD-1 monoclonal antibody and the original Pembrolizumab antibody of the present invention.
  • the ordinate is the percentage of Raji-PD-1 cell lysis and the abscissa is the sample concentration.
  • Pembrolizumab monoclonal antibody was used as a positive control for the ADCC experiment, and H1L0 was a modified PD-1 monoclonal antibody of the present invention.
  • FIG. 12 The experimental results of PD-1 monoclonal antibodies and original Pembrolizumab antibodies modified by the present invention to inhibit PD-L1 and PD-1 active cells.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the mean unit of luciferase activity.
  • FIG. 13 Sequence alignment results of the modified CTLA4 monoclonal antibody of the present invention and the original Ipilimumab antibody heavy chain.
  • Figure 14 Affinity evaluation results of the modified CTLA4 monoclonal antibody of the present invention and the original Ipilimumab antibody.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the absorbance at 450 nm.
  • Figure 15 ADCC activity experiment of the CTLA4 monoclonal antibody and the original Ipilimumab antibody after the transformation of the present invention.
  • the ordinate is the percentage of Raji-CTLA4 cell lysis and the abscissa is the sample concentration.
  • CTLA4 monoclonal antibody is the sample concentration.
  • FIG. 16 Inhibition of CTLA4 and CD80 and CD86 binding activity experiments of the modified CTLA4 monoclonal antibody and original Ipilimumab antibody of the present invention.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the absorbance at 450 nm.
  • FIG. 17 The results of the modified CTLA4 monoclonal antibody and the original Ipilimumab antibody of the present invention competing with CD80 and CD86 for binding to human CTLA4 transfected CHO cells.
  • the abscissa is the concentration (M) of the antibody, and the ordinate is the absorbance at 450 nm.
  • Antibody refers to any form of an antibody that exhibits a desired biological activity, such as inhibiting the binding of a ligand to its receptor or by inhibiting receptor-induced receptor signaling. Therefore, the antibodies are used in the broadest sense and explicitly include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies.
  • a "Fab fragment” consists of a light chain and a heavy chain CH1 and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc” region is two heavy chain fragments containing the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.
  • a "Fab 'fragment” contains a light chain and a portion of a heavy chain containing a region between the VH and CH1 domains and between the CH1 and CH2 domains, so that it can be between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F (ab ') 2 molecules.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the properties of an antibody obtained from a substantially homogeneous antibody population and cannot be understood as requiring the antibody to be prepared by any particular method.
  • flexible amino acid sequence refers to a segment of linked polypeptide consisting of amino acids with smaller side chains, which is used to prevent or reduce mutual interference in spatial conformation between the domains at both ends.
  • the sequence includes, but is not limited to, GGSGGS , GSGGSGG, GSGGSGGG, GGGGSGGG, GSGSG, GGSGG, GGS, GGSGS, and GGGS.
  • Immune cell includes cells that have a hematopoietic origin and play a role in the immune response.
  • Immune cells include: lymphocytes, such as lymph B cells and lymph T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in significant amounts.
  • administering refers to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, subjects Healer, cell, tissue, organ or biological fluid contact.
  • administering may refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, for example, by agents, diagnostics, binding compositions, or by other cells.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject can vary depending on a variety of factors, such as the disease to be treated, the overall health of the patient, the method and route of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.
  • the antibody DNA of the present invention can be placed in an expression vector, and the vector can be transfected into a host cell to achieve the synthesis of monoclonal antibodies in a recombinant host cell, such as E. coli Cells, Chinese hamster eggs (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. Recombinant production of antibodies will be described in more detail below.
  • Suitable routes of administration include parenteral (e.g., intramuscular, intravenous or subcutaneous) and oral administration.
  • Antibodies for use in pharmaceutical compositions or for practicing the methods of the invention can be administered in a variety of conventional ways, such as by oral inhalation, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, parenteral, intraarterial or Intravenous injection.
  • one can administer antibodies in a targeted drug delivery system.
  • inhibit or “treat or treatment” includes delaying the development of symptoms associated with a disease and / or reducing the severity of those symptoms that the disease is or is expected to develop.
  • the term also includes alleviating pre-existing symptoms, preventing additional symptoms, and underlying causes that slow or prevent these symptoms. Thus, the term indicates that beneficial results have been imparted to a vertebrate subject with a disease.
  • terapéuticaally effective amount refers to an antibody or fragment thereof that is effective to prevent or slow the disease or to be treated when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. Amount of illness.
  • a therapeutically effective dose further refers to an amount of the compound sufficient to cause a reduction in symptoms, such as treating, curing, preventing, or slowing a related medical condition, or increasing the rate of treatment, cure, prevention, or slowing of the condition .
  • a therapeutically effective amount refers to that individual ingredient.
  • a therapeutically effective amount refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of whether they are actually administered in combination, continuously, or simultaneously.
  • a therapeutically effective amount will generally reduce symptoms by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.
  • the pharmaceutical composition of the present invention may also contain other agents, including but not limited to cytotoxic agents, cytostatic agents, anti-angiogenesis drugs or anti-metabolic drugs, tumor-targeting drugs, immunostimulants or immunomodulators or with cytotoxic agents, cells Produce inhibitors or other toxic drug-conjugated antibodies.
  • the pharmaceutical composition may also be administered with other treatment modalities, such as surgery, chemotherapy, and radiation therapy.
  • Typical veterinary, experimental, or research subjects include monkeys, dogs, cats, rats, mice, guinea pigs, rabbits, horses, and humans.
  • the therapeutic applications of the antibodies of the present invention are as follows:
  • Antibodies of the invention that specifically bind to human PD-1 / L1 and CTLA4 can be used to increase, enhance, stimulate or up-regulate the immune response.
  • the antibodies of the invention are particularly suitable for treating a subject suffering from a disease that can be treated by increasing a T cell-mediated immune response.
  • Preferred subjects include human patients in need of an enhanced immune response.
  • the antibodies of the present invention are useful for treating cancer (ie, inhibiting the growth or survival of tumor cells).
  • Preferred cancers that can be inhibited by the antibodies of the present invention include cancers that generally respond to immunotherapy, but also cancers that have hitherto not been associated with immunotherapy.
  • Non-limiting examples of preferred cancers for treatment include melanoma (e.g., malignant metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., prostate hormone adenocarcinoma with difficult hormone control), pancreatic adenocarcinoma , Breast cancer, colon cancer, lung cancer (e.g.
  • non-small cell lung cancer non-small cell lung cancer
  • esophageal cancer head and neck squamous cell carcinoma
  • liver cancer ovarian cancer
  • cervical cancer thyroid cancer
  • glioblastoma glioma
  • leukemia lymph Tumors and other malignancies
  • the invention includes refractory or recurrent cancers that can be inhibited by the antibodies of the invention.
  • antitumor drugs or immunogenic agents e.g., attenuated cancer cells, tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), antigen-presenting cells such as those derived from tumors Antigen or nucleic acid-stimulated dendritic cells, immunostimulatory cytokines (such as IL-2, IFNa2, GM-CSF) and cells transfected with genes encoding immunostimulatory cytokines (such as, but not limited to, GM-CSF); Standard cancer treatment (such as chemotherapy, radiation or surgery); or other antibodies (including but not limited to antibodies against: VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CTLA- 4. OX-40, 4-IBB and ICOS).
  • antitumor drugs or immunogenic agents e.g., attenuated cancer cells, tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules),
  • the immunocompromised diseases, the antibodies of the invention can also be used to prevent or treat immunocompromised diseases.
  • Antibodies can be used alone or in combination with drugs to stimulate immune responses against pathogens and multimers.
  • Antibodies can be used to stimulate an immune response to human-infected viruses such as, but not limited to, human immunodeficiency virus, hepatitis A, B, C virus, Epstein-Barr virus, human cytomegalovirus, human papilloma virus, herpes virus.
  • Antibodies can be used to stimulate the immune response to infections caused by bacterial or fungal parasites and other pathogens.
  • Antibodies can be used to stimulate the immune response and removal of multimers in the body, to treat diseases such as dementia.
  • the antibodies targeted by the present invention include but are not limited to PD-L1, PD-1, CTLA4, TNF- ⁇ , PCSK9, NGF, C5, A ⁇ , IL-6, IL-17A, IL23A, HGF, CMET, Notch1, CCL11, IL6R, IL-31R, IL-1B, IL-20, CD40, CD47, DKK1 / 2, TIGIT, 4-1BB, IGF-1R, LL4, PDGFR2, HER3, IGF1 / 2, RANKL, sclerostin, GCGR, CGRP, ANGPTL3, IL-13, IL-4R, CSF-1R, TFPI, FCGRT, CD47, etc., can be used to treat cancer, asthma, autoimmune disease, migraine, dementia, hyperlipidemia, osteoarthritis, Cluster headache, osteoporosis and other diseases.
  • the invention uses Pymol software to analyze the structure of Atezolizumab of Genentech, find a relatively flexible region between the antibody variable region and the constant region, and insert a flexible amino acid sequence in the corresponding region to cut off the binding of the variable region of the antibody.
  • the mechanical stress transmission generated after the antigen prevents the constant region of the antibody drug heavy chain and the Fc receptor and / or complement complement site from being fully exposed, weakening the expression of IgG by the antibody drug and NK cells, macrophages and neutrophils. Fc receptor killer cell binding or complement complement cannot or reduce the signal that induces ADCC and CDC.
  • Atezolizumab antibody 297 amino acid A was mutated back to N to restore glycosylation, and a flexible amino acid sequence was inserted between the heavy chain CDR3 and CH2 regions of the IgG1 antibody to block antibody binding. Post-antigen stress transmission in the variable and constant regions of the antibody, thereby reducing ADCC / CDC function without significantly increasing multimer formation.
  • the full length of the heavy chain and the amino acid sequence of the light chain variable region of the original Atezolizumab sequence are shown in SEQ ID Nos. 1, 2 respectively.
  • the low immunogenic anti-PD-L1 monoclonal antibody provided by the present invention has the amino acid sequence of its heavy chain as shown in SEQ ID Nos. 3 and 4, and the amino acid sequence of its light chain is SEQ ID NO. 2 and is named H -1L0, H-2L0.
  • the present invention performs full gene synthesis on the corresponding modified sequence, sequencing the synthesized gene and selecting the correct sequence for the next operation, and designing the light chain digestion site It is EcoR I + Hind III, and the design site for the heavy chain is EcoR I + Hind III, which is linked to the expression vectors pEE6.4 and pEE12.4 respectively, and transformed into E. coli DH5 ⁇ to obtain antibody heavy and light chain expression vectors. .
  • the constructed antibodies were sequenced and sequenced.
  • the sequence comparison results of the modified PD-L1 monoclonal antibody and the original Atezolizumab antibody heavy chain are shown in Figure 1.
  • the present invention transforms Pembrolizumab of Merck Company in the United States.
  • the heavy chain subtype is changed from IgG4 to IgG1, and structural analysis is performed by using Pymol software to find a relatively flexible region between the variable region and the constant region of the antibody.
  • a flexible amino acid sequence was inserted in the corresponding region, gene synthesis was performed on the corresponding sequence, sequencing was performed, the correct sequence was selected, and the monoclonal antibody H1L0 was selected.
  • the present invention attempts to insert a flexible amino acid sequence between the CDR3 and CH2 regions of the heavy chain of the original IgG1 subtype antibody.
  • the inserted flexible amino acid sequence blocks the mechanical stress transmission generated after the antibody variable region binds to the antigen, making the antibody heavy.
  • a flexible amino acid sequence is a linking polypeptide composed of amino acids with smaller side chains, which is used to prevent or reduce the spatial conformational interference between the domains at both ends. Its sequence includes but is not limited to GGSGGS, GSGGSGG, GSGGSGGG, GGGGSGGG, GSGSG, GGSGG, GGS, GGSGS, and GGGS.
  • the amino acid sequence of the heavy chain after being modified according to the method of the present invention is shown in SEQ ID No.7.
  • the corresponding modified sequence is subjected to whole gene synthesis, the synthesized gene is sequenced, and the correct sequence is selected for the next operation.
  • the light chain design digestion site is EcoR I + Hind III, and the heavy chain design digestion is performed.
  • the site is EcoR I + Hind III, which were linked to the expression vectors pEE6.4 and pEE12.4 respectively, and transformed into E. coli DH5 ⁇ at the same time to obtain antibody heavy and light chain expression vectors.
  • the constructed antibodies were sequenced and sequenced.
  • the sequence comparison results of the modified PD-1 monoclonal antibody and the original Pembrolizumab antibody heavy chain are shown in Figure 9.
