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WO2025237372A1 - Anticorps anti-egfr et/ou anti-pd-l1 et son utilisation - Google Patents

Anticorps anti-egfr et/ou anti-pd-l1 et son utilisation

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Publication number
WO2025237372A1
WO2025237372A1 PCT/CN2025/095121 CN2025095121W WO2025237372A1 WO 2025237372 A1 WO2025237372 A1 WO 2025237372A1 CN 2025095121 W CN2025095121 W CN 2025095121W WO 2025237372 A1 WO2025237372 A1 WO 2025237372A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
seq
sequence shown
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/095121
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English (en)
Chinese (zh)
Inventor
宁金鹰
彭浩
吴国进
郝锋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyinno Biotechnology (beijing) Co Ltd
Original Assignee
Kyinno Biotechnology (beijing) Co Ltd
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Publication of WO2025237372A1 publication Critical patent/WO2025237372A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K39/12—Viral antigens
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • This invention relates to the field of biomedicine, and more specifically, to a peptide and its use as an antibody light chain, and to antibodies against EGFR and/or PD-L1 comprising the peptide as a common light chain.
  • PD-L1 programmed death ligand 1
  • Somatic activation mutations of the epidermal growth factor receptor are among the most common oncogenic drivers in cancers such as non-small cell lung cancer (NSCLC), metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer, with the highest prevalence in NSCLC.
  • NSCLC non-small cell lung cancer
  • glioblastoma glioblastoma
  • pancreatic cancer pancreatic cancer
  • breast cancer with the highest prevalence in NSCLC.
  • Molecularly targeted drugs targeting the EGFR signaling pathway have shown strong clinical efficacy; however, acquired resistance has been found to be inevitable in patients.
  • the EGFR ⁇ PD-L1 bispecific antibody is designed to simultaneously bind to the tumor-associated antigen, epidermal growth factor receptor (EGFR), and programmed death ligand 1 (PD-L1).
  • EGFR epidermal growth factor receptor
  • PD-L1 programmed death ligand 1
  • the EGFR ⁇ PD-L1 bispecific antibody can mediate effective antibody-dependent cytotoxicity (ADCC) and activate T-cell antitumor immunity by blocking the interaction between programmed death receptor 1 (PD-1) and its corresponding ligand PD-L1. Therefore, the EGFR ⁇ PD-L1 bispecific antibody can achieve enhanced therapeutic effects by directly inhibiting tumor growth and by blocking T-cell activation through tumor-targeted immune checkpoints.
  • ADCC effective antibody-dependent cytotoxicity
  • bispecific antibodies often require the simultaneous transfection of four plasmids to express two different heavy chains and two different light chains.
  • these four polypeptide chains are assembled into an antibody, mismatch between the heavy and light chains may occur. Therefore, it has been proposed that a single polypeptide can be used as a universal light chain to pair with the heavy chain of any antibody that has binding affinity and specificity for a specific target protein (e.g., EGFR, PD-L1), thereby constructing an antibody (homodimer).
  • a specific target protein e.g., EGFR, PD-L1
  • this polypeptide can also be used as a universal light chain to pair with two or more antibody heavy chains, thus constructing bispecific or even multispecific antibodies (heterodimers, heteromultimers) with affinity and binding specificity for different targets.
  • the present invention aims to provide a polypeptide, which, as a light chain variable region or light chain of an antibody, can be constructed into an antibody with a heavy chain variable region or heavy chain of an antibody having binding affinity and/or specificity to a specific target protein (antigen), or with two or more heavy chain variable regions or heavy chains of antibodies having binding affinity and/or specificity to different target proteins (antigens).
  • Another object of the present invention is to provide an antibody comprising the said polypeptide as a light chain variable region or light chain, for example, in the form of a homodimer or heterodimer.
  • the present invention provides a polypeptide whose amino acid sequence comprises: the amino acid sequence shown in SEQ ID NO. 29; the amino acid sequence shown in SEQ ID NO. 30; and the amino acid sequence shown in SEQ ID NO. 31.
  • polypeptide comprises the amino acid sequence shown in SEQ ID NO.11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.15.
  • the “at least 75% identity” as described in this invention covers any percentage of identity between two amino acid sequences, ranging from at least 75% to 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.
  • polypeptide comprises the amino acid sequence shown in SEQ ID NO.38 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22.
  • the polypeptide provided by the present invention can be used to construct antibodies by combining with the heavy chain variable region or heavy chain of antibodies that have binding affinity and/or specificity to different target proteins (antigens).
  • the antibodies retain or have stronger binding affinity and/or specificity to the target protein (antigen) and corresponding biological activity.
  • the constructed antibody can have any structural form, such as a monoclonal antibody or antibody forms like scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.
  • the polypeptide as the light chain variable region or light chain of the antibody, can be combined with the heavy chain variable region or heavy chain of an antibody that has binding affinity and/or specificity to a certain target protein (antigen) to construct an antibody that still has binding affinity and/or specificity to that target protein (antigen) (a monospecific antibody).
  • This antibody is a homodimer with two identical heavy chains and two identical light chains.
  • the polypeptide as the light chain variable region or light chain of the antibody, can be combined with the heavy chain variable regions or heavy chains of two antibodies that have binding affinity and/or specificity to two target proteins (antigens) respectively to construct an antibody that still has binding affinity and/or specificity to each of the two target proteins (antigens) (a bispecific antibody).
  • This antibody is a heterodimer with two different heavy chains and two identical light chains.
  • the polypeptides provided by this invention can also be used to construct multispecific antibodies with binding affinity and/or specificity to more target proteins (antigens).
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide described in the first aspect.
