WO2019196716A1 - 具有延长的半衰期和增强的抗肿瘤效果的双特异性抗体 - Google Patents

具有延长的半衰期和增强的抗肿瘤效果的双特异性抗体 Download PDF

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WO2019196716A1
WO2019196716A1 PCT/CN2019/081164 CN2019081164W WO2019196716A1 WO 2019196716 A1 WO2019196716 A1 WO 2019196716A1 CN 2019081164 W CN2019081164 W CN 2019081164W WO 2019196716 A1 WO2019196716 A1 WO 2019196716A1
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fab
antigen
bispecific antibody
binding fragment
constant region
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French (fr)
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马梵辛
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非同（成都）生物科技有限公司
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to bispecific antibodies, and in particular to PEGylated bispecific antibodies having an extended half-life and enhanced anti-tumor effects.
  • Bispecific antibodies are a powerful tool in cancer immunotherapy that binds immune cells directly to cancer cells.
  • Bispecific antibodies have two distinct antigen binding sites, one that recognizes tumor cells and the other that recognizes immune cells (usually T cells or natural killer (NK) cells).
  • Bispecific antibodies exist in a variety of forms, including full-length forms based on IgG (eg, intact bispecific antibodies), single-stranded forms (including tandem single-chain variable fragments (ScFv)), and bispecific T cell junctions Body (BiTE).
  • S-Fab single domain antibody linked Fab
  • Single domain antibodies derived from natural camelid heavy chain antibodies lack the first constant (CH1) domain and light chain and are therefore referred to as Nanobodies or VHH.
  • Nanobodies are small and generally more stable than traditional scFv or BiTE, making them a good property for constructing bispecific antibodies.
  • a bispecific S-Fab antibody was constructed by ligating a single domain Nanobody anti-CEA to a CD3-Fab. S-Fab exhibits excellent tumor cell killing activity in vitro (see L. Li, P.
  • Fab fragments including S-Fabs
  • Fc constant regions
  • the invention provides a pegylated bispecific antibody (also referred to herein as PEG-S-Fab) comprising (a) an antigen binding fragment (Fab) having a light chain variable region (VL) And a light chain constant region (CL), and a heavy chain variable region (VH) and a partial heavy chain constant region (CH1); (b) a partial heavy chain constant region (CH1) fused to the antigen-binding fragment (Fab) a C-terminal single domain antigen-binding fragment (VHH), and (c) a C-terminal polyethylene glycol (PEG) fused to the light chain constant region (CL) of the antigen-binding fragment (Fab).
  • PEG-S-Fab pegylated bispecific antibody
  • the polyethylene glycol has a molecular weight of from 10,000 to 30,000. In other embodiments, the polyethylene glycol has a molecular weight of 20,000.
  • the invention provides a pharmaceutical composition comprising the PEGylated bispecific antibody, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient. Accordingly, the invention provides a host cell comprising one or more polynucleotides encoding said bispecific antibody.
  • Another aspect of the invention provides an engineered bispecific antibody comprising (a) an antigen binding fragment (Fab) having a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain a variable region (VH) and a partial heavy chain constant region (CH1); and (b) a single domain antigen-binding fragment (VHH) fused to the C-terminus of a portion of the heavy chain constant region (CH1) of the antigen-binding fragment (Fab); Wherein the C-terminus of the light chain constant region (CL) of the antigen-binding fragment (Fab) is engineered to have at least one cysteine residue. Accordingly, the invention provides the use of the engineered bispecific antibody in the preparation of the PEGylated bispecific antibody of the invention.
  • Fab antigen binding fragment having a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain a variable region (VH) and a partial heavy chain constant region (CH1)
  • VHH single domain antigen
  • Another aspect of the invention provides a method of treating cancer comprising contacting a PEGylated bispecific antibody of the invention with a cancer cell. Accordingly, the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a PEGylated bispecific antibody of the invention.
  • Another aspect of the invention provides a host cell comprising one or more polynucleotides encoding the bispecific antibody of any of the preceding claims.
  • the host cell is E. coli.
  • the manipulation of polynucleotides involves knowledge and experimental manipulations in the fields of molecular biology, genetic engineering, protein engineering, and the like, which are well known to those skilled in the art.
  • PEG-S-Fab thiol site-specific PEGylation to improve the half-life (t 1/2 ) of S-Fab bispecific antibodies.
  • MAL-PEG-OMe a functionalized 20 kDa linear methoxy PEG maleimide
  • PEG-S-Fab The pegylated S-Fab (PEG-S-Fab) retains the binding of tumor cells and T cells.
  • PEG-S-Fab enhances plasma stability and increases the half-life of S-Fab by a factor of 12.
  • PEG-S-Fab also has more potent tumor suppressive potency in a xenograft mouse model.
  • FIG. 1 Expression and purification of S-Fab from E. coli.
  • A. The bacterial S-Fab expression construct contains the pelB signal sequence, anti-CD3 (human UTCH1 clone) VH (or VL) and CH1 (CL) and anti-CEA-VHH. In order to facilitate antibody detection and purification, a flag tag and a his6 tag are added to the C-terminus of the heavy and light chains, respectively;
  • B Schematic diagram of the S-Fab after co-expression;
  • C Coomass of the purified S-Fab after two-step purification Blue stained SDS-PAGE; D, gel filtration analysis showed that the molecular weight of S-Fab was approximately 130 kD.
  • FIG. 1 PEGylation of bispecific S-Fabs using 20 kDa linear methoxy PEG maleimide (MAL-PEG-OMe).
  • S-Fab was reacted with functionalized PEG in a series of PEG:S-Fab molar equivalent ratios (0:1, 10:1, 20:1, 40:1, and 60:1) and shaken at 22 °C for 2 hours, then The sample was examined by SDS-PAGE (5 ul/sample).
  • A Coomassie blue staining of S-Fabs of different molar ratios of PEG:S-Fab during PEGylation.
  • FIG. 1 Purification of PEG-S-Fab. Gel filtration was used to separate the PEGylated mixture (fractions 1, 2, 3). A. Chromatogram of PEG-S-Fab. B. Coomassie blue staining of the PEG-S-Fab purified fraction. C. PEG-S-Fab purified fractions stained with cesium iodide complex.
  • PEG-S-Fab can bind to CD3 + on CEA and T cells on tumor cells.
  • Flow cytometry analysis using PEG-S-Fab and S-Fab was performed on CEA-positive LS174T cells (A), CEA-negative SKOV3 cells (B) and CD3 + T cells (C).
  • the positive control anti-CD3 antibody OKT3 was used for T cell flow cytometry.
  • Confocal microscopy of immunofluorescence staining of S-Fab (D) and PEG-S-Fab (E) was performed in LS174T cells (top panel) and SKOV3 cells (bottom panel), respectively.
  • the scale bar represents 30 ⁇ m.
  • PEG-S-Fab has potent specific cytotoxicity against tumor cells.
  • B. Cytotoxicity assay using CEA-negative SKOV3 cells. Different concentrations of S-Fab or PEG-S-Fab were incubated with tumor cells and effector T cells (E/T 10). All data are shown as triplicate averages and error bars indicate SD.
  • FIG. 1 Pharmacokinetic and stability analysis of plasma concentrations of PEG-S-Fab and S-Fab.
  • FIG. 7 PEGylation of S-Fab induces more potent in vivo anti-tumor activity.
  • a entity refers to one or more such entities, for example, "a bispecific antibody” is understood to mean one or more bispecific antibodies. Likewise, the terms “a”, “one or more” and “sai” are used interchangeable.
  • antibody or “antigen binding polypeptide” refers to a polypeptide or polypeptide complex that specifically recognizes and binds to one or more antigens.
  • An antibody is a whole antibody and any antigen-binding fragment or single strand thereof.
  • antibody includes any protein or peptide comprising at least a molecule of an immunoglobulin molecule, a portion of which has biological activity for binding to an antigen.
  • Such examples include, but are not limited to, a complementarity determining region (CDR), a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region of a heavy/light chain or a ligand binding portion thereof Or any part thereof, or at least a portion of a binding protein.
  • CDR complementarity determining region
  • FR framework
  • the term antibody also encompasses a polypeptide or polypeptide complex that possesses antigen binding ability upon activation.
  • certain immunoglobulin molecules derived from Camelidae or Camelidae immunoglobulin-based engineering intact immunoglobulin molecules may consist solely of heavy chains without a light chain, see for example Hamers -Casterman et al., Nature 363:446-448 (1993).
  • Specific binding or “specific to” generally refers to the binding of an antibody to an epitope by its antigen binding domain, and such binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody is said to "specifically bind” to an epitope when it is capable of binding to a particular epitope more rapidly via its antigen binding domain than to a random, unrelated epitope.
  • the term “specificity” is used to quantify the relative affinity of a particular antibody for binding to a particular epitope. For example, for a given epitope, antibody “A” is considered to be more specific than antibody "B", or antibody "A” binds to epitope "C” more specifically than its binding to the relevant The specificity of epitope "D”.
  • treating refers to a therapeutic or prophylactic means in which a subject is prevented or slows (mitigates) an undesired pathological change or disorder, such as the progression of cancer.
  • Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization of disease states (ie, no deterioration), delay or slowing of disease progression, improvement or mitigation of disease states, and remission ( Whether local or holistic, whether or not these results are detectable or undetectable.
  • Treatment also refers to prolonged survival as compared to the expected survival without treatment. Subjects in need of treatment include those already with the disease or condition as well as those prone to have the disease or condition or those in need of prevention of the disease or condition.
  • Subject or “individual” or “animal” or “patient” or “mammal” refers to any subject, particularly a mammalian subject, in whom a diagnosis, prognosis or treatment is desired.
  • Mammalian subjects include humans, domesticated animals, farm animals, and zoo animals, competitive animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.
  • patient in need of treatment or “subject in need of treatment” includes a subject, such as a mammalian subject, that can benefit from administration of an antibody or composition of the invention to achieve, for example, detection, diagnostic procedures, and/or therapeutic purposes. .
  • the schematic type of bispecific antibody is an antibody that targets two different antigens, one of which is present on tumor cells or microorganisms and the other on immune cells.
  • bispecific antibodies When administered to an individual, such bispecific antibodies specifically bind to tumor cells or microorganisms while specifically binding to immune cells (eg, cytotoxic cells). This dual binding can result in the bound tumor or microorganism being killed by the host's immune system.
  • a “single domain antigen binding fragment” or “single domain antibody fragment” or “VHH” is an antigen binding fragment that is capable of binding to an antigen without the need for a light chain.
  • VHH was originally isolated from a single domain antibody (sdAb) as a single antigen-binding fragment.
  • the first known single domain antibody was isolated from camel (Hamers-Casterman et al., Nature 363:446-8 (1993)) and thereafter isolated from cartilage fish.
  • Camels produce functional antibodies without light chains, their single N-terminal domain (VHH) binding antigen without domain pairing (see Harmsen and Haard, App Microbiol Biotechnol., 77: 13-22 (2007)).
  • Single domain antibodies do not include the CH1 domain, and in conventional antibodies, the CH1 domain interacts with the light chain.
  • VHH comprises four framework regions (FR1-FR4) constituting the core structure of the immunoglobulin domain and three complementarity determining regions (CDR1-CDR3) involved in antigen binding.
  • the VHH framework region shows high sequence homology (>80%) to the human VH domain compared to the human VH domain. See Harmsen and Haard, 2007, which further describes: "The most characteristic feature of VHH is the amino acid substitutions at the four FR2 positions (positions 37, 44, 45 and 47; Kabat numbering), which are in the conventional VH structure.
  • VHH typically has different amino acids (such as Leu11Ser, Val37Phe or Tyr, Gly44Glu, Leu45Arg or Cys, Trp47Gly) at these and other positions that are highly conserved in conventional VH.
  • VHH3 has an extended CDR3 that is normally stabilized by the formation of additional disulfide bonds with cysteines in CDR1 or FR2, resulting in the CDR3 loop folding over the entire interface of the previous VL.
  • the specific subfamily of llama VHH (VHH3) also contains an extended CDR3 that is stabilized by the formation of additional disulfide bonds with the cysteine at position 50 of FR2.
  • sdAbs single domain antibodies
  • VHH can be easily identified and prepared.
  • Table 1 lists non-limiting examples of many VHHs and sdAbs. Accordingly, in some embodiments, the invention provides polypeptides comprising each such disclosed sequence or equivalent thereof, as well as polynucleotides encoding each polypeptide.
  • VHH single domain antigen binding fragments
  • sdAb single domain antibodies
  • a Fab or VHH (or VHH') fragment of a bispecific antibody has immunospecificity for a tumor antigen.
  • Tumor antigen is an antigenic substance produced in a tumor cell, that is, it causes an immune response of the host. Tumor antigens can be used to identify tumor cells and are potential candidates for cancer therapy. Normal proteins in the body are not antigenic. However, certain proteins are produced or overexpressed during tumorigenesis and thus appear to be "exogenous" to the body. This may include proteins that are well evaded from the immune system, proteins that are normally produced in very small amounts, proteins that are normally produced only at a particular developmental stage, or proteins whose structure is modified by mutation.
  • tumor antigens are known in the art and many new tumor antigens are readily found by screening.
  • tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2 , VEGF, VEGFR, integrin, ⁇ V ⁇ 3, ⁇ 5 ⁇ 1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.
  • the Fab or VHH fragment has specificity for a protein that is overexpressed on tumor cells compared to the corresponding non-tumor cells.
  • “Corresponding non-tumor cells” refers to non-tumor cells having the same cell type as cells from which tumor cells are derived. It should be noted that such proteins are not necessarily different from tumor antigens.
  • Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon cancer, rectal cancer, breast cancer, lung cancer, pancreatic cancer, and gastrointestinal cancer; modulin receptor (HER-2, neu or c-) erbB-2), which is commonly overexpressed in breast, ovarian, rectal, lung, prostate, and cervical cancers; epidermal growth factor receptor, which is involved in a range of solid tumors (including breast cancer, head and neck cancer, non-small cells) Overexpression in sexual lung cancer and prostate cancer; asialoglycoprotein receptor; transferrin receptor; serine protease inhibitor enzyme complex receptor expressed on hepatocytes; overexpressed on islet ductal adenocarcinoma cells Fibroblast growth factor receptor (FGFR); vascular endothelial growth factor receptor (VEGFR) for anti-angiogenic gene therapy; folate receptor selectively overexpressed in 90% of non-mucinous ovarian cancer; cells Surface polysaccharide-protein complex (glycocaly
  • the Fab portion comprises: one or two amino acid sequences selected from the group consisting of SEQ ID NOs: 14-25 (Table 2), or optionally one or two or three insertions, deletions or substitutions.
  • any of the antibodies or polypeptides described above may further comprise additional polypeptides, for example, signal peptides that direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
  • antibodies disclosed herein can be modified such that they differ in amino acid sequence from the native binding polypeptide from which they are derived.
  • a polypeptide or amino acid sequence derived from a particular protein may be similar, for example, having a certain percentage of identity to the starting sequence, for example it may be 60%, 70%, 75%, 80% identical to the starting sequence. , 85%, 90%, 95%, 98% or 99% consistency.
  • nucleotides or amino acids can be made in the "non-essential" amino acid region.
  • a polypeptide or amino acid derived from a particular protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions (eg, one, two, three, four, five, six, Seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions or deletions).
  • the polypeptide or amino acid sequence derived from a particular protein has from one to five, one to ten, or one to twenty individual amino acid substitutions, insertions or deletions relative to the starting sequence.
  • an antigen binding polypeptide of the invention may comprise a conservative amino acid substitution.
  • one amino acid residue is substituted with an amino acid residue having a similar side chain.
  • a family of amino acid residues having similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) Uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg alanine, guanamine) Acid, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), beta branch side chain (eg threonine, valine, isoleucine) and aromatic side Chain (eg tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg,
  • non-essential amino acid residues in the immunoglobulin polypeptide are more suitably substituted with other amino acid residues from the same side chain family.
  • the amino acid chain can be substituted with a structurally similar but sequential or compositionally different chain of side chain family members.
  • the following table provides non-limiting examples of conservative amino acid substitutions.
  • a similarity score of 0 or higher in the table indicates a conservative substitution at both amino acids.
  • an antibody can be bound to a therapeutic formulation, prodrug, peptide, protein, enzyme, virus, lipid, biological effect modifier, or pharmaceutical formulation.
  • a therapeutic preparation which can include a detectable label (eg, a radiolabel), an immunomodulator, a hormone, an enzyme, an oligonucleotide, a phototherapeutic or diagnostic agent, a cell.
  • a detectable label eg, a radiolabel
  • an immunomodulator e.g., a hormone, an enzyme, an oligonucleotide, a phototherapeutic or diagnostic agent, a cell.
  • Toxic agents drug or toxins
  • ultrasound enhancers e.g., ultrasound enhancers, non-radioactive labels, combinations thereof, and other formulations known in the art.
  • the antibody By labeling the antibody with a compound that is coupled to chemiluminescence, the antibody can be detected. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of fluorescence (during the course of a chemical reaction).
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridine salt and oxalate.
  • bispecific antibodies, variants or derivatives thereof of the invention are useful in certain therapeutic and diagnostic methods associated with cancer or infectious diseases.
  • the invention further relates to antibody-based therapies which involve administering a bispecific antibody of the invention to a patient, such as an animal, a mammal and a human, to treat one or more of the diseases or conditions described herein.
  • Therapeutic compositions of the invention include, but are not limited to, antibodies of the invention (including variants and derivatives thereof described herein) and antibodies encoding the invention (including variants and derivatives thereof described herein) Nucleic acid or polynucleotide.
  • the antibodies of the invention can be used to treat, inhibit or prevent a disease, disorder, or condition, including a malignant disease, disorder, or condition associated with, for example, a disease or disorder (such as cancer) associated with increased cell survival or inhibition of apoptosis
  • cancers include, but are not limited to, follicular lymphoma, cancer with p53 mutations, and hormone-dependent tumors (including but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinal neoplasia, glioblastoma, Lung cancer, colon cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, fibroids, lymphoma, endothelial tumor, osteoblastoma, osteoclast, osteosarcoma, chondrosarcoma, adenocarcinoma, breast cancer, Prostate cancer, Kaposi sarcoma; autoimmune disorders (eg multiple sclerosis, Sjogren syndrome, Grave disease, Hashi
  • antigen binding polypeptides, variants or derivatives thereof of the invention are used to inhibit the development, progression and/or metastasis of cancer, particularly the cancers listed above or in subsequent paragraphs.
  • the antibodies of the present invention can also be used to treat infectious diseases caused by microorganisms or to kill microorganisms by targeting microorganisms and immune cells to affect the elimination of microorganisms.
  • the microorganism is a virus (including RNA and DNA viruses), a Gram-positive bacterium, a Gram-negative bacterium, a protozoa or a fungus.
  • the dosage and treatment regimen specific for any particular patient will depend on a variety of factors (including the specific antigen binding polypeptide used, variants or derivatives thereof, age, weight, overall health, sex, diet, time of administration, excretion of the patient) Rate, combination of drugs, and severity of the particular disease being treated). The determination of such factors by a medical professional is within the judgment of one of ordinary skill in the art.
  • the dosage will also be based on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the composition employed, the severity of the disease, and the desired effect.
  • the dosage employed can be determined by the principles of pharmacology and pharmacokinetics well known in the art.
  • bispecific antibodies include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the antigen binding polypeptide or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption of the epithelial or mucosal protective layer (eg, oral mucosa, rectal and intestinal mucosa, etc.); Formulations are used together.
  • compositions comprising an antigen binding polypeptide of the invention may be administered orally, rectally, parenterally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops or dermal patches) Spray application of the mouth or nose.
  • parenteral refers to the manner of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injection and infusion.
  • Administration can be systemic or topical.
  • the antibodies of the invention can be introduced into the central nervous system by any suitable route, including: intracerebroventricular and intrathecal injection; promotion by intraventricular catheters (eg, attachment to a reservoir (eg, Ommaya reservoir)) Intraventricular injection.
  • Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and a formulation with an aerosol.
  • a bispecific antibody or composition of the invention may be topically administer to a region in need of treatment; this may be accomplished, for example, but not limited to, by local infusion, topical administration (eg, post-operative combination) Use a wound dressing), inject, through a catheter, through a suppository, or through an implant (the implant is a porous, non-porous, non-permeable or gel-like material, including a film (eg, a silicone membrane) or fiber) .
  • a protein including an antibody
  • care should be taken to use a material that the protein does not adsorb.
  • an effective amount of an antibody of the invention in the treatment, inhibition, and prevention of an inflammatory, immune or malignant disease, disorder, or condition can be determined by standard clinical techniques.
  • in vitro assays can be optionally employed to help identify the optimal dose range.
  • the exact dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined in accordance with the judgment of the practitioner and the condition of each patient. Effective doses can be deduced from dose-response curves derived from in vitro or animal model test systems.
  • the dose of the antigen-binding polypeptide of the present invention administered to a patient is usually from 0.1 mg/kg to 100 mg/kg of patient body weight, from 0.1 mg/kg to 20 mg/kg of patient body weight, or from 1 mg/kg to 10 mg/kg of patient. body weight.
  • human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to exogenous polypeptides. Therefore, lower doses and lower doses of human antibodies are generally possible.
  • the frequency and dosage of administration of the antibodies of the present invention can be reduced by modification (e.g., lipidation) to enhance the uptake of these antibodies and tissue penetration (e.g., into the brain).
  • a method for treating an infection or a malignant disease, disorder or disorder comprising administering an antibody, variant or derivative thereof, for use in a human body, usually in vitro, and then in vivo in an acceptable animal model Test to obtain the desired therapeutic or prophylactic activity.
  • Suitable animal models, including transgenic animals are well known to those of ordinary skill in the art.
  • in vitro assays demonstrating the therapeutic utility of the antigen binding polypeptides described herein include the effect of antigen binding polypeptides on cell lines or patient tissue samples. The effect of an antigen binding polypeptide on a cell line and/or tissue sample can be determined using techniques known to those of skill in the art, such as those disclosed elsewhere herein.
  • In vitro assays that can be used to determine whether a specific antigen-binding polypeptide is required for use in accordance with the present invention include in vitro cell culture assays (where patient tissue samples are grown in culture and exposed or otherwise administered to the antibody) and observed The effect of antibodies on this tissue sample.
  • compositions of the invention are administered in combination with an anti-neoplastic, anti-viral, antibacterial or antibiotic or anti-fungal formulation. Any of these formulations known in the art can be administered in the compositions of the present invention.
  • compositions of the invention are administered in combination with a chemotherapeutic agent.
  • Chemotherapeutic agents that can be administered with the compositions of the present invention include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, actinomycin), antiestrogens (eg, tamoxifen) ), antimetabolites (eg, fluorouracil, 5-FU, methotrexate, fluorouridine, interferon alpha-2b, glutamate, pucamycin, guanidine, and 6-mercaptoguanine), cytotoxic agents (eg, Carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, benzamidine, mitomycin, busulfan, cisplatin and vincristine Sulfate), hormones (eg medroxyprogesterone, estramustine sodium phosphate
  • compositions of the invention are administered in combination with a cytokine.
  • Cytokines that can be administered with the compositions of the invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L and TNF- ⁇ .
  • compositions of the invention are administered in combination with other therapeutic or prophylactic therapies, such as radiation therapy.
  • compositions comprise an effective amount of an antibody and an acceptable carrier.
  • pharmaceutically acceptable means approved by a federal or state government regulatory agency, or listed in the US Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more specifically For people.
  • a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of excipient.
  • carrier refers to a diluent, adjuvant, excipient or carrier with which the drug is used.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Salt solutions and aqueous dextrose and glycerol solutions can also be employed as carriers for the liquid phase, especially for injectable solutions.
  • Suitable pharmaceutical excipients include: starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, c Triol, propylene, ethylene glycol, water, ethanol, and the like.
  • the compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents such as, for example, acetate, citrate or phosphate.
  • compositions may take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • the composition can be formulated as a suppository using conventional binders and carriers such as triglycerides.
  • Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described by E. W. Martin in Remington's Pharmaceutical Sciences (which is incorporated herein by reference). Such compositions will contain a therapeutically effective amount of the antigen binding polypeptide (preferably in a purified form) with a suitable amount of carrier to provide a suitable mode of administration for the patient. This formulation should be suitable for the mode of administration.
  • Such parental preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.
  • the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans.
  • the composition for intravenous administration is a solution in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic (e.g., lidocaine to reduce pain at the injection site), if necessary.
  • the components are usually provided, either alone or in combination, in unit dosage form, for example, as a lyophilized powder or a water-free concentrate in a closed container, such as an ampoule or sachette indicating the amount of active agent.
  • the composition When the composition is administered by infusion, it can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline for injection can be provided so that the ingredients can be mixed prior to administration.
  • compositions of the invention may be formulated in a neutral or salt form.
  • Pharmaceutically acceptable salts include those formed from anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed from cations such as those derived from sodium, potassium, ammonium, calcium, and hydrogen. Salts of iron oxide, isopropylamine, triethylamine, ethylhydroxyethylamine, histidine, procaine, and the like.
  • S-Fab The structure of the S-Fab is shown in Figure 1A.
  • Anti-CD3 VH-CH1 and VL-CL fragments were synthesized and cloned by standard DNA cloning techniques.
  • the signal sequence pelB was added to the N-terminus for periplasmic expression.
  • a cysteine residue is added to the C-terminus of the light chain, followed by a short linker and another cysteine (CGGGGC) and his6 tag.
  • S-Fab was constructed by heterodimerization of VL-CL/VH-CH1 (anti-CD3 Fab) with anti-CEA VHH Nanobody.
  • a Flag-tag was added to the C-terminus of the heavy chain for detection (Fig. 1B).
  • the cell pellet was resuspended in a pre-cooled sucrose solution (20 mM Tris-HCl pH 7.5; 25% (w/v) sucrose; 1 mM EDTA) at 1:4 (g: mL).
  • Periplasmic extraction After incubating for 15 minutes on ice, the suspension was centrifuged at 10,000 g for 20 minutes, and the supernatant fraction was collected as a sucrose fraction. The pellet was then resuspended in ice-cold periplasmic solution (5 mM MgCl 2 ) and centrifuged at 10,000 g for 20 minutes. The supernatant was collected as a periplasmic fraction.
  • the S-Fab protein was then purified from the combined sucrose and periplasmic fraction by two-step purification: first by immobilized Ni-NTA affinity chromatography (GE Health, USA), then by IgG-CH1 affinity matrix (Lot 194320005; ThermoFisher) Scientific Inc, USA) (Fig. 1C). Gel filtration analysis was performed using a Bio-Rad FPLP system and a GE Superdex 200TM Incremental 10/300 GL column at a flow rate of 0.5 ml/min. Fractions (0.5 ml per fraction) were collected and then subjected to SDS-PAGE analysis under reducing conditions. Coomassie blue staining.
  • Protein markers (Lot MWGF200; Sigma Aldrich Co., Ltd, USA) were used as standard controls for gel filtration analysis.
  • Gel filtration analysis revealed a complete S-Fab antibody with a molecular weight of ⁇ 130 kD (Fig. 1D), indicating dimerization of S-Fab (approximately 65 kD monomer), which may be due to the cysteine residue at the C-terminus of VL-CL The disulfide bond formed by the group.
  • the S-Fab was engineered to have two terminal cysteine residues at the C-terminus of CL for use as a site for conjugation to a 20 kDa linear MAL-PEG-OMe.
  • S-Fab (about 1.35 mg/mL (about 20 ⁇ M)) in 5.0 mL phosphate buffered saline (PBS, pH 7.4) and three molar equivalents of 1 mM tris(2-carboxyethyl)phosphine (TCEP, final 60 ⁇ M) , about 300 ⁇ L) were mixed and incubated at 22 ° C for 2 hours to obtain a reduced S-Fab fragment.
  • PBS phosphate buffered saline
  • TCEP tris(2-carboxyethyl)phosphine
  • MAL-PEG-OMe was dissolved in sterile water to obtain a working concentration of 20 mg/mL (1 mM).
  • PEGylation of S-Fab was carried out by mixing MAL-PEG-OMe (at working concentration) with reduced S-Fab and shaking at 22 °C for 2 hours.
  • the resulting sample was subjected to 12% SDS-PAGE electrophoresis (5 ul/sample/PAGE), and then PEG was subjected to Coomassie blue and cesium iodide dye according to the reference (Figs. 2A and 2B). After electrophoresis, the PEGylated strand was detected using a Western blot assay. Briefly, the other two gels were transferred to a polyvinylidene fluoride membrane (Millipore, USA). After blocking with 5% skim milk for 2 hours, the membrane was separately treated with mouse monoclonal anti-Flag HRP (1:2000 for heavy chain) and mouse monoclonal anti-his IgG (1:3000) in 5% skim milk.
  • the PEGylated S-Fab was purified using an AKTA york 25 FPLC purification system (GE Healthware, USA) and a Superdex 10/300 GL column at a flow rate of 0.8 ml/min. The column was first equilibrated with two column volumes (CV) of distilled water and then equilibrated with two CV PBSs prior to application of the sample. After the SDS-PAGE under reducing conditions, all collected fractions were analyzed by Coomassie Blue and cesium iodide complex dyes. The fractions of the purified PEGylated S-Fab were combined together.
  • the higher molecular weight PEGylated bands also increased slightly, indicating further pegylation on other cysteine residues on VH-CH1 or VL-CL.
  • the PEG:S-Fab was then selected to have a molar ratio of 20 for conjugation because it has a higher VL-CL conjugation without higher molecular weight conjugation.
  • PEG-S-Fab binds to tumor antigens CEA and CD3 + T cells
  • the bispecific S-Fab has two distinct binding sites, recognizing CEA-resistant anti-CEA VHH on tumor cells and recognizing anti-CD3 recognition of CD3 + on T cells.
  • flow cytometry analysis was performed using LS174T cells with CEA overexpression.
  • the CEA negative cell line SKOV3 was used as a negative control.
  • 1x10 6 (for LS174T and SKOV3) or 5x10 5 (for T cells) cells/samples were collected by centrifugation at 1000 rpm for 5 minutes, then washed once with 1.0 mL of ice-cold PBS containing 0.2% bovine serum albumin (BSA).
  • Primary antibodies including S-Fab, PEG-S-Fab and blank control (vector, PBS only) were added to a final concentration of 10 ⁇ g/mL, then incubated on ice for 1 hour, followed by washing with ice-cold PBS containing 0.1% BSA 2 Times.
  • Anti-CD3 FITC (OKT3, final concentration 10 ⁇ g/mL) was used as a positive control for CD3 + antigen binding assay.
  • Goat anti-human IgG (H+L)-AlexaFluor 488 antibody was then added to a final concentration of 5 [mu]g/mL. The cells were incubated on ice for 1 hour. After washing the cells twice, flow cytometry was performed.
  • PEG-S-Fab has potent specific cytotoxicity against tumor cells
  • LS174T and SKOV3 cells were used as target cells (T), and unstimulated freshly prepared human CD3 + T cells were used as effector cells (E).
  • In vitro cytotoxicity assays were performed in triplicate in 5,000 target cells per well in 100 ⁇ L of the corresponding medium in 96-well microplates. After 6 hours of incubation, an equal volume of CD3 + T cells was added to each well at a 10:1 E:T ratio and then a series of concentrations (0.033, 0.1, 0.33, 1, 3.3, 10, 33) were added accordingly.
  • S-Fab and PEG-S-Fab were effective in killing CEA-positive LS174T cells, even at 0.033 nM (Fig. 5A), but not cytotoxic to CEA-negative SKOV3 cells (Fig. 5B).
  • PEG-S-Fab has slightly reduced cytotoxicity compared to S-Fab (Fig. 5A). This may be due to a slight decrease in antigen binding or steric interference with PEGylation. However, the level of reduced activity appears to be much lower than many other PEGylated proteins.
  • the intravenous PK profiles of S-Fab and PEG-S-Fab in rats were analyzed.
  • the PK assay used SPF male SD rats (250-300 g). Control the food to maintain the animal's weight below 350 grams.
  • S-Fab (1.0 mg/kg), PEG-S-Fab (1.0 mg/kg) or volume equivalent of carrier PBS was administered via the tail vein.
  • blood samples were taken from the orbital veins using a capillary tube under isoflurane anesthesia (approximately 150- each) 200 ⁇ L). All blood samples were collected into heparinized tubes. Plasma was obtained by centrifugation at 3,500 g for 30 minutes and then stored at -80 °C until further analysis.
  • Samples and standards (100, 80, 50, 40, 30, 20, 10, 5, 1 and 0.1 ⁇ g/mL) were prepared in blocking buffer, where the standard (S-Fab) was 1:10 using PBS Prepared in diluted plasma, which is important to avoid matrix effects in the assay. For plasma samples, a 1:3 dilution was used. A 100 [mu]L sample or standard aliquot was then added and incubated for 1 hour at 37[deg.] C. in triplicate.
  • the inventors also evaluated the stability of S-Fab and PEG-S-Fab in human fresh plasma for two weeks. Briefly, human fresh plasma (without platelets) diluted S-Fab and PEG-S-Fab, resulting in an initial concentration of 100 ⁇ g/mL. The samples were incubated at 37 ° C for two weeks. At time intervals of 0, 24, 48, 72, 96, 168, 264 and 336 hours, 40 ⁇ L of sample was collected and stored directly at -80 ° C until further analysis. The sample was thawed on ice and then centrifuged at 14,000 rpm for 10 minutes at 4 °C. The supernatant was then electrophoresed on a 12% SDS-PAGE (5 ul sample per well).
  • PEG-S-Fab induces more potent antitumor activity in vivo

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Abstract

本发明公开了一种聚乙二醇化的双特异性抗体，包含(a)抗原结合片段(Fab)，其具有轻链可变区(VL)和轻链恒定区(CL)，以及重链可变区(VH)和部分重链恒定区(CH1)；(b)融合至所述抗原结合片段(Fab)的部分重链恒定区(CH1)的C端的单域抗原结合片段(VHH)，以及(c)融合至所述抗原结合片段(Fab)的轻链恒定区(CL)的C端的聚乙二醇(PEG)。聚乙二醇化的S-Fab(PEG-S-Fab)保留了肿瘤细胞和T细胞的结合。PEG-S-Fab增强了血浆稳定性并使S-Fab的半衰期增加了12倍。PEG-S-Fab在异种移植小鼠模型中也具有更有效的肿瘤抑制效力。这些数据表明聚乙二醇化可能是增强双特异性抗体的抗肿瘤特性的有效方法。

Description

具有延长的半衰期和增强的抗肿瘤效果的双特异性抗体 技术领域
本发明涉及双特异性抗体，具体涉及具有延长的半衰期和增强的抗肿瘤效果的聚乙二醇化的双特异性抗体。
背景技术
双特异性抗体是癌症免疫治疗中的一种强有力的手段，其将免疫细胞直接与癌细胞结合。双特异性抗体有两个不同的抗原结合位点，一个识别肿瘤细胞，另一个识别免疫细胞(通常是T细胞或自然杀伤(NK)细胞)。双特异性抗体存在多种形式，包括基于IgG的全长形式(例如完整的双特异性抗体)、基于单链的形式(包括串联单链可变片段(ScFv))和双特异性T细胞衔接体(BiTE)。
为了增强全长IgG抗体的肿瘤组织渗透并提高ScFv和BiTE的稳定性，开发了双特异性单域抗体连接的Fab(S-Fab)形式，其可以结合不同的表位并使用原核表达系统。衍生自天然骆驼重链抗体的单结构域抗体缺乏第一个恒定(CH1)结构域和轻链，因此被称为纳米抗体或VHH。纳米抗体体积小，一般比传统的scFv或BiTE更稳定，使其成为构建双特异性抗体的良好特性。在之前的研究中，通过将单域纳米抗体抗CEA与CD3-Fab连接构建了双特异性S-Fab抗体。S-Fab在体外表现出优异的肿瘤细胞杀伤活性(参见L.Li,P.He,C.Zhou,L.Jing,B.Dong,S.Chen,N.Zhang,Y.Liu,J.Miao,Z.Wang,Q.Li,A novel bispecific antibody,S-Fab,induces potent cancer cell killing,Journal of immunotherapy,38(2015)350-356)。
然而，由于缺乏恒定区(Fc)，Fab片段(包括S-Fab)由于体内快速降解而仍具有短的血浆半衰期，这使得它们不适合长期临床使用。
发明内容
本发明一方面提供一种聚乙二醇化的双特异性抗体(在本文中也称PEG-S-Fab)，包含(a)抗原结合片段(Fab)，其具有轻链可变区(VL)和轻链恒定区(CL)，以及重链可变区(VH)和部分重链恒定区(CH1)；(b)融合至所述抗原结合片段(Fab)的部分重链恒定区(CH1)的C端的单域抗原结合片段(VHH)，以及(c)融合至所述抗原结合片段(Fab)的轻链恒定区(CL)的C端的聚乙二醇(PEG)。本领域技术人员可以根据本领域常规试验操作来测试合适的PEG分子量。在一些实施方式中，所述聚乙二醇的分子量为10000至30000。在另一些实施方式中，所述聚乙二醇的分子量为20000。
相应地，本发明提供包含所述聚乙二醇化的双特异性抗体的药物组合物，所述药物组合物还包含药学上可接受的载体或赋形剂。相应地，本发明提供一种宿主细胞，其包含一个或 多个编码所述的双特异性抗体的多核苷酸。
本发明另一方面提供一种工程化的双特异性抗体，包含(a)抗原结合片段(Fab)，其具有轻链可变区(VL)和轻链恒定区(CL)，以及重链可变区(VH)和部分重链恒定区(CH1)；以及(b)融合至所述抗原结合片段(Fab)的部分重链恒定区(CH1)的C端的单域抗原结合片段(VHH)；其中所述抗原结合片段(Fab)的轻链恒定区(CL)的C端被工程化改造而具有至少一个半胱氨酸残基。相应地，本发明提供所述工程化的双特异性抗体在制备本发明的所述聚乙二醇化的双特异性抗体中的应用。
本发明另一方面提供一种治疗癌症的方法，其包括将本发明所述聚乙二醇化的双特异性抗体与癌细胞接触。相应地，本发明提供一种治疗对象的癌症的方法，其包括将治疗有效量的本发明所述聚乙二醇化的双特异性抗体施用至该对象。
本发明另一方面提供一种宿主细胞，其包含一个或多个编码上述权利要求任一项所述的双特异性抗体的多核苷酸。在一些实施方式中，所述宿主细胞是E.coli。对于多核苷酸的操作涉及分子生物学、基因工程、蛋白质工程等领域的知识和实验操作，这些都是本领域技术人员所熟知的。
发明人在此探索了硫醇位点特异性PEG化以改善S-Fab双特异性抗体的半衰期(t 1/2)。在该研究中，使用官能化的20kDa线性甲氧基PEG马来酰亚胺(MAL-PEG-OMe)缀合S-Fab。聚乙二醇化的S-Fab(PEG-S-Fab)保留了肿瘤细胞和T细胞的结合。PEG-S-Fab增强了血浆稳定性并使S-Fab的半衰期增加了12倍。PEG-S-Fab在异种移植小鼠模型中也具有更有效的肿瘤抑制效力。这些数据表明聚乙二醇化可能是增强双特异性抗体的抗肿瘤特性的有效方法。
附图说明
图1.来自大肠杆菌的S-Fab的表达和纯化。A.细菌S-Fab表达构建体含有pelB信号序列，抗CD3(人类UTCH1克隆)VH(或VL)和CH1(CL)以及抗CEA-VHH。为了便于抗体检测和纯化，分别在重链和轻链的C端添加flag标签和his6标签；B.共表达后S-Fab的示意图；C.两步纯化后纯化的S-Fab的考马斯蓝染色的SDS-PAGE；D，凝胶过滤分析显示S-Fab的分子量约为130kD。
图2.使用20kDa线性甲氧基PEG马来酰亚胺(MAL-PEG-OMe)对双特异性S-Fab进行PEG化。以一系列PEG:S-Fab摩尔当量比(0:1、10:1、20:1、40:1和60:1)将S-Fab与官能化PEG反应并在22℃振荡2小时，然后用SDS-PAGE(5ul/样品)检查样品。A.聚乙二醇化过程中不同摩尔比的PEG:S-Fab的S-Fab的考马斯蓝染色。B.具有不同摩尔比的PEG:S-Fab的S-Fab的碘化钡复合物染料染色。C.使用抗Flag抗体检测VH-CH-CEA链的Western印 迹。D.使用抗his6抗体检测VL-CL链的Western印迹。注意:0、10、20、40和60表示PEG:Fab的摩尔比的0:1、10:1、20:1、40:1和60:1。
图3.PEG-S-Fab的纯化。使用凝胶过滤来分离聚乙二醇化混合物(级分1、2、3)。A.PEG-S-Fab的色谱图。B.PEG-S-Fab纯化的级分的考马斯蓝染色。C.PEG-S-Fab纯化的级分的碘化钡复合物染色。
图4.PEG-S-Fab可以结合肿瘤细胞上的CEA和T细胞上的CD3 +。在CEA阳性LS174T细胞(A)，CEA阴性SKOV3细胞(B)和CD3 +T细胞(C)上进行使用PEG-S-Fab和S-Fab的流式细胞术分析。阳性对照抗CD3抗体OKT3用于T细胞流式细胞术。分别在LS174T细胞(上图)和SKOV3细胞(下图)中进行S-Fab(D)和PEG-S-Fab(E)的免疫荧光染色的共聚焦显微镜。比例尺代表30μm。
图5.PEG-S-Fab对肿瘤细胞具有有效的特异性细胞毒性。A.使用CEA阳性LS174T细胞的细胞毒性测定。B.使用CEA阴性SKOV3细胞的细胞毒性测定。用肿瘤细胞和效应T细胞(E/T＝10)孵育不同浓度的S-Fab或PEG-S-Fab。所有的数据都显示为一式三份的平均值，误差线表示SD。
图6.PEG-S-Fab和S-Fab的血浆浓度的药代动力学和稳定性分析。A.定量ELISA的标准曲线。B.静脉内给药后的血清蛋白浓度-时间曲线。(注:每个数据点表示为平均值±SEM)。C-D.使用抗Flag抗体进行Western印迹以检测产生的S-Fab(C)和PEG-S-Fab(D)。E-F.使用抗his抗体进行Western印迹以检测产生的S-Fab(E)和PEG-S-Fab(F)(注:M、P-S-Fab和P分别表示标记物、PEG-S-Fab和仅血浆)。
图7.S-Fab的聚乙二醇化诱导更有效的体内抗肿瘤活性。B-NDG小鼠(每组n＝6)皮下移植LS174T细胞和人PBMC。然后在6天内每天仅用PBS、0.3nmol的S-Fab或S-Fab-PEG20K每天处理小鼠。数据代表6只小鼠的平均肿瘤体积。误差线表示SEM。使用GraphPad Prism 5软件通过双向ANOVA分析分析数据，并且***表示当将载体组与S-Fab或PEG-S-Fab组进行比较时p<0.01。
具体实施方式
定义
应当注意的是术语“一种”实体是指一个或多个该实体，例如，“一种双特异性抗体”被理解为代表一个或多个双特异性抗体。同样地，这些术语“一种”、“一个或多个”和“至少一个”在此可互换使用。
本文所用的“抗体”或“抗原结合多肽”是指特异性地识别和结合到一种或多种抗原的多肽或多肽复合物。抗体是全抗体和其任何抗原结合片段或单链。因此，术语“抗体”包括 至少含有免疫球蛋白一部分的分子的任何蛋白或肽，此免疫球蛋白分子的一部分具有结合至抗原的生物活性。这样的例子包括，但不限于，重/轻链或其配体结合部分的互补性决定区(CDR)、重链或轻链可变区、重链或轻链恒定区、构架(FR)区或其任何部分、或结合蛋白的至少一部分。术语抗体也包含一旦激活即具备抗原结合能力的多肽或多肽复合物。在一些例子中，例如，源自骆驼科或基于骆驼科免疫球蛋白的工程化的某些免疫球蛋白分子，完整免疫球蛋白分子可能仅由重链组成，而不具有轻链，参见例如Hamers-Casterman et al.,Nature 363:446-448(1993)。
“特异性结合”或“对……有特异性”通常是指抗体通过其抗原结合域结合至表位，并且该结合需要抗原结合域和表位之间的一定的互补性。根据这个定义，当抗体经由其抗原结合域相比于结合至随机的不相关的表位而言能够更快速地结合至特定表位，则称该抗体“特异性结合”至该表位。术语“特异性”被用来定量特定抗体结合特定表位的相对亲和性。例如，对于给定的表位，抗体“A”被视为比抗体“B”具有更高的特异性，或者抗体“A”结合至表位“C”的特异性高于其结合至相关的表位“D”的特异性。
本文使用的术语“治疗”是指治疗性或预防性手段，其中对象被预防或减慢(缓解)不期望的病理学变化或紊乱，例如癌症的进展。有益的或期望的临床结果包括，但不限于，症状的减轻、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和以及缓解(无论是局部的或整体的)，无论这些结果是否是可检测的或不可检测的。“治疗”还指与不接受治疗预期的存活期相比存活期延长。需要治疗的对象包括已患有疾病或病症的对象以及倾向于患有该疾病或病症的对象或需要预防该疾病或病症的对象。
“对象”或“个体”或“动物”或“患者”或“哺乳动物”是指任何期望诊断、预后或治疗的对象，特别是哺乳动物对象。哺乳动物对象包括人、驯养动物、农畜以及动物园动物、竞技动物或宠物，例如狗、猫、豚鼠、兔、大鼠、小鼠、马、牛、奶牛等。
术语“有治疗需要的患者”或“有治疗需要的对象”包括可从施用本发明的抗体或组合物中受益的对象，例如哺乳动物对象，以实现例如检测、诊断程序和/或治疗的目的。
如试验例中所证实的，双特异性抗体的示意性类型是靶定两个不同抗原的抗体，其中一个抗原存在于肿瘤细胞或微生物上，另一个存在于免疫细胞上。当施用至个体时，这种双特异性抗体特异性地结合至肿瘤细胞或微生物，同时特异性地结合至免疫细胞(如细胞毒细胞)。这种双重结合可导致所结合的肿瘤或微生物被宿主的免疫系统杀死。
“单域抗原结合片段”或“单域抗体片段”或“VHH”是一种能够结合至抗原而不需配备轻链的抗原结合片段。VHH最初以单个抗原结合片段的形式分离自单域抗体 (sdAb)。第一个已知的单域抗体分离自骆驼(Hamers-Casterman et al.,Nature 363:446-8(1993))，之后分离自软骨鱼。骆驼产生没有轻链的功能性抗体，它们的单个N端结构域(VHH)结合抗原而无需结构域配对(见Harmsen and Haard,App Microbiol Biotechnol.,77:13-22(2007)综述)。单域抗体不包括CH1域，在常规抗体中，CH1域与轻链相互作用。
VHH包含构成免疫球蛋白结构域的核心结构的四个框架区(FR1-FR4)以及涉及抗原结合的三个互补决定区(CDR1-CDR3)。相比于人VH结构域，VHH框架区显示出与人VH结构域的高序列同源性(>80％)。参见Harmsen and Haard,2007，其进一步描述称：“VHH的最特征性的特点在于在四个FR2位置(第37、44、45和47位；Kabat编号)处的氨基酸取代，它们在常规VH结构域中是保守的，并且涉及与VL结构域的疏水相互作用”。VHH通常具有在这些以及其他在常规VH中高度保守的位置处的不同氨基酸(如Leu11Ser、Val37Phe或Tyr、Gly44Glu、Leu45Arg或Cys、Trp47Gly)。
Harmsen and Haard,2007还描述了：VHH的CDR具有某些已知的特征性特点。例如，CDR1的N端部分比常规抗体更加可变。而且，某些VHH具有延长的CDR3，其通常通过与CDR1或FR2中的半胱氨酸形成额外的二硫键而稳定，导致CDR3环在之前的VL的整个界面上折叠。美洲驼VHH的特定亚家族(VHH3)还含有一个延长的CDR3，其通过与FR2的第50位的半胱氨酸形成额外的二硫键而稳定。
本领域已知有许多单域抗体(sdAb)，并且能够从例如骆驼等动物中容易制备。基于这些sdAb，它们的VHH可轻易被识别和制备。表1列出了许多VHH和sdAb的非限制性例子。因此，在一些实施方式中，本发明提供包含每个这样的公开序列或其等同物的多肽以及编码每个多肽的多核苷酸。
表1.示例性的单域抗原结合片段(VHH)和单域抗体(sdAb)
Figure PCTCN2019081164-appb-000001
在一些实施方式中，双特异性抗体的Fab或VHH(或VHH’)片段具有对肿瘤抗原的免疫特异性。
“肿瘤抗原”是在肿瘤细胞中产生的抗原物质，即，它引起宿主的免疫响应。肿瘤抗原可用于识别肿瘤细胞并且是癌症治疗的潜在候选物质。体内的正常蛋白不是抗原性的。但是，某些蛋白在肿瘤生成过程中产生或过度表达，因而对身体来说显得是“外源”的。这可包括很好地躲避了免疫系统的正常蛋白、通常以极小量产生的蛋白、通常仅在特定发育阶段产生的蛋白或结构由于突变而被修饰的蛋白。
本领域已知有许多肿瘤抗原，通过筛选可容易发现许多新的肿瘤抗原。肿瘤抗原的非限制性例子包括EGFR、Her2、EpCAM、CD20、CD30、CD33、CD47、CD52、CD133、CEA、gpA33、黏蛋白、TAG-72、CIX、PSMA、叶酸结合蛋白、GD2、GD3、GM2、VEGF、VEGFR、整合素、αVβ3、α5β1、ERBB2、ERBB3、MET、IGF1R、EPHA3、TRAILR1、TRAILR2、RANKL、FAP和Tenascin。
在一些方面，Fab或VHH片段具有对相比于相应非肿瘤细胞在肿瘤细胞上过度表达的蛋白的特异性。“相应非肿瘤细胞”是指与肿瘤细胞来源的细胞具有相同细胞类型的非肿瘤细胞。要注意的是，这样的蛋白不必然不同于肿瘤抗原。非限制性的例子包括癌胚抗原(CEA)，其过度表达于大多数结肠癌、直肠癌、乳癌、肺癌、胰腺癌和胃肠道癌；调蛋白受体(HER-2,neu或c-erbB-2)，其通常在乳癌、卵巢癌、直肠癌、肺癌、前列腺癌和宫颈癌中过度表达的；表皮生长因子受体，其在一系列实体肿瘤(包括乳癌、头颈癌、非小细胞性肺癌和前列腺癌)内过量表达；脱唾液酸糖蛋白受体；转铁蛋白受体；在肝细胞上表达的丝氨酸蛋白酶抑制剂酶复合体受体；在胰岛导管腺癌细胞上过度表达的成纤维细胞生长因子受体(FGFR)；用于抗血管新生基因治疗的血管内皮生长因子受体(VEGFR)；选择性地过表达于90％的非黏液型的卵巢癌的叶酸受体；细胞表面的多糖-蛋白复合物(glycocalyx)；碳水化合物受体；以及多聚免疫球蛋白受体，其有益于基因转运至呼吸道上皮细胞，并且对肺病(例如囊胞性纤维症)治疗有吸引力。
在一些方面，Fab部分包括：一个或两个选自SEQ ID NO:14-25(表2)的氨基酸序列，或者任选地具有一个或两个或三个插入、缺失或取代。
表2.Fab序列示例
Figure PCTCN2019081164-appb-000002
Figure PCTCN2019081164-appb-000003
Figure PCTCN2019081164-appb-000004
以上描述的任何的抗体或多肽可进一步地包括额外的多肽，例如，指导所编码的多肽的分泌的信号肽、如本文所述的抗体恒定区或其他如本文所述的异源多肽。
本领域普通技术人员将理解，本文所公开的抗体可被修饰，使得它们在氨基酸序列上不同于它们所其衍生的天然结合多肽。例如，源自一种特定蛋白的多肽或氨基酸序列可以是相似的，例如，对起始序列具有一定百分数的一致性，例如它可能和起始序列具有60％、70％、75％、80％、85％、90％、95％、98％或99％的一致性。
此外，可在“非必需”氨基酸区进行核苷酸或氨基酸的保守性取代、缺失或插入。例 如，源自特定蛋白多肽或氨基酸可以同起始序列一样，除了一个或多个单个的氨基酸取代、插入、或缺失(例如，一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十五个、二十个或更多个单独的氨基酸取代、插入或缺失)。在某些实施方式中，相对于起始序列，源自特定蛋白的多肽或氨基酸序列具有一至五、一至十、或一至二十个单独的氨基酸取代、插入或缺失。
在其他实施方式中，本发明的抗原结合多肽可以包含保守的氨基酸取代。
在“保守的氨基酸取代”中，一个氨基酸残基被具有相似侧链的氨基酸残基取代。具有相似侧链的氨基酸残基家族已经在本领域被定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)、β分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，在免疫球蛋白多肽的非必须氨基酸残基更适宜由来自同样侧链家族的其他氨基酸残基取代。在另一个实施方式中，氨基酸链可以被结构相似但侧链家族成员的顺序或组成上不同的链取代。
下表提供了保守氨基酸取代的非限制的例子。在表中相似性分数0或更高来表明在两种氨基酸的保守取代。
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
在一些实施方式中，抗体可以被结合至治疗性的制剂、前药、肽、蛋白、酶、病毒、脂类、 生物效应调节物或药物制剂。
这些抗体可以被结合或融合至治疗性的制剂，这种治疗性制剂可包括可检测标记(例如放射性标记)、免疫调节剂、激素、酶、寡核苷酸、光敏治疗剂或诊断剂、细胞毒性制剂(药物或毒素)、超声增强剂、非放射性标记、它们的组合和其他本领域已知的制剂。
通过耦合至化学发光的化合物来标记抗体，则该抗体可以被检测。然后，通过检测荧光(出现在化学反应过程中)的存在，来确定化学发光标记的抗原结合多肽的存在。极其有用的化学发光标记化合物的例子是鲁米诺、异鲁米诺、theromatic吖啶酯、咪唑、吖啶盐和草酸酯。
如上所述，本发明的双特异性抗体、其变体或衍生物可用于与癌症或感染性疾病相关的某些治疗和诊断方法中。
治疗及疗法
本发明进一步涉及基于抗体的疗法，其涉及将本发明的双特异性抗体施用至患者，例如动物、哺乳动物和人体，以治疗本文所述的一种或多种疾病或症状。本发明的治疗性组合物包括，但不限于，本发明的抗体(包括本文所述的其变体和衍生物)和编码本发明的抗体(包括本文所述的其变体和衍生物)的核酸或多核苷酸。
本发明的抗体可被用来治疗、抑制或预防以下疾病、失调或病症，包括与例如与增加细胞存活或抑制细胞凋亡相关的疾病或失调(如癌症)相关的恶性疾病、失调或病症，所述癌症包括但不限于滤泡性淋巴瘤、带有p53突变的癌症以及激素依赖的肿瘤(包括但不限于结肠癌、心脏肿瘤、胰腺癌、黑色素瘤、成视网膜瘤、恶性胶质瘤、肺癌、大肠癌、睾丸癌、胃癌、成神经母细胞瘤、粘液瘤、肌瘤、淋巴瘤、内皮瘤、成骨细胞瘤、破骨细胞瘤、骨肉瘤、软骨肉瘤、腺癌、乳腺癌、前列腺癌、Kaposi肉瘤)；自体免疫失调(例如多发性硬化症、Sjogren综合征、Grave病、Hashimoto甲状腺炎、自体免疫糖尿病、胆汁性肝硬变、Behcet病、Crohn病、多肌炎、系统性红斑狼疮、免疫相关的肾小球肾炎、自体免疫的胃炎、自体免疫的血小板减少性紫癜及类风湿性关节炎)；和病毒性感染(例如疱疹病毒、痘病毒及腺病毒)、炎症、移植物抗宿主病(急性和/或慢性)、急性移植物排斥和慢性移植物排斥。本发明的抗原结合多肽、其变体或衍生物被用于抑制癌症的发展、演进和/或转移，特别是在上文或随后的段落中列出的癌症。
本发明的抗体也可通过靶定微生物和免疫细胞来影响微生物的消除而用于治疗由微生物引起的感染性疾病或杀灭微生物。在一个方面，该微生物是病毒(包括RNA和DNA病毒)、革兰氏阳性菌、革兰氏阴性菌、原生动物或真菌。
对于任何特定病人特异的剂量和治疗方案将取决于多种因素(包括所使用的特异抗原结 合多肽、其变体或衍生物，患者的年龄、体重、总体健康、性别、饮食、施用时间、排泄率、药物的联合以及被治疗的特定疾病的严重程度)。医护人员对这样的因素的判断属于本领域普通技术人员判断范围内。该剂量还会基于被治疗的个体病人、施用途径、配方的类型、所使用的组合物的特点、疾病的严重程度及所期待的效果。使用的剂量可通过本领域所公知的药理学和药代动力学的原则决定。
双特异抗体、变体的施用方法包括但不限于：皮内的、肌内的、腹腔的、静脉的、皮下的、鼻内的、硬膜外的和口腔途径。抗原结合多肽或组合物可以通过任何方便途径施用，例如，通过输注或弹丸式注射、通过上皮或黏膜保护层的吸收(例如口腔黏膜、直肠和肠黏膜等等)；它可同其他生物活性制剂一起使用。因此，包含本发明的抗原结合多肽的药用组合物可以经口腔、直肠、肠外、阴道内、腹腔、局部(如通过粉剂、软膏、滴剂或皮肤药贴)、含服施用或是通过口腔或鼻的喷雾施用。
本文所用的术语“肠胃外的”是指施用的方式，这种方式包括静脉内的、肌内的、腹腔的、胸骨内的、皮下的和关节内的注射和输注。
施用可以是系统的或局部的。此外，可预期通过任何合适途径将本发明的抗体引入至中枢神经系统，这些途径包括：脑室内和鞘内的注射；通过脑室内导管(例如连接至储存器(例如Ommaya储存器))可以促进脑室内的注射。也可采用肺部给药，例如通过使用吸入器或喷雾器及带有气雾剂的配方给药。
将本发明的双特异抗体或组合物局部施用至需要治疗的区域可能是合乎需要的；这可通过例如(但不限于)以下方式实现：手术中局部输注、局部施用(例如在术后联合使用伤口敷料)、注射、通过导管、通过栓剂或通过植入物(植入物为多孔的、非多孔的、非渗透的或凝胶状的材料，包括薄膜(例如硅胶膜)或纤维))。优选地，当给予包括本发明的蛋白(包括抗体)时，需注意使用此蛋白不吸附的材料。
本发明的抗体在炎症、免疫或恶性疾病、失调或病症的治疗、抑制和预防中有效量，可以通过标准的临床技术来确定。此外，可以选择性地采用体外测定来帮助识别最佳剂量范围。在配方中采用的准确的剂量也将取决于给药的途径以及疾病、失调、或病症的严重性，并且应当根据执业医生的判断和每一个患者的情况来决定。有效剂量可以从源自体外的或动物模型测试系统的剂量-反应曲线推论出。
作为一般性的建议，给予患者的本发明的抗原结合多肽的剂量通常为0.1mg/kg至100mg/kg患者体重、0.1mg/kg至20mg/kg患者体重，或1mg/kg至10mg/kg患者体重。总体来说，由于对外源的多肽的免疫反应，在人体内，人的抗体比来自其他种属的抗体具有更长的半衰期。因此，人抗体的较低给药剂量和更低给药频率通常是可能的。而且，本发明的抗 体的给药频率和剂量可通过修饰(例如脂化)而增强这些抗体的摄入和组织穿透力(例如进入大脑)来降低。
用于治疗感染或恶性疾病、病症或失调的方法(包含给予本发明的抗体、其变体或衍生物)，在用于人体前，通常在体外被测试，然后在可接受动物模型中进行体内测试，以获得期待的治疗或预防活性。合适的动物模型(包括转基因动物)对本领域普通技术人员是熟知的。例如，表明本文所述的抗原结合多肽的治疗效用的体外实验包括抗原结合多肽在细胞系或病人组织样品上的效果。利用本领域技术人员所知的技术(例如在本文其他地方公开的试验)可确定抗原结合多肽在细胞系和/或组织样品上的效果。根据本发明，可被用来确定是否需要使用特异性的抗原结合多肽的体外试验包括体外细胞培养试验(其中病人组织样品在培养基中生长并被暴露于或以其他方式给予抗体)以及观察这种抗体在该组织样品上的效果。
在进一步的实施方式中，本发明的组合物与抗肿瘤剂、抗病毒剂、抗菌剂或抗生素制剂或抗真菌制剂联合施用。任何这些在本领域已知的制剂都可以在本发明的组合物中被给予。
在另一个实施方式中，本发明的组合物与化疗制剂联合给予。可与本发明的组合物一起给予的化疗制剂包括但不限于：抗生素衍生物(例如阿霉素、博来霉素、道诺霉素、防线菌素)、抗雌激素(例如他莫昔芬)、抗代谢物(例如氟尿嘧啶、5-FU、氨甲嘌呤、氟尿苷、干扰素α-2b、谷氨酸、普卡霉素、巯嘌呤和6-巯基鸟嘌呤)、细胞毒剂(例如卡莫司汀、BCNU、洛莫司汀、CCNU、阿糖胞苷、环磷酰胺、雌莫司汀、羟基脲、甲苄肼、丝裂霉素、白消安、顺铂及长春新碱硫酸盐)、激素(例如甲羟孕酮、雌莫司汀磷酸钠、乙炔雌二醇、雌二醇、醋酸甲地孕酮、甲羟睾酮、二磷酸己烯雌酚、氯烯雌醚及睾内酯)、氮芥衍生物(苯丙氨酸氮芥、苯丁酸氮芥、二氯甲基二乙胺(氮芥)、噻替哌)、类固醇类及制品(倍他米松磷酸钠)及其他(例如达卡巴嗪、天冬酰胺酶、米托坦、长春新碱硫化物、长春花碱硫化物和依托泊苷)。
在另外的实施方式中，本发明的组合物与细胞因子联合给予。可与本发明的组合物一起被给予的细胞因子包括但不限于：IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、抗-CD40、CD40L和TNF-α。
在另外的实施方式中，本发明的组合物与其他治疗或预防疗法(例如放射治疗)联合给予。
药物组合物
本发明还提供了药物组合物。这样的组合物包含有效量的抗体及可接受的载体。在一个具体实施方式中，术语“药学上可接受的”意为由联邦的或州政府的监管机构许可的、或在美国 药典或其他通常公认的药典所列的用于动物，更具体来说，用于人的。进一步地，“药学上可接受载体”通常将是无毒的固态的、半固态的或液态的填充剂、稀释剂、包封材料或任何类型的辅料。
术语“载体”是指药物使用所借助的稀释剂、佐剂、赋形剂或载体。这样的药物载体可以是无菌的液体，例如水和油，包括石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。当药物组合物被静脉注射地给药时，水是首选载体。盐溶液及水性的葡萄糖与甘油溶液也可以作为液相的载体被采用，特别是对可注射的溶液来说。合适的药物赋形剂包括：淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、丙三醇、丙烯、乙二醇、水、乙醇等。如果需要，组合物也可以包含少量的润湿或乳化剂或pH缓冲剂，例如醋酸盐、柠檬酸盐或磷酸盐。也可预期加入抗菌制剂(例如苄醇或苯甲酸甲酯)；抗氧化剂(例如抗坏血酸或亚硫酸氢钠)；螯合剂(乙二胺四乙酸)和用于等张性调节的制剂(例如氯化钠或右旋糖)。这些组合物可以采取溶液、悬浮液、药片、药丸、胶囊、粉末、缓释配方等形式。该组合物可使用惯常的粘合剂和载体(例如甘油三酯)而作为栓剂配制。口服配方可包括标准的载体，例如，药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等等。在Remington's Pharmaceutical Sciences中由E.W.Martin描述合适的药物载体的例子(通过引用将其合并至本中)。这样的组合物将包含治疗上有效量的抗原结合多肽(较好为纯化的形式)与合适数量的载体，以便为患者提供合适的给药方式。这种配方应适合给药的方式。这种亲代(parental)制剂可装于安瓿瓶、一次性注射器或是玻璃或塑料制成的多剂量瓶中。
在一种实施方式中，根据例行程序将该组合物配制成适于静脉给药于人类的药物组合物。通常，用于静脉给药的组合物为在无菌等渗的水性缓冲液中的溶液。在必要时，组合物也可包括增溶剂和局部麻醉药(例如利多卡因，以减轻注射部位疼痛)。组分通常是单独或是混合在一起以单位剂量形式提供，例如，在密闭容器(例如标明活性制剂的数量的安瓿或sachette)中的冻干粉剂或无水浓缩物。当组合物通过输注给予时，它可分散于含有无菌的药物级水或盐水的输液瓶中。当组合物通过注射给予时，可提供一个安瓿的用于注射的无菌水或盐水，以便在给药前此成分可以被混合。
本发明的组合物可以被配制成中性的或盐的形式。药学上可接受的盐包括由阴离子形成的盐，例如那些源于盐酸、磷酸、乙酸、草酸、酒石酸等的盐，及由阳离子形成的盐，例如那些源于钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、乙基羟乙胺、组氨酸、普鲁卡因等的盐。
实施例
S-Fab设计和蛋白质纯化
S-Fab的结构如图1A所示。合成抗CD3的VH-CH1和VL-CL片段并通过标准DNA克隆技术克隆。将信号序列pelB添加至N端用于周质表达。对于PEG的位点特异性缀合，在轻链的C端添加半胱氨酸残基，接着是短接头及另一个半胱氨酸(CGGGGC)和his6标签。通过VL-CL/VH-CH1(抗CD3Fab)与抗CEA VHH纳米抗体的异源二聚化构建S-Fab。Flag-tag被添加到重链的C-末端用于检测(图1B)。
为了产生S-Fab，将编码各自VH-CH1-VHH和VL-CL多肽的两种质粒共转化到具有适当抗生素的BL21(DE3，codon plus)感受态细胞中。当细胞培养物的吸光度(OD600)达到0.8时，加入0.2mM异丙基-β-D-硫代半乳糖苷(IPTG)以诱导蛋白质表达。收获前将细胞在16℃再培养40小时。通过离心收获细胞后，通过将细胞沉淀物以1:4(g:mL)重悬于预先冷却的蔗糖溶液(20mM Tris-HCl pH 7.5；25％(w/v)蔗糖；1mM EDTA)中进行周质提取。在冰上温育15分钟后，将悬浮液以10,000g离心20分钟，并收集上清液部分作为蔗糖部分。然后将沉淀重新悬浮在冰冷的周质溶液(5mM MgCl 2)中并以10,000g离心20分钟。收集上清液作为周质部分。然后通过两步纯化从合并的蔗糖和周质部分纯化S-Fab蛋白:首先通过固定化Ni-NTA亲和层析(GE Health，USA)，然后通过IgG-CH1亲和基质(Lot 194320005；ThermoFisher Scientific Inc，USA)(图1C)。使用Bio-Rad FPLP系统和GE Superdex 200TM增量10/300GL柱以0.5ml/min的流速进行凝胶过滤分析。收集级分(每个级分0.5ml)，然后在还原条件下进行SDS-PAGE分析。考马斯蓝染色。蛋白质标记物(Lot MWGF200；Sigma Aldrich Co.，Ltd，USA)作为凝胶过滤分析的标准对照。凝胶过滤分析显示分子量为～130kD的完整S-Fab抗体(图1D)，表明S-Fab的二聚化(约65kD单体)，这可能是由VL-CL的C端的半胱氨酸残基的二硫键形成的。
S-Fab与PEG的缀合(聚乙二醇化)
对S-Fab进行工程改造，使其在CL的C末端具有两个末端半胱氨酸残基，用作与20kDa线性MAL-PEG-OMe缀合的位点。在5.0mL磷酸盐缓冲盐水(PBS，pH7.4)中的S-Fab(约1.35mg/mL(约20μM))和三摩尔当量的1mM三(2-羧乙基)膦(TCEP，最终60μM，约300μL)混合并在22℃下温育2小时以获得还原的S-Fab片段。
为了探索聚乙二醇化过程中MAL-PEG-OMe和S-Fab的最佳摩尔比，我们进行了一系列反应，其中PEG:S-Fab的摩尔当量为0:1,10:1,20:1,40:1和60:1(图2A)。将MAL-PEG-OMe溶于无菌水中以获得20mg/mL(1mM)的工作浓度。S-Fab的聚乙二醇化通过将MAL-PEG-OMe(在工作浓度下)与还原的S-Fab混合并在22℃振荡2小时来进行。将所得样品进行12％SDS-PAGE电泳(5ul/样品/PAGE)，然后根据参考文献对PEG进行考马斯蓝和碘化钡染 色(图2A和2B)。电泳后，使用Western印迹试验检测聚乙二醇化链。简言之，将另外两种凝胶转移至聚偏氟乙烯膜(Millipore，USA)。用5％脱脂牛奶封闭2小时后，将膜分别与5％脱脂牛奶中的小鼠单克隆抗Flag HRP(1:2000，对于重链)和小鼠单克隆抗his IgG(1:3000，对于轻链)温育。用TBST缓冲液洗涤后，将二抗(山羊抗小鼠HRP-缀合的IgG，1:3000)与轻链膜温育另外1小时。用TBST缓冲液洗涤后，用Pierce's West Pico化学发光底物(Millipore，USA)使膜显影。使用AKTA avant25FPLC纯化系统(GE Healthware,USA)和Superdex 10/300GL柱以0.8ml/min的流速纯化聚乙二醇化的S-Fab。该柱首先用两个柱体积(CV)的蒸馏水平衡，然后在施加样品之前用两个CV的PBS平衡。在还原条件下的SDS-PAGE后，通过考马斯蓝和碘化钡复合物染料分析所有收集的级分。将纯化的聚乙二醇化S-Fab的级分合并在一起。
在用MAL-PEG-OMe对S-Fab进行聚乙二醇化中，增加的PEG:S-Fab的比例导致形成较高的分子量带(～107kD和～45kD)，表明马来酰亚胺官能化的PEG与S-Fab缀合。45kD条带提示在单个C端半胱氨酸处的单一偶联(图2B和2D)。～107kD指示在VL-CL末端处的两个半胱氨酸残基都被聚乙二醇化(图2B和2D)。～107kD条带首先出现并占大部分轻链，表明在两个半胱氨酸残基上优选的缀合(图2B和2D)。由于没有观察到VH-CH1-VHH的减少(图2A和2C)，并且基于Western印迹未观察到高分子量的VH-CH1-VHH(图2C)，表明在VH-CH1-VHH链上没有PEG化。由于VL-CL链的条带减少(图2A和2D)，并且基于抗-his Western印迹PEG化对应于VL-CL链(图2B和2D)，它表明只有VL-CL具有聚乙二醇化。随着PEG:S-Fab的摩尔比增加，较高分子量PEG化条带也略有增加，表明在VH-CH1或VL-CL上的其他半胱氨酸残基上进一步聚乙二醇化。然后选择PEG:S-Fab的摩尔比为20时用于缀合，因为它具有较高VL-CL缀合而没有更高分子量缀合。
为除去游离PEG、游离S-Fab和高分子量蛋白质，对缀合反应混合物进行尺寸排阻分析。基于SDS-PAGE及考马斯蓝染色(图3B)和聚乙二醇化碘化钡染色(图3C)，高分子量级分1(图3A-3C)含有大比例的多种缀合物质。组分2(图3A-3C)主要含有双位点和单位点缀合的PEG-S-Fab，而组分3含有游离S-Fab、游离PEG和单位点缀合的S-Fab(图3A-3C)。合并主要含有双位点和单位点缀合的PEG-S-Fab(图3中的级分2)的级分用于进一步研究。
PEG-S-Fab可以结合肿瘤抗原CEA和CD3 +T细胞
双特异性S-Fab具有两个不同的结合位点，识别肿瘤细胞上的CEA的抗CEA VHH和识别T细胞上的抗CD3识别CD3 +。为了检查S-Fab的聚乙二醇化是否影响CEA阳性癌细胞的结合，使用具有CEA超表达的LS174T细胞进行流式细胞术分析。使用CEA阴性细胞系SKOV3作为阴性对照。
简言之，通过以1000rpm离心5分钟收集1x10 6(对于LS174T和SKOV3)或5x10 5(对于T细胞)细胞/样品，然后用1.0mL含有0.2％牛血清白蛋白(BSA)的冰冷PBS洗涤一次。加入包括S-Fab、PEG-S-Fab和空白对照(载体，仅PBS)的初级抗体至最终浓度为10μg/mL，然后在冰上孵育1小时，随后用含有0.1％BSA的冰冷PBS洗涤2次。使用抗CD3FITC(OKT3，终浓度10μg/mL)作为CD3 +抗原结合分析的阳性对照。然后加入山羊抗人IgG(H+L)-AlexaFluor 488抗体至终浓度为5μg/mL。将细胞在冰上温育1小时。将细胞洗涤两次后，进行流式细胞术检测。
S-Fab和PEG-S-Fab都表现出明显的特异性荧光强度变化，表明PEG-S-Fab仍然可以与LS174T细胞结合(图4A)。PEG-S-Fab稍微左移至S-Fab的值(图4B)，表明S-Fab的PEG化可能略微降低S-Fab对其抗原的结合亲和力。对于SKOV3细胞，由于细胞表面缺乏CEA抗原，S-Fab和PEG-S-Fab都不能结合细胞，表明PEG-S-Fab保留了特异性抗原结合(图4B)。同样，对于T细胞上的CD3 +、S-Fab和PEG-S-Fab还显示了明显的荧光强度变化。聚乙二醇化也可以略微降低对T细胞上CD3 +抗原的结合活性(图4C)。PEG-S-Fab的仅轻微降低的结合活性表明，在S-Fab中间的位点特异性偶联对双特异性抗体结合亲和力具有最小影响。
为了进一步分析S-Fab和PEG-S-Fab与细胞表面CEA的结合，进行免疫荧光测定。简言之，将LS174T和SKOV3细胞(分别2.5x10 5个细胞/1.0mL)接种在30mm共聚焦玻璃底部培养皿上(NEST，目录号801002)至80％汇合。然后用冷PBS洗涤细胞三次，然后用4％多聚甲醛固定。将固定的细胞分别与20μg S-Fab或PEG-S-Fab温育，然后与10μg山羊抗人IgG(H+L)-AlexaFluor 488抗体在4℃温育2小时。细胞核用DAPI复染。用PBS洗涤后，使用Olympus FV3000激光扫描共聚焦显微镜检查样品，并通过Olympus FV31S-SW_V2.1软件分析。
S-Fab(图4D中的上图)和PEG-S-Fab(图4E中的上图)都可以结合CEA阳性LS174T细胞。对于CEA阳性SKOV3细胞，S-Fab(图4D中的下图)或PEG-S-Fab(图4E中的下图)都没有观察到结合，进一步支持了PEG化保持S-Fab对其抗原的特异性结合亲和力。
PEG-S-Fab对肿瘤细胞具有有效的特异性细胞毒性
使用CEA阳性人类LS174T细胞和CEA阴性人类SKOV3细胞来评估S-Fab和PEG-S-Fab的体外生长抑制作用。简言之，将LS174T和SKOV3细胞用作靶细胞(T)，并将未经刺激的新制备的人CD3 +T细胞用作效应细胞(E)。在96孔微量培养板中每孔接种在100μL相应培养基中的5,000个靶细胞，进行体外细胞毒性测定，一式三份。孵育6小时后，将等体积的CD3 +T细胞以10:1的E:T比率加入到每个孔中，然后相应地加入一系列浓度(0.033、0.1、0.33、1、3.3、10、33和100nM)的S-Fab或PEG-S-Fab。培养72小时后，根据制造商方案 通过CCK8测定评估细胞活力。使用TECAN酶标仪在450nm处检测吸光度值。存活率(100％)计算为:[(As-Ab)/(A0-Ab)]×100％，其中As为测量组的吸收值，Ab为介质的吸收值，A0为测量组在0nM的吸光度值。
S-Fab和PEG-S-Fab都可以有效杀伤CEA阳性LS174T细胞，即使在0.033nM(图5A)，而对CEA阴性SKOV3细胞没有细胞毒性(图5B)。PEG-S-Fab比S-Fab具有略微降低的细胞毒性(图5A)。这可能是由于抗原结合力略微降低或聚乙二醇化的空间干扰导致的。然而，活性降低的水平似乎远低于许多其他PEG化蛋白。
聚乙二醇化延长了S-Fab的体内半衰期
为了确定PEG-S-Fab的循环半衰期，分析了S-Fab和PEG-S-Fab在大鼠中的静脉内PK曲线。PK测定使用SPF雄性SD大鼠(250-300g)。控制食物以维持动物体重低于350克。通过尾静脉施用S-Fab(1.0mg/kg)、PEG-S-Fab(1.0mg/kg)或体积当量的载体PBS。在给药后0、0.5、1、2、4、8、16、24、36、48、72、96和144小时，在异氟烷麻醉下，使用毛细管从眼窝静脉取血样(各约150-200μL)。将所有血液样品收集到肝素化的管中。通过在3,500g离心30分钟获得血浆，然后储存在-80℃直至进一步分析。
使用上述ELISA测定法定量血浆样品中的S-Fab和PEG-S-Fab。简言之，将100μL的6D6等分试样(小鼠抗人IgG Fab抗体)(PBS中1.0μg/mL)涂布于96孔ELISA微孔板(ThermoFisher，USA)的每个孔中，37℃下孵育2小时。然后用200μL的PBST(PBS+0.05％Tween-20)洗涤孔两次。然后将孔用200μL封闭缓冲液(含有1％牛血清白蛋白的PBST)在37℃封闭2小时。然后在添加100μL样品或标准品之前，每个孔用PBST洗涤5次。在封闭缓冲液中制备样品和标准品(100、80、50、40、30、20、10、5、1和0.1μg/mL)，其中标准品(S-Fab)在使用PBS以1:10稀释的血浆中制备，这对于避免测定中的基质效应很重要。对于血浆样品，使用1:3稀释液。然后加入100μL样品或标准等分试样，并在37℃下孵育1小时，一式三份。每孔加入100μL第二抗体(1:500稀释度的小鼠单克隆抗Flag过氧化物酶(HRP)抗体)，每孔再次用PBST洗涤1小时。洗涤5次后，每孔加入100μL等分的TMB底物溶液。孵育10分钟后，加入100μL 2M H 2SO 4以终止反应。然后使用TECAN ELISA酶标仪在450nm处检测吸光度。使用标准公式通过3p97药代动力学软件计算血清消除t 1/2和清除率。结果以平均值±SEM表示，组间比较采用不成对的Student's t-检验。如果p<0.05，则差异被认为是统计学显著的。
使用ELISA方法定量S-Fab和PEG-S-Fab。一系列标准品的定量显示标准曲线为y＝0.0934x+0.0748(0≤x≤25μg/mL)，R 2值为0.9982(图6A)。基于直至144小时的血清蛋白浓度，S-Fab的体内半衰期约为3.0小时，与报道的其他Fab体内半衰期相似。聚乙二醇化 显着延长了PEG-S-Fab的半衰期至36.0小时，这是S-Fab的12倍(图6B)。
发明人还评估了S-Fab和PEG-S-Fab在人新鲜血浆中在两周内的稳定性。简言之，人新鲜血浆(无血小板)稀释S-Fab和PEG-S-Fab，产生100μg/mL的初始浓度。样品在37℃孵育两周。在0、24、48、72、96、168、264和336小时的时间间隔，收集40μL样品，然后直接在-80℃下储存直至进一步分析。将样品在冰上解冻，然后在4℃以14,000rpm离心10分钟。然后将上清液在12％SDS-PAGE(每孔5ul样品)上进行电泳。电泳后，进行western印迹以分析蛋白质水平。当与人血浆在37℃体外孵育时，72小时后S-Fab水平急剧下降(图6C和6E)。对于PEG-S-Fab，降低速度要慢得多(图6D和6F)。因此，聚乙二醇化也可以提高血浆中S-Fab的稳定性，这可能是由于保护其不受血浆中的酶消化导致的。
PEG-S-Fab诱导更有效的体内抗肿瘤活性
PEG-S-Fab体内半衰期的增加和相当的体外细胞毒性促使我们评估了PEG-S-Fab在过继性异种移植模型中的体内抗肿瘤活性。使用LS174T细胞皮下移植的NOD/SCID小鼠研究S-Fab和PEG-S-Fab的体内抗肿瘤活性。简言之，收获LS174T细胞并用PBS洗涤一次，然后与从健康供体新鲜分离的人类PBMC混合。将1×10 6个LS174T细胞和5×10 6个人类PBMC的混合物皮下注射到NOD/SCID小鼠的右侧，总体积为每只小鼠0.2mL。植入后1小时，腹膜内注射～0.3nmol S-Fab(每只小鼠20.0μg)和～0.3nmol PEG-S-Fab(每只小鼠32.0μg)或载体对照(PBS)。然后在接下来的六天中每天处理动物(每组中0.3nM每只小鼠)。用两个垂直尺寸的游标卡尺测量肿瘤体积，并使用公式(宽度 2×长度)/2计算肿瘤体积。所有数据均表示为每组的平均值±SE，组间差异通过使用GraphPad Prism 5软件的双向ANOVA来确定。
结果显示，当NOD/SCID小鼠移植LS174T细胞和新鲜分离的人PBMC时，观察到快速的肿瘤生长。与载体组相比，当在人PBMC存在下用S-Fab或PEG-S-Fab处理小鼠时观察到显著的肿瘤生长抑制(p<0.01)(图7)。与S-Fab相比，在存在PEG-S-Fab时肿瘤生长抑制更显著(p<0.01)，表明S-Fab蛋白的聚乙二醇化可增强S-Fab在异种移植小鼠模型中的治疗功效。
说明书中引用的所有专利和其他参考文献都是本发明所属领域普通技术人员的水平的表示，通过引用将它们以整体形式合并至本文中，包括其中的任何表格和附图，就如同每个参考文献都单独通过引用以其整体形式合并至本文中一样。
本领域技术人员会容易意识到，本发明可容易改造而获得本文所述的那些目的和优点以及隐含在本文中的那些目的和优点。在本文中以当前优选实施方式的代表的形式描述的方法、变体和组合物是示例性的，并不意在限制本发明的范围。对于本领域技术人员来说，可对它们做出改变或将其用于其他用途，但这都包括在如所附权利要求定义的本发明的范围 内。
此外，当以马库什群组或其他替代物组群形式描述本发明的一些特征或方面时，本领域技术人员会意识到，本发明也以该马库什群组或其他群组的任何单个成员或成员的子群组的形式被描述。
而且，除非有相反表示，否则当一些实施方式中提供了各种数量值时，通过取任何两个不同的值作为一个范围值的端点，来描述其他实施方式。这些范围值也在本发明的范围之内。
在适当时候，本文中解释性地描述的内容可在缺少未在本文具体公开的任何一个或多个元件、一个或多个限制条件的情况下实施。因此，例如，在本文的每种情况下，术语“包括”、“实质上由…组成”以及“由…组成”中的任何一个都可被其他两个术语替代。因此，要理解的是，虽然本发明已通过优选实施方式和任选的特征具体公开，但本领域技术人员可对本文公开的想法做出修改和变化，而这些修改和变化仍属于所附权利要求定义出的本发明的范围之内。

Claims (15)

  1. 一种聚乙二醇化的双特异性抗体，包含
    (a)抗原结合片段(Fab)，其具有轻链可变区(VL)和轻链恒定区(CL)，以及重链可变区(VH)和部分重链恒定区(CH1)；
    (b)融合至所述抗原结合片段(Fab)的部分重链恒定区(CH1)的C端的单域抗原结合片段(VHH)，以及
    (c)融合至所述抗原结合片段(Fab)的轻链恒定区(CL)的C端的聚乙二醇(PEG)。
  2. 根据权利要求1所述的聚乙二醇化的双特异性抗体，其中所述聚乙二醇通过所述轻链恒定区(CL)的C端的至少一个半胱氨酸残基连接至所述轻链恒定区(CL)的C端。
  3. 根据权利要求2所述的聚乙二醇化的双特异性抗体，其中所述聚乙二醇通过所述轻链恒定区(CL)的C端具有两个半胱氨酸残基连接至所述轻链恒定区(CL)的C端时，所述两个半胱氨酸残基之间间隔有接头序列。
  4. 根据权利要求1至3中的任一项所述的聚乙二醇化的双特异性抗体，其中所述聚乙二醇的分子量为10000至30000。
  5. 根据权利要求4所述的聚乙二醇化的双特异性抗体，其中所述聚乙二醇的分子量为20000。
  6. 根据权利要求1至3中的任一项所述的聚乙二醇化的双特异性抗体，其中所述抗原结合片段对肿瘤抗原具有特异性，并且所述单域抗原结合片段对免疫细胞具有特异性。
  7. 根据权利要求6所述的聚乙二醇化的双特异性抗体，其中所述肿瘤抗原选自CEA、EGFR、Her2、EpCAM、CD20、CD30、CD33、CD47、CD52、CD133、CEA、gpA33、黏蛋白、TAG-72、CIX、PSMA、叶酸结合蛋白、GD2、GD3、GM2、VEGF、VEGFR、整合素、αVβ3、α5β1、ERBB2、ERBB3、MET、IGF1R、EPHA3、TRAILR1、TRAILR2、RANKL、FAP和Tenascin。
  8. 根据权利要求7所述的聚乙二醇化的双特异性抗体，其中所述肿瘤抗原为CEA或Her2。
  9. 根据权利要求6所述的聚乙二醇化的双特异性抗体，其中所述单域抗原结合片段对哺乳动物T细胞或哺乳动物NK细胞具有特异性。
  10. 根据权利要求6所述的聚乙二醇化的双特异性抗体，其中所述单域抗原结合片段对选自CD3、CD16、CD19、CD28和CD64的任一种抗原具有特异性。
  11. 根据权利要求10所述的聚乙二醇化的双特异性抗体，其中所述抗原为CD16或CD3。
  12. 一种工程化的双特异性抗体，包含
    (a)抗原结合片段(Fab)，其具有轻链可变区(VL)和轻链恒定区(CL)，以及重链可变区(VH)和部分重链恒定区(CH1)；以及
    (b)融合至所述抗原结合片段(Fab)的部分重链恒定区(CH1)的C端的单域抗原结合片段(VHH)；
    其中所述抗原结合片段(Fab)的轻链恒定区(CL)的C端被工程化改造而具有1或2个半胱氨酸残基。
  13. 根据权利要求12所述的工程化的双特异性抗体，其中当具有2个半胱氨酸残基时，它们之间间隔有接头序列。
  14. 一种宿主细胞，其包含一个或多个编码权利要求1-13任一项所述的双特异性抗体的多核苷酸。
  15. 一种药物组合物，其包含权利要求1-13任一项所述的双特异性抗体以及药学上可接受的载体。
PCT/CN2019/081164 2018-04-09 2019-04-03 具有延长的半衰期和增强的抗肿瘤效果的双特异性抗体 WO2019196716A1 (zh)

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HAITAO PAN: "Site-specific PEGylation of an anti-CEA/ CD 3 bispecific an- tibody improves its antitumor efficacy", INTERNATIONAL JOURNAL OF NANOMEDICINE, vol. 13, 30 May 2018 (2018-05-30), pages 3189 - 3201, XP055643317 *

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