  • the invention transforms ipilimumab of Bristol-Myers Squibb (BMS) company, uses Pymol software to analyze the structure, finds a relatively flexible region between the variable region and the constant region of the antibody, and inserts a flexible amino acid sequence in the corresponding region.
  • the sequence was gene synthesized and sequenced. The correct sequence was selected and screened to obtain the monoclonal antibody H1L0.
  • an attempt is made to insert a flexible amino acid sequence between the CDR3 and CH2 regions of the heavy chain of ipilimumab monoclonal antibody IgG1 subtype antibody.
  • the inserted flexible amino acid sequence blocks the mechanical stress transmission generated after the antibody variable region binds to the antigen.
  • a flexible amino acid sequence is a linking polypeptide composed of amino acids with smaller side chains, which is used to prevent or reduce the spatial conformational interference between the domains at both ends. Its sequence includes but is not limited to GGSGGS, GSGGSGG, GSGGSGGG, GGGGSGGG, GSGSG, GGSGG, GGS, GGSGS, and GGGS.
  • the amino acid sequence of the modified heavy chain is shown in SEQ ID NO.10.
  • the corresponding modified sequence is subjected to whole gene synthesis, the synthesized gene is sequenced, and the correct sequence is selected for the next operation.
  • the light chain design restriction site is EcoR I + Hind III, and the heavy chain design restriction enzyme The site is EcoR I + Hind III, which were linked to the expression vectors pEE6.4 and pEE12.4 respectively, and transformed into E. coli DH5 ⁇ at the same time to obtain antibody heavy and light chain expression vectors.
  • the constructed antibodies were sequenced and sequenced.
  • the sequence comparison results of the modified CTLA4 monoclonal antibody and the original Ipilimumab antibody heavy chain are shown in Figure 13.
  • the present invention evaluates the ADCC activity of the antibody by detecting the killing ability of FcR-TANK cells to target protein (PD-L1, PD-1, CTLA4) overexpressing cells.
  • ADCC activity detection of PD-L1 antibody Take the ADCC activity detection of PD-L1 antibody as an example: After using the target protein overexpressing cells to stain with CFSE, adjust the cell density to 6 ⁇ 10 5 / mL, and modify the modified antibodies (H-1L0, H-2L0) and Atezolizumab And the positive control antibody IMM25 (purchased from Yiming Angco) was diluted with a certain gradient (maximum concentration of 4 ⁇ g / mL, 12 gradients were diluted three times with the medium), at the same time, FcR-TANK cells were counted and cell density Adjust to 6 ⁇ 10 5 / mL; co-incubate the antibody, target cells and effector cells: take 50 ⁇ L each of the above-mentioned different dilution gradients of antibodies, 50 ⁇ L of target cells, and 100 ⁇ L of FcR-TANK cells into a 96-well plate, and perform each gradient Repeat the well operation and set a blank control
  • ADCC% [(Sample% PI Positive Cell-No Antibody% PI Positive Cell) / (100-No Antibody % PI Positive Cell)] ⁇ 100, and plot the relationship between ADCC% and concentration.
  • the ADCC activity experiments of the modified PD-L1 monoclonal antibody of the present invention and the original research drug Atezolizumab antibody show that the ADC-1 activities of the two PD-L1 monoclonal antibodies H-1L0 and H-2L0 of the present invention are consistent with the original research Atezolizumab antibody, see Figure 3 . Since H-1L0 and H-2L0 both proved that the ADCC effect was removed, subsequent studies mainly used H-1L0 and the original drug Atezolizumab for comparison and verification.
  • the ADCC activity experiment of the modified PD-1 monoclonal antibody of the present invention and the original research drug Pembrolizumab antibody shows that the ADCC activity of the modified PD-1 monoclonal antibody H1L0 disappears, and the original research drug Pembrolizumab antibody has a weaker ADCC activity. 11.
  • the ADCC activity test of the modified CTLA4 monoclonal antibody of the present invention and the original research drug Ipilimumab antibody showed that the ADCC activity of the CTLA4 monoclonal antibody H1L0 of the present invention disappeared, and the ADCC activity of the original research drug Ipilimumab antibody was normal, as shown in FIG. 15.
  • the plasmid construction of the vectors constructed in Examples 1-3 was performed, and Qiagen's endotoxin-free plasmid extraction kit was selected; the selected plasmids were optimized and combined, and CHO cells were used for transient transfection expression. Protein-based ELISA experiments Used to determine apparent binding affinity (reported as EC 50 value). Compare the binding ability of the improved antibody with the original antibody.
  • Protein ELISA is used to determine the relative binding of antibodies to human PD-L1.
  • PD-L1 was immobilized on an enzyme plate at 4 ° C overnight, and non-specific binding sites were blocked by incubation with 3% BSA in PBS for 1 hour at room temperature. After coating, the plate was washed with PBS, and a dilution of the anti-PD-L1 antibody was prepared, and it was incubated with the immobilized protein at 25 ° C for 1 hour. After binding, the plates were washed 3 times with PBS, incubated at 25 ° C for 1 hour with a peroxidase-labeled goat anti-human IgG binding buffer diluted to 1/2000, and washed again using TMB for color development.
  • the ELISA results are shown in FIG. 2.
  • the magnitude of the relative binding affinity is expressed as the half-maximum binding concentration.
  • the expressed antibodies were tested for affinity and EC50 (see Table 1).
  • Affinity ELISA results showed that the affinity of H-1L0 and PD-L1 was similar to that of the original drug Atezolizumab antibody, as shown in Figure 2.
  • Protein ELISA is used to determine the relative binding of antibodies to human PD-1.
  • PD-1 was immobilized on a microplate at 4 ° C overnight and non-specific binding sites were blocked by incubation with PBS containing 3% BSA for 1 hour at room temperature. After coating, the plate was washed with PBS, and a dilution of the anti-PD-1 antibody was prepared and incubated with the immobilized protein at 25 ° C for 1 hour. After binding, the plates were washed 3 times with PBS, incubated at 25 ° C for 1 hour with a peroxidase-labeled goat anti-human IgG binding buffer diluted to 1/2000, and washed again using TMB for color development. The ELISA results are shown in FIG. 10.
  • the magnitude of the relative binding affinity is expressed as the half-maximum binding concentration.
  • the expressed antibodies were tested for affinity and EC50 (see Table 2). Affinity ELISA results showed that the affinity of H1L0 and PD-1 was similar to that of the original drug Pembrolizumab antibody.
  • Protein ELISA is used to determine the relative binding of antibodies to human CTLA4.
  • PD-1 was immobilized on a microplate at 4 ° C overnight and non-specific binding sites were blocked by incubation with PBS containing 3% BSA for 1 hour at room temperature. After coating, the plate was washed with PBS, and a dilution of the anti-CTLA4 antibody was prepared, and it was incubated with the immobilized protein at 25 ° C for 1 hour. After binding, the plates were washed 3 times with PBS, incubated at 25 ° C for 1 hour with a peroxidase-labeled goat anti-human IgG binding buffer diluted to 1/2000, and washed again using TMB for color development. The ELISA results are shown in FIG. 14.
  • the magnitude of the relative binding affinity is expressed as the half-maximum binding concentration.
  • the expressed antibodies were tested for affinity and EC50 (see Table 3). Affinity ELISA results showed that the affinity of H1L0 and CTLA4 was similar to that of the original drug Ipilimumab antibody, as shown in Figure 14.
  • This method is based on two cell lines, the CHO-PD-L1-CD3L cell line and the Jurkat-PD-1-NFAT cell line.
  • CHO-PD-L1-CD3L cell line stably expresses PD-L1 and anti-CD3-scFv as target cells;
  • Jurkat-PD-1-NFAT cell line stably expresses PD-1 and luciferase, luciferase Genes are regulated by NFAT elements (transcription factors) (IL-2 promoter) as effector cells.
  • NFAT elements transcription factors
  • the combination of PD-1 and PD-L1 can block the downstream signal transduction of CD3, thereby inhibiting the expression of luciferase.
  • PD-1 antibody or PD-L1 antibody is added, this Block effect is reversed and luciferase Can be expressed so that a fluorescent signal is detected.
  • the specific experimental steps are as follows:
  • Jurkat-PD-1-NFAT cells use RPMI1640 medium containing 10% FBS, 1% NEAA and antibiotics; CHO-PD-L1-CD3L cells use DMEM containing 10% FBS, 1% NEAA and antibiotics Or F12 medium.
  • CHO-PD-L1-CD3L cells were seeded at 50,000 cells / well in 96-well plates and cultured at 37 ° C and 5% CO2 for 12-14 hours. After removing the culture medium, 50 ⁇ L of an analytical culture solution (RPMI1640 medium containing 2% FBS) containing 100,000 Jurkat-PD-1-NFAT cells was added to each well.
  • an analytical culture solution RPMI1640 medium containing 2% FBS
  • the modified antibody or the original antibody drug of the present invention is diluted at a ratio of 1: 3 from a concentration of 10 ⁇ g / mL (using an analysis culture solution). The diluted antibody was then added to each well and incubated for 6 hours at 37 ° C, 5% CO 2 .
  • the ELISA method was used to detect the efficiency of the monoclonal antibody of the present invention in inhibiting the binding of CTLA4 to CD80 or CD86.
  • HCD80 or hCD86 was immobilized on the enzyme plate overnight at 4 ° C, and non-specific binding sites were blocked by incubation with 3% BSA in PBS for 1 hour at room temperature. After coating, the plate was washed with PBS, and a mixed dilution of anti-CTLA4 antibody and Biotin-CTLA4 was prepared (Biotin-CTLA4 was a fixed concentration of 1 ⁇ g / mL.
  • the anti-CTLA4 antibody of the present invention was diluted by gradient with the highest concentration of 1 ⁇ g / mL.
  • the lowest concentration is 0), and it is incubated with the immobilized protein at 25 ° C for 1 hour. After binding, the plates were washed 3 times with PBS, incubated at 25 ° C for 1 hour with a binding buffer containing peroxidase-labeled avidin diluted to 1/8000, and washed with TMB after washing again.
  • the ELISA results are shown in Figure 16, showing that the CTLA4 mAb of the present invention is capable of inhibiting the binding of CTLA4 to CD80 or CD86.
  • the present invention uses the CELISA method to evaluate the effect of antibodies competing with CD80 and CD86 for binding to human CTLA4 transfected CHO cells.
  • CHO cells expressing hCTLA4 were constructed, and CHO-hCTLA4 cells were cultured in 50 ⁇ L to 80% -100% coverage.
  • the modified antibody H-1L0 and original research Atezolizumab were immunized to the mice to evaluate the titer of ADA produced by the mice.
  • Balb / c mice were used for group experiments.
  • the specific immunization methods were as follows: 1) Basic immunization: the antigen and Freund's complete adjuvant were mixed in an equal volume and fully emulsified, and subcutaneously injected at different points. 2) Strengthening immunity: The strengthening of immunity uses an emulsion of antigen and Freund's incomplete adjuvant. The total injection volume per Balb / c mouse was 70 ⁇ g.
  • the modified PD-L1 antibody and the original Atezolizumab antibody of the present invention were simultaneously constructed with a CHO stable cell bank, and 5 g of small-scale expression and purification of the modified PD-L1 antibody and the original Atezolizumab were performed in accordance with well-known antibody drug production specifications in the art.
  • the obtained antibodies were compared by HPLC-SEC, as shown in Figs. 6A and 6B.
  • the loading amount (50 ⁇ L) and concentration (60 mg / mL) of the original research Atezolizumab and the modified PD-L1 antibody H-1L0 were the same.
  • the modified PD-L1 antibody was further verified for its glycoform. It is mainly carried out by a method known in the art: after digestion and collection of oligosaccharides on the antibody, analysis and identification of glycoforms by mass spectrometry. It was identified that the original research Atezolizumab did not detect N sugar modification, and the modified PD-L1 antibody was a normal glycosylation modification, as shown in Figure 7. Table 7 shows the glycoform analysis results of the modified PD-L1 antibody.
  • the stability of the modified PD-L1 monoclonal antibody and the original Atezolizumab antibody of the present invention was verified.
  • the improved PD-L1 antibody and the original Atezolizumab antibody were subjected to rapid heat treatment at the same time, and the stability was verified.
  • the main operation process is as follows:
  • the present invention screens improved monoclonal antibodies against ADCC function against multiple different targets. They not only retain the original affinity for binding to the target protein, but also remove their ADCC function, which can specifically block the target protein and Binding of the corresponding ligand.
  • the improved PD-L1 antibody by using the method for removing ADCC function of the present invention, not only the purpose of removing the ADCC effect is achieved, but also the original antibody drug with high polymer content and strong immunogenicity caused by transformation is solved. problem.
  • the invention provides a low ADCC / CDC functional monoclonal antibody and a preparation method and application thereof.
  • the invention modifies antibody sequences of multiple targets such as PD-L1, PD-1, CTLA4, TNF- ⁇ , PCSK9, NGF, CD47, and C5, and inserts a flexible segment between the heavy chain CDR3 and CH2 regions of the IgG1 subtype antibody.
  • Amino acid sequence can reduce ADCC / CDC function without significantly increasing multimer formation.
  • the modified antibody and the original antibody of the present invention are directed to the same site, but the antibody subtype or glycosylation is different, the stability is significantly improved or / and the immunogenicity is significantly reduced, and the half-life is prolonged.
  • the modified antibody removes the ADCC / CDC function. Therefore, the antibody modified by the technology of the present invention can effectively avoid the side effects caused by the ADCC / CDC function, improve the curative effect, has excellent clinical application value, is suitable for popularization, and has high Economic value and broad market prospects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Dermatology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种低ADCC/CDC功能性单抗及其制备方法与应用。对PD-L1、PD-1、CTLA4、TNF-α、PCSK9、NGF、CD47和C5等多靶点的抗体序列进行改造，在IgG1亚型抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，可降低ADCC/CDC功能，同时不显著增加多聚体形成。上述改造抗体和原始抗体针对相同位点，但抗体亚型或糖基化不同，稳定性显著提高或/和免疫原性显著降低，半衰期延长。

