WO2021031784A1 - 靶向cd3和cd20的双特异性抗体及其应用 - Google Patents

靶向cd3和cd20的双特异性抗体及其应用 Download PDF

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WO2021031784A1
WO2021031784A1 PCT/CN2020/103731 CN2020103731W WO2021031784A1 WO 2021031784 A1 WO2021031784 A1 WO 2021031784A1 CN 2020103731 W CN2020103731 W CN 2020103731W WO 2021031784 A1 WO2021031784 A1 WO 2021031784A1
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chain
fab
antibody
bispecific antibody
connecting peptide
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WO2021031784A9 (zh
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杨洋
尹伟
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杨洋
尹伟
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Publication of WO2021031784A1 publication Critical patent/WO2021031784A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to anti-CD3 and anti-CD20 single-chain Fab molecules, as well as bispecific antibodies targeting CD3 and CD20 and their applications.
  • B-cell lymphoma is a common blood system disease, divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma. Its etiology is unknown. Immunodeficiency and environmental factors are considered as possible pathogenic factors. The treatment and prognosis of B-cell lymphoma depend on the specific type and stage of the lymphoma. According to different clinical progress, B-cell lymphoma is divided into indolent lymphoma and aggressive lymphoma. Indolent lymphoma usually develops slowly and can maintain stable disease and long-term survival for many years, but it cannot be cured; while aggressive lymphoma usually requires immediate emergency treatment. Due to the lack of effective therapeutic drugs, the current therapeutic effect of B lymphocyte tumors is not ideal.
  • CD3 is a differentiation antigen that is connected to T cell receptor (TCR) and expressed only on the surface of T cells, and is a molecule necessary for T cell activation.
  • CD3 includes four chains of ⁇ , ⁇ , ⁇ and ⁇ , and functional CD3 is a dimer formed by two of these chains.
  • Anti-CD3 antibodies can bind to CD3 on the surface of T cells to produce an effect similar to TCR-CD3 molecules, thereby activating T lymphocytes.
  • CD20 is a B-cell marker, expressed in mature and activated B lymphocytes. It is widely expressed in B-cell non-Hodgkin’s lymphoma and other B-cell malignancies, but it is expressed in precursor B lymphocytes, plasma cells and lymphocytes. It is not expressed in cells such as competent stem cells. In addition, the CD20 antigen is obvious, easy to identify, and there is no free CD20 in the serum of the body. Therefore, CD20 is a good therapeutic target for B lymphoma.
  • CD3 and CD20 are the specific targets of T lymphocytes and B lymphocytes and have good drug-making potential
  • many research teams have already or are constructing bispecific antibodies targeting CD3 and CD20, and some have been applied for Patent (such as CN104640881A and CN104558191A), but due to the limitations of molecular structure and development technology, bispecific antibodies usually have problems such as low expression, mismatching of heavy chains, decreased binding force of antibody and antigen, and complicated purification process.
  • the present invention provides an anti-CD3 single-chain Fab molecule comprising a Fab light chain and a Fab heavy chain, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain through a connecting peptide, wherein The connecting peptide has a length of 45 to 80 amino acids.
  • the connecting peptide is 60 amino acids in length.
  • the connecting peptide includes a tandemly repeated GGGGS sequence.
  • the anti-CD3 single-chain Fab molecule includes an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO: 6 or 9.
  • the present invention provides an anti-CD20 single-chain Fab molecule comprising a Fab light chain and a Fab heavy chain, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain through a connecting peptide, wherein The connecting peptide has a length of 45 to 80 amino acids.
  • the connecting peptide is 60 amino acids in length.
  • the connecting peptide includes a tandemly repeated GGGGS sequence.
  • the anti-CD20 single-chain Fab molecule includes an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO: 10.
  • the present invention provides the use of the anti-CD3 single-chain Fab molecule or the anti-CD20 single-chain Fab molecule for preparing bispecific antibodies.
  • the present invention provides a bispecific antibody comprising a first part that binds CD3 and a second part that binds CD20, wherein the first part is an anti-CD3 single-chain Fab molecule, the anti-CD3 single-chain Fab
  • the molecule includes a Fab light chain and a Fab heavy chain.
  • the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain through a connecting peptide with a length of 45 to 80 amino acids; the second part includes the anti-CD20 antibody Light chain and heavy chain.
  • the connecting peptide is 60 amino acids in length.
  • the connecting peptide includes a tandemly repeated GGGGS sequence.
  • the first part that binds to CD3 includes an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO: 6 or 9.
  • the light chain of the anti-CD20 antibody includes an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO: 7 and the heavy chain of the anti-CD20 antibody includes the amino acid sequence shown in SEQ ID NO: 8. Shown are amino acid sequences with at least 90% identity.
  • the C-terminus of the light chain of the anti-CD20 antibody is connected to the N-terminus of the heavy chain of the anti-CD20 antibody through the connecting peptide.
  • the second part that binds to CD20 includes an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID NO: 10.
  • the present invention provides the use of the bispecific antibody in the preparation of drugs for treating B cell-related diseases.
  • the B-cell related disease is B-cell leukemia or B-cell lymphoma.
  • the present invention provides a pharmaceutical composition, which comprises the bispecific antibody of the claims and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating a B-cell-related disease in a patient, which comprises administering to the patient a therapeutically effective amount of the bispecific antibody or the pharmaceutical composition.
  • Fig. 1 is a schematic diagram of the structure of a candidate molecule prepared in Example 1 for evaluating the influence of a connecting peptide on the properties of a single-chain Fab molecule.
  • Figure 2 shows the results of SDS-PAGE electrophoresis analysis after the above candidate molecules with different lengths of connecting peptides are expressed and purified in cells.
  • Figure 3 shows the detection results of the above-mentioned candidate molecules with different length connecting peptides for CD3 binding ability.
  • Figure 4 is a schematic diagram of the structure of the bispecific antibodies YY0421 and YY0422 of the present invention.
  • ⁇ -CD3 refers to the CD3 binding portion
  • ⁇ -CD20 refers to the CD20 binding portion.
  • Figure 5 shows the Western blot detection results of the binding of the bispecific antibody of the present invention and the bispecific antibody REGN1979 (Con in the figure) as a control to CD3 ( Figure 5a) and CD20 ( Figure 5b).