  • the present invention provides the use of the polypeptide described in the first aspect or the nucleic acid molecule described in the second aspect in the construction of antibodies.
  • the polypeptide provided in the first aspect of the present invention can be used with the heavy chain or antibody heavy chain variable region of an antibody that has binding affinity and/or specificity to different target proteins (antigens) to construct new antibodies, wherein the antibodies retain (or even have stronger) binding affinity and/or specificity to the target protein (antigen) and biological activity, etc.
  • the target protein may be epidermal growth factor receptor (EGFR) or programmed death ligand 1 (PD-L1).
  • EGFR epidermal growth factor receptor
  • PD-L1 programmed death ligand 1
  • the epidermal growth factor receptor or programmed death ligand 1 may be mammalian, such as primate or rodent EGFR or PD-L1, more preferably human.
  • the constructed antibody can be a homodimeric antibody having two identical heavy chains and two identical light chains, or a heterodimeric antibody having two different heavy chains and two identical light chains.
  • the polypeptide can serve as the light chain or a variable region of the light chain of the antibody.
  • the constructed antibody is a bispecific antibody, and the polypeptide provided in the first aspect of the present invention serves as the common light chain of the bispecific antibody; the bispecific antibody is such as the EGFR ⁇ PDL1 bispecific antibody provided by the present invention.
  • the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a polypeptide provided in the first aspect of the present invention as a light chain or a light chain variable region; in other words, the polypeptide may serve as a light chain variable region or a light chain of the antibody or the antigen-binding fragment thereof.
  • the antigen-binding fragment is any functional fragment of an antibody capable of specifically binding to a target protein.
  • target protein and “antigen” are used interchangeably.
  • the antibodies or antigen-binding fragments provided by this invention can be IgG-like antibodies, or antibody forms such as scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.
  • IgG-like antibody refers to an antibody or antigen-binding fragment having two Fab arms and an optional Fc region, and is a traditional monoclonal antibody (mAb) structure or a similar structure.
  • Fab arm refers to a Fab fragment (Antigen-binding fragment), which consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and a constant region fragment), with the light chain and heavy chain linked by disulfide bonds.
  • target protein (antigen) binding properties the antibodies or antigen-binding fragments provided by this invention can be bispecific antibodies.
  • the antibody or antigen-binding fragment provided by the present invention contains the amino acid sequence provided by the first aspect of the present invention as its light chain CDRs in its light chain variable region; or, the antibody or antigen-binding fragment provided by the present invention contains the amino acid sequence shown in SEQ ID NO. 11 or SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence as its light chain variable region (VL); or, the antibody or antigen-binding fragment provided by the present invention contains the amino acid sequence shown in SEQ ID NO. 38 or SEQ ID NO. 22 or an amino acid sequence having at least 75% identity with said amino acid sequence as its light chain.
  • the antibody or its antigen-binding fragment may further include a heavy chain variable region (VH), which, together with the light chain variable region, forms a binding domain targeting the target protein.
  • VH heavy chain variable region
  • the target protein may be epidermal growth factor receptor (EGFR) or programmed cell death ligand 1 (PD-L1).
  • the antibody or its antigen-binding fragment may comprise heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown below:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31;
  • HCDR1 containing the amino acid sequence shown in SEQ ID NO.34 HCDR2 containing the amino acid sequence shown in SEQ ID NO.37, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31.
  • the antibody or its antigen-binding fragment may comprise a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and the framework regions (FRs) therebetween, and the arrangement of the regions may be FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the heavy chain variable region and the light chain variable region may respectively comprise:
  • amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11;
  • the "at least 75% identity" of the present invention results in a maximum 25% difference between two amino acid sequences, which may exist in any frame region within the heavy chain variable region or the light chain variable region, or in any structural domain or sequence outside the heavy chain variable region and the light chain variable region.
  • the antibody or its antigen-binding fragment provided by this invention can be a mouse antibody, a chimeric antibody, or a humanized antibody; and may further comprise a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region can be the heavy chain constant region of IgG1 or IgG4; the light chain constant region can be the kappa or lambda light chain constant region.
  • the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 18 (hIgG1) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.
  • the heavy chain constant region of the antibody contains the amino acid sequence shown in SEQ ID NO.20 (hIgG1; Knob) or SEQ ID NO.21 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with the amino acid sequence;
  • the light chain constant region of the antibody contains the amino acid sequence shown in SEQ ID NO.19 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • the antibody or antigen-binding fragment provided by the present invention may have at least two VH+VL domain combinations (further, at least two Fab arms), said domain combinations may be identical to each other, thereby binding the same target protein (antigen).
  • the antibody or antigen-binding fragment contains the same amino acid sequence combination selected from HCDR1 to HCDR3 and LCDR1 to LCDR3 provided above in its at least two VH+VL domain combinations, or contains the same amino acid sequence combination selected from the heavy chain variable region and light chain variable region provided above in (1) to (6).
  • the antibody or antigen-binding fragment also contains a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO.
  • the antibody or antigen-binding fragment has a heavy chain and a light chain, for example, a monoclonal antibody having the same two heavy chains and the same two light chains.
  • the at least two VH+VL domain combinations (further, at least two Fab arms) of the antibody or its antigen-binding fragment provided by the present invention may be different from each other, thereby binding to different target proteins (antigens).
  • the antibody contains different amino acid sequence combinations selected from HCDR1 to HCDR3 and LCDR1 to LCDR3 provided above (1) to (4), or contains different amino acid sequence combinations selected from the heavy chain variable region and light chain variable region provided above (1) to (6).
  • the at least two VH+VL domain combinations (further, at least two Fab arms) of the antibody or its antigen-binding fragment contain the same light chain CDRs sequence combination, or contain the same light chain VL sequence.
