WO2025190332A1 - 特异性结合nkg2a抗原和pd-l1抗原的多特异性抗体及其应用 - Google Patents

特异性结合nkg2a抗原和pd-l1抗原的多特异性抗体及其应用

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Publication number
WO2025190332A1
WO2025190332A1 PCT/CN2025/082284 CN2025082284W WO2025190332A1 WO 2025190332 A1 WO2025190332 A1 WO 2025190332A1 CN 2025082284 W CN2025082284 W CN 2025082284W WO 2025190332 A1 WO2025190332 A1 WO 2025190332A1
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Prior art keywords
antibody
antigen
nkg2a
amino acid
seq
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PCT/CN2025/082284
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English (en)
French (fr)
Inventor
韩荣荣
李忠
唐巧朋
张思
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Beijing Solobio Genetechnology Co Ltd
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Beijing Solobio Genetechnology Co Ltd
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Publication of WO2025190332A1 publication Critical patent/WO2025190332A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present application relates to multispecific antibodies that specifically bind to NKG2A and PD-L1, as well as pharmaceutical compositions comprising multispecific antibodies that specifically bind to NKG2A and PD-L1, as well as preparation methods and uses thereof, including methods for preventing and treating HBV infection or diseases associated with HBV infection using the same.
  • NKG2A is primarily expressed on the surface of NK cells and can also be expressed on some T cells.
  • this protein is disulfide-linked to the co-expressed CD94 molecule on the NK cell surface to form the heterodimeric complex NKG2A-CD94.
  • This complex is then recognized by the non-classical major histocompatibility complex class I (MHC I) human leukocyte antigen alpha chain E (HLA-E) on target cells.
  • MHC I major histocompatibility complex class I
  • HLA-E human leukocyte antigen alpha chain E
  • the present application provides a multispecific antibody (e.g., a bispecific antibody) that binds to NKG2A and PDL1, comprising a first antigen-binding domain that specifically binds to NKG2A, and a second antigen-binding domain that specifically binds to PD-L1, wherein the first antigen-binding domain that specifically binds to NKG2A comprises: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementary determining region (HC-CDR) 1 comprising SNTMS (SEQ ID NO: 1); HC-CDR2 comprising NINTGGNTYYANWAKG (SEQ ID NO: 2); and HC-CDR3 comprising GSTIDSSGLSL (SEQ ID NO: 3); and a light chain variable region ( VL ), the VL comprising: a light chain complementary determining region (LC-CDR) 1 comprising QASQNIGSDLA (SEQ ID NO: 4); LC-CDR2 comprising L
  • VH
  • the second antigen-binding domain that specifically binds PD-L1 comprises: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising GFTFGGFG (SEQ ID NO:18); an HC-CDR2 comprising ITGDSSTI (SEQ ID NO:19); and an HC-CDR3 comprising VRGPPGTWAY (SEQ ID NO:20); and a light chain variable region ( VL ), the VL comprising: a light chain complementarity determining region (LC-CDR) 1 comprising ESVEFYGTTL (SEQ ID NO:21); an LC-CDR2 comprising GAS (SEQ ID NO:22); and an LC-CDR3 comprising QQIRKVPWT (SEQ ID NO:23).
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • the two polypeptide chains comprise V L 1 -CL from N-terminus to C-terminus, wherein V L 1 is a light chain variable region that specifically binds to NKG2A, and CL is a light chain constant region; and
  • One polypeptide chain comprises VH1 - CH1 from N-terminus to C-terminus, wherein VH1 is a heavy chain variable region that specifically binds to the NKG2A antigen; CH1 is a heavy chain constant region CH1 domain; preferably, the polypeptide chain further comprises an Fc comprising CH2 and CH3 domains;
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) that can specifically bind to NKG2A
  • VH2 -L3- VL2 constitutes the antigen-binding domain (scFv) that can specifically bind to PD-L1.
  • the multispecific antibody that specifically binds to NKG2A and PDL1 described herein comprises: the amino acid sequence SEQ ID NO: 52 or a variant thereof, the variant having 80% sequence identity with the amino acid sequence SEQ ID NO: 52; and/or the amino acid sequence SEQ ID NO: 53 or a variant thereof, the variant having at least 80% sequence identity with the amino acid sequence SEQ ID NO: 53; and/or the amino acid sequence SEQ ID NO: 54 or a variant thereof, the variant having at least 80% sequence identity with the amino acid sequence SEQ ID NO: 54.
  • the multispecific antibody (e.g., bispecific antibody) has an IgG-(scFv)2 structure.
