CN114736303A - Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof - Google Patents

Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof Download PDF

Info

Publication number
CN114736303A
CN114736303A CN202210266264.6A CN202210266264A CN114736303A CN 114736303 A CN114736303 A CN 114736303A CN 202210266264 A CN202210266264 A CN 202210266264A CN 114736303 A CN114736303 A CN 114736303A
Authority
CN
China
Prior art keywords
ser
gly
bifunctional antibody
antibody
thr
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
CN202210266264.6A
Other languages
Chinese (zh)
Inventor
邱均专
陈均勇
王振生
孙锴
孙键
李忠良
区日山
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yingnuohu Pharmaceutical Hangzhou Co ltd
Original Assignee
Yingnuohu Pharmaceutical Hangzhou Co ltd
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 Yingnuohu Pharmaceutical Hangzhou Co ltd filed Critical Yingnuohu Pharmaceutical Hangzhou Co ltd
Priority to CN202210266264.6A priority Critical patent/CN114736303A/en
Publication of CN114736303A publication Critical patent/CN114736303A/en
Priority to PCT/CN2023/080387 priority patent/WO2023174140A1/en
Pending legal-status Critical Current

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
    • C07K16/28—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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention relates to the technical field of antibodies, in particular to a bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof. The 4-1BB antibody selected by the invention needs the support of crosslinking action in the aspect of immune activity stimulation, and has no crosslinking action, and the 4-1BB antibody has no activity, so that toxic and side effects cannot be generated in the liver. In the tumor milieu, tumor cells have a higher expression level of PD-L1. The PD-L1/4-1BB bifunctional antibody is partially combined with PD-L1 on the surface of a tumor cell through a PD-L1 antibody, and provides a strong cross-linking effect for the 4-1BB antibody. Therefore, the immunostimulation activity of the PD-L1/4-1BB bifunctional antibody can be obviously improved in a tumor microenvironment specifically, so that toxic and side effects of the 4-1BB antibody in the liver are avoided or reduced.