Description

低ADCC/CDC功能性单抗及其制备方法与应用
交叉引用
本申请要求于2018年7月6日提交的专利名称为“低ADCC/CDC功能性抗PD-L1全人源单抗及其应用”的第201810738565.8号中国专利申请、2018年8月14日提交的专利名称为“针对人程序性死亡受体PD-1的IgG1亚型低ADCC/CDC功能性抗体”的第201810924249.X号中国专利申请以及2019年5月7日提交的专利名称为“针对人CTLA4的IgG1亚型低ADCC/CDC功能性抗体”的第201910376852.3号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
[根据细则91更正 20.11.2019]　
本发明涉及治疗用抗体的制备及应用，主要涉及低ADCC/CDC功能性单抗的应用，以及降低抗体的ADCC/CDC等功能的方法。
背景技术
抗体依赖的细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity，ADCC)是指表达IgG Fc受体的NK细胞、巨噬细胞和中性粒细胞等，通过与已结合在靶细胞表面的IgG抗体的Fc段结合，而杀伤这些靶细胞的作用。IgG抗体可介导这些细胞发挥ADCC作用，其中NK细胞是能发挥ADCC作用的主要细胞。在抗体介导的ADCC作用的发生过程中，抗体只能与靶细胞上的相应抗原表位特异性结合，而NK细胞等效应细胞可杀伤任何已与抗体结合的靶细胞，故抗体与靶细胞上的抗原结合是特异性的，NK细胞等对靶细胞的杀伤作用是非特异性的。
CDC指补体系统被激活后，在靶细胞表面形成MAC，导致靶细胞溶解，这种效应称为补体依赖的细胞毒作用。补体能导致多种细菌和其他病原生物细胞的溶解，是机体抵抗病原生物感染的重要防御机制。尤其是对防止革兰阴性菌的感染有重要作用。在某些病例情况下，补体系统可引起机体组织或细胞损伤，参与超敏反应和自身免疫病的致病过程。
抗体药的应用并不局限于传染病和肿瘤，为了避免抗体结合靶蛋白后诱导不利的ADCC和CDC功能，目前采取的主要方法有对抗体重链进行了糖基化位点氨基酸N(297)突变成A的去糖基化突变或将抗体重链改为IgG2/4亚型。但是抗体重链去糖基化，导致了产品中多聚体含量过高，药物免疫原性显著增强，病人多次给药后产生抗药抗体(ADA)发生率高。IgG2亚型的抗体仍能够通过与FcγRIIa的结合引发单 核细胞介导的ADCC与巨噬细胞介导的ADCP。此外，IgG2的铰链区上游存在额外的Cys残基，因此会形成二硫键异构体影响IgG2分子的空间构象与功能。而将抗体重链改为IgG4亚型，IgG4亚型的抗体药重链在血液中会自动发生和其他人IgG4重链交换，形成半分子以及双特异的功能单价的抗体。同时，IgG2/4亚型抗体半衰期比IgG1亚型显著缩短。
PD-1主要在激活的T细胞和B细胞中表达，功能是抑制免疫细胞的激活，这是免疫系统的一种正常的自稳机制，因为过度的T/B细胞激活会引起自身免疫病，所以PD-1是我们人体的一道护身符。但是，肿瘤微环境会诱导浸润的T细胞高表达PD-1分子，肿瘤细胞会高表达PD-1的配体PD-L1和PD-L2，导致肿瘤微环境中PD-1通路持续激活，T细胞功能被抑制，无法杀伤肿瘤细胞。
PD-1和PD-L1的抗体都可以阻断这一通路，部分恢复T细胞的功能，使这些细胞能够继续杀伤肿瘤细胞。美国FDA于2016年5月18日批准基因泰克(Genentech)公司的Atezolizumab(商品名：Tecentriq)注射液上市，用于治疗接受含铂化疗期间/之后或接受含铂化疗的新辅助疗法/辅助疗法12个月内疾病进展的局部晚期或转移性尿路上皮癌。但是该抗体药为了避免诱导抗体依赖的细胞毒性作用(ADCC)和补体依赖的细胞毒性作用(CDC)功能，对抗体重链进行了去糖基化突变，结果导致了产品中多聚体含量过高，药物免疫原性显著增强，病人多次给药后产生抗药抗体(ADA)发生率高达41.5％。
2014年9月4日美国食品药品监督管理局(FDA)授权加速批准美国默克(Merck)公司Pembrolizumab(商品名：Keytruda)上市，用于治疗对其他不再反应的晚期或不可切除黑色素瘤。2017年5月27日，美国食品药品监督管理局(FDA)批准Keytruda用于治疗微卫星不稳定的实体瘤，成为首个可以治疗多种癌症的抗体药物。该抗体药为了避免诱导抗体依赖的细胞毒性作用(ADCC)和补体依赖的细胞毒性作用(CDC)功能，使用了对于FcγR受体具有较弱亲和力且缺乏激活补体能力的IgG4亚型。由于IgG4亚型抗体本身存在Fab臂置换，因此有形成半抗体或双特异抗体的几率，增加用药的潜在风险，且IgG4亚型的抗体的生产工艺不如IgG1亚型抗体成熟(主要表现在结构稳定性、理化特性、抗聚合能力、表达量等方面)。
这给临床患者的使用带来了风险和不便，同时药物半衰期也相对较短，使用的过程中也需要增大药物剂量来弥补该方面的缺陷。基于此，若能够在不影响抗体亲和力和特异性的前提下，通过对抗体恒定区域进行改造，在降低抗体的ADCC/CDC功能的同时，避免抗体药形成大量多聚体，减少抗体药的免疫原性，则一方面可使单抗 的安全性得到提高；另一方面可增加药物半衰期，在减少其用剂量同时也能起到提高疗效的作用。
CTLA-4，又名CD152，由CTLA-4基因编码的一种跨膜蛋白质，表达于活化的CD4+和CD8+T细胞，与T细胞表面的协同刺激分子受体(CD28)具有高度的同源性。CTLA-4和CD28均为免疫球蛋白超家族成员，二者与相同的配体CD86(B7-2)和CD80(B7-1)结合。CTLA-4的免疫调控功能的关键体现在控制CD4+FoxP3-、CD8+T细胞以及调节性T细胞(Treg)。CTLA-4能够中止激活的T细胞的反应(T cell response)以及介导Treg的抑制功能。目前的研究表明CTLA-4抑制T细胞的反应主要是通过两种途径：一是通过与CD28竞争性的结合B7或者招募磷酸酶到CTLA-4的胞内结构域部分从而降低TCR(T cell receptor)和CD28的信号。另一种是降低CD80和CD86在抗原呈递细胞(APC)的表达水平或者通过转胞吞作用(transendocytosis)将它们从APC移除，这样就减少了CD28参与进行T细胞激活。此外，CTLA-4还会介导树突细胞结合CD80/CD86并诱导色氨酸降解酶IDO的表达，从而导致TCR的抑制。CTLA-4抗体通过结合CTLA-4来减少Treg，激活TCR。
2011年4月美国食品药品监督管理局(FDA)授权批准美国百时美施贵宝(Bristol-Myers Squibb，BMS)公司ipilimumab(商品名：Yervoy)上市，用于治疗晚期黑色素瘤，使其成为第一个上市的免疫检查点抑制剂。但由于其适用症很少，虽然在上市时间等方面拔得头筹，但被PD-1/PD-L1相关抗体药后来居上，占据了更大的市场，相比于PD-1/PD-L1的350亿美元市场份额，Ipilimumab抗体药2017年全年销售额仅为12亿美元。迄今为止，PD-1和PD-L1抗体获批的适应证包括晚期黑色素瘤、头颈鳞癌、非小细胞肺癌(NSCLC)、尿路上皮癌、霍奇金淋巴瘤、默克尔细胞癌(Merkel's cell carcinoma)和微卫星不稳定(MSI-H)实体瘤，而CTLA-4抗体ipilimumab的适应证仅限于晚期黑色素瘤和高危复发黑色素瘤。另外，与PD-1抗体相比，ipilimumab治疗晚期黑色素瘤的有效率低，免疫相关副作用发生率却更高。这给临床患者的使用带来了风险和不便，极大限制了该抗体药物的市场占有率及销售份额。
目前针对CTLA4抗体的主要推进方向为针对多靶点的更有效的联合用药，例如Ipilimumab和Nivolumab(抗PD-1的抗体)强强联合后抗肿瘤效果大增。因此，在联合用药领域，CTLA4抗体同诸多明星抗体药物(PD-1抗体Nivolumab，PD-L1抗体Atezolizumab等)的联合治疗有效地增大了其市场占有份额。然而，一半以上的患者产生了3-4级严重的副作用，再次成为了限制抗CTLA-4抗体的研发与应用的最主 要因素。
CTLA4抗体Ipilimumab使用的是IgG1亚型，其具有较强的ADCC/CDC功能，在一定程度上这种强ADCC效应会造成免疫治疗诱导的副作用的产生，这主要通过FcR介导的特异性清除肿瘤微环境中的Treg细胞产生的。因此，低ADCC/CDC效应抗体将可有效降低ADCC造成相应的副作用产生几率。
BMS的伊匹单抗为抗CTLA-4人源IgG1，剂量为3mg/kg，静脉给药，每3周总共四剂。伊匹抗体副作用非常强，单独用药，10-20％的病人因为T细胞过度激活和繁殖产生严重不良反应甚至死亡。和O药联用抗肿瘤效果大增，然而，一半以上的患者产生了3-4级严重的副作用。对严重不良反应永久终止用药。现发现伊匹单抗不仅暂时阻断了CTLA-4信号通路对免疫系统的抑制，而且因它自带的ADCC功能把T调控细胞杀死，一劳永逸地解除了病人的免疫抑制调控，导致免疫系统过度激活。若能够对伊匹单抗进行去ADCC/CDC改造，获得既保留原研药的中和作用，又去除ADCC毒副作用，将可大大降低免疫治疗相关的免疫毒性，显著减少3-4级严重的副作用发生率，更加安全。
目前未见通过插入氨基酸序列来减少抗体药中多聚体含量和它的免疫原性的报道。
发明内容
本发明的第一目的在于提供一种降低抗体的ADCC/CDC功能的方法及其应用。
本发明的第二目的在于提供了一系列低ADCC/CDC、低多聚体、低免疫原性的治疗性抗体包括抗PD-L1、抗PD-1和抗CTLA-4等多种单克隆抗体及其应用。
为完成上述发明目的，本发明采取如下技术方案。
本发明提供一种降低或去除IgG1亚型抗体的ADCC/CDC功能的方法，该方法在原始IgG1抗体重链的恒定区插入一段柔性氨基酸序列。
在本发明的一些具体实施方案中，该方法在原始IgG1亚型抗体重链的CDR3与CH2区之间插入一段柔性氨基酸序列。
进一步的，所述插入一段柔性氨基酸序列，阻断了抗体可变区结合抗原后产生的机械应力传递，使抗体重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化抗体与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。
在本发明的一些具体实施方案中，所述IgG1抗体单抗为PD-L1、PD-1、CTLA4、TNF-α、PCSK9、NGF、C5、Aβ、IL-6、IL-17A、IL23A、HGF、CMET、Notch1、 CCL11、IL6R、IL-31R、IL-1B、IL-20、CD40、CD47、DKK1/2、TIGIT、4-1BB、IGF-1R、LL4、PDGFR2、HER3、IGF1/2、RANKL、sclerostin、GCGR、CGRP、ANGPTL3、IL-13、IL-4R、CSF-1R、TFPI、FCGRT、CD47抗体，例如Atezolizumab单抗、Pembrolizumab单抗、Ipilimumab单抗、Eculizumab单抗、evolocumab单抗、erenumab单抗、fulranumab单抗、crenezumab单抗、brodalumab单抗、ixekizumab单抗、satralizumab单抗、gevokizumab单抗、denosumab单抗、blosozumab单抗、crotedumab单抗、fasinumab单抗、fremanezumab单抗、evinacumab单抗、lebrikizumab单抗、dupilumab单抗、cabiralizumab单抗、concizumab单抗、rozanolixizumab单抗、magrolimab单抗等抗体药的IgG1亚型改进。
在本发明的一些具体实施方案中，所述柔性氨基酸序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS。
在本发明的一些具体实施方案中，所述柔性氨基酸序列插入位点包括但不限于GG(138/139)、SS(177/178)、SG(178/179)、SS(184/185)、SSS(191/192/193)、LL(235/236)、GG(237/238)。
本发明还提供一种柔性氨基酸序列在提高抗体稳定性/降低抗体免疫原性/延长抗体半衰期中的应用。
在本发明的一些具体实施方案中，所述柔性氨基酸序列插入原始IgG1抗体重链的恒定区中。
在本发明的一些具体实施方案中，所述柔性氨基酸序列插入原始IgG1抗体重链的CDR3与CH2之间。
本发明还提供一类低ADCC/CDC、低多聚体、低免疫原性的治疗性抗体包括抗PD-L1的单克隆抗体。
本发明的还提供一类制备低ADCC/CDC、低多聚体、低免疫原性治疗性抗体包括抗PD-L1的单克隆抗体的方法。
[根据细则91更正 20.11.2019]　
本发明基于抗体诱导ADCC/CDC的作用机理，通过在抗体重链的恒定区插入一段柔性氨基酸序列，切断抗体可变区结合抗原后产生的机械应力传递，使抗体药重链恒定区与Fc受体和/或结补体合位点不能充分暴露出来，弱化抗体药与NK细胞、巨噬细胞和中性粒细胞等表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC等的信号。
本发明还通过在基因泰克(Genentech)公司的Atezolizumab抗体药的298位氨基酸A突变回N恢复糖基化的基础上，在IgG1抗体重链CDR3与CH2区之间插入一段柔 性氨基酸序列来阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，从而达到在降低ADCC/CDC功能的同时不显著增加多聚体形成。
本发明的低免疫原性抗PD-L1单克隆抗体，是在原始Atezolizumab序列中非抗原结合区进行氨基酸改造，降低其免疫原性，所述原始Atezolizumab序列的重链全长和轻链可变区的氨基酸序列分别如SEQ ID NO.1、2所示。
进一步地，本发明提供的低免疫原性抗PD-L1单克隆抗体，其重链的氨基酸序列为SEQ ID NO.3或4所示，其轻链的氨基酸序列为SEQ ID NO.2。
本发明提供的抗人PD-L1单克隆抗体，其轻链如L0(SEQ ID NO.2)所示，重链全长具有H-1(SEQ ID NO.3)，H-2(SEQ ID NO.4)这2条碱基序列所示的任一个序列。
具备上述轻链可变区和重链可变区的组合中，本发明筛选得到2个单抗序列，它们既保持了原有与人PD-L1结合的亲和力，可特异性地阻断PD-1与PD-L1的结合，又降低了多聚体的含量，这可使它们的免疫原性得到有效的降低，同时在改造过程中ADCC活性验证结果显示本发明筛选获得的2个单抗序列的ADCC活性与原始Atezolizumab一致。
本发明得到的2个低免疫原性的抗PD-L1的单克隆抗体，分别为H-1L0、H-2L0。
本发明提供的上述单克隆抗体或含有该抗体的生物材料在治疗以PD-L1或PD-1为靶标的疾病中的应用。
发明提供的上述单克隆抗体或含有该抗体的生物材料在杀伤肿瘤细胞或治疗免疫能力退行疾病中的应用。
所述生物材料为表达盒、表达载体、工程菌或细胞。
本发明提供上述2个低免疫原性的抗PD-L1的单克隆抗体在制备治疗疾病药物中的应用。
所述的疾病为肿瘤，免疫功能退下等。