  • Figure 6 shows the detection results of the binding ability of the bispecific antibody of the present invention and the bispecific antibody REGN1979 as a control to Raji cells.
  • Figure 7 shows the detection results of the binding ability of the bispecific antibody of the present invention and the bispecific antibody REGN1979 as a control to Jurkat cells.
  • Figure 8 shows the results of killing human Daudi cells (Figure 8) by the bispecific antibody of the present invention and the bispecific antibody REGN1979 as a control.
  • the ordinate represents the relative change value of proliferation.
  • Figure 9 shows the results of the bispecific antibody of the present invention and the bispecific antibody REGN1979 as a control inhibiting the growth of subcutaneous tumors in nude mice induced by human Daudi cells.
  • the abscissa represents the number of days, and the ordinate represents the tumor volume (cubic millimeters).
  • Antibody refers to immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is correspondingly called an antigen.
  • the basic structure of a classic antibody molecule is a 4-mer composed of two identical heavy chains and two identical light chains. According to the conservative difference in amino acid sequence, the heavy chain is divided into the variable region (VH) at the amino terminal and the constant region (CH) at the carboxy terminal. The light chain is similarly divided into a variable region (VL) at the amino terminus and a constant region (CL) at the carboxy terminus. The variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv).
  • the constant region of the light chain includes only one Ig domain, while the constant region of the heavy chain includes a different number of Ig domains depending on the type of antibody (Isotype).
  • IgG, IgA and IgD include 3 Ig domains: CH1, CH2 and CH3, IgM and IgE include 4 Ig domains: CH1, CH2, CH3 and CH4.
  • Fab refers to the fragment of antigen binding, including the VH and CH1 domains of a complete light chain and heavy chain of an antibody.
  • the C-terminus of the light chain in the Fab is connected to the N-terminus of the heavy chain through a short connecting peptide to form a "single chain Fab (scFab) molecule".
  • scFab single chain Fab
  • the light chain part in the single-chain Fab molecule is referred to as "Fab light chain” and the heavy chain part is referred to as "Fab heavy chain”.
  • the Fab heavy chain may also include an Fc segment linked at the C-terminus.
  • connecting peptide can have a length of 40 to 80 (for example, 50, 52, 55, 58, 60, 62, 65, 70, 75, or 78) amino acids.
  • the use of connecting peptides helps stabilize the molecular structure and prevent light chain mismatches in bispecific antibodies.
  • the "Fc segment” is a fragment crystallizable region, which corresponds to the CH2 and CH3 domains of an IgG antibody.
  • IgG can bind to cells with corresponding receptors on the surface through its Fc segment, and produce different biological effects, such as opsonization and antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the antibody Fc segment can be obtained by hydrolyzing the antibody molecule by a proteolytic enzyme (such as papain).
  • the "Fc segment” may also include the hinge region of the antibody heavy chain.
  • the "Fc segment” is derived from IgG, IgA, IgD, or IgM antibodies.
  • a single chain Fab molecule that binds CD3 binds as a CD3 binding portion to another antigen binding portion to form a bispecific antibody that targets CD3 and the other antigen.
  • the single chain Fab molecule binds to the CD20 binding portion as a CD3 binding portion to form a bispecific antibody targeting CD3 and CD20.
  • the single chain Fab molecule acts as a CD3 binding moiety and binds to multiple additional antigen binding moieties to form a multispecific antibody.
  • the CD20-binding single-chain Fab molecule binds to another antigen-binding moiety as a CD20-binding moiety to form a bispecific antibody that targets CD20 and the other antigen.
  • the single chain Fab molecule binds to the CD3 binding portion as a CD20 binding portion to form a bispecific antibody targeting CD3 and CD20.
  • the single chain Fab molecule acts as a CD20 binding moiety and binds to multiple additional antigen binding moieties to form a multispecific antibody.
  • the binding of the CD20 binding portion and the CD3 binding portion of the bispecific antibody of the present invention can be covalent binding (for example, through the formation of disulfide bonds between Fc segments) or non-covalent binding.
  • the light chain and heavy chain variable domains of the CD3 binding portion of the bispecific antibody are derived from OKT3 monoclonal antibody or L2K monoclonal antibody (WO2004106380). In some embodiments, the light chain and heavy chain variable domains of the CD20 binding portion of the bispecific antibody are derived from Rituximab or Ofatumumab.
  • an antibody “binding" to an antigen when referring to an antibody "binding" to an antigen, it means that the antibody can recognize and detectably bind to the antigen.
  • the binding of the antibody to the antigen can be measured by a known antigen-antibody binding assay method, such as ELISA.
  • the dissociation equilibrium constant KD of antibody antigen binding can be used to express the binding affinity of the antibody to the corresponding antigen.
  • the "Knobs-into-holes" of the Fc segment is used to prevent heavy chain mismatches in bispecific antibodies.
  • This technology was developed by Genentech (see US Patent 5,731,168). The specific method is to mutate the smaller threonine (T) at position 366 of the CH3 region of one of the antibodies to the larger tyrosine (Y) to form a prominent "Knobs" structure (T366Y , (Kabat numbering system)); At the same time, the larger tyrosine (Y) residue at position 407 in the CH3 region of the other antibody heavy chain was mutated to a smaller threonine (T) to form concave "holes" ⁇ )” structure (Y407T).
  • sequence identity refers to the degree of similarity between amino acid sequences or nucleotide sequences, generally expressed as a percentage of identity, which can be determined by visual inspection or by computer programs (such as BLAST).
  • sequence identity refers to the degree of similarity between amino acid sequences or nucleotide sequences, generally expressed as a percentage of identity, which can be determined by visual inspection or by computer programs (such as BLAST).
  • the CD3 binding single-chain Fab molecule includes the amino acid sequence shown in SEQ ID NO: 6, or includes at least 80% (for example, at least 90%, Amino acid sequences with at least 95%, at least 98%, or at least 99% or 100%) sequence identity.
  • the CD3 binding portion of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO: 6, or at least 80% (e.g., at least 90% of the sequence shown in SEQ ID NO: 6). , At least 95%, at least 98%, at least 99% or 100%) sequence identity.