  • each of the at least two VH+VL domain combinations comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29; LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 31.
  • each of the at least two VH+VL domain combinations comprises: a light chain variable region containing the amino acid sequence shown in SEQ ID NO. 11 or SEQ ID NO. 15, or an amino acid sequence having at least 75% identity with said amino acid sequence.
  • the antibody or its antigen-binding fragment comprises:
  • the first VH+VL domain combination of EGFR (further, the Fab arm), wherein the first VH+VL domain combination comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 26, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 27, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising SEQ ID NO. 31; and
  • the second VH+VL domain combination of PD-L1 (further, the Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 37, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 containing SEQ ID NO. 31; or
  • the first VH+VL domain assembly comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 32; HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 33; and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29; LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; and
  • the second VH+VL domain combination of PD-L1 (further, the Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 37, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29; LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 31.
  • the antibody or its antigen-binding fragment further comprises:
  • the first VH+VL domain combination of EGFR (further, the Fab arm), wherein the first VH+VL domain combination comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity with said amino acid sequence; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; and
  • the second VH+VL domain combination of PD-L1 (further, the Fab arm) comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 17 or an amino acid sequence having at least 75% identity with said amino acid sequence; and a light chain variable region containing the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; or
  • the first VH+VL domain combination of EGFR (further, the Fab arm), wherein the first VH+VL domain combination comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence having at least 75% identity with said amino acid sequence; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; and
  • the second VH+VL domain combination of PD-L1 (further, the Fab arm) comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 15.
  • the antibody or its antigen-binding fragment further comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity with said amino acid sequence
  • the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity with said amino acid sequence.
  • the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 20 (hIgG1; Knob) or SEQ ID NO.
  • the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity with said amino acid sequence.
  • the antibody is a bispecific antibody, which has two different heavy chains but contains the same two light chains.
  • the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO. 22 or an amino acid sequence having at least 75% identity with the amino acid sequence as the light chain.
  • the bispecific antibodies provided by the present invention are shown in Table 15 of the "Best Modes for Carrying Out the Invention".
  • the bispecific antibodies in Table 15 include the following structural domains:
  • the bispecific antibody H22A12x58E3V4-hIgG1-new comprises two Fab arms targeting EGFR and PD-L1 respectively.
  • One Fab arm contains a heavy chain variable region (SEQ ID NO.14) and a light chain variable region (SEQ ID NO.15) targeting EGFR, and the other Fab arm contains a heavy chain variable region (SEQ ID NO.17) and a light chain variable region (SEQ ID NO.15) targeting PD-L1.
  • the bispecific antibody contains two identical light chain variable regions (SEQ ID NO.15) and two corresponding identical light chains (SEQ ID NO.22).
  • the bispecific antibody H103G9x58E3V4-hIgG1-new comprises two Fab arms targeting EGFR and PD-L1, respectively.
  • One Fab arm contains a heavy chain variable region (SEQ ID NO.16) and a light chain variable region (SEQ ID NO.15) targeting EGFR, and the other Fab arm contains a heavy chain variable region (SEQ ID NO.17) and a light chain variable region (SEQ ID NO.15) targeting PD-L1.
  • the bispecific antibody contains two identical light chain variable regions (SEQ ID NO.15) and two corresponding identical light chains (SEQ ID NO.22).
  • the bispecific antibody H22A12x58E3V4-hIgG1-new contains the amino acid sequence shown in SEQ ID NO.23 as heavy chain 1, the amino acid sequence shown in SEQ ID NO.24 as heavy chain 2, and the amino acid sequence shown in SEQ ID NO.22 as the same light chain.
  • the bispecific antibody H103G9x58E3V4-hIgG1-new comprises the amino acid sequence shown in SEQ ID NO.25 as heavy chain 1, the amino acid sequence shown in SEQ ID NO.24 as heavy chain 2, and the amino acid sequence shown in SEQ ID NO.22 as the same light chain.
  • the present invention provides a composition comprising the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, or the antibody or antigen-binding fragment thereof described in the fourth aspect.
  • the composition is preferably a pharmaceutical composition, which may further optionally comprise pharmaceutically acceptable excipients, carriers, or excipients.
  • the pharmaceutical composition can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.
  • the present invention provides the use of the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the antibody described in the fourth aspect or its antigen-binding fragment, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament.
  • the drug may be an antibody-based drug used to treat diseases related to the expression of its target protein or antigen, such as EGFR and/or PD-L1 expression-related diseases.
  • the drug may be used to treat tumors or cancers such as lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), head and neck cancer, and colon cancer.
  • the present invention provides a method of treating a disease, the method comprising administering to a subject in need the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the antibody described in the fourth aspect or an antigen-binding fragment thereof, or the pharmaceutical composition described in the fifth aspect of the present invention.
  • the method provided by this invention is used to treat tumors or cancer.
  • the tumor or cancer is lung cancer (such as non-small cell lung cancer, small cell lung cancer), head and neck cancer, or colon cancer.
  • the subject is a mammal, preferably a primate or rodent, and more preferably a human.
  • the present invention also provides an antibody or antigen-binding fragment thereof that binds to EGFR, said antibody or antigen-binding fragment comprising heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown below:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 26, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 27, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or
  • the antibody binding to EGFR or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise:
  • amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11;
  • the EGFR-binding antibody or its antigen-binding fragment provided by this invention can be an IgG-like antibody, or an antibody in the form of scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.
  • the antibody or its antigen-binding fragment may be a mouse antibody, a chimeric antibody, or a humanized antibody; and may further comprise a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region may be the heavy chain constant region of IgG1 or IgG4; the light chain constant region may be the kappa or lambda light chain constant region.