本发明提供了上述抗PD-L1单克隆抗体在制备以PD-L1为靶标的疾病治疗药物中的应用。
所述的药物为抗肿瘤，免疫功能退下疾病的药物。
本发明提供了含有上述抗PD-L1单克隆抗体的药物或检测试剂。
本发明提供了上述低ADCC/CDC功能性抗PD-L1单抗在治疗以PD-L1或PD-1为靶标的疾病中的应用。
本发明提供了上述低ADCC/CDC功能性抗PD-L1单抗在杀伤肿瘤细胞或治疗免 疫能力退行疾病中的应用。
本发明的抗PD-L1单抗可与第二治疗剂或治疗形式组合。抗PD-L1抗体可与包括施用CTLA-4抗体或CD47抗体的癌症治疗组合。组合的非限制性实例包括抗PD-L1抗体与抗CTLA-4的抗体联合用于治疗黑素瘤或非小细胞肺癌。
用本发明的抗PD-L1抗体的治疗可与化疗组合。使用细胞毒剂的化疗将导致癌细胞死亡，由此增加肿瘤抗原的释放。所述增加肿瘤抗原的有效性可导致与抗PD-L1治疗的协同作用。
用本发明的PD-L1单抗治疗可与手术组合以从受治疗者中去除癌细胞。
本发明的抗PD-L1抗体可与能同PD-L1阻断产生协同作用的疗法组合，所述疗法包括用于激素消除或抑制血管生成的靶向药物，或靶向在肿瘤细胞中具有活性的蛋白质的靶向药物，所有这些均导致肿瘤细胞死亡增加和免疫刺激性肿瘤抗原的有效性提高。
本发明的抗PD-L1单抗可与用于治疗癌症或感染性疾病的另一种治疗性抗体联合。具体实例可有：抗PD-L1抗体与靶向PD-1或靶向CTLA-4的抗体的组合。
本发明提供了制备上述抗PD-L1单克隆抗体的方法，包括以下步骤：
(1)本发明利用Pymol软件对基因泰克(Genentech)公司的Atezolizumab的结构进行分析，在抗体可变区与恒定区之间寻找相对柔性的区域，并在相应区域插入柔性氨基酸序列，对相应序列进行基因合成，测序，选择测序正确的序列。
(2)根据已知的抗体重链糖基化信息，本发明对相应修改后的序列进行全基因合成，对合成的基因进行测序同时选择测序正确的序列进行下一步操作，将轻链可变区设计酶切位点为Kpn I+BamH I，重链可变区设计酶切位点为KpnI+AgeI，分别与表达载体pJH16载体连接，同时转化大肠杆菌DH5α，得到重链、轻链嵌合抗体表达载体。同时对构建的抗体进行测序和序列对比；
(3)对上述表达的载体进行质粒大提工作，选取Qiagen的无内毒素质粒大提试剂盒；
(4)对选取的质粒进行优化组合，同时利用CHO细胞进行瞬时转染表达，对表达的抗体进行亲和力和EC50检测(详见图2)根据检测结果来选定哪些组合进行稳定转染；
(5)根据上述检测结果，本发明选取相应的组合利用电转染方式构建稳定株，同时利用MTX进行抗体表达程度的筛选，对于加压后的稳定株进行单克隆筛选，最终挑选出抗体产量较高的稳定株，用于后续实验所用；
(6)本发明生产的单克隆抗体在小鼠体内的免疫反应比Atezolizumab低。
本发明提供的抗PD-L1抗体和Atezolizumab针对的是PD-L1相同位点，但其免疫原性和抗体构象同Atezolizumab不同，同Atezolizumab相比其稳定性显著提高、免疫原性大大降低、动物体内药物半衰期延长，有望成为非常理想的生物靶向治疗抗体。本发明通过对Atezolizumab单抗恒定区序列进行改造，将显著降低该抗体药在病人体内产生免疫原性的风险。本发明实施例显示本发明的改进型Atezolizumab抗体与PD-L1结合的亲和力和原始Atezolizumab相近，并显著降低了ADA滴度，延长抗体药的半衰期，提高疗效。
本发明还提供针对人PD-1的IgG1亚型低ADCC/CDC功能性单克隆抗体及该单克隆抗体的应用。
本发明基于抗体诱导ADCC/CDC的作用机理，通过对原始Pembrolizumab单抗氨基酸序列进行改造，将抗体由IgG4亚型更换为IgG1亚型，并在IgG1抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，使单抗重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化单抗与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号，所述原始Pembrolizumab单抗的重链全长的氨基酸序列如SEQ ID NO.5所示，轻链全长的氨基酸序列如SEQ ID NO.6所示。
进一步地，本发明提供的IgG1亚型低ADCC/CDC功能性PD-1单抗，其重链全长含有SEQ ID NO.7所示的氨基酸序列或SEQ ID NO.7所示的氨基酸序列的变体，其轻链全长含有SEQ ID NO.6所示的氨基酸序列或SEQ ID NO.6所述氨基酸序列的变体。
在上述任何实施方案中，抗体重链恒定区或其变体为IgG1。
本发明抗体可包含：IgG1抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，达到在降低ADCC/CDC功能的同时降低抗体后续生产工艺开发的难度，提高抗体的抗聚合性、表达量及稳定性。
具体地，本发明提供的IgG1亚型低ADCC/CDC功能性PD-1单抗，其轻链全长氨基酸如L0(SEQ ID NO.6)所示，重链全长氨基酸序列具有H1(SEQ ID NO.7)所示的序列。
具备上述轻链可变区和重链可变区的组合中，本发明筛选得到H2L0单抗序列。它既保持了原有与人PD-1结合的亲和力，可特异性地阻断PD-1与PD-L1的结合， 又降低了ADCC和CDC功能活性，且由于为IgG1亚型单抗，相对于作为IgG4亚型的原始Pembrolizumab单抗，本发明单抗生产工艺成熟，难度低，抗体的抗聚合性、表达量和稳定性均得以提高。
本发明提供了含有上述任一个IgG1亚型低ADCC/CDC功能性PD-1单抗轻链和重链基因的表达载体。含有所述表达载体的宿主菌、宿主细胞或表达盒也在本发明的保护范围内。
本发明提供了提高免疫细胞活性的方法，所述方法包括将治疗有效量的本发明抗体给予需要治疗的对象。所述方法可用于治疗癌症、可用于治疗免疫能力低下疾病。本发明还包括进一步给予第二种治疗剂或治疗形式。
本发明还包括上述抗PD-1单抗在用于制备提高免疫功能的药物中的用途。
本发明的抗PD-1单抗可与第二治疗剂或治疗形式组合。抗PD-1抗体可与包括施用重组细胞因子或分泌免疫因子的癌症治疗组合。组合的非限制性实例包括抗PD-1抗体与重组IL-2或重组IFN2联合用于治疗黑素瘤或肾细胞癌。重组IL-2增加癌症患者中的T细胞产生。重组IFN2在被治疗患者中不但抑制癌细胞生长，同时还提高癌细胞、抗原呈递细胞和其他体细胞上的PD-1的抑制性配体的表达。本发明的抗PD-1单抗可与被认为可用于治疗癌症或感染性疾病的其他细胞因子组合。
本发明的抗PD-1单抗或抗体片段可与疫苗组合以预防或治疗癌症或感染性疾病。作为非限制性实例，抗PD-1单抗可与以下物质组合：含有与待治疗的癌症或感染有关的一种或多种抗原的蛋白质、肽或DNA疫苗或由用所述抗原刺激的树突细胞组成的疫苗。本发明提供了抗PD-1与(弱化的)癌细胞或全病毒疫苗的用途。本发明提供了抗PD-1疗法与经基因工程改造而分泌GM-CSF的全细胞癌症疫苗组合。
本发明的抗PD-1单抗可与被认为是癌症或感染性疾病中的护理标准的治疗组合。所述组合的基本原理是，由抗PD-1并行提高的免疫活化作用将诱导或促进对护理治疗标准的初始临床反应、诱导长期的临床反应和对疾病的长期免疫控制。
用本发明的抗PD-1抗体的治疗可与化疗组合。使用细胞毒剂的化疗将导致癌细胞死亡，由此增加肿瘤抗原的释放。所述增加肿瘤抗原的有效性可导致与抗PD-1治疗的协同作用。
用本发明的PD-1单抗治疗可与手术组合以从受治疗者中去除癌细胞。
本发明的抗PD-1抗体可与能同PD-1阻断产生协同作用的疗法组合，所述疗法包括用于激素消除或抑制血管生成的靶向药物，或靶向在肿瘤细胞中具有活性的蛋白质的靶向药物，所有这些均导致肿瘤细胞死亡增加和免疫刺激性肿瘤抗原的有效性提 高。在于抗PD-1抗体联合的情况下，T细胞活化作用的提高可导致对癌症的持久免疫控制。
本发明的抗PD-1单抗可与用于治疗癌症或感染性疾病的另一种治疗性抗体联合。具体实例可有：抗PD-1抗体与靶向Her2/neu或靶向EGF受体的抗体的组合；抗PD-1抗体与抗CTLA-4组合；抗PD-1抗体或抗体片段与靶向OX40的抗体组合。
本发明提供的上述单克隆抗体或含有该单抗的生物材料在治疗以PD-L1或PD-1为靶标的疾病中的应用。
发明提供的上述单克隆抗体或含有该单抗的生物材料在杀伤肿瘤细胞或治疗免疫能力退行疾病中的应用。
所述生物材料为表达盒、表达载体、工程菌或细胞。
本发明提供上述IgG1亚型低ADCC/CDC功能性PD-1单抗在制备治疗疾病药物或提高免疫功能药物中的应用。
所述的疾病为肿瘤，免疫功能退下等。
本发明提供了上述IgG1亚型低ADCC/CDC功能性PD-1单抗在制备以PD-L1为靶标的疾病治疗药物中的应用。
所述的药物为抗肿瘤，免疫功能退下疾病的药物。
本发明提供了含有上述IgG1亚型低ADCC/CDC功能性PD-1单抗的药物或检测试剂。
本发明提供了上述IgG1亚型低ADCC/CDC功能性PD-1单抗在治疗以PD-L1或PD-1为靶标的疾病中的应用。
本发明提供了上述IgG1亚型低ADCC/CDC功能性PD-1单抗在杀伤肿瘤细胞或治疗免疫能力退行疾病中的应用。
本发明利用Pymol软件对美国默克(Merck)公司的Pembrolizumab的结构进行分析，将抗体由IgG4亚型更换为IgG1亚型，并在IgG1抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，达到在降低ADCC/CDC功能的同时降低抗体后续生产工艺难度，提高抗体的抗聚合性、表达量和稳定性，避免IgG4亚型的重链置换特性导致的半抗体和双特异性抗体的形成。本发明的单抗与人PD-1结合的亲和力和原始Pembrolizumab抗体相近，可特异性地阻断PD-1与细胞表面PD-L1的结合，延长抗体药的半衰期，提高疗效，具有优异的临床应用价值。
本发明还提供针对人CTLA4的IgG1亚型低ADCC/CDC功能性单克隆抗体及其该单克隆抗体的应用。
本发明基于抗体诱导ADCC/CDC的作用机理，通过对原始Ipilimumab单抗氨基酸序列进行改造，在IgG1亚型抗体Ipilimumab重链CDR3与CH2区之间插入一段柔性氨基酸序列，使单抗重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化单抗与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号，所述原始Ipilimumab单抗的重链全长的氨基酸序列如SEQ ID NO.8所示，轻链全长的氨基酸序列如SEQ ID NO.9所示。
本发明利用Pymol软件对美国百时美施贵宝(BMS)公司的ipilimumab的结构进行分析，在IgG1抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，达到在降低ADCC/CDC功能的同时降低抗体在治疗中的副作用，提高抗体的抗聚合性、表达量和稳定性。本发明的单抗与人CTLA4结合的亲和力和原始ipilimumab抗体相近，可特异性地阻断CTLA4与CD80及CD86的结合，降低抗体药治疗副作用，提高抗体药疗效，具有优异的临床应用价值。
进一步地，本发明提供的IgG1亚型低ADCC/CDC功能性CTLA4单抗，其重链全长含有SEQ ID NO.10所示的氨基酸序列，其轻链全长含有SEQ ID NO.9所示的氨基酸序列。
在上述任何实施方案中，抗体重链恒定区为IgG1亚型。
本发明抗体可包含：IgG1抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，达到在降低ADCC/CDC功能的同时降低抗体后续生产工艺开发的难度，提高抗体的抗聚合性、表达量及稳定性。
具体地，本发明提供的IgG1亚型低ADCC/CDC功能性CTLA4单抗，其轻链全长氨基酸如L0(SEQ ID NO.9)所示，重链全长氨基酸序列如H1(SEQ ID NO.10)序列所示。
本发明筛选H1L0单抗序列。它既保持了原有与人CTLA4结合的亲和力，可特异性地阻断CTLA4与其配体的结合，又降低了ADCC和CDC功能活性。
本发明提供了含有上述任一个IgG1亚型低ADCC/CDC功能性CTLA4单抗轻链和重链基因的表达载体。含有所述表达载体的宿主菌、宿主细胞或表达盒也在本发明的保护范围内。
本发明提供了提高免疫细胞活性的方法，所述方法包括将治疗有效量的本发明抗体给予需要治疗的对象。所述方法可用于治疗癌症、可用于治疗免疫能力低下疾病。本发明还包括进一步给予第二种治疗剂或治疗形式。
本发明还包括上述抗CTLA4单抗在用于制备提高免疫功能的药物中的用途。
本发明的抗CTLA4单抗可与第二治疗剂或治疗形式组合。抗CTLA4抗体可与包括施用PD-1抗体或PD-L1抗体的癌症治疗组合。组合的非限制性实例包括抗CTLA4抗体与抗PD-1的抗体联合用于治疗黑素瘤或非小细胞肺癌。
用本发明的抗CTLA4抗体的治疗可与化疗组合。使用细胞毒剂的化疗将导致癌细胞死亡，由此增加肿瘤抗原的释放。所述增加肿瘤抗原的有效性可导致与抗CTLA4治疗的协同作用。
用本发明的CTLA4单抗治疗可与手术组合以从受治疗者中去除癌细胞。
本发明的抗CTLA4抗体可与能同CTLA4阻断产生协同作用的疗法组合，所述疗法包括用于激素消除或抑制血管生成的靶向药物，或靶向在肿瘤细胞中具有活性的蛋白质的靶向药物，所有这些均导致肿瘤细胞死亡增加和免疫刺激性肿瘤抗原的有效性提高。在于抗CTLA4抗体联合的情况下，T细胞活化作用的提高可导致对癌症的持久免疫控制。
本发明的抗CTLA4单抗可与用于治疗癌症或感染性疾病的另一种治疗性抗体联合。具体实例可有：抗CTLA4抗体与靶向PD-1或靶向PD-L1的抗体的组合。
本发明提供的上述单克隆抗体或含有该单抗的生物材料在治疗以CTLA4为靶标的疾病中的应用。
发明提供的上述单克隆抗体或含有该单抗的生物材料在杀伤肿瘤细胞或治疗免疫能力退行疾病中的应用。
所述生物材料为表达盒、表达载体、工程菌或细胞。
本发明提供上述IgG1亚型低ADCC/CDC功能性CTLA4单抗在制备治疗疾病药物或提高免疫功能药物中的应用。
所述的疾病为肿瘤，免疫功能退行等。
本发明提供了上述IgG1亚型低ADCC/CDC功能性CTLA4单抗在制备以CTLA4为靶标的疾病治疗药物中的应用。
所述的药物为抗肿瘤，免疫功能退行疾病的药物。
本发明提供了上述IgG1亚型低ADCC/CDC功能性CTLA4单抗在治疗以PD-L1或PD-1为靶标的疾病中的联合应用。
本发明提供了上述IgG1亚型低ADCC/CDC功能性CTLA4单抗在杀伤肿瘤细胞或治疗免疫能力退行疾病中的应用。
综上，本发明的降低抗体的ADCC/CDC功能的方法，可降低ADCC/CDC功能， 同时不显著增加多聚体形成。改造后单抗和原始单抗针对相同位点，均去除了ADCC功能，但抗体构象不同，稳定性显著提高，免疫原性显著降低，半衰期延长。有效避免因ADCC/CDC功能造成的副反应，提高疗效，具有优异的临床应用价值。
附图说明
