WO2019042153A1 - 重组双特异性抗体 - Google Patents
重组双特异性抗体 Download PDFInfo
- Publication number
- WO2019042153A1 WO2019042153A1 PCT/CN2018/100971 CN2018100971W WO2019042153A1 WO 2019042153 A1 WO2019042153 A1 WO 2019042153A1 CN 2018100971 W CN2018100971 W CN 2018100971W WO 2019042153 A1 WO2019042153 A1 WO 2019042153A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- seq
- scfv
- bispecific antibody
- pdl
- Prior art date
Links
- 0 CC[C@](C(*)C1*)C(C)(CC(*)C(CC[N+]([O-])=O)O)C(C(*)C*)C1[O+]CC Chemical compound CC[C@](C(*)C1*)C(C)(CC(*)C(CC[N+]([O-])=O)O)C(C(*)C*)C1[O+]CC 0.000 description 2
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- 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
-
- 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
- C07K2317/52—Constant or Fc region; Isotype
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
-
- 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
- C07K2317/71—Decreased effector function due to an Fc-modification
-
- 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
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present invention relates to the field of biomedicine, in particular to the field of prevention and/or treatment of diseases associated with CTLA-4 and/or PDL-1.
- the invention relates to bispecific antibodies that specifically bind to CTLA-4 and PDL-1, or PDL-1 and CTLA-4.
- the PD-1/PDL-1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune escape.
- the expression of PD-1 (programmed cell death 1, programmed cell death factor 1) is mainly in immune cells such as T cells, and the ligand PDL-1 of PD-1 is highly expressed in many human tumor tissues.
- Application of immunohistochemical methods has been in breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma and other human tumors
- the expression of PDL-1 protein was detected in the tissues, and the expression level of PDL-1 was closely related to the clinical and prognosis of the patients.
- Blocking the PD-1/PDL-1 signaling pathway activates suppressed T cells and attacks cancer cells.
- Blocking PD-1/PDL-1 signaling can promote the proliferation of tumor antigen-specific T cells and play a role in killing tumor cells, thereby inhibiting local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455) -2465); PDL-1 mAb up-regulates the secretion of IFN- ⁇ in infiltrating CD8+ T cells, suggesting that blockade of the PD-1/PDL-1 signaling pathway plays a role in tumor immune responses for the purpose of inducing immune responses ( Blank C et al., 2006, Int. J. Cancer. 119:317-327).
- PDL-1 can also bind to B7-1 in vivo. Studies have shown that the PDL-1/B7-1 complex is also a negative signal for T cell activation, and the combination of the two can lead to decreased expression of T cell surface activation markers and inhibition of T cell proliferation.
- the cytotoxic T lymphocyte associated antigen-4 (also referred to as CTLA-4) has a close relationship with the CD28 molecule in gene structure, chromosomal location, sequence homology and gene expression. It is a receptor for the costimulatory molecule B7 and is mainly expressed on the surface of activated T cells. When CTLA-4 binds to B7, it inhibits the activation of mouse and human T cells and plays a negative regulatory role in T cell activation.
- Ipilimumab Antibodies are approved by the FDA for the treatment of advanced melanoma, but this antibody injection has serious and even fatal immune-mediated adverse effects. These immune-mediated adverse effects may involve any organ system; however, the most common serious immune-mediated adverse effects are enterocolitis, hepatitis, dermatitis (including toxic epidermal necrosis), neuropathy, and endocrine diseases. Most of these immune-mediated responses initially appear in the course of treatment; another small fraction occurs within a few weeks after discontinuation. For severe immune-mediated responses, it should be permanently disabled And began a systemic large dose of glucocorticoid treatment.
- Bispecific antibodies are a class of dual-homophilic combinatorial antibodies, usually bivalent (also tetravalent and hexavalent), ie, two antigen-binding arms with binding to two different specific antigens.
- Chinese patent application CN106967172A discloses anti-CTLA-4-anti-PD-1 bifunctional antibody, its pharmaceutical composition and use thereof, and CN104987421A also discloses a dual variable domain immunoglobulin which specifically binds PD-1 and/or CTLA-4. protein.
- a vector comprising a nucleic acid molecule, a method of preparing a bispecific antibody, a pharmaceutical composition comprising a bispecific antibody, a use of the pharmaceutical composition in the preparation of a medicament, a bispecific antibody in diagnosis/treatment/prevention with CTLA-4 and Use or method in a PDL-1 related disease (eg, an autoimmune disease or tumor), and a kit comprising a bispecific antibody.
- a PDL-1 related disease eg, an autoimmune disease or tumor
- the invention provides a bispecific antibody comprising a protein or polypeptide that binds to both the antigen PDL-1 and the antigen CTLA-4.
- the bispecific antibody comprises:
- a first antibody that specifically binds to a first antigen comprising a heavy chain (HC) and a light chain (LC);
- an antibody fragment eg, Fv, scFv, di-scFv
- VH heavy chain variable region
- VL light chain variable region
- antibody fragment is linked to the N-terminus or C-terminus of the heavy or light chain of the first antibody
- the first antigen is CTLA-4 and the second antigen is PDL-1; alternatively, the first antigen is PDL-1 and the second antigen is CTLA-4.
- the antibody fragment is a scFv.
- the bispecific antibody comprises 1 of the first antibody and 2 of the scFv; and the first antibody comprises two HCs and two LCs, wherein the The heavy chain variable region (VH) of one HC of an antibody forms an antigen binding site with the light chain variable region (VL) of one LC, and the heavy chain variable region (VH) of another HC is linked to the light chain of another LC The variable region (VL) forms an antigen binding site.
- each of the scFvs is linked to the N-terminus or C-terminus of the two heavy or two light chains of the first antibody, respectively.
- each of the scFvs is linked to the N-terminus of the two heavy chains of the first antibody, respectively. In certain preferred embodiments, each of the scFvs is linked to the C-terminus of the two heavy chains of the first antibody, respectively.
- the first antigen is PDL-1
- the second antigen is CTLA-4
- the scFv that binds to the second antigen is linked to the C of the two heavy chains of the first antibody end.
- each of the scFvs is linked to the N-terminus of the two light chains of the first antibody, respectively. In certain preferred embodiments, each of the scFvs is linked to the C-terminus of the two light chains of the first antibody, respectively.
- one of the scFvs is linked to the N-terminus of the heavy or light chain of the first antibody, and the other of the scFvs is linked to the heavy or light chain of the first antibody. end.
- the bispecific antibodies of the invention comprise:
- a first antibody that specifically binds to a first antigen the first antibody comprising a heavy chain (HC) and a light chain (LC); and 2) an scFv that specifically binds to a second antigen
- the bispecific antibody comprises 1 said first antibody and 2 said scFv
- said first antibody comprises two HCs and two LCs, wherein a VH of one HC of said first antibody forms an antigen binding site with a VL of one LC Another VH of HC forms an antigen binding site with the VL of another LC; each of said scFvs is linked to the N-terminus of the two heavy chains of said first antibody, respectively; or each of said scFvs is linked to The C-terminus of the two heavy chains of the first antibody; the first antigen is PDL-1 and the second antigen is CTLA-4.
- each scFv is linked to the N-terminus or C-terminus of each heavy chain of the first antibody via linker S1.
- the scFv is linked by VH and VL via an S2 linker.
- the structure of the scFv is NH2-VL-S2-VH-COOH, wherein the S2 is a linker.
- the heavy chain of the first antibody comprises a heavy chain variable region (VH) and a CH1 domain, and the light chain comprises a light chain variable region (VL) and a light chain constant region (CL).
- the first antibody can be a Fab fragment, a Fab' fragment or a F(ab') 2 fragment.
- the heavy chain of the first antibody comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and Light chain constant region (CL).
- the first antibody can be a full length antibody.
- the heavy chain of the first antibody is an IgG isotype, such as IgGl, IgG2, IgG3 or IgG4; preferably a human IgG isotype. In certain embodiments, the heavy chain of the first antibody is a human IgGl isotype. In certain preferred embodiments, the light chain of the first antibody is a Kappa isoform, preferably a human Kappa isoform.
- the two HCs of the first antibody comprise the same CDR; and/or the two LCs of the first antibody comprise the same CDR.
- the two HCs of the first antibody comprise the same VH; and/or the two LCs of the first antibody comprise the same VL.
- the two HCs of the first antibody have the same amino acid sequence; and/or the two LCs of the first antibody have the same amino acid sequence.
- the two scFvs have the same or different amino acid sequences. In certain preferred embodiments, the two scFvs have the same amino acid sequence.
- the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each of the polypeptide chains:
- said first polypeptide chains each independently comprise a heavy chain (HC) of said first antibody and said scFv;
- said second polypeptide chain each independently comprising a light chain (LC) of said first antibody
- scFv is linked to the N-terminus or C-terminus of the HC of the first antibody via a linker S1.
- the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each of the polypeptide chains:
- said first polypeptide chains each independently comprise a light chain (LC) of said first antibody and said scFv;
- said second polypeptide chain each independently comprising a heavy chain (HC) of said first antibody
- scFv is linked to the N-terminus or C-terminus of the LC of the first antibody via a linker S1.
- the N-terminus or C-terminus of the scFv is linked to the C-terminus or N-terminus of the linker S1.
- the scFv has the structure: NH 2 -VH-S2-VL -COOH or NH 2 -VL-S2-VH- COOH, wherein said S2 is a linker.
- the linker S1 and/or S2 is a peptide linker, for example having an amino acid sequence as shown by (G m S n ) x , wherein m, n are each independently selected from 1 to 8 An integer (eg, 1, 2, 3, 4, 5, 6, 7, or 8), x is independently selected from an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
- the linker S1 and/or S2 has an amino acid sequence as shown by (G 4 S) x , and x is independently selected from an integer from 1 to 6.
- the linker S1 and/or S2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
- the linker S2 has an amino acid sequence as shown by (G 4 S) 4 , namely GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 25).
- the linker S1 when the scFv is linked to the N-terminus of the heavy or light chain of the first antibody, the linker S1 has an amino acid sequence as shown by (G 4 S) 3 , ie GGGGSGGGGSGGGGS (SEQ ID NO: 26); when the scFv is linked to the C-terminus of the heavy or light chain of the first antibody, the linker S1 has an amino acid sequence as shown by (G 4 S) 2 , ie GGGGSGGGGS (SEQ ID NO: 27).
- a disulfide bond is present between the VH and VL of the scFv.
- Methods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, for example, see U.S. Patent Application No. 5,747,654; Rajagopal et al, Prot. Engin. 10 (1997) 1453-1459; Reiter et al.
- the amino acid at position 44 of VH and the amino acid at position 100 of VL of the scFv are respectively cysteine, wherein the amino acid position referred to is according to the position of the Kabat numbering system;
- the VH and VL of the scFv are linked by a disulfide bond formed between two cysteine residues at the 44th position of VH and the 100th position of VL, respectively.
- the first antibody specifically binds to CTLA-4 and the scFv specifically binds to PDL-1, wherein the scFv comprises:
- HCDR1 as shown in SEQ ID NO: 1
- HCDR2 as shown in SEQ ID NO: 2
- HCDR3 as shown in SEQ ID NO: 3
- LCDR1 as shown in SEQ ID NO: 4
- LCDR2 shown in :5
- LCDR3 as shown in SEQ ID NO: 6.
- the first antibody specifically binds to CTLA-4 and the scFv specifically binds to PDL-1, wherein:
- the first antibody comprises:
- HCDR1 as shown in SEQ ID NO: 13; HCDR2 as shown in SEQ ID NO: 14; and HCDR3 as shown in SEQ ID NO: 15; LCDR1 as shown in SEQ ID NO: 16; LCDR2 shown in 17; and LCDR3 as shown in SEQ ID NO: 18;
- the scFv contains:
- HCDR1 as shown in SEQ ID NO: 1
- HCDR2 as shown in SEQ ID NO: 2
- HCDR3 as shown in SEQ ID NO: 3
- LCDR1 as shown in SEQ ID NO: 4
- LCDR2 shown in :5
- LCDR3 as shown in SEQ ID NO: 6.
- the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 13; HCDR2 as set forth in SEQ ID NO: 14; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 16; LCDR2 as shown in SEQ ID NO: 17; and LCDR3 as shown in SEQ ID NO: 18; and, the scFv comprises: as SEQ ID NO: 1 HCDR1 is shown; HCDR2 as shown in SEQ ID NO: 2; and HCDR3 as shown in SEQ ID NO: 3; LCDR1 as shown in SEQ ID NO: 4; LCDR2 as shown in SEQ ID NO: And, LCDR3 as shown in SEQ ID NO: 6.
- the amino acid sequence of the heavy chain variable region (VH) of the first antibody has at least 90%, at least 91 of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 19. %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the first antibody
- the amino acid sequence of the light chain variable region (VL) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the amino acid sequence of the light chain variable region set forth in SEQ ID NO:21.
- amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 8 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99%, or 100% sequence identity
- amino acid sequence of the light chain variable region (VL) of the scFv The amino acid sequence of the light chain variable region of SEQ ID NO: 9 or SEQ ID NO: 10 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% At least 97%, at least 98%, at least 99%, or 100% sequence identity.
- the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 7; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: The light chain variable region shown by 9.
- the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 8; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: The light chain variable region shown by 10.
- the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 19; and the light chain variable region of the first antibody is selected From the light chain variable region of SEQ ID NO: 21; and, the heavy chain variable region of the scFv is selected from the heavy chain variable region of SEQ ID NO: 7; and, the light of the scFv
- the chain variable region is selected from the light chain variable region set forth in SEQ ID NO:9.
- the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 19; and the light chain variable region of the first antibody is selected From the light chain variable region of SEQ ID NO: 21; and, the heavy chain variable region of the scFv is selected from the heavy chain variable region of SEQ ID NO: 8; and, the light of the scFv
- the chain variable region is selected from the light chain variable region set forth in SEQ ID NO: 10.
- the first antibody comprises: VH as set forth in SEQ ID NO: 19 and VL as set forth in SEQ ID NO: 21;
- the scFv includes:
- VH as shown in SEQ ID NO: 7 and VL as shown in SEQ ID NO: 9;
- VH as shown in SEQ ID NO: 8 and VL as shown in SEQ ID NO: 10.
- the first antibody specifically binds to PDL-1 and the scFv specifically binds to CTLA-4, wherein:
- the first antibody comprises:
- HCDR1 as shown in SEQ ID NO: 1
- HCDR2 as shown in SEQ ID NO: 2
- HCDR3 as shown in SEQ ID NO: 3
- LCDR1 as shown in SEQ ID NO: 4
- LCDR2 shown in SEQ ID NO: 5
- LCDR3 as shown in SEQ ID NO: 6;
- the scFv contains:
- HCDR1 as shown in SEQ ID NO: 13; HCDR2 as shown in SEQ ID NO: 14; and HCDR3 as shown in SEQ ID NO: 15; LCDR1 as shown in SEQ ID NO: 16; LCDR2 shown in 17; and LCDR3 as shown in SEQ ID NO: 18.
- the first antibody specifically binds to PDL-1 and the scFv specifically binds to CTLA-4, wherein the first antibody comprises:
- HCDR1 as shown in SEQ ID NO: 1
- HCDR2 as shown in SEQ ID NO: 2
- HCDR3 as shown in SEQ ID NO: 3
- LCDR1 as shown in SEQ ID NO: 4
- LCDR2 shown in :5
- LCDR3 as shown in SEQ ID NO: 6.
- the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 1; HCDR2 as set forth in SEQ ID NO: 2; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 4; LCDR2 as shown in SEQ ID NO: 5; and LCDR3 as shown in SEQ ID NO: 6; and, the scFv comprises: as SEQ ID NO: 13 HCDR1 is shown; HCDR2 as shown in SEQ ID NO: 14; and HCDR3 as shown in SEQ ID NO: 15; LCDR1 as shown in SEQ ID NO: 16; LCDR2 as shown in SEQ ID NO: And, LCDR3 as shown in SEQ ID NO: 18.
- the amino acid sequence of the heavy chain variable region (VH) of the first antibody has at least 90%, at least 91 of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7. %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the first antibody
- the amino acid sequence of the light chain variable region (VL) and the amino acid sequence of the light chain variable region of SEQ ID NO: 9 are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the amino acid sequence of the heavy chain variable region (VH) of the scFv and SEQ ID NO:
- the amino acid sequence of the heavy chain variable region of 19 or SEQ ID NO: 20 has at least 90%, at least 91%, at least 91%
- the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 7; and the light chain variable region of the first antibody is selected From the light chain variable region set forth in SEQ ID NO:9.
- the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 7; and the light chain variable region of the first antibody is selected The light chain variable region shown in SEQ ID NO: 9; and the heavy chain variable region of the scFv is selected from the heavy chain variable region shown in SEQ ID NO: 19; and the light of the scFv The chain variable region is selected from the light chain variable region set forth in SEQ ID NO:21.
- the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 7; and the light chain variable region of the first antibody is selected From the light chain variable region of SEQ ID NO: 9; and, the heavy chain variable region of the scFv is selected from the heavy chain variable region of SEQ ID NO: 20; and, the light of the scFv The chain variable region is selected from the light chain variable region set forth in SEQ ID NO:22.
- the first antibody comprises: VH as set forth in SEQ ID NO: 7 and VL as set forth in SEQ ID NO: 9;
- the scFv includes:
- VH as shown in SEQ ID NO: 19 and VL as shown in SEQ ID NO: 21;
- VH as shown in SEQ ID NO: 20 and VL as shown in SEQ ID NO: 22.
- the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.
- the first polypeptide chain has an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in any one of SEQ ID NOs: 28, 29; and/or the second polypeptide The chain has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs: 11, 23.
- the bispecific antibody comprises:
- the first antibody of the bispecific antibody of the invention comprises: CH as indicated by Entry No. P01857 of Uniprot; and/or CL as indicated by Entry No. P01834 of Uniprot.
- the CH may further comprise a mutation in which the 117th, 118th, and 120th mutations in the CH of the Entry No. P01857 of UniProt are A; or the mutation of the 97th in the CH of the Entry No. P01857 of UniProt is R.
- the bispecific antibodies of the invention have antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In certain embodiments, the bispecific antibodies of the invention have antibody-dependent cell-mediated cytotoxicity (ADCC) activity and also have complement dependent cytotoxicity (CDC) activity. In certain preferred embodiments, the bispecific antibodies of the invention have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or have enhanced complement-dependent cytotoxicity (CDC) activity. In certain preferred embodiments, the enhanced ADCC and/or CDC activity is caused by a mutation in the first antibody CH of the bispecific antibody.
- the bispecific antibody of the invention has an equal or weaker binding activity to the first antigen than the parent antibody of the first antibody; in certain preferred embodiments, the bispecific of the invention An antibody having an equal or weaker binding activity to a second antigen than the parent antibody of the scFv; in certain preferred embodiments, the bispecific antibody of the invention has a first antibody The parent antibody is equivalent or weaker in binding to the activity of the first antigen and has an equal or weaker binding activity to the second antigen than the parent antibody of the scFv; in certain preferred embodiments, the invention a bispecific antibody having an activity of binding to a first antigen that is comparable to a parent antibody of the first antibody, and having a weaker binding activity to a second antigen than a parent antibody of the scFv; In some preferred embodiments, the bispecific antibodies of the invention have equivalent binding activity as compared to the parent antibody that binds PDL-1; in certain preferred embodiments, the bispecific antibodies of the invention, There is a weaker binding activity compared
- the bispecific antibodies of the invention have an affinity for CTLA-4 and PDL-1.
- the bispecific antibodies of the invention have comparable affinities for CTLA-4 and PDL-1 as compared to their respective parent antibodies.
- the bispecific antibodies of the invention have an affinity for PDL-1 that is comparable to that of their parent antibody.
- the bispecific antibodies of the invention have comparable affinities for CTLA-4 as compared to their parental antibodies.
- the bispecific antibodies of the invention have a weaker affinity for CTLA-4 than for their parent antibody.
- the bispecific antibodies of the invention have an affinity for PDL-1 that is comparable to that of their parent antibody, and CTLA-4 has a weaker affinity than its parent antibody.
- the bispecific antibodies of the invention have good thermal stability. In certain preferred embodiments, the bispecific antibodies of the invention have substantially the same thermal stability as compared to the parent antibody.
- the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody of the invention.
- the isolated nucleic acid molecule encodes a bispecific antibody of the invention.
- the isolated nucleic acid molecule comprises a nucleotide sequence encoding a first polypeptide chain of the invention. In certain preferred embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a second polypeptide chain of the invention. In certain preferred embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a first polypeptide chain of the invention and a nucleotide sequence encoding a second polypeptide chain of the invention.
- the invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention.