  • the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 18 (hIgG1) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.
  • the heavy chain constant region of the antibody contains the amino acid sequence shown in SEQ ID NO.20 (hIgG1; Knob) or SEQ ID NO.21 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with the amino acid sequence;
  • the light chain constant region of the antibody contains the amino acid sequence shown in SEQ ID NO.19 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • the present invention also provides an antibody or antigen-binding fragment thereof that binds to PD-L1, said antibody or antigen-binding fragment comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and light chain CDRs (LCDR1, LCDR2 and LCDR3) as shown below:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31;
  • the antibody binding to PD-L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise:
  • the antibody or antigen-binding fragment of PD-L1 provided by this invention can be an IgG-like antibody, or an antibody in the form of scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.
  • the antibody or its antigen-binding fragment may be a mouse antibody, a chimeric antibody, or a humanized antibody; and may further comprise a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region may be the heavy chain constant region of IgG1 or IgG4; the light chain constant region may be the kappa or lambda light chain constant region.
  • the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 18 (hIgG1) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.
  • the heavy chain constant region of the antibody contains the amino acid sequence shown in SEQ ID NO.20 (hIgG1; Knob) or SEQ ID NO.21 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with the amino acid sequence;
  • the light chain constant region of the antibody contains the amino acid sequence shown in SEQ ID NO.19 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • the present invention provides the use of the antibody or antigen-binding fragment thereof that binds to EGFR as described in the eighth aspect or the antibody or antigen-binding fragment thereof that binds to PD-L1 as described in the ninth aspect in the preparation of a medicament.
  • the drug may be an antibody-based drug used to treat diseases related to the expression of its target protein or antigen, such as EGFR and/or PD-L1 expression-related diseases.
  • the drug may be used to treat tumors or cancers, such as, preferably, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), head and neck cancer, or colon cancer.
  • This invention provides a polypeptide, which, experimentally demonstrated, can serve as a common light chain or light chain variable region of an antibody, combined with the heavy chain or heavy chain variable region of an antibody possessing binding affinity and/or specificity to other target proteins (antigens), to construct a bispecific antibody.
  • This antibody retains or even exhibits binding affinity and biological activity against two different target proteins (antigens). Therefore, the polypeptide provided by this invention has the potential to be used as a common light chain variable region or light chain for constructing bispecific antibodies.
  • this invention also provides antibodies comprising the polypeptide as a common light chain variable region or light chain, such as anti-EGFR antibodies, anti-PD-L1 antibodies, and EGFR ⁇ PD-L1 bispecific antibodies.
  • Figure 1 shows the binding activity of the murine anti-PD-L1 antibody and the control antibody to PD-L1 overexpressing cells
  • Figure 2 shows the blocking activity of the murine anti-PD-L1 antibody and the control antibody against the binding of PD-L1 to PD-1;
  • Figure 3 shows the blocking activity of the anti-PD-L1 murine antibody and the control antibody against PD-L1/PD-1 signaling
  • Figure 4 shows the affinity of the murine anti-PD-L1 antibody for human PD-L1
  • Figure 5 shows the binding activity of the humanized anti-PD-L1 antibody and the control antibody to PD-L1 overexpressing cells
  • Figure 6 shows the blocking activity of humanized anti-PD-L1 antibody and control antibody against PD-L1 binding to PD-1;
  • Figure 7 shows the blocking activity of humanized anti-PD-L1 antibodies and control antibodies against PD-L1/PD-1 signaling
  • Figure 8 shows the binding activity of the anti-EGFR murine antibody and the control antibody to EGFR-overexpressing cells
  • Figure 9 shows the blocking activity of the anti-EGFR murine antibody and the control antibody against the binding of EGFR to EGF
  • Figure 10 shows the blocking activity of anti-EGFR murine antibody and control antibody against EGFR signaling
  • Figure 11 shows the affinity of the anti-EGFR murine antibody for EGFR
  • Figure 12 shows the binding activity of the humanized anti-EGFR antibody and the control antibody to EGFR-overexpressing cells
  • Figure 13 shows the blocking activity of the humanized anti-EGFR antibody and the control antibody against the binding of EGFR to EGF;
  • Figure 14 shows the blocking activity of humanized anti-EGFR antibodies and control antibodies against EGFR signaling
  • Figure 15 shows the binding activity of the EGFR ⁇ PDL1 bispecific antibody and the control antibody to PD-L1 overexpressing cells
  • Figure 16 shows the blocking activity of EGFR ⁇ PDL1 bispecific antibody and control antibody against PD-L1 binding to PD-1;
  • Figure 17 shows the blocking activity of the EGFR ⁇ PDL1 bispecific antibody and the control antibody against PD-L1/PD-1 signaling
  • Figure 18 shows the binding activity of the EGFR ⁇ PDL1 bispecific antibody and the control antibody to EGFR-overexpressing cells
  • Figure 19 shows the blocking activity of the EGFR ⁇ PDL1 bispecific antibody and the control antibody against the binding of EGFR to EGF;
  • Figure 20 shows the blocking activity of the EGFR ⁇ PDL1 bispecific antibody and the control antibody against EGFR signaling.
  • Figure 21 shows the killing activity of EGFR ⁇ PDL1 bispecific antibody and control antibody against tumor cells.
  • Atezolizumab Self-made by the applicant (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., "Kangyuan Bochuang"), Cat#: KA-1303-02;
  • Cetuximab Self-made by the applicant, Cat#: KA-1398-01.
  • target proteins EGFR and PD-L1 involved in the examples are human proteins.
  • KV4-type germlines are expressed at a relatively high frequency in mice and pair with mouse heavy chains at a higher frequency. Therefore, this type of germline sequence was selected, and the following mouse germline sequence KV4-59 was chosen from it because it is relatively easy to humanize, has good solubility, low immunogenicity, and no potential modification sites.
  • variable region sequences of the antibody light chain were used as the variable region sequences of the antibody light chain, and chimeric antibodies were constructed by combining them with the variable region sequences of the heavy chain of the mouse anti-chicken egg lysozyme antibody and the constant regions of the human heavy and light chains.
  • the heavy chain variable region sequence of mouse anti-chicken egg lysozyme antibody (SEQ ID NO.5):
  • sgRNAs were designed and validated in vitro using a Cas9-gRNA target efficiency assay kit (Vishanglide, Cat#: VK007-30T). Highly efficient sgRNA sequences were selected: TGAATGCCATGTACTTATGG (SEQ ID NO. 6) and AAGTTCCAGCTCCTAAAATGG (SEQ ID NO. 7). These sgRNA sequences were synthesized and microinjected into mouse zygotes along with spCas91.1 protein. The sgRNA mediated the Cas9 protein to cleave both ends of the target gene fragment to be deleted, resulting in a DNA double-strand break.
  • NHEJ non-homologous end joining
  • sgRNAs were designed and validated in vitro using a Cas9-gRNA target efficiency assay kit (Vishanglide, Cat#: VK007-30T). Highly efficient sgRNA sequences were selected: IGK-L1: GTGAATGCCATGTACTTATGG (SEQ ID NO. 8); IGK-R1: CAAGTTCAGCTCCTAAAATGG (SEQ ID NO. 9).
  • the synthesized sgRNA sequences were microinjected into mouse zygotes along with spCas91.1 protein. The sgRNA mediated the Cas9 protein to create a gap in the DNA at the target site.
  • homologous recombination was used to knock the DNA fragment into the target site. Genotyping was performed on mice two weeks after birth, confirming that the common light chain gene had been knocked in.
  • mice with lambda gene knockout were mated with heterozygous mice with common light chain knock-in. Genotyping was performed on each offspring 2 weeks after birth to detect common light chain gene knock-in and lambda knockout, until mice with all alleles of common light chain gene knock-in and both lambda and kappa knockout were obtained.
  • Example 3 Preparation of hybridoma cells secreting anti-EGFR and anti-PD-L1 antibodies
  • mice constructed in Example 2 were immunized using EGFR-mFc (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. (“Kangyuan Bochuang”), Cat#: KP-1151) as an immunogen. Mice with high titers were selected, and serum was collected. After dissection, the spleen was harvested, and spleen cells were isolated. These spleen cells were fused with cultured myeloma cells to obtain hybridoma cells.
  • the binding activity of the hybridoma cell supernatant to the antigen protein EGFR-His was detected using ELISA, yielding multiple positive hybridoma cell lines secreting anti-EGFR antibodies.
  • a murine antibody against EGFR was obtained from a positive hybridoma cell line. Its variable region sequence is as follows (where the heavy chain and light chain CDRs are underlined and obtained according to the KABAT definition method, the same below): 22A12-2 (murine antibody against EGFR 1; also named "KD-22-0198")
  • VH SEQ ID NO.10; HCDR1/HCDR2/HCDR3: SEQ ID NO.26/SEQ ID NO.27/SEQ ID NO.28:
  • VL SEQ ID NO.11; LCDR1/LCDR2/LCDR3: SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31:
  • VH SEQ ID NO.12; HCDR1/HCDR2/HCDR3: SEQ ID NO.32/SEQ ID NO.33/SEQ ID NO.28):
  • VL SEQ ID NO.11; LCDR1/LCDR2/LCDR3: SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31:
  • mice constructed in Example 2 were immunized using PDL1-mFc (Kangyuan Bochuang, Cat#: KP-1008) as an immunogen. Mice with high titers were selected, and serum was collected. After dissection, the spleen was harvested, and spleen cells were isolated. These spleen cells were fused with cultured myeloma cells to obtain hybridoma cells. The binding activity of the hybridoma cell supernatant to the antigen protein PDL1-His (Kangyuan Bochuang, Cat#: KP-1002) was detected using ELISA, yielding multiple positive hybridoma cell lines secreting anti-PD-L1 antibodies.
  • Anti-PD-L1 murine antibodies were obtained from positive hybridoma cell lines, and their variable region sequences are shown below (heavy chain and light chain CDRs are underlined):
  • VH SEQ ID NO.13; HCDR1/HCDR2/HCDR3: SEQ ID NO.34/SEQ ID NO.35/SEQ ID NO.36:
  • VL SEQ ID NO.11; LCDR1/LCDR2/LCDR3: SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31:
  • Anti-EGFR murine antibodies 22A12-2 and 103G9-3, and anti-PD-L1 murine antibody 58E3-2 have the same light chain variable region.
  • the heavy chain and light chain variable regions of two anti-EGFR murine antibodies were humanized to obtain humanized antibody sequences.
  • the coding sequences of these humanized antibody sequences were then linked to the coding sequences of the heavy chain constant region (SEQ ID NO. 18) of human IgG1 and the light chain constant region (SEQ ID NO. 19) of human kappa, respectively.
  • the resulting coding genes were cloned into a eukaryotic expression vector, expressed, and two anti-EGFR humanized antibodies were obtained, named H22A12-2V1-hIgG1 and H103G9-3V2-hIgG1, respectively.
  • Their heavy chain and light chain variable region sequences and full-length heavy and light chains are shown below (where the heavy chain and light chain CDRs are underlined).
  • the coding sequences of the heavy chain variable region and light chain variable region of two anti-EGFR murine antibodies were linked to the coding sequences of the heavy chain constant region of human IgG1 (SEQ ID NO.18) and the light chain constant region of human kappa (SEQ ID NO.19), respectively.
  • the resulting coding genes were cloned into a eukaryotic expression vector, expressed, and used to obtain anti-EGFR chimeric antibodies.
  • H22A12-2V1-hIgG1 anti-EGFR humanized antibody 1; also named "KA-1063"
  • H103G9-3V2-hIgG1 humanized anti-EGFR antibody 2; also named "KA-1083"
  • the heavy chain and light chain variable regions of a murine anti-PD-L1 antibody were humanized to obtain a humanized antibody sequence.
  • the coding sequences of the humanized antibody sequence were then linked to the coding sequences of the heavy chain constant region (SEQ ID NO. 18) of human IgG1 and the light chain constant region (SEQ ID NO. 19) of human kappa, respectively.
  • the resulting coding genes were cloned into a eukaryotic expression vector, expressed, and a humanized anti-PD-L1 antibody was obtained, named H58E3-2V4-2-hIgG1. Its heavy chain and light chain variable region sequences and full-length heavy and light chains are shown below (where the heavy chain and light chain CDRs are underlined).
  • the coding sequences of the heavy chain variable region and light chain variable region of a murine anti-PD-L1 antibody were linked to the coding sequences of the heavy chain constant region (SEQ ID NO.18) of human IgG1 and the light chain constant region (SEQ ID NO.19) of human kappa, respectively.
  • the resulting coding genes were cloned into a eukaryotic expression vector, expressed, and a chimeric anti-PD-L1 antibody was obtained.
  • H58E3-2V4-2-hIgG1 humanized antibody against PD-L1; also named "KA-2016"
  • the anti-EGFR humanized antibodies H22A12-2V1-hIgG1 and H103G9-3V2-hIgG1, and the anti-PD-L1 humanized antibody H58E3-2V4-2-hIgG1 have the same light chain variable region (SEQ ID NO.15) and the same light chain (SEQ ID NO.22).
  • SEQ ID NO.22 (VL: SEQ ID NO.15; LCDR1/LCDR2/LCDR3: SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31; LC: SEQ ID NO.19)
  • Two anti-EGFR chimeric antibodies and one anti-PD-L1 chimeric antibody have the same light chain variable region (SEQ ID NO.14) and the same light chain (SEQ ID NO.38).
  • SEQ ID NO.38 (VL: SEQ ID NO.11; LCDR1/LCDR2/LCDR3: SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31; LC: SEQ ID NO.19)
  • Example 5 Binding activity of murine anti-PD-L1 antibody to PD-L1 overexpressing cells
  • the antibodies to be tested were serially diluted and co-incubated with PD-L1-overexpressing 293T cells (Kangyuan Bochuang, Cat#: KC-0205), and then co-incubated with the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307) or the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signals bound to the cells. The results are shown in Table 1 and Figure 1.
  • Example 6 Blocking activity of murine anti-PD-L1 antibody against PD-L1 binding to PD-1
  • the antibodies to be tested were serially diluted and co-incubated with 2 ⁇ g/ml of PDL1-mFc (Kangyuan Biotech, Cat#: KP-1008) or PDL1-hFc (Kangyuan Biotech, Cat#: KP-1009), then co-incubated with PD-1 overexpressing 293T cells (Kangyuan Biotech, Cat#: KC-0204), and finally co-incubated with the anti-human secondary antibody Goat anti-human IgG-PE (Southern Biotech, Cat#: 2010-09) or the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307).
  • the PDL1-Fc signal bound to the cells was detected by flow cytometry. The results are shown in Table 2 and Figure 2.
  • Example 7 Blocking activity of murine anti-PD-L1 antibody against PD-L1/PD-1 signaling
  • the antibodies to be tested were serially diluted and co-incubated with Jurkat NFAT-PD1-luc cells (Kangyuan Bochuang, Cat#: KC-1503, expressing luciferase after NFAT signal activation) and 293T OS8-hPDL1 cells (Kangyuan Bochuang, Cat#: KC-1148, expressing OKT3 antibody on the cell surface). Activation of luciferase signal was detected; cells without antibody incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after antibody addition and the antibody concentration, and the EC50 of the antibody was calculated. The results are shown in Table 3 and Figure 3.
  • Example 8 Affinity of mouse anti-PD-L1 antibody to PD-L1
  • the affinity of the antibody for PD-L1 was detected using the ForteBio Octet.
  • concentrations of the mobile phase hPDL1-His were 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, respectively.
  • the affinity test results are shown in Table 4 and Figure 4.
  • Example 9 Binding activity of humanized anti-PD-L1 antibody to PD-L1 overexpressing cells
  • the antibodies to be tested were serially diluted and co-incubated with PD-L1-overexpressing 293T cells (Kangyuan Biotech, Cat#: KC-0205), and then co-incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signals bound to the cells; the results are shown in Table 5 and Figure 5.
  • Example 10 Blocking activity of humanized anti-PD-L1 antibody against PD-L1 binding to PD-1
  • the antibody to be tested was serially diluted and co-incubated with 1.2 ⁇ g/ml PDL1-mFc (Kangyuan Bochuang, Cat#: KP-1008), then co-incubated with PD-1 overexpressing 293T cells (Kangyuan Bochuang, Cat#: KC-0204), and finally co-incubated with anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307). Flow cytometry was used to detect the PDL1-mFc signal bound to the cells. The results are shown in Table 6 and Figure 6.
  • Example 11 Blocking activity of humanized anti-PD-L1 antibody against PD-L1/PD-1 signaling
  • the antibodies to be tested were serially diluted and co-incubated with Jurkat NFAT-PD1-luc cells (Kangyuan Bochuang, Cat#: KC-1503, expressing luciferase after NFAT signal activation) and 293T OS8-hPDL1 cells (Kangyuan Bochuang, Cat#: KC-1148, expressing OKT3 antibody on the cell surface). Activation of luciferase signal was detected; cells without antibody incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after antibody addition and the antibody concentration, and the EC50 of the antibody was calculated. The results are shown in Table 7 and Figure 7.
  • the antibodies to be tested were serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Bochuang, Cat#: KC-1451), and then co-incubated with the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307) or the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signals bound to the cells. The results are shown in Table 8 and Figure 8.
  • Example 13 Blocking activity of murine anti-EGFR antibody against EGFR-EGF binding
  • the antibodies to be tested were serially diluted and co-incubated with 0.4 ⁇ g/ml EGFR-mFc (Kangyuan Bochuang, Cat#: KP-1148) or EGFR-hFc (Kangyuan Bochuang, Cat#: KP-1149), then incubated with ELISA plates coated with EGF-His (2 ⁇ g/ml, Acro, Cat#: EGF-H52H3), and finally co-incubated with anti-mouse secondary antibody HRP anti-mouse IgG (SIGMA, Cat#: A9309) or anti-human secondary antibody HRP anti-human IgG (SIGMA, Cat#: A0170).
  • the binding signal was detected by an ELISA reader, and the IC50 of the antibody was calculated. The results are shown in Table 9 and Figure 9.
  • Example 14 Blocking activity of murine anti-EGFR antibodies against EGFR signaling
  • the antibody to be tested was serially diluted and co-incubated with 293T-NFAT-Luc2-EGFR cells (Kangyuan Bochuang, Cat#: KC-2952, expressing luciferase after EGFR signal activation) for 30 minutes. Then, EGF-His (20 ng/ml, Acro, Cat#: EGF-H52H3) was added and the cells were cultured overnight. The activated luciferase signal was detected. Cells without EGF incubation were considered to have basal activation. The fold increase in activation intensity after adding EGF was plotted against the antibody concentration, and the IC50 of the antibody was calculated. The results are shown in Table 10 and Figure 10.
  • Example 15 Affinity of anti-EGFR murine antibody to EGFR
  • the ForteBio Octet was used to detect the affinity of the antibody for EGFR.
  • concentrations of the mobile phase EGFR-His were 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, respectively.
  • the affinity test results are shown in Table 11 and Figure 11 (22A12-2).
  • Example 16 Binding activity of humanized anti-EGFR antibody to EGFR-overexpressing cells
  • the antibodies to be tested were serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Biotech, Cat#: KC-1451), and then co-incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signals bound to the cells; the results are shown in Table 12 and Figure 12.
  • Example 17 Blocking activity of humanized anti-EGFR antibodies against EGFR-EGF binding.
  • the antibody to be tested was serially diluted and co-incubated with 0.4 ⁇ g/ml EGFR-mFc (Kangyuan Bochuang, Cat#: KP-1148), then incubated with an ELISA plate coated with EGF-His (2 ⁇ g/ml, Acro, Cat#: EGF-H52H3), and finally co-incubated with the anti-mouse secondary antibody HRP anti-mouse IgG (SIGMA, Cat#: A9309).
  • the binding signal was detected by a microplate reader, and the results are shown in Table 13 and Figure 13.
  • Example 18 Blocking activity of humanized anti-EGFR antibodies against EGFR signaling
  • the antibody to be tested was serially diluted and co-incubated with 293T-NFAT-Luc2-EGFR (Kangyuan Bochuang, Cat#: KC-2952, expressing luciferase after EGFR signal activation) for 30 minutes. Then, EGF-His (20 ng/ml, Acro, Cat#: EGF-H52H3) was added and the mixture was incubated overnight. The activated luciferase signal was detected. The absence of EGF incubation indicated basal activation. A correlation curve was plotted between the fold increase in activation intensity after EGF addition and the antibody concentration, and the IC50 of the antibody was calculated. The results are shown in Table 14 and Figure 14.
  • the heavy chain variable region (SEQ ID NO. 16) of the anti-EGFR humanized antibody 2, namely H103G9-3V2-hIgG1 was linked to the coding sequence of the human heavy chain Knob CH1 and FC sequence (SEQ ID NO. 20).
  • the obtained coding sequences were cloned into eukaryotic expression vectors to obtain four recombinant expression plasmids (one plasmid encoding a common light chain, two plasmids encoding heavy chains targeting EGFR, and one plasmid encoding heavy chains targeting PD-L1).
  • Transient co-transfection of the three plasmids (selecting one of the two plasmids encoding the heavy chain targeting EGFR) using the HEK293F expression system was performed to produce EGFR ⁇ PD-L1 bispecific antibodies.
  • HEK293F cells were seeded at a density of 1 ⁇ 106 cells/mL in 250mL of culture medium and incubated at 110rpm in 5% CO2 .
  • the three pre-prepared expression vectors and transfection reagents were mixed in a certain ratio, and the transfection complex was added to the cells at a cell density of 2 ⁇ 106 cells/mL.
  • nutrients and DNA inhibitors were added.
  • the expression supernatant was collected, centrifuged, filtered, and purified using a MabSelectSure affinity chromatography column (GE Healthcare). The purity of the purified antibody was detected by SDS-PAGE electrophoresis, and the antibody concentration was detected by Nanodrop.
  • H22A12x58E3V4-hIgG1-new (EGFR ⁇ PD-L1 bispecific antibody 1; also named "KA-2888")
  • H103G9x58E3V4-hIgG1-new EGFR ⁇ PD-L1 bispecific antibody 2; also named "KA-2887"
  • PDL1-58E3-2-Hole (Heavy chain 2, SEQ ID NO.24; VH: SEQ ID NO.17; HCDR1/HCDR2/HCDR3: SEQ ID NO.34/SEQ ID NO.37/SEQ ID NO.36; CH [Hole]: SEQ ID NO.21)
  • Example 20 Binding activity of EGFR ⁇ PDL1 bispecific antibody to PD-L1 overexpressing cells
  • the antibodies to be tested were serially diluted and co-incubated with PD-L1-overexpressing 293T cells (Kangyuan Biotech, Cat#: KC-0205), and then co-incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signals bound to the cells; the results are shown in Table 16 and Figure 15.
  • Example 21 Blocking activity of EGFR ⁇ PDL1 bispecific antibody against PD-L1 and PD-1 binding.
  • the antibody to be tested was serially diluted and co-incubated with 1.2 ⁇ g/ml PDL1-mFc (Kangyuan Bochuang, Cat#: KP-1008), then co-incubated with PD-1 overexpressing 293T cells (Kangyuan Bochuang, Cat#: KC-0204), and finally co-incubated with anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307). Flow cytometry was used to detect the PDL1-mFc signal bound to the cells. The results are shown in Table 17 and Figure 16.
  • Example 22 Blocking activity of EGFR ⁇ PDL1 bispecific antibody against PD-L1/PD-1 signaling
  • the antibodies to be tested were serially diluted and co-incubated with Jurkat NFAT-PD1-luc cells (Kangyuan Bochuang, Cat#: KC-1503, expressing luciferase after NFAT signal activation) and 293T OS8-hPDL1 cells (Kangyuan Bochuang, Cat#: KC-1148, expressing OKT3 antibody on the cell surface). Activation of luciferase signal was detected; cells without antibody incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after antibody addition and antibody concentration, and the EC50 of the antibody was calculated. The results are shown in Table 18 and Figure 17.
  • the antibodies to be tested were serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Biotech, Cat#: KC-1451), and then co-incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signals bound to the cells; the results are shown in Table 19 and Figure 18.
  • Example 24 Blocking activity of EGFR ⁇ PDL1 bispecific antibody against EGFR-EGF binding
  • the antibody to be tested was serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Bochuang, Cat#: KC-1451), then co-incubated with 0.05 ⁇ g/ml EGF-mFc (Acro, Cat#: EGF-H525b), and finally co-incubated with the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307). Flow cytometry was used to detect the EGF-mFc signal bound to the cells; the results are shown in Table 20 and Figure 19.
  • Example 25 Blocking activity of EGFR ⁇ PDL1 bispecific antibody against EGFR signaling
  • the antibody to be tested was serially diluted and co-incubated with 293T-NFAT-Luc2-EGFR cells (Kangyuan Bochuang, Cat#: KC-2952, expressing luciferase after EGFR signal activation) for 30 minutes. Then, EGF-His (20 ng/ml, Acro, Cat#: EGF-H52H3) was added and the cells were cultured overnight. The activated luciferase signal was detected. Cells without EGF incubation were considered to have basal activation. The fold increase in activation intensity after adding EGF was plotted against antibody concentration, and the results are shown in Table 21 and Figure 20.
  • the ForteBio Octet was used to detect the affinity of the antibody for EGFR and PD-L1.
  • the mobile phase consisted of human EGFR-His (Kangyuan Bochuang, Cat#: KP-1150) and human PDL1-His (Kangyuan Bochuang, Cat#: KP-1002), diluted sequentially to 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM. The results are shown in Table 22.
  • Lung cancer cells NCI-H292 (Kangyuan Bochuang, Cat#: KC-0578; expressing EGFR and PD-L1) were suspended in DMEM medium at a density of 5.0 ⁇ 105 cells/mL, and then 80 ⁇ L of the cell suspension was added to each well of a 96-well plate.
  • the antibody to be tested was serially diluted (starting concentration 200 ⁇ g/mL, 3.16-fold serial dilution) and added to each well at 10 ⁇ L, and incubated for 30 min. Then, 10 ⁇ L of human AB serum (ORi CELLS, Cat#: 20230419-Z0575) was added to each well, mixed, and incubated for 3 h.
  • the cells were transferred to ice, centrifuged at low temperature to remove the supernatant, resuspended in FACS buffer, and then incubated with 7-AAD for 10 min.
  • Flow cytometry was used to analyze 5000 cells per well. Data analysis was performed using Flowjo software to calculate the 7-AAD+ proportion of the target cell population, and GraphPad Prism was used to plot antibody vs. cell death ratio curves and calculate IC50. The results are shown in Table 23 and Figure 21.

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Abstract

La présente invention concerne un polypeptide. Le polypeptide peut être utilisé en tant que chaîne légère commune pour des anticorps bispécifiques, et peut être combiné à une région variable de chaîne lourde ou à une chaîne lourde d'un anticorps ayant une affinité de liaison et/ou une spécificité pour différentes protéines cibles afin de construire un anticorps bispécifique. La présente invention concerne en outre un anticorps anti-EGFR, un anticorps anti-PD-L1 et un anticorps bispécifique EGFR × PD-L1, les anticorps comprenant une région variable de chaîne légère ou une chaîne légère commune.
PCT/CN2025/095121 2024-05-16 2025-05-15 Anticorps anti-egfr et/ou anti-pd-l1 et son utilisation Pending WO2025237372A1 (fr)

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