图1本发明改造后的PD-L1单抗(H-1L0)与原始Atezolizumab抗体重链的序列比对结果。
图2本发明改造后的PD-L1单抗和原始Atezolizumab抗体亲和力评价结果。横坐标为抗体的浓度(M)，纵坐标为450nm下的吸光度。
图3本发明改造后的两个PD-L1单抗(H-1L0、H-2L0)和原始Atezolizumab抗体ADCC活性实验，纵坐标为Raji-PDL1细胞裂解百分率，横坐标为样品浓度，IMM25为ADCC阳性抗体作为阳性对照，Atezolizumab原研药作为ADCC实验阴性对照。
图4本发明改造后的PD-L1单抗抑制PD-L1与PD-1活性细胞实验结果。横坐标为抗体的浓度(M)，纵坐标为荧光素酶活性单位均值。
图5本发明改造后的PD-L1单抗和原始Atezolizumab抗体ADA评价对比。横坐标为血清稀释比例，纵坐标为450nm下的吸光度。
图6A和图6B分别为原始Atezolizumab抗体和本发明改造后PD-L1单抗的HPLC-SEC结果图。
图7A和图7B为本发明改造后PD-L1单抗的糖型分析。其中图7A为原研Atezolizumab的糖型分析质谱结果，图7B为改造后PD-L1单抗的糖型分析质谱结果。
图8本发明改造后PD-L1单抗和原始Atezolizumab抗体热稳定性实验结果。图中横坐标为抗体的浓度(M)，纵坐标为450nm下的吸光度。
图9本发明改造后的PD-1单抗与原始Pembrolizumab抗体重链的序列比对结果。
图10本发明改造后的PD-1单抗与原始Pembrolizumab抗体亲和力评价结果。横坐标为抗体的浓度(M)，纵坐标为450nm下的吸光度。
图11本发明改造后的PD-1单抗与原始Pembrolizumab抗体ADCC活性实验，纵坐标为Raji-PD-1细胞裂解百分率，横坐标为样品浓度，Pembrolizumab原研药作为ADCC实验对照，替换为IgG1亚型的Pembrolizumab单抗作为ADCC实验阳性对照，H1L0为本发明改造后的PD-1单抗。
图12本发明改造后的PD-1单抗与原始Pembrolizumab抗体抑制PD-L1与PD-1活性细胞实验结果。横坐标为抗体的浓度(M)，纵坐标为荧光素酶活性单位均值。
图13本发明改造后的CTLA4单抗与原始Ipilimumab抗体重链的序列比对结果。
图14本发明改造后的CTLA4单抗与原始Ipilimumab抗体亲和力评价结果。横坐标为抗体的浓度(M)，纵坐标为450nm下的吸光度。
图15本发明改造后的CTLA4单抗与原始Ipilimumab抗体ADCC活性实验，纵坐标为Raji-CTLA4细胞裂解百分率，横坐标为样品浓度，Ipilimumab原研药作为ADCC实验阳性对照，H1L0为本发明改造后的CTLA4单抗。
图16本发明改造后的CTLA4单抗与原始Ipilimumab抗体抑制CTLA4与CD80及CD86结合活性实验。横坐标为抗体的浓度(M)，纵坐标为450nm下的吸光度。
图17本发明改造后的CTLA4单抗和原始Ipilimumab抗体与CD80和CD86竞争结合人CTLA4转染的CHO细胞结果。横坐标为抗体的浓度(M)，纵坐标为450nm下的吸光度。
具体实施方式
缩写和定义
“抗体”，是指表现所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号传递)的抗体的任何形式。因此，所述抗体以最广泛的意义使用，并明确包括但不限于单克隆抗体、多克隆抗体和多特异性抗体。
“Fab片段”由一条轻链和一条重链的CH1及可变区构成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区域为含有抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。
本申请所用术语“单克隆抗体”是指从基本上同种抗体群中获得的抗体，即除了可能少量存在的可能的天然突变体外，构成所述群的各个抗体是一致的。单克隆抗体具有高度特异性，可针对单个的抗原位点。此外，与通常包括针对多个不同的决定簇(表位)的多种不同抗体的常规(多克隆)抗体制备物相反，每种单克隆抗体仅针对抗原上的单个决定簇。修饰语“单克隆”表示从基本上同种抗体群获得的抗体的特性，不能理解为需要通过任何特定方法来制备所述抗体。
本文所用术语“柔性氨基酸序列”是指由侧链较小的氨基酸组成的一段连接多肽，用于防止或降低其两端的结构域之间在空间构象上的相互干扰，其序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS 和GGGS。
本文所用术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞。免疫细胞包括：淋巴细胞，例如淋巴B细胞和淋巴T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
当提及配体/受体、抗体/抗原或其他结合对时，“特异性”结合是指在蛋白和/或其他生物试剂的异质群体中确定是否存在所述蛋白例如PD-1的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著量与样品中存在的其他蛋白结合。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
“有效量”包括足以改善或防止医学疾病的症状或病症的量。有效量还意指足以使得可以诊断或促进诊断的量。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。单克隆抗体制备，可将本发明所涉及到抗体DNA置于表达载体中，然后将载体转染到宿主细胞中以在重组宿主细胞中实现单克隆抗体的合成，所述宿主细胞例如有大肠杆菌细胞、中国仓鼠卵(CHO)细胞或不会另外产生免疫球蛋白的骨髓瘤细胞。抗体的重组制备将在下文更详细地阐述。
合适的给药途径包括胃肠外给药(例如肌肉注射、静脉内或皮下给药)及口服给药。可按多种常规方式给予用于药物组合物或用于实践本发明方法的抗体，这些方 法例如有经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。人们可以以局部而非全身方式(通常为长效制剂或缓释制剂)给予抗体，例如经由将抗体直接注射到作用位点。此外，人们可在靶向药物递送系统中给予抗体。
本文所用“抑制”或“治疗(treat或treatment)”包括延缓与疾病有关的症状的发展和/或减轻所述疾病将要或预期发展的这些症状的严重程度。所述术语还包括减缓已有症状、防止另外的症状和减缓或防止这些症状的潜在原因。因此，所述术语表示业已将有益结果赋予患有疾病的脊椎动物对象。
本文所用术语“治疗有效量”或“有效量”是指当将抗体或其片段单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时，其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述化合物足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。当施用给个体单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其实联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；优选至少约30％；更优选至少40％和最优选至少50％。
本发明药物组合物还可含有其他药剂，包括但不限于细胞毒剂、细胞生长抑制剂、抗血管形成药物或抗代谢药物、靶向肿瘤药物、免疫刺激剂或免疫调节剂或与细胞毒剂、细胞生产抑制剂或其它毒性药物缀合的抗体。也可与其它治疗形式(例如手术、化疗及放射疗法)一起施用所述药物组合物。
典型的兽医、实验或研究对象包括猴、狗、猫、大鼠、小鼠、豚鼠、兔、马和人。
本发明抗体的治疗应用如下：
与人PD-1/L1及CTLA4特异性结合的本发明抗体可用于增加、提高、刺激或上调免疫应答。本发明抗体尤其适用于治疗罹患可通过提高T细胞介导的免疫应答来治疗的疾病的受治疗者。优选的受治疗者包括需要提高免疫应答的人患者。
癌症，本发明抗体可用于治疗癌症(即抑制肿瘤细胞的生长或存活)。可用本发明抗体抑制其生长的优选的癌症包括通常对免疫疗法有反应的癌症，但还包括迄今与免疫疗法尚无关联的癌症。用于治疗的优选癌症的非限制性实例包括黑素瘤(例如恶性转移性黑素瘤)、肾癌(例如透明细胞癌)、前列腺癌(例如激素难控制的前列腺腺癌)、胰腺腺癌、乳腺癌、结肠癌、肺癌(例如非小细胞肺癌)、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、胶质母细胞瘤、神经胶质瘤、白血病、淋 巴瘤和其它恶性肿瘤。另外，本发明包括可用本发明抗体抑制其生长的难治性或复发性癌。
本发明抗体可单独使用或与以下其它物质联合使用：抗肿瘤药或免疫原剂(例如减弱的癌细胞、肿瘤抗原(包括重组蛋白质、肽和糖类分子)、抗原呈递细胞例如用来源于肿瘤的抗原或核酸刺激的树突细胞、免疫刺激细胞因子(例如IL-2、IFNa2、GM-CSF)和用编码免疫刺激细胞因子(例如但不限于GM-CSF)的基因转染的细胞)；标准癌症治疗(例如化疗、放疗或手术)；或其它抗体(包括但不限于针对以下物质的抗体：VEGF、EGFR、Her2/neu、VEGF受体、其它生长因子受体、CD20、CD40、CTLA-4、OX-40、4-IBB和ICOS)。
免疫低下性疾病，本发明抗体还可用于防止或治疗免疫低下疾病。抗体可单独使用，或与药物联合使用，以刺激针对病原体、多聚体的免疫应答。抗体可用于刺激对感染人的病毒的免疫应答，这些病毒例如但不限于人免疫缺陷病毒、甲、乙、丙型肝炎病毒、爱泼斯坦巴尔病毒、人巨细胞病毒、人乳头瘤病毒、疱疹病毒。抗体可用于刺激对细菌或真菌寄生虫及其它病原体引起的感染的免疫应答。抗体可用于刺激对体内多聚体的免疫应答和去除，治疗老年痴呆等疾病。
本发明所涉及到的抗体针对靶点包括但不限于PD-L1、PD-1、CTLA4、TNF-α、PCSK9、NGF、C5、Aβ、IL-6、IL-17A、IL23A、HGF、CMET、Notch1、CCL11、IL6R、IL-31R、IL-1B、IL-20、CD40、CD47、DKK1/2、TIGIT、4-1BB、IGF-1R、LL4、PDGFR2、HER3、IGF1/2、RANKL、sclerostin、GCGR、CGRP、ANGPTL3、IL-13、IL-4R、CSF-1R、TFPI、FCGRT、CD47等，可用于治疗癌症、哮喘、自身免疫疾病、偏头痛、老年痴呆、高血脂症、骨关节炎、丛集性头痛、骨质疏松等疾病。
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1低ADCC/CDC功能的降低免疫原性的PD-L1单抗的分析及设计
本发明利用Pymol软件对基因泰克(Genentech)公司的Atezolizumab的结构进行分析，在抗体可变区与恒定区之间寻找相对柔性的区域，并在相应区域插入柔性氨基酸序列，切断抗体可变区结合抗原后产生的机械应力传递，使抗体药重链恒定区与Fc受体和/或结补体合位点不能充分暴露出来，弱化抗体药与NK细胞、巨噬细胞和中性 粒细胞等表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。
具体是在基因泰克(Genentech)公司的Atezolizumab抗体药的297位氨基酸A突变回N恢复糖基化的基础上，在IgG1抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列来阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，从而达到在降低ADCC/CDC功能的同时不显著增加多聚体形成。原始Atezolizumab序列的重链全长和轻链可变区的氨基酸序列分别如SEQ ID NO.1、2所示。
本发明提供的低免疫原性抗PD-L1单克隆抗体，其重链的氨基酸序列为SEQ ID NO.3、4所示，其轻链的氨基酸序列为SEQ ID NO.2，分别命名为H-1L0、H-2L0。
根据已知的抗体重链糖基化信息，本发明对相应修改后的序列进行全基因合成，对合成的基因进行测序同时选择测序正确的序列进行下一步操作，将轻链设计酶切位点为EcoR I+Hind III，重链设计酶切位点为EcoR I+Hind III，分别与表达载体pEE6.4及pEE12.4载体连接，同时转化大肠杆菌DH5α，得到抗体重链、轻链表达载体。同时对构建的抗体进行测序和序列对比，改造后的PD-L1单抗与原始Atezolizumab抗体重链的序列比对结果见图1。
实施例2低ADCC/CDC功能的PD-1单抗的分析及设计
本发明对美国默克(Merck)公司Pembrolizumab进行改造，首先将其重链亚型由IgG4变更为IgG1，利用Pymol软件进行结构分析，在抗体可变区与恒定区之间寻找相对柔性的区域，并在相应区域插入柔性氨基酸序列，对相应序列进行基因合成，测序，选择测序正确的序列，筛选得到单抗H1L0。本发明尝试在原始IgG1亚型抗体重链的CDR3与CH2区之间插入一段柔性氨基酸序列，所插入的柔性氨基酸序列，阻断了抗体可变区结合抗原后产生的机械应力传递，使抗体重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化抗体与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。柔性氨基酸序列由侧链较小的氨基酸组成的一段连接多肽，用于防止或降低其两端的结构域之间在空间构象上的相互干扰，其序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS。按照本发明所述方法进行改造后重链的氨基酸序列如SEQ ID NO.7所示。
本发明对相应修改后的序列进行全基因合成，对合成的基因进行测序同时选择测序正确的序列进行下一步操作，将轻链设计酶切位点为EcoR I+Hind III，重链设计酶切位点为EcoR I+Hind III，分别与表达载体pEE6.4及pEE12.4载体连接，同时转 化大肠杆菌DH5α，得到抗体重链、轻链表达载体。同时对构建的抗体进行测序和序列比对，改造后的PD-1单抗与原始Pembrolizumab抗体重链的序列比对结果见图9。
实施例3低ADCC/CDC功能的CTLA4单抗的分析及设计
本发明对美国百时美施贵宝(BMS)公司ipilimumab进行改造，利用Pymol软件进行结构分析，在抗体可变区与恒定区之间寻找相对柔性的区域，并在相应区域插入柔性氨基酸序列，对相应序列进行基因合成，测序，选择测序正确的序列，筛选得到单抗H1L0。本实施例尝试在ipilimumab单抗IgG1亚型抗体重链的CDR3与CH2区之间插入一段柔性氨基酸序列，所插入的柔性氨基酸序列，阻断了抗体可变区结合抗原后产生的机械应力传递，使抗体重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化抗体与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。柔性氨基酸序列由侧链较小的氨基酸组成的一段连接多肽，用于防止或降低其两端的结构域之间在空间构象上的相互干扰，其序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS。改造后重链的氨基酸序列如SEQ ID NO.10所示。