Description

Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof
Technical Field
The invention relates to the technical field of antibodies, in particular to a bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof.
Background
PD-L1 (programmed death ligand) is an I-type transmembrane protein with a molecular weight of 40kDa, the receptor of which is programmed cell death protein 1(PD-1, also known as CD 279). PD-L1 consists of an IgV-like domain, an IgC-like domain, transmembrane region, and a short cytoplasmic domain (Keir et al, (2008) Annu Rev Immunol 26: 677-. Through interactions with PD-1 and CD80, PD-L1 plays an important role in the regulation of the immune system in the body, such as the inhibition of T Cell Receptor (TCR) -mediated signals for IL-2 production and activation of T cell proliferation. The deletion of the PD-L1 gene resulted in an upregulation of the T cell response, resulting in autoreactive T cells (Latchman et al, (2004) PNAS 101: 10691-10696). Abnormalities in PD-L1 and PD-1 expression levels or signaling are associated with the pathogenesis of a variety of diseases, including cancer, inflammation, and autoimmune diseases, among others. Preclinical data indicate that PD-L1 is highly expressed in a variety of tumor cells, such as melanoma, gastrointestinal Cancer, lung Cancer, kidney Cancer, head and neck Cancer, bladder Cancer, ovarian Cancer, and hematologic malignancies (Jung et al, (2017) Cancer Res Treat 49: 246-. In these tumor tissues, PD-L1 exerts immunosuppressive effects by binding to PD-1, such as inhibiting T cell activation mediated by T Cell Receptor (TCR), reducing the expression of cytokines such as interleukin-2 (IL-2), interferon-gamma (IFN-. gamma.), and blocking the proliferation of activated T cells (Fife et al (2011) Nature Immunology 10: 1185-. Therefore, the expression level of PD-L1 is closely related to the prognosis of cancer patients. Blocking the interaction of PD-L1 and PD-1 inhibits tumor tissue growth and increases the survival of experimental animals in a variety of tumor models (Iwai et al, (2005) Int Immunol 17: 133-.
4-1BB (CD137, TNFRSF9) is an immune checkpoint member of the TNF receptor superfamily, expressed predominantly on activated T cells and NK cells. As a costimulatory molecule, activation of the 4-1BB signaling pathway significantly enhances T cell proliferation and survival, cytokine secretion (e.g., IL-2, TNFa, and INFy), and cytotoxic activity. In a mouse model, activation of 4-1BB significantly enhanced the anti-tumor immunity of mice (Croft et al, (2009) Nat Rev Immunol 9: 271-285; Lynch et al, (2008) immunological revision 22: 277-286), and higher 4-1BB expression levels of tumor-infiltrating T Cells (CTL) corresponded to stronger anti-tumor activity (Ye et al, Clin Cancer Res; 20 (1): 44-55). The ligand of 4-1BB is 4-1BBL, and the cross-linked 4-1BB ligand or 4-1BB trigger antibody can rapidly activate 4-1BB on the surface of T cells, so as to stimulate the proliferation and activation of the T cells. Activation of 4-1BB promotes T cell production of Th1 type cytokines such as IL-2, IFN-. gamma.and TNF-. alpha.and inhibits the production of immunomodulatory factors IL-4 and TGF-. beta.by synergistic action with CD28 co-stimulatory molecules. 4-1BB breaks the anergy of Cytotoxic T Lymphocytes (CTL) by inducing Th1 type effector T cells to differentiate. 4-1BB may also promote the proliferation, survival and cytokine secretion of B cells by interacting with its ligands. In addition, 4-1BB also enhances NK cell killing. Two 4-1BB antibodies (Urelumab and Utomillab) are currently in clinical trials. Urelumab showed antitumor activity in the treatment of melanoma, renal and ovarian cancer patients (Sznol et al, (2008) j. clin. oncol.26(supp15):3007), but dose-dependent hepatotoxic side effects were seen in some patients. Compared with Urelumab, Utomillumab has good safety, but the antitumor effect of Utomillab is relatively weak.
In recent years, monoclonal antibody drugs blocking the interaction between PD-L1 and PD-1 have achieved encouraging efficacy in clinical treatment of some cancer patients, but still face the problems of limited efficacy and drug resistance in most tumor patients. The results of animal experiments and clinical trials before clinic show that the combined use of the PD-1 or PD-L1 antibody and other immune checkpoint antibodies can obviously improve the inhibition effect on tumor growth and enhance the clinical curative effect.
Disclosure of Invention
The invention aims to provide a bifunctional antibody against PD-L1 and 4-1BB, which comprises a first protein functional region targeting PD-L1 and a second protein functional region targeting 4-1 BB;
the first functional region comprises heavy chain CDRs shown as SEQ ID NO. 1-3 and light chain CDRs shown as SEQ ID NO. 4-6;
the second functional region comprises heavy chain CDRs shown as SEQ ID NO 7-9 and light chain CDRs shown as SEQ ID NO 10-12.
The invention also relates to nucleic acids, vectors and host cells related to the bifunctional antibodies as described above.
The invention also relates to a method for producing a bifunctional antibody comprising culturing a host cell as described above under suitable conditions and recovering the bifunctional antibody from the cell culture.
The invention also relates to conjugates which are bifunctional antibodies as described above bound to a therapeutic agent or a detection agent.
The present invention also relates to a pharmaceutical composition comprising a bifunctional antibody as described above or a conjugate as described above, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
The invention also relates to the use of a bifunctional antibody as described above or a conjugate as described above for the preparation of a medicament for the treatment of a tumor.
The PD-L1/4-1BB bifunctional antibody is constructed, so that the breadth and the depth of the immune reaction of an organism are improved and expanded, and the toxic and side effects of the 4-1BB antibody are reduced. The 4-1BB antibody selected by the invention needs the support of crosslinking action in the aspect of immune activity stimulation, and has no crosslinking action, and the 4-1BB antibody has no activity, so that toxic and side effects cannot be generated in the liver. In the tumor milieu, tumor cells have a higher expression level of PD-L1. The PD-L1/4-1BB bifunctional antibody is combined with PD-L1 on the surface of a tumor cell through a PD-L1 antibody part, and provides stronger crosslinking action for the 4-1BB antibody. Therefore, the immunostimulation activity of the PD-L1/4-1BB bifunctional antibody can be obviously improved in a tumor microenvironment specifically, so that toxic and side effects of the 4-1BB antibody in the liver are avoided or reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic molecular diagram of a humanized PD-L1/4-1BB bifunctional antibody;
FIG. 2 shows the measurement of the binding of the humanized PD-L1/4-1BB bifunctional antibody to PD-L1 protein by ELISA;
FIG. 3 shows the measurement of binding of the humanized PD-L1/4-1BB bifunctional antibody to 4-1BB protein by ELISA;
FIG. 4 is a FACS method to determine the binding of the humanized PD-L1/4-1BB bifunctional antibody to 293T-PD-L1 cells;
FIG. 5 is a FACS method to determine the binding of humanized PD-L1/4-1BB bifunctional antibody to Jurkat-4-1BB cells;
FIG. 6 is a FACS method used to determine the blocking effect of the humanized PD-L1/4-1BB bifunctional antibody on the binding of PD-1 to 293T-PD-L1 cells;
FIG. 7A is a graph showing the determination of NF-. kappa.B mediated reporter activation by a humanized PD-L1/4-1BB bifunctional antibody using 293T-CD32a for cross-linking;
FIG. 7B is a graph showing the determination of NF-. kappa.B mediated reporter activation by humanized PD-L1/4-1BB bifunctional antibody using 293T-PD-L1 for cross-linking;
FIG. 8A is a graph showing the activation of IL2 production and release from Jurkat cells by a humanized PD-L1/4-1BB bifunctional antibody, as measured by crosslinking 293T-PD-L1;
FIG. 8B is a graph showing the activation of IL2 release from Jurkat cells by a humanized PD-L1/4-1BB bifunctional antibody, using 293T-PD-L1 for cross-linking;
FIG. 9 shows the effect of humanized PD-L1/4-1BB bifunctional antibody on the production and release of IL2 cytokines by primary CD 4T cells;