  • the multispecific antibody e.g., bispecific antibody
  • the two polypeptide chains comprise a VH1 - CH1 - CH2 - CH3 - LVH2 -L3- VL2 structure from N-terminus to C-terminus, wherein VH1 is a heavy chain variable region that specifically binds to NKG2A; VH2 is a heavy chain variable region that specifically binds to PD-L1; VL2 is a light chain variable region that specifically binds to PD-L1; L and L3 are connecting peptides; CH1 is the CH1 domain of the heavy chain constant region; CH2 is the CH2 domain of the heavy chain constant region; CH3 is the CH3 domain of the heavy chain constant region; and
  • the other two polypeptide chains comprise a VL1 - CL structure from N-terminus to C-terminus, where VL1 is the light chain variable region that specifically binds to NKG2A, and CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) that specifically binds to NKG2A
  • VH2 -L3- VL2 constitutes the antigen-binding domain (scFv) that specifically binds to PD-L1.
  • the multispecific antibody that specifically binds to NKG2A and PDL1 described herein comprises: the amino acid sequence SEQ ID NO: 54 or a variant thereof, the variant having 80% sequence identity with the amino acid sequence SEQ ID NO: 54; and/or the amino acid sequence SEQ ID NO: 58 or a variant thereof, the variant having at least 80% sequence identity with the amino acid sequence SEQ ID NO: 58.
  • a method for treating a desired individual disease or condition comprising administering an effective amount of any multispecific antibody (e.g., bispecific antibody) as described above or a pharmaceutical composition comprising the same to the individual.
  • any multispecific antibody e.g., bispecific antibody
  • any multispecific antibody as described above for the preparation of a pharmaceutical composition for treating a desired individual disease or condition.
  • any multispecific antibody e.g., bispecific antibody
  • the disease or condition includes HBV infection or a disease or condition associated with HBV infection.
  • the disease or condition includes hepatitis B, liver failure, cirrhosis or liver cancer.
  • the present application provides a method for treating and/or preventing a disease or disorder in an individual in need thereof, comprising administering to the individual an effective amount of a multispecific antibody (e.g., a bispecific antibody) and/or any pharmaceutical composition described herein.
  • a multispecific antibody e.g., a bispecific antibody
  • the disease or condition comprises HBV infection or a disease associated with HBV infection.
  • the disease or condition comprises hepatitis B, liver failure, cirrhosis, or liver cancer.
  • the present application provides an isolated nucleic acid molecule encoding any multispecific antibody (e.g., a bispecific antibody) as described above.
  • a vector is provided, comprising any nucleic acid molecule as described above.
  • a host cell is provided, comprising any multispecific antibody (e.g., a bispecific antibody), any nucleic acid molecule, or any vector as described above.
  • a multispecific antibody e.g., a bispecific antibody
  • compositions, kits, and articles of manufacture comprising any of the multispecific antibodies (eg, bispecific antibodies), nucleic acid molecules, vectors, or host cells described above.
  • Figure 1A shows an exemplary structural schematic diagram of an IgG-scFv multispecific antibody
  • Figure 1B shows an exemplary structural schematic diagram of an IgG-(scFv)2 multispecific antibody
  • Figure 1C shows an exemplary structural schematic diagram of a Bs4Ab-scFv multispecific antibody
  • Figure 1D shows an exemplary structural schematic diagram of a DVD-Ig (Dual-variable domain-Ig) multispecific antibody
  • Figure 1E shows an exemplary structural schematic diagram of a Hetero H, CrossMab multispecific antibody
  • Figure 1F shows an exemplary structural schematic diagram of an scFv-Fab IgG multispecific antibody.
  • the present application provides an antibody or antigen-binding fragment that specifically binds to NKG2A.
  • the present application provides a multispecific antibody (eg, a bispecific antibody) that specifically binds to NKG2A and PDL1.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a multispecific antibody (eg, a bispecific antibody) that specifically binds to NKG2A and PDL1.
  • a multispecific antibody eg, a bispecific antibody
  • the present application also provides a method for treating a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of a multispecific antibody (eg, a bispecific antibody) that specifically binds to NKG2A and PDL1, or a pharmaceutical composition comprising the same.
  • a multispecific antibody eg, a bispecific antibody
  • the present application provides use of a multispecific antibody (eg, a bispecific antibody) that specifically binds to NKG2A and PDL1, or a pharmaceutical composition comprising the same, in the preparation of a medicament for preventing or treating HBV infection.
  • a multispecific antibody eg, a bispecific antibody
  • a pharmaceutical composition comprising the same
  • the present application also provides nucleic acids encoding multispecific antibodies (eg, bispecific antibodies) that specifically bind to NKG2A and PDL1, vectors and host cells containing the nucleic acids, and methods for preparing the multispecific antibodies.
  • multispecific antibodies eg, bispecific antibodies
  • treatment is a method for obtaining beneficial or desired results, including clinical results.
  • the beneficial or desired clinical results including but not limited to one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (for example, preventing or delaying disease worsening), preventing or delaying the spread of the disease (for example, metastasis), preventing or delaying disease recurrence, delaying or slowing down disease progression, improving the disease state, alleviating the disease (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying disease progression, improving or improving quality of life, gaining weight, and/or prolonging life.
  • treatment also includes the reduction of disease pathology results (for example, for HBV virus infection, viral load, degree of liver damage). The method of the present application takes into account any one or more aspects of these treatments.
  • prevent and similar words, such as “prevented”, “preventing”, “prevention” or “prophylactic”, etc., refer to a method for preventing, inhibiting or reducing the likelihood of the occurrence or recurrence of a disease or condition (such as HBV infection). It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, impact, symptoms and/or burden of a disease or condition before it occurs or recurs. As used herein, “prevention” and similar words also include reducing the risk and susceptibility of a disease or condition to occur or recur, such as HBV infection.
  • antibody is broad and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity.
  • a full-length antibody includes two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding.
  • variable regions in the two chains typically include three highly variable loops, known as complementary determining regions (CDRs), light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3.
  • CDRs complementary determining regions
  • LC light chain
  • HC heavy chain
  • CDR boundaries of the antibodies or antigen-binding fragments disclosed herein may be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDR regions of the heavy or light chain are interposed between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops.
  • the constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions.
  • Antibodies are classified based on the amino acid sequence of their heavy chain constant regions.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by heavy chains of type ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • the term "antigen-binding fragment” includes an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab') 2 , an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , a multispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody (scFv), an scFv dimer (divalent diabody), a multispecific antibody composed of an antibody fragment comprising one or more CDRs, a single-domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment capable of binding to an antigen but not comprising a complete antibody structure.
  • an antibody fragment including, for example, a diabody, Fab, Fab', F(ab') 2 , an Fv fragment, a disulfide-stabilized
  • Fab fragment antigen-binding
  • Antigen-binding fragments also include fusion proteins comprising the above-mentioned antibody fragments.
  • Fab can be combined with the same antigen as the parent antibody or parent antibody fragment (such as parent scFv).
  • Fab may include one or more CDRs from a specific human antibody, and these CDRs are transplanted to the framework region from one or more different human antibodies.
  • multispecific antibody refers to an antibody molecule (e.g., bispecific antibody, trispecific antibody) that has binding specificity for at least two different antigens or epitopes in one molecule.
  • the multispecific antibody is a bispecific antibody.
  • the term "bispecific antibody” refers to an antibody molecule that has binding specificity for two different antigens or epitopes in one molecule.
  • the structure of the multispecific antibody e.g., bispecific antibody
  • the production process of the multispecific antibody includes the design of the complete molecule, the synthesis and cloning of the nucleotide sequence of each domain, the expression of mammalian cells, and the purification of the final product.
  • the structures of exemplary multispecific antibodies are known in the art, for example, IgG-scFv structure, DVD-Ig structure, Bs4Ab structure, Hetero H, CrossMab structure, IgG-(scFv) 2 structure or scFv-Fab IgG structure, etc. (for example, see the review document Labrijn AF, et al. Nat Rev Drug Discov. 2019 Aug; 18(8): 585-608).
  • the term "antigen binding domain” refers to the portion of an antigen binding molecule that specifically binds to an antigen. More specifically, the term “antigen binding domain” refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. In the case of a large antigen, an antigen binding molecule may only bind to a specific portion of the antigen, which is called an antigenic epitope.
  • an antigen binding domain can be provided by one or more variable regions (also referred to as variable domains).
  • the antigen binding domain includes an antibody light chain variable region ( VL ) and an antibody heavy chain variable region ( VH ).
  • an antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of the antigen.
  • Antigen binding domains that specifically bind to NKG2A antigen or PDL1 antigen include antibodies and antigen-binding fragments further defined herein.
  • epitope refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.
  • a first antibody "competes" for binding to an NKG2A antigen target with a second antibody when the first antibody, at equimolar concentrations, inhibits binding of the second antibody to the NKG2A antigen target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa.
  • PCT Publication WO 03/48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.
  • the terms “specifically bind,” “specifically recognize,” or “specific for” refer to a measurable and reproducible interaction, such as the binding of a target to an antibody that can confirm the presence of the target in a heterogeneous population of molecules, including biomolecules.
  • an antibody's ability to specifically recognize a target means that the antibody binds to the target with greater affinity, avidity, greater ease, and/or greater persistence than it binds to other targets.
  • an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.
  • an “isolated” antibody is an antibody that (1) is not related to naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not exist in nature.
  • isolated nucleic acid refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid” (1) is unrelated to all or part of a polynucleotide found in nature, (2) is operably linked to a polynucleotide to which it is not naturally associated, or (3) does not occur in nature as part of a longer sequence.
  • CDR complementarity determining region
  • chimeric antibody refers to an antibody in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • Fv is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable region and a light chain variable region tightly non-covalently linked.
  • Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and give the antibody specificity for binding to the antigen.
  • a single variable region or half of an Fv fragment, which contains only 3 CDRs specific for the antigen
  • Single-chain Fv also abbreviated as “sFv” or “scFv” is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain.
  • the scFv polypeptide further comprises a linker polypeptide between the VH and VL domains that enables the scFv to form an ideal structure for antigen binding.
  • diabodies refers to small antibody fragments prepared by constructing scFv fragments (see above) with a short linker (e.g., 5-10 residues) between the VH and VL chains. This allows the variable regions to pair between the chains rather than within the chains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites.
  • Multispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the VH and VL domains of the two antibodies are located on different polypeptide chains.
  • Diabodies are fully described in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • the "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as a mouse, rat, rabbit or non-human primate with ideal antibody specificity, affinity and performance (donor antibody).
  • residues in the human immunoglobulin framework region (FR) are replaced by corresponding non-human residues.
  • a humanized antibody can include residues that are not present in either the recipient antibody or the donor antibody.
  • a humanized antibody will comprise essentially at least one, usually two, variable regions, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences.
  • the human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the "percent (%) amino acid sequence homology” or “homology” or “identity” of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues in the candidate sequence and the polypeptide sequence being compared.
  • the percentage of amino acid sequence homology or identity can be determined by a variety of alignment methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software.
  • One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to maximize alignment over the full length of the compared sequences.
  • Fc (fragment crystallizable)" or “Fc region” refers to a polypeptide comprising the constant region of an intact antibody, excluding the CH1 domain and, in some cases, a portion of the hinge, whether in monomeric or multimeric form.
  • the original immunoglobulin source of the native Fc is preferably human and can be any immunoglobulin, for example, IgG1, IgG2, IgG3, or IgG4.
  • the native Fc region is composed of monomeric polypeptides that can be linked into dimers or multimers by covalent (i.e., disulfide bonds) and non-covalent associations.
  • the Fc region of an immunoglobulin generally comprises the CH2 and CH3 domains of the heavy chain constant region, and optionally comprises a CH4 domain.
  • each of the two Fc monomers in the Fc dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers.
  • heterodimerization of the Fc monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole” residue pairs into the two Fc monomers.
  • the "knob-into-hole” technology is also disclosed in U.S. Patent Publication No. 8,216,805.
  • one Fc monomer comprises a knob mutation T366W
  • the other Fc monomer comprises a hole mutation T366S, L358A, and Y407V.
  • two Cys residues (S354C on the "knob” side and Y349C on the "hole” side) that form a stabilized disulfide bridge are introduced.
  • Fc receptor or "FcR” is used to describe a receptor that binds to the Fc region of an antibody.
  • the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (activating receptor) and Fc ⁇ RIIB (inhibiting receptor), which have similar amino acid sequences and differ primarily in the cytoplasmic domain.
  • the cytoplasmic domain of the activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM).
  • the cytoplasmic domain of the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997).
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the term also includes allotypes, such as the Fc ⁇ RIIIA allotypes: Fc ⁇ RIIIA-Phe158, Fc ⁇ RIIIA-Val158, Fc ⁇ RIIA-R131, and/or Fc ⁇ RIIA-H131.
  • FcRs are described in Ravetch and Kinet, Annu. Rev.
  • FcR in this application encompasses other types of FcRs, including those identified in the future.
  • FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
  • FcRn refers to the neonatal Fc receptor (FcRn).
  • FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an ⁇ chain non-covalently bound to ⁇ 2 microglobulin.
  • MHC major histocompatibility complex
  • FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels.
  • FcRn acts as a salvage receptor, binding and transporting endocytosed IgGs intact within and between cells, protecting them from default degradation pathways.
  • the " CH1 domain" of the human IgG heavy chain constant region typically stretches from amino acid 118 to amino acid 215 (EU numbering system).
  • the "hinge region” is generally defined as extending from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG subtypes can be aligned with the IgG1 sequence.
  • the " CH2 domain" of the human IgG Fc region typically extends from amino acids 231 to 340.
  • the CH2 domain is unique in that it does not pair closely with another domain. Instead, two N-terminally linked branched sugar chains are inserted between the two CH2 domains in the intact native IgG molecule. It is hypothesized that the carbohydrates may serve as a substitute for interdomain pairing, helping to maintain CH2 domain stability. Burton, Molec Immunol. 22:161-206 (1985).
  • Antibodies with IgG Fc variants having "altered" FcR binding affinity or ADCC activity have enhanced or diminished FcR binding activity and/or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence.
  • Fc variants that exhibit "enhanced binding" to an FcR have a higher binding affinity (e.g., a lower apparent Kd or IC50 value) for at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence.
  • the binding ability is enhanced by 3-fold, e.g., 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500-fold, or the binding ability is increased by 25% to 1000%, compared to the parent polypeptide.
  • Fc variants that exhibit "decreased binding" to an FcR have a lower affinity (e.g., a higher apparent Kd or IC50 value) for at least one FcR compared to the parent polypeptide.
  • the binding ability is decreased by 40% or more compared to the parent polypeptide.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Igs immunoglobulins
  • FcRs Fc receptors
  • cytotoxic cells such as natural killer (NK) cells, neutrophils, and macrophages
  • NK cells express only Fc ⁇ RIII
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • ADCC activity of the target molecule can also be assessed in vivo, for example, in an animal model as described in Clynes et al. PNAS (USA) 95: 652-656 (1998).
  • Polypeptides comprising Fc variants exhibit "enhanced ADCC activity" or are capable of mediating ADCC effects more effectively in the presence of human effector cells compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides, and the polypeptides comprising Fc variants are capable of mediating ADCC more effectively both in vitro and in vivo when the amount of the polypeptides comprising the Fc variants is substantially the same as that comprising the wild-type IgG Fc polypeptide (or parent polypeptide) during the experiment.
  • Such variants are generally identified using any in vitro ADCC experimental method known in the art, such as an experiment or method for identifying ADCC activity, such as in an animal model. In some embodiments, such variants mediate ADCC 5 to 100 times more efficiently, for example, 25 to 50 times more efficiently, than wild-type Fc (or parent polypeptide).
  • “Complement-dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to the cognate antigen.
  • C1q the first component of the complement system
  • an antibody of the appropriate structural subclass
  • a CDC assay such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99/51642. The contents of these patent publications are expressly incorporated into this application by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • a nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.
  • operably linked refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter.
  • a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.
  • “Homologous” refers to the sequence similarity or sequence homology between two polypeptides or between two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, adenine is present at the same position in two DNA molecules, then the two DNA molecules are homologous at that position.
  • the percentage homology between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the homology between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.
  • an “effective amount” of an antibody (including a multispecific antibody) or composition disclosed herein is an amount sufficient to achieve a specific purpose.
  • An “effective amount” can be determined empirically and by known methods related to the purpose.
  • therapeutically effective amount refers to the amount of the antibody (including multispecific antibodies) or composition described herein that can effectively treat an individual's disease or symptoms. That is, an amount sufficient to reduce or improve the severity and/or duration of the disease or one or more of its symptoms; to prevent the development of the disease, cause the symptoms to subside, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, detect the disease, or enhance/improve the preventive or therapeutic effect of another therapy (such as a prophylactic or therapeutic agent).
  • the therapeutically effective amount of the antibody or composition disclosed herein is the amount of the binding molecule that can effectively prevent or treat the condition caused by HBV infection.
  • a therapeutically effective amount refers to an amount that can prolong the patient's survival. In some embodiments, a therapeutically effective amount refers to an amount that can improve the patient's progression-free survival.
  • pharmaceutically acceptable or “pharmacologically compatible” refers to a material that is free of biological activity or other undesirable properties, e.g., a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition.
  • Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and/or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.
  • reference to a value or parameter "not” generally refers to and describes "other than” that value or parameter.
  • the method cannot be used to treat type X infection means that the method is generally used to treat infection other than type X.
  • the present application provides antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy and/or light chain CDRs as described herein.
  • the antibody or antigen-binding domain is an isolated antibody or antigen-binding domain that binds to NKG2A or PDL1.
  • Antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1 include all or fragments of full-length antibodies (such as full-length IgG1, IgG2, or IgG4) that specifically bind to NKG2A or PDL1, single-chain antibodies that specifically bind to NKG2A or PDL1, multispecific (such as bispecific) antibodies that bind to NKG2A and PDL1, immunoconjugates that specifically bind to NKG2A or PDL1, and the like.
  • the antibody or antigen-binding domain that specifically binds to NKG2A or PDL1 is a Fab, Fab', F(ab)' 2 , Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, or diabody.
  • the antibody or antigen-binding domain that specifically binds to NKG2A or PDL1 refers to an antibody or antigen-binding domain that binds to NKG2A or PDL1 with an affinity that is at least 10 times greater (including, for example, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times greater) than the affinity for binding to a non-target.
  • a non-target is an antigen that is not NKG2A or PDL1.
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA).
  • Kd values can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interferometry (BLI) technology.
  • non-human antibodies are also contemplated.
  • non-human antibodies comprise human CDR sequences and non-human framework region sequences for antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1 as described herein.
  • non-human framework region sequences include any sequence used to generate heavy and/or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc.
  • mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc.
  • non-human antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1 comprise antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1 generated by grafting one or more human CDR sequences described herein into non-human framework region sequences (e.g., mouse or chicken framework region sequences).
  • Antibodies or antigen-binding domains that specifically bind to NKG2A are antibodies or antigen-binding domains that specifically bind to NKG2A
  • the present application provides an antibody or antigen binding domain that specifically binds to NKG2A.
  • the antibody or antigen binding domain that specifically binds NKG2A binds to the NKG2A antigen. In some embodiments, the antibody or antigen binding domain that specifically binds NKG2A is specific for NKG2A and does not cross-react with species or other non-NKG2A antigens. In some embodiments, the antibody or antigen binding domain that specifically binds NKG2A cross-reacts with other non-NKG2A antigens.
  • the antibody or antigen binding domain that specifically binds NKG2A comprises: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SNTMS (SEQ ID NO: 1); HC-CDR2 comprising NINTGGNTYYANWAKG (SEQ ID NO: 2); and HC-CDR3 comprising GSTIDSSGLSL (SEQ ID NO: 3); and a light chain variable region ( VL ), the VL comprising: a light chain complementarity determining region (LC-CDR) 1 comprising QASQNIGSDLA (SEQ ID NO: 4); LC-CDR2 comprising LASTLAS (SEQ ID NO: 5); and LC-CDR3 comprising QQSWSSSNVDNV (SEQ ID NO: 6).
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • HC-CDR2 comprising NINTGGNTYYANWAKG
  • the antibody or antigen binding domain that specifically binds to NKG2A comprises:
  • VH comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13; and VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 16; or
  • VH comprising the amino acid sequence of SEQ ID NO:14 or a variant thereof having at least 80% sequence identity thereto; and VL comprising the amino acid sequence of SEQ ID NO:16 or a variant thereof having at least 80% sequence identity thereto.
  • the antibody or antigen-binding domain that specifically binds to PDL1 binds to the PDL1 antigen. In some embodiments, the antibody or antigen-binding domain that specifically binds to PDL1 is specific for PDL1 and does not cross-react with species or other non-PDL1 species. In some embodiments, the antibody or antigen-binding domain that specifically binds to PDL1 cross-reacts with other non-PDL1 species.
  • the antibody or antigen binding domain that specifically binds to PDL1 comprises: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising GFTFGGFG (SEQ ID NO: 18); HC-CDR2 comprising ITGDSSTI (SEQ ID NO: 19); and HC-CDR3 comprising VRGPPGTWAY (SEQ ID NO: 20); and a light chain variable region ( VL ), the VL comprising: a light chain complementarity determining region (LC-CDR) 1 comprising ESVEFYGTTL (SEQ ID NO: 21); LC-CDR2 comprising GAS (SEQ ID NO: 22); and LC-CDR3 comprising QQIRKVPWT (SEQ ID NO: 23).
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • HC-CDR2 comprising ITGDSSTI
  • HC-CDR3 comprising VRGPPGTWAY
  • the antibody or antigen-binding domain that specifically binds to PDL1 comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 34, or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 30 and 34; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 32 and 36, or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 32 and 36.
  • the present application provides antibodies or antigen-binding domains that specifically bind to preS1.
  • the specific amino acid sequences of the antibodies are shown in Tables 4-1, 4-2, and 4-3, wherein the CDRs are numbered according to the Kabat definition.
  • amino acid substitutions described above are limited to the "exemplary substitutions” shown in Table 11 of this application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 10 of this application.
  • the present application provides antibodies or antigen-binding domains that can competitively bind to NKG2A or PDL1 with any of the above-mentioned antibodies that specifically bind to NKG2A or PDL1. In some embodiments, the present application provides antibodies or antigen-binding domains that competitively bind to the same epitope as any of the above-mentioned antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1.
  • competition assays can be used to identify monoclonal antibodies or antigen-binding domains that compete with the antibodies or antigen-binding domains that specifically bind to NKG2A or PDL1 described herein for binding to NKG2A or PDL1.
  • Competition assays can determine whether two antibodies bind to the same epitope by recognizing the same or spatially overlapping epitopes or by competitively inhibiting binding of one antibody to the antigen by the other. In certain embodiments, such competing antibodies bind to the same epitope as the antibodies described herein.
  • Some exemplary competition assays include, but are not limited to, conventional assays such as those described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.
  • each antibody is said to bind to the same epitope if it blocks 50% or more of the binding of the other antibody.
  • an antibody that competes with an antibody described herein that specifically binds to NKG2A or PDL1 is a chimeric, humanized, or fully human antibody.
  • the antibody or antigen-binding domain that specifically binds to PDL1 can be selected from the antibodies or antigen-binding fragments that specifically bind to PDL1 described in patent application WO2024040212A2.
  • the sequences of exemplary antibodies or antigen-binding domains that specifically bind to PDL1 are shown in Tables 3-1, 3-2, and 3-3, where CDR numbering is performed according to Kabat (S70) or IMGT (SBT451) definitions.
  • the antibody or antigen-binding domain that specifically binds to preS1 can be selected from the antibodies or antigen-binding fragments that specifically bind to preS1 described in patent application WO2023/066171A. Sequences of exemplary antibodies or antigen-binding domains that specifically bind to preS1 are shown in Tables 4-1, 4-2, and 4-3, where CDR numbering is performed according to the Kabat definition.
  • the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to NKG2A, and a second antigen-binding domain that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody
  • the first antigen binding domain that specifically binds NKG2A comprises: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SNTMS (SEQ ID NO: 1); HC-CDR2 comprising NINTGGNTYYANWAKG (SEQ ID NO: 2); and HC-CDR3 comprising GSTIDSSGLSL (SEQ ID NO: 3); and a light chain variable region ( VL ), the VL comprising: a light chain complementarity determining region (LC-CDR) 1 comprising QASQNIGSDLA (SEQ ID NO: 4); LC-CDR2 comprising LASTLAS (SEQ ID NO: 5); and LC-CDR3 comprising QQSWSSSNVDNV (SEQ ID NO: 6).
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • HC-CDR2 comprising NINTGGNTYYANWAKG
  • the second antigen binding domain that specifically binds PDL1 comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising GFTFGGFG (SEQ ID NO: 18); HC-CDR2 comprising ITGDSSTI (SEQ ID NO: 19); and HC-CDR3 comprising VRGPPGTWAY (SEQ ID NO: 20); and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising ESVEFYGTTL (SEQ ID NO: 21); LC-CDR2 comprising GAS (SEQ ID NO: 22); and LC-CDR3 comprising QQIRKVPWT (SEQ ID NO: 23).
  • V H heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • HC-CDR2 comprising ITGDSSTI
  • HC-CDR3 comprising VRGPPGTWAY
  • the first antigen binding domain that specifically binds NKG2A comprises:
  • a VH comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 13; and a VL comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 16; or
  • a VH comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 14; and a VL comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 16.
  • the second antigen-binding domain that specifically binds to PDL1 comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 34, or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30 and 34; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 32 and 36, or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 32 and 36.
  • the present application provides a multispecific antibody (preferably, a bispecific antibody), comprising a first antigen-binding domain that specifically binds to NKG2A, and a second antigen-binding domain that specifically binds to PDL1, wherein the first antigen-binding domain comprises: a VH comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 14; and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 16; and wherein the second antigen-binding domain comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 34, or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 30 and 34; and a VL comprising the amino acid sequence of any one
  • the present application provides a multispecific antibody (preferably, a trispecific antibody), which comprises a first antigen-binding domain that specifically binds to NKG2A, a second antigen-binding domain that specifically binds to PDL1, and a third antigen-binding domain that specifically binds to preS1.
  • a multispecific antibody preferably, a trispecific antibody
  • Multispecific antibodies that specifically bind to preS1 and PDL1
  • the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to PDL1, and a second antigen-binding domain that specifically binds to preS1.
  • a multispecific antibody preferably, a bispecific antibody
  • the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to NKG2A, and a second antigen-binding domain that specifically binds to preS1.
  • a multispecific antibody preferably, a bispecific antibody
  • the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to NKG2A and a second antigen-binding domain that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody
  • the structure of the multispecific antibody is selected from IgG-scFv, IgG-(scFv) 2 , Bs4Ab-scFv, DVD-Ig, Hetero H, CrossMab, or scFv-Fab IgG.
  • an IgG-scFv multispecific antibody structure (preferably, a bispecific antibody) comprises an scFv fragment attached to the Fc region of one of the heavy chains of an IgG antibody, forming an antigen-binding domain capable of specifically binding to a different antigen, thereby achieving multispecific (preferably, bispecific) binding.
  • the scFv is linked to the C-terminus of the Fc region via a linker peptide (L).
  • L linker peptide
  • a knob-in-hole (KIH) structure can be designed in the Fc region (which comprises the CH2 and CH3 domains).
  • amino acid residues in the CH3 domain of one Fc region are replaced with amino acid residues with larger side chain volume, forming a "knob”
  • amino acid residues in the CH3 domain of the other Fc region are replaced with amino acid residues with smaller side chain volume, forming a "hole.”
  • two Cys residue mutations S354C on the "knob” side and Y349C on the "hole” side
  • FIG1A A schematic diagram of the typical structure of this multispecific antibody is shown in FIG1A .
  • any multispecific antibody (preferably, a bispecific antibody) described herein has an IgG-scFv structure, which is a heterodimer and a trivalent multispecific antibody composed of two different monomers, wherein one monomer is composed of two polypeptide chains (hereinafter referred to as the first heavy chain and the first light chain, respectively), which contain two antigen-binding domains, one of which is a Fab and the other is an scFv, each binding to a different antigen; the other monomer is composed of two polypeptide chains (hereinafter referred to as the second heavy chain and the second light chain, respectively), which contain one antigen-binding domain (Fab).
  • the sequences of the first and second light chains are identical.
  • the first heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises, from N-terminus to C-terminus, the structure VH1-CH1-CH2-CH3-LVH2 - L3 - VL2 .
  • the first heavy chain of a multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, the structure VH1 - CH1 - CH2 - CH3 - LVL2 -L3- VH2 .
  • the first light chain of a multispecific antibody comprises, from N-terminus to C-terminus, the structure VL1 - CL .
  • VH1 and VL1 are the heavy chain variable region and light chain variable region that specifically bind to a first antigen; VH2 and VL2 are the heavy chain variable region and light chain variable region that specifically bind to a second antigen; CH1 is the CH1 domain of the heavy chain constant region; CH2 is the CH2 domain of the heavy chain constant region; CH3 is the CH3 domain of the heavy chain constant region; CL is the light chain constant region; and L and L3 are connecting peptides.
  • the second heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises, from N-terminus to C-terminus: VH1 - CH1 .
  • the second light chain of any multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus: VL1 - CL structure.
  • the second heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the second heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, a VH1 - CH1 - CH2 - CH3 structure; in some embodiments, the second light chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, a VL1 - CL structure.
  • VH1 and VL1 are the heavy and light chain variable regions, respectively, that bind to the first antigen; CH1 is the heavy chain constant region CH1 domain; CH2 is the heavy chain constant region CH2 domain; CH3 is the heavy chain constant region CH3 domain; and CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL comprise the antigen-binding domain (Fab) that binds to the first antigen.
  • sequences of the first light chain and the second light chain are identical.
  • an amino acid residue in the CH3 domain of the first heavy chain is replaced with an amino acid residue with a larger side chain volume to form a "knob”
  • an amino acid residue in the CH3 domain of the second heavy chain is replaced with an amino acid residue with a smaller side chain volume to form a "hole.”
  • an amino acid residue in the CH3 domain of the second heavy chain is replaced with an amino acid residue with a larger side chain volume to form a "knob”
  • an amino acid residue in the CH3 domain of the first heavy chain is replaced with an amino acid residue with a smaller side chain volume to form a "hole.”
  • the CH3 domain comprises, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.
  • the leucine (L) at position 234 and the leucine (L) at position 235 of the hinge region of the heavy chain are replaced with alanine (A), and the proline (P) at position 331 is replaced with serine (S), forming the combined mutation LALAPS.
  • This combination of mutations can weaken the binding of the antibody Fc to the FcR receptors CD64, CD32A, CD16, and human complement component C1q, thereby weakening antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • the numbering is according to the EU index as in Kabat.
  • the present application relates to a multispecific antibody (preferably, a bispecific antibody) that can bind to NKG2A and PDL1, which has the IgG-scFv structure as described above.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to NKG2A
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to PDL1.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) that specifically binds to NKG2A
  • VH2 -L3- VL2 or VL2 -L3- VH2 constitute the antigen-binding domain (scFv) that specifically binds to PDL1.
  • the multispecific antibody (preferably, a bispecific antibody) can simultaneously bind to NKG2A and PDL1.
  • the antigen-binding domain scFv that specifically binds to PD-L1 includes genetically engineered cysteine mutations. By introducing two cysteine mutations at