本发明对相应修改后的序列进行全基因合成，对合成的基因进行测序同时选择测序正确的序列进行下一步操作，将轻链设计酶切位点为EcoR I+Hind III，重链设计酶切位点为EcoR I+Hind III，分别与表达载体pEE6.4及pEE12.4载体连接，同时转化大肠杆菌DH5α，得到抗体重链、轻链表达载体。同时对构建的抗体进行测序和序列比对，改造后的CTLA4单抗与原始Ipilimumab抗体重链的序列比对结果见图13。
实施例4改良型抗体的ADCC功能验证
为了评估本发明中抗体的ADCC功能，本发明通过检测FcR-TANK细胞对靶蛋白(PD-L1、PD-1、CTLA4)过表达细胞的杀伤能力来评价抗体ADCC活性。
以PD-L1抗体的ADCC活性检测为例：使用靶蛋白过表达细胞用CFSE染色后，将细胞密度调整至6×10 5/mL，将改良型抗体(H-1L0，H-2L0)与Atezolizumab以及阳性对照抗体IMM25(购自宜明昂科公司)以一定比例梯度(最高浓度为4μg/mL，用培养基三倍稀释12个梯度)进行稀释，同时对FcR-TANK细胞进行计数并将细胞密度调整至6×10 5/mL；将抗体、靶细胞和效应细胞共孵育：取上述不同稀释梯度的抗体各50μL，靶细胞50μL，FcR-TANK细胞100μL加入到96孔板中，每个梯度进行复孔操作，同时设置空白对照(培养基150μL+靶细胞50μL及培养基50μL+靶细胞50μL+FcR-TANK细胞100μL)。于37℃，5％CO 2条件下孵育4h。孵育结束后，将细 胞培养板于室温放置10min，加入PI染色液(终浓度为5μg/mL)并混合均匀。流式细胞术分析不同浓度下细胞PI染色阳性率，进而计算抗体介导的ADCC强度，计算公式为ADCC％＝【(Sample％PI Positive Cell-No Antibody％PI Positive Cell)/(100-No Antibody％PI Positive Cell)】×100，并绘制ADCC％与浓度的关系曲线。
本发明改造后的PD-L1单抗与原研药Atezolizumab抗体ADCC活性实验显示，本发明的两个PD-L1单抗H-1L0、H-2L0与原研药Atezolizumab抗体的ADCC活性一致，见图3。由于H-1L0及H-2L0均证明去除了ADCC效应，因此后续研究主要使用H-1L0同原研药Atezolizumab进行对比验证。
本发明改造后的PD-1单抗与原研药Pembrolizumab抗体ADCC活性实验显示，本发明的改造后PD-1单抗H1L0的ADCC活性消失，而原研药Pembrolizumab抗体存在较弱的ADCC活性，见图11。
本发明改造后的CTLA4单抗与原研药Ipilimumab抗体ADCC活性实验显示，本发明的CTLA4单抗H1L0的ADCC活性消失，而原研药Ipilimumab抗体的ADCC活性正常，见图15。
实施例5改良型抗体的亲和力评价
对实施例1-3构建的载体进行质粒大提工作，选取Qiagen的无内毒素质粒大提试剂盒；对选取的质粒进行优化组合，同时利用CHO细胞进行瞬时转染表达，基于蛋白质的ELISA实验用于测定表观结合亲和力(报告为EC 50值)。比较改良型抗体与原研抗体的结合能力。
蛋白质ELISA用于测定抗体与人PD-L1的相对结合。通过4℃过夜将PD-L1固定化在酶标版上，通过与含3％BSA的PBS室温孵育1小时来阻断非特异性结合位点。包被后，用PBS洗涤所述板，并制备抗PD-L1抗体的稀释液，将其与固定化的蛋白在25℃下孵育1小时。结合后用PBS洗涤所述板3次，在25℃下用含稀释至1/2000的过氧化物酶标记的山羊抗人IgG的结合缓冲液孵育1小时，再次洗涤后使用TMB显色。ELISA结果示于图2中。用半最大结合浓度表示相对结合亲和力的量值。对表达的抗体进行亲和力和EC50检测(见表1)，亲和力ELISA结果显示H-1L0与PD-L1的亲和力同原研药Atezolizumab抗体相近，见图2。
表1抗体亲和力评价结果
mAb EC 50(pM)
Atezolizumab 320.3
H-1L0 219.4
蛋白质ELISA用于测定抗体与人PD-1的相对结合。通过4℃过夜将PD-1固定化在酶标版上，通过与含3％BSA的PBS室温孵育1小时来阻断非特异性结合位点。包被后，用PBS洗涤所述板，并制备抗PD-1抗体的稀释液，将其与固定化的蛋白在25℃下孵育1小时。结合后用PBS洗涤所述板3次，在25℃下用含稀释至1/2000的过氧化物酶标记的山羊抗人IgG的结合缓冲液孵育1小时，再次洗涤后使用TMB显色。ELISA结果示于图10中。用半最大结合浓度表示相对结合亲和力的量值。对表达的抗体进行亲和力和EC50检测(见表2)，亲和力ELISA结果显示H1L0与PD-1的亲和力同原研药Pembrolizumab抗体相近，见图10。
表2抗体亲和力评价结果
mAb EC 50(pM)
Pembrolizumab 54.5
H1L0 43.7
蛋白质ELISA用于测定抗体与人CTLA4的相对结合。通过4℃过夜将PD-1固定化在酶标版上，通过与含3％BSA的PBS室温孵育1小时来阻断非特异性结合位点。包被后，用PBS洗涤所述板，并制备抗CTLA4抗体的稀释液，将其与固定化的蛋白在25℃下孵育1小时。结合后用PBS洗涤所述板3次，在25℃下用含稀释至1/2000的过氧化物酶标记的山羊抗人IgG的结合缓冲液孵育1小时，再次洗涤后使用TMB显色。ELISA结果示于图14中。用半最大结合浓度表示相对结合亲和力的量值。对表达的抗体进行亲和力和EC50检测(见表3)，亲和力ELISA结果显示H1L0与CTLA4的亲和力同原研药Ipilimumab抗体相近，见图14。
表3抗体亲和力评价结果
mAb EC 50(pM)
Ipilimumab 155.0
H1L0 134.9
实施例6改良型PD-1、PD-L1抗体的生物活性检测
本方法基于两种细胞系，CHO-PD-L1-CD3L细胞系及Jurkat-PD-1-NFAT细胞系。CHO-PD-L1-CD3L细胞系稳定表达PD-L1和anti-CD3-scFv，作为靶细胞；Jurkat-PD-1-NFAT细胞系稳定表达PD-1和荧光素酶(luciferase)，荧光素酶基因受NFAT元件(转录因子)调控(IL-2启动子)，作为效应细胞。PD-1与PD-L1结合可以阻断CD3下游信号的转导，从而抑制荧光素酶的表达，当加入PD-1抗体或者PD-L1抗体时，这种Block效应被反转，荧光素酶可以表达，从而检测到荧光信号。 具体实验步骤如下：
1、细胞培养：Jurkat-PD-1-NFAT细胞使用含有10％FBS，1％NEAA以及抗生素的RPMI1640培养基；CHO-PD-L1-CD3L细胞使用含有10％FBS，1％NEAA以及抗生素的DMEM或F12培养基。
2、细胞接种：CHO-PD-L1-CD3L细胞以50000个/孔接种于96孔板中，在37℃，5％CO2条件下培养12-14小时。之后去除培养基后，将含有100000个Jurkat-PD-1-NFAT细胞的50μL分析培养液(含有2％FBS的RPMI1640培养基)加入各孔。
3、抗体条件筛选：将本发明改造抗体或原研抗体药从10μg/mL浓度以1：3的比例梯度稀释(使用分析培养液)。之后将稀释后的抗体加入各孔，并于37℃，5％CO 2孵育6小时。
4、加入100μL荧光底物(Promega Bio-GloTM Luciferase Assay)并用荧光光度计(SpectraMax M5)计算相对荧光素酶活性单位。
结果显示本发明改造PD-L1抗体H-1L0与原研药Atezolizumab抗体生物活性对比显示H-1L0与原研Atezolizumab抑制PD-1/PD-L1效果相近(如图4)，绘制表格并计算抗体抑制PD-1、PD-L1的IC50数值如表4所示。
表4抗体抑制力评价结果
mAb IC 50(nM)
Atezolizumab 1.988
H-1L0 1.945
结果显示本发明改造PD-1抗体H1L0与原研药Pembrolizumab抗体生物活性对比显示H1L0和原研Pembrolizumab抑制PD-1/PD-L1效果相近(如图12)，绘制表格并计算抗体抑制PD-1、PD-L1的IC50数值如表5所示。
表5抗体抑制力评价结果
mAb IC 50(nM)
Pembrolizumab 1.986
H1L0 1.943
实施例7改良型CTLA4抗体抑制CTLA4与CD80或CD86结合活性验证及竞争性实验
使用ELISA方法检测本发明单抗抑制CTLA4与CD80或CD86结合效率。通过4℃过夜将hCD80或hCD86固定化在酶标版上，通过与含3％BSA的PBS室温孵育1 小时来阻断非特异性结合位点。包被后，用PBS洗涤所述板，并制备抗CTLA4抗体与Biotin-CTLA4混合稀释液(Biotin-CTLA4为1μg/mL的固定浓度，本发明抗CTLA4抗体梯度稀释，最高浓度为1μg/mL，最低浓度为0)，将其与固定化的蛋白在25℃下孵育1小时。结合后用PBS洗涤所述板3次，在25℃下用含稀释至1/8000的过氧化物酶标记的亲和素的结合缓冲液孵育1小时，再次洗涤后使用TMB显色。ELISA结果示于图16中，显示本发明的CTLA4单抗能够抑制CTLA4与CD80或CD86的结合。
本发明使用CELISA方法来评估抗体与CD80及CD86竞争结合人CTLA4转染的CHO细胞的效果。首先构建表达hCTLA4的CHO细胞，在50μL培养及中将CHO-hCTLA4细胞培养到80％-100％覆盖度。之后加入50μL含有固定浓度(1μg/mL)Biotin-CD80或Biotin-CD86及不同稀释浓度(最高浓度为1μg/mL，最低浓度为0，梯度稀释)的抗CTLA4抗体的混合培养基，在37℃孵育1小时。之后用PBS洗涤3次后，加入100μL的1/8000稀释比例的过氧化物酶标记的亲和素，于37℃孵育1小时。再用PBS洗涤数次后使用TMB显色。ELISA结果示于图17中。
实施例8改良型PD-L1抗体及原研Atezolizumab抗体小鼠体内免疫原性评价
将改良型抗体H-1L0及原研Atezolizumab分别免疫小鼠评价小鼠产生ADA的滴度。使用Balb/c小鼠进行分组实验，每组5只，具体免疫方法如下：1)基础免疫：将抗原与福氏完全佐剂等体积混合并充分乳化，分点皮下注射。2)加强免疫：加强免疫采用抗原与福氏不完全佐剂的乳化液。每只Balb/c小鼠总注射量为70μg。
对免疫后小鼠分别取免疫前、免疫后7天、免疫后14天、免疫后21天血清，评价小鼠ADA的滴度，结果见图5。结果显示本发明生产的单克隆抗体H-1L0在小鼠体内的ADA滴度比Atezolizumab降低了80％。
实施例9改良型PD-L1抗体的稳定细胞株构建及小试表达
对本发明改造的PD-L1抗体及原研Atezolizumab抗体同时进行CHO稳定细胞库的构建并按照本领域公知的抗体药物生产规范对改良PD-L1抗体及原研Atezolizumab进行了5g的小试表达及纯化。并对获得的抗体进行了HPLC-SEC的对比，见图6A和图6B。原研Atezolizumab及改良型PD-L1抗体H-1L0的上样量(50μL)及浓度(60mg/mL)均相同。从图中可见，在上样量相同的情况下，原研Atezolizumab抗体的主峰面积较改良型PD-L1抗体H-1L0减少了近10％，因此，可以判断原研Atezolizumab中多聚体含量较多，因此在进行HPLC-SEC上样时，多聚体被滤膜除去而导致峰面积下降。两个抗体的HPLC-SEC的峰面积计算结果见表6。同时， HPLC-SEC结果显示改良型PD-L1抗体H-1L0出峰位置较原研Atezolizumab靠前，可以判断是由于恢复了抗体的糖基化导致抗体的分子量增大，进而出峰位置更为靠前。
表6抗体HPLC-SEC结果
Figure PCTCN2019094928-appb-000001
实施例10改良型PD-L1抗体的糖型分析
基于实施例9得到的结果，进一步将改良型PD-L1抗体进行了糖型的验证。主要采用该领域公知的方法进行：将抗体上的寡糖进行酶切及收集后，利用质谱的方式进行糖型的分析及鉴定。经鉴定原研Atezolizumab未检测到N糖修饰，而改良型PD-L1抗体是正常糖基化修饰，见图7。改良型PD-L1抗体的糖型分析结果如表7所示。
表7改良型PD-L1抗体糖型分析结果
Figure PCTCN2019094928-appb-000002
实施例11改良型PD-L1抗体及原研Atezolizumab抗体的稳定性验证
对本发明改造的PD-L1单抗及原研Atezolizumab抗体进行稳定性的对比验证。将改良性PD-L1抗体及原研Atezolizumab抗体同时进行快速热处理，并验证其稳定性主要操作流程如下：
将两个不同抗体分别取等量体积，并在4℃静置10小时后取小样；第一次取样后，将两个抗体放入60℃恒温水浴10分钟，观察外观变化并第二次取小样；取样后将两个抗体于4℃，10000rpm离心5分钟，观察离心管管底是否出现肉眼可见的沉淀，并再次对上清取样；对第三次取样后的上清使用0.2μm的滤膜进行过滤处理后，再次取样；取样完成后，将过滤后的上清放置于4℃下，持续观察；对所有获得的样本进行抗体浓度及抗体结合PD-L1能力的检测。
改良型PD-L1抗体及原研Atezolizumab的结合PD-L1能力的ELISA检测结果见图8，两个抗体各样本浓度数据见表8。可见改良型PD-L1抗体较原研Atezolizumab稳定性更好。
表8改良型PD-L1抗体糖型分析结果
Figure PCTCN2019094928-appb-000003
可见，本发明筛选得到针对多个不同靶点的改良型去ADCC功能单抗，它们既保持了原有与靶蛋白结合的亲和力，同时去除了其ADCC功能，可特异性地阻断靶蛋白与其相应配体的结合。同时对于改良型PD-L1抗体，通过利用本发明的去ADCC功能方法，不仅达到了去除ADCC效应的目的，又解决了原研抗体药由于改造导致的多聚体含量较高、免疫原性强的问题。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
工业实用性
本发明提供了一种低ADCC/CDC功能性单抗及其制备方法与应用。本发明对PD-L1、PD-1、CTLA4、TNF-α、PCSK9、NGF、CD47和C5等多靶点的抗体序列进行改造，在IgG1亚型抗体重链CDR3与CH2区之间插入一段柔性氨基酸序列，可降低ADCC/CDC功能，同时不显著增加多聚体形成。本发明的改造抗体和原始抗体针对相同位点，但抗体亚型或糖基化不同，稳定性显著提高或/和免疫原性显著降低，半衰期延长。改造抗体去除了ADCC/CDC功能，因此，经本发明的技术改造后的抗体能有效避免因ADCC/CDC功能造成的副反应，提高疗效，具有优异的临床应用价值，适于推广，具有较高的经济价值和广阔的市场前景。