  • the light chain of the CD20 binding portion of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO: 7, or at least 80% of the sequence shown in SEQ ID NO: 7 (for example, At least 90%, at least 95%, at least 98%, at least 99% or 100%) sequence identity;
  • the heavy chain of the CD20 binding portion of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO: 8, or It includes an amino acid sequence that has at least 80% (for example, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) sequence identity with the sequence shown in SEQ ID NO: 8.
  • the CD3 binding portion of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO: 9, or includes at least 80% (for example, at least 90% of the sequence shown in SEQ ID NO: 9). , At least 95%, at least 98%, at least 99% or 100%) sequence identity.
  • the CD20 binding portion of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO: 10, or includes at least 80% (for example, at least 90% of the sequence shown in SEQ ID NO: 10). , At least 95%, at least 98%, at least 99% or 100%) sequence identity.
  • the present invention can be obtained by replacing, deleting, adding and verifying or screening the binding ability or biological activity of the resulting product with the corresponding antigen on the basis of a few amino acids.
  • Corresponding variants of single-chain Fab molecules or bispecific antibodies that bind CD3 or CD20 are provided, and these variants should also be included in the scope of the present invention.
  • the "pharmaceutically acceptable carrier” used refers to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered. These substances are suitable for humans and/or animals. It can be administered without excessive side effects and is suitable for maintaining the vitality of the drugs or active agents located therein.
  • “Therapeutically effective amount” refers to the amount of the active compound sufficient to cause the biological or medical response desired by the clinician in the subject.
  • the “therapeutically effective amount” of the bispecific antibody of the present invention can be determined by those skilled in the art according to factors such as the route of administration, the subject's weight, age, and disease conditions. For example, a typical daily dosage may range from 0.01 mg to 100 mg of active ingredient per kg body weight.
  • the present inventors designed a single-chain Fab molecule targeting CD3, that is, the light chain and its corresponding heavy chain variable region, and the CH1 segment and the "knob" Fc segment are connected through a flexible connecting peptide.
  • the light chain and heavy chain variable region sequences are derived from L2K antibodies.
  • the formed single-chain Fab molecule has an amino acid sequence as shown in SEQ ID NO: 6.
  • the coding nucleic acid sequence of the single-stranded Fab molecule and the coding nucleic acid sequence of the "hole-shaped" Fc segment were synthesized by overlapping PCR technology, and cloned into the eukaryotic expression vector pcDNA (purchased from Life Technologies).
  • the expression vector including the coding nucleic acid sequence of the single-stranded Fab molecule and the expression vector including the "hole-shaped" Fc segment coding sequence were mixed at a ratio of 1.5:1 to a total mixture of 200 ⁇ g, and the transfection reagent PEI
  • the mass-to-volume ratio 1:2.5 was mixed to prepare a DNA-transfection reagent complex and added dropwise to 200 mL of 293F cells in logarithmic growth phase. Collect the cell supernatant 5-7 days after transfection.
  • Lanes 1 to 5 correspond to candidate molecules including flexible linking peptides of 26, 32, 45, 60, and 80 amino acids in length, respectively. It can be seen from the figure that the candidate molecules (the bands marked by the black boxes in the figure and the Fab single-chain molecules containing the Fc segment) constructed using the 60-amino acid-long flexible linking peptide have a slightly higher expression level, but have fewer impurity bands.
  • the protein (candidate molecule) purified in section 1.1 above was heat-treated at 37°C for 24 hours and 50°C for 2 hours, respectively, and subjected to SEC-HPLC detection with the antibody placed at 4°C for the same time.
  • the SEC-HPLC detection operation is briefly described as follows: select TSK-GELG3000SWXL gel chromatography column (TOSOH company), use mobile phase (50mM phosphate buffer, 150mM sodium chloride, pH 7.0) to equilibrate the column, and then use UV detection. Take 50 ⁇ L of the proteins treated at different temperatures and pass them through the chromatographic column at a flow rate of 1 mL/min. After each sample runs for 20 minutes, the result graph is fitted and the purity of the target protein is calculated.
  • the binding ability of the antibody (ie candidate molecule) purified in section 1.1 above to CD3 antigen was tested by ELISA.
  • the brief steps are as follows: Coat 100 ⁇ L of CD3 antigen (Sino Biologics, SEK10981) at a concentration of 0.5 ⁇ g/mL on an ELISA plate and place it at 4°C overnight. After washing with PBS buffer containing 2% BSA, blocking with PBS containing 2% BSA for 1 hour at room temperature and washing 3 times.
  • CD3 binding part (anti-CD3 single-chain Fab molecule, amino acid sequence is SEQ ID NO: 6) coding DNA fragment and anti-CD20 antibody light chain and heavy chain (amino acid sequence are SEQ ID NO: 7 and SEQ ID NO: 8), using recombinant DNA such as overlapping PCR technology to clone them into the eukaryotic expression vector pcDNA (purchased from Life Technologies), and then the obtained expression vector containing the coding DNA sequence of the CD3 binding part, containing The expression vector containing the DNA sequence encoding the light chain of the anti-CD20 antibody and the expression vector containing the DNA sequence encoding the heavy chain of the anti-CD20 antibody are mixed in a mass ratio of 1.5:2:1.
  • a total of 200 ⁇ g of the above-mentioned expression vector mixture and the transfection reagent PEI were mixed in a mass-volume ratio of 1:2.5 to prepare a DNA-transfection reagent complex, and added dropwise to 200 mL of 293F cells in the logarithmic growth phase. Collect the cell supernatant 5-7 days after transfection.
  • the binding buffer (12.15g Tris plus appropriate amount of sterile water to dissolve, adjust the pH to 7.0, add 8.78g NaCl , Dilute to 1L) the washed affinity chromatography gel column (Protein A), then dissolve it with the eluent (7.5g glycine in an appropriate amount of double distilled water, adjust the pH to 3.5, add 8.78g NaCl, and dilute to 1L) Elution.
  • the eluted fraction was neutralized with 1M Tris-HCl pH 9.0 to obtain the bispecific antibody YY0421 of the present invention.
  • the bispecific antibody YY0422 of the present invention was prepared, in which the light chain and heavy chain of the CD20 binding part were connected by a connecting peptide (the amino acid sequence of the CD3 binding part is shown in SEQ ID NO: 9, and the CD20 binding part The amino acid sequence is shown in SEQ ID NO: 10).
  • the schematic structure of the bispecific antibodies YY0421 and YY0422 is shown in Figure 4.
  • the Western blot detection results are shown in Figure 5a.
  • the results showed that the bispecific antibodies YY0421 and YY0422, which simultaneously target human CD3 and CD20, can effectively recognize the CD3 antigen at the protein level.
  • the Western blot detection results are shown in Figure 5b.
  • the results showed that the bispecific antibodies YY0421 and YY0422, which target human CD3 and CD20, can effectively recognize the CD20 antigen at the protein level.
  • Example 4 Flow cytometry to detect the binding of the bispecific antibody of the present invention to CD3 and CD20 on the cell surface
  • Example 6 The bispecific antibody of the present invention mediates the killing of tumor cells by CD8 + T cells
  • the number of healthy cells stained with CFSE at a final concentration of 5 ⁇ M was 5 ⁇ 10 3 human Daudi cells in a 96-well plate. After starving overnight with 0.5% fetal bovine serum medium, CD8 + T cells were added at an E/T ratio of 5:1. The experiment was performed in two batches, each of which divided the cells into three groups (each group contained three wells). In the first batch, medium (Blank in Figure 8a), control antibody REGN1979 (Control in Figure 8a), and antibody YY0421 of the present invention were added separately. In the second batch, culture medium (Blank in Figure 8b), control antibody REGN1979 (Control in Figure 8b) and the antibody YY0422 of the present invention were added respectively.
  • the killing result is shown in Figure 8. It can be seen from the results that the bispecific antibodies YY0421 and YY0422 that simultaneously target human CD3 and CD20 of the present invention can effectively mediate the killing of Daudi cells by T cells.
  • Figure 9 shows the test results of transplanted tumors in nude mice.
  • the results show that the bispecific antibody simultaneously targeting human CD3 and CD20 of the present invention has good biological activity and can significantly inhibit the proliferation of subcutaneous tumors in nude mice induced by human Daudi cells.
  • amino acid sequences mentioned in this article are as follows:

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Abstract

公开了抗CD3单链Fab分子和抗CD20单链Fab分子以及它们在制备双特异性抗体中的应用。还提供了靶向CD3和CD20的双特异性抗体及其在制备用于治疗B细胞相关疾病的药物中的应用。

Description

靶向CD3和CD20的双特异性抗体及其应用 技术领域
本发明涉及抗CD3和抗CD20单链Fab分子，还涉及靶向CD3和CD20的双特异性抗体以及它们的应用。
背景技术
B细胞淋巴瘤是常见的血液***疾病，分为霍奇金淋巴瘤和非霍奇金淋巴瘤，其病因未明，免疫缺陷和环境因素被认为是可能的发病因素。B细胞淋巴瘤的治疗和预后取决于淋巴瘤的具体类型以及分期分级。根据临床进展的不同，B细胞淋巴瘤分为惰性淋巴瘤和侵袭性淋巴瘤。惰性淋巴瘤通常发展缓慢，可保持多年疾病稳定及长期生存，但无法治愈；而侵袭性淋巴瘤通常需要立即进行紧急治疗。由于缺乏有效治疗药物，目前B淋巴细胞瘤的治疗效果并不理想。
CD3是与T细胞受体(T cell receptor，TCR)相连接的仅表达于T细胞表面的分化抗原，是T细胞活化所必需的分子。CD3包括ε、ζ、δ和γ四条链，功能性CD3则是由其中的二条链形成的二聚体。抗CD3抗体可以与T细胞表面的CD3相结合，产生类似TCR-CD3分子的效应，从而激活T淋巴细胞。
CD20是B细胞标记物，表达于成熟和激活的B淋巴细胞，在B细胞非霍奇金淋巴瘤和其他B-细胞恶性肿瘤中广泛表达，但在前体B淋巴细胞、浆细胞和淋巴多能干细胞等细胞中不表达。此外，CD20抗原显露明显，易于识别且体内血清中无游离的CD20存在。因此，CD20是良好的B淋巴细胞瘤的治疗靶点。
由于CD3和CD20是T淋巴细胞和B淋巴细胞的特异性靶点，具有良好的成药潜能，因此，已有多个研究团队已经或正在构建靶向CD3和CD20的双特异性抗体，部分已申请专利(例如CN104640881A和CN104558191A)，但由于分子结构和开发技术的限制，双特异性抗体通常存在表达量较低、重链错配、抗体和抗原的结合力下降、纯化工艺繁琐等方面的问题。
发明内容
在一方面，本发明提供了一种抗CD3单链Fab分子，其包括Fab轻链和Fab重链，所述Fab轻链的C末端通过连接肽与所述Fab重链的N末端连接，其中所述连接肽的长度为45至80个氨基酸。
在一些实施方案中，所述连接肽的长度为60个氨基酸。
在一些实施方案中，所述连接肽包括串联重复的GGGGS序列。
在一些实施方案中，所述抗CD3单链Fab分子包括与SEQ ID NO：6或9所示序列有至少90％一致性的氨基酸序列。
另一方面，本发明提供了一种抗CD20单链Fab分子，其包括Fab轻链和Fab重链，所述Fab轻链的C末端通过连接肽与所述Fab重链的N末端连接，其中所述连接肽的长度为45至80个氨基酸。
在一些实施方案中，所述连接肽的长度为60个氨基酸。
在一些实施方案中，所述连接肽包括串联重复的GGGGS序列。
在一些实施方案中，所述抗CD20单链Fab分子包括与SEQ ID NO：10所示序列有至少90％一致性的氨基酸序列。
另一方面，本发明提供了所述抗CD3单链Fab分子或所述抗CD20单链Fab分子用于制备双特异性抗体的用途。
另一方面，本发明提供了一种双特异性抗体，其包括结合CD3的第一部分和结合CD20的第二部分，其中所述第一部分为抗CD3单链Fab分子，所述抗CD3单链Fab分子包括Fab轻链和Fab重链，所述Fab轻链的C末端通过长度为45至80个氨基酸的连接肽与所述Fab重链的N末端连接；所述第二部分包括抗CD20抗体的轻链和重链。
在一些实施方案中，所述连接肽的长度为60个氨基酸。
在一些实施方案中，所述连接肽包括串联重复的GGGGS序列。
在一些实施方案中，所述结合CD3的第一部分包括与SEQ ID NO：6或9所示序列有至少90％一致性的氨基酸序列。
在一些实施方案中，所述抗CD20抗体的轻链包括与SEQ ID NO：7所示序列有至少90％一致性的氨基酸序列，所述抗CD20抗体的重链包括与SEQ ID NO：8所示序列有至少90％一致性的氨基酸序列。
在一些实施方案中，所述抗CD20抗体的轻链的C末端通过所述连接肽与所述抗CD20抗体的重链的N末端连接。
在一些实施方案中，所述结合CD20的第二部分包括与SEQ ID NO：10所示序列有至少90％一致性的氨基酸序列。
另一方面，本发明提供了所述双特异性抗体在制备治疗B细胞相关疾病的药物中的用途。
在一些实施方案中，所述B细胞相关疾病为B细胞白血病或B细胞淋巴瘤。
另一方面，本发明提供了药物组合物，其包括权利要求所述双特异性抗体和药学上可接受的载体。
另一方面，本发明提供了在患者中治疗B细胞相关疾病的方法，其包括以治疗有效量的所述双特异性抗体或所述药物组合物向所述患者给药。
附图说明
图1为实施例1中所制备的用于评估连接肽对单链Fab分子性质影响的候选分子的结构示意图。
图2显示了具有不同长度连接肽的上述候选分子在细胞中表达并纯化后进行SDS-PAGE电泳分析的结果。
图3显示了具有不同长度连接肽的上述候选分子对CD3结合能力的检测结果。
图4为本发明双特异性抗体YY0421和YY0422的结构示意图。α-CD3指CD3结合部分，α-CD20指CD20结合部分。
图5显示了本发明双特异性抗体以及作为对照的双特异性抗体REGN1979(图中Con)与CD3(图5a)和CD20(图5b)结合的蛋白印迹检测结果。
图6显示了本发明双特异性抗体以及作为对照的双特异性抗体REGN1979与Raji细胞结合能力的检测结果。
图7显示了本发明双特异性抗体以及作为对照的双特异性抗体REGN1979与Jurkat细胞结合能力的检测结果。
图8显示了本发明双特异性抗体以及作为对照的双特异性抗体REGN1979对人Daudi细胞(图8)杀伤的结果。纵坐标表示增殖的相对变化值。
图9显示了本发明双特异性抗体以及作为对照的双特异性抗体REGN1979抑制人Daudi细胞诱导的裸鼠皮下瘤体生长的结果。横坐标表示天数，纵坐标表示肿瘤体积(立方毫米)。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
“抗体”指由浆细胞(效应B细胞)分泌、被机体免疫***用来中和外来物质(多肽、病毒、细菌等)的免疫球蛋白。该外来物质相应地称作抗原。经典抗体分子的基本结构是由2条相同重链和2条相同轻链组成的4聚体。根据氨基酸序列的保守性差异，将重链分为位于氨基端的可变区(VH)和位于羧基端的恒定区(CH)。轻链也类似地分为位于氨基端的可变区(VL)和位于羧基端的恒定区(CL)。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。轻链的恒定区仅包括一个Ig结构域，而重链的恒定区随抗体种类(Isotype)的不同而包括不同数量的Ig结构域，例如IgG、IgA和IgD包括 3个Ig结构域：CH1、CH2、和CH3，IgM和IgE则包括4个Ig结构域：CH1、CH2、CH3和CH4。
“Fab”指抗原结合片段(fragment of antigen binding)，包括抗体的一条完整轻链和重链的VH和CH1结构域。在本发明的一些实施方案中，通过短的连接肽将Fab中轻链的C末端与重链的N末端连接，形成“单链Fab(scFab)分子”。为了方便描述，在一些述及Fab的上下文中，将该单链Fab分子中的轻链部分，称为“Fab轻链”，重链部分称为“Fab重链”。在一些实施方案中，Fab重链也可以包括在C末端连接的Fc段。
关于“连接肽”，本文在实施例中有更详细的说明，其可以具有40到80个(例如50、52、55、58、60、62、65、70、75或78个)氨基酸长度。连接肽的使用有助于稳定分子结构和防止双特异性抗体中的轻链错配。
“Fc段”为可结晶段(fragment crystallizable region)，对应于IgG抗体的CH2和CH3结构域。IgG可通过其Fc段与表面具有相应受体的细胞结合，产生不同的生物学作用，例如调理作用(opsonization)和抗体依赖的细胞介导的细胞毒性作用(ADCC)。可通过蛋白水解酶(如木瓜蛋白酶)水解抗体分子而得到抗体Fc段。在一些实施方案中，“Fc段”还可以包括抗体重链的铰链区(hinge region)。在一些实施方案中，“Fc段”源于IgG、IgA、IgD、或IgM抗体。
在一些实施方案中，结合CD3的单链Fab分子作为CD3结合部分与另一抗原结合部分结合，形成靶向CD3和该另一抗原的双特异性抗体。在一些实施方案中，该单链Fab分子作为CD3结合部分与CD20结合部分结合，形成靶向CD3和CD20的双特异性抗体。在一些实施方案中，该单链Fab分子作为CD3结合部分与多种另外的抗原结合部分结合，形成多特异性抗体。
在一些实施方案中，结合CD20的单链Fab分子作为CD20结合部分与另一抗原结合部分结合，形成靶向CD20和该另一抗原的双特异性抗体。在一些实施方案中，该单链Fab分子作为CD20结合部分与CD3结合部分结合，形成靶向CD3和CD20的双特异性抗体。在一些实施方案中，该单链Fab分子作为CD20结合部分与多种另外的抗原结合部分结合，形成多特异性抗体。
本发明双特异性抗体中CD20结合部分与CD3结合部分的结合可以为共价结合(例如通过Fc段之间形成二硫键)或非共价结合。
在一些实施方案中，双特异性抗体中CD3结合部分的轻链和重链可变结构域源于OKT3单抗或L2K单抗(WO2004106380)。在一些实施方案中，双特异性抗体中CD20结合部分的轻链和重链可变结构域源于利妥昔单抗(Rituximab)或奥法木单抗(Ofatumumab)。
提及抗体“结合”抗原时，指该抗体能够识别并可检测地结合该抗原。抗体与抗原的结合可通过公知的抗原抗体结合测定方法测定，例如ELISA。另外，抗体抗原结合的解离平衡常数KD可用于表示抗体与相应抗原的结合亲和力。
在本发明的一些实施方案中，利用Fc段的“杵臼结构(Knobs-into-holes)”来防止双特异性抗体中的重链错配。这项技术由Genentech公司开发(参见美国专利5,731,168)。具体方法是将其中一个抗体的重链CH3区366位体积较小的苏氨酸(T)突变为体积较大的酪氨酸(Y)，形成突出的“Knobs(杵状)”结构(T366Y，(Kabat编号***))；同时将另一个抗体重链CH3区407位较大的酪氨酸(Y)残基突变成较小的苏氨酸(T)，形成凹陷的“holes(臼状)”结构(Y407T)。利用“Knobs-into-holes”结构的空间位阻效应实现两种不同抗体重链间的正确装配。突变后，产品正确装配率有明显提高，能够满足规模化生产的要求。但重链CH3的这一改构方式降低了抗体结构的稳定性，为了克服这一缺点，有研究者通过噬菌体展示技术进行随机突变筛选，构建了更为稳定的“3+1”模式即T366W突变形成突出的“Knobs”型，3个氨基酸突变(T366S，L368A和Y407V)形成凹陷的“holes”型。Knobs-holes结构设计有利于2种异源抗体重链的装配。
“序列一致性(Sequence identity)”指氨基酸序列或核苷酸序列之间的相似程度，一般以一致性百分比表示，可通过目视检查或通过计算机程序(如BLAST)来确定。在本发明的一些实施方案中，结合CD3的单链Fab分子包括SEQ ID NO：6所示的氨基酸序列，或者包括与SEQ ID NO：6所示序列有至少80％(例如，至少90％、至少95％、至少98％、或至少99％或100％)序列一致性的氨基酸序列。
在本发明的一些实施方案中，双特异性抗体中CD3结合部分包括SEQ ID NO：6所示的氨基酸序列，或者包括与SEQ ID NO：6所示序列有至少80％(例如，至少90％、至少95％、至少98％、至少99％或100％)序列一致性的氨基酸序列。在本发明的一些实施方案中，双特异性抗体中CD20结合部分的轻链包括SEQ ID NO：7所示的氨基酸序列，或者包括与SEQ ID NO：7所示序列有至少80％(例如，至少90％、至少95％、至少98％、至少99％或100％)序列一致性的氨基酸序列；双特异性抗体中CD20结合部分的重链包括SEQ ID NO：8所示的氨基酸序列，或者包括与SEQ ID NO：8所示序列有至少80％(例如，至少90％、至少95％、至少98％、至少99％或100％)序列一致性的氨基酸序列。
在本发明的一些实施方案中，双特异性抗体中CD3结合部分包括SEQ ID NO：9所示的氨基酸序列，或者包括与SEQ ID NO：9所示序列有至少80％(例如，至少90％、至少95％、至少98％、至少99％或100％)序列一致性的氨基酸序列。在本发明的一些实施方案中，双特异性抗体中CD20结合部分包括SEQ ID NO：10所示的氨基酸序列， 或者包括与SEQ ID NO：10所示序列有至少80％(例如，至少90％、至少95％、至少98％、至少99％或100％)序列一致性的氨基酸序列。
本领域技术人员可以理解的是，在本文提供的具体序列基础上，可以通过对少数氨基酸进行替换、删除、添加并验证或筛选所得产物与相应抗原的结合能力或生物学活性，从而获得本发明提供的结合CD3或CD20的单链Fab分子或双特异性抗体的相应变体，这些变体也应包括在本发明的范围内。
提及药物组合物，所使用的“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。
“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明双特异性抗体的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。
以下结合具体实施例进一步描述本发明。
实施例1.对构建抗CD3单链Fab分子所采用的柔性连接肽长度的筛选
基于Fab分子的空间结构特点，本发明人设计了靶向CD3的单链Fab分子，即通过柔性连接肽将轻链和与其对应的重链可变区以及CH1段和“杵状”Fc段连接，其中轻链和重链可变区序列源于L2K抗体。所形成的单链Fab分子具有如SEQ ID NO：6所示的氨基酸序列。
考虑到柔性连接肽的长度应不小于3.5纳米以及氨基酸相邻肽键的距离为0.38纳米，并考虑到scFab的空间结构需要保持一定的柔性，我们设计了多个长度在26至80个氨基酸之间的柔性连接肽，其中5个代表性连接肽的长度和序列显示在表1中。
表1 柔性连接肽的长度和序列
Figure PCTCN2020103731-appb-000001
1.1不同长度柔性连接肽对表达水平的影响
通过重叠PCR技术分别合成上述单链Fab分子的编码核酸序列以及“臼状”Fc段的编码核酸序列，并分别克隆到真核表达载体pcDNA(购自Life Technologies)中。然后，将包括该单链Fab分子的编码核酸序列的表达载体与包括“臼状”Fc段编码序列的表达载体按1.5:1的比例混合成总量为200μg的混合物，与转染试剂PEI按质量体积比1:2.5混匀制备成DNA-转染试剂复合体，逐滴添加至200mL处于对数生长期的293F细胞。转染后5-7天收集细胞上清液，经0.45μm滤膜过滤后，将上清液加入经结合缓冲液(12.15g Tris加适量灭菌水溶解，调pH 7.0，加8.78g NaCl，定容至1L)冲洗过的亲和层析凝胶柱(Protein A)，接着用洗脱液(7.5g甘氨酸加入适量双蒸水溶解，调pH 3.5，加8.78g NaCl，定容至1L)洗脱。洗脱组分用1M Tris-HCl，pH 9.0进行中和，得到具有不同长度柔性连接肽连接的候选分子(结构示意图显示在图1中)。
取20μL洗脱液，加入上样缓冲液后，95℃变性10分钟，以聚丙烯酰胺凝胶电泳分离蛋白，考马斯亮蓝染色显示条带。染色结果显示在图2中。泳道1至5分别对应包括26、32、45、60和80个氨基酸长度柔性连接肽的候选分子。从图中可见，采用60个氨基酸长的柔性连接肽构建的候选分子(图中黑色方框标注的条带，包含Fc段的Fab单链分子)表达量略高，而杂质条带较少。
1.2不同长度柔性连接肽对热稳定性的影响
将上文1.1部分纯化后的蛋白(候选分子)分别进行37℃24小时和50℃2小时热处理，并与放置于4℃处理同样时间的抗体进行SEC-HPLC检测。SEC-HPLC检测操作简述如下：选择TSK-GELG3000SWXL凝胶色谱柱(TOSOH公司)，使用流动相(50mM磷酸盐缓冲液，150mM氯化钠，pH 7.0)平衡色谱柱后以UV检测。取不同温度处理后的蛋白各50μL以1mL/min的流速通过色谱柱，每个样品运行20分钟后拟合结果图并计算出目的蛋白的纯度。
结果如表2所示。总体上，随着柔性连接肽长度的延长，抗体的热稳定性也越好，即与4℃状态下蛋白的差值越小。在50℃处理时，60个氨基酸长度的柔性连接肽构建的候选分子表现出最佳的热稳定性。
表2 不同长度柔性连接肽构建的候选分子的热稳定性比较
Figure PCTCN2020103731-appb-000002
Figure PCTCN2020103731-appb-000003
1.3不同长度柔性连接肽对抗原结合能力的影响
通过ELISA检测上文1.1部分纯化后的抗体(即候选分子)与CD3抗原的结合能力。简要步骤如下：在ELISA板上包被100μL浓度为0.5μg/mL的CD3抗原(Sino Biologics公司，SEK10981)，置于4℃过夜。以含2％BSA的PBS缓冲液洗涤后，使用含有2％BSA的PBS室温封闭1小时后洗涤3次。然后，加入100μL含有梯度稀释的抗体室温孵育1小时后洗涤3次，再加入100μL辣根过氧化酶标记的羊抗人IgG的二抗(Brthyl公司，1:5000稀释)室温孵育1小时。洗涤3次后，加入100μL TMB显色液显色，于酶标仪读取450纳米波长的吸光度值。
结果如图3所示，大体上随着柔性连接肽长度的增加，EC50值减小，即结合能力增强。其中，60个氨基酸长度的柔性连接肽构建的候选分子表现出最佳的抗原结合能力(EC50值最小)。
综合考虑不同长度柔性连接肽对表达水平、热稳定性以及结合抗原能力的影响，我们选择将60个氨基酸长度的柔性连接肽用于后续双特异性抗体的构建。
实施例2.双特异性抗体的制备
分别合成CD3结合部分(抗CD3单链Fab分子，氨基酸序列为SEQ ID NO：6)的编码DNA片段以及抗CD20抗体轻链和重链(氨基酸序列分别为SEQ ID NO：7和SEQ ID NO：8)的编码DNA片段，利用重组DNA如重叠PCR技术将它们分别克隆到真核表达载体pcDNA(购自Life Technologies)中，然后将所获得的包含CD3结合部分的编码DNA序列的表达载体、包含抗CD20抗体轻链的编码DNA序列的表达载体和包含抗CD20抗体重链的编码DNA序列的表达载体按质量比1.5:2:1的比例混匀。将总量为200μg的上述表达载体混合物与转染试剂PEI按质量体积比1:2.5混匀制备成DNA-转染试剂复合体，逐滴添加至200mL处于对数生长期的293F细胞。转染后5-7天收集细胞上清液，经0.45μm滤膜过滤后，将上清液加入经结合缓冲液(12.15g Tris加适量灭菌水溶解，调pH7.0，加8.78g NaCl，定容至1L)冲洗过的亲和层析凝胶柱(Protein A)，然后用洗脱液(7.5g甘氨酸加入适量双蒸水溶解，调pH3.5，加8.78g NaCl，定容至1L)洗脱。洗脱组分用1M Tris-HCl pH9.0进行中和，得到本发明的双特异性抗体YY0421。采用类似方式，制备获得本发明的双特异性抗体YY0422，其中CD20结合部分的轻链和重链通过连接肽连接(其CD3结合部分的氨基酸序列如SEQ ID NO：9所示，CD20结合部分的氨基酸序列如SEQ ID NO：10所示)。双特异性抗体YY0421和YY0422的结构示意图显示在图4中。
实施例3 蛋白印迹检测
3.1检测本发明双特异性抗体识别CD3的能力
收集细胞形态健康、融合率达80％的Jurkat细胞，离心后弃上清。加入细胞裂解液，95℃变性10分钟，再以聚丙烯酰胺凝胶电泳分离细胞蛋白。利用半干转法将蛋白转移至硝酸纤维素膜(NC膜)，以5％脱脂牛奶室温下封闭1小时。加入本发明的抗体YY0421、YY0422或对照抗体REGN1979(CD3和CD20双特异性抗体，参见WO2014047231和US20170355767)，室温下孵育1小时，PBST洗涤后，加入带辣根过氧化物酶标签的二抗，室温下孵育1小时。PBST洗涤后，利用化学发光试剂显色，暗室显影。
蛋白印迹检测结果见图5a。结果表明，同时靶向人CD3和CD20的双特异性抗体YY0421和YY0422，能够在蛋白水平有效识别CD3抗原。
3.2检测本发明双特异性抗体识别CD20的能力
收集细胞形态健康、融合率达80％的人Raji细胞，离心后弃上清。加入细胞裂解液，95℃变性10分钟，再以聚丙烯酰胺凝胶电泳分离细胞蛋白。利用半干转法将蛋白转移至硝酸纤维素膜(NC膜)，以5％脱脂牛奶室温下封闭1小时，加入本发明的抗体YY0421、YY0422或对照抗体REGN1979，室温下孵育1小时，PBST洗涤后，加入带辣根过氧化物酶标签的二抗，室温下孵育1小时。PBST洗涤后，利用化学发光试剂显色，暗室显影。
蛋白印迹检测结果见图5b。结果表明，靶向人CD3和CD20的双特异性抗体YY0421和YY0422能够在蛋白水平有效识别CD20抗原。
实施例4.流式细胞术检测本发明双特异性抗体与细胞表面CD3和CD20的结合
接种人Raji细胞和Jurkat细胞于6孔板，培养20小时后胰酶(不含EDTA)消化收集细胞，以1000rpm室温离心5分钟并PBS洗涤两次，与YY0421、YY0422或对照抗体REGN1979在冰上孵育1小时。PBS洗涤后冰上与FITC标记的兔抗人IgG(Fab)孵育1小时，流式细胞仪检测。
流式细胞仪检测结果见图6和图7。结果表明，本发明靶向人CD3和CD20的双特异性抗体YY0421和YY0422与细胞表面的人CD3(图6)和CD20(图7)具有高度亲和力。
实施例6.本发明双特异性抗体介导CD8 +T细胞对肿瘤细胞的杀伤
接种形态健康的经终浓度5μM CFSE染色的细胞数量为5x10 3人Daudi细胞于96孔板。以含0.5％胎牛血清培养基饥饿过夜后，按E/T比为5:1加入CD8 +T细胞。实验 分两批进行，每批均将细胞分为三组(每组包含三个孔)。第一批中分别加入培养基(图8a中Blank)、对照抗体REGN1979(图8a中Control)、本发明的抗体YY0421。第二批中分别加入培养基(图8b中为Blank)、对照抗体REGN1979(图8b中为Control)以及本发明的抗体YY0422。然后，继续在细胞培养箱中培养4小时。收集上清和细胞悬液，离心后弃上清用PBS重悬，接着加入1μg/mL PI，进流式细胞仪检测CFSE、PI双阳性细胞占CFSE阳性细胞的比例，以检测双特异性抗体介导CD8 +T杀伤肿瘤细胞的能力。
杀伤结果见图8。从结果可知，本发明的同时靶向人CD3和CD20的双特异性抗体YY0421和YY0422，可以有效介导T细胞对Daudi细胞的杀伤。
实施例7.裸鼠移植瘤检测
将5x10 7个对数生长期且形态良好的人Daudi细胞注射于SPF级BALB/C裸鼠前腋皮下，注射后第6日注射PBMC细胞(2.5x10 8，购自ATCC)及本发明的双特异性抗体或对照抗体REGN1979各10μg。每日测量皮下肿瘤的最大长径和最大横径，计算肿瘤的体积，并观察记录裸鼠饮食和体重等情况。第28天时以颈椎脱离法处死全部小鼠，随即分离肿瘤，拍照并称重。
裸鼠移植瘤检测结果见图9。结果表明，本发明的同时靶向人CD3和CD20的双特异性抗体具有良好的生物活性，能够显著抑制人Daudi细胞诱导的裸鼠皮下瘤体的增殖。
本文中所提及的一些氨基酸序列如下：
YY0421 CD3结合部分的氨基酸序列SEQ ID NO：6
Figure PCTCN2020103731-appb-000004
Figure PCTCN2020103731-appb-000005
YY0421 CD20结合部分的轻链氨基酸序列SEQ ID NO：7
Figure PCTCN2020103731-appb-000006
YY0421 CD20结合部分的重链氨基酸序列SEQ ID NO：8
Figure PCTCN2020103731-appb-000007
YY0422 CD3结合部分的氨基酸序列SEQ ID NO：9
Figure PCTCN2020103731-appb-000008
YY0422 CD20结合部分的氨基酸序列SEQ ID NO：10
Figure PCTCN2020103731-appb-000009

Claims (20)

  1. 抗CD3单链Fab分子，包括Fab轻链和Fab重链，所述Fab轻链的C末端通过连接肽与所述Fab重链的N末端连接，其中所述连接肽的长度为45至80个氨基酸。
  2. 如权利要求1所述的抗CD3单链Fab分子，其中所述连接肽的长度为60个氨基酸。
  3. 如权利要求1或2所述的抗CD3单链Fab分子，其中所述连接肽包括串联重复的GGGGS序列。
  4. 如权利要求1-3任一项所述的抗CD3单链Fab分子，其包括与SEQ ID NO：6或9所示序列有至少90％一致性的氨基酸序列。
  5. 抗CD20单链Fab分子，包括Fab轻链和Fab重链，所述Fab轻链的C末端通过连接肽与所述Fab重链的N末端连接，其中所述连接肽的长度为45至80个氨基酸。
  6. 如权利要求5所述的抗CD20单链Fab分子，其中所述连接肽的长度为60个氨基酸。
  7. 如权利要求5或6所述的抗CD20单链Fab分子，其中所述连接肽包括串联重复的GGGGS序列。
  8. 如权利要求5-7任一项所述的抗CD20单链Fab分子，其包括与SEQ ID NO：10所示序列有至少90％一致性的氨基酸序列。
  9. 权利要求1-4任一项所述的抗CD3单链Fab分子或权利要求5-8任一项所述的抗CD20单链Fab分子用于制备双特异性抗体的用途。
  10. 双特异性抗体，包括结合CD3的第一部分和结合CD20的第二部分，其中所述第一部分为抗CD3单链Fab分子，所述抗CD3单链Fab分子包括Fab轻链和Fab重链，所述Fab轻链的C末端通过长度为45至80个氨基酸的连接肽与所述Fab重链的N末端连接；所述第二部分包括抗CD20抗体的轻链和重链。
  11. 如权利要求10所述的双特异性抗体，其中所述连接肽的长度为60个氨基酸。
  12. 如权利要求10或11所述的双特异性抗体，其中所述连接肽包括串联重复的GGGGS序列。
  13. 如权利要求10-12任一项所述的双特异性抗体，其中所述结合CD3的第一部分包括与SEQ ID NO：6或9所示序列有至少90％一致性的氨基酸序列。
  14. 如权利要求10-13任一项所述的双特异性抗体，其中所述抗CD20抗体的轻链包括与SEQ ID NO：7所示序列有至少90％一致性的氨基酸序列，所述抗CD20抗体的重链包括与SEQ ID NO：8所示序列有至少90％一致性的氨基酸序列。
  15. 如权利要求10-14任一项所述的双特异性抗体，其中所述抗CD20抗体的轻链的C末端通过所述连接肽与所述抗CD20抗体的重链的N末端连接。
  16. 如权利要求10-15任一项所述的双特异性抗体，其中所述结合CD20的第二部分包括与SEQ ID NO：10所示序列有至少90％一致性的氨基酸序列。
  17. 权利要求10-16任一项所述的双特异性抗体在制备治疗B细胞相关疾病的药物中的用途。
  18. 如权利要求17所述的用途，其中所述B细胞相关疾病为B细胞白血病或B细胞淋巴瘤。
  19. 药物组合物，包括权利要求10-16任一项所述的双特异性抗体和药学上可接受的载体。
  20. 在患者中治疗B细胞相关疾病的方法，包括以治疗有效量的权利要求10-16任一项所述的双特异性抗体或权利要求19所述的药物组合物向所述患者给药。
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