- a vector eg, a cloning vector or an expression vector
- the vector comprises a nucleotide sequence encoding a first polypeptide chain of the invention. In certain preferred embodiments, the vector comprises a nucleotide sequence encoding a second polypeptide chain of the invention. In certain preferred embodiments, the vector comprises a nucleotide sequence encoding a first polypeptide chain of the invention and a nucleotide sequence encoding a second polypeptide chain of the invention.
- the vectors of the invention are, for example, plasmids, cosmids, phages, and the like.
- the vector is capable of expressing a bispecific antibody, a first polypeptide chain, or a second polypeptide chain of the invention in a subject (eg, a mammal, eg, a human).
- the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention.
- host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
- the host cell of the invention is a mammalian cell, such as CHO (eg, CHO-K1, CHO-S, CHO DG44) or HEK293.
- the invention provides a method of making a bispecific antibody of the invention, comprising culturing a host cell of the invention under conditions permitting expression of the bispecific antibody, and culturing from the cultured host cell The bispecific antibody is recovered in the substance.
- the method comprises:
- an expression vector comprising a nucleotide sequence encoding a first polypeptide chain and a nucleotide sequence encoding a second polypeptide chain; or constructing a first nucleotide sequence comprising a first polypeptide chain
- An expression vector and a second expression vector comprising a nucleotide sequence encoding a second polypeptide chain;
- the bispecific antibodies of the invention can be used to inhibit CTLA-4 and PDL-1 activity, block CTLA-4 and/or PDL-1 signaling pathways, and for prevention and/or treatment in vitro or in a subject.
- a disease associated with CTLA-4 and/or PDL-1 eg, an autoimmune disease or a tumor or an infectious disease.
- the invention provides a pharmaceutical composition comprising a bispecific antibody of the invention, and a pharmaceutically acceptable carrier and/or excipient.
- the pharmaceutical composition may also comprise additional pharmaceutically active agents.
- the additional pharmaceutically active agent is for use in preventing and/or treating a disease associated with CTLA-4 and/or PDL-1 (eg, an autoimmune disease or a tumor or an infectious disease) a drug, such as an anti-inflammatory drug or an immunosuppressive agent, such as a non-steroidal anti-inflammatory drug (eg, ibuprofen, diclofenac, naproxen, indomethacin, piroxicam, meloxicam, nabumetone or nimese) Shuli), steroidal anti-inflammatory drugs (such as prednisone, dexamethasone or hydrocortisone), antibodies or antagonists of inflammatory cytokines.
- a disease associated with CTLA-4 and/or PDL-1 eg, an autoimmune disease or a tumor or an infectious disease
- the invention provides the use of a bispecific antibody of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for use in the prevention and/or treatment of a subject (eg, a human) CTLA-4 and/or PDL-1 related diseases (such as autoimmune diseases or tumors or infectious diseases), and/or for inhibiting CTLA-4 and/or PDL in vitro or in a subject (eg, a human) 1 activity.
- a subject eg, a human
- CTLA-4 and/or PDL-1 related diseases such as autoimmune diseases or tumors or infectious diseases
- the invention provides a method for preventing and/or treating a disease associated with CTLA-4 and/or PDL-1 in a subject (eg, a human) (eg, an autoimmune disease or tumor or infection) And/or a method for inhibiting the activity of CTLA-4 and/or PDL-1 in vitro or in a subject (eg, a human), wherein the method comprises administering to a subject in need thereof an effective A quantity of a bispecific antibody of the invention, or a pharmaceutical composition of the invention.
- the diseases associated with CTLA-4 and/or PDL-1 include, but are not limited to, autoimmune diseases or tumors or infectious diseases, such as tumors, including but not limited to: adenocarcinoma, leukemia, lymphoid Tumor, melanoma, sarcoma, or include but not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, Prostate, skin, salivary gland, spleen, testis, thymus, thyroid, and uterus-related tumors, including but not limited to hepatitis B, hepatitis A, and HIV.
- the bispecific antibody of the present invention or the pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules. Agents, tinctures, lozenges, suppositories, injections (including injections, sterile powder for injection and concentrated solutions for injection), inhalants, sprays, and the like.
- the preferred dosage form will depend on the intended mode of administration and therapeutic use.
- the pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage.
- a preferred dosage form is an injectable. Such an injection may be a sterile injectable solution.
- a sterile injectable solution can be prepared by incorporating the required amount of the bispecific antibody of the invention in a suitable solvent, and optionally, incorporating other desired ingredients (including but not limited to, A pH adjusting agent, a surfactant, an adjuvant, an ionic strength enhancer, an isotonicity agent, a preservative, a diluent, or any combination thereof, followed by filtration sterilization.
- sterile injectable solutions can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.
- the bispecific antibodies of the invention may be present in a pharmaceutical composition in unit dosage form for ease of administration.
- the unit dose is at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 6 mg, at least 7 mg, at least 8 mg, at least 9 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg At least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.
- the pharmaceutical composition is in the form of a liquid (eg, injection) dosage form, it may comprise a concentration of at least 0.1 mg/ml, such as at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg. /ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml, or at least 100 mg/ml of the bispecific of the invention antibody.
- concentration of at least 0.1 mg/ml such as at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg. /ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml, or
- the bispecific antibodies or pharmaceutical compositions of the invention may be administered by any suitable method known in the art including, but not limited to, orally, buccally, sublingually, ocular, topical, parenteral, rectal, intrathecal, intrathecal, In the cytoplasmic trough, in the groin, intravesical, local (eg, powder, ointment or drops), or nasal route.
- the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
- the bispecific antibody or pharmaceutical composition of the invention is administered by intravenous infusion or injection.
- the medicament, pharmaceutical composition or bispecific antibody provided by the present invention may be used singly or in combination, or may be used in combination with another pharmaceutically active agent such as an autoimmune disease or a tumor or an infectious disease.
- the bispecific antibodies of the invention are used in combination with other anti-inflammatory drugs or immunosuppressive agents to prevent and/or treat diseases associated with CTLA-4 and/or PDL-1 (eg, Autoimmune disease or tumor or infectious disease).
- additional pharmaceutically active agents can be administered prior to, concurrently with, or subsequent to administration of the bispecific antibodies of the invention or the pharmaceutical compositions of the invention.
- compositions of the invention may comprise a "therapeutically effective amount” or a "prophylactically effective amount” of a bispecific antibody of the invention.
- prophylactically effective amount is meant an amount sufficient to prevent, arrest, or delay the onset of a disease, such as a disease associated with CTLA-4 and/or PDL-1.
- therapeutically effective amount is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
- the therapeutically effective amount of the bispecific antibody of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, and the manner in which the drug is administered. , as well as other treatments administered at the same time, and the like.
- the dosage regimen can be adjusted to achieve the best purpose response (e.g., therapeutic or prophylactic response).
- the best purpose response e.g., therapeutic or prophylactic response
- it may be administered in a single administration, may be administered multiple times over a period of time, or may be proportionally reduced or increased depending on the urgency of the treatment.
- a typical non-limiting range of therapeutically or prophylactically effective amounts of the bispecific antibodies of the invention is from 0.02 to 50 mg/kg, such as from 0.1 to 50 mg/kg, from 0.1 to 25 mg/kg, or from 1 to 10 mg/kg. It should be noted that the dosage may vary depending on the type and severity of the condition to be treated. Moreover, those skilled in the art understand that for any particular patient, the particular dosage regimen should be adjusted over time according to the needs of the patient and the professional evaluation of the physician; the dosage ranges given herein are for illustrative purposes only and are not limiting Use or range of the pharmaceutical compositions of the invention.
- the subject may be a mammal, such as a human.
- the bispecific antibody of the present invention is capable of specifically binding to CTLA-4 and/or PDL-1, thereby being useful for detecting the presence or level of CTLA-4 and/or PDL-1 in a sample, and for diagnosing whether the subject is suffering from There are diseases associated with CTLA-4 and/or PDL-1 (such as autoimmune diseases or tumors or infectious diseases).
- the invention provides a kit comprising a bispecific antibody of the invention.
- the bispecific antibodies of the invention carry a detectable label.
- the kit further comprises a second antibody that specifically recognizes the first antibody or scFv of the bispecific antibody of the invention.
- the second antibody further comprises a detectable label.
- the detectable label may be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such markers are suitable for use in immunological assays (eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.).
- immunological assays eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.
- Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Red albumin (PE), Texas Red, rhodamine, quantum dot or cyanine dye derivatives (eg Cy7, Alexa 750), acridinium ester compounds, magnetic beads (eg, a heat-sensitive marker such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex, etc.) beads, and avidin modified for binding to the above markers (for example, streptavidin) Biotin.
- enzymes e.
- markers that teach the use of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241 each incorporated herein by reference.
- the markers encompassed in the present invention can be detected by methods known in the art.
- the radioactive label can be detected using a photographic film or a scintillation counter
- the fluorescent label can be detected using a photodetector to detect the emitted light.
- Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and the calorimetric label is detected by simply visualizing the stained label.
- a detectable label as described above can be linked to a bispecific antibody of the invention by a linker of varying length to reduce potential steric hindrance.
- the invention provides a method of detecting the presence or level of CTLA-4 and/or PDL-1 in a sample comprising the step of using a bispecific antibody of the invention.
- the bispecific antibodies of the invention also carry a detectable label.
- the method further comprises detecting a bispecific antibody segment of the invention using an agent with a detectable label. The method can be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).
- the invention provides a method of diagnosing whether a subject has a disease associated with CTLA-4 and/or PDL-1, such as an autoimmune disease or a tumor or an infectious disease, comprising: using the invention
- the bispecific antibody detects the presence or level of CTLA-4 and/or PDL-1 in a sample from the subject.
- the bispecific antibodies of the invention also carry a detectable label.
- the method further comprises the step of detecting the bispecific antibody of the invention using an agent with a detectable label.
- a bispecific antibody of the invention in a kit for detecting the presence or level of CTLA-4 and/or PDL-1 in a sample, or for The subject is diagnosed as having a disease associated with CTLA-4 and/or PDL-1 (such as an autoimmune disease or a tumor or infectious disease).
- PDL-1 is also referred to as “Programmed death-ligand 1", “Programmed cell death ligand 1”, “Protein PD-” L1", “PD-L1”, “PDL1”, “PDCDL1”, “hPD-L1”, “hPD-LI”, “CD274", and “B7-H1” are used interchangeably.
- antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
- Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains.
- Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
- the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
- Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
- the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
- Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
- the light chain constant region consists of one domain CL.
- the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
- the VH and VL regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FR).
- CDRs complementarity determining regions
- VH and VL each heavy/light chain pair form an antigen binding site, respectively.
- the assignment of amino acids to regions or domains can be followed by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; Chothia et al. (1989) Nature 342: 878-883.
- CDR complementarity determining region
- LCDR1 ⁇ , 50-56 ⁇ LCDR2 ⁇ , 89-97 ⁇ LCDR3 ⁇ , and residues 31-35 ⁇ HCDR1 ⁇ , 50-65 ⁇ HCDR2 ⁇ , 95-102 ⁇ HCDR3 ⁇ in the heavy chain variable region see, for example, Kabat et al, Sequences of Proteins of lmmunological lnterest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), or residues in the light chain variable region 26-32 ⁇ Ll ⁇ , 50-52 ⁇ L2 ⁇ , 91-96 ⁇ L3 ⁇ and residues 26-32 ⁇ H1 ⁇ , 53-55 ⁇ H2 ⁇ , 96-101 ⁇ H3 ⁇ in the heavy chain variable region (see, Chothia) And Lesk J.
- the CDRs can be obtained by technical means well known to those skilled in the art, for example, by analyzing the amino acid sequence of the heavy chain variable region or the amino acid sequence of the variable region of the light chain according to the IMGT definition by the VBASE2 database.
- framework region or "FR” residue refers to those amino acid residues in the variable regions of an antibody other than the CDR residues as defined above.
- antibody is not limited by any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
- the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
- antigen binding site refers to a portion of the heavy and light chain variable regions (VH and VL) that are formed by amino acid residues that participate in antigen binding, including those An amino acid residue that interacts with an antigen and determines its specificity and affinity for the antigen.
- the antigen binding site may also comprise amino acid residues of the framework regions necessary for maintaining the proper conformation of the amino acid residues directly binding to the antigen described above.
- full length antibody means an antibody consisting of two “full length heavy chains” and two “full length light chains”.
- full-length heavy chain refers to a polypeptide chain which is composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain in the N-terminal to C-terminal direction.
- VH heavy chain variable region
- HR hinge region
- the constant region CH2 domain, the heavy chain constant region CH3 domain is composed; and, when the full length antibody is of the IgE isotype, optionally further comprises a heavy chain constant region CH4 domain.
- the "full length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
- a “full length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
- VL light chain variable region
- CL light chain constant region
- Two pairs of full length antibody chains are joined by a disulfide bond between the disulfide bond between CL and CH1 and the HR of the two full length heavy chains.
- the full length antibodies of the invention may be from a single species, such as a human; or may be chimeric or humanized antibodies.
- the full-length antibody of the present invention comprises two antigen-binding sites formed by a pair of VH and VL, respectively, which specifically recognize/bind the same antigen.
- Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains;
- Fab'fragment means reducing two residues in a F(ab') 2 fragment.
- the fragment obtained after the disulfide bond of the strand fragment consists of a complete Fd fragment of the light and heavy chains (consisting of the VH and CH1 domains);
- F(ab') 2 fragment means the inclusion of the region through the hinge
- An antibody fragment of two Fab fragments joined by a disulfide bridge Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody for specific binding to the antigen.
- scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, eg, Bird et al., Science 242: 423). -426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
- Such scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
- Disulfide bonds may also be present between the VH and VL of the scFv of the invention. Methods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, for example, see U.S. Patent Application No. 5,747,654; Rajagopal et al, Prot. Engin. 10 (1997) 1453-1459; Reiter et al.
- di-scFv refers to an antibody fragment formed by the joining of two scFvs.
- Fv fragment means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
- the term "parent antibody” refers to an anti-PDL-1 antibody or an anti-CTLA-4 antibody used to prepare the bispecific antibody of the present invention, which antibody has an amino acid sequence which can be replaced by, for example, amino acid substitution. Or a structural alteration or the like for the preparation of a first antibody or scFv comprised by the bispecific antibody of the invention.
- the CDRs, VH, VL, CH, CL, HC, and LC contained in the bispecific antibody of the present invention may also be derived from other antibodies or antibody fragments thereof known in the art capable of specifically binding PDL-1 or CTLA-4. Or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with the above-mentioned known antibodies, antibody fragments thereof or CDRs thereof, VH, VL, CH, CL, HC, LC An antibody that is at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
- linker refers to a linear polypeptide formed by the joining of multiple amino acid residues by peptide bonds.
- the linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region.
- linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121- 1123)).
- the term “specifically binds” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen to which it is directed.
- an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
- K D refers to a particular antibody - antigen interaction solutions dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen.
- the antibody e.g., an antibody of the invention
- the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
- the dissociation equilibrium constant (K D ) binds to an antigen (eg, HBsAg), for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument.
- vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
- a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
- the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
- Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
- Phage such as lambda phage or M13 phage and animal virus.
- Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
- a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
- the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
- a prokaryotic cell such as Escherichia coli or Bacillus subtilis
- a fungal cell such as a yeast cell or an Aspergillus.
- S2 Drosophila cells or insect cells such as Sf9
- animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
- identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
- a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
- Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
- the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
- the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
- the comparison is made when the two sequences are aligned to produce maximum identity.
- Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
- the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
- the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
- amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
- alanine can be represented by A or Ala.
- pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancement. Agents, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives.
- pH adjusting agents include, but are not limited to, phosphate buffers.
- Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
- Ionic strength enhancers include, but are not limited to, sodium chloride.
- Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
- Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
- Agents that delay absorption include, but are not limited to, monostearate and gelatin.
- the term "subject” refers to a mammal, such as a primate mammal, such as a human.
- the subject eg, a human
- diseases or disease states are characterized by their benefit from a reduction in CTLA-4 and/or PDL-1 levels or inhibition of CTLA-4 and/or PDL-1 activity resulting in amelioration or cure.
- an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
- an effective amount to prevent a disease eg, a disease associated with CTLA-4 and/or PDL-1
- a disease eg, a disease associated with CTLA-4 and/or PDL-1
- Amount that occurs; an effective amount to treat a disease refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
- the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
- ADCC antibody-dependent cell-mediated cytotoxicity
- cytotoxic cells eg, natural killer (NK) cells, neutrophils.
- FcR Fc receptor
- Methods for detecting ADCC activity of an antibody are known in the art and can be evaluated, for example, by measuring the binding activity between an antibody to be tested and an Fc receptor (e.g., CD16a).
- complement dependent cytotoxicity refers to the activation of a cytotoxic form of the complement cascade by binding complement component Clq to antibody Fc.
- Methods for detecting the CDC activity of an antibody are known in the art and can be evaluated, for example, by measuring the binding activity between an antibody to be tested and an Fc receptor (e.g., C1q).
- the bispecific antibody of the present invention not only specifically recognizes/binds CTLA-4 and PDL-1, but also has affinity for CTLA-4 and PDL-1 comparable to that of its respective parent antibody, or affinity for CTLA-4.
- the parent antibody is attenuated and can significantly and simultaneously inhibit the activity of CTLA-4 and/or PD-L1 in vitro and in the subject, blocking the CTLA-4 and/or PD-L1 signaling pathway.
- the bispecific antibody of the present invention has an excellent thermal stability equivalent to that of the parent antibody of the first antibody. It is particularly surprising that in some cell-level and in vivo experiments, the bispecific antibodies of the present invention exhibit even greater inhibitory activity and reduced antibody toxicity than their parental antibodies, increasing the safety of the drug. Therefore, the bispecific antibody of the present invention has potential for treating diseases associated with CTLA-4 and PDL-1, such as autoimmune diseases or tumors or infectious diseases, and has great clinical value.
- Figure 1 shows a schematic representation of the structure of anti-PDL-1 and CTLA-4 recombinant bispecific antibodies AB03 and AB04.
- Figure 2 shows the reduced SDS-PAGE electropherogram of AB03 and AB04.
- the lanes from left to right are: AB01 reduction, AB02 reduction, AB03 reduction, AB04 reduction.
- Figure 3 shows the binding activity of the antibody to PDL-1.
- FIGS 4A and 4B in Figure 4 show the binding activity profiles of antibodies to different batches of CTLA-4.
- Figure 5 shows the binding activity profile of the antibody to both PDL-1 and CTLA-4.
- Figure 6 shows the antibody blocking PD-1 to PDL-1 binding activity curve.
- Figure 7 shows the binding activity of antibodies to CD16a (Val).
- Figure 8 shows the binding activity curve of the antibody to C1q.
- Figure 9 shows the antibody blocking PD1/PDL-1 cell binding activity curve.
- Figure 10 shows a graph of the activity of an antibody to enhance IL-2 secretion by T cells under SEB-stimulated PBMC.
- Figure 11 shows the efficacy of AB01 in a non-small cell lung cancer model.
- Figure 12 shows the efficacy of antibodies in a mouse model of colon cancer.
- Figure 13 shows the efficacy of antibodies in a mouse model of non-small cell lung cancer.
- AB01-scFv indicates the parent antibody AB01-derived scFv, the variable region of which differs from AB01 in that the VH at position 44 and the VL at position 100 of the scFv are respectively cysteine;
- AB02-scFv indicates the parent antibody
- the AB02-derived scFv differs in its variable region from AB02 in that the VH at position 44 and the VL at position 100 of the scFv are respectively cysteine; other similar expressions have similar meanings.
- Example 1 Construction of an expression vector encoding an anti-PDL-1/CTLA-4 recombinant bispecific antibody
- the anti-CTLA-4 parent antibody (AB02) shown in Table 2 was purchased from Squibb, and the anti-PDL-1 parent antibody (AB01) shown in Table 2 was first obtained by DNA recombination technique, and then An expression vector comprising a nucleotide sequence encoding a first polypeptide chain and an expression vector encoding a nucleotide sequence of a second polypeptide chain are separately constructed to obtain recombinant antibodies AB03 and AB04 of the present invention.
- each parent antibody AB01 and AB02. The nucleic acid sequence encoding the parent antibody (AB01) was synthesized by Nanjing Kingsray Biotechnology Service Co., Ltd. and cloned into the PUC19 vector.
- Table 2 Variable and constant region sequences of parental antibodies and PDL-1/CTLA-4 recombinant bispecific antibodies
- the database number is the index number of Uniprot.
- the nucleotide sequence encoding the first polypeptide chain of the recombinant antibody and the nucleotide sequence encoding the second polypeptide chain were constructed in accordance with the construction of each recombinant antibody shown in Table 3.