Claims (40)

  1. 一种降低或去除IgG1亚型抗体的ADCC/CDC功能的方法，其特征在于，在原始IgG1亚型抗体重链的恒定区插入一段柔性氨基酸序列。
  2. 如权利要求1所述的方法，其特征在于，在原始IgG1亚型抗体重链的CDR3与CH2区之间插入一段柔性氨基酸序列。
  3. 如权利要求1-2任一所述的方法，其特征在于，所述插入一段柔性氨基酸序列，阻断了抗体可变区结合抗原后产生的机械应力传递，使抗体重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化抗体与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。
  4. 如权利要求1-2任一所述的方法，其特征在于，所述原始IgG1亚型抗体为PD-L1、PD-1、CTLA4、TNF-α、PCSK9、NGF、C5、Aβ、IL-6、IL-17A、IL23A、HGF、CMET、Notch1、CCL11、IL6R、IL-31R、IL-1B、IL-20、CD40、CD47、DKK1/2、TIGIT、4-1BB、IGF-1R、LL4、PDGFR2、HER3、IGF1/2、RANKL、sclerostin、GCGR、CGRP、ANGPTL3、IL-13、IL-4R、CSF-1R、TFPI、FCGRT、CD47抗体，优选Atezolizumab单抗、Pembrolizumab单抗、Ipilimumab单抗、Eculizumab单抗、Evolocumab单抗、Erenumab单抗、Fulranumab单抗、Crenezumab单抗、Brodalumab单抗、Ixekizumab单抗、Satralizumab单抗、Gevokizumab单抗、Denosumab单抗、Blosozumab单抗、Crotedumab单抗、Fasinumab单抗、Fremanezumab单抗、Evinacumab单抗、Lebrikizumab单抗、Dupilumab单抗、Cabiralizumab单抗、Concizumab单抗、Rozanolixizumab单抗、Magrolimab单抗或上述抗体药的IgG1亚型的改进型单抗。
  5. 如权利要求3所述的方法，其特征在于，所述柔性氨基酸序列由侧链较小的氨基酸组成的一段连接多肽，用于防止或降低其两端的结构域之间在空间构象上的相互干扰，其序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS。
  6. 一种低ADCC/CDC功能性单抗，其特征在于，是由权利要求1-5任一所述方法制备的单抗。
  7. 权利要求6所述的低ADCC/CDC功能性单抗，其特征在于，所述单抗为嵌合单抗、人源化单抗、全人源单抗、双特异抗体或融合蛋白。
  8. 权利要求6-7任一所述一种低ADCC/CDC功能性单抗，其特征在于， 所述单抗为PD-L1、PD-1、CTLA4、TNF-α、PCSK9、NGF、C5、Aβ、IL-6、IL-17A、IL23A、HGF、CMET、Notch1、CCL11、IL6R、IL-31R、IL-1B、IL-20、CD40、CD47、DKK1/2、TIGIT、4-1BB、IGF-1R、LL4、PDGFR2、HER3、IGF1/2、RANKL、sclerostin、GCGR、CGRP、ANGPTL3、IL-13、IL-4R、CSF-1R、TFPI、FCGRT、CD47的单抗。
  9. 权利要求8所述低ADCC/CDC功能性单抗，其特征在于，所述单抗是在原始IgG1亚型抗体重链的CDR3与CH2区之间插入一段柔性氨基酸序列得到，所述柔性氨基酸序列由侧链较小的氨基酸组成的一段连接多肽，用于防止或降低其两端的结构域之间在空间构象上的相互干扰，其序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS在降低或去除IgG1亚型抗体的ADCC/CDC功能中的应用。
  10. 柔性氨基酸序列在降低或去除IgG1亚型抗体的ADCC/CDC功能中的应用。
  11. 权利要求10所述的应用，其特征在于，所述柔性氨基酸序列插入原始IgG1亚型抗体重链的恒定区中。
  12. 权利要求11所述的应用，其特征在于，所述柔性氨基酸序列插入原始IgG1抗体重链的CDR3与CH2之间。
  13. 如权利要求10-12任一所述的应用，其特征在于，所述柔性氨基酸序列插入位点包括但不限于GG(138/139)、SS(177/178)、SG(178/179)、SS(184/185)、SSS(191/192/193)、LL(235/236)、GG(237/238)。
  14. 权利要求13所述的应用，其特征在于，所述柔性氨基酸序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS。
  15. 柔性氨基酸序列在提高抗体稳定性/降低抗体免疫原性/延长抗体半衰期中的应用。
  16. 权利要求15所述的应用，其特征在于，所述柔性氨基酸序列插入原始IgG1抗体重链的恒定区中，优选地，所述柔性氨基酸序列插入原始IgG1抗体重链的CDR3与CH2之间，更优选地，所述柔性氨基酸序列包括但不限于GGSGGS、GSGGSGG、GSGGSGGG、GGGGSGGG、GSGSG、GGSGG、GGS、GGSGS和GGGS。
  17. 一种低ADCC/CDC功能的抗PD-L1单抗，其特征在于，在原始 Atezolizumab单抗重链的恒定区插入一段柔性氨基酸序列，切断抗体可变区结合抗原后产生的机械应力传递，使单抗重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化单抗与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。
  18. 如权利要求17所述的低ADCC/CDC功能性抗PD-L1单抗，其特征在于，在原始Atezolizumab单抗重链的CDR3与CH2区之间插入一段柔性氨基酸序列来阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，所述原始Atezolizumab单抗重链全长的氨基酸序列如SEQ ID NO.1所示，其轻链全长的氨基酸序列如SEQ ID NO.2所示。
  19. 如权利要求17所述的低ADCC/CDC功能性抗PD-L1单抗，其特征在于，其轻链全长含有SEQ ID NO.2所示的氨基酸序列或其变体，其重链全长含有SEQ ID NO.3～4任一所示的氨基酸序列。
  20. 编码权利要求17～19任一所述低ADCC/CDC功能性抗PD-L1单抗的基因。
  21. 含有权利要求20所述基因的生物材料，所述生物材料为表达盒、表达载体、工程菌或细胞。
  22. 权利要求20所述的基因或权利要求21所述的生物材料的以下任一应用：(1)在制备治疗以PD-L1或PD-1为靶标的疾病药物中的应用；
    (2)在治疗以PD-L1或PD-1为靶标的疾病中的应用；
    (3)在治疗肿瘤或免疫能力退行疾病中的应用。
  23. 权利要求17～19任一所述低ADCC/CDC功能性抗PD-L1单抗的以下任一应用：
    (1)在制备治疗以PD-L1或PD-1为靶标的疾病药物中的应用；
    (2)在制备治疗肿瘤药物中的应用；
    (3)在制备治疗免疫能力退行疾病药物中的应用；
    (4)在治疗以PD-L1或PD-1为靶标的疾病中的应用；
    (5)在治疗肿瘤或免疫能力退行疾病中的应用。
  24. 含有权利要求17～19任一所述低ADCC/CDC功能性抗PD-L1单抗的药物或检测试剂。
  25. 一种低ADCC/CDC功能性抗PD-1单抗，其特征在于，在原始Pembrolizumab单抗重链的恒定区插入一段柔性氨基酸序列，切断抗体可变区 结合抗原后产生的机械应力传递，使单抗重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化单抗与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。
  26. 如权利要求25所述的单抗，其特征在于，在原始Pembrolizumab单抗重链的CDR3与CH2区之间插入一段柔性氨基酸序列来阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，所述原始Pembrolizumab单抗重链全长的氨基酸序列如SEQ ID NO.5所示，其轻链全长的氨基酸序列如SEQ ID NO.6所示。
  27. 如权利要求25所述的单抗，其特征在于，其轻链全长含有SEQ ID NO.6所示的氨基酸序列，其重链全长含有SEQ ID NO.7所示的氨基酸序列。
  28. 编码权利要求25～27任一所述低ADCC/CDC功能性抗PD-1单抗的基因。
  29. 含有权利要求28所述基因的生物材料，所述生物材料为表达盒、表达载体、工程菌或细胞。
  30. 权利要求28所述的基因或权利要求29所述的生物材料的以下任一应用：(1)在制备治疗以PD-L1或PD-1为靶标的疾病药物中的应用；
    (2)在治疗以PD-L1或PD-1为靶标的疾病中的应用；
    (3)在治疗肿瘤或免疫能力退行疾病中的应用。
  31. 权利要求25～27任一所述低ADCC/CDC功能性抗PD-1单抗的以下任一应用：
    (1)在制备治疗以PD-L1或PD-1为靶标的疾病药物中的应用；
    (2)在制备治疗肿瘤药物中的应用；
    (3)在制备治疗免疫能力退行疾病药物中的应用；
    (4)在治疗以PD-L1或PD-1为靶标的疾病中的应用；
    (5)在治疗肿瘤或免疫能力退行疾病中的应用。
  32. 含有权利要求25～27任一所述低ADCC/CDC功能性抗PD-1单抗的药物或检测试剂。
  33. 一种低ADCC/CDC功能性抗CTLA-4单抗，其特征在于，在原始Ipilimumab单抗重链的恒定区插入一段柔性氨基酸序列，切断抗体可变区结合抗原后产生的机械应力传递，使单抗重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，弱化单抗与NK细胞、巨噬细胞和中性粒细胞表达IgG Fc 受体的杀伤细胞结合或和补体结合，无法或降低诱导ADCC和CDC的信号。
  34. 如权利要求33所述的低ADCC/CDC功能性抗CTLA-4单抗，其特征在于，在原始Ipilimumab单抗重链的CDR3与CH2区之间插入一段柔性氨基酸序列来阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，所述原始Ipilimumab单抗重链全长的氨基酸序列如SEQ ID NO.8所示，其轻链全长的氨基酸序列如SEQ ID NO.9所示。
  35. 如权利要求34所述的低ADCC/CDC功能性抗CTLA-4单抗，其特征在于，其轻链全长含有SEQ ID NO.9所示的氨基酸序列，其重链全长含有SEQ ID NO.10所示的氨基酸序列。
  36. 编码权利要求33～35任一所述低ADCC/CDC功能性抗CTLA-4单抗的基因。
  37. 含有权利要求36所述基因的生物材料，所述生物材料为表达盒、表达载体、工程菌或细胞。
  38. 权利要求36所述的基因或权利要求37所述的生物材料的以下任一应用：(1)在制备治疗以CTLA-4为靶标的疾病药物中的应用；
    (2)在治疗以CTLA-4为靶标的疾病中的应用；
    (3)在治疗肿瘤或提高免疫能力中的应用。
  39. 权利要求33～35任一所述低ADCC/CDC功能性抗CTLA-4单抗的以下任一应用：
    (1)在制备治疗以CTLA-4为靶标的疾病药物中的应用；
    (2)在制备治疗肿瘤药物中的应用；
    (3)在制备强化免疫能力药物中的应用；
    (4)在治疗以CTLA-4为靶标的疾病中的应用；
    (5)在治疗肿瘤细胞或免疫能力退行疾病中的应用。
  40. 含有权利要求33～35任一所述低ADCC/CDC功能性抗CTLA-4单抗的药物或检测试剂。
PCT/CN2019/094928 2018-07-06 2019-07-05 低adcc/cdc功能性单抗及其制备方法与应用 WO2020007368A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2021521872A JP7193628B2 (ja) 2018-07-06 2019-07-05 低ａｄｃｃ／ｃｄｃ機能性モノクローナル抗体、及びその調製方法と使用
US17/258,097 US20210380700A1 (en) 2018-07-06 2019-07-05 De-adcc/cdc functions of antibodies and their applications
EP19831301.7A EP3825333A4 (en) 2018-07-06 2019-07-05 LOW FUNCTIONALITY ADCC/CDC MONOCLONAL ANTIBODY, METHOD FOR PREPARATION AND USE

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201810738565.8 2018-07-06
CN201810738565 2018-07-06
CN201810924249.X 2018-08-14
CN201810924249 2018-08-14
CN201910376852 2019-05-07
CN201910376852.3 2019-05-07

Publications (1)

Publication Number Publication Date
WO2020007368A1 true WO2020007368A1 (zh) 2020-01-09

Family

ID=68218156

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/094928 WO2020007368A1 (zh) 2018-07-06 2019-07-05 低adcc/cdc功能性单抗及其制备方法与应用

Country Status (5)

Country Link
US (1) US20210380700A1 (zh)
EP (1) EP3825333A4 (zh)
JP (1) JP7193628B2 (zh)
CN (2) CN114044827B (zh)
WO (1) WO2020007368A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11643463B2 (en) 2017-05-19 2023-05-09 Wuxi Biologics (Shanghai) Co., Ltd. Monoclonal antibodies to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112210005B (zh) * 2019-07-11 2024-03-26 京天成生物技术(北京)有限公司 低免疫原性低adcc/cdc功能的抗c5人源化单抗及其应用
CN113440601B (zh) * 2021-02-08 2022-01-21 中山大学 Nk细胞和tfpi联用在防治鼻咽癌中的应用
EP4184168A1 (en) * 2021-11-22 2023-05-24 Ustav organicke chemie a biochemie AV CR, v.v.i. Probe and method for detecting membrane-associated molecules in living cells

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101466733A (zh) * 2006-04-14 2009-06-24 特鲁比昂药品公司 包含免疫球蛋白铰链区和Fc效应子功能改变了的Fc区的结合蛋白
CN103201289A (zh) * 2010-04-30 2013-07-10 阿雷克森制药公司 抗-c5a抗体和使用所述抗体的方法
CN103690947A (zh) * 2006-01-12 2014-04-02 阿莱克申药物公司 抗ox-2/cd200的抗体及其用途
EP2862875A1 (en) * 2012-06-14 2015-04-22 Chugai Seiyaku Kabushiki Kaisha ANTIGEN-BINDING MOLECULE CONTAINING MODIFIED Fc REGION
CN104788565A (zh) * 2003-05-02 2015-07-22 赞科股份有限公司 优化的Fc变体及其产生方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1234731C (zh) * 2003-01-30 2006-01-04 旭华（上海）生物研发中心有限公司 具有高度生物活性的人促红细胞生成素的Fc融合蛋白
WO2014066532A1 (en) * 2012-10-23 2014-05-01 Bristol-Myers Squibb Company Combination of anti-kir and anti-ctla-4 antibodies to treat cancer
CN103880963A (zh) * 2014-04-08 2014-06-25 江苏匡亚生物医药科技有限公司 半乳糖凝集素-3C（Galectin-3C) 抗体融合蛋白的制备方法和用途
CN107250157B (zh) * 2014-11-21 2021-06-29 百时美施贵宝公司 包含修饰的重链恒定区的抗体
CN107206088A (zh) * 2014-12-05 2017-09-26 豪夫迈·罗氏有限公司 使用pd‑1轴拮抗剂和hpk1拮抗剂用于治疗癌症的方法和组合物
WO2018069927A1 (en) * 2016-10-10 2018-04-19 The National Institute for Biotechnology in the Negev Ltd. Non-cytotoxic modified cells and use thereof
CN108623692B (zh) * 2017-03-20 2020-11-17 徐寒梅 一种融合蛋白及其制备方法和其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104788565A (zh) * 2003-05-02 2015-07-22 赞科股份有限公司 优化的Fc变体及其产生方法
CN103690947A (zh) * 2006-01-12 2014-04-02 阿莱克申药物公司 抗ox-2/cd200的抗体及其用途
CN101466733A (zh) * 2006-04-14 2009-06-24 特鲁比昂药品公司 包含免疫球蛋白铰链区和Fc效应子功能改变了的Fc区的结合蛋白
CN103201289A (zh) * 2010-04-30 2013-07-10 阿雷克森制药公司 抗-c5a抗体和使用所述抗体的方法
EP2862875A1 (en) * 2012-06-14 2015-04-22 Chugai Seiyaku Kabushiki Kaisha ANTIGEN-BINDING MOLECULE CONTAINING MODIFIED Fc REGION

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHUNPENG YANG; GAO XINYU; GONG RUI: "Engineering of Fc Fragments with Optimized Physicochemical Properties Implying Improvement of Clinical Potentials for Fc-Based Thera- peutics", FRONTIERS IN IMMUNOLOGY, vol. 8, 1860, 8 January 2018 (2018-01-08), pages 1 - 14, XP055671009, DOI: 10.3389/fimmu.2017.01860 *
ELISABETH LOBNER; MICHAEL W TRAXLMAYR; CHRISTIAN OBINGER; CHRISTOPH HASENHINDL: "Engineered IgGl-Fc - one fragment to bind them all", IMMUNOLOGICAL REVIEWS, vol. 270, no. 1, 1 March 2016 (2016-03-01), pages 113 - 131, XP055416736, ISSN: 0105-2896, DOI: 10.1111/imr.12385 *
See also references of EP3825333A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11643463B2 (en) 2017-05-19 2023-05-09 Wuxi Biologics (Shanghai) Co., Ltd. Monoclonal antibodies to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)

Also Published As

Publication number Publication date
JP2021531819A (ja) 2021-11-25
JP7193628B2 (ja) 2022-12-20
EP3825333A1 (en) 2021-05-26
CN114044827B (zh) 2024-06-07
CN114044827A (zh) 2022-02-15
EP3825333A4 (en) 2022-04-06
CN110357962B (zh) 2021-11-26
US20210380700A1 (en) 2021-12-09
CN110357962A (zh) 2019-10-22

Similar Documents

Publication Publication Date Title
JP7450592B2 (ja) 癌治療におけるｆｇｆｒ２阻害剤単独または免疫刺激剤との組み合わせ
KR102515509B1 (ko) 결장직장암을 갖는 환자의 치료에서의 항-pd-1 항체의 용도
WO2020007368A1 (zh) 低adcc/cdc功能性单抗及其制备方法与应用
US10550196B2 (en) Humanized antibodies against CEACAM1
CN111094982A (zh) 用于治疗和诊断癌症的tim-3拮抗剂
EP4001308A1 (en) Anti-tigit antibodies and application thereof
JP2023554422A (ja) がんの治療のための多重特異性抗体
EP4274850A1 (en) Combination therapy using an anti-fucosyl-gm1 antibody
US11427647B2 (en) Polynucleotides encoding humanized antibodies against CEACAM1
CA3178649A1 (en) Dosing and administration of activatable anti-ctla-4 antibody
US20240092912A1 (en) Novel combinations of antibodies and uses thereof
CN114630839B (zh) 抗ox40抗体及其用途
US20240092934A1 (en) Assessment of ceacam1 expression on tumor infiltrating lymphocytes
CN112672759B (zh) 抗程序性死亡配体-1(pd-l1)抗体的抗肿瘤用途
CN118806890A (zh) 抗体的新型组合及其用途
JP2024515879A (ja) 抗ｓｉｇｌｅｃ組成物及びその使用
EA045980B1 (ru) Антитела против рецептора полиовируса (pvr) и их применение
CN111447952A (zh) 用于细胞毒性t细胞耗竭的组合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19831301

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021521872

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE