WO2021063330A1 - 靶向cd3的抗体、双特异性抗体及其用途 - Google Patents

靶向cd3的抗体、双特异性抗体及其用途 Download PDF

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WO2021063330A1
WO2021063330A1 PCT/CN2020/118606 CN2020118606W WO2021063330A1 WO 2021063330 A1 WO2021063330 A1 WO 2021063330A1 CN 2020118606 W CN2020118606 W CN 2020118606W WO 2021063330 A1 WO2021063330 A1 WO 2021063330A1
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amino acid
acid sequence
antibody
human
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何云
石磊
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和铂医药(苏州)有限公司
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Priority to JP2022520343A priority Critical patent/JP2022550832A/ja
Priority to KR1020227014723A priority patent/KR20220071263A/ko
Priority to CN202080006173.8A priority patent/CN113015749B/zh
Priority to CA3152438A priority patent/CA3152438A1/en
Priority to US17/764,284 priority patent/US20220348661A1/en
Priority to AU2020359928A priority patent/AU2020359928A1/en
Priority to EP20872856.8A priority patent/EP4039707A4/en
Publication of WO2021063330A1 publication Critical patent/WO2021063330A1/zh
Priority to JP2024000181A priority patent/JP2024036342A/ja

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • 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
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    • 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
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    • C07K2319/00—Fusion polypeptide
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular to a CD3-targeting antibody, bispecific antibody and uses thereof.
  • T lymphocytes are an important cell type involved in adaptive immune responses, and T cells recognize antigens through T cell receptors (TCR).
  • TCR does not directly recognize antigen surface epitopes, but specifically recognizes antigen-presenting cells (APC) or antigen peptide-MHC molecular complexes (pMHC) presented on the surface of target cells.
  • APC antigen-presenting cells
  • pMHC antigen peptide-MHC molecular complexes
  • the specificity of T cell response is mediated by the recognition of pMHC by the molecular complex of TCR and CD3.
  • TCR is a heterodimer composed of two different transmembrane polypeptide chains. There are four peptide chains: ⁇ , ⁇ , ⁇ , and ⁇ . According to different combinations of peptide chains, TCR is divided into TCR ⁇ and TCR ⁇ .
  • CD3 has different transmembrane polypeptide chains, namely ⁇ , ⁇ , ⁇ , and ⁇ . These peptide chains interact to form homodimers or heterodimers as part of the TCR-CD3 complex.
  • the TCR-CD3 complex includes TCR ⁇ dimer, CD3 ⁇ dimer, CD3 ⁇ dimer, and CD3 ⁇ dimer. Because the cytoplasmic region of the TCR peptide chain is very short, it is generally believed that the activation signal generated by the TCR recognition antigen is transduced into the T cell by the CD3 peptide chain.
  • OKT3 is the first approved therapeutic antibody.
  • OKT3 was first approved by the US FDA in 1985 for the treatment of acute rejection after organ transplantation.
  • the immunosuppressive ability brought about by repeated administration of OKT3 provides an effective treatment for rejection after renal transplantation, its application is limited by the first toxic dose response syndrome; this syndrome is considered to be related to OKT3 mediated T cells Activation is related to the release of cytokines. Later, due to severe cytokine storm and immunogenicity problems caused by mouse resistance and other factors, OKT3 withdrew from the market in 2010.
  • CD3 antibodies have been found to be species-specific.
  • OKT3 reacts with chimpanzee CD3 but does not react with other primates such as the CD3 homologs of macaques, or the CD3 homologs of mice.
  • the species specificity of CD3 monoclonal antibody is a significant obstacle to its development as an antibody drug for the treatment of human diseases. Any new drug candidate must undergo rigorous preclinical verification before it can be used in human patients for clinical trials. The purpose of preclinical testing is to confirm that the candidate drug has the desired activity and most importantly, the candidate drug is safe.
  • Preclinical safety testing is to administer candidate drugs to related species, preferably non-human primates (Non-Human Primates).
  • the species described in the art that are suitable for safety evaluation testing may be rhesus monkeys, particularly cynomolgus monkeys.
  • CD3 antibodies that lack primate species-specific cross-reactivity are difficult to provide effective preclinical safety assessment data.
  • SP34 is one of the very few antibodies that can bind to a variety of primate CD3 (such as human and cynomolgus CD3) (see, Salmeron, A. et al, J Immunol 147 (1991) 3047-3052; Conrad ML, et.al, Cytometry A 71 (2007) 925-933).
  • CD3 monoclonal antibodies have been clinically verified for their effectiveness in certain disease areas, in recent years, CD3 antibodies have been more used in the development of bispecific antibody drugs.
  • CD3-based bi-specific T-cell bridge antibody BsTCE, Bi-specific T-cell engager
  • CD3 bispecific antibody BsTCE shows strong curative effect like CAR-T cell therapy, on the other hand, it can be produced and commercialized like traditional monoclonal antibodies.
  • BsTCEs are both BsTCEs.
  • CD3 antibody is an important part of the construction of BsTCE.
  • the BsTCE bispecific antibody can simultaneously bind to two targets, one end of which can recognize tumor-associated antigen (TAA) on the surface of tumor cells, and the other end can bind to CD3 molecules on T cells.
  • TAA tumor-associated antigen
  • the BsTCE bispecific antibody binds to the surface of tumor cells and can recruit and activate T cells near the tumor cells, thereby killing the tumor cells.
  • CD3 antibodies When designing and constructing the various structures of BsTCE bispecific antibodies, the selection and optimization of CD3 antibodies are crucial. First, the species specificity of the CD3 monoclonal antibody is extremely important, especially the monkey cross-reactivity. Second, the affinity of the CD3 antibody to the CD3 complex is also very important. CD3 antibodies with too high affinity may restrict the antibody to the spleen and other parts, making it difficult to reach the tumor; and too high affinity may also overstimulate T cells and bring high levels Cytokine release. Third, the binding valence of CD3 antibodies also has an important influence. Previously, it was found that the multivalent form of CD3 bispecific antibodies may activate T cells without binding to tumor-associated antigens and cause side reactions. Therefore, most of the CD3 bispecific antibodies in research are bispecific. The sex antibody is in the form of a monovalent CD3.
  • BsTCE bispecific antibodies In addition to the CD3 antibody, the structural design of the BsTCE bispecific antibody is also very important.
  • the structures of BsTCE bispecific antibodies are diverse and can be divided into two main categories: Fc-containing IgG-like structures and Fc-free antibody fragment structures.
  • Blinatumomab is a single polypeptide chain structure composed of two single-chain variable region antibody fragments (scFv) in series, but this structure has a short half-life and requires continuous intravenous infusion, which is very inconvenient to use. Therefore, many BsTCE bispecific antibodies adopt Fc-containing structures to improve molecular stability and pharmacokinetic properties.
  • the structure containing Fc is often an asymmetric structure.
  • Fc-containing asymmetric structures have many technical difficulties that need to be overcome, such as heavy chain homodimerization problems in asymmetric structures, light chain mismatch problems, molecular cross-linking caused by Fc ⁇ receptors, and ADCC or CDC effects. Role and so on.
  • To construct a BsTCE bispecific antibody from anti-TAA IgG antibody and anti-CD3 IgG antibody [ Figure 16(A)] different asymmetric structures can be selected.
  • One of the common structures is an IgG-like structure that retains two independent Fab domains.
  • This structure contains four different polypeptide chains [two different heavy chains and two different light chains, the structure shown in Figure 16(B)], which has a similar molecular weight to traditional monoclonal antibodies; however, this structure is due to Containing multiple different polypeptide chains, which may bring many kinds of combined by-products, which brings huge challenges to the expression, purification and production process of antibodies.
  • the Fab of the CD3 antibody is transformed into a scFv structure, the "four-chain” structure can be changed to a "three-chain” structure [structure shown in Figure 16(C)] to further reduce the number of by-product combinations, thereby reducing its production Complexity.
  • the present inventors tried to convert SP34 mouse anti-IgG into scFv, but no matter which (VH/VL) arrangement mode was adopted or the length of the connecting peptide was changed, a stable scFv could not be obtained.
  • the field urgently needs a stable anti-CD3 monoclonal antibody, especially its stable scFv structure.
  • the present invention provides a CD3 targeting antibody, bispecific antibody and use thereof.
  • the technical solution of the first aspect of the present invention is to provide an antibody targeting CD3, wherein the antibody targeting CD3 comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL is the amino acid sequence shown in SEQ ID NO: 56 or a mutation thereof; the VH has a mutation in the amino acid sequence shown in SEQ ID NO: 42, and the mutation is selected from one or more The following positions: 30th, 73rd, 76th, 78th, 93rd and 94th amino acid residues (the positions are numbered using Chothia coding rules). The mutation is the addition, deletion or substitution of one or more amino acid residues in the original amino acid sequence.
  • the CD3 targeting antibody of the present invention changes the binding ability with T cells and reduces the level of cytokine release, thereby reducing the toxicity caused by the cytokine release syndrome.
  • the mutations that occur on the VH are selected from the following combinations:
  • the mutations that occur on the VH are selected from the following combinations:
  • the antibody of the present invention is on the VL of the amino acid sequence shown in SEQ ID NO: 56, or on the VH of the amino acid sequence shown in SEQ ID NO: 42.
  • Make mutations so that the mutated amino acid sequence has 80%, 85%, 90%, 95%, 98%, 99% or more identity with the original amino acid sequence, and the amino acid sequence that maintains or improves the function of the antibody is also The scope of protection of the present invention.
  • amino acid sequence of the VH is shown in any one of SEQ ID NO: 43-55, and/or the amino acid sequence of the VL is shown in SEQ ID NO: 57-60 As shown in any sequence.
  • amino acid sequence of the VH is shown in SEQ ID NO: 44, and the amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 51
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 44, and the amino acid sequence of the VL is shown in SEQ ID NO: 60; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 51
  • amino acid sequence of the VL is shown in SEQ ID NO: 60; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 45, and the amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 52
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 43
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 43, and the amino acid sequence of the VL is shown in SEQ ID NO: 60; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 50, and the amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 47
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 48, and the amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 49
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 53
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 54 and the amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 43, and the amino acid sequence of the VL is shown in SEQ ID NO: 57; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 44, and the amino acid sequence of the VL is shown in SEQ ID NO: 57; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 43, and the amino acid sequence of the VL is shown in SEQ ID NO: 59; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 44, and the amino acid sequence of the VL is shown in SEQ ID NO: 59; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 51
  • amino acid sequence of the VL is shown in SEQ ID NO: 57; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 55
  • amino acid sequence of the VL is shown in SEQ ID NO: 58; or,
  • amino acid sequence of the VH is shown in SEQ ID NO: 46
  • amino acid sequence of the VL is shown in SEQ ID NO: 58.
  • the antibody includes VL-Linker-VH, or VH-Linker-VL single-chain variable fragment (scFv).
  • the Linker ie connecting peptide
  • the Linker is (GGGGS) n [abbreviation (G 4 S) n ] or a variant thereof, wherein n is a non-zero natural number, preferably 1-20, more preferably as SEQ ID NO : 65, SEQ ID NO: 66, and the amino acid sequence shown in SEQ ID NO: 67.
  • the amino acid sequence of the scFv is as shown in SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 80. More preferably, the antibody further includes a fragment crystallizable (Fc), and the Fc is connected to the scFv through a hinge region (Hinge).
  • Fc fragment crystallizable
  • the antibody also includes a constant region, preferably a human constant region.
  • the human constant region includes a human light chain constant region and a human heavy chain constant region, and the human light chain constant region is preferably the human kappa light chain constant region shown in SEQ ID NO: 61 or The human lambda light chain constant region shown in SEQ ID NO: 62.
  • the human heavy chain constant region is hlgG1, hlgG2, hlgG3, hlgG4 or mutations thereof, preferably the heavy chain constant region shown in SEQ ID NO: 63 or SEQ ID NO: 64.
  • the technical solution of the second aspect of the present invention is to provide a bispecific antibody.
  • the bispecific antibody of the present invention has a three-chain structure, which can reduce the number of by-product combinations, thereby reducing the complexity of its production; however, its development cannot be obtained with a little modification using the antibody of the prior art.
  • the inventors tried to convert SP34 mouse anti-IgG into scFv, but no matter which arrangement mode (VH/VL) is adopted or the length of the connecting peptide is changed, the stability cannot be obtained. ScFv. After repeated mutation design and verification, the inventors found that only some mutations can make scFv maintain a stable structure.
  • the bispecific antibody of the present invention includes a first protein functional region and a second protein functional region, wherein the first protein functional region comprises the CD3 targeting antibody as described in the first aspect of the present invention; preferably,
  • the bispecific antibody includes the following three chains: (1) VL1-Linker-VH1-Hinge-CH2-CH3 (knob) or VH1-Linker-VL1-Hinge-CH2-CH3 (knob) of the first protein functional region, (2) VH2-CH1-Hinge-CH2-CH3 (hole) in the second protein functional area and (3) VL2-CL in the second protein functional area; the second protein functional area is targeted to another target
  • the antibody is preferably an antibody targeting B7H4 or an antibody targeting ROR1;
  • the linker is preferably (G 4 S) n , where n is a non-zero natural number, preferably 1-20, more preferably as SEQ ID NO: 65, SEQ ID NO: 66, the amino acid sequence shown in SEQ ID NO: 67;
  • the technical solution of the third aspect of the present invention is to provide an isolated nucleic acid that encodes the CD3-targeting antibody as described in the first aspect of the present invention or the antibody described in the second aspect of the present invention. Bispecific antibodies.
  • the technical solution of the fourth aspect of the present invention is to provide an expression vector comprising the isolated nucleic acid as described in the third aspect of the present invention; preferably, the expression vector is selected from retroviruses Vectors, lentiviral vectors, adenovirus vectors and adeno-associated virus vectors.
  • the technical solution of the fifth aspect of the present invention is to provide a genetically modified cell, wherein it is transfected with the expression vector as described in the fourth aspect of the present invention; preferably, the genetically modified cell
  • the cell is a eukaryotic cell.
  • the technical solution of the sixth aspect of the present invention is to provide a pharmaceutical composition, wherein the pharmaceutical composition comprises the CD3 targeting antibody as described in the first aspect of the present invention, The bispecific antibody described in the second aspect, the genetically modified cell described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further includes an immune checkpoint antibody.
  • the technical solution of the seventh aspect of the present invention is to provide a CD3 targeting antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, and the second aspect of the present invention.
  • the isolated nucleic acid described in the third aspect, the expression vector described in the fourth aspect of the present invention, the genetically modified cell described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention are used in the preparation of drugs for treating tumors. In the application.
  • the technical solution of the eighth aspect of the present invention is to provide a kit combination comprising a kit A and a kit B;
  • the kit A contains the target described in the first aspect of the invention.
  • the kit B contains other antibodies, bispecific antibodies , Genetically modified cells or pharmaceutical compositions, the other antibodies, bispecific antibodies, genetically modified cells or pharmaceutical compositions target CD3, B7H4, ROR1 or other targets.
  • the use of the medicine box A and the medicine box B is in no particular order, or the medicine box A is used first and then the medicine box B, or the medicine box B is used first and then the medicine box A is used.
  • the drug in the kit A is in an injectable form, such as an injection
  • the drug in the kit B is in an injectable form, such as an injection, or in a swallowable form, such as a tablet or pill.
  • the CD3 targeting antibody of the first aspect of the present invention, the bispecific antibody of the second aspect, the genetically modified cell of the fifth aspect, the pharmaceutical composition of the sixth aspect, or the eighth aspect of the present invention can be administered to patients for the treatment of related tumors.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the monoclonal antibody of the present invention changes the ability to bind to T cells and reduces the level of cytokine release, thereby reducing the toxicity caused by cytokine release syndrome;
  • the bispecific antibody prepared therefrom overcomes the instability of the single-chain antibody arm targeting CD3, and is stable and has T cell binding ability;
  • Bispecific antibodies containing only three chains are easy to prepare, which reduces the difficulty of production.
  • Figure 1 shows the HPLC-SEC results of CD3 single-chain antibody after one-step purification: (A) PR000275, (B) PR000276, (C) PR000307, (D) PR000308;
  • Figure 2 is a sequence alignment of SP34VH humanized variants
  • Figure 3 is a sequence alignment of SP34VL humanized variants
  • Figure 4 shows the differences in important positions of different VH/VL variant sequences, where (A) is the VH variant sequence and (B) is the VL variant sequence;
  • Figure 5 shows the results of (A) SDS-PAGE and (B) HPLC-SEC results of CD3 single-chain antibody PR000510 after one-step purification;
  • Figure 6 shows the binding ability of CD3 antibody PR000260 with (A) recombinant CHOK1 cells overexpressing human CD3 and (B) recombinant CHOK1 cells overexpressing cynomolgus CD3;
  • Figure 7 shows the binding ability of CD3 antibody to human T cells, including the binding curve and the relative intensity of MFI (the fluorescence intensity MFI of antibody binding to human T cells at a specific concentration, and the relative ratio to the initial antibody PR000260 (SP34)) or MFI The maximum value, where (A) PR000511, PR000512, PR000513, PR000514 and PR000260 bind human T cells, (B) PR001848, PR001849 and PR000260 bind human T cells, (C) PR002467, PR002468, PR002469, PR002470, PR002471, PR002472, PR001848 And PR000260 bind to human T cells, (D) PR001848, PR002742, PR002743 and PR000260 bind to human T cells, (E) PR002833, PR002834, PR002835, PR002836, PR002837, PR002742, PR001848, PR002469 and PR000260 bind to human T cells, (F)
  • Figure 8 shows the binding ability of CD3 single-chain antibody to human T cells, including the binding curve and the relative intensity of MFI (the fluorescence intensity MFI of the antibody binding to human T cells at a specific concentration, and the relative ratio relative to the initial antibody PR000260 (SP34)) , wherein (A) PR000510, PR000624, PR000627 and PR000260 bind human T cells, (B) PR001850 and PR000260 bind human T cells;
  • MFI the fluorescence intensity MFI of the antibody binding to human T cells at a specific concentration, and the relative ratio relative to the initial antibody PR000260 (SP34)
  • Figure 9 shows the binding ability of CD3 antibody to cynomolgus monkey T cells
  • Figure 10 shows the ability of CD3 antibody to activate human T cells to produce cytokine IFN- ⁇ , in which (A) PR000511, PR000512, PR000513, PR000514 and PR000260 activate T cells, (B) PR001848, PR001849 and PR000260 activate T cells, (C) PR002468, PR002469, PR002471 and PR001848 activate T cells, (D) PR002742, PR001848 and PR000260 activate T cells, (E) PR002833, PR002834, PR002835, PR002836, PR002837 and PR000260 activate T cells, (F) PR003886, PR001848 and PR002742 activate T cells, (G) PR001848, PR002469 and PR004616 activate T cells;
  • Figure 11 shows the ability of CD3 single-chain antibodies (PR000510, PR000623, PR000624, PR000627 and PR000260) to activate human T cells to produce cytokine IFN- ⁇ ;
  • Figure 12 shows the SDS-PAGE results of the samples after one-step purification of the bispecific antibodies (A) PR002883 and (B) PR002885;
  • Figure 13 shows the binding ability of monoclonal antibodies and bispecific antibodies to (A) SK-BR-3 cells and (B) human T cells;
  • Figure 14 shows the target cell killing ability mediated by bispecific antibody PR002883 in vitro (A) SK-BR-3 cell killing and (B) IFN- ⁇ release level;
  • Figure 15 shows the binding ability of monoclonal antibodies and bispecific antibodies to (A) Panc-1 cells and (B) human T cells;
  • Figure 16 shows the structure of a monoclonal antibody or bispecific antibody (A) IgG structure, (B) an asymmetric "four-chain” structure, and (C) an asymmetric "three-chain” structure containing a single-chain antibody.
  • the term "antibody” generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antibody to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antibodies include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and/or dAb), immunoconjugates, Multispecific antibodies (for example, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they show the desired antigen-binding activity.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light chain and heavy chain variable regions, which are called complementarity determining regions (CDR) or hypervariable regions (HVR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • the variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a ⁇ -sheet configuration, connected by three CDRs to form a loop connection, and in some cases form part of a ⁇ -sheet structure.
  • the CDRs in each chain are close together through the FR region, and together with the CDR from the other chain form the antigen binding site of the antibody.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions. , Such as participating in the antibody-dependent cytotoxicity of the antibody.
  • the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, Al-Lazikani et al., JMol Biol 273:927-48, 1997).
  • the combined definition rule including Kabat definition and Chothia definition is also used to determine the amino acid residues in the variable domain sequence and the full-length antibody sequence (Table 1).
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rules) to position bb (Chothia coding rules);
  • Haa-Hbb can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain , The amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule).
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and according to the Chothia coding rules The amino acid sequence from position 26 to position 32.
  • the effector functions mediated by the Fc domain of antibodies such as ADCC and CDC also have very important biological functions.
  • Different IgG subtypes have different ADCC or CDC functions.
  • IgG1 and IgG3 have strong ADCC and CDC functions, while IgG2
  • the effect of IgG4 and IgG4 is relatively weak.
  • changing the binding ability of Fc to Fc receptors through amino acid mutations or modifications can also modulate the original effector functions of Fc.
  • the "LALA" double mutant (L234A/L235A) in IgG1 can significantly reduce the affinity with Fc ⁇ RIIIA (CD16A), thereby reducing the ADCC effect.
  • the P329G mutation can significantly reduce the binding to multiple Fc ⁇ receptors (see, Schlothauer T, Herter S, Koller CF, et al. Protein Eng Des Sel. 2016 Oct; 29(10):457-466).
  • the Fc of these CD3 antibodies introduced "LALA” double mutants (L234A/L235A) or "LALAPG” triple mutants (L234A/L235A/P329G).
  • VH antibody heavy chain variable domain sequence
  • the antibody light chain variable domain sequence (VL) is synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody ⁇ light chain constant domain sequence (SEQ ID NO: 61) to encode the full length of the antibody Kappa light chain; or VL is synthesized and cloned into a mammalian cell expression plasmid vector encoding a human antibody lambda light chain constant domain sequence (SEQ ID NO: 62) to encode a full-length lambda light chain for antibody production.
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, adjust the cell concentration to 6-8 ⁇ 10 5 cells/ml, and incubate for 24 hours at 37°C in an 8% CO 2 shaker. The cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • VH sequence and the VL sequence of the antibody are connected by a flexible peptide (Linker) to obtain a single polypeptide chain that simultaneously encodes VH and VL, namely, a single-chain antibody variable region fragment (scFv).
  • a connecting peptide of appropriate length such as (G 4 S) 3 (SEQ ID NO: 65) or (G 4 S) 4 (SEQ ID NO: 66)
  • VH and VL can be correctly folded and assembled into functional Antibody.
  • different scFv structures VH-linker-VL or VL-linker-VH
  • a single scFv contains an antigen binding region composed of a pair of VH and VL, and usually only binds one antigen molecule, so it is called a monovalent binding molecule.
  • a His tag composed of 6 histidines was fused to the C-terminus of the scFv.
  • the polypeptide sequences encoding scFv and His tags are gene-synthesized and cloned into mammalian cell expression plasmid vectors to obtain plasmids encoding scFv-his, transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and expressed by conventional recombinant proteins And purification technology, you can get purified recombinant protein.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504).
  • the cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • a human IgG1 constant region Fc sequence (Glu216-Lys447, including hinge region, CH2 domain and CH3 domain) was fused to the C-terminus of scFv to construct a scFv-Fc recombinant molecule, using Fc homodimerization , Forming a bivalent scFv-Fc dimer molecule capable of binding two antigen molecules at the same time.
  • the “LALA” double mutant (L234A/L235A) or the “LALAPG” triple mutant was introduced into the Fc to reduce the binding of the antibody to the Fc ⁇ receptor.
  • the polypeptide sequence encoding scFv-Fc was gene synthesized and cloned into mammalian cell expression plasmid vector to obtain the plasmid encoding scFv-Fc transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and then used as described in Example 1.1
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the protein expression and purification method to obtain purified recombinant protein.
  • Analytical size exclusion chromatography was used to analyze the purity and aggregate form of protein samples. Connect the analytical column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8mm ⁇ 30cm) to a high pressure liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II), equilibrate with PBS buffer at room temperature for at least 1 hour . An appropriate amount of protein sample (at least 10 ⁇ g) is filtered with a 0.22 ⁇ m filter membrane and injected into the system, and the HPLC program is set: the sample is flowed through the chromatographic column at a flow rate of 1.0ml/min with PBS buffer for a maximum time of 20 minutes. HPLC will generate an analysis report, reporting the residence time of different molecular size components in the sample.
  • HPLC high pressure liquid chromatograph
  • SP34 is a mouse-derived anti-human CD3e antibody that can bind to a variety of primate CD3 and has the function of activating T cells.
  • the variable region sequences VH and VL of SP34 have been disclosed in WO2016071004A1.
  • the amino acid sequence of the VH of SP34 is SEQ ID NO: 42, and its corresponding mouse germline V gene is IGHV10-1;
  • the amino acid sequence of VL of SP34 is SEQ ID NO: 56, and its corresponding mouse
  • the germline V gene is IGLV1.
  • the VH sequence of SP34 was fused with the human IgG1 antibody heavy chain constant domain sequence (SEQ ID NO: 63) containing the "LALA" double mutant (L234A/L235A) to produce SP34 mouse-human chimera
  • the full-length heavy chain of an IgG1 antibody the VL amino acid sequence of SP34 is fused with the human antibody lambda light chain constant domain sequence (SEQ ID NO: 62) to generate the full-length lambda light chain of the SP34 mouse-human chimeric antibody.
  • the SP34 mouse-human chimeric recombinant antibody PR000260 was prepared according to the method of Example 1.1. Table 2 below shows the recombinant expression data of PR000260.
  • VH sequence (SEQ ID NO: 42) and the VL sequence (SEQ ID NO: 56) of SP34 are connected through a flexible peptide (Linker) to obtain a single polypeptide chain encoding VH and VL at the same time, that is, a single chain antibody variable region fragment (scFv).
  • Linker flexible peptide
  • scFv single chain antibody variable region fragment
  • different scFv structures can be constructed, and the C-terminal of scFv is fused with 6 histidines.
  • the His tag is used for purification.
  • the connecting peptide shown in SEQ ID NO: 67 can also be used in the construction of the scFv of this application.
  • scFv antibody molecules PR000275, PR000276, PR000307, PR000308, were prepared according to the method of Example 1.2.
  • Table 3 lists the sequence numbers of the four recombinant scFv antibody molecules;
  • Table 4 shows the recombinant expression data of these four molecules;
  • Figure 1 shows the HPLC-SEC results of these four molecules after one-step purification, where ( A) is PR000275, (B) is PR000276, (C) is PR000307, (D) is the result of PR000308. It can be seen that using the VH and VL sequences of SP34 to construct scFv, no matter which arrangement mode (VH/VL) is adopted or the length of the connecting peptide is changed, stable scFv cannot be obtained.
  • This example uses the "CDR grafting" method to humanize the sequence, that is: transplanting the CDR of the mouse anti-VH to the framework region of the human antibody VH, and transplanting the CDR of the mouse anti-VL to the framework region of the human antibody VL .
  • the sequence of the framework region of the human antibody VH or VL can be derived from the human germline gene sequence or the antibody sequence after V(D)J rearrangement or the consensus sequence of the specific VH or VL gene family of the human antibody.
  • the framework region sequence provided by the human germline gene sequence is used as the humanized template sequence, that is, the human germline V gene fragment provides the framework region FR1, FR2, FR3 sequence, and the human germline J gene fragment provides Sequence of FR4 framework region.
  • the humanized variable region (VH or VL) was constructed in an arrangement of (human) FR1- (mouse) CDR1- (human) FR2- (mouse) CDR2- (human) FR3- (mouse) CDR3- (human) FR4 )sequence.
  • human germline V gene fragment IGHV3-73*01 or human germline V gene fragment IGHV3-23*01 combined with the sequence of human germline J gene fragment IGHJ1*01 were used as humanized templates to provide framework region sequences. And introduce one or more amino acid mutations at position 30, 73, 76, 78, 93 or 94 (according to Chothia coding rules) to obtain multiple different VH variants sequence.
  • human germline V gene fragment IGLV7-46*02 combined with the sequence of human germline J gene fragment IGLJ2*01 or human germline V gene fragment IGKV1-39*01 combined with human germline J gene fragment IGKJ4*01
  • the sequence serves as a template for humanization to provide the framework region sequence.
  • introduce zero or more amino acid mutations at position 2, 36, 46, 49, 66, 69, 71 or 87 (according to Chothia coding rules) Get a number of different VL variant sequences.
  • Table 5 lists the sequence numbers of antibody variable regions and optimized variant sequences (FV) and CDR and FR region sequences defined by CHOTHIA.
  • VH3731 51 7 1 10 3 18 4 twenty two VH3732 52 7 2 10 3 18 4 twenty two VH3733 53 7 2 10 3 19 4 twenty two VH3734 54 7 2 10 3 20 4 twenty two VH3735 55 7 2 10 3 17 4 twenty two VH3230 43 6 1 9 3 12 4 twenty two VH3231 44 6 1 9 3 13 4 twenty two VH3232 45 6 2 9 3 13 4 twenty two VH3233 46 6 2 9 3 12 4 twenty two VH3234 47 6 2 9 3 14 4 twenty two VH3235 48 6 2 9 3 15 4 twenty two VH3236 49 6 2 9 3 16 4 twenty two SP34_VL 56 26 twenty three 30 twenty four 35 25 40 VL7460 57 27 twenty three 33 twenty four 38 25 40 VL7461 58 27 twenty three 34 twenty four 39 25 40 VK1392 59 28 twenty three 31 twenty four 36 25 41 VK1393 60 29 twenty three 32 twenty four 37 25 41
  • Figure 2 lists the sequence comparison of the VH variants.
  • Figure 3 lists the alignment of the VL variant sequences.
  • Fig. 4 (A) and (B) respectively list the difference between the VH variant sequence and the VL variant sequence in important positions. It can be seen from Figures 2 to 4 that the mutations on the VH of the antibody of the present invention are in the 30th, 73rd, 76th, 78th, 93rd and 93rd positions of the amino acid sequence shown in SEQ ID NO:42. One or more amino acid residue mutations occurred at position 94. The mutations that occur on the VL are at positions 2, 36, 46, 49, 66, 69, 71, and/ of the sequence shown in SEQ ID NO: 56. Or mutation of the 87th amino acid residue.
  • Example 4.1 The VH variant sequence and the VL variant sequence obtained in Example 4.1 were paired and combined, and the IgG recombinant antibody was constructed according to the method in Example 1.1, and the "LALA” double mutant or “LALAPG” was introduced into the constant region of the IgG1 heavy chain. "Three mutants to reduce the Fc effector function.
  • Table 6 lists the sequence table of the recombinant antibody molecule optimized by the sequence.
  • Table 7 lists the expression data of the recombinant antibody. Except for the low expression yield of the three IgG molecules constructed by the VH variant VH3230, other IgG molecules have reasonable expression yields.
  • Example 4.1 The VH variant sequence and the VL variant sequence obtained in Example 4.1 were paired and combined, and a plurality of recombinant bivalent scFv antibody molecules were prepared according to the method in Example 1.3.
  • Table 8 and 9 respectively list the sequence information and protein expression of scFv. It can be seen from Table 9 that especially PR000510 and PR000627 can get better expression and stable molecules.
  • Figure 5 shows the results of (A) SDS-PAGE and (B) HPLC-SEC of PR000510. It can be seen that it has very good monomer purity and no obvious aggregates.
  • Table 8 The structure and sequence information of the scFv molecule constructed based on the sequence-optimized variant sequence
  • the CD3 expressing cells can be: CHOK1 cells overexpressing human CD3 or HEK293 cells (encoding the ⁇ , ⁇ , ⁇ , and ⁇ chains of human CD3)
  • the ORF plasmid and the plasmid encoding the human TCR ⁇ and ⁇ chain ORF are co-transfected into host cells CHOK1 (ATCC, CCL-61) or HEK293 (ATCC, CRL-1573) to construct a structure expressing the human TCR/CD3 complex Stable cell line); CHOK1 or HEK293 cells overexpressing cynomolgus CD3; human pan-T cells (isolated from PBMC with human pan-T cell isolation kit (Miltenyi, #130-096-535)); crab-eating Monkey pan-T cells.
  • the collected cells were washed twice with PBS (FACS buffer) containing 2% FBS, then resuspended in FACS buffer, and divided into 96-well plates, 1 ⁇ 10 5 cells per well, centrifuged at 500 g speed 5 minutes, discard the supernatant, add 100 ⁇ l of pre-diluted CD3 antibody, incubate for 1 hour at room temperature, wash twice with FACS buffer, and add the secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fc ⁇ fragment specific( Jackson ImmunoResearch, #109-545-098) resuspend the cells, incubate in the dark at room temperature for 30 minutes, wash twice with FACS buffer, and then resuspend with 200 ⁇ l FACS buffer.
  • PBS FACS buffer
  • Figure 6 shows the binding ability of the CD3 antibody obtained in Example 2 with recombinant CHOK1 cells overexpressing human CD3 ( Figure 6(A)) and recombinant CHOK1 cells overexpressing cynomolgus CD3 ( Figure 6(B)).
  • the results indicate that the SP34 chimeric antibody PR000260 has a strong binding ability to human CD3 and cyno CD3.
  • Figure 7 (A) ⁇ (G) respectively show the binding ability of the CD3 antibody (including PR000260 and its variants) obtained in Example 4.2 to human pan-T cells, and the calculated CD3 antibody concentration is 7.4 or 10 ⁇ g
  • PR000512, PR000513, PR001849, and PR002837 have the same binding capacity as PR000260 (ie SP34 chimeric antibody); and the binding capacity of PR000514 is slightly higher than that of PR000260; PR000511, PR001848, PR002469, PR002472, PR002742 , PR002833, PR002834, PR002835, PR002836, PR003886, and PR004616 have low ability to bind to T cells; PR002467, PR002468, PR002470, PR002471, and PR002743 hardly bind to T cells (or no signal can be detected at the current antibody concentration).
  • the above results indicate that the present invention has obtained a number of new antibodies by optimizing the sequence of the CD3 antibody, which have different binding capabilities with human T cells and can be applied to different application scenarios.
  • Figure 8 (A) and (B) show the binding ability of the anti-CD3 scFv-Fc single chain antibody obtained in Example 4.3 to human pan-T cells, and it is calculated that the CD3 antibody is at an antibody concentration of 7.4 or 10 ⁇ g/ml
  • PR000624 has a binding capacity equivalent to or slightly higher than that of PR000260
  • PR000510 and PR000627 have a binding capacity equivalent to or slightly lower than that of PR000260
  • the ability of PR001850 to bind to T cells is significantly lower than PR000260.
  • the above results indicate that the present invention has also obtained several stable scFv single-chain antibodies by optimizing the sequence of the CD3 antibody, which can bind to human T cells and are suitable for application scenarios such as the construction of bispecific antibodies.
  • Figure 9 shows the binding ability of part of the CD3 antibody obtained from Example 4.2 to cynomolgus monkey pan-T cells. It can be seen that different molecules have different binding abilities to cynomolgus monkey pan-T cells, and are positively correlated with their ability to bind human pan-T cells; that is, molecules with strong binding to human pan-T cells have a positive effect on cynomolgus monkeys. Pan-T cell binding ability is also strong, and vice versa.
  • the CD3 antibody (such as 50, 10, 5, 1, 0.5, 0.05 ⁇ g/ml) with gradient dilutions (eg, 50, 10, 5, 1, 0.5, 0.05 ⁇ g/ml) was coated in a 96-well cell culture plate at a concentration of three replicates and 50 ⁇ l per well, overnight at 4°C.
  • Figure 10 (A) to (G) respectively show the ability of each CD3 antibody (including SP34 chimeric antibody) obtained in Example 4.2 to activate human T cells.
  • the antibody concentration is 1 ⁇ g/mL
  • PR000511, PR000512, PR000513, and PR000514 activate T cells to produce IFN- ⁇ levels significantly lower than PR000260
  • the antibody concentration is 10 ⁇ g/mL
  • PR000512, PR000513, PR000514 activate IFN- ⁇ levels Slightly lower than PR000260
  • Figure 10(B) the level of IFN- ⁇ activated by PR001848 was significantly lower than that of PR000260
  • Figure 11 shows the ability of the anti-CD3 scFv-Fc antibody obtained in Example 4.3 to activate human T cells.
  • the above results indicate that the present invention has also obtained several stable scFv single-chain antibodies by optimizing the sequence of the CD3 antibody. They have weaker ability to activate human T cells and have a lower level of cytokine release for application. Used in application scenarios such as the construction of bispecific antibodies.
  • B7H4 is a member of the B7 family of transmembrane proteins. It is highly expressed in breast cancer, ovarian cancer, endometrial cancer and other solid tumor tissues, but not expressed or very weakly expressed in normal tissues, so B7H4 is specific Very good tumor-related target antigen.
  • the construction of bispecific antibody molecules that simultaneously target B7H4 and CD3 can selectively activate T cells near tumor cells by targeting and binding to B7H4 on the surface of tumor cells, thereby specifically killing tumor cells.
  • variable region sequence of the B7H4 antibody is derived from WO2016040724, and the recombinant IgG antibody PR000014 against B7H4 was constructed according to the method of Example 1.1. Table 10 below lists the sequence information of the B7H4 antibody PR000014.
  • Antibody number Target Heavy chain SEQ ID NO: Light chain SEQ ID NO: PR000014 B7H4 82 83
  • the sequence of the B7H4 antibody PR000014 obtained in Example 7.1 and the sequence of the CD3 single-chain antibody PR000627 obtained in Example 4.3 were used to construct a bispecific antibody molecule PR002883 targeting B7H4 ⁇ CD3, which contains three polypeptide chains, respectively: containing CD3
  • the molecule has a special asymmetric structure, in order to reduce the production of homologous heavy chain dimers, different amino acid mutations have been introduced in the constant regions of the two heavy chains.
  • the "LALAPG" triple mutant in order to prevent cross-linking caused by Fc ⁇ receptor binding and reduce effector functions, was introduced into the constant region of the heavy chain.
  • the recombinant protein of the bispecific antibody PR002883 was prepared by using the method described in Example 1.1 and combining the plasmid ratio (such as 1:1:1 or other ratio) and undergoing one-step affinity purification.
  • Table 11 lists the sequence listing of the bispecific antibody PR002883;
  • Table 12 lists the expression of the bispecific antibody.
  • Figure 12(A) shows the results of SDS-PAGE analysis of the bispecific antibody PR002883 after one-step purification. It shows that the main by-products are incompletely assembled molecules, and high polymer components are less. The by-products can be reduced by optimizing the purification steps or optimizing the plasmid transfection ratio.
  • This example studies the ability of bispecific antibodies to bind to tumor cells SK-BR-3 (ATCC, HTB-30) expressing human B7H4. Specifically, the SK-BR-3 cell suspension was collected, the cell density was adjusted to 1 ⁇ 10 6 /ml, and 100 ⁇ l/well was seeded on 96-well V bottom plate (Corning, #3894); then 100 ⁇ l/well was added The antibody to be tested is diluted in a 3-fold concentration gradient of 2 times the final concentration. Place at 4°C and incubate in the dark for 2 hours. After that, 100 ⁇ l/well was added with pre-cooled PBS to rinse the cells twice, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded.
  • Figure 13(A) shows the binding ability of the monoclonal antibody obtained in Example 7.1 and the bispecific antibody obtained in Example 7.2 to SK-BR-3 cells. It can be seen that the bispecific antibody PR002883 has comparable or even better binding capacity than the monoclonal antibody PR000014.
  • Example 5 The method described in Example 5 was used to detect the ability of the bispecific antibody PR002883 to bind to human pan-T cells. As shown in Figure 13(B), PR002883 can bind to human pan-T cells.
  • a blank control is set in the plate: SKBR3+PBMC+RPMI1640/10% FBS medium; E-plate is placed in a 37°C 5% CO 2 incubator and incubated for 24 hours. After the incubation is complete, put the E-plate into the xCELLigence RTCA instrument to detect the cell index.
  • Cell killing% (1-test sample/blank control)*100%.
  • the cell culture supernatant was collected and used to detect the release of cytokine IFN- ⁇ .
  • the ELISA detection method refers to the operating instructions of the IFN- ⁇ kit (IFN gamma Human Uncoated ELISA Kit, Thermo, #88-7316-77).
  • the bispecific antibody PR002883 can activate T cells to release cytokines such as IFN- ⁇ and effectively kill tumor cells SK-BR-3.
  • concentration of the bispecific antibody was 0.01 ⁇ g/ml, almost 100% of the tumor cells were killed ( Figure 14(A)).
  • ROR1 is an inactive tyrosine protein kinase transmembrane protein, which is overexpressed in many tumors, but hardly expressed in normal tissues. After ROR1 interacts with Wnt5a as a receptor, it transduce the Wnt signaling pathway, thereby contributing to cell proliferation and migration in chronic lymphocytic leukemia, and contributing to epithelial-mesenchymal-transition (EMT) in solid tumors.
  • EMT epithelial-mesenchymal-transition
  • the tumor-specific expression of ROR1 makes it a suitable tumor-associated antigen target for the development of therapeutic drugs.
  • the construction of bispecific antibody molecules that simultaneously target ROR1 and CD3 can selectively activate T cells near tumor cells by targeting and binding to ROR1 on the surface of tumor cells, thereby specifically killing tumor cells.
  • variable region sequence of the ROR1 antibody is derived from WO2016094873, and the recombinant IgG antibody PR000374 against ROR1 was constructed according to the method of Example 1.1.
  • Table 13 lists the sequence listing of the ROR1 antibody PR000374.
  • Antibody number Target Heavy chain SEQ ID NO: Light chain SEQ ID NO: PR000374 ROR1 84 85
  • the sequence of the ROR1 antibody PR000374 obtained in Example 8.1 and the sequence of the CD3 single-chain antibody PR000627 obtained in Example 4.3 were used to construct a bispecific antibody molecule PR002885 targeting ROR1 ⁇ CD3, which contains three polypeptide chains, respectively: containing CD3
  • the molecule has a special asymmetric structure, in order to reduce the production of homologous heavy chain dimers, different amino acid mutations have been introduced in the constant regions of the two heavy chains.
  • the "LALAPG" triple mutant in order to prevent cross-linking caused by Fc ⁇ receptor binding and reduce effector functions, was introduced into the constant region of the heavy chain.
  • the recombinant protein of the bispecific antibody PR002885 was prepared by using the method described in Example 1.1 and combining the plasmid ratio (such as 1:1:1 or other ratio) and undergoing one-step affinity purification.
  • Table 14 lists the sequence information of the bispecific antibody PR002885;
  • Table 15 lists the expression of the bispecific antibody.
  • Figure 12(B) shows the results of SDS-PAGE analysis of the bispecific antibody PR002885 after one-step purification. It shows that the main by-products are incompletely assembled molecules, and high polymer components are less. The by-products can be reduced by optimizing the purification steps or optimizing the plasmid transfection ratio.
  • the ability of the bispecific antibody to bind to the tumor cell Panc-1 (ATCC, CRL-1469) expressing human ROR1 was investigated. Specifically, the Panc-1 cell suspension was collected, the cell density was adjusted to 1 ⁇ 10 6 /ml, and 100 ⁇ l/well was seeded in 96-well V bottom plate (Corning, #3894); then 2 was added at a volume of 100 ⁇ l/well. 3 times the final concentration of the antibody to be tested diluted in a concentration gradient. Place at 4°C and incubate in the dark for 2 hours. After that, 100 ⁇ l/well was added with pre-cooled PBS to rinse the cells twice, centrifuged at 500 g for 5 minutes, and the supernatant was discarded.
  • Figure 15(A) shows the binding ability of the monoclonal antibody obtained in Example 8.1 and the bispecific antibody obtained in Example 8.2 to Panc-1 cells. It can be seen that both the bispecific antibody PR002885 and the monoclonal antibody PR000374 can bind to Panc-1.
  • Example 5 The method described in Example 5 was used to detect the ability of the bispecific antibody PR002885 to bind to human pan-T cells. As shown in Figure 15(B), PR002885 can bind to human pan-T cells.

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Abstract

公开了一种靶向CD3的抗体、双特异性抗体及其用途。所述的靶向CD3的抗体包含轻链可变区(VL)和重链可变区(VH)，所述VL为如SEQ ID NO:56所示的氨基酸序列或其突变；所述VH在如SEQ ID NO:42所示的氨基酸序列上发生突变，所述突变选自一个或多个以下位点：第30位、第73位、第76位、第78位、第93位和第94位的氨基酸残基。所述双特异性抗体包括第一蛋白功能区和第二蛋白功能区，其中，所述第一蛋白功能区包含如上所述的靶向CD3的抗体。所述靶向CD3的抗体降低细胞因子释放综合征带来的毒性，由其制备的双特异性抗体稳定且具有T细胞结合能力，降低了生产难度。

Description

靶向CD3的抗体、双特异性抗体及其用途
本申请要求申请日为2019/9/30的中国专利申请2019109413286的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物制药领域，尤其涉及一种靶向CD3的抗体、双特异性抗体及其用途。
背景技术
T淋巴细胞是重要的参与适应性免疫应答的细胞类别，T细胞通过T细胞受体(TCR)识别抗原。TCR并不能直接识别抗原表面表位，而是特异性地识别抗原呈递细胞(APC)或靶细胞表面提呈的抗原肽-MHC分子复合物(pMHC)。T细胞应答的特异性通过由TCR和CD3的分子复合物对pMHC的识别来介导。TCR是由两条不同跨膜多肽链构成的异二聚体，肽链有α、β、γ、δ四种；根据肽链的不同组合，TCR分为TCRαβ和TCRγδ。CD3有不同的跨膜多肽链即γ、δ、ε、ζ，这些肽链相互作用形成同源二聚体或异源二聚体，作为TCR-CD3复合物的一部分。例如，TCR-CD3复合物包括TCRαβ二聚体、CD3γε二聚体、CD3δε二聚体，CD3ζζ二聚体。由于TCR肽链的胞质区很短，一般认为TCR识别抗原所产生的活化信号由CD3肽链转导至T细胞内。
由于CD3在启动免疫应答中的重要作用，靶向TCR-CD3信号传导尤其是针对CD3的单克隆抗体被认为是可以调节免疫过程并用于治疗炎性疾病或自身免疫性疾病的有效药物。事实上，抗CD3抗体Orthoclone OKT3是第一个被批准的治疗性抗体。OKT3最先在1985年由美国FDA批准用于治疗器官移植后的急性排斥反应。尽管OKT3的重复施用带来的免疫抑制能力为肾移植之后的排斥反应提供了有效治疗，然而其应用却被首次毒性剂量反应综合征所限制；该综合征被认为是与OKT3介导的T细胞活化和细胞因子释放相关。后来，由于严重的细胞因子风暴和鼠抗带来的免疫原性问题以及其他因素，OKT3于2010年退出市场。
CD3抗体的另一个问题是已发现许多CD3抗体为物种特异性的，例如OKT3与黑猩猩CD3反应但不与其它灵长类诸如猕猴的CD3同系物、或鼠的CD3同系物反应。CD3单克隆抗体的物种特异性是将其开发为治疗人类疾病的抗体药物的显著障碍。任何新的候选药物必须经过严格的临床前验证以后才能被用于人类患者进行临床试验。临床前测 试的目的是证实候选药物具有期望的活性和最重要地是候选药物是安全的。临床前安全性测试是将候选药物施用于相关物种，优选地为非人类灵长类动物(Non-Human Primates)。但是高等灵长类动物，尤其是黑猩猩，被认为是濒危物种，使用这种动物进行药物安全性测试是被高度限制的。本领域描述的适用于安全性评价测试的物种可以是猕猴，特别地是食蟹猴。然而，缺乏灵长类物种特异性交叉反应的CD3抗体难以提供有效的临床前安全性评估数据。在已知的结合人CD3的抗体中，SP34是极少数的可以结合多种灵长类CD3(例如人和食蟹猴CD3)的抗体(参见，Salmeron,A.et al,J Immunol 147(1991)3047-3052；Conrad M.L.,et.al,Cytometry A 71(2007)925-933)。
尽管CD3的单抗已经在临床上验证了其在某些疾病领域的有效性，然而近年来，CD3抗体更多地是应用到双特异性抗体药物的开发中。目前，在全球处于临床阶段或临床前阶段的双特异性抗体项目中，基于CD3的双特异性T细胞桥接器抗体(BsTCE，Bi-specific T-cell engager)的项目占到一半以上。CD3双特异性抗体BsTCE一方面如CAR-T细胞治疗一样显示强劲疗效，另一方面又可以像传统单抗那样进行生产和产品化。目前全球批准上市的双特异性抗体药物中，最先上市的Catumaxomab(2009年欧洲EMA批准上市，2013年从美国退市)和Blinatumomab(2014年美国FDA批准上市)都是BsTCE。CD3抗体是构建BsTCE的重要组成部分。BsTCE双特异性抗体可以同时结合两个靶点，其中一端可以识别肿瘤细胞表面的抗原分子(Tumor-associated antigen，TAA)，而另一端可以结合T细胞上的CD3分子。在肿瘤细胞存在下，BsTCE双特异性抗体结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，进而杀死肿瘤细胞。在设计和构建BsTCE双特异性抗体的各种结构的时候，CD3抗体的选择和优化至关重要。第一，CD3单抗的种属特异性及其重要，尤其是猴类交叉反应。第二，CD3抗体对CD3复合体的亲和力也非常重要，过高亲和力的CD3抗体可能将把抗体限制在脾脏等部位，难以接触到肿瘤；而且过高亲和力也可能过度刺激T细胞带来高水平细胞因子释放。第三，CD3抗体结合价键也具有重要影响，以前发现多价形式的CD3双特异性抗体可能会在没有结合肿瘤相关抗原情况下激活T细胞导致副反应，因而绝大多数在研CD3双特异性抗体是以单价CD3形式的。
除了CD3抗体以外，BsTCE双特异性抗体的结构设计也非常重要。BsTCE双特异性抗体的结构多种多样，主要可分为两大类：含有Fc的类IgG结构和不含Fc的抗体片段结构。例如，Blinatumomab就是由两个单链可变区抗体片段(scFv)串联组成的单个多肽链结构，但是这种结构半衰期很短，需要连续的静脉灌注，使用起来非常不方便。因而许多BsTCE双特异性抗体采用含有Fc的结构以改善分子稳定性和药代动力学性能。但 是由于在BsTCE中CD3结合域通常需要单价形式，因此含有Fc的结构往往是非对称结构。这些含有Fc的非对称结构有许多技术困难需要克服，例如非对称结构中的重链同源二聚化问题、轻链错配问题、Fcγ受体引起的分子交联和ADCC或CDC等效应功能作用等等。从抗TAA的IgG抗体和抗CD3的IgG抗体[图16(A)]构建BsTCE双特异性抗体可以选择不同的非对称结构，其中一种常用结构是保留两个独立Fab结构域的类IgG结构，这种结构含有四条不同的多肽链[两条不同的重链和两条不同的轻链，图16(B)所示结构]，与传统单抗有近似的分子量；但是，这种结构由于含有多个不同的多肽链，其可能带来很多种组合的副产物，这给抗体的表达纯化和生产工艺带来巨大的挑战。如果把其中CD3抗体的Fab改造成scFv结构，则可以将“四链”结构改变为“三链”结构[图16(C)所示结构]，进一步减少副产物组合的数量，进而降低其生产的复杂性。为构建BsTCE双特异性抗体，本发明人尝试将SP34鼠抗IgG转变为scFv，但是无论采用哪一种(VH/VL)排列模式或者改变连接肽的长度，都不能得到稳定的scFv，因此本领域急需一种稳定的抗CD3单抗，尤其是其稳定的scFv结构。
综上所述，本领域急需一种能够结合灵长类动物的CD3，有合适的CD3结合能力，且具有稳定的单链scFv结构的CD3抗体。
发明内容
为克服本领域缺乏低抗原性、有效且安全的抗CD3抗体和双特异性抗体非对称结构的技术问题，本发明提供了一种靶向CD3的抗体、双特异性抗体及其用途。
为解决上述技术问题，本发明第一方面的技术方案为：提供一种靶向CD3的抗体，其中，所述的靶向CD3的抗体包含轻链可变区(VL)和重链可变区(VH)，所述VL为如SEQ ID NO:56所示的氨基酸序列或其突变；所述VH在如SEQ ID NO:42所示的氨基酸序列上发生突变，所述突变选自一个或多个以下位点：第30位、第73位、第76位、第78位、第93位和第94位的氨基酸残基(所述位点使用Chothia编码规则的位置编号)。所述突变即在原氨基酸序列上发生一个或多个氨基酸残基的增添、缺失或取代。本发明的靶向CD3的抗体改变了与T细胞的结合能力，降低了细胞因子释放水平，从而预期降低细胞因子释放综合征带来的毒性。
在一较佳的具体实施例中，在所述VH上发生的突变选自以下组合：
(a)第30位；
(b)第30位、第73位和第76位；
(c)第30位、第93位和第94位；
(d)第30位、第73位和第93位；
(e)第30位、第93位；
(f)第30位、第76位和第78位；
(g)第73位、第76位、第93位和第94位；
(h)第76位、第78位和第93位；
(i)第30位、第73位、第76位、第93位和第94位；
(j)第30位、第76位、第78位和第93位。
在一较佳的具体实施例中，在所述VH上发生的突变选自以下组合：
(a)N30S；
(b)N30S、D73N和S76N；
(c)N30S、V93A和R94K；
(d)N30S、D73N和V93A；
(e)N30S和V93T；
(f)N30S、S76N和L78A；
(g)D73N、S76N、V93A和R94K；
(h)S76N、L78A和V93T；
(i)N30S、D73N、S76N、V93A和R94K；
(j)N30S、S76N、L78A和V93T。
在抗体的VH具有上述限定的突变的前提下，本发明的抗体在如SEQ ID NO:56所示的氨基酸序列的VL上，或在如SEQ ID NO:42所示的氨基酸序列的VH上进一步进行突变，使得突变后的氨基酸序列与原氨基酸序列具有80％、85％、90％、95％、98％、99％或以上的同一性，且保持或改善了抗体的功能的氨基酸序列也在本发明保护的范畴。
在一较佳的具体实施例中，所述的VH的氨基酸序列如SEQ ID NO:43–55中任一序列所示，和/或，所述VL的氨基酸序列如SEQ ID NO:57-60中任一序列所示。
在一较佳的具体实施例中，
所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:51所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:60所示；或，
所述VH的氨基酸序列如SEQ ID NO:51所示，所述VL的氨基酸序列如SEQ ID NO:60所示；或，
所述VH的氨基酸序列如SEQ ID NO:45所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:52所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:60所示；或，
所述VH的氨基酸序列如SEQ ID NO:50所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:47所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:48所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:49所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:53所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:54所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:57所示；或，
所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:57所示；或，
所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:59所示；或，
所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:59所示；或，
所述VH的氨基酸序列如SEQ ID NO:51所示，所述VL的氨基酸序列如SEQ ID NO: 57所示；或，
所述VH的氨基酸序列如SEQ ID NO:55所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
所述VH的氨基酸序列如SEQ ID NO:46所示，所述VL的氨基酸序列如SEQ ID NO:58所示。
在一较佳的具体实施例中，所述抗体包括VL-Linker-VH，或VH-Linker-VL的单链可变区抗体(single-chain variable fragment,scFv)。较佳地，所述Linker(即连接肽)为(GGGGS) n[缩写(G 4S) n]或其变体，其中n为非0自然数，优选1～20，更优选为如SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67所示的氨基酸序列。更佳地，所述scFv的氨基酸序列如SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:78、SEQ ID NO:79或SEQ ID NO:80所示。进一步更佳地，所述抗体还包括可结晶片段(fragment crystallizable,Fc)，所述Fc通过铰链区(Hinge)和scFv相连。
在一较佳的具体实施例中，所述抗体还包括恒定区，优选人源恒定区。较佳地，所述人源恒定区包括人源轻链恒定区和人源重链恒定区，所述人源轻链恒定区优选如SEQ ID NO:61所示的人κ轻链恒定区或如SEQ ID NO:62所示的人λ轻链恒定区。更佳地，所述人源重链恒定区为hIgG1、hIgG2、hIgG3、hIgG4或其突变，优选如SEQ ID NO:63或SEQ ID NO:64所示的重链恒定区。
为解决上述技术问题，本发明第二方面的技术方案为：提供一种双特异性抗体。本发明的双特异性抗体为三链结构，其能减少副产物组合的数量，进而降低其生产的复杂性；但其研发并不是利用现有技术的抗体稍加改造就能够获得的。如背景所述，为构建BsTCE双特异性抗体，本发明人尝试将SP34鼠抗IgG转变为scFv，但是无论采用哪一种(VH/VL)排列模式或者改变连接肽的长度，都不能得到稳定的scFv。发明人经多番突变设计和验证，发现其中仅有一些突变能使scFv保持稳定结构。本发明的双特异性抗体包括第一蛋白功能区和第二蛋白功能区，其中，所述第一蛋白功能区包含如本发明的第一方面所述的靶向CD3的抗体；较佳地，所述双特异性抗体包括以下三条链：(1)第一蛋白功能区的VL1-Linker-VH1-Hinge-CH2-CH3(knob)或VH1-Linker-VL1-Hinge-CH2-CH3(knob)，(2)第二蛋白功能区的VH2-CH1-Hinge-CH2-CH3(hole)和(3)第二蛋白功能区的VL2-CL；所述第二蛋白功能区为靶向另一个靶点的抗体，优选靶向B7H4的抗体或靶向ROR1的抗体；所述linker优选为(G 4S) n，其中n为非0自然数，优选1～20，更优选为如SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67所示的氨基酸序列；更佳地，所述双特异性抗体包括如SEQ ID NO:88所示的VL1-Linker-VH1-Hinge-CH2-CH3 (knob)、如SEQ ID NO:86所示的VH2-CH1-Hinge-CH2-CH3(hole)和如SEQ ID NO:83所示的VL2-CL，或，如SEQ ID NO:88所示的VL1-Linker-VH1-Hinge-CH2-CH3(knob)、如SEQ ID NO:87所示的VH2-CH1-Hinge-CH2-CH3(hole)和如SEQ ID NO:85所示的VL2-CL。本发明的双特异性抗体克服了靶向CD3的单链抗体臂不稳定的缺陷，其稳定且具有T细胞结合能力。仅包含三条链的双特异性抗体容易制备，降低了生产难度。
为解决上述技术问题，本发明第三方面的技术方案为：提供一种分离的核酸，其编码如本发明的第一方面所述的靶向CD3的抗体或本发明的第二方面所述的双特异性抗体。
为解决上述技术问题，本发明第四方面的技术方案为：提供一种表达载体，其包含如本发明的第三方面所述的分离的核酸；优选地，所述表达载体选自逆转录病毒载体、慢病毒载体、腺病毒载体和腺相关病毒载体。
为解决上述技术问题，本发明第五方面的技术方案为：提供一种基因修饰的细胞，其中，其转染有如本发明的第四方面所述的表达载体；优选地，所述基因修饰的细胞为真核细胞。
为解决上述技术问题，本发明第六方面的技术方案为：提供一种药物组合物，其中，所述药物组合物包含如本发明的第一方面所述的靶向CD3的抗体、本发明的第二方面所述的双特异性抗体、本发明的第五方面所述的基因修饰的细胞以及药学上可接受的载体；较佳地，所述药物组合物还包括免疫检查点抗体。
为解决上述技术问题，本发明第七方面的技术方案为：提供一种如本发明第一方面所述的靶向CD3的抗体、本发明第二方面所述的双特异性抗体、本发明第三方面所述的分离的核酸、本发明第四方面所述的表达载体、本发明第五方面所述的基因修饰的细胞或本发明第六方面所述的药物组合物在制备治疗肿瘤的药物中的应用。
此外，为解决上述技术问题，本发明第八方面的技术方案为：提供一种药盒组合，其包括药盒A和药盒B；所述药盒A包含本发明第一方面所述的靶向CD3的抗体、第二方面所述的双特异性抗体、第五方面所述的基因修饰的细胞或第六方面所述的药物组合物；所述药盒B包含其它抗体、双特异性抗体、基因修饰的细胞或药物组合物，所述其它抗体、双特异性抗体、基因修饰的细胞或药物组合物靶向CD3、B7H4、ROR1或其它靶点。所述药盒A和药盒B的使用不分先后顺序，或先使用药盒A再使用药盒B，或先使用药盒B再使用药盒A。所述药盒A中的药物以可注射的形式例如针剂存在，所述药盒B中的药物以可注射的形式例如针剂存在，或可吞服的形式例如药片或药丸存在。
本发明的第一方面所述的靶向CD3的抗体、第二方面所述的双特异性抗体、第五方 面所述的基因修饰的细胞、第六方面所述的药物组合物或第八方面所述的药盒组合可施用于病人，用于治疗相关肿瘤。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
1、本发明的单克隆抗体改变了与T细胞的结合能力，降低了细胞因子释放水平，从而预期降低细胞因子释放综合征带来的毒性；
2、由其制备的双特异性抗体克服了靶向CD3的单链抗体臂不稳定的缺陷，其稳定且具有T细胞结合能力；
3、仅包含三条链的双特异性抗体容易制备，降低了生产难度。
附图说明
图1为CD3单链抗体经过一步纯化后的HPLC-SEC结果：(A)PR000275，(B)PR000276，(C)PR000307，(D)PR000308；
图2为SP34VH人源化变体序列比对；
图3为SP34VL人源化变体序列比对；
图4为不同VH/VL变体序列在重要位点上的差异，其中(A)为VH变体序列，(B)为VL变体序列；
图5为CD3单链抗体PR000510经过一步纯化后的(A)SDS-PAGE结果，(B)HPLC-SEC结果；
图6为CD3抗体PR000260与(A)过表达人CD3的重组CHOK1细胞和(B)过表达食蟹猴CD3的重组CHOK1细胞的结合能力；
图7为CD3抗体与人T细胞的结合能力，包括结合曲线和MFI相对强度(抗体在特定浓度时结合人T细胞的荧光强度MFI，以及相对于初始抗体PR000260(SP34)的相对比值)或MFI最大值，其中(A)PR000511、PR000512、PR000513、PR000514和PR000260结合人T细胞，(B)PR001848、PR001849和PR000260结合人T细胞，(C)PR002467、PR002468、PR002469、PR002470、PR002471、PR002472、PR001848和PR000260结合人T细胞，(D)PR001848、PR002742、PR002743和PR000260结合人T细胞，(E)PR002833、PR002834、PR002835、PR002836、PR002837、PR002742、PR001848、PR002469和PR000260结合人T细胞，(F)PR003886、PR001848和PR002742结合人T细胞，(G)PR001848、 PR002469和PR004616结合人T细胞；
图8为CD3单链抗体与人T细胞的结合能力，包括结合曲线和MFI相对强度(抗体在特定浓度时结合人T细胞的荧光强度MFI，以及相对于初始抗体PR000260(SP34)的相对比值)，其中(A)PR000510、PR000624、PR000627和PR000260结合人T细胞，(B)PR001850和PR000260结合人T细胞；
图9为CD3抗体与食蟹猴T细胞的结合能力；
图10为CD3抗体激活人T细胞产生细胞因子IFN-γ的能力，其中(A)PR000511、PR000512、PR000513、PR000514和PR000260激活T细胞，(B)PR001848、PR001849和PR000260激活T细胞，(C)PR002468、PR002469、PR002471和PR001848激活T细胞，(D)PR002742、PR001848和PR000260激活T细胞，(E)PR002833、PR002834、PR002835、PR002836、PR002837和PR000260激活T细胞，(F)PR003886、PR001848和PR002742激活T细胞，(G)PR001848、PR002469和PR004616激活T细胞；
图11为CD3单链抗体(PR000510、PR000623、PR000624、PR000627和PR000260)激活人T细胞产生细胞因子IFN-γ的能力；
图12为双特异性抗体(A)PR002883和(B)PR002885经过一步纯化后的样品的SDS-PAGE结果；
图13为单抗和双特异性抗体与(A)SK-BR-3细胞和(B)人T细胞的结合能力；
图14为双特异性抗体PR002883在体外介导的靶细胞杀伤能力(A)SK-BR-3细胞杀伤和(B)IFN-γ释放水平；
图15为单抗和双特异性抗体与(A)Panc-1细胞和(B)人T细胞的结合能力；
图16为单抗或双特异性抗体结构(A)IgG结构，(B)非对称“四链”结构，(C)含有单链抗体的非对称“三链”结构。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
在本申请中，术语“抗体”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗体与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每 个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见，Al-Lazikani等人，JMol Biol 273:927-48,1997)。在本申请中，还使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基(表1)。
表1本申请抗体CDR定义方法(可参见http://bioinf.org.uk/abs/)
CDR区 Kabat定义 Chothia定义 Combined定义
LCDR1 L24--L34 L24--L34 L24--L34
LCDR2 L50--L56 L50--L56 L50--L56
LCDR3 L89--L97 L89--L97 L89--L97
HCDR1 H31--H35 H26--H32 H26--H35
HCDR2 H50--H65 H52--H56 H50--H65
HCDR3 H95--H102 H95--H102 H95--H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可 以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
抗体Fc结构域介导的效应子功能如ADCC和CDC也有非常重要的生物学功能，不同的IgG亚型有着不同的ADCC或CDC功能，例如IgG1和IgG3有较强的ADCC和CDC作用，而IgG2和IgG4的作用相对较弱。另外，通过氨基酸突变或者修饰来改变Fc与Fc受体的结合能力也可以调节Fc原有的效应子功能。例如，IgG1中的“LALA”双突变体(L234A/L235A)能够显著降低与FcγRIIIA(CD16A)的亲和力，进而降低ADCC作用。另外，P329G突变能够显著降低与多种Fcγ受体的结合(参见，Schlothauer T,Herter S,Koller CF,et al.Protein Eng Des Sel.2016Oct；29(10):457-466)。在申请中，为了减少CD3抗体与Fcγ受体的结合，这些CD3抗体的Fc引入了“LALA”双突变体(L234A/L235A)或者“LALAPG”三突变体(L234A/L235A/P329G)。
实施例1重组抗体的制备和表征分析
1.1制备IgG重组抗体
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG1抗体的全长重链，并且在该IgG1重链恒定区引入“LALA”双突变体(L234A/L235A)(SEQ ID NO:63)或者“LALAPG”三突变体(L234A/L235A/P329G)(SEQ ID NO:64)以减少抗体与Fcγ受体的结合。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体κ轻链恒定结构域序列(SEQ ID NO:61)的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长κ轻链；或者将VL通过基因合成并克隆到编码人抗体λ轻链恒定结构域序列(SEQ ID NO:62)的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长λ轻链。
将编码抗体重链的质粒和编码抗体轻链的质粒同时转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到具有轻重链正确配对组装的纯化的重组抗体。具体来说，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6～8×10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3的比例混合共计 30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo,#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelect  TM(GE Healthcare Life Science,#71-5020-91AE)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,#UFC901024)浓缩换液至PBS缓冲液，得到纯化的重组抗体溶液。最后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
1.2制备单价scFv-his重组抗体
将抗体的VH序列和VL序列通过柔性肽段(Linker)连接起来得到同时编码VH和VL的单一多肽链即单链抗体可变区片段(scFv)。如果选择合适长度的连接肽，例如(G 4S) 3(SEQ ID NO:65)或(G 4S) 4(SEQ ID NO:66)，VH和VL可以正确地折叠和组装成有功能的抗体。根据VH、VL的不同的排列和连接肽的不同，可以构建不同的scFv结构(VH-linker-VL或VL-linker-VH)。单个scFv包含由一对VH和VL组成的抗原结合区，通常只能结合一个抗原分子，因而称为单价结合分子。
为了便于纯化，在本实施例中，在scFv的C末端融合有由6个组氨酸组成的His标签。将编码scFv和His标签的多肽序列通过基因合成并克隆到哺乳动物细胞表达质粒载体中得到编码scFv-his的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到纯化的重组蛋白。具体说来，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6～8×10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将上述30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo,#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS缓冲液(pH7.4)平衡含有Ni Sepharose excel(GE Healthcare Life Science,#17-3712-01)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，先用缓冲液A(含有20 mM咪唑，150mM磷酸盐，pH8.0)洗脱非特异性吸附的杂蛋白，然后用缓冲液B(含有500mM咪唑，150mM磷酸盐，pH8.0)洗脱目的蛋白。最后用超滤管(Millipore,#UFC901024)浓缩换液至PBS缓冲液，得到纯化的重组抗体溶液。最后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
1.3制备双价scFv-Fc重组抗体
在本实施例中，在scFv的C末端融合人IgG1恒定区Fc序列(Glu216-Lys447，包含铰链区、CH2结构域和CH3结构域)构建scFv-Fc重组分子，利用Fc的同源二聚化，形成能够同时结合两个抗原分子的双价的scFv-Fc二聚体分子。并且在Fc引入“LALA”双突变体(L234A/L235A)或者“LALAPG”三突变体(L234A/L235A/P329G)以减少抗体与Fcγ受体的结合。将编码scFv-Fc的多肽序列通过基因合成并克隆到哺乳动物细胞表达质粒载体中得到编码scFv-Fc的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，然后利用实施例1.1所述的蛋白表达纯化方法得到纯化的重组蛋白。
1.4利用HPLC-SEC分析蛋白纯度
使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience,#08541,5μm,7.8mm×30cm)连接到高压液相色谱仪(HPLC)(Agilent Technologies,Agilent 1260Infinity II)，用PBS缓冲液室温下平衡至少1小时。适量蛋白样品(至少10μg)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用PBS缓冲液将样品以1.0ml/min的流速流过色谱柱，最长时间为20分钟。HPLC将生成分析报告，报告出样品内不同分子尺寸组份的滞留时间。
实施例2 CD3抗体SP34的鼠-人嵌合抗体重组表达
SP34是来源于小鼠的抗人CD3e的抗体，可以结合多种灵长类CD3，具有激活T细胞的功能。SP34的可变区序列VH和VL已经公开于WO2016071004A1。在本申请中，SP34的VH的氨基酸序列是SEQ ID NO:42，其对应的小鼠胚系V基因是IGHV10-1；SP34的VL的氨基酸序列是SEQ ID NO:56，其对应的小鼠胚系V基因是IGLV1。在本实施例中，将SP34的VH序列与包含“LALA”双突变体(L234A/L235A)的人IgG1抗体重链恒定结构域序列(SEQ ID NO:63)进行融合产生SP34鼠-人嵌合IgG1抗体的全长重链；将SP34的VL的氨基酸序列人抗体λ轻链恒定结构域序列(SEQ ID NO:62)进行融合产生SP34鼠-人嵌合抗体的全长λ轻链。
根据实施例1.1的方法制备SP34鼠-人嵌合重组抗体PR000260。下表2为PR000260的重组表达的数据。
表2重组抗体PR000260的表达和纯化
抗体编号 表达系统(体积) 纯化方法 产量(mg/L) HPLC-SEC(单体纯度％)
PR000260 HEK293(100ml) MabSelect 19.30 99.75％
实施例3将CD3抗体SP34鼠抗转变为重组scFv抗体
将SP34的VH序列(SEQ ID NO:42)和VL序列(SEQ ID NO:56)通过柔性肽段(Linker)连接起来得到同时编码VH和VL的单一多肽链即单链抗体可变区片段(scFv)。根据VH、VL的不同的排列和不同长度的连接肽(SEQ ID NO:65、SEQ ID NO:66)，可以构建不同的scFv结构，并且在scFv的C末端融合有由6个组氨酸组成的His标签用以纯化。如SEQ ID NO:67所示的连接肽也可以用于本申请scFv的构建。
本实施例根据实施例1.2的方法制备了四个重组scFv抗体分子(PR000275,PR000276,PR000307,PR000308)。下表3列出了这四个重组scFv抗体分子的序列编号；下表4为这四个分子的重组表达的数据；图1为这四个分子经过一步纯化后的HPLC-SEC结果，其中(A)为PR000275，(B)为PR000276，(C)为PR000307，(D)为PR000308的结果。可以看出，利用SP34的VH和VL序列构建scFv，无论采用哪一种(VH/VL)排列模式或者改变连接肽的长度，都不能得到稳定的scFv。
表3四个重组抗体分子的结构与序列编号
Figure PCTCN2020118606-appb-000001
表4重组scFv抗体分子的表达和纯化
Figure PCTCN2020118606-appb-000002
Figure PCTCN2020118606-appb-000003
实施例4 SP34的序列优化
4.1可变区序列的人源化和框架区突变
本实施例使用“CDR移植”的方法进行序列的人源化，即：将鼠抗的VH的CDR移植到人抗体VH的框架区，将鼠抗的VL的CDR移植到人抗体VL的框架区。人抗体VH或VL的框架区的序列可以来源于人的胚系基因序列或者经过V(D)J重排后的抗体序列或者人抗体特定VH或VL基因家族的一致性(consensus)序列。本实施例使用人的胚系基因序列提供的框架区序列作为人源化模板序列，即：人的胚系V基因片段提供框架区FR1，FR2，FR3的序列，人的胚系J基因片段提供框架区FR4的序列。最后以(人)FR1-(鼠)CDR1-(人)FR2-(鼠)CDR2-(人)FR3-(鼠)CDR3-(人)FR4的排列方式构建人源化可变区(VH或VL)序列。
本实施例使用人胚系V基因片段IGHV3-73*01或人胚系V基因片段IGHV3-23*01结合人胚系J基因片段IGHJ1*01的序列作为人源化模板提供框架区序列。并且在第30位、第73位、第76位、第78位、第93位或第94位(按照Chothia编码规则)引入一个或者多个位点的氨基酸突变，得到多个不同的VH变体序列。
本实施例使用人胚系V基因片段IGLV7-46*02结合人胚系J基因片段IGLJ2*01的序列或者人胚系V基因片段IGKV1-39*01结合人胚系J基因片段IGKJ4*01的序列作为人源化模板提供框架区序列。并且在第2位、第36位、第46位、第49位、第66位、第69位、第71位或第87位(按照Chothia编码规则)引入零个或者多个位点的氨基酸突变，得到多个不同的VL变体序列。
下表5列出了抗体可变区及优化变体序列(FV)和CHOTHIA定义的CDR、FR区序列的序列编号。
表5 SP34抗体可变区及其可变区优化变体序列(FV)和CHOTHIA定义的CDR、FR区的序列表
ID FV FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
SP34_VH 42 5 1 8 3 11 4 21
VH3730 50 7 1 10 3 17 4 22
VH3731 51 7 1 10 3 18 4 22
VH3732 52 7 2 10 3 18 4 22
VH3733 53 7 2 10 3 19 4 22
VH3734 54 7 2 10 3 20 4 22
VH3735 55 7 2 10 3 17 4 22
VH3230 43 6 1 9 3 12 4 22
VH3231 44 6 1 9 3 13 4 22
VH3232 45 6 2 9 3 13 4 22
VH3233 46 6 2 9 3 12 4 22
VH3234 47 6 2 9 3 14 4 22
VH3235 48 6 2 9 3 15 4 22
VH3236 49 6 2 9 3 16 4 22
SP34_VL 56 26 23 30 24 35 25 40
VL7460 57 27 23 33 24 38 25 40
VL7461 58 27 23 34 24 39 25 40
VK1392 59 28 23 31 24 36 25 41
VK1393 60 29 23 32 24 37 25 41
图2列出了VH变体序列的对比。图3列出了VL变体序列的对比。图4的(A)和(B)分别列出了VH变体序列和VL变体序列在重要位点上的差异。由图2～4可知，本发明抗体在VH上发生的突变为在如SEQ ID NO:42所示的氨基酸序列的第30位、第73位、第76位、第78位、第93位和第94位的一个或多个位点上发生了氨基酸残基的突变。在所述VL上发生的突变为在如SEQ ID NO:56所示的序列的第2位、第36位、第46位、第49位、第66位、第69位、第71位和/或第87位氨基酸残基的突变。更详细的突变信息可见表5中VH3730、VH3731、VH3732、VH3733、VH3734、VH3735、VH3230、VH3231、VH3232、VH3233、VH3234、VH3235、VH3236、VL7460、VL7461、VK1392和VK1393序列的具体内容。
4.2序列优化变体的重组抗体分子
将实施例4.1中得到的VH变体序列和VL变体序列进行配对组合，并按照实施例1.1中的方法构建IgG重组抗体，并且在IgG1重链恒定区引入“LALA”双突变体或者 “LALAPG”三突变体以降低Fc效应功能。表6列出了经过序列优化的重组抗体分子的序列表。表7列出了重组抗体的表达的数据。除了VH变体VH3230构建的三个IgG分子的表达产量很低以外，其他IgG分子都有合理的表达产量。
表6 SP34嵌合抗体或者序列优化抗体的序列表
Figure PCTCN2020118606-appb-000004
表7重组抗体表达产量和纯度
Figure PCTCN2020118606-appb-000005
Figure PCTCN2020118606-appb-000006
4.3序列优化变体的重组scFv分子
将实施例4.1中得到的VH变体序列和VL变体序列进行配对组合，并按照实施例1.3中的方法制备了多个重组双价scFv抗体分子。下表8、9分别列出了scFv的序列信息和蛋白表达情况。从表9可知，尤其是PR000510和PR000627可以得到较好的表达和稳定的分子。图5显示了PR000510的(A)SDS-PAGE和(B)HPLC-SEC的结果，可以看出其有很好的单体纯度，没有明显的聚体。
表8基于序列优化后的变体序列构建的scFv分子的结构与序列信息
Figure PCTCN2020118606-appb-000007
表9序列优化后scFv抗体的表达的数据
Figure PCTCN2020118606-appb-000008
Figure PCTCN2020118606-appb-000009
实施例5利用FACS测定CD3抗体和表达CD3的细胞的结合能力
利用流式分析法FACS分析CD3抗体与表达CD3的细胞的结合情况，其中表达CD3的细胞可以是：过表达人CD3的CHOK1细胞或者HEK293细胞(编码人源CD3的γ、δ、ε、ζ链ORF的质粒和编码人TCR的α、β链ORF的质粒共同转染宿主细胞CHOK1(ATCC，CCL-61)或者HEK293(ATCC，CRL-1573)，以构建出表达人TCR/CD3复合物结构的稳定细胞系)；过表达食蟹猴CD3的CHOK1或者HEK293细胞；人pan-T细胞(用人pan-T细胞分离试剂盒(Miltenyi,#130-096-535)从PBMC中分离出)；食蟹猴pan-T细胞。具体地，将收集的细胞用含有2％FBS的PBS(FACS缓冲液)洗两遍，再加FACS缓冲液重悬，分至96孔板中，每孔1×10 5个细胞，500g转速离心5分钟，弃上清，加入100μl预先梯度稀释的CD3抗体，室温孵育1小时，FACS缓冲液洗两遍，再加入FACS缓冲液稀释的二抗Alexa Fluor 488 AffiniPure Goat Anti-Human IgG,Fcγfragment specific(Jackson ImmunoResearch，#109-545-098)重悬细胞，室温避光孵育30分钟，FACS缓冲液洗两遍，再用200μl FACS缓冲液重悬。使用流式细胞仪(BD FACS CANTOII或ACEA NovoCyte)读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
图6显示实施例2得到的CD3抗体与过表达人CD3的重组CHOK1细胞(图6(A))和过表达食蟹猴CD3的重组CHOK1细胞的结合能力(图6(B))。其结果说明SP34嵌合抗体PR000260对人CD3和食蟹猴CD3都有较强的结合能力。
图7的(A)～(G)分别显示了实施例4.2得到的CD3抗体(包括PR000260及其变体)与人pan-T细胞的结合能力，且计算出CD3抗体在抗体浓度为7.4或10μg/ml时结 合人pan-T细胞的荧光强度MFI以及相对于初始抗体PR000260的相对比值。具体地，SP34 IgG抗体序列优化以后，PR000512、PR000513、PR001849、PR002837具有与PR000260(即SP34嵌合抗体)相当的结合能力；而PR000514结合能力略高于PR000260；PR000511、PR001848、PR002469、PR002472、PR002742、PR002833、PR002834、PR002835、PR002836、PR003886和PR004616与T细胞结合的能力较低；PR002467、PR002468、PR002470、PR002471、PR002743则几乎不结合T细胞(或者说无法在目前抗体浓度下检测到信号)。以上结果说明，本发明通过对CD3抗体的序列优化得到了多个新的抗体，它们与人的T细胞有不同的结合能力，可以适用于不同的应用场景。
图8的(A)和(B)显示实施例4.3得到的抗CD3的scFv-Fc单链抗体与人pan-T细胞的结合能力，且计算出CD3抗体在抗体浓度为7.4或10μg/ml时结合人pan-T细胞的荧光强度MFI以及相对于初始抗体PR000260的相对比值。具体地，SP34scFv抗体人源化优化以后，PR000624具有与PR000260相当的或略高的结合能力；PR000510、PR000627具有与PR000260相当的或略低的结合能力；而PR001850和T细胞结合的能力明显低于PR000260。以上结果说明，本发明还通过对CD3抗体的序列优化得到了几个稳定的scFv形式的单链抗体，它们能够与人的T细胞结合，以适用于构建双特异性抗体等应用场景。
图9显示了部分从实施例4.2得到的CD3抗体与食蟹猴pan-T细胞的结合能力。可以看出，不同分子对食蟹猴pan-T细胞结合能力不同，而且与其对人pan-T细胞的结合能力呈正相关性；即，对人pan-T细胞结合较强的分子对食蟹猴pan-T细胞结合能力也较强，反之亦然。
实施例6测定CD3抗体对人T细胞的激活作用
将梯度稀释的CD3抗体(如50、10、5、1、0.5、0.05μg/ml)以每个浓度三个复孔且每孔50μl包被于96孔细胞培养板中，于4℃过夜。将人PBMC(妙通生物)或者人pan-T细胞(用人pan-T细胞分离试剂盒(Miltenyi,#130-096-535)从PBMC中分离出)的细胞密度调整至7.5×10 5/ml，加入人CD28抗体浓度至1μg/ml，再以每孔200μl加入到细胞培养板中，置于CO 2培养箱培养。培养72小时后，取上清，使用IFN-γELISA试剂盒(Thermo，#88-7316-77)测定上清中的IFN-γ含量。用GraphPad Prism软件进行数据分析和制图。
图10的(A)～(G)分别显示了实施例4.2得到的各CD3抗体(包括SP34嵌合抗体)激活人T细胞的能力。当抗体浓度为1μg/mL时，PR000511、PR000512、PR000513、PR000514激活T细胞后产生IFN-γ水平明显低于PR000260；当抗体浓度为10μg/mL 时，PR000512、PR000513、PR000514激活的IFN-γ水平略低于PR000260(图10(A))。当抗体浓度为0.5μg/mL和5μg/mL时，PR001848激活的IFN-γ水平明显低于PR000260(图10(B))。此外，还检测了0.5μg/mL、5μg/mL和50μg/mL的抗体PR002468、PR002469、PR002471、PR002742、PR002833、PR002834、PR002835、PR002836、PR002837、PR001848、PR003886和PR004616等激活T细胞的效果(图10的(C)～(G))，其结果说明这些抗体激活T细胞产生的IFN-γ水平远低于PR000260，其中PR002468和PR002471未检测到IFN-γ的释放，而PR002469和PR002835仅在50μg/mL时能够检测到微弱的IFN-γ水平；PR002742和PR003886相当，略弱于PR001848；PR002469和PR004616相当，明显弱于PR001848。以上结果说明，本发明通过对CD3抗体的序列优化得到了多个新的抗体，它们对人T细胞的激活能力不同，可以控制细胞因子不同的释放水平，以适用于不同的应用场景。
图11显示实施例4.3得到的抗CD3的scFv-Fc抗体激活人T细胞的能力。1μg/mL和10μg/mL浓度的PR000510、PR000623、PR000624和PR000627均表现出低于PR000260但高于同型对照抗体的IFN-γ水平，说明这4个分子通过调节T细胞的激活水平来限制细胞因子的释放。以上结果说明，本发明还通过对CD3抗体的序列优化得到了几个稳定的scFv形式的单链抗体，它们对人的T细胞的激活能力较弱，有较低的细胞因子释放水平，以适用于构建双特异性抗体等应用场景。
实施例7含有抗CD3 scFv抗体的靶向B7H4的双特异性抗体
B7H4是B7家族跨膜蛋白的成员，它在乳腺癌、卵巢癌、子宫内膜癌等多种实体肿瘤组织内有高表达，而在正常组织不表达或非常微弱的表达，因此B7H4是特异性非常好的肿瘤相关靶标抗原。构建同时靶向B7H4和CD3的双特异性抗体分子，可以通过靶向和结合到肿瘤细胞表面的B7H4来选择性地激活肿瘤细胞附近的T细胞，从而对肿瘤细胞进行特异性的杀伤。
7.1制备B7H4抗体
B7H4抗体的可变区序列来源于WO2016040724，根据实施例1.1的方法构建针对B7H4的重组IgG抗体PR000014。下表10列出了B7H4抗体PR000014的序列信息。
表10 B7H4抗体PR000014的轻、重链序列信息
抗体编号 靶点 重链SEQ ID NO: 轻链SEQ ID NO:
PR000014 B7H4 82 83
7.2制备含有抗CD3 scFv抗体的靶向B7H4的双特异性抗体
用实施例7.1得到的B7H4抗体PR000014的序列和实施例4.3得到的CD3单链抗体PR000627的序列来构建靶向B7H4×CD3的双特异性抗体分子PR002883，其含有三条多肽链，分别是：含有CD3单链抗体scFv的重链(SEQ ID NO:88)，含有B7H4抗体VH的重链(SEQ ID NO:86)，含有B7H4抗体VL的轻链(SEQ ID NO:83)。其结构如图16(C)所示。由于该分子有特殊的非对称结构，为了减少同源重链二聚体的产生，在两条重链的恒定区引入了不同的氨基酸突变。同时，为了防止由Fcγ受体结合引起的交联和降低效应子功能，在重链恒定区引入“LALAPG”三突变体(L234A/L235A/P329G)。
用实施例1.1所述方法并结合质粒配比(如1:1:1或其他比例)并经过一步亲和纯化制备双特异性抗体PR002883的重组蛋白。表11列出了双特异性抗体PR002883的序列表；表12列出该双特异性抗体的表达情况。
表11双特异性抗体的链以及对应的序列信息
Figure PCTCN2020118606-appb-000010
表12双特异性抗体的表达情况
双特异性抗体 HEK293中的产量(mg/L) SDS-PAGE纯度(％)
PR002883 94.0 70
图12(A)显示出了双特异性抗体PR002883经过一步纯化后经SDS-PAGE分析的结果。显示出其主要的副产物是未完全组装的分子，高聚体组分较少，可以通过优化纯化步骤或者优化质粒转染比例来减少副产物。
7.3结合表达B7H4的肿瘤细胞
本实施例研究双特异性抗体结合表达人B7H4的肿瘤细胞SK-BR-3(ATCC,HTB-30)的能力。具体说来，收集SK-BR-3细胞悬液，将细胞密度调整为1×10  6/ml，以100μl/孔接种于96孔V底板(Corning,#3894)；随后以100μl/孔体积加入2倍于终浓度的3倍浓度梯度稀释的待测抗体。放置于4℃避光孵育2小时。之后，以100μl/孔加入预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再以100μl/孔加入荧光二抗Alexa Fluor 488 AffiniPure Goat Anti-Human IgG,Fcγfragment specific(Jackson ImmunoResearch，#109-545-098)，于4℃避光孵育1小时。再以100μl/孔加入预冷PBS 洗涤细胞两次，于500g离心5分钟后弃上清。最后，以200μL/孔加入预冷PBS重悬细胞。使用流式细胞仪(BD FACS CANTOII或ACEA NovoCyte)读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
图13(A)显示实施例7.1得到的单抗和实施例7.2得到的双特异性抗体与SK-BR-3细胞的结合能力。可以看出双特异性抗体PR002883与单抗PR000014相比有相当甚至更好的结合能力。
7.4结合人T细胞
用实施例5所述方法检测双特异性抗体PR002883与人pan-T细胞结合的能力。如图13(B)所示，PR002883可以与人pan-T细胞结合。
7.5双特异性抗体对B7H4高表达细胞系SK-BR-3的体外杀伤以及细胞因子的释放
为了研究B7H4×CD3双特异性抗体在体外介导的靶细胞杀伤能力，采用人PBMC作为效应细胞，高表达B7H4的细胞系SK-BR-3(ATCC,HTB-30)作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的，在E-plate(ACEA Biosciences Inc.,#05232368001)里每孔加入50μL RPMI1640/10％FBS培养基，放置在37℃，5％CO 2培养箱内平衡30分钟，然后将E-plate放入仪器xCELLigence RTCA(ACEA Biosciences)上检测是否正常。用RPMI1640/10％FBS培养基将SK-BR-3的密度调整为0.4×10 6细胞/mL,50μL细胞/孔接种于E-plate内，然后将E-plate放在xCELLigence RTCA上过夜检测细胞指数(cell index)。用RPMI1640/10％FBS培养基将PBMC的密度调整为4×10 6细胞/mL，50μL细胞/孔接种于E-plate内，随后加入50μL/孔，4倍于终浓度的5倍浓度梯度稀释的待测抗体，其中抗体最高终浓度为0.2nM，每个抗体共7个浓度，最终效应细胞与靶细胞的比例为10:1，设置两个重复。同时，在板内设置空白对照：SKBR3+PBMC+RPMI1640/10％FBS培养基；E-plate置于37℃5％CO 2培养箱孵育24小时。孵育完成后，将E-plate放入xCELLigence RTCA仪器上检测细胞指数。
将检测的细胞指数按照如下公式计算抗体对细胞的特异性杀伤：
细胞杀伤％＝(1-检测样品/空白对照)*100％。
收集细胞培养上清，用于检测细胞因子IFN-γ的释放。ELISA检测方法参照IFN-γ试剂盒(IFN gamma Human Uncoated ELISA Kit,Thermo,#88-7316-77)的操作说明。
如图14的(A)和(B)所示，双特异性抗体PR002883可以激活T细胞释放出细胞因子如IFN-γ，并对肿瘤细胞SK-BR-3进行有效地杀伤。当双特异性抗体的浓度在0.01 μg/ml时，几乎100％的肿瘤细胞被杀伤(图14(A))。
实施例8含有抗CD3 scFv抗体的靶向ROR1的双特异性抗体
ROR1是一种无活性的酪氨酸蛋白激酶跨膜蛋白，其在许多肿瘤中过表达，但在正常组织中几乎不表达。ROR1作为受体与Wnt5a相互作用后，转导Wnt信号通路，从而有助于慢性淋巴细胞白血病中的细胞增殖和迁移，并且有助于实体瘤中的上皮-间充质-转变(EMT)。ROR1的肿瘤特异性表达使其成为用于开发治疗药物的合适的肿瘤相关抗原靶标。构建同时靶向ROR1和CD3的双特异性抗体分子，可以通过靶向和结合到肿瘤细胞表面的ROR1来选择性地激活肿瘤细胞附近的T细胞，从而对肿瘤细胞进行特异性的杀伤。
8.1制备ROR1抗体
ROR1抗体的可变区序列来源于WO2016094873，根据实施例1.1的方法构建针对ROR1的重组IgG抗体PR000374。表13列出了ROR1抗体PR000374的序列表。
表13 ROR1抗体PR000374的序列表。
抗体编号 靶点 重链SEQ ID NO: 轻链SEQ ID NO:
PR000374 ROR1 84 85
8.2制备含有抗CD3 scFv抗体的靶向ROR1的双特异性抗体
用实施例8.1得到的ROR1抗体PR000374的序列和实施例4.3得到的CD3单链抗体PR000627的序列来构建靶向ROR1×CD3的双特异性抗体分子PR002885，其含有三条多肽链，分别是：含有CD3单链抗体scFv的重链(SEQ ID NO:88)，含有ROR1抗体VH的重链(SEQ ID NO:87)，含有ROR1抗体VL的轻链(SEQ ID NO:85)。其结构如图16(C)所示。由于该分子有特殊的非对称结构，为了减少同源重链二聚体的产生，在两条重链的恒定区引入了不同的氨基酸突变。同时，为了防止由Fcγ受体结合引起的交联和降低效应子功能，在重链恒定区引入“LALAPG”三突变体(L234A/L235A/P329G)。
用实施例1.1所述方法并结合质粒配比(如1:1:1或其他比例)并经过一步亲和纯化制备双特异性抗体PR002885的重组蛋白。表14列出了双特异性抗体PR002885的序列信息；表15列出该双特异性抗体的表达情况。
表14双特异性抗体的链以及对应的序列编号
Figure PCTCN2020118606-appb-000011
Figure PCTCN2020118606-appb-000012
表15双特异性抗体的表达情况
双特异性抗体 HEK293中的产量(mg/L) SDS-PAGE纯度(％)
PR002885 90.0 70
图12(B)显示出了双特异性抗体PR002885经过一步纯化后经SDS-PAGE分析的结果。显示出其主要的副产物是未完全组装的分子，高聚体组分较少，可以通过优化纯化步骤或者优化质粒转染比例来减少副产物。
8.3结合表达ROR1的肿瘤细胞
本实施例研究双特异性抗体结合表达人ROR1的肿瘤细胞Panc-1(ATCC,CRL-1469)的能力。具体说来，收集Panc-1细胞悬液，将细胞密度分别调整为1×10  6/ml，以100μl/孔接种于96孔V底板(Corning,#3894)；随后以100μl/孔体积加入2倍于终浓度的3倍浓度梯度稀释的待测抗体。放置于4℃避光孵育2小时。之后，以100μl/孔加入预冷PBS漂洗细胞两次，于500g下离心5分钟，弃上清。再以100μl/孔加入荧光二抗Alexa Fluor 488 AffiniPure Goat Anti-Human IgG,Fcγfragment specific(Jackson ImmunoResearch，#109-545-098)，于4℃避光孵育1小时。再以100μl/孔加入预冷PBS洗涤细胞两次，于500g下离心5分钟，弃上清。最后，以200μL/孔加入预冷PBS重悬细胞。使用流式细胞仪(BD FACS CANTOII或ACEA NovoCyte)读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
图15(A)显示实施例8.1得到的单抗和实施例8.2得到的双特异性抗体与Panc-1细胞的结合能力。可以看出双特异性抗体PR002885与单抗PR000374都可以结合Panc-1。
8.4结合人T细胞
用实施例5所述方法检测双特异性抗体PR002885与人pan-T细胞结合的能力。如图15(B)所示，PR002885可以与人pan-T细胞结合。

Claims (15)

  1. 一种靶向CD3的抗体，其特征在于，所述的靶向CD3的抗体包含轻链可变区(VL)和重链可变区(VH)，所述VL为如SEQ ID NO:56所示的氨基酸序列或其突变；所述VH在如SEQ ID NO:42所示的氨基酸序列上发生突变，所述突变选自一个或多个以下位点：第30位、第73位、第76位、第78位、第93位和第94位的氨基酸残基；所述位点使用Chothia编码规则的位置编号。
  2. 如权利要求1所述的靶向CD3的抗体，其特征在于，在所述VH上发生的突变选自以下组合：
    (a)第30位；
    (b)第30位、第73位和第76位；
    (c)第30位、第93位和第94位；
    (d)第30位、第73位和第93位；
    (e)第30位、第93位；
    (f)第30位、第76位和第78位；
    (g)第73位、第76位、第93位和第94位；
    (h)第76位、第78位和第93位；
    (i)第30位、第73位、第76位、第93位和第94位；
    (j)第30位、第76位、第78位和第93位。
  3. 如权利要求1所述的靶向CD3的抗体，其特征在于，在所述VH上发生的突变选自以下组合：
    (a)N30S；
    (b)N30S、D73N和S76N；
    (c)N30S、V93A和R94K；
    (d)N30S、D73N和V93A；
    (e)N30S和V93T；
    (f)N30S、S76N和L78A；
    (g)D73N、S76N、V93A和R94K；
    (h)S76N、L78A和V93T；
    (i)N30S、D73N、S76N、V93A和R94K；
    (j)N30S、S76N、L78A和V93T。
  4. 如权利要求1-3任一项所述的靶向CD3的抗体，其特征在于，所述的VH的氨基酸序列如SEQ ID NO:43-55中任一序列所示，和/或，所述VL的氨基酸序列如SEQ ID NO:57-60中任一序列所示。
  5. 如权利要求4所述的靶向CD3的抗体，其特征在于，
    所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:51所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:60所示；或，
    所述VH的氨基酸序列如SEQ ID NO:51所示，所述VL的氨基酸序列如SEQ ID NO:60所示；或，
    所述VH的氨基酸序列如SEQ ID NO:45所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:52所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:60所示；或，
    所述VH的氨基酸序列如SEQ ID NO:50所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:47所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:48所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:49所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:53所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:54所示，所述VL的氨基酸序列如SEQ ID NO: 58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:57所示；或，
    所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:57所示；或，
    所述VH的氨基酸序列如SEQ ID NO:43所示，所述VL的氨基酸序列如SEQ ID NO:59所示；或，
    所述VH的氨基酸序列如SEQ ID NO:44所示，所述VL的氨基酸序列如SEQ ID NO:59所示；或，
    所述VH的氨基酸序列如SEQ ID NO:51所示，所述VL的氨基酸序列如SEQ ID NO:57所示；或，
    所述VH的氨基酸序列如SEQ ID NO:55所示，所述VL的氨基酸序列如SEQ ID NO:58所示；或，
    所述VH的氨基酸序列如SEQ ID NO:46所示，所述VL的氨基酸序列如SEQ ID NO:58所示。
  6. 如权利要求1-5中任一项所述的抗体，其特征在于，所述抗体包含VL-Linker-VH或VH-Linker-VL的单链抗体(scFv)；
    较佳地，所述Linker为(G 4S) n或其变体，其中n为非0自然数，优选1～20，更优选为如SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67所示的氨基酸序列；
    更佳地，所述scFv的氨基酸序列如SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:78、SEQ ID NO:79或SEQ ID NO:80所示；
    进一步更佳地，所述抗体还包括Fc，所述Fc通过铰链区(Hinge)和scFv相连。
  7. 如权利要求1-6中任一项所述的抗体，其特征在于，所述抗体还包括恒定区，优选人源恒定区；
    较佳地，所述人源恒定区包括人源轻链恒定区和人源重链恒定区，所述人源轻链恒定区优选如SEQ ID NO:61所示的人κ轻链恒定区或如SEQ ID NO:62所示的人λ轻链恒定区；
    更佳地，所述人源重链恒定区为hIgG1、hIgG2、hIgG3、hIgG4或其突变，优选如SEQ ID NO:63或SEQ ID NO:64所示的重链恒定区。
  8. 一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中，所述第一蛋白功能区包含如权利要求1～7中任一项所述的靶向CD3的抗体。
  9. 如权利要求8所述的双特异性抗体，其特征在于，所述双特异性抗体包括以下三条链：(1)第一蛋白功能区的VL1-Linker-VH1-Hinge-CH2-CH3(knob)或VH1-Linker-VL1-Hinge-CH2-CH3(knob)，(2)第二蛋白功能区的VH2-CH1-Hinge-CH2-CH3(hole)和(3)第二蛋白功能区的VL2-CL；所述第二蛋白功能区为靶向非CD3靶点的抗体，优选靶向B7H4的抗体或靶向ROR1的抗体，所述linker优选为(G 4S) n，其中n为非0自然数，优选1～20，更优选为如SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67所示的氨基酸序列。
  10. 如权利要求9所述的双特异性抗体，其特征在于，所述双特异性抗体包括如SEQ ID NO:88所示的VL1-Linker-VH1-Hinge-CH2-CH3(knob)、如SEQ ID NO:86所示的VH2-CH1-Hinge-CH2-CH3(hole)和如SEQ ID NO:83所示的VL2-CL，或，如SEQ ID NO:88所示的VL1-Linker-VH1-Hinge-CH2-CH3(knob)、如SEQ ID NO:87所示的VH2-CH1-Hinge-CH2-CH3(hole)和如SEQ ID NO:85所示的VL2-CL。
  11. 一种分离的核酸，其编码如权利要求1-7中任一项所述的靶向CD3的抗体或如权利要求8-10中任一项所述的双特异性抗体。
  12. 一种表达载体，其包含如权利要求11所述的分离的核酸；较佳地，所述表达载体选自逆转录病毒载体、慢病毒载体、腺病毒载体和腺相关病毒载体。
  13. 一种基因修饰的细胞，其特征在于，其转染有如权利要求12所述的表达载体；较佳地，所述基因修饰的细胞为真核细胞。
  14. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-7中任一项所述的靶向CD3的抗体、权利要求8-10中任一项所述的双特异性抗体、权利要求13所述的基因修饰的细胞以及药学上可接受的载体；较佳地，所述药物组合物还包括免疫检查点抗体。
  15. 一种如权利要求1-7中任一项所述的靶向CD3的抗体、权利要求8-10中任一项所述的双特异性抗体、权利要求11所述的分离的核酸、权利要求12所述的表达载体、权利要求13所述的基因修饰的细胞或权利要求14所述的药物组合物在制备治疗肿瘤的药物中的应用。
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