FIG. 10 shows the effect of humanized PD-L1/4-1BB bifunctional antibody on the production and release of IFN- γ cytokines by primary CD 4T cells.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, the term "antibody" refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments that comprise complementarity determining regions (CDR regions), particularly full-length antibodies or functional fragments of antibodies. The term "full-length antibody" includes both polyclonal and monoclonal antibodies, and the term "antibody functional fragment" is a substance that comprises part or all of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including whole antibodies) for binding to a given epitope.
In some particular embodiments, the antibody is specifically directed to a bifunctional antibody or a bispecific antibody (BiAb). The term "bifunctional" refers herein to specifically binding two different antigens.
In the present invention, the term "humanized antibody", also referred to as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a framework sequence of a different type of human germline antibody; the term "humanized antibody" also includes humanized antibodies such as resurfaced antibodies and fully humanized antibodies.
The term "complementarity determining regions" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest,5th Ed, "US Department of Health and Human Services, NIH,1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Herein, the terms "CDR" and "CDRs" are used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to the antigen or epitope it recognizes, depending on the circumstances. In another embodiment, the CDR regions or CDRs refer to the highly variable regions of the heavy and light chains of an immunoglobulin as defined by IMGT.
As used herein, the phrase "therapeutic agent" generally refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent when it exhibits a statistically significant effect in the appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, pre-existing clinical condition, and the like. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, reduce, inhibit, prevent, delay onset, reduce severity, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a "therapeutic agent" is a drug that has been or requires approval by a governmental agency before it is commercially available for administration to humans. In some embodiments, a "therapeutic agent" is a drug required for a medical prescription for administration to a human.
As used herein, the term "detection agent" refers to any detectable ingredient, molecule, functional group, compound, fragment or moiety. In some embodiments, the detection entity is provided separately or used. In some embodiments, the detection entity is provided and/or used in conjunction with (e.g., conjugated to) another reagent. Examples of detection entities include (but are not limited to): various ligands, radionuclides (e.g.3H、14C、18F、19F、32P、35S、135I、125I、123I、64Cu、187Re、111In、90Y、99mTc、177Lu、89Zr, etc.), fluorescent dyes (see below for specific exemplary fluorescent dyes), chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (e.g., dyes, colloidal gold, etc.), biotin, digoxigenin (digoxigenin), haptens, and proteins to which antisera or monoclonal antibodies are applicable.
The term "and/or", "and/or" as used herein is intended to be inclusive of any one of the two or more items listed in association, and also to include any and all combinations of the items listed in association, including any two or more of the items listed in association, any more of the items listed in association, or all combinations of the items listed in association. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or/and", "and/or", it should be understood that, in the present application, the technical solutions definitely include the technical solutions all connected by "logic and", and also the technical solutions all connected by "logic or". For example, "A and/or B" includes A, B and A + B. For example, embodiments of "a, and/or, B, and/or, C, and/or, D" include A, B, C, D (i.e., embodiments all connected by "logical or"), A, B, C, D includes any and all combinations of any two or any three of A, B, C, D, and A, B, C, D includes four combinations (i.e., embodiments all connected by "logical and").
As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.
The present invention relates to concentration values, which include fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may be allowed to fluctuate within ± 0.1%. For values that are larger or do not require more fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, may allow fluctuations within the range of. + -. 1%,. + -. 2%,. + -. 5%, etc. The molecular weight is referred to, allowing the meaning to include fluctuations of ± 10%.
In the present invention, the terms "plurality", and the like mean, unless otherwise specified, 2 or more in number.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, "preferably", "better" and "preferably" are only used for describing better embodiments or examples, and it should be understood that the scope of the present invention is not limited by the claims. In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one of two juxtapositions selected from "present" and "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. The citation referred to herein is incorporated by reference in its entirety for all purposes unless otherwise in conflict with the present disclosure's objectives and/or technical solutions. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art features are also incorporated by reference into the present application, but the present invention is not limited to the embodiments. It should be understood that where a reference conflicts with the description herein, the application is controlling or adaptively modified in accordance with the description herein.
The present invention relates to bifunctional antibodies against PD-L1 and 4-1BB comprising a first protein functional region targeting PD-L1 and a second protein functional region targeting 4-1 BB;
the first functional region comprises heavy chain CDRs shown as SEQ ID NO. 1-3 and light chain CDRs shown as SEQ ID NO. 4-6;
the second functional region comprises heavy chain CDRs shown as SEQ ID NO 7-9 and light chain CDRs shown as SEQ ID NO 10-12.
In some embodiments, the first functional region comprises the heavy chain variable region of SEQ ID NO 13 and the light chain variable region of SEQ ID NO 14.
In some embodiments, the second functional region comprises the heavy chain variable region shown in SEQ ID NO. 15 and the light chain variable region shown in SEQ ID NO. 16.
In some embodiments, the second functional region is sc-Fv as shown in SEQ ID NO: 17.
In some embodiments, the first functional region further comprises a light chain constant region and a heavy chain constant region Fc comprising CH2, CH3, and optionally CH 4.
The heavy chain constant region can be from IgG, IgE, IgD, IgA, IgM. IgG can be further classified into IgG1, IgG2, IgG3, and IgG4 subtypes.
In some embodiments, the C-terminus of the antibody heavy chain of the first protein functional region and the N-terminus of the second protein functional region are linked by a linking peptide.
The sc-Fv of the second domain may be 2 and are linked to the C-termini of the two constant domains of the first domain.
In some embodiments, the number of amino acids of the linker peptide is 1 to 30; may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.
In some embodiments, the amino acids of the linker peptide are nonsense polypeptides that do not have additional functions (e.g., protein localization, cleavage sites, etc.) other than linking.
In some embodiments, the linker peptide is a flexible linker peptide;
in some embodiments, the amino acid sequence of the linking peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.
In some embodiments, the amino acid sequence of the linker peptide is selected from (GGGGS) n, (GGGS) n, (GGS) n, (GS) n, or (G) n, wherein n is selected from 1, 2, 3, 4, 5, or 6.
In some embodiments, the amino acid sequence of the linker peptide is set forth in SEQ ID NO 18.
In some embodiments, the amino acid sequence of the heavy chain of the bifunctional antibody resulting from the fusion of the heavy chain of PD-L1 and the functional region of the second protein is set forth in SEQ ID NO 19.
In some embodiments, the second functional region heavy chain variable region (i.e., relative to the sequence shown in SEQ ID NO: 15) has a mutation at amino acid position 44 to C.
In some embodiments, the second domain light chain variable region (i.e., relative to the sequence set forth in SEQ ID NO: 16) has the amino acid at position 105 mutated to a C.
The above mutation in the framework region can enhance the stability of the antibody.
Variants of the above amino acid sequences are also within the scope of the present invention, the variants comprising up to 3 amino acid mutations compared to any one of the polypeptides of SEQ ID NO 1 to SEQ ID NO 12, respectively; the variant may comprise less than 3 or more mutations compared to any of the polypeptides of SEQ ID NO 13 to SEQ ID NO 17, 19, e.g. a sequence having at least 80%, 85%, 90%, 93%, 95%, 97% or 99% identity to the variant compared to SEQ ID NO 13 to SEQ ID NO 17, 19. The mutation may be a substitution, deletion or addition of an amino acid or any combination thereof; preferably, the mutation is a conservative substitution. "conservative substitutions" refer to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein.
Substitutions which are generally regarded as conservative substitutions are substitutions for one another in the aliphatic amino acids Ala, Val, Leu and Ile, for the hydroxyl residues Ser and Thr, for the acidic residues Asp and Glu, for the amide residues Asn and Gln, for the basic residues Lys and Arg and for the aromatic residues Phe, Tyr. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity.
It is also possible for the person skilled in the art to avoid heavy chain mismatches by introducing mutations in the Fc, which is easy for the person skilled in the art.
In some embodiments, the Fc-introduced mutation is based on KiH technology, ART-lg technology, SEED technology, or a combination thereof.
The invention also relates to an isolated nucleic acid encoding a bifunctional antibody as described above.
The term "isolated nucleic acid" refers herein to a polymer of deoxyribonucleic acid or ribonucleic acid in either single-or double-stranded form. The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, the polypeptide also includes naturally occurring polynucleotides, sugars, or analogs of altered bases. According to one aspect of the invention, the polynucleotide is a light chain polynucleotide.
The isolated nucleic acid includes a nucleotide sequence encoding an amino acid sequence of a protein complex and also includes a nucleotide sequence complementary thereto. The complementary sequences include fully complementary sequences and substantially complementary sequences, which refers to sequences that hybridize under stringent conditions known in the art to nucleotide sequences encoding amino acid sequences of protein complexes.
Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such alterations include additions, deletions, or non-conservative or conservative substitutions. Polynucleotides encoding the amino acid sequences of protein complexes can be interpreted to include nucleotide sequences that are substantially identical to the isolated nucleic acid. The substantial identity aligns the nucleotide sequence with additional random sequences in a manner that maximizes their correspondence, which sequences may exhibit greater than 80% homology, greater than 90% homology, or greater than 95% homology when the aligned sequences are analyzed using algorithms common in the art.
The invention also relates to a vector comprising a nucleic acid as described above.
The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.
In the present invention, the vector may be a composition, for example, a mixture of plasmids, different plasmids carrying a portion of an antibody or antigen-binding fragment thereof.
The invention also provides a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.
Suitable host cells or cell lines for expressing the antigen binding proteins of the invention include: mammalian cells such as NS0, Sp2/0, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells may be used, thus allowing the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.
It may be demonstrated that bacterial cells may be used as host cells, which are suitable for expression of the humanized antibodies or other embodiments of the present invention. However, since proteins expressed in bacterial cells tend to be in unfolded or incorrectly folded or unglycosylated form, any humanized antibodies produced in bacterial cells must be screened to retain antigen binding ability. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be the desired host, or, in an alternative embodiment, the molecule may be expressed in a bacterial host, followed by refolding. For example, various strains of E.coli for expression are well known host cells in the biotechnology arts. Various strains of Bacillus subtilis, Streptomyces, other Bacillus species, and the like can also be used in the method.
If desired, yeast cell strains known to those skilled in the art, as well as insect cells, such as Drosophila and Lepidoptera insect and viral expression systems, can also be used as host cells.
In some embodiments, the nucleic acid is inserted into the genome of the cell and is stably expressed.
The insertion may be carried out by using a vector as described above, or the nucleic acid may be directly transferred into the cell without being incorporated into the vector (e.g., liposome-mediated transfection technique).
The present invention also relates to a method for producing a bifunctional antibody comprising culturing a host cell as described above under suitable conditions and recovering the bifunctional antibody from the cell culture.
For example, nucleic acid encoding one or both polypeptide chains of a bispecific antibody can be introduced into a cultured host cell by a variety of known methods (e.g., transformation, transfection, electroporation, bombardment with nucleic acid-coated microparticles, etc.). In some embodiments, the nucleic acid encoding the bispecific antibody may be inserted into a vector suitable for expression in a host cell prior to introduction into the host cell. Typically the vector will contain sequence elements that enable expression of the inserted nucleic acid at the RNA and protein levels.
The invention also relates to conjugates which are bifunctional antibodies as described above bound to a therapeutic agent or a detection agent.
The therapeutic agent may be or comprise any class of chemical entity including, for example (but not limited to), proteins, carbohydrates, lipids, nucleic acids, small organic molecules, non-biological polymers, metals, ions, radioisotopes, and the like. In some embodiments, therapeutic agents used in accordance with the present invention may have biological activity associated with treating one or more symptoms or causes of cancer. In some embodiments, the therapeutic agents used according to the present invention have one or more other activities.
In some embodiments of the invention, the conjugated therapeutic agent is a radioisotope, drug conjugate, nanoparticle, immunotoxin, or any other therapeutic cargo.
The detection agent comprises any moiety that can be detected using the assay, for example due to its specific functional properties and/or chemical characteristics. Non-limiting examples of such agents include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands (e.g., biotin). In some embodiments of the invention, the bound detection agent is a diagnostic agent or an imaging agent.
The present invention also relates to a pharmaceutical composition comprising a bifunctional antibody as described above or a conjugate as described above, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
The bifunctional antibodies or conjugates of the present invention may be used in the preparation of pharmaceutical or sterile compositions, e.g., by mixing the bifunctional antibody with a pharmaceutically acceptable carrier, excipient or stabilizer.
The term "pharmaceutically acceptable" means that the molecular entity, molecular fragment, or composition does not produce an adverse, allergic, or other untoward reaction when properly administered to an animal or human. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof include phosphoric acid, citric acid, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); antibacterial agents (e.g., octadecyl dimethyl benzene ammonium chloride, hexa-hydrocarbyl quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol); a low molecular weight (less than about 10kDa) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including, for example, glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions; a metal composite; and/or a non-ionic surfactant (e.g., including TWEENTM, PLURONICSTM, or polyethylene glycol). In addition, a commonly used filler, diluent, binder, moisturizer, disintegrant, and/or surfactant may be appropriately selected by one of ordinary skill in the art according to the formulation method.
The bifunctional antibody activator contained in the pharmaceutical composition may be contained in microcapsules, or in a drug delivery system of a colloidal nature (such as liposomes, albumin spheroids, microemulsions, nanoparticles and nanocapsules), or in macroemulsions (macroemulsions), which may be prepared, for example, by coacervation (coacervation) techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively.
The pharmaceutical compositions of the present invention may be administered by any route, as will be appreciated by those skilled in the art. In some embodiments, the pharmaceutical compositions of the invention are administered orally (PO), Intravenously (IV), Intramuscularly (IM), intraarterially, intramedullary, intrathecally, Subcutaneously (SQ), intraventricularly, transdermally, intradermally, rectally (PR), vaginally, Intraperitoneally (IP), Intragastrically (IG), topically (e.g., using powders, ointments, creams, gels, lotions, and/or drops), mucosally, intranasally, intrabuccally, enterally, vitreally, sublingually; instilling into trachea, bronchus and/or inhaling; as an oral spray, nasal spray and/or aerosol and/or administered via a portal vein catheter.
The invention also relates to the use of a bifunctional antibody as described above or a conjugate as described above for the preparation of a medicament for the treatment of a tumor.
In some embodiments, the cancer cells in the tumor express PD-L1 and/or 4-1 BB.
"tumour" in the context of the present invention includes hematological and solid tumours, preferably hematological tumours, such as leukaemia.
The patient with the tumor may be selected from the group consisting of human, dog, cat, chimpanzee, orangutan, gibbon, macaque, marmoset, pig, horse, panda and elephant.
The present invention also relates to a method for reducing cytotoxicity to non-target sites during tumor therapy comprising administering to a patient having a tumor a safe and effective amount of a bifunctional antibody or conjugate or pharmaceutical composition as described above.
The phrase "safe and effective amount". As used herein, means that the amount of drug within a reasonable range of medical adjustment is large enough to significantly effectively alleviate the symptoms or conditions being treated, but small enough to avoid serious side effects (at a reasonable benefit/risk ratio). The safe and effective amount of bifunctional antibody or conjugate or active ingredient in the pharmaceutical composition used in the methods of the present invention will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the disease, the time of treatment, the concurrent treatment, the particular active ingredient employed, the particular pharmaceutically acceptable excipient employed and such factors as the knowledge and skill of the attending physician.
In some embodiments, the method is for reducing toxicity to the liver.
Embodiments of the present invention will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the conditions not specified in the following examples, preferably with reference to the guidelines given in the present invention, may also be performed according to the experimental manual or the conventional conditions in the art, and may also be performed according to other experimental procedures known in the art, or according to the conditions suggested by the manufacturer.
In the following specific examples, the measurement parameters relating to the components of the raw materials, if not specified otherwise, may be subject to slight deviations within the accuracy of the weighing. Temperature and time parameters are involved to allow for acceptable deviation due to instrument test accuracy or operational accuracy.
The PD-L1/4-1BB bifunctional antibody is constructed by using the hu6F9B1A6 antibody in the patent CN201911023312.3 and the hu10B2 antibody in the patent CN202011555299.9, and the schematic construction form of the bifunctional antibody is shown in FIG. 1. Compared with corresponding PD-L1 and 4-1BB monoclonal antibodies, the antibodies in the bifunctional antibody hu6F9B1A6/4-1BB retain the binding ability with respective antigens, and the bifunctional antibody shows higher biological activity in promoting the production and secretion of cytokines by human immune cells.
Example 1 PD-L1/4-1BB bifunctional antibody construction
The hu6F9B1A6/hu10B2-scFv bifunctional antibody was constructed.
In construction, the 4-1BB humanized antibody was designed as a single chain scFv (SEQ ID NO:17) linked via Linker (G4S)2 to the C-terminus of the hu6F9B1A6 heavy chain (as shown in FIG. 1), and co-transfected with the light chain vector into 293 cells. Shake flask culture at 37 ℃ for 6 days, centrifuge and collect supernatant, purify with Protein A, and perform various assays including binding and functional activity after purification.
Example 2 determination of binding of the humanized PD-L1/4-1BB bifunctional antibody to PD-L1 and 4-1BB proteins by ELISA
Coating 96-well ELISA plates (Corning, Cat No:9018) with PD-L1-ECD-His or 4-1BB-ECD-His, standing overnight at 4 degrees, washing 3 times with washing buffer (PBS + 0.05% Tween20), adding blocking buffer (PBS + 1% BSA (Sigma, Cat No: V90093)) and incubating for 1 hour; washing the 96-well plate for 3 times; adding a serial diluted sample to be detected comprising a positive control and a negative control, then incubating for 1 hour, and washing for 3 times; adding 100 μ L of goat anti-mouse IgG secondary antibody (Thermo, Cat No:31432) diluted 1:10000 times to each well, incubating for 1 hour at room temperature, and washing for 3 times; 100. mu.L of TMB (Cat No: ES-002) was added to each well for color development for 3 minutes, and 100. mu.L/well of stop buffer (2N H)2SO4) The reaction was stopped and the OD450nm signal was measured for each sample using a Tecan Spark plate reader.
The test results are shown in fig. 2 and 3; the PD-L1/4-1BB bifunctional antibody maintains the binding activity with PD-L1-ECD-His or 4-1 BB-ECD-His.
Example 3 measurement of binding of humanized PD-L1/4-1BB bifunctional antibody to 293T-PD-L1 and Jurkat-4-1BB cells by FACS method
Adding 50 μ L of serially diluted sample to be tested including positive and negative controls and mixing with 50 μ L of 293T-PD-L1 or Jurkat-4-1BB cells: (1×105Cells/well) were added to a U-bottom 96-well plate and incubated for 1 hour, washed 2 times by centrifugation with FACS buffer (PBS + 3% FCS), added with a 1: 400-fold dilution of PE-labeled goat anti-mouse secondary antibody (Biolegged, Cat No:405307), incubated for 30 minutes, washed with FACS buffer, and detected for PE signal from 293T-PD-L1 cells using a BD C6 flow cytometer.
As shown in FIGS. 4 and 5, the PD-L1/4-1BB bifunctional antibody retained the binding activity to 293T-PD-L1 and Jurkat-4-1BB cells.
Example 4 measurement of blocking Effect of the humanized PD-L1/4-1BB bifunctional antibody on the binding of PD-1 to 293T-PD-L1 cells by FACS method
The blocking effect of the PD-L1/4-1BB bifunctional antibody on the binding of PD-1 and PD-L1 is detected by a competitive FACS method. In a U-bottom 96-well plate, serial dilutions of the samples to be tested, including positive and negative controls and 293T-PD-L1 cells (1X 10)5Cells/well) were incubated for 1 hour, 0.05. mu.g/mL of PD-1-biotin was added for 1 hour, washed 2 times with FACS buffer after centrifugation, added with anti-biotin-PE (Biolegend, Cat No:409004) for 30 minutes, and PE signals of 293T-PD-L1 cells were detected by a BDC6 flow cytometer after centrifugation and washing.
As shown in FIG. 6, the PD-L1/4-1BB bifunctional antibody maintained the activity of blocking the binding of PD-1 to 293T-PD-L1 cells.
Example 5 determination of the NF-. kappa.B-mediated activation of reporter genes by humanized PD-L1/4-1BB bifunctional antibody
The reporter gene method is adopted to measure the activation activity of the humanized PD-L1/4-1BB bifunctional antibody on 4-1 BB. Add 50. mu.l (5X 10) to 96-well plates4Cell/well) Jurkat-4-1BB-NFkB-luc cells and 50. mu.l of serially diluted sample to be tested including positive control and negative control, 293T-CD32a or 293T-PD-L1 cells (2X 10)4Cells/well), mixed and incubated at 37 ℃ for 4 hours. 25 μ l of Bright Glo (Promega, Cat No: E2620) was added, incubated at room temperature for 5 minutes, and the chemiluminescent signal of each sample was measured using a Tecan Spark plate reader.
As shown in FIGS. 7A and 7B, when 293T-CD32a cells were added as a cross-linking donor, only hu10B2 had 4-1BB activating signal, while PD-L1/4-1BB bifunctional antibody had no activating activity; while the PD-L1/4-1BB bifunctional antibody exhibited significant 4-1BB activation activity when the cross-linked donor was 293T-PD-L1 cells. This result indicates that the activation of the 4-1BB signaling pathway by the PD-L1/4-1BB bifunctional antibody is dependent on the binding of its PD-L1 antibody to cell surface PD-L1.
Example 6 determination of the Effect of humanized PD-L1/4-1BB bifunctional antibody on cytokine secretion by Jurkta-4-1BB cells
Adding serial diluted samples to be detected including positive control and negative control, co-culturing with 293T-OKT3 and 293T-CD32a or 293T-PD-L1 for 48 hours, collecting cell supernatant, and detecting the expression level of IL-2, wherein the specific determination method is referred to example 2.
As shown in FIGS. 8A and 8B, when 293T-CD32a cells were added as a cross-linking donor, only hu10B 24-1 BB antibody stimulated IL-2 secretion by Jurkat cells, while PD-L1/4-1BB bifunctional antibody was inactive; while the PD-L1/4-1BB bifunctional antibody exhibited significant IL-2 production by Jurkat cells when the cross-linked donor was 293T-PD-L1 cells. The results are consistent with the results of the reporter gene assay, indicating that the activation of Jurkat by PD-L1/4-1BB bifunctional antibody is dependent on the binding of PD-L1 antibody to PD-L1.
Example 7 promotion of production and Release of IL2 or IFN- γ cytokines by humanized PD-L1/4-1BB bifunctional antibodies on Primary CD 4T cells, and affinity information for related antibodies
In a U-bottom 96-well plate, 50. mu.L of CD4+ T cells, 50. mu.L of differentiated Dendritic Cells (DCs) and 100. mu.L of samples to be tested including positive and negative controls (20. mu.g/mL, 2. mu.g/mL …) were added to each well, incubated at 37 ℃ for 5 days, and the supernatant was collected and subjected to cytokine concentration measurement using IL-2(R & DSystems, Cat No: DY202) and IFN-. gamma. (R & D Systems, Cat No: DY285) ELISA kits, as shown in FIGS. 9 and 10. The experimental result of the mixed lymphocyte reaction shows that compared with humanized PD-L1 and 4-1BB monoclonal antibody, the PD-L1/4-1BB bifunctional antibody has stronger stimulation activity on the generation and secretion of IL-2 and IFN-gamma of primary CD 4T cells. Therefore, the PD-L1/4-1BB bifunctional antibody can further improve the activation effect on immune cells.
Binding of each humanized antibody sample to a 4-1BB or PD-L1 antigen sample was determined using Biacore. The determination method comprises the following steps: the anti-human IgG was immobilized on CM5 chip by amino coupling followed by capture of the humanized antibody at a flow rate of 10. mu.L/min. The flow rate was switched to 30. mu.L/min, and different concentrations of histidine-tagged 4-1BB or PD-L1 antigen (100nM,50nM,25nM,12.5nM,6.25nM,3.125nM, etc.) were flowed through the sample and reference channels in sequence, with a binding time of 3min and a dissociation time of 10 min. Finally, the chip was regenerated with glycine buffer solution of pH 1.7.
The detection results are shown in tables 1 and 2, and are consistent with the results of ELISA and FACS detection in general, and show that the PD-L1/4-1BB bifunctional antibody has high affinity with PD-L1-ECD-His or 4-1 BB-ECD-His.
TABLE 1, 4-1BB-mFc multiple cycle kinetic testing (SPR assay)
Antibodies Ka(1/MS) Kd(1/S) KD(M)
4-1BB(hu10B2) 6.02E+05 2.04E-04 3.38E-10
hu6F9B1A6-10B2-CC 2.89E+05 1.22E-04 4.23E-10
TABLE 2 PD-L1 his Multi-cycle kinetics test (SPR measurement)
Figure BDA0003551929120000191
Figure BDA0003551929120000201
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.
SEQUENCE LISTING
<110> Inonolake medicine (Hangzhou) Co., Ltd
<120> bifunctional antibody against PD-L1 and 4-1BB and medical application thereof
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 HCDR1
<400> 1
Gly Tyr Thr Val His
1 5
<210> 2
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 HCDR2
<400> 2
Trp Ile Tyr Pro Gly Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 3
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 HCDR3
<400> 3
His Gly Thr Gly Lys Phe Asn Tyr Phe Asp Val
1 5 10
<210> 4
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 LCDR1
<400> 4
Arg Ala Ser Ser Thr Val Asn Tyr Met His
1 5 10
<210> 5
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 LCDR2
<400> 5
Ala Thr Phe Asn Leu Ala Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 LCDR3
<400> 6
Gln Thr Trp Ser Ser Tyr Pro Leu Thr
1 5
<210> 7
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB HCDR1
<400> 7
Thr Phe Gly Leu His
1 5
<210> 8
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB HCDR2
<400> 8
Tyr Ile Ser Ser Asp Ser Asn Thr Ile Tyr Tyr Ala Asp Thr Met Lys
1 5 10 15
Gly
<210> 9
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB HCDR3
<400> 9
Gly Asp Pro Ala Tyr Tyr Gly Tyr Gly Gly Arg Phe Val Tyr Ala Leu
1 5 10 15
Asp Phe
<210> 10
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB LCDR1
<400> 10
Arg Ser Ser Gln Ser Leu Thr Asn Ser Tyr Gly His Thr Tyr Leu Ser
1 5 10 15
<210> 11
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB LCDR2
<400> 11
Gly Ile Ser Ile Arg Phe Ser
1 5
<210> 12
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB LCDR3
<400> 12
Leu Gln Gly Thr His Gln Pro Trp Thr
1 5
<210> 13
<211> 120
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 VH
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Gly Tyr
20 25 30
Thr Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg His Gly Thr Gly Lys Phe Asn Tyr Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 14
<211> 106
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 VL
<400> 14
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Thr Val Asn Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Phe Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Thr Trp Ser Ser Tyr Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 15
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB VH
<400> 15
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe
20 25 30
Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Asp Ser Asn Thr Ile Tyr Tyr Ala Asp Thr Met
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Asp Pro Ala Tyr Tyr Gly Tyr Gly Gly Arg Phe Val Tyr
100 105 110
Ala Leu Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 112
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB VL
<400> 16
Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser
20 25 30
Tyr Gly His Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gly Ile Ser Ile Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly
85 90 95
Thr His Gln Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 17
<211> 254
<212> PRT
<213> artificial sequence
<220>
<223> 4-1BB sc-Fv
<400> 17
Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser
20 25 30
Tyr Gly His Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gly Ile Ser Ile Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly
85 90 95
Thr His Gln Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser
130 135 140
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe Gly
145 150 155 160
Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
165 170 175
Tyr Ile Ser Ser Asp Ser Asn Thr Ile Tyr Tyr Ala Asp Thr Met Lys
180 185 190
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
195 200 205
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr
210 215 220
Arg Gly Asp Pro Ala Tyr Tyr Gly Tyr Gly Gly Arg Phe Val Tyr Ala
225 230 235 240
Leu Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
245 250
<210> 18
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> Linker
<400> 18
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 19
<211> 711
<212> PRT
<213> artificial sequence
<220>
<223> Heavy chain full length
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Gly Tyr
20 25 30
Thr Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg His Gly Thr Gly Lys Phe Asn Tyr Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220
Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
260 265 270
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Val Thr Gln Thr
450 455 460
Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys
465 470 475 480
Arg Ser Ser Gln Ser Leu Thr Asn Ser Tyr Gly His Thr Tyr Leu Ser
485 490 495
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly
500 505 510
Ile Ser Ile Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
515 520 525
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
530 535 540
Val Gly Val Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Trp Thr Phe
545 550 555 560
Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
565 570 575
Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly
580 585 590
Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala
595 600 605
Ser Gly Phe Thr Phe Ser Thr Phe Gly Leu His Trp Val Arg Gln Ala
610 615 620
Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Asn
625 630 635 640
Thr Ile Tyr Tyr Ala Asp Thr Met Lys Gly Arg Phe Thr Ile Ser Arg
645 650 655
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
660 665 670
Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Gly Asp Pro Ala Tyr Tyr
675 680 685
Gly Tyr Gly Gly Arg Phe Val Tyr Ala Leu Asp Phe Trp Gly Gln Gly
690 695 700
Thr Leu Val Thr Val Ser Ser
705 710

Claims (17)

1. A bifunctional antibody against PD-L1 and 4-1BB comprising a first protein functional region targeting PD-L1 and a second protein functional region targeting 4-1 BB;
the first functional region comprises heavy chain CDRs shown as SEQ ID NO. 1-3 and light chain CDRs shown as SEQ ID NO. 4-6;
the second functional region comprises heavy chain CDRs shown as SEQ ID NO 7-9 and light chain CDRs shown as SEQ ID NO 10-12.
2. The bifunctional antibody of claim 1, wherein the first functional region comprises the heavy chain variable region of SEQ ID NO 13 and the light chain variable region of SEQ ID NO 14.
3. The bifunctional antibody of claim 1, wherein the second functional region comprises the heavy chain variable region of SEQ ID NO. 15 and the light chain variable region of SEQ ID NO. 16; preferably, sc-Fv represented by SEQ ID NO. 17.
4. The bifunctional antibody of any one of claims 1-3, wherein the first functional region further comprises a light chain constant region and a heavy chain constant region Fc, said Fc comprising CH2, CH3, and optionally CH 4.
5. The bifunctional antibody according to any one of claims 1-3, wherein the C-terminus of the antibody heavy chain of the first protein functional region and the N-terminus of the second protein functional region are linked via a linker peptide.
6. The bifunctional antibody of claim 5, wherein the amino acid sequence of the linker peptide is shown in SEQ ID NO 18.
7. The bifunctional antibody of claim 6, wherein the amino acid sequence of the heavy chain obtained by fusing the heavy chain of PD-L1 and the functional region of the second protein is shown as SEQ ID NO. 19.
8. The bifunctional antibody of any one of claims 3, 6 and 7, wherein the amino acid at position 44 of the heavy chain variable region of the second functional region is mutated to C; and/or; and the 105 th amino acid of the light chain variable region of the second functional region is mutated into C.
9. An isolated nucleic acid encoding the bifunctional antibody of any one of claims 1-8.
10. A vector comprising the nucleic acid of claim 9.
11. A host cell comprising the nucleic acid of claim 9, or transformed with the vector of claim 10.
12. A method for producing a bifunctional antibody comprising culturing the host cell of claim 11 under suitable conditions and recovering the bifunctional antibody from the cell culture.
13. A conjugate which is a bifunctional antibody according to any one of claims 1 to 8 conjugated to a therapeutic agent or a detection agent.
14. A pharmaceutical composition comprising the bifunctional antibody of any one of claims 1-8 or the conjugate of claim 12, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
15. Use of a bifunctional antibody according to any one of claims 1 to 8 or a conjugate according to claim 12 for the manufacture of a medicament for the treatment of a tumour.
16. The use of claim 15, wherein the cancer cells in the tumor express PD-L1 and/or 4-1 BB.
17. The use according to claim 15 or 16, wherein the tumour is leukaemia.
CN202210266264.6A 2022-03-17 2022-03-17 Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof Pending CN114736303A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210266264.6A CN114736303A (en) 2022-03-17 2022-03-17 Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof
PCT/CN2023/080387 WO2023174140A1 (en) 2022-03-17 2023-03-09 Anti-pd-l1 and 4-1bb bifunctional antibody and medical use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210266264.6A CN114736303A (en) 2022-03-17 2022-03-17 Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof

Publications (1)

Publication Number Publication Date
CN114736303A true CN114736303A (en) 2022-07-12

Family

ID=82277965

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210266264.6A Pending CN114736303A (en) 2022-03-17 2022-03-17 Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof

Country Status (2)

Country Link
CN (1) CN114736303A (en)
WO (1) WO2023174140A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023174140A1 (en) * 2022-03-17 2023-09-21 英诺湖医药(杭州)有限公司 Anti-pd-l1 and 4-1bb bifunctional antibody and medical use thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110903391A (en) * 2019-10-25 2020-03-24 东大生物技术(苏州)有限公司 Group of PD-L1 monoclonal antibodies and medical application thereof
CN111196855A (en) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 anti-EGFR/PD-1 bispecific antibodies
WO2020107715A1 (en) * 2018-11-30 2020-06-04 Abl Bio Inc. Anti-pd-l1/anti-4-1bb bispecific antibodies and uses thereof
CN112538116A (en) * 2020-12-24 2021-03-23 东大生物技术(苏州)有限公司 Group of 4-1BB monoclonal antibodies and medical application thereof
CN112830972A (en) * 2019-11-25 2021-05-25 中山康方生物医药有限公司 Bispecific antibodies against PD-1-anti-VEGFA, pharmaceutical compositions thereof and uses thereof
CN114106190A (en) * 2020-08-31 2022-03-01 普米斯生物技术(珠海)有限公司 anti-VEGF/PD-L1 bispecific antibody and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115991778A (en) * 2018-04-09 2023-04-21 原启生物科技(上海)有限责任公司 anti-PD-L1 antibodies and uses thereof
TW202035447A (en) * 2018-07-04 2020-10-01 瑞士商赫孚孟拉羅股份公司 Novel bispecific agonistic 4-1bb antigen binding molecules
KR20210040103A (en) * 2018-07-31 2021-04-12 피어이스 파마슈티컬즈 게엠베하 Novel fusion proteins specific for CD137 and PD-L1
CN112646031B (en) * 2019-10-10 2021-12-07 天境生物科技(上海)有限公司 Anti-4-1 BB nanobody and application thereof
CN114736303A (en) * 2022-03-17 2022-07-12 英诺湖医药(杭州)有限公司 Bifunctional antibody for resisting PD-L1 and 4-1BB and medical application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111196855A (en) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 anti-EGFR/PD-1 bispecific antibodies
WO2020107715A1 (en) * 2018-11-30 2020-06-04 Abl Bio Inc. Anti-pd-l1/anti-4-1bb bispecific antibodies and uses thereof
CN113286825A (en) * 2018-11-30 2021-08-20 爱必乐生物公司 anti-PD-L1/anti-4-1 BB bispecific antibody and application thereof
CN110903391A (en) * 2019-10-25 2020-03-24 东大生物技术(苏州)有限公司 Group of PD-L1 monoclonal antibodies and medical application thereof
CN112830972A (en) * 2019-11-25 2021-05-25 中山康方生物医药有限公司 Bispecific antibodies against PD-1-anti-VEGFA, pharmaceutical compositions thereof and uses thereof
CN114106190A (en) * 2020-08-31 2022-03-01 普米斯生物技术(珠海)有限公司 anti-VEGF/PD-L1 bispecific antibody and application thereof
CN112538116A (en) * 2020-12-24 2021-03-23 东大生物技术(苏州)有限公司 Group of 4-1BB monoclonal antibodies and medical application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023174140A1 (en) * 2022-03-17 2023-09-21 英诺湖医药(杭州)有限公司 Anti-pd-l1 and 4-1bb bifunctional antibody and medical use thereof

Also Published As

Publication number Publication date
WO2023174140A1 (en) 2023-09-21

Similar Documents

Publication Publication Date Title
US20190309069A1 (en) Pd-1 antibody, antigen-binding fragment thereof, and medical application thereof
CN106029697B (en) Canine antibodies with modified CH2-CH3 sequences
WO2021188599A1 (en) Novel antigen binding domains and synthetic antigen receptors incorporating the same
CN110396129B (en) Humanized CD19 antigen-binding single-chain antibody and chimeric antigen receptor, immune cell and application thereof
CN111432831A (en) I L-2 mutant protein and application thereof
CN113614109A (en) Bifunctional anti-PD-1/IL-7 molecules
JP2021533755A (en) Chimeric antigen receptor and CAR-T cells that bind to HLA-DR
JP2020503860A (en) Conditional agonist of the immune response
US20210070860A1 (en) Fc variant compositions and methods of use thereof
JP2022532173A (en) Humanized anti-CD137 antibody and its use
TW201522373A (en) Anti-CD52 antibodies
KR20210030406A (en) FC binding fragment comprising the CD137 antigen-binding site
WO2023174140A1 (en) Anti-pd-l1 and 4-1bb bifunctional antibody and medical use thereof
CN112794911B (en) Humanized anti-folate receptor 1 antibody and application thereof
KR20190095943A (en) Humanized CXCR3 Antibody Having Depleting Activity and Method of Use thereof
WO2023020459A1 (en) MONOCLONAL ANTIBODY TARGETING SIRPα AND USE THEREOF
TW202330612A (en) Compositions targeting bcma and methods of use thereof
WO2018119288A1 (en) Anti-human cxcr3 antibodies for treatment of vitiligo
CA3041717A1 (en) Treatment of allergic diseases with chimeric protein
KR20230169936A (en) GARP protein antibodies and their applications
CN114685659A (en) CD 22-specific humanized antibody and chimeric antigen receptor using same
KR20210118116A (en) LILRB3 binding molecules and uses thereof
CN114616243A (en) CAR-T cell compositions and methods of use thereof
CN114174336A (en) Caninized antibodies to human and canine CTLA-4
CN117659206A (en) PD-L1 antibody/VEGF-Trap bifunctional fusion protein and medical application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20220712

WD01 Invention patent application deemed withdrawn after publication