- VL and VH are linked by a peptide linker S2 (SEQ ID NO: 25, amino acid sequence: GGGGSGGGGSGGGGSGGGGS), and the VH of the scFv parent antibody is determined by a PCR site-directed mutagenesis method.
- the amino acid at position 100 and VL is mutated to cysteine (Cys, C), respectively, such that a disulfide bond is formed between VH and VL of the scFv.
- the amino acid sequence of the mutated AB01-scFv is shown in SEQ ID NO: 31, the amino acid sequence of the VL after mutation is shown in SEQ ID NO: 10, and the amino acid sequence of VH after mutation is shown in SEQ ID NO: 8.
- the amino acid sequence of the mutated AB02-scFv is shown in SEQ ID NO: 32, the amino acid sequence of the VL after mutation is shown in SEQ ID NO: 22, and the amino acid sequence of VH after mutation is shown in SEQ ID NO: 20.
- nucleic acid construct of a partially recombinant antibody as an example is constructed as follows:
- the nucleotide sequence encoding AB02-scFv is ligated to the 5' end of the nucleotide sequence encoding VH of AB01 by the nucleotide sequence encoding the linker S1 of SEQ ID NO: 26, and then ligated to the heavy chain constant region UniProt
- the 5' end of the coding nucleotide sequence of P01857 constructs a nucleotide sequence encoding the first polypeptide chain of AB03; wherein the sequence of AB02-scFv from N-terminus to C-terminus is VL-S2-VH, and the encoded amino acid sequence thereof As shown in SEQ ID NO:32.
- the nucleotide sequence encoding the AB01 light chain is used as the coding nucleotide sequence of the second polypeptide chain of ABO3.
- the nucleotide sequence of the VH of AB01 was ligated to the 5' end of the coding nucleotide sequence of the heavy chain constant region UniProt P01857, and the nucleotide sequence encoding the linker S1 of SEQ ID NO: 27 was ligated to the coding AB02-scFv.
- a nucleotide sequence encoding the first polypeptide chain of AB04 is constructed; wherein the sequence of the AB02-scFv from the N-terminus to the C-terminus is VL-S2-VH, and the encoded amino acid sequence is SEQ. ID NO: 32.
- the nucleotide sequence encoding the AB01 light chain is used as the coding nucleotide sequence of the second polypeptide chain of ABO4.
- a nucleotide sequence encoding each of the first polypeptide chain or the second polypeptide chain described above is linked to a nucleotide sequence encoding an IgG-Kappa signal peptide (amino acid sequence SEQ ID NO: 30), and by homologous recombination
- the pTT5 plasmid was introduced separately to construct an expression vector encoding the first polypeptide chain and an expression vector pTT5 encoding the second polypeptide chain.
- the amino acid sequences of the finally obtained recombinant antibodies are shown in Table 4.
- CHO-S cells purchased from Thermo Corporation, Cat. No. A1155701
- CHOgro medium purchased from MIRUS
- the plasmid to be transfected obtained in Example 1 was sterilized by filtration through a 0.22 ⁇ m filter, and 125 ⁇ g of the first polypeptide chain recombinant plasmid and 125 ⁇ g of the corresponding second polypeptide chain recombinant plasmid were added, and 25 ml of CHOgro complex was added to form a solution (CHOgro).
- the CHO-S cell culture expressed in Example 2 for 7 days was centrifuged at a low speed to separate the supernatant from the cell pellet; and then centrifuged at high speed to obtain a clear liquid.
- the recombinant antibody was purified by affinity chromatography (Protein A) and ion exchange two-step method.
- the medium used in the purification was Mab Select SuRe produced by GE Company and Eshmuno CPX produced by Millipore Company.
- the expression levels of each recombinant bispecific antibody were basically consistent, all in the range of 40-85 mg/L, and consistent with the expression of the same condition anti-PDL-1 parental antibody AB01, indicating that each recombinant antibody can be successfully expressed and has a higher expression.
- the expression efficiency, the specific expression level is shown in Table 5.
- the isolated and purified recombinant antibody was concentrated by ultrafiltration tube and exchanged into PBS solution.
- the reduction SDS-PAGE electrophoresis showed that as shown in Fig. 2, AB01 and AB02 were reduced to 50kDa (heavy chain) and 25kDa (light chain);
- the antibody AB03, AB04 reduced band size was 75 kDa (first peptide chain) and 25 kDa (second peptide chain), and the SEC purity was 96.34% and 96.04%, respectively.
- the size of the bands was in line with expectations, indicating that the recombinant bispecific antibodies were highly expressed, assembled correctly, and showed no obvious aggregation degradation, indicating good stability.
- Antibody name Obtain protein amount Expression yield AB01 20.5mg 60mg/L AB03 22.6mg 83mg/L AB04 12.2mg 42mg/L
- Example 4 Detection of antigen binding activity of anti-PDL-1/CTLA-4 recombinant bispecific antibody
- the difference in affinity of each recombinant bispecific antibody to the same antigen as its parent antibody is detected by ELISA, and the relative affinity of the recombinant bispecific antibody to simultaneously bind the two antigens is verified to verify the ability to block an antigen alone. Whether it is lower than the parent antibody; whether it can block both antigens at the same time, blocking the two signaling pathways, thereby producing a synergistic effect.
- the recombinant PDL-1-mFc protein (obtained from Columboate uniport No.: Q9NZQ7) was added to a 96-well microtiter plate (purchased from Thermo Corporation) at 100 ng/well, and coated at 4 ° C overnight; the next day, the solution in the well was discarded.
- the EC50 of the recombinant bispecific antibody AB03 combined with CTLA-4 was slightly increased compared with the parent antibody AB02, and the EC50 of the recombinant bispecific antibody AB04 binding to CTLA-4 was about 10 of the parent antibody AB02. -12 times, it can be seen that the activity of AB04 in combination with CTLA-4 is significantly weaker than that of the parent antibody, but still has a high binding activity at the pM level.
- the recombinant PDL-1-mFc protein was coated in a 96-well microtiter plate at 100 ng/well at 4° C.; the next day, the solution was discarded and washed with a washing buffer (phosphate buffer containing 0.05% Tween-20).
- a washing buffer phosphate buffer containing 0.05% Tween-20
- the EC50 results are shown in Table 8.
- the recombinant bispecific antibodies AB03 and AB04 bind to both PDL-1 and CTLA-4 antigens at the pM level.
- the above results indicate that the recombinant bispecific antibody of the present invention binds to an antigen.
- recombinant bispecific antibodies such as AB03 and AB04 can simultaneously bind the two antigens efficiently, and can simultaneously block the PDL-1 and CTLA-4 targets that play a key role in tumor immunity. Simultaneous inhibition of the two signaling pathways, thereby playing a synergistic role in the treatment of tumors.
- Example 5 Recombinant bispecific antibody blocks PD-1/PDL-1 binding activity by competitive ELISA assay
- the recombinant PD1-hFc protein (purchased from Beijing Yishen Shenzhou Co., Ltd.) was added to a 96-well microtiter plate (purchased from Thermo Corporation) at 100 ng/well, and coated at 4 ° C overnight; the next day, the solution in the well was discarded, and the washing buffer was used ( Washed with 0.05% Tween-20 phosphate buffer), patted dry; added 2% BSA in PBS solution, 100 ⁇ l/well, blocked at 37 °C for 2h and patted dry; recombinant bispecific antibody AB03, AB04 and parent Antibody AB01 was started at 10 ⁇ g/ml, diluted 1.5 times in 11 gradients, and an equal volume of 1.6 ⁇ g/ml PDL-1-mFC (obtained from Columboate) was added and allowed to stand at room temperature for 30 min, then 100 ⁇ l/well was added.
- the plate was incubated in the plate at 37 ° C for 2 h, patted dry, washed 3 times with washing buffer; HRP-conjugated Goat Anti-mouse IgG (H + L) (purchased from Thermo) solution, 100 ⁇ l /
- HRP-conjugated Goat Anti-mouse IgG H + L
- the wells were incubated at 37 ° C for 1 h; 100 ⁇ l/well was added to the TMB solution, and reacted at room temperature for about 5 min; 100 ⁇ l/well was added to the stop solution, and placed in a microplate reader to read the OD450 absorbance.
- the experimental data was fitted by curves, as shown in Figure 6, and the EC50 was calculated.
- Example 6 ELISA detection of CDC and ADCC activities of recombinant bispecific antibodies
- the activity of the recombinant bispecific antibody ADCC and CDC was verified, and the binding of the recombinant bispecific antibody to CD16a (Val) and C1q was detected by an ELISA assay.
- the recombinant CD16a (Val) protein (purchased from Yishen Shenzhou Co., Ltd.) was added to a 96-well microtiter plate (purchased from Thermo Corporation) at 100 ng/well, and coated at 4 ° C overnight; the next day, the solution in the well was discarded, and the washing buffer was used ( Washed with 0.05% Tween-20 phosphate buffer), patted dry; added 2% BSA in PBS solution, 100 ⁇ l/well, blocked at 25°C for 2h, patted dry; recombinant bispecific antibody AB03, AB04 and parent Antibody AB01 and positive control antibody Human IgG1 (obtained from Columboate) were added to Goat F(ab) 2 anti-Human Kappa (purchased from BIORAD) at 37 ° C for 1 h at 50 ⁇ g/ml, then divided into 11 gradients.
- the detected recombinant bispecific antibodies AB03 and AB04 have the activity of binding to CD16a (Val) protein and are equivalent to Human IgG1, while AB01 almost does not bind to CD16a (Val), indicating that the recombinant bispecific of the present application
- the antibody has ADCC activity.
- Recombinant bispecific antibodies AB03, AB04 and parental antibody AB01 and positive control antibody Human IgG1 were started at 500 ⁇ g/mL, diluted 3 times in 11 gradients, 100 ⁇ l/well, and added to 96 wells. The plate was plated overnight at 4 °C. The next day, the solution in the well was discarded, washed once with washing buffer, and photographed dry; PBS solution containing 2% BSA was added, 100 ⁇ l/well, blocked at 37 ° C for 2 h, and patted dry.
- the activity of the detected recombinant bispecific antibody AB03, AB04 in binding to the C1q protein was stronger than that of Human IgG1, indicating that it had stronger CDC activity.
- the recombinant bispecific antibody and the parent antibody AB01 were diluted to 1 mg/ml with PBS solution, Orange Protein Gel Stain (available from Thermo Company, Cat. No. S6651) was diluted 40 times with distilled water. Take a 0.2 ml centrifuge tube and add 12.5 ⁇ l of the diluted sample in turn. 4.2 ⁇ l of orange protein gel staining solution and 8.3 ⁇ l of distilled water. After mixing, the sample was placed in a real-time PCR instrument (purchased from Thermo Corporation, Model 7500), and the reaction parameters were set at 25 ° C for 3 min, and increased to 95 ° C at 1 ° rate, and 95 ° C for 2 min.
- Orange Protein Gel Stain available from Thermo Company, Cat. No. S6651
- the recombinant bispecific antibody was diluted to 1 mg/ml with PBS solution, 9 ⁇ l/well, and placed in a high-throughput protein stability analyzer (UNCHAINED LABS, model Uncle), and the reaction parameters were set at 25 ° C to 95 ° C, at a rate of 0.3 ° C / Min.
- the recombinant bispecific antibodies AB03 and AB04 have good thermal stability. Among them, AB03 has better thermal stability than AB04, and the Tm values of parental antibodies AB01 and AB02 are more similar. The recombinant bispecific antibody of the invention has good thermal stability as a whole.
- Example 8 Blocking of PD1/PDL-1 cell activity by recombinant bispecific antibodies
- a reporter gene method was used to detect the biological activity of the PD-1/PDL-1 antibody.
- CHO-PDL-1-CD3L cell line stably expresses PDL-1 and anti-CD3-scFv as target cells;
- Jurkat-PD-1-NFAT cell line stably expresses PD-1 and luciferase, luciferase
- the gene is regulated by the NFAT element (transcription factor) (IL-2 promoter) as an effector cell.
- IL-2 promoter transcription factor
- PD-1 binds to PDL-1 to block the transduction of CD3 downstream signals, thereby inhibiting the expression of luciferase.
- IL-2 promoter transcription factor
- CHO-S-OKT3-PDL1 cells purchased from Promega
- assay buffer RPMI1640+1% FBS resuspended CHO-S-OKT3-PDL1 cells were plated at 5 x 10 5 / 40 ⁇ L/well.
- Jurkat-PD1-NFAT cells 2.5 ⁇ 10 6 /40 ⁇ L/well were added to wells pre-plated CHO-S-OKT3-PDL1 cells, and 40 ⁇ L of 100 ⁇ g/mL starting 5-fold diluted recombinant bispecific antibody AB03, AB04 and AB01 antibodies.
- Example 9 Recombinant bispecific antibody enhances IL-2 secretion by T cells under SEB stimulation of PBMC
- Superantigen can cross-link the TCR of MHC II and T cells of APC cells to activate T cells and promote IL-2 cytokine expression. Binding of PD1 to PDL-1 on T cells and binding of CDLA-4 to CD80/CD86 on APC cells on T cells inhibited T cell activation and decreased IL-2 cytokine expression.
- the recombinant bispecific antibody of the present invention can block PD1/PDL-1 and CTLA-4/CD80 (CD86), and further promote SEB stimulation of IL-2 expression by PBMC.
- the detected recombinant bispecific antibodies AB03 and AB04 significantly increased the secretion of IL-2 by PB stimulated by SEB as compared with the parental antibodies AB01 and AB02.
- the recombinant bispecific antibody and the parent antibody exerted the maximum response value at high concentration.
- the recombinant bispecific antibodies AB03 and AB04 have a certain enhancement function compared with the parental antibodies AB01 and AB02 (marking asterisk) At).
- Example 10 In vivo efficacy of PDL-1 antibody AB01 on colon cancer
- Human IgG is provided by Sichuan Kelun Pharmaceutical Research Institute Co., Ltd.; Purchased from Roche, human IgG was purchased from Chengdu Rongsheng Pharmaceutical Co., Ltd.
- MC-38/H-11 cells are mouse colon cancer MC-38 (purchased from Cobioer, Cat. No. CBP60825) cells knocked out mouse endogenous PDL-1, transfected and expressed human PDL-1 monoclonal cells, therefore MC-38/H-11 cells only highly expressed human PDL-1 protein.
- mice C57BL/6 mice, 7-8 weeks old, purchased from Shanghai Slack Laboratory Animals Co., Ltd.
- mice were subcutaneously inoculated with 1 ⁇ 10 5 MC-38/H-11 cells, and the next day after inoculation (D0), randomized and intraperitoneally injected (IP) drugs once every other day (Q2D); Volume of human IgG (15mg/kg), AB01 (1.5, 5, 15mg/kg), (15 mg/kg), injection volume 0.1 mL/10 g body weight. Each group was 10 mice.
- the experimental index is to investigate the effect of drugs on tumor growth, and the specific indicators are T/C% or tumor inhibition rate TGI (%).
- the tumor diameter was measured twice a week with a vernier caliper.
- the tumor volume (V) was calculated as:
- V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
- T/C% T/C ⁇ 100
- C and T are the tumor volume or tumor weight of the solvent group and the treatment group, respectively.
- Tumor inhibition rate (TGI) (%) (C-T) / C ⁇ 100, C, T are the tumor volume or tumor weight of the solvent group and the treatment group, respectively.
- Table 12 AB01 (1.5, 5, 15 mg/kg), Efficacy of subcutaneous xenografts in mouse colon cancer MC-38/H-11 mice
- the tumor inhibition rates of AB01 (1.5, 5, 15 mg/kg) on subcutaneous xenografts of MC-38/H-11 mice were 63.9%, 75.8% and 68.6%, respectively, based on the mean tumor volume; Individual differences are large, and the median tumor volume is calculated to be more reasonable, and the tumor inhibition rate is adjusted to 100%, 100%, and 100%; reference drug (15mg/kg)
- the inhibition rate of MC-38/H-11 was 93.8% (calculated according to the median tumor volume); the tumor inhibition rate was calculated by the median tumor weight, AB01 (1.5, 5, 15mg/kg)
- the inhibition rate of MC-38/H-11 was 100%, 100%, 100%, respectively.
- the tumor inhibition rate was 93.7%; the median tumor volume was very consistent with the tumor weight calculated from the median tumor weight, indicating the reliability of the tumor volume measurement method.
- AB01 (1.5, 5, 15mg/kg) not only inhibited tumor growth, but also inhibited tumor formation.
- the dose ratio of AB01 (1.5, 5, 15mg/kg) was 40%, 40% and 40%, The tumor formation rate of the group was 50%.
- the tumor-bearing mice were well tolerated by the above drugs, and no obvious weight loss and other symptoms occurred.
- AB01 (1.5, 5, 15 mg/kg) compared to subcutaneous xenografts in mouse colon cancer MC-38/H-11 mice. Has a stronger anti-tumor effect.
- Example 11 In vivo efficacy of PDL-1 antibody AB01 on lung cancer
- Non-small cell lung cancer cells HCC827 (purchased from ATCC No. CRL-2868) were subcutaneously inoculated with NOG mice to construct a lung cancer-bearing mouse model, until the tumor grew to about 100 mm 3 , and the mice were intravenously activated before administration.
- Human PBMCs mimic the human immune system and are then administered.
- Dosing schedule The dose was 10 mg/kg, intravenously, once every two days, for a total of four doses. Tumor volume was measured twice weekly after administration. Divided into control IgG, AB01, Three groups of six each.
- the tumor growth curve is shown in Figure 11.
- the tumor volume of the AB01 group was significantly smaller than that on the fourth day.
- the tumor growth of AB01 was almost completely inhibited.
- the tumors in the group and the IgG control group were continuously growing, which proved that the AB01 antibody was more Has a stronger anti-tumor effect in vivo.
- Example 12 Testing of recombinant bispecific antibodies in a transgenic colon cancer mouse model
- MC38-1F3 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37 ° C, 5% CO 2 , and MC38-1F3 cells in exponential growth phase were collected, resuspended in PBS to the appropriate concentration, and inoculated into females.
- -C57BL/6J-huCTLA-4 mice Jiji Keikang
- tumor size When the average volume of tumors was about 82 mm 3 , they were randomly grouped according to tumor size, which were intravenous immunoglobulin (negative control) group, AB01 control group, AB04 antibody low-dose group and high-dose group, intraperitoneal injection, 2 per week. The total dose was 3 weeks.
- the tumor volume and body weight of the mice were observed and periodically measured after administration. The specific results are shown in Table 13, Figure 12.
- the anti-tumor effect of AB04 antibody low-dose group (3mg/kg) on MC38-1F3 colon cancer xenograft model is better than AB01, and its high-dose group (10mg/kg) has significantly better anti-tumor effect than tumor.
- anti-tumor efficacy was significant. All the treatment groups showed no animal death and significant animal weight loss during the observation period, and no significant drug toxicity was observed. The mice were well tolerated by AB04 antibody during the treatment period.
- Example 13 Testing of recombinant bispecific antibodies in a transgenic non-small cell lung cancer mouse model
- the human non-small cell lung cancer cell line HCC827 was cultured in RPMI1640 medium containing 10% fetal bovine serum at 37 ° C under 5% CO 2 .
- HCC827 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female NSG immunodeficient mice to establish a xenograft model (hPBMC-NSG-HCC827 human immunoreconstruction model).
- hPBMC-NSG-HCC827 human immunoreconstruction model When the average tumor volume was about 77 mm 3 , they were randomly grouped according to tumor size, which were normal saline (negative control) group, AB01 control group, low dose group of AB04 antibody, and high dose group.
- PBMC Human peripheral blood mononuclear cells PBMC were cultured in RPMI 1640 medium containing 10% fetal bovine serum, CD28/CD3 and DNase at 37 ° C, 5% CO 2 . After 3 days of stimulation with CD3 and CD28, activated PBMCs were harvested, resuspended in PBS to a suitable concentration, and tail veins were inoculated into female NSG mice that had been included in the group to reconstitute their immune system. After the group, the tail vein was injected twice a week for a total of 3 weeks. The tumor volume and body weight of the mice were observed and periodically measured after administration. The specific results are shown in Table 14, Figure 13.
- the low dose group of AB04 antibody (1mg/kg) has a certain inhibitory effect on the tumor growth of subcutaneously transplanted human non-small cell lung cancer HCC827 in NSG-immunized mice, and the high-dose group (10mg/kg) has anti-tumor efficacy.
- All treatment groups showed no animal death and significant animal weight loss during the observation period, and no significant drug toxicity was observed. The mice were well tolerated by antibodies during the treatment period.
- this experiment was intravenous cynomolgus monkeys (one male and one female), single administration, set administration group AB04, control group was normal saline, recovery The period is 14 days.
- the experimental results showed that a single intravenous injection of 500 mg/kg of AB04, cynomolgus monkeys were well tolerated by drugs, with an MTD of 500 mg/kg; repeated doses were given at a dose of 100 mg/kg once a week for 5 consecutive doses. There was no significant toxicity to the target organ and the site of administration, and NOAEL (no toxicity dose) was 100 mg/kg.
- the anti-CTLA-4 antibody Ipilimumab (trade name: Yervoy) was administered with a NOAEL of 10 mg/kg, (at this dose, the spleen weight was reduced after administration, and multiple organ lymphocytes were observed.
- a mild toxicity such as infiltration), MTD is 30 mg/kg.
- AB04 reduces the toxicity of CTLA-4 antibody and improves the safety of the drug to some extent.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
提供了一种针对CTLA-4和PDL-1的重组双特异性抗体,编码抗体的核酸分子,包含该核酸分子的载体和宿主细胞,以及制备该抗体的方法。还提供了包含该双特异性抗体的药物组合物、以及该双特异性抗体在制备药物组合物中的用途。
Description
本发明涉及生物医药领域,特别是预防和/或治疗与CTLA-4和/或PDL-1相关的疾病的领域。具体而言,本发明涉及特异性结合CTLA-4和PDL-1,或PDL-1和CTLA-4的双特异性抗体。
PD-1/PDL-1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥重要作用。PD-1(programmed cell death 1,程序性细胞死亡因子1)的表达主要在T细胞等免疫细胞,而PD-1的配体PDL-1主要在许多人类肿瘤组织呈高表达。应用免疫组织化学方法,已先后在乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤等多种人类肿瘤组织中检测到PDL-1蛋白的表达,且PDL-1的表达水平和患者的临床及预后紧密相关。
阻断PD-1/PDL-1信号通路可使被抑制的T细胞激活,进而攻击癌细胞。阻断PD-1/PDL-1信号可以促进肿瘤抗原特异性T细胞的增殖,发挥杀伤肿瘤细胞的作用,进而抑制局部肿瘤生长(Julie R et al.,2012,N Engl J Med.366:2455–2465);PDL-1单抗可上调浸润CD8+T细胞IFN-γ的分泌,表明PD-1/PDL-1信号通路的阻断在以诱导免疫应答为目的的肿瘤免疫应答中发挥作用(Blank C et al.,2006,Int.J.Cancer.119:317–327)。
另外,PDL-1在体内还可以与B7-1结合。已有研究表明PDL-1/B7-1复合物也是T细胞活化的负信号,二者的结合可以导致T细胞表面活化标记物的表达下降,抑制T细胞增殖等。
细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte associated antigen-4,亦简称为CTLA-4)与CD28分子在基因结构、染色体定位、序列的同源性及基因表达具有十分相近的关系,都是共刺激分子B7的受体,主要表达于被激活T细胞表面。CTLA-4与B7结合后能抑制小鼠和人T细胞 的激活,在T细胞活化中起负调节作用。
目前,药品审批部门在CTLA-4靶点上批准了Ipilimumab
在PD-1和PDL-1靶点上批准了Nivolumab
和Pembrolizumab
Atezolizumab
Durvalumab
等。
Ipilimumab
抗体被FDA批准用于治疗晚期黑色素瘤,但该抗体注射液存在严重的甚至致命的免疫介导性不良反应。这些免疫介导性不良反应可能涉及到任何器官系统;然而,最常见的严重的免疫介导性不良反应为小肠结肠炎、肝炎、皮炎(包括中毒性表皮坏死松懈症)、神经病及内分泌病。大多数这些免疫介导性反应最初出现在治疗过程中;而另外一小部分则发生于停药后的几星期内。对于严重的免疫介导性反应,应永久停用
并开始进行全身性大剂量的进行糖皮质激素治疗。
双特异性抗体(BsAb)是一类具有双亲嗜性的组合抗体,通常是双价的(也有四价和六价的),即有两条抗原结合臂,具有结合两种不同特异性抗原的功能。中国专利申请CN106967172A公开了抗CTLA-4-抗PD-1双功能抗体、其药物组合物及其用途,CN104987421A也公开了特异结合PD-1和/或CTLA-4的双重可变结构域免疫球蛋白。与此同时施贵宝2017年8月公布了其Opdivo/Yervoy组合作为一线疗法在晚期肾癌(RCC)病人与Sutent(舒尼替尼)比较的三期临床CM214的顶层数据。Opdivo/Yervoy组合的应答率为41.6%,Sutent为26.5%,达到这个一级终点。但是这个组合错过mPFS(中位无进展生存期)这个一级终点(11.6vs 8.4个月,但未达到统计显著)。阿斯列康2017年7月公布了其PDL-1抗体Durvalumab(商品名Imfinzi)与CTLA-4抗体Tremelimumab组合在一个叫做Mystic的三期临床中期分析结果。这个试验比较Durva/Treme组合和标准化疗在PDL-1>25%一线NSCLC病人对PFS(无进展生存期)和OS(总生存期)的影响,结果这个组合没有改善PFS(试验一级终点之一)。已有数据显示Durvalumab单方改善PFS机会也很小(试验二级终点之一)。
因此,对于自身免疫性疾病或肿瘤等疾病仍有未满足的需求,发展创新的、能更为有效、且副作用更小的治疗方法和药物是迫切而必要的。
发明内容
在本申请中,发明人通过大量的研究开发了一种双特异性结合CTLA-4和PDL-1,或PDL-1和CTLA-4的双特异性抗体,编码双特异性抗体的核酸分子,包含核酸分子的载体,制备双特异性抗体的方法,包含双特异性抗体的药物组合物,药物组合物的在制备药物中的用途,双特异性抗体在诊断/治疗/预防与CTLA-4和PDL-1相关的疾病(例如自身免疫性疾病或肿瘤)中的用途或方法,以及包含双特异性抗体的试剂盒。
双特异性抗体
因此,在一个方面,本发明提供了一种双特异性抗体,其包含同时结合抗原PDL-1和抗原CTLA-4的蛋白或多肽。
在某些优选的实施方案中,所述双特异性抗体包含:
1)特异性结合第一抗原的第一抗体,所述第一抗体包括重链(HC)和轻链(LC);和
2)特异性结合第二抗原的包含重链可变区(VH)和轻链可变区(VL)的抗体片段(例如Fv,scFv,di-scFv);
其中,所述抗体片段连接于第一抗体的重链或轻链的N端或C端;
所述第一抗原为CTLA-4,并且所述第二抗原为PDL-1;或者,第一抗原为PDL-1,并且所述第二抗原为CTLA-4。
在某些优选的实施方案中,所述抗体片段为scFv。
在某些优选的实施方案中,所述双特异性抗体包含1个所述第一抗体和2个所述scFv;并且,所述第一抗体包括两条HC和两条LC,其中所述第一抗体的一条HC的重链可变区(VH)与一条LC的轻链可变区(VL)形成抗原结合部位,另一条HC的重链可变区(VH)与另一条LC的轻链可变区(VL)形成抗原结合部位。
在某些优选的实施方案中,每个所述scFv分别连接于所述第一抗体的两条重链或两条轻链的N端或C端。
在某些优选的实施方案中,每个所述scFv分别连接于所述第一抗体的两条重链的N端。在某些优选的实施方案中,每个所述scFv分别连接于所述第一抗体的两条重链的C端。在某些优选的实施方案中,第一抗原为PDL-1,所述第二抗原为CTLA-4,且所述结合第二抗原的scFv连接于所述第一抗体 的两条重链的C端。
在某些优选的实施方案中,每个所述scFv分别连接于所述第一抗体的两条轻链的N端。在某些优选的实施方案中,每个所述scFv分别连接于所述第一抗体的两条轻链的C端。
在某些优选的实施方案中,一个所述scFv连接于所述第一抗体的重链或轻链的N端,另一个所述scFv连接于所述第一抗体的重链或轻链的C端。
在某些优选的实施方案中,本发明双特异性抗体,包含:
1)特异性结合第一抗原的第一抗体,所述第一抗体包括重链(HC)和轻链(LC);和2)特异性结合第二抗原的scFv;所述双特异性抗体包含1个所述第一抗体和2个所述scFv;并且,所述第一抗体包括两条HC和两条LC,其中所述第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位,另一条HC的VH与另一条LC的VL形成抗原结合部位;每个所述scFv分别连接于所述第一抗体的两条重链的N端;或者,每个所述scFv分别连接于所述第一抗体的两条重链的C端;所述第一抗原为PDL-1,并且所述第二抗原为CTLA-4。在某些优选的实施方案中,所述每个scFv通过接头S1与所述第一抗体的每条重链的N端或C端连接。在某些优选的实施方案中,所述scFv的由VH与VL通过S2接头连接。在某些优选的实施方案中,所述scFv的结构为NH2-VL-S2-VH-COOH,其中所述S2为接头。
在某些优选的实施方案中,所述第一抗体的重链包含重链可变区(VH)和CH1结构域,并且所述轻链包含轻链可变区(VL)和轻链恒定区(CL)。在此类实施方案中,所述第一抗体可以为Fab片段、Fab’片段或F(ab’)
2片段。在某些优选的实施方案中,所述第一抗体的重链包含重链可变区(VH)和重链恒定区(CH),并且所述轻链包含轻链可变区(VL)和轻链恒定区(CL)。在此类实施方案中,所述第一抗体可以为全长抗体。
在某些优选的实施方案中,所述第一抗体的重链为IgG同种型,例如IgG1、IgG2、IgG3或IgG4;优选为人IgG同种型。在某些实施方案中,所述第一抗体的重链为人IgG1同种型。在某些优选的实施方案中,所述第一抗体的轻链为Kappa同种型,优选为人Kappa同种型。
在某些优选的实施方案中,所述第一抗体的两条HC包含相同的CDR; 和/或,所述第一抗体的两条LC包含相同的CDR。
在某些优选的实施方案中,所述第一抗体的两条HC包含相同的VH;和/或,所述第一抗体的两条LC包含相同的VL。
在某些优选的实施方案中,所述第一抗体的两条HC具有相同的氨基酸序列;和/或,所述第一抗体的两条LC具有相同的氨基酸序列。
在某些优选的实施方案中,两个所述scFv具有相同或不相同的氨基酸序列。在某些优选的实施方案中,两个所述scFv具有相同的氨基酸序列。
在某些优选的实施方案中,所述双特异性抗体包含两条第一多肽链和两条第二多肽链,其中对于所述每条多肽链:
a)所述第一多肽链各自独立地包含所述第一抗体的重链(HC)和所述scFv;和
b)所述第二多肽链各自独立地包含所述第一抗体的轻链(LC);
其中,所述scFv通过接头S1与所述第一抗体的HC的N端或C端相连。
在某些优选的实施方案中,所述双特异性抗体包含两条第一多肽链和两条第二多肽链,其中对于所述每条多肽链:
i)所述第一多肽链各自独立地包含所述第一抗体的轻链(LC)和所述scFv;和
ii)所述第二多肽链各自独立地包含所述第一抗体的重链(HC);
其中,所述scFv通过接头S1与所述第一抗体的LC的N端或C端相连。
在某些优选的实施方案中,所述scFv的N端或C端与接头S1的C端或N端连接。
在某些优选的实施方案中,所述scFv具有结构:NH
2-VH-S2-VL-COOH或NH
2-VL-S2-VH-COOH,其中所述S2为接头。
在某些优选的实施方案中,所述接头S1和/或S2为肽接头,例如具有如(G
mS
n)
x所示的氨基酸序列,其中m、n各自独立地选自1~8的整数(例如,1、2、3、4、5、6、7或8),x独立地选自1~20的整数(例如,1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在某些优选的实施方案中,所述接头S1和/或S2具有如(G
4S)
x所示的氨基酸序列,x独立地选自1-6的整数。
在某些优选的实施方案中,所述接头S1和/或S2具有选自下列的氨基酸序列:SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27。
在某些优选的实施方案中,所述接头S2具有如(G
4S)
4所示的氨基酸序列,即GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:25)。在某些优选的实施方案中,当所述scFv与所述第一抗体的重链或轻链的N端连接时,所述接头S1具有如(G
4S)
3所示的氨基酸序列,即GGGGSGGGGSGGGGS(SEQ ID NO:26);当所述scFv与所述第一抗体的重链或轻链的C端连接时,所述接头S1具有如(G
4S)
2所示的氨基酸序列,即GGGGSGGGGS(SEQ ID NO:27)。
在某些优选的实施方案中,所述scFv的VH与VL之间存在二硫键。在抗体的VH和VL之间引入二硫键的方法是本领域熟知的,例如可参见美国专利申请US5,747,654;Rajagopal等人,Prot.Engin.10(1997)1453-1459;Reiter等人,Nature Biotechnology 14(1996)1239-1245;Reiter等人,Protein Engineering 8(1995)1323-1331;Webber等人,Molecular Immunology 32(1995)249-258;Reiter等人,Immunity 2(1995)281-287;Reiter等人,JBC 269(1994)18327-18331;Reiter等人,Inter.J.of Cancer 58(1994)142-149;或,Reiter等人,Cancer Res.54(1994)2714-2718;其通过引用并入本文。
在某些优选的实施方案中,所述scFv的位于VH第44位的氨基酸和位于VL第100位的氨基酸分别为半胱氨酸,其中提及的氨基酸位置是根据Kabat编号系统的位置;并且,所述scFv的VH与VL通过分别位于VH第44位和VL第100位的2个半胱氨酸残基之间所形成的二硫键连接。
在某些优选的实施方案中,所述第一抗体特异性结合CTLA-4,并且所述scFv特异性结合PDL-1,其中所述scFv包含:
如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
在某些优选的实施方案中,所述第一抗体特异性结合CTLA-4,并且所述scFv特异性结合PDL-1,其中:
所述第一抗体包含:
如SEQ ID NO:13所示的HCDR1;如SEQ ID NO:14所示的HCDR2; 以及,如SEQ ID NO:15所示的HCDR3;如SEQ ID NO:16所示的LCDR1;如SEQ ID NO:17所示的LCDR2;以及,如SEQ ID NO:18所示的LCDR3;
和/或,
所述scFv包含:
如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
在某些优选的实施方案中,所述第一抗体包含:如SEQ ID NO:13所示的HCDR1;如SEQ ID NO:14所示的HCDR2;以及,如SEQ ID NO:15所示的HCDR3;如SEQ ID NO:16所示的LCDR1;如SEQ ID NO:17所示的LCDR2;以及,如SEQ ID NO:18所示的LCDR3;并且,所述scFv包含:如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
在某些优选的实施方案中,所述第一抗体的重链可变区(VH)的氨基酸序列与SEQ ID NO:19所示的重链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性;并且,所述第一抗体的轻链可变区(VL)的氨基酸序列与SEQ ID NO:21所示的轻链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性;和,所述scFv的重链可变区(VH)的氨基酸序列与SEQ ID NO:7或SEQ ID NO:8所示的重链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性;并且,所述scFv的的轻链可变区(VL)的氨基酸序列与SEQ ID NO:9或SEQ ID NO:10所示的轻链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性。
在某些优选的实施方案中,所述scFv的重链可变区选自SEQ ID NO:7 所示的重链可变区;并且,所述scFv的的轻链可变区选自SEQ ID NO:9所示的轻链可变区。
在某些优选的实施方案中,所述scFv的重链可变区选自SEQ ID NO:8所示的重链可变区;并且,所述scFv的的轻链可变区选自SEQ ID NO:10所示的轻链可变区。
在某些优选的实施方案中,所述第一抗体的重链可变区选自SEQ ID NO:19所示的重链可变区;并且,所述第一抗体的轻链可变区选自SEQ ID NO:21所示的轻链可变区;和,所述scFv的重链可变区选自SEQ ID NO:7所示的重链可变区;并且,所述scFv的的轻链可变区选自SEQ ID NO:9所示的轻链可变区。
在某些优选的实施方案中,所述第一抗体的重链可变区选自SEQ ID NO:19所示的重链可变区;并且,所述第一抗体的轻链可变区选自SEQ ID NO:21所示的轻链可变区;和,所述scFv的重链可变区选自SEQ ID NO:8所示的重链可变区;并且,所述scFv的的轻链可变区选自SEQ ID NO:10所示的轻链可变区。
在某些优选的实施方案中,所述第一抗体包含:如SEQ ID NO:19所示的VH和如SEQ ID NO:21所示的VL;
并且,所述scFv包含:
(1)如SEQ ID NO:7所示的VH和如SEQ ID NO:9所示的VL;或,
(2)如SEQ ID NO:8所示的VH和如SEQ ID NO:10所示的VL。
在某些优选的实施方案中,所述第一抗体特异性结合PDL-1,并且所述scFv特异性结合CTLA-4,其中:
所述第一抗体包含:
(a)如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3;
和/或,
所述scFv包含:
如SEQ ID NO:13所示的HCDR1;如SEQ ID NO:14所示的HCDR2; 以及,如SEQ ID NO:15所示的HCDR3;如SEQ ID NO:16所示的LCDR1;如SEQ ID NO:17所示的LCDR2;以及,如SEQ ID NO:18所示的LCDR3。
在某些优选的实施方案中,所述第一抗体特异性结合PDL-1,并且所述scFv特异性结合CTLA-4,其中所述第一抗体包含:
如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
在某些优选的实施方案中,所述第一抗体包含:如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3;并且,所述scFv包含:如SEQ ID NO:13所示的HCDR1;如SEQ ID NO:14所示的HCDR2;以及,如SEQ ID NO:15所示的HCDR3;如SEQ ID NO:16所示的LCDR1;如SEQ ID NO:17所示的LCDR2;以及,如SEQ ID NO:18所示的LCDR3。
在某些优选的实施方案中,所述第一抗体的重链可变区(VH)的氨基酸序列与SEQ ID NO:7所示的重链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性;并且,所述第一抗体的的轻链可变区(VL)的氨基酸序列与SEQ ID NO:9所示的轻链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性;和,所述scFv的重链可变区(VH)的氨基酸序列与SEQ ID NO:19或SEQ ID NO:20所示的重链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性;并且,所述scFv的的轻链可变区(VL)的氨基酸序列与SEQ ID NO:21或SEQ ID NO:22所示的轻链可变区的氨基酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性。
在某些优选的实施方案中,所述第一抗体的重链可变区选自SEQ ID NO: 7所示的重链可变区;并且,所述第一抗体的轻链可变区选自SEQ ID NO:9所示的轻链可变区。
在某些优选的实施方案中,所述第一抗体的重链可变区选自SEQ ID NO:7所示的重链可变区;并且,所述第一抗体的轻链可变区选自SEQ ID NO:9所示的轻链可变区;和,所述scFv的重链可变区选自SEQ ID NO:19所示的重链可变区;并且,所述scFv的的轻链可变区选自SEQ ID NO:21所示的轻链可变区。
在某些优选的实施方案中,所述第一抗体的重链可变区选自SEQ ID NO:7所示的重链可变区;并且,所述第一抗体的轻链可变区选自SEQ ID NO:9所示的轻链可变区;和,所述scFv的重链可变区选自SEQ ID NO:20所示的重链可变区;并且,所述scFv的的轻链可变区选自SEQ ID NO:22所示的轻链可变区。
在某些优选的实施方案中,所述第一抗体包含:如SEQ ID NO:7所示的VH和如SEQ ID NO:9所示的VL;
并且,所述scFv包含:
(1)如SEQ ID NO:19所示的VH和如SEQ ID NO:21所示的VL;或,
(2)如SEQ ID NO:20所示的VH和如SEQ ID NO:22所示的VL。
在某些优选的实施方案中,所述双特异性抗体包含两条相同的第一多肽链和两条相同的第二多肽链。
在某些优选的实施方案中,所述第一多肽链具有选自下列的氨基酸序列:SEQ ID NOs:28,29任一项所示的氨基酸序列;和/或,所述第二多肽链具有选自下列的氨基酸序列:SEQ ID NOs:11,23任一项所示的氨基酸序列。
在某些优选的实施方案中,所述双特异性抗体包含:
(1)如SEQ ID NO:28所示的第一多肽链和如SEQ ID NO:11所示的第二多肽链;
(2)如SEQ ID NO:29所示的第一多肽链和如SEQ ID NO:11所示的第二多肽链;
(3)如SEQ ID NO:28所示的第一多肽链和如SEQ ID NO:23所示的第二多肽链;
(4)如SEQ ID NO:29所示的第一多肽链和如SEQ ID NO:23所示的第二多肽链;
在某些实施方式中,本发明双特异性抗体中所述第一抗体包含:如Uniprot的Entry号P01857所示的CH;和/或,如Uniprot的Entry号P01834所示的CL。可选的,所述CH还可以包含如下突变:在UniProt的Entry号P01857的CH中第117,118,120位突变为A;或,在UniProt的Entry号P01857的CH中第97位突变为R。
在某些实施方案中,本发明的双特异性抗体具有抗体依赖性细胞介导的细胞毒性(ADCC)活性。在某些实施方案中,本发明的双特异性抗体具有抗体依赖性细胞介导的细胞毒性(ADCC)活性,并且还具有补体依赖的细胞毒性(CDC)活性。在某些优选的实施方案中,本发明的双特异性抗体具有增强的抗体依赖性细胞介导的细胞毒性(ADCC)活性,和/或具有增强的补体依赖性细胞毒性(CDC)活性。在某些优选的实施方案中,所述增强的ADCC和/或CDC活性是由双特异性抗体的第一抗体CH发生突变导致。
另一方面,本发明的双特异性抗体,具有与所述第一抗体的亲本抗体相比同等或更弱的结合第一抗原的活性;在某些优选的实施方案中,本发明的双特异性抗体,具有与所述scFv的亲本抗体相比同等或更弱的结合第二抗原的活性;在某些优选的实施方案中,本发明的双特异性抗体,具有与所述第一抗体的亲本抗体相比同等或更弱的结合第一抗原的活性,且具有与所述scFv的亲本抗体相比同等或更弱的结合第二抗原的活性;在某些优选的实施方案中,本发明的双特异性抗体,具有与所述第一抗体的亲本抗体相比同等的结合第一抗原的活性,且具有与所述scFv的亲本抗体相比更弱的结合第二抗原的活性;在某些优选的实施方案中,本发明的双特异性抗体,具有与结合PDL-1的亲本抗体相比同等的结合活性;在某些优选的实施方案中,本发明的双特异性抗体,具有与结合CTLA-4的亲本抗体相比更弱的结合活性;在某些优选的实施方案中,本发明的双特异性抗体,具有与结合PDL-1的亲本抗体相比同等的结合活性,且具有与结合CTLA-4的亲本抗体相比更弱的结合活性。
另一方面,本发明的双特异性抗体对CTLA-4和PDL-1具有亲和力。在 某些优选的实施方案中,本发明的双特异性抗体对CTLA-4和PDL-1具有与其各自的亲本抗体相比同等亲和力。在某些优选的实施方案中,本发明的双特异性抗体对PDL-1具有与其亲本抗体相比同等的亲和力。在某些优选的实施方案中,本发明的双特异性抗体对CTLA-4具有与其亲本抗体相比同等的亲和力。在某些优选的实施方案中,本发明的双特异性抗体对CTLA-4具有与其亲本抗体相比更弱的亲和力。在某些优选的实施方案中,本发明的双特异性抗体对PDL-1具有与其亲本抗体相比同等的亲和力,对CTLA-4具有与其亲本抗体相比更弱的亲和力。
另一方面,本发明的双特异性抗体具有良好的热稳定性。在某些优选的实施方案中,本发明的双特异性抗体具有与亲本抗体相比基本相同的热稳定性。
双特异性抗体的表达
在另一个方面,本发明提供了一种分离的核酸分子,其包含编码本发明的双特异性抗体的核苷酸序列。在某些优选的实施方案中,所述分离的核酸分子编码本发明的双特异性抗体。
在某些优选的实施方案中,所述分离的核酸分子包含编码本发明的第一多肽链的核苷酸序列。在某些优选的实施方案中,所述分离的核酸分子包含编码本发明的第二多肽链的核苷酸序列。在某些优选的实施方案中,所述分离的核酸分子包含编码本发明的第一多肽链的核苷酸序列和编码本发明的第二多肽链的核苷酸序列。
在另一个方面,本发明提供了一种载体(例如克隆载体或表达载体),其包含本发明的分离的核酸分子。
在某些优选的实施方案中,所述载体包含编码本发明的第一多肽链的核苷酸序列。在某些优选的实施方案中,所述载体包含编码本发明的第二多肽链的核苷酸序列。在某些优选的实施方案中,所述载体包含编码本发明的第一多肽链的核苷酸序列和编码本发明的第二多肽链的核苷酸序列。
在某些优选的实施方案中,本发明的载体是例如质粒,粘粒,噬菌体等。在某些优选的实施方案中,所述载体能够在受试者(例如哺乳动物,例如人) 体内表达本发明的双特异性抗体、第一多肽链或第二多肽链。
在另一个方面,本发明提供了一种宿主细胞,其包含本发明的分离的核酸分子或本发明的载体。此类宿主细胞包括但不限于,原核细胞例如大肠杆菌细胞,以及真核细胞例如酵母细胞,昆虫细胞,植物细胞和动物细胞(如哺乳动物细胞,例如小鼠细胞、人细胞等)。在某些优选的实施方案中,本发明的宿主细胞是哺乳动物细胞,例如CHO(例如CHO-K1、CHO-S、CHO DG44)或HEK293。
在另一个方面,本发明提供了制备本发明的双特异性抗体的方法,其包括,在允许所述双特异性抗体表达的条件下,培养本发明的宿主细胞,和从培养的宿主细胞培养物中回收所述双特异性抗体。
在某些优选的实施方案中,所述方法包括:
(1)构建包含编码第一多肽链的核苷酸序列和编码第二多肽链的核苷酸序列的表达载体;或,构建包含编码第一多肽链的核苷酸序列的第一表达载体和包含编码第二多肽链的核苷酸序列的第二表达载体;
(2)将步骤(1)中所述的表达载体转化至宿主细胞;或,将步骤(1)中所述的第一表达载体和第二表达载体转化至宿主细胞;
(3)在允许本发明的双特异性抗体表达的条件下,培养步骤(2)中所述的宿主细胞;和
(4)从培养的宿主细胞培养物中回收所述双特异性抗体。
治疗方法和药物组合物
本发明的双特异性抗体可用于体外或在受试者体内中抑制CTLA-4和PDL-1的活性,阻断CTLA-4和/或PDL-1信号通路,以及用于预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(例如自身免疫性疾病或肿瘤或传染病)。
因此,在另一个方面,本发明提供了一种药物组合物,其含有本发明的双特异性抗体,以及药学上可接受的载体和/或赋形剂。在某些优选的实施方案中,所述药物组合物还可以包含另外的药学活性剂。在某些优选的实施方案中,所述另外的药学活性剂是用于预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(例如自身免疫性疾病或肿瘤或传染病)的药物,例如抗炎药物 或免疫抑制剂,例如非甾体抗炎药(如布洛芬、双氯芬酸、萘普生、吲哚美辛、吡罗昔康、美洛昔康、萘丁美酮或尼美舒利)、甾体抗炎药(如强的松、地塞米松或氢化考的松)、致炎性细胞因子的抗体或拮抗剂等。
在另一个方面,本发明提供了本发明的双特异性抗体或本发明的药物组合物在制备药物中的用途,所述药物用于在受试者(例如人)中预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病),和/或用于体外或在受试者(例如人)体内抑制CTLA-4和/或PDL-1的活性。
在另一个方面,本发明提供了一种用于在受试者(例如人)中预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病),和/或用于体外或在受试者(例如人)体内抑制CTLA-4和/或PDL-1的活性的方法,其中所述方法包括,给有此需要的受试者施用有效量的本发明的双特异性抗体,或者本发明的药物组合物。
在本发明中,所述与CTLA-4和/或PDL-1相关的疾病包括但不限于如自身免疫性疾病或肿瘤或传染病病,例如肿瘤,包括但不限于:腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤,或包括但不限于肾上腺、胆囊、骨、骨髓、脑、乳腺、胆管、胃肠道、心脏、肾脏、肝脏、肺、肌肉、卵巢、胰腺、甲状旁腺、阴茎、前列腺、皮肤、唾液腺、脾脏、睾丸、胸腺、甲状腺和子宫相关的肿瘤,传染病包括但不限于乙肝,甲肝,HIV。
本发明的双特异性抗体或者本发明的药物组合物可以配制成医学领域已知的任何剂型,例如,片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如,可通过下述方法来制备无菌注射溶液:在适当的溶剂中掺入必需剂量的本发明的双特异性抗体,以及任选地,同时掺入其他期望的成分(包括但不限于,pH调节剂,表面活性剂,佐剂,离子强度增强剂,等渗剂、防腐剂、稀释剂,或其任何组合),随后过滤除菌。此外,可以将无菌注射溶液制备为无菌冻干粉剂 (例如,通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中,例如无菌无热原水。
此外,本发明的双特异性抗体可以以单位剂量形式存在于药物组合物中,以便于施用。在某些实施方案中,所述单位剂量为至少1mg,至少2mg,至少3mg,至少4mg,至少5mg,至少6mg,至少7mg,至少8mg,至少9mg,至少10mg,至少15mg,至少20mg,至少25mg,至少30mg,至少45mg,至少50mg,至少75mg,或至少100mg。在所述药物组合物为液体(例如,注射剂)剂型的情况下,其可以包含浓度至少为0.1mg/ml,如至少0.25mg/ml,至少0.5mg/ml,至少1mg/ml,至少2.5mg/ml,至少5mg/ml,至少8mg/ml,至少10mg/ml,至少15mg/ml,至少25mg/ml,至少50mg/ml,至少75mg/ml,或至少100mg/ml的本发明的双特异性抗体。
本发明的双特异性抗体或药物组合物可以通过本领域已知的任何合适的方法来施用,包括但不限于,口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如,粉剂、药膏或滴剂),或鼻腔途径。但是,对于许多治疗用途而言,优选的给药途径/方式是胃肠外给药(例如静脉注射,皮下注射,腹膜内注射,肌内注射)。技术人员应理解,给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中,本发明的双特异性抗体或药物组合物通过静脉输注或注射给予。
本发明所提供的药物、药物组合物或双特异性抗体可以单独使用或联合使用,也可以与另外的药学活性剂(如自身免疫性疾病或肿瘤或传染病)联合使用。在某些优选的实施方案中,将本发明的双特异性抗体与其它抗炎药物或免疫抑制剂联合使用,以预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病)。这种另外的药学活性剂可以在施用本发明的双特异性抗体或本发明的药物组合物之前、同时或之后施用。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明的双特异性抗体。“预防有效量”是指,足以预防,阻止,或延迟疾病(例如与CTLA-4和/或PDL-1相关的疾病)的发生的量。“治疗有效量”是指,足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明的双特异性抗体的治疗有效量可根据如下因素发生变化:待治疗的疾病的严重 度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄,体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
在本发明中,可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如,可以单次给药,可以在一段时间内多次给药,或者可以随治疗情况的紧急程度按比例减少或增加剂量。
本发明的双特异性抗体的治疗或预防有效量的典型非极限范围是0.02~50mg/kg,例如0.1~50mg/kg,0.1~25mg/kg,或1~10mg/kg。应注意的是,剂量可随需要治疗的症状的类型和严重性不同而发生变化。此外,本领域技术人员理解,对于任一特定患者,特定的给药方案应根据患者需要和医生的专业评价而随时间调整;此处给出的剂量范围只用于举例说明目的,而不限定本发明药物组合物的使用或范围。
在本发明中,所述受试者可以为哺乳动物,例如人。
检测/诊断方法和试剂盒
本发明的双特异性抗体能够特异性结合CTLA-4和/或PDL-1,从而可用于检测CTLA-4和/或PDL-1在样品中的存在或其水平,以及诊断受试者是否患有与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病)。
因此,在另一个方面,本发明提供了一种试剂盒,其包括本发明的双特异性抗体。在某些优选的实施方案中,本发明的双特异性抗体带有可检测的标记。在一个优选的实施方案中,所述试剂盒还包括第二抗体,其特异性识别本发明的双特异性抗体的第一抗体或scFv。优选地,所述第二抗体还包括可检测的标记。
在本发明中,所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是,此类标记能够适用于免疫学检测(例如,酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的,包括但不限于,酶(例如,辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶,等)、放射性核素(例如,
3H、
125I、
35S、
14C或
32P)、荧光染料(例如,异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明 (TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如,
)、测热标记物例如胶体金或有色玻璃或塑料(例如,聚苯乙烯、聚丙烯、乳胶,等)珠、以及用于结合上述标记物修饰的亲和素(例如,链霉亲和素)的生物素。教导该标记物的使用的专利包括,但不限于,美国专利3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149;及4,366,241(全部通过引用并入本文)。本发明中涵盖的标记物可通过本领域已知的方法检测。例如,放射性标记可使用摄影胶片或闪烁计算器检测,荧光标记物可使用光检测器检测,以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测,及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中,可通过不同长度的接头将如上所述的可检测的标记连接至本发明的双特异性抗体,以降低潜在的位阻。
在另一个方面,本发明提供了检测CTLA-4和/或PDL-1在样品中的存在或其水平的方法,其包括使用本发明的双特异性抗体的步骤。在一个优选的实施方案中,本发明的双特异性抗体还带有可检测的标记。在另一个优选的实施方案中,所述方法还包括,使用带有可检测的标记的试剂来检测本发明的双特异性抗体段。所述方法可以用于诊断目的,或者非诊断目的(例如,所述样品是细胞样品,而非来自患者的样品)。
在另一个方面,本发明提供了诊断受试者是否患有与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病)的方法,其包括:使用本发明的双特异性抗体检测CTLA-4和/或PDL-1在来自所述受试者的样品中的存在或其水平。在一个优选的实施方案中,本发明的双特异性抗体还带有可检测的标记。在另一个优选的实施方案中,所述方法还包括,使用带有可检测的标记的试剂来检测本发明的双特异性抗体的步骤。
在另一个方面,提供了本发明的双特异性抗体在制备试剂盒中的用途,所述试剂盒用于检测CTLA-4和/或PDL-1在样品中的存在或其水平,或用于诊断受试者是否患有与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病)。
术语定义
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
在本文中,“PDL-1”也被称为“程序性死亡配体1(Programmed death-ligand 1)”、“程序性细胞死亡配体1(Programmed cell death ligand 1)”、“蛋白质PD-L1”、“PD-L1”、“PDL1”、“PDCDL1”、“hPD-L1”、“hPD-LI”、“CD274”及“B7-H1”,并且可互换的使用。
如本文中所使用的,术语“抗体”是指,通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε,并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内,可变区和恒定区通过大约12或更多个氨基酸的“J”区连接,重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子,包括免疫系统的各种细胞(例如,效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR)),其间散布有较保守的称为构架区(FR)的区域。各V
H和V
L由按下列顺序:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸至各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)),或Chothia&Lesk(1987)J.Mol.Biol.196:901-917;Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的,术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基,其通常可包括,轻链可变区中的残基24-34{LCDR1}、50-56{LCDR2}、89-97{LCDR3}以及重链可变区中的残基31-35{HCDR1}、50-65{HCDR2}、95-102{HCDR3}(参见例如,Kabat等,Sequences of Proteins of lmmunological lnterest,第五版,Public Health Service,美国国立卫生研究院,贝塞斯达、马里兰州(1991)),或者轻链可变区中的残基26-32{Ll}、50-52{L2}、91-96{L3}以及重链可变区中的残基26-32{H1}、53-55{H2}、96-101{H3}(参见,Chothia和Lesk J.Mol.Biol.196:901-917(1987))。或,CDR可以通过本领域技术人员所熟知的技术手段,例如通过VBASE2数据库根据IMGT定义分析下面的重链可变区的氨基酸序列或者轻链可变区的氨基酸序列得到。
如本文中所使用的,术语“构架区”或“FR”残基是指,抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如,其包括,重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体,例如,IgG(例如,IgG1,IgG2,IgG3或IgG4亚型),IgA1,IgA2,IgD,IgE或IgM抗体。
如本文中所使用的,术语“抗原结合部位”是指,由重链以及轻链的可变区(VH和VL)的氨基酸残基所形成的、参与进行抗原结合的部分,其包含那些与抗原相互作用且决定了该抗体对抗原的特异性及亲和力的氨基酸残基。所述抗原结合部位还可以包含那些对维持上述直接结合抗原的氨基酸残基的合适构象所必需的框架区的氨基酸残基。
如本文中所使用的,术语“全长抗体”意指,由两条“全长重链”和两条“全长轻链”组成的抗体。其中,“全长重链”是指这样的多肽链,其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成;并且,当所述全长抗体为IgE同种型时,任选地还包括重链恒定区CH4结构域。优选地,“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上 由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种,例如人;也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位,这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的,术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段;术语“Fab'片段”意指还原连接F(ab')
2片段中两个重链片段的二硫键后所获片段,由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成;术语“F(ab')
2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力,和/或与全长抗体竞争对抗原的特异性结合。
如本文中所使用的,术语“scFv”是指,包含VL和VH结构域的单个多肽链,其中所述VL和VH通过接头(linker)相连(参见,例如,Bird等人,Science 242:423-426(1988);Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988);和Pluckthun,The Pharmacology of Monoclonal Antibodies,第113卷,Roseburg和Moore编,Springer-Verlag,纽约,第269-315页(1994))。此类scFv分子可具有一般结构:NH
2-VL-接头-VH-COOH或NH
2-VH-接头-VL-COOH。本发明的scFv的VH与VL之间还可以存在二硫键。在抗体的VH和VL之间引入二硫键的方法是本领域熟知的,例如可参见美国专利申请US5,747,654;Rajagopal等人,Prot.Engin.10(1997)1453-1459;Reiter等人,Nature Biotechnology 14(1996)1239-1245;Reiter等人,Protein Engineering 8(1995)1323-1331;Webber等人,Molecular Immunology 32(1995)249-258;Reiter等人,Immunity 2(1995)281-287;Reiter等人,JBC 269(1994)18327-18331;Reiter等人,Inter.J.of Cancer 58(1994)142-149;或,Reiter等人,Cancer Res.54(1994)2714-2718;其通过引用并入本文。如本文中所使用的,术语“di-scFv”是指,由两个scFv连接形成的抗体片段。
如本文中所使用的,术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段。
如本文中所使用的,术语“亲本抗体”是指,用于制备本发明的双特异性抗体的抗PDL-1抗体或抗CTLA-4抗体,该抗体所具有的氨基酸序列可通过例如氨基酸置换或结构改变等方式以用于制备本发明的双特异性抗体所包含的第一抗体或scFv。
本发明的双特异性抗体所包含的CDR、VH、VL、CH、CL、HC、LC还可以来自其他被本领域所知晓的能够特异性结合PDL-1或CTLA-4的抗体或其抗体片段,或者与上述已知抗体、其抗体片段或其CDR、VH、VL、CH、CL、HC、LC具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性的抗体。
如本文中所使用的,术语“接头”是指,由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列,或天然存在的多肽序列,例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如,Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448;Poljak,R.J.等人(1994)Structure 2:1121-1123))。
如本文中所使用的,术语“特异性结合”是指,两分子间的非随机的结合反应,如抗体和其所针对的抗原之间的反应。在某些实施方式中,特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指,抗体以小于大约10
-5M,例如小于大约10
-6M、10
-7M、10
-8M、10
-9M或10
-10M或更小的亲和力(K
D)结合该抗原。在本发明中,术语“K
D”是指特定抗体-抗原相互作用的解离平衡常数,其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小,抗体-抗原结合越紧密,抗体与抗原之间的亲和力越高。通常,抗体(例如,本发明的抗体)以小于大约10
-5M,例如小于大约10
-6M、10
-7M、10
-8M、10
-9M或10
-10M或更小的解离平衡常数(K
D)结合抗原(例如,HBsAg),例如,如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的,术语“载体(vector)”是指,可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时,载体称为表达载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒;噬菌粒;柯斯质粒;人工染色体,例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于,逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件,包括但不限于,启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。
如本文中所使用的,术语“宿主细胞”是指,可用于导入载体的细胞,其包括但不限于,如大肠杆菌或枯草菌等的原核细胞,如酵母细胞或曲霉菌等的真菌细胞,如S2果蝇细胞或Sf9等的昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的,术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如,两个DNA分子的每一个中的某个位置都被腺嘌呤占据,或两个多肽的每一个中的某个位置都被赖氨酸占据),那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如,如果两个序列的10个位置中有6个匹配,那么这两个序列具有60%的同一性。例如,DNA序列CTGACT和CAGGTT共有50%的同一性(总共6个位置中有3个位置匹配)。通常,在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用,例如,可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48:443-453的方法来实现。还可使用已整合入 ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.,4:11-17(1988))的算法,使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外,可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法,使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如,Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),其以引用的方式并入本文中。在本发明中,术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中,氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如,丙氨酸可用A或Ala表示。
如本文中所使用的,术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂,其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995),并且包括但不限于:pH调节剂,表面活性剂,佐剂,离子强度增强剂,稀释剂,维持渗透压的试剂,延迟吸收的试剂,防腐剂。例如,pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子,阴离子或者非离子型表面活性剂,例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂,例如对羟苯甲酸酯,三氯叔丁醇,苯酚,山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。
如本文中使用的,术语“受试者”是指哺乳动物,例如灵长类哺乳动物,例如人。在某些实施方式中,所述受试者(例如人)患有与CTLA-4和/或PDL-1相关的疾病,或者,具有患有上述疾病的风险。通常而言,这 类疾病或疾病状态的特征是其将受益于CTLA-4和/或PDL-1水平的降低或CTLA-4和/或PDL-1活性的抑制从而得到缓解或治愈。
如本文中所使用的,术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如,预防疾病(例如与CTLA-4和/或PDL-1相关的疾病)有效量是指,足以预防,阻止,或延迟疾病(例如与CTLA-4和/或PDL-1相关的疾病)的发生的量;治疗疾病有效量是指,足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如,对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄,体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
如本文中所使用的,术语“抗体依赖性细胞介导的细胞毒性(ADCC)”是指,一种细胞毒性形式,Ig通过与细胞毒性细胞(例如自然杀伤(NK)细胞、中性粒细胞或巨噬细胞)上存在的Fc受体(FcR)结合,使这些细胞毒性效应细胞特异性结合到抗原附着的靶细胞上,然后通过分泌细胞毒素杀死靶细胞。检测抗体的ADCC活性的方法是本领域已知的,例如可通过测定待测抗体与Fc受体(例如CD16a)之间的结合活性来评价。
如本文中所使用的,术语“补体依赖的细胞毒性(CDC)”是指,通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的CDC活性的方法是本领域已知的,例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价。
发明的有益效果
与现有技术相比,本发明的技术方案至少具有以下有益效果:
本发明的双特异性抗体不仅能够特异性识别/结合CTLA-4和PDL-1,而且其对CTLA-4和PDL-1的亲和力与其各自的亲本抗体相当,或对CTLA-4的亲和力比其亲本抗体减弱,在体外和受试者体内能够显著且同时抑制CTLA-4和/或PD–L1的活性,阻断CTLA-4和/或PD–L1信号通路。并且,本发明的双特异性抗体还具有与第一抗体的亲本抗体同等优良的热稳定性。 尤其令人意外的是,在部分细胞水平及体内实验中,本发明的双特异性抗体甚至展现出了明显优于其亲本抗体的抑制活性和降低的抗体毒性反应,提升了药物的安全性。因此,本发明的双特异性抗体具有用于治疗与CTLA-4和PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病)的潜力,具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述,但是本领域技术人员将理解,下列附图和实施例仅用于说明本发明,而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述,本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
图1显示了抗PDL-1和CTLA-4重组双特异抗体AB03和AB04结构示意图。
图2显示了AB03和AB04的还原SDS-PAGE电泳图,从左至右泳道为:AB01还原,AB02还原,AB03还原,AB04还原。
图3显示了抗体与PDL-1结合活性曲线。
图4中的图4A和图4B显示了抗体与不同批次CTLA-4结合活性曲线。
图5显示了抗体同时与PDL-1和CTLA-4结合活性曲线。
图6显示了抗体阻断PD-1与PDL-1结合活性曲线。
图7显示了抗体与CD16a(Val)结合活性曲线。
图8显示了抗体与C1q结合活性曲线。
图9显示了抗体阻断PD1/PDL-1细胞结合活性曲线。
图10显示了抗体在SEB刺激PBMC下增强T细胞分泌IL-2的活性图。
图11显示了AB01对非小细胞肺癌模型的疗效。
图12显示了抗体在结肠癌小鼠模型中的疗效。
图13显示了抗体在非小细胞肺癌小鼠模型中的疗效。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1:序列的描述
SEQ ID NO: | 序列名称/描述 | SEQ ID NO: | 序列名称/描述 |
1 | AB01 HCDR1 | 17 | AB02 LCDR2 |
2 | AB01 HCDR2 | 18 | AB02 LCDR3 |
3 | AB01 HCDR3 | 19 | AB02VH |
4 | AB01 LCDR1 | 20 | AB02突变型VH |
5 | AB01 LCDR2 | 21 | AB02VL |
6 | AB01 LCDR3 | 22 | AB02突变型VL |
7 | AB01 VH | 23 | AB02轻链 |
8 | AB01突变型VH | 24 | AB02重链 |
9 | AB01 VL | 25 | S2 |
10 | AB01突变型VL | 26 | S1 |
11 | AB01轻链 | 27 | S1 |
12 | AB01重链 | 28 | AB03第一多肽链氨基酸序列 |
13 | AB02 HCDR1 | 29 | AB04第一多肽链氨基酸序列 |
14 | AB02 HCDR2 | 30 | IgG-Kappa信号肽氨基酸 |
15 | AB02 HCDR3 | 31 | AB01-scFv氨基酸序列* |
16 | AB02 LCDR1 | 32 | AB02-scFv氨基酸序列* |
*注:AB01-scFv表示亲本抗体AB01来源的scFv,其可变区与AB01的区别在于:该scFv的VH第44位和VL第100位氨基酸分别为半胱氨酸;AB02-scFv表示亲本抗体AB02来源的scFv,其可变区与AB02的区别在于:该scFv的VH第44位和VL第100位氨基酸分别为半胱氨酸;其他类似的表述具有类似的含义。
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明,本发明中所使用的分子生物学实验方法和免疫检测 法,基本上参照J.Sambrook等人,分子克隆:实验室手册,第2版,冷泉港实验室出版社,1989,以及F.M.Ausubel等人,精编分子生物学实验指南,第3版,John Wiley&Sons,Inc.,1995中所述的方法进行;限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓,实施例以举例方式描述本发明,且不意欲限制本发明所要求保护的范围。
实施例1:编码抗PDL-1/CTLA-4重组双特异性抗体的表达载体的构建
在本实施例中,表2中所示的抗CTLA-4亲本抗体(AB02)从施贵宝公司购买,首先通过DNA重组技术,获得表2中所示的抗PDL-1亲本抗体(AB01),然后分别构建包含编码第一多肽链的核苷酸序列的表达载体和编码第二多肽链的核苷酸序列的表达载体,以获得本发明的重组抗体AB03和AB04。
各亲本抗体AB01和AB02可变区及恒定区序列参照表2。亲本抗体(AB01)的编码核酸序列由南京金斯瑞生物技术服务公司全基因合成,克隆到PUC19载体上。
表2:亲本抗体及PDL-1/CTLA-4重组双特异性抗体的可变区及恒定区序列
注:数据库收录号为Uniprot的收录号。
按照表3中所示各重组抗体的构建方式构建编码该重组抗体的第一多肽链的核苷酸序列和编码第二多肽链的核苷酸序列。其中,对于各重组抗体中的scFv,其VL、VH通过肽接头S2(SEQ ID NO:25,氨基酸序列为:GGGGSGGGGSGGGGSGGGGS)相连接,并且通过PCR定点突变方法将该 scFv的亲本抗体的VH第44位和VL第100位氨基酸分别突变为半胱氨酸(Cys,C),从而使得该scFv的VH与VL之间形成二硫键。其中突变后的AB01-scFv的氨基酸序列如SEQ ID NO:31所示,突变后VL的氨基酸序列如SEQ ID NO:10所示,突变后VH的氨基酸序列如SEQ ID NO:8所示。其中突变后的AB02-scFv的氨基酸序列如SEQ ID NO:32所示,突变后VL的氨基酸序列如SEQ ID NO:22所示,突变后VH的氨基酸序列如SEQ ID NO:20所示。
表3中所示各重组抗体的构建方式例举见图1。
表3:各重组抗体的构建方式
具体而言,作为示例地部分重组抗体的核酸构建体的构建方式如下:
将编码AB02-scFv的核苷酸序列通过编码SEQ ID NO:26所示接头S1的核苷酸序列连接于编码AB01的VH的核苷酸序列的5’端,然后连接于重链恒定区UniProt P01857的编码核苷酸序列的5’端,构建编码AB03的第一多肽链的核苷酸序列;其中AB02-scFv从N端到C端的顺序为VL-S2-VH,其编码的氨基酸序列如SEQ ID NO:32所示。
将编码AB01轻链的核苷酸序列作为ABO3的第二多肽链的编码核苷酸序列。
将AB01的VH的核苷酸序列连接于重链恒定区UniProt P01857的编码核苷酸序列的5’端,通过编码SEQ ID NO:27所示接头S1的核苷酸序列连接 于编码AB02-scFv的核苷酸序列的5’端,构建编码AB04的第一多肽链的核苷酸序列;其中AB02-scFv从N端到C端的顺序为VL-S2-VH,其编码的氨基酸序列如SEQ ID NO:32所示。
将编码AB01轻链的核苷酸序列作为ABO4的第二多肽链的编码核苷酸序列。
将编码上述各第一多肽链或第二多肽链的核苷酸序列与编码IgG-Kappa信号肽的核苷酸序列连接(氨基酸序列SEQ ID NO:30),并通过同源重组的方式分别导入pTT5质粒,以构建编码第一多肽链的表达载体及编码第二多肽链的表达载体pTT5。最终获得的各重组抗体的氨基酸序列如表4所示。
表4:各重组抗体的氨基酸序列
实施例2:抗PDL-1/CTLA-4重组双特异性抗体的表达
将生长状态良好、处于对数期的CHO-S细胞(购自Thermo公司,货号A1155701),离心并按3.5×10
6细胞/ml接种250mlCHOgro培养基(购自MIRUS公司)。将实施例1中获得的待转染质粒通过0.22μm滤膜除菌过滤,取125μg第一多肽链重组质粒及125μg相应的第二多肽链重组质粒,加入25ml CHOgro复合物形成溶液(CHOgro Complex Formation Solution,购自MIRUS公司),加入1mg/ml PEIMAX(购自polysciencse公司)1.25ml,震荡混匀三次,静置10min,加入到250ml上述细胞培养物中。将培养物置于37℃、5%CO
2摇床培养,24h后加入20ml 10%Sheff-CHO PF ACF(购自KERRY公司),继续培养7天后收获细胞培养物。
实施例3:抗PDL-1/CTLA-4重组双特异性抗体的纯化
取实施例2中表达7天的CHO-S细胞培养物,先低速离心分离上清与 细胞沉淀;再高速离心,得到澄清的料液。通过亲和层析法(Protein A)和离子交换两步法进行重组抗体的纯化,纯化中使用的介质分别是GE公司生产的Mab Select SuRe和Millipore公司生产的Eshmuno CPX。各重组双特异性抗体表达产量基本一致,均在40-85mg/L区间,且和同等条件抗PDL-1亲本抗体AB01的表达水平保持一致,说明各重组抗体均可成功表达,且具有较高的表达效率,具体表达水平见表5。分离纯化的重组抗体通过超滤管浓缩并换液到PBS溶液中,还原SDS-PAGE电泳显示如图2所示,AB01、AB02还原后为50kDa(重链)及25kDa(轻链);双特异抗体AB03、AB04还原条带大小为75kDa(第一肽链)及25kDa(第二肽链),并且SEC纯度分别为96.34%和96.04%。条带大小符合预期,说明重组各双特异性抗体均能高效表达,正确装配,且无明显聚集降解,说明稳定性良好。
表5:重组双特异性抗体的表达水平
抗体名称 | 获得蛋白量 | 表达产量 |
AB01 | 20.5mg | 60mg/L |
AB03 | 22.6mg | 83mg/L |
AB04 | 12.2mg | 42mg/L |
实施例4:抗PDL-1/CTLA-4重组双特异性抗体的抗原结合活性检测
本实施例以ELISA的方式分别检测各重组双特异性抗体与其亲本抗体结合相同抗原的亲和力的差异,以及重组双特异性抗体同时结合两种抗原的相对亲和力高低,以验证单独封闭一种抗原能力比亲本抗体是否有所下降;是否能同时封闭两种抗原,阻断两种信号通路,从而产生协同作用。
4.1重组双特异性性抗体对PDL-1的结合活性检测
将重组PDL-1-mFc蛋白(获得自科伦博泰uniport号:Q9NZQ7)以100ng/孔加入96孔酶标板(购自thermo公司),4℃包被过夜;次日,弃孔内溶液,用洗涤缓冲液(含有0.05%Tween-20的磷酸盐缓冲液)洗1次,拍干;加入含2%BSA的PBS溶液,100μl/孔,37℃封闭2h后拍干;重组双特异性抗 体AB03、AB04以及亲本抗体AB01以1000ng/ml起始,分11个浓度点做3倍稀释,100μl/孔;将酶标板放入37℃孵育2h,拍干,洗涤缓冲液洗涤3次;加入HRP偶联Goat Anti-Human IgG(H+L)溶液,100μl/孔,37℃孵育1h;100μl/孔加入TMB溶液,室温反应约5min;100μl/孔加入终止液,并放入酶标仪,读OD450吸光值。实验数据通过曲线拟合,如图3所示,并计算EC50。
EC50结果如表6所示,所检测的重组双特异抗体结合PDL-1-mFc的活性EC50与亲本抗体AB01抗PDL-1-mFc的EC50相当,表明本发明的重组抗体整体上保持了与亲本抗体同等优良的对PDL-1-mFc的结合活性。
表6:重组抗体对PDL-1-mFc的结合活性
重组抗体/亲本抗体 | EC50(pM) |
AB01 | 15.55 |
AB03 | 21.46 |
AB04 | 32.43 |
4.2重组双特异性抗体对CTLA-4的结合活性检测
分别将两批次的重组CLTA-4-His蛋白(获得自科伦博泰,uniport号:P16410)用PBS稀释,100ng/孔加入96孔酶标板,4℃包被过夜;次日,弃孔内溶液,用洗涤缓冲液洗1次,拍干;加入含2%BSA的PBS溶液,100μl/孔,37℃封闭2h后拍干;重组双特异性抗体AB03、AB04以及抗CTLA-4亲本抗体AB02以30nM起始,分11个梯度做4倍稀释,100μl/孔;将酶标板放入37℃孵育2h,拍干,洗涤缓冲液洗涤3次;加入HRP偶联Goat Anti-Human IgG(H+L)溶液,37℃孵育1h;加入TMB溶液,室温反应约8min;加入终止液,并放入酶标仪,读OD450吸光值。实验数据通过曲线拟合,两批次的重组CLTA-4-His结合活性检测结果分别如图4A和图4B所示,并计算EC50。
EC50结果如表7所示。
表7:重组抗体对CTLA-4的结合活性
CLTA-4-His蛋白批号 | 批次一 | 批次二 |
重组抗体/亲本抗体 | EC50(pM) | EC50(pM) |
AB02 | 55.93 | 42.38 |
AB03 | 107.5 | 86.17 |
AB04 | 600.2 | 531.3 |
根据实验结果可知,所检测的的重组双特异抗体AB03结合CTLA-4的EC50与亲本抗体AB02相比略有升高,而重组双特异抗体AB04结合CTLA-4的EC50约是亲本抗体AB02的10-12倍,可见AB04结合CTLA-4的活性明显弱于亲本抗体,但仍然具有pM级别的高效结合活性。
4.3重组双特异性抗体同时结合PDL-1及CTLA-4的活性检测
将重组PDL-1-mFc蛋白以100ng/孔,4℃过夜包被于96孔酶标板;次日,弃孔内溶液,用洗涤缓冲液(含有0.05%Tween-20的磷酸盐缓冲液)洗1次,拍干;加入含2%BSA的PBS溶液,100μl/孔,37℃封闭2h后拍干;重组双特异性抗体以10000ng/ml起始,分11个梯度做5倍稀释,100μl/孔;将酶标板放入37℃孵育2h,拍干,洗涤缓冲液洗涤3次;每孔加CTLA-4-His蛋白1.5μg/ml,100μl/孔,37℃孵育2h,拍干,洗涤缓冲液洗涤3次;加入HRP偶联Goat Anti-His(购自BioLegend公司)溶液,37℃孵育1h;加入TMB溶液,室温反应约5min;加入终止液,并放入酶标仪,读OD450吸光值。实验数据通过曲线拟合,如图5所示,并计算EC50。
EC50结果如表8所示,重组双特异性抗体AB03及AB04同时结合PDL-1及CTLA-4两种抗原EC50都在pM级,上述结果表明本发明的重组双特异性抗体结合一种抗原后,不影响第二种抗原结合,AB03及AB04等重组双特异性抗体都能同时高效的结合两种抗原,可以通过同时封闭肿瘤免疫中起关键作用的PDL-1及CTLA-4靶点,以同时抑制两条信号通路,从而在肿瘤治疗中起到药效协同作用。
表8:重组双特异性抗体同时结合PDL-1及CTLA-4的活性
重组双特异抗体名称 | EC50(pM) |
AB03 | 16.24 |
AB04 | 12.98 |
实施例5:重组双特异性抗体阻断PD-1/PDL-1结合活性竞争ELISA检测
本实施例中,通过竞争ELISA检测重组双特异性抗体是否能阻断PD1和PDL-1的结合。
将重组PD1-hFc蛋白(购自北京义翘神州公司)以100ng/孔加入96孔酶标板(购自thermo公司),4℃包被过夜;次日,弃孔内溶液,用洗涤缓冲液(含有0.05%Tween-20的磷酸盐缓冲液)洗1次,拍干;加入含2%BSA的PBS溶液,100μl/孔,37℃封闭2h后拍干;重组双特异性抗体AB03、AB04以及亲本抗体AB01以10μg/ml起始,分11个梯度做1.5倍稀释,加入等体积1.6μg/ml PDL-1-mFC(获得自科伦博泰)混匀室温放置30min,然后加入100μl/孔至酶标板中;将酶标板放入37℃孵育2h,拍干,洗涤缓冲液洗涤3次;加入HRP偶联Goat Anti-mouse IgG(H+L)(购自thermo公司)溶液,100μl/孔,37℃孵育1h;100μl/孔加入TMB溶液,室温反应约5min;100μl/孔加入终止液,并放入酶标仪,读OD450吸光值。实验数据通过曲线拟合,如图6所示,并计算EC50。
EC50结果如表9所示,所检测的重组双特异抗体AB03、AB04阻断PDL-1结合PD1的活性EC50与抗PDL-1亲本抗体AB01相比表现更强,表明本发明的重组双特异抗体整体上保持了与亲本抗体同等或更优良的对PDL-1结合PD1的阻断活性。
表9:重组抗体对PDL-1/PD1的阻断活性
重组抗体/亲本抗体 | EC50(nM) |
AB01 | 3.82 |
AB03 | 2.60 |
AB04 | 1.84 |
实施例6:重组双特异性抗体的CDC、ADCC活性ELISA检测
本实施例为验证重组双特异性抗体ADCC、CDC活性,通过ELISA实验检测重组双特异性抗体分别与CD16a(Val)、C1q的结合。
6.1重组双特异性抗体对CD16a(Val)的结合活性检测
将重组CD16a(Val)蛋白(购自义翘神州公司)以100ng/孔加入96孔酶标板(购自thermo公司),4℃包被过夜;次日,弃孔内溶液,用洗涤缓冲液(含有0.05%Tween-20的磷酸盐缓冲液)洗1次,拍干;加入含2%BSA的PBS溶液,100μl/孔,25℃封闭2h后拍干;重组双特异性抗体AB03、AB04以及亲本抗体AB01和阳性对照抗体Human IgG1(获得自科伦博泰)以50μg/ml,加入Goat F(ab)
2anti-Human Kappa(购自BIORAD公司)37℃交联反应1h,然后分11个梯度做3倍稀释,100μl/孔;将酶标板放入25℃孵育2h,拍干,洗涤缓冲液洗涤3次;加入1:10000HRP偶联F(ab)
2亲和纯化F(ab’)
2片段化山羊抗人IgG二抗(购自Jackson公司)溶液,100μl/孔,25℃孵育1h;拍干,洗涤缓冲液洗涤5次;100μl/孔加入TMB溶液,室温反应约30min;100μl/孔加入终止液,并放入酶标仪,读OD450吸光值。实验数据通过曲线拟合,如图7。
结果如图7所示,所检测的重组双特异抗体AB03、AB04具有结合CD16a(Val)蛋白的活性,并且与Human IgG1相当,而AB01几乎与CD16a(Val)不结合,表明本申请重组双特异抗体具有ADCC活性。
6.2重组双特异性抗体对C1q的结合活性检测
将重组双特异性抗体AB03、AB04以及亲本抗体AB01和阳性对照抗体Human IgG1(获得自科伦博泰)以500μg/mL起始,分11个梯度做3倍稀释,100μl/孔,加入96孔酶标板,4℃包被过夜。次日,弃孔内溶液,用洗涤缓冲液洗1次,拍干;加入含2%BSA的PBS溶液,100μl/孔,37℃封闭2h后拍干。含2%BSA的PBS溶液稀释C1q(购买PROSPEC公司)3μg/mL,100μl/孔;将酶标板放入37℃孵育2h,拍干,洗涤缓冲液洗涤3次;加入1:200稀释HRP抗C1q抗体(购买Abcam公司)溶液,100μl/孔,37℃孵育1h;拍干,洗涤缓冲液洗涤5次,加入TMB溶液,室温反应约5min;加入终止 液,并放入酶标仪,读OD450吸光值。实验数据通过曲线拟合,如图8。
结果如图8所示,所检测的重组双特异抗体AB03、AB04结合C1q蛋白的活性与Human IgG1相比更强,表明其具有更强的CDC活性。
实施例7:热变性温度Tm检测
7.1DSF(差示扫描荧光法)测定重组双特异性抗体的Tm值
将重组双特异性抗体及亲本抗体AB01用PBS溶液稀释至1mg/ml,将
橙色蛋白凝胶染色液(Orange Protein Gel Stain,购自Thermo公司,货号S6651)用蒸馏水稀释40倍。取0.2ml离心管,依次加入稀释样品12.5μl,
橙色蛋白凝胶染色液4.2μl,蒸馏水8.3μl。混匀后放入荧光定量PCR仪(购自Thermo公司,型号7500),设置反应参数25℃ 3min,以1%速率上升至95℃,95℃ 2min。
7.2 DSC(差示扫描量热法)测定重组双特异性抗体的Tm值
将重组双特异性抗体用PBS溶液稀释至1mg/ml,9μl/孔,放入高通量蛋白稳定分析仪(UNCHAINED LABS公司,型号Uncle),设置反应参数25℃到95℃,速率0.3℃/min。
DSF与DSC结果如表10所示,所述重组双特异性抗体AB03、AB04的热稳定性良好,其中,AB03热稳定性优于AB04,与亲本抗体AB01、AB02的Tm值更为相近,本发明所述重组双特异性抗体整体上具有良好热稳定性。
表10:重组双特异性抗体的热变性温度
重组抗体/亲本抗体 | AB01 | AB02 | AB03 | AB04 |
DSF Tm(℃) | 77.6 | 75.2 | 71.8 | 68.7 |
DSC Tm1(℃) | 72.0 | 未检测 | 74.6 | 71.0 |
DSC Tm2(℃) | 81.3 | 未检测 | 82.1 | 79.8 |
实施例8:重组双特异性抗体的阻断PD1/PDL-1细胞活性
报告基因法来检测PD-1/PDL-1抗体的生物活性。CHO-PDL-1-CD3L细胞系稳定表达PDL-1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PDL-1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PDL-1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:取Jurkat-PD1NFAT细胞、CHO-S-OKT3-PDL1细胞(购买Promega公司),200g离心5min,检测缓冲液RPMI1640+1%FBS重悬。CHO-S-OKT3-PDL1细胞5×10
5个/40μL/孔铺板。Jurkat-PD1-NFAT细胞2.5×10
6个/40μL/孔加入至预先铺好CHO-S-OKT3-PDL1细胞的孔中,加入40μL 100μg/mL起始5倍稀释的重组双特异性抗体AB03、AB04以及AB01抗体。共孵育6小时后取出,室温放置20min,加入60μL检测试剂Bright-Glo Luciferase(购自Promega公司),室温震荡5min,酶标仪(BMG公司,PHEARstar FS)上机检测。实验数据通过GraphPad prism5拟合曲线并计算EC50,如图9所示。
结果如表11所示,所检测的重组双特异抗体AB03、AB04阻断PDL-1结合PD1的细胞活性EC50与抗PDL-1亲本抗体AB01相当,表明本发明的重组双特异抗体整体上保持了与亲本抗体同等优良的对PDL-1结合PD1的阻断细胞活性。
表11:重组抗体对PDL-1/PD1的阻断活性
重组抗体/亲本抗体 | EC50(nM) |
AB01 | 1.68 |
AB03 | 1.82 |
AB04 | 1.53 |
实施例9:重组双特异性抗体在SEB刺激PBMC下增强T细胞分泌IL-2的活性
超抗原(SEB)可以交联APC细胞的MHC II与T细胞的TCR来激活T细胞,促进IL-2细胞因子表达。T细胞上PD1与APC细胞上PDL-1结合、 T细胞上CTLA-4与APC细胞上CD80/CD86结合会抑制T细胞激活,降低IL-2细胞因子表达。本发明所述重组双特异性抗体能阻断PD1/PDL-1及CTLA-4/CD80(CD86),能进一步促进SEB刺激PBMC分泌IL-2表达。
具体实验步骤:复苏PBMC细胞,使用5mL RPMI1640+10%FBS(胎牛血清)重悬,细胞活率85%,活细胞密度8×10
5细胞/ml,37℃CO
2培养箱培养大于2小时;120uL细胞悬液,10
5细胞/孔;配制RPMI1640+10%FBS含2.5ng/mL SEB(来自中科院)抗体稀释液,分别稀释重组双特异性抗体AB03、AB04以及对照抗体AB01、AB02,加入80uL至对应孔中;混匀,放于37℃CO
2培养箱培养3天;取上清,采用IL-2检测试剂盒(购自CisBio公司)进行IL-2表达量检测。
结果如图10所示,所检测的重组双特异抗体AB03、AB04与亲本抗体AB01、AB02相比,整体上均能大幅提升SEB刺激PBMC分泌IL-2表达。同时在高浓度时,重组双特异抗体和亲本抗体都发挥了最大响应值,在低浓度抗体条件下,重组双特异抗体AB03、AB04比亲本抗体AB01、AB02作用功能有一定的增强(标记星号处)。
实施例10:PDL-1抗体AB01对结肠癌的体内疗效
1.受试药物
配制方法:三药均用含0.1%BSA生理盐水稀释成所需浓度。
1.试验细胞和动物
MC-38/H-11细胞是小鼠结肠癌MC-38(购自Cobioer,货号CBP60825)细胞敲除了小鼠内源性PDL-1,转染并表达人PDL-1的单克隆细胞,因此,MC-38/H-11细胞只高表达人PDL-1蛋白。
C57BL/6小鼠,7-8周,♀,购自上海斯莱克实验动物有限责任公司。
2.实验步骤
每只小鼠皮下接种1×10
5MC-38/H-11细胞,接种后第二天(D0)随机分组并腹腔注射(IP)药物,隔1天1次(Q2D);溶剂组注射相同体积的人IgG (15mg/kg),
AB01(1.5、5、15mg/kg)、
(15mg/kg),注射体积0.1mL/10g体重。各组均为10只小鼠。
3.实验指标
实验指标为考察药物对肿瘤生长的影响,具体指标为T/C%或抑瘤率TGI(%)。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为:
V=1/2×a×b
2,其中a、b分别表示长、宽。
T/C%=T/C×100,C、T分别为溶剂组和治疗组的肿瘤体积或肿瘤重量。
抑瘤率(TGI)(%)=(C-T)/C×100,C、T分别为溶剂组和治疗组的肿瘤体积或肿瘤重量。
4.实验结果
如下面的表12所示。
表12:AB01(1.5、5、15mg/kg)、
对小鼠结肠癌MC-38/H-11小鼠皮下移植瘤的疗效
注:随机分组,第一次给药时间为D0;D27为给药后第27天。
AB01(1.5、5、15mg/kg)对MC-38/H-11小鼠皮下移植瘤的抑瘤率分别为 63.9%、75.8%和68.6%(根据平均肿瘤体积计算);鉴于每组肿瘤的个体差异较大,采用中位肿瘤体积计算抑瘤率比较合理,则抑瘤率调整为100%、100%和100%;参比药物
(15mg/kg)对MC-38/H-11的抑瘤率为93.8%(根据中位肿瘤体积计算);采用中位肿瘤重量计算抑瘤率,则AB01(1.5、5、15mg/kg)对MC-38/H-11的抑瘤率分别为100%、100%、100%,
的抑瘤率为93.7%;中位肿瘤体积与中位肿瘤重量计算所得抑瘤率非常一致,说明肿瘤体积测量方法的可靠性。AB01(1.5、5、15mg/kg)不但抑制肿瘤生长,还抑制肿瘤成瘤,实验结束时(D27),AB01(1.5、5、15mg/kg)剂量组成瘤率分别为40%、40%和40%,
组的成瘤率为50%。荷瘤小鼠对以上药物均能很好耐受,没有明显体重减轻等症状发生。相比较,AB01(1.5、5、15mg/kg)对小鼠结肠癌MC-38/H-11小鼠皮下移植瘤较
具有更强的抑瘤作用。
实施例11:PDL-1抗体AB01对肺癌的体内疗效
建模方法:将非小细胞肺癌细胞HCC827(购自ATCC货号CRL-2868)皮下接种NOG鼠,构建肺癌荷瘤鼠模型,待肿瘤长到约100mm
3,给药前小鼠静脉注射激活后的人PBMCs模拟人免疫系统,然后给药。
肿瘤生长曲线见图11。
实施例12.重组双特异性抗体在转基因结肠癌小鼠模型的测试
用含10%胎牛血清的RPMI1640培养液,在37℃,5%CO
2的条件下培养MC38-1F3细胞,收集指数生长期的MC38-1F3细胞,PBS重悬至适合浓度,接种于雌性研-C57BL/6J-huCTLA-4小鼠(集萃药康)皮下建立结肠癌模型。 待肿瘤平均体积约82mm
3时,根据肿瘤大小随机分组,分别为静注人免疫球蛋白(阴性对照)组、AB01对照组、AB04抗体的低剂量组和高剂量组,腹腔注射,每周2次,共给药3周。给药后观察并定期测量小鼠肿瘤体积及体重,具体结果见表13,图12。
由试验结果可知,AB04抗体低剂量组(3mg/kg)对MC38-1F3结肠癌移植瘤模型的抗肿瘤药效好于AB01,且其高剂量组(10mg/kg)抗肿瘤药效显著好于低剂量组,抗肿瘤药效显著。所有治疗组在观察期内均无动物死亡及显著动物体重降低,没有表现明显的药物毒性,治疗期间小鼠对AB04抗体耐受性良好。
表13.重组双特异性抗体对小鼠结肠癌疗效
实施例13.重组双特异性抗体在转基因非小细胞肺癌小鼠模型的测试
用含10%胎牛血清的RPMI1640培养液,在37℃,5%CO2的条件下培养人非小细胞肺癌细胞株HCC827。收集指数生长期的HCC827细胞,PBS重悬至适合浓度,接种于雌性NSG免疫缺陷小鼠皮下建立移植瘤模型(hPBMC-NSG(百奥赛图)-HCC827人免疫重建模型)。待肿瘤平均体积约77mm
3时,根据肿瘤大小随机分组,分别为生理盐水(阴性对照)组、AB01对照组、AB04抗体的低剂量组和高剂量组。用含10%胎牛血清、CD28/CD3和DNase的RPMI1640培养液,在37℃,5%CO2的条件下培养人外周血单核细胞PBMC。经CD3和CD28刺激培养3天后,收集活化的PBMC,用 PBS重悬至适合浓度,尾静脉接种于已纳入分组的雌性NSG小鼠体内以重建其免疫系统。分组后尾静脉注射,每周2次,共给药3周。给药后观察并定期测量小鼠肿瘤体积及体重,具体结果见表14,图13。
表14.重组双特异性抗体对转基因非小细胞肺癌小鼠疗效
由试验结果可知,AB04抗体低剂量组(1mg/kg)对皮下移植人非小细胞肺癌HCC827的NSG免疫重建小鼠的肿瘤生长有一定抑制作用,高剂量组(10mg/kg)抗肿瘤药效显著。所有治疗组在观察期内均无动物死亡及显著动物体重降低,没有表现明显的药物毒性,治疗期间小鼠对抗体耐受性良好。
实施例14.重组双特异性抗体的毒理试验
为检测重组双特性抗体在食蟹猴体内的毒理情况,本次试验为静脉注射食蟹猴(雌雄各一只),单次给药,设置给药组AB04,对照组为生理盐水,恢复期14天。实验结果表明:单次静脉注射给予500mg/kg的AB04,食蟹猴对药物耐受性良好,MTD为500mg/kg;重复给药按100mg/kg的剂量,每周1次,连续给药5次,未见明显毒性靶器官和给药部位刺激性,NOAEL(未见毒性反应剂量)为100mg/kg。根据现有FDA公布的研究资料显示,抗CTLA-4抗体Ipilimumab(商品名:Yervoy)的重复给药NOAEL为10mg/kg,(在该剂量下,给药后可见脾脏重量降低,多器官淋巴细胞浸润等轻微毒性反应),MTD为30mg/kg。AB04降低了CTLA-4抗体毒性作用,一定程度上提升了药物的安全性。
尽管本发明的具体实施方式已经得到详细的描述,但本领域技术人员将理解:根据已经公布的所有教导,可以对细节进行各种修改和变动,并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
Claims (29)
- 一种双特异性抗体,其包含:1)特异性结合第一抗原的第一抗体,所述第一抗体包括重链(HC)和轻链(LC);和2)特异性结合第二抗原的scFv;其中,所述scFv连接于第一抗体的重链或轻链的N端或C端;所述第一抗原为CTLA-4,并且所述第二抗原为PDL-1;或者,第一抗原为PDL-1,并且所述第二抗原为CTLA-4;优选地,所述双特异性抗体包含1个所述第一抗体和2个所述scFv;并且,所述第一抗体包括两条HC和两条LC,其中所述第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位,另一条HC的VH与另一条LC的VL形成抗原结合部位;优选地,一个所述scFv连接于所述第一抗体的重链或轻链的N端,另一个所述scFv连接于所述第一抗体的重链或轻链的C端;优选地,每个所述scFv分别连接于所述第一抗体的两条重链或两条轻链的N端;或者,每个所述scFv分别连接于所述第一抗体的两条重链或两条轻链的C端。
- 权利要求1所述的双特异性抗体,其包含:1)特异性结合第一抗原的第一抗体,所述第一抗体包括重链(HC)和轻链(LC);和2)特异性结合第二抗原的scFv;所述双特异性抗体包含1个所述第一抗体和2个所述scFv;并且,所述第一抗体包括两条HC和两条LC,其中所述第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位,另一条HC的VH与另一条LC的VL形成抗原结合部位;每个所述scFv分别连接于所述第一抗体的两条重链的N端;或者,每个所述scFv分别连接于所述第一抗体的两条重链的C端;所述第一抗原为PDL-1,并且所述第二抗原为CTLA-4;优选地,所述每个scFv通过接头S1与所述第一抗体的每条重链的N端或C端连接;更优选的,所述scFv的由VH与VL通过S2接头连接。
- 权利要求2所述的双特异性抗体,所述scFv的结构为NH2-VL-S2-VH-COOH。
- 权利要求1-3所述任一项的双特异性抗体,其中所述第一抗体的重链包含重链可变区(VH)和重链恒定区(CH),并且所述轻链包含轻链可变区(VL)和轻链恒定区(CL);优选地,所述第一抗体为全长抗体。
- 权利要求1-4所述任一项的双特异性抗体,其中所述第一抗体的重链为IgG同种型,例如IgG1、IgG2、IgG3或IgG4,例如人IgG同种型;和/或,所述第一抗体的轻链为Kappa同种型,例如人Kappa同种型。
- 权利要求1-5任一项所述的双特异性抗体,其中所述第一抗体的两条HC包含相同的CDR;和/或,所述第一抗体的两条LC包含相同的CDR;优选地,所述第一抗体的两条HC包含相同的VH;和/或,所述第一抗体的两条LC包含相同的VL;优选地,所述第一抗体的两条HC具有相同的氨基酸序列;和/或,所述第一抗体的两条LC具有相同的氨基酸序列;优选地,两个所述scFv具有相同或不相同的氨基酸序列;更优选地,两个所述scFv具有相同的氨基酸序列。
- 权利要求1-6任一项所述的双特异性抗体,其中所述双特异性抗体包含两条第一多肽链和两条第二多肽链,其中对于所述每条多肽链:a)所述第一多肽链各自独立地包含所述第一抗体的重链(HC)和所述scFv;和b)所述第二多肽链各自独立地包含所述第一抗体的轻链(LC);其中,所述scFv通过接头S1与所述第一抗体的HC的N端或C端相连;或者,i)所述第一多肽链各自独立地包含所述第一抗体的轻链(LC)和所述 scFv;和ii)所述第二多肽链各自独立地包含所述第一抗体的重链(HC);其中,所述scFv通过接头S1与所述第一抗体的LC的N端或C端相连;优选地,所述scFv具有结构:NH 2-VH-S2-VL-COOH或NH 2-VL-S2-VH-COOH,其中所述S2为接头;优选地,所述双特异性抗体包含两条相同的第一多肽链和两条相同的第二多肽链。
- 权利要求1-7所述的双特异性抗体,具有与所述第一抗体的亲本抗体相比同等或更弱的结合第一抗原的活性;优选地,所述的双特异性抗体,具有与所述scFv的亲本抗体相比同等或更弱的结合第二抗原的活性;更优选地,所述的双特异性抗体,具有与所述第一抗体的亲本抗体相比同等或更弱的结合第一抗原的活性,且具有与所述scFv的亲本抗体相比同等或更弱的结合第二抗原的活性;更优选地,所述的双特异性抗体,具有与所述第一抗体的亲本抗体相比同等的结合第一抗原的活性,且具有与所述scFv的亲本抗体相比更弱的结合第二抗原的活性;更优选地,所述的双特异性抗体,具有与结合PDL-1的亲本抗体相比同等的结合活性;更优选地,所述的双特异性抗体,具有与结合CTLA-4的亲本抗体相比更弱的结合活性;更优选地,所述的双特异性抗体,具有与结合PDL-1的亲本抗体相比同等的结合活性,且具有与结合CTLA-4的亲本抗体相比更弱的结合活性。
- 权利要求2,3,7或8所述的双特异性抗体,其中所述接头S1和/或S2为肽接头,例如具有如(G mS n) x所示的氨基酸序列,其中m、n各自独立地选自1~8的整数(例如,1、2、3、4、5、6、7或8),x独立地选自1~20的整数(例如,1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20);优选地,所述所述接头S1和/或S2具有如(G 4S) x所示的氨基酸序列,x 独立地选自1-6的整数;优选地,所述S1和/或S2具有选自下列的氨基酸序列:SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27;优选地,所述接头S2具有如SEQ ID NO:25所示的氨基酸序列,且当所述scFv与所述第一抗体的重链或轻链的N端连接时,所述接头S1具有如SEQ ID NO:26所示的氨基酸序列,当所述scFv与所述第一抗体的重链或轻链的C端连接时,所述接头S1如SEQ ID NO:27所示的氨基酸序列。
- 权利要求1-9任一项所述的双特异性抗体,其中所述scFv的VH与VL之间存在二硫键;优选地,所述scFv的位于VH第44位的氨基酸和位于VL第100位的氨基酸分别为半胱氨酸,其中提及的氨基酸位置是根据Kabat编号系统的位置;并且,所述scFv的VH与VL通过分别位于VH第44位和VL第100位的2个半胱氨酸残基之间所形成的二硫键连接。
- 权利要求1-10任一项所述的双特异性抗体,其中所述第一抗体特异性结合CTLA-4,并且所述scFv特异性结合PDL-1,其中:所述scFv包含:如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
- 权利要求11所述的双特异性抗体,其中所述第一抗体特异性结合CTLA-4,并且所述scFv特异性结合PDL-1,其中:所述第一抗体包含:如SEQ ID NO:13所示的HCDR1;如SEQ ID NO:14所示的HCDR2;以及,如SEQ ID NO:15所示的HCDR3;如SEQ ID NO:16所示的LCDR1;如SEQ ID NO:17所示的LCDR2;以及,如SEQ ID NO:18所示的LCDR3;并且,所述scFv包含:如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
- 权利要求1-12任一项所述的双特异性抗体,其中,所述scFv包含:(1)如SEQ ID NO:7所示的VH和如SEQ ID NO:9所示的VL;或,(2)如SEQ ID NO:8所示的VH和如SEQ ID NO:10所示的VL。
- 权利要求1-13任一项所述的双特异性抗体,其中:所述第一抗体包含如SEQ ID NO:19所示的VH和如SEQ ID NO:21所示的VL;并且,所述scFv包含:(1)如SEQ ID NO:7所示的VH和如SEQ ID NO:9所示的VL;或,(2)如SEQ ID NO:8所示的VH和如SEQ ID NO:10所示的VL。
- 权利要求1-10任一项所述的双特异性抗体,其中所述第一抗体特异性结合PDL-1,并且所述scFv特异性结合CTLA-4,其中:所述第一抗体包含:如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3。
- 权利要求1-10或15任一项所述的双特异性抗体,其中所述第一抗体特异性结合PDL-1,并且所述scFv特异性结合CTLA-4,其中:所述第一抗体包含:如SEQ ID NO:1所示的HCDR1;如SEQ ID NO:2所示的HCDR2;以及,如SEQ ID NO:3所示的HCDR3;如SEQ ID NO:4所示的LCDR1;如SEQ ID NO:5所示的LCDR2;以及,如SEQ ID NO:6所示的LCDR3;并且,所述scFv包含:如SEQ ID NO:13所示的HCDR1;如SEQ ID NO:14所示的HCDR2;以及,如SEQ ID NO:15所示的HCDR3;如SEQ ID NO:16所示的LCDR1;如SEQ ID NO:17所示的LCDR2;以及,如SEQ ID NO:18所示的LCDR3。
- 权利要求1-10,15或16任一项所述的双特异性抗体,其中:所述第一抗体包含如SEQ ID NO:7所示的VH和如SEQ ID NO:9所示的VL。
- 权利要求1-10,15-17任一项所述的双特异性抗体,其中:所述第一抗体包含如SEQ ID NO:7所示的VH和如SEQ ID NO:9所示的VL;并且, 所述scFv包含:(1)如SEQ ID NO:19所示的VH和如SEQ ID NO:21所示的VL;或,(2)如SEQ ID NO:20所示的VH和如SEQ ID NO:22所示的VL。
- 根据权利要求1-18任一项所述的双特异性抗体,具有抗体依赖性细胞介导的细胞毒性(ADCC)活性;优选地,所述的双特异性抗体具有补体依赖性细胞毒性(CDC)活性;更优选地,其中所述第一抗体CH含有突变,所述突变后双特异性抗体具有增强的抗体依赖性细胞介导的细胞毒性(ADCC)活性;更优选地,其中所述第一抗体CH含有突变,所述突变后双特异性抗体具有增强的补体依赖性细胞毒性(CDC)活性。
- 权利要求1-19任一项所述的双特异性抗体,其中所述第一抗体包含:如Uniprot的收录号P01857所示的CH;和/或,如Uniprot的收录号P01834所示的CL,可选地,所述CH含有突变,为在UniProt的收录号P01857的CH中第117,118,120位突变为A;或,在UniProt的收录号P01857的CH中第97位突变为R。
- 权利要求1-20任一项所述的双特异性抗体,其中所述双特异性抗体包含:(1)如SEQ ID NO:28所示的第一多肽链和如SEQ ID NO:11所示的第二多肽链;或(2)如SEQ ID NO:29所示的第一多肽链和如SEQ ID NO:11所示的第二多肽链。
- 权利要求1-21任一项所述的双特异性抗体,其中所述双特异性抗体具有与亲本抗体相比基本相同的热稳定性。
- 一种分离的核酸分子,其包含编码权利要求1-22任一项所述的双特异性抗体的核苷酸序列;优选地,所述分离的核酸分子包含编码权利要求1-22任一项所述的双特异性抗体的第一多肽链的核苷酸序列;优选地,所述分离的核酸分子包含编码权利要求1-22任一项所述的双特 异性抗体的第二多肽链的核苷酸序列。
- 一种载体,其包含权利要求23所述的分离的核酸分子。
- 宿主细胞,其包含权利要求23所述的分离的核酸分子或权利要求24所述的载体。
- 制备权利要求1-22任一项所述的双特异性抗体的方法,其包括,在允许所述双特异性抗体表达的条件下,培养权利要求25所述的宿主细胞,和从培养的宿主细胞培养物中回收所述双特异性抗体。
- 药物组合物,其含有权利要求1-22任一项所述的双特异性抗体,以及药学上可接受的载体和/或赋形剂;优选地,所述药物组合物还包含另外的药学活性剂,例如用于预防和/或治疗与CTLA-4和/或PDL-1相关的疾病的药物,例如抗炎药物或免疫抑制剂;优选地,所述与CTLA-4和/或PDL-1相关的疾病为自身免疫性疾病或肿瘤或传染病病,例如肿瘤,包括但不限于:腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤,或包括但不限于肾上腺、胆囊、骨、骨髓、脑、乳腺、胆管、胃肠道、心脏、肾脏、肝脏、肺、肌肉、卵巢、胰腺、甲状旁腺、阴茎、前列腺、皮肤、唾液腺、脾脏、睾丸、胸腺、甲状腺和子宫相关的肿瘤,传染病包括但不限于乙肝,甲肝,HIV。
- 权利要求1-22任一项所述的双特异性抗体或权利要求27所述的药物组合物在制备药物中的用途,所述药物用于在受试者(例如人)中预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(如自身免疫性疾病或肿瘤或传染病),和/或用于体外或在受试者(例如人)体内抑制CTLA-4和/或PDL-1的活性;优选地,所述与CTLA-4和/或PDL-1相关的疾病为自身免疫性疾病或肿瘤或传染病病,例如肿瘤,包括但不限于:腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤,或包括但不限于肾上腺、胆囊、骨、骨髓、脑、乳腺、胆管、胃肠道、心脏、肾脏、肝脏、肺、肌肉、卵巢、胰腺、甲状旁腺、阴茎、前列腺、皮肤、唾液腺、脾脏、睾丸、胸腺、甲状腺和子宫相关的肿瘤,传染病包括但不限于乙肝,甲肝,HIV;优选地,所述受试者可以为哺乳动物,例如人。
- 一种用于在受试者(例如人)中预防和/或治疗与CTLA-4和/或PDL-1相关的疾病(例如炎性疾病或自身免疫性疾病),和/或用于体外或在受试者(例如人)体内抑制CTLA-4和/或PDL-1的活性的方法,其中所述方法包括,给有此需要的受试者施用有效量的权利要求1-22任一项所述双特异性抗体,或者权利要求27所述的药物组合物;优选地,所述与CTLA-4和/或PDL-1相关的疾病为自身免疫性疾病或肿瘤或传染病病,例如肿瘤,包括但不限于:腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤,或包括但不限于肾上腺、胆囊、骨、骨髓、脑、乳腺、胆管、胃肠道、心脏、肾脏、肝脏、肺、肌肉、卵巢、胰腺、甲状旁腺、阴茎、前列腺、皮肤、唾液腺、脾脏、睾丸、胸腺、甲状腺和子宫相关的肿瘤,传染病包括但不限于乙肝,甲肝,HIV;优选地,所述受试者可以为哺乳动物,例如人。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019572582A JP7145895B2 (ja) | 2017-09-01 | 2018-08-17 | 組換え二重特異性抗体 |
KR1020197038632A KR102678252B1 (ko) | 2017-09-01 | 2018-08-17 | 재조합 이중특이적 항체 |
CN201880041765.6A CN110799546B (zh) | 2017-09-01 | 2018-08-17 | 重组双特异性抗体 |
US16/624,408 US11421029B2 (en) | 2017-09-01 | 2018-08-17 | Recombinant bispecific antibodies to PD-L1 and CTLA-4 |
EP18851360.0A EP3626745A4 (en) | 2017-09-01 | 2018-08-17 | RECOMBINANT BISPECIFIC ANTIBODY |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710781402.3 | 2017-09-01 | ||
CN201710781402 | 2017-09-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2019042153A1 true WO2019042153A1 (zh) | 2019-03-07 |
Family
ID=65524891
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2018/100971 WO2019042153A1 (zh) | 2017-09-01 | 2018-08-17 | 重组双特异性抗体 |
Country Status (6)
Country | Link |
---|---|
US (1) | US11421029B2 (zh) |
EP (1) | EP3626745A4 (zh) |
JP (1) | JP7145895B2 (zh) |
KR (1) | KR102678252B1 (zh) |
CN (1) | CN110799546B (zh) |
WO (1) | WO2019042153A1 (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020025659A1 (en) * | 2018-07-31 | 2020-02-06 | Pieris Pharmaceuticals Gmbh | Novel fusion protein specific for cd137 and pd-l1 |
WO2020227431A1 (en) * | 2019-05-06 | 2020-11-12 | Brown University | Bispecific antibodies against chi3l1 and pd1 with enhanced t cell-mediated cytotoxic effects on tumor cells |
EP4039704A4 (en) * | 2019-09-30 | 2023-09-20 | Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. | ANTI-PD-1 ANTIBODIES AND ITS USE |
EP4069746A4 (en) * | 2019-12-04 | 2023-12-27 | Jiangsu Alphamab Biopharmaceuticals Co., Ltd. | BISPECIFIC FUSION PROTEIN FOR TUMOR TREATMENT |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG10201912560YA (en) | 2016-12-07 | 2020-02-27 | Agenus Inc | Anti-ctla-4 antibodies and methods of use thereof |
WO2021226984A1 (zh) * | 2020-05-15 | 2021-11-18 | 三生国健药业(上海)股份有限公司 | 一种抗pd-1和pd-l1的四价双特异性抗体 |
CN116514974A (zh) * | 2022-01-30 | 2023-08-01 | 百奥泰生物制药股份有限公司 | 抗cd28抗体及其应用 |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3817837A (en) | 1971-05-14 | 1974-06-18 | Syva Corp | Enzyme amplification assay |
US3850752A (en) | 1970-11-10 | 1974-11-26 | Akzona Inc | Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically |
US3939350A (en) | 1974-04-29 | 1976-02-17 | Board Of Trustees Of The Leland Stanford Junior University | Fluorescent immunoassay employing total reflection for activation |
US3996345A (en) | 1974-08-12 | 1976-12-07 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4275149A (en) | 1978-11-24 | 1981-06-23 | Syva Company | Macromolecular environment control in specific receptor assays |
US4277437A (en) | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
US4366241A (en) | 1980-08-07 | 1982-12-28 | Syva Company | Concentrating zone method in heterogeneous immunoassays |
US5747654A (en) | 1993-06-14 | 1998-05-05 | The United States Of America As Represented By The Department Of Health And Human Services | Recombinant disulfide-stabilized polypeptide fragments having binding specificity |
CN104974253A (zh) * | 2014-04-01 | 2015-10-14 | 上海中信国健药业股份有限公司 | 抗ctla-4/pd-1双特异性抗体、其制备方法及应用 |
CN104987421A (zh) | 2015-05-13 | 2015-10-21 | 北京比洋生物技术有限公司 | 抗ctla-4和pd-1的双重可变结构域免疫球蛋白 |
CN106967172A (zh) | 2016-08-23 | 2017-07-21 | 中山康方生物医药有限公司 | 抗ctla4‑抗pd‑1 双功能抗体、其药物组合物及其用途 |
CN107082812A (zh) * | 2017-03-29 | 2017-08-22 | 上海科医联创生物科技有限公司 | 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用 |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2690312T3 (es) * | 2012-07-13 | 2018-11-20 | Roche Glycart Ag | Anticuerpos biespecíficos anti-VEGF/anti-ANG-2 y su uso en el tratamiento de enfermedades vasculares oculares |
US20160145355A1 (en) | 2013-06-24 | 2016-05-26 | Biomed Valley Discoveries, Inc. | Bispecific antibodies |
US10570204B2 (en) | 2013-09-26 | 2020-02-25 | The Medical College Of Wisconsin, Inc. | Methods for treating hematologic cancers |
CN105820251B (zh) * | 2015-01-08 | 2019-10-15 | 江苏康宁杰瑞生物制药有限公司 | 具有共同轻链的双特异性抗体或抗体混合物 |
GB201500319D0 (en) * | 2015-01-09 | 2015-02-25 | Agency Science Tech & Res | Anti-PD-L1 antibodies |
JP6955507B2 (ja) * | 2015-12-14 | 2021-10-27 | マクロジェニクス,インコーポレーテッド | Pd‐1及びctla‐4との免疫応答性を有する二重特異性並びにその使用方法 |
JP2019506863A (ja) * | 2016-02-02 | 2019-03-14 | カドモン コーポレイション,リミティド ライアビリティ カンパニー | Pd−l1及びkdrに対する二重結合タンパク質 |
IL260937B2 (en) * | 2016-02-06 | 2024-07-01 | Epimab Biotherapeutics Inc | FABS antibodies one by one and their uses |
UA126905C2 (uk) * | 2016-06-13 | 2023-02-22 | Ай-Маб Байофарма Юес Лімітед | Антитіло до pd-l1 та його застосування |
US10793632B2 (en) * | 2016-08-30 | 2020-10-06 | Xencor, Inc. | Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors |
GB201619652D0 (en) * | 2016-11-21 | 2017-01-04 | Alligator Bioscience Ab | Novel polypeptides |
-
2018
- 2018-08-17 CN CN201880041765.6A patent/CN110799546B/zh active Active
- 2018-08-17 WO PCT/CN2018/100971 patent/WO2019042153A1/zh unknown
- 2018-08-17 US US16/624,408 patent/US11421029B2/en active Active
- 2018-08-17 JP JP2019572582A patent/JP7145895B2/ja active Active
- 2018-08-17 EP EP18851360.0A patent/EP3626745A4/en active Pending
- 2018-08-17 KR KR1020197038632A patent/KR102678252B1/ko active IP Right Grant
Patent Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3850752A (en) | 1970-11-10 | 1974-11-26 | Akzona Inc | Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically |
US3817837A (en) | 1971-05-14 | 1974-06-18 | Syva Corp | Enzyme amplification assay |
US3939350A (en) | 1974-04-29 | 1976-02-17 | Board Of Trustees Of The Leland Stanford Junior University | Fluorescent immunoassay employing total reflection for activation |
US3996345A (en) | 1974-08-12 | 1976-12-07 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4277437A (en) | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
US4275149A (en) | 1978-11-24 | 1981-06-23 | Syva Company | Macromolecular environment control in specific receptor assays |
US4366241A (en) | 1980-08-07 | 1982-12-28 | Syva Company | Concentrating zone method in heterogeneous immunoassays |
US4366241B1 (zh) | 1980-08-07 | 1988-10-18 | ||
US5747654A (en) | 1993-06-14 | 1998-05-05 | The United States Of America As Represented By The Department Of Health And Human Services | Recombinant disulfide-stabilized polypeptide fragments having binding specificity |
CN104974253A (zh) * | 2014-04-01 | 2015-10-14 | 上海中信国健药业股份有限公司 | 抗ctla-4/pd-1双特异性抗体、其制备方法及应用 |
CN104987421A (zh) | 2015-05-13 | 2015-10-21 | 北京比洋生物技术有限公司 | 抗ctla-4和pd-1的双重可变结构域免疫球蛋白 |
CN106967172A (zh) | 2016-08-23 | 2017-07-21 | 中山康方生物医药有限公司 | 抗ctla4‑抗pd‑1 双功能抗体、其药物组合物及其用途 |
CN107082812A (zh) * | 2017-03-29 | 2017-08-22 | 上海科医联创生物科技有限公司 | 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用 |
Non-Patent Citations (24)
Title |
---|
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426 |
BLANK C ET AL., INT. J. CANCER., vol. 119, 2006, pages 317 - 327 |
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883 |
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917 |
E. MEYERSW. MILLER, COMPUT. APPLBIOSCI., vol. 4, 1988, pages 11 - 17 |
F/M AUSUBEL ET AL.: "Remington's Pharmaceutical Sciences Edited by Gennaro AR", 1995, JOHN WILEY AND SONS, INC. |
HOLLIGER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448 |
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883 |
J SAMBROOK ET AL.: "Molecular Cloning: a laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS |
JIN ET AL.: "Chromosomal engineering", CURRENT BIOTECHNOLOGY, vol. 5, no. 6, 31 December 2015 (2015-12-31), pages 420 - 424, XP022350912, ISSN: 2095-2341 * |
JULIE R ET AL., N ENGL J MED., vol. 366, 2012, pages 2455 - 2465 |
KABAT ET AL.: "Sequences of Proteins of lmmunologicallnterest", 1991, SINAUER ASSOCIATES, SUNDERLAND, MASS |
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 443 - 453 |
NEEDLEMANWUNSCH, J MOL BIOL., vol. 48, 1970, pages 444 - 453 |
POLJAK, RJ ET AL., STRUCTURE, vol. 113, 1994, pages 1121 - 1123 |
RAJAGOPAL ET AL., PROT. ENGIN., vol. 10, 1997, pages 1453 - 1459 |
REITER ET AL., CANCER RES., vol. 54, 1994, pages 2714 - 2718 |
REITER ET AL., IMMUNITY, vol. 2, 1995, pages 281 - 287 |
REITER ET AL., INTER. J. OF CANCER, vol. 58, 1994, pages 142 - 149 |
REITER ET AL., JBC, vol. 269, 1994, pages 18327 - 18331 |
REITER ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 1239 - 1245 |
REITER ET AL., PROTEIN ENGINEERING, vol. 8, 1995, pages 1323 - 1331 |
See also references of EP3626745A4 |
WEBBER ET AL., MOLECULAR IMMUNOLOGY, vol. 32, 1995, pages 249 - 258 |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020025659A1 (en) * | 2018-07-31 | 2020-02-06 | Pieris Pharmaceuticals Gmbh | Novel fusion protein specific for cd137 and pd-l1 |
EP4249064A3 (en) * | 2018-07-31 | 2023-12-06 | Pieris Pharmaceuticals GmbH | Novel fusion protein specific for cd137 and pd-l1 |
WO2020227431A1 (en) * | 2019-05-06 | 2020-11-12 | Brown University | Bispecific antibodies against chi3l1 and pd1 with enhanced t cell-mediated cytotoxic effects on tumor cells |
CN114008078A (zh) * | 2019-05-06 | 2022-02-01 | 布朗大学 | 具有增强的t细胞介导的对肿瘤细胞的细胞毒性作用的针对chi3l1和pd1的双特异性抗体 |
EP4039704A4 (en) * | 2019-09-30 | 2023-09-20 | Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. | ANTI-PD-1 ANTIBODIES AND ITS USE |
EP4069746A4 (en) * | 2019-12-04 | 2023-12-27 | Jiangsu Alphamab Biopharmaceuticals Co., Ltd. | BISPECIFIC FUSION PROTEIN FOR TUMOR TREATMENT |
Also Published As
Publication number | Publication date |
---|---|
EP3626745A4 (en) | 2021-03-17 |
US20200216538A1 (en) | 2020-07-09 |
CN110799546B (zh) | 2023-01-24 |
EP3626745A1 (en) | 2020-03-25 |
KR20200041835A (ko) | 2020-04-22 |
CN110799546A (zh) | 2020-02-14 |
KR102678252B1 (ko) | 2024-06-26 |
US11421029B2 (en) | 2022-08-23 |
JP2020534791A (ja) | 2020-12-03 |
JP7145895B2 (ja) | 2022-10-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7488323B2 (ja) | 抗lag-3抗体および組成物 | |
JP7143452B2 (ja) | CD47とSIRPaの相互作用を遮断できる抗体及びその応用 | |
JP6875385B2 (ja) | 抗pd−1抗体および組成物 | |
JP7425604B2 (ja) | 抗ctla4-抗pd-1二機能性抗体、その医薬組成物および使用 | |
TWI752950B (zh) | 抗tim-3抗體及組成物 | |
JP7145895B2 (ja) | 組換え二重特異性抗体 | |
WO2020043184A1 (zh) | 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途 | |
WO2019141092A1 (zh) | 抗lag-3抗体及其用途 | |
WO2022171080A1 (zh) | 抗cd112r抗体及其用途 | |
TW202118787A (zh) | 抗4-1bb抗體、其抗原結合片段及雙特異性抗體 | |
KR20220167331A (ko) | 항-flt3 항체 및 조성물 | |
WO2019238074A1 (zh) | 一种高亲和力高生物活性的lag-3抗体及其应用 | |
WO2022188721A1 (zh) | 抗pvrig蛋白抗体或抗体片段及其用途 | |
WO2019011167A1 (zh) | 双特异性重组蛋白 | |
US12030942B2 (en) | Anti-PD-1 antibodies and compositions | |
WO2022127066A9 (zh) | 一种特异性中和辅助性T细胞TGF-β信号的双特异性抗体、其药物组合及其用途 | |
WO2024007672A1 (zh) | 特异性结合cd24的抗体及其用途 | |
WO2023186063A1 (zh) | 抗pvrig抗体、其药物组合物及用途 | |
WO2024022478A1 (zh) | 结合大麻素受体cb1的抗体及其用途 | |
WO2022262749A1 (zh) | 靶向pd1和/或ox40的特异性结合蛋白 | |
CN117730101A (zh) | 抗nectin4抗体和包含其的多特异性蛋白质复合物 |
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: 18851360 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2019572582 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2018851360 Country of ref document: EP Effective date: 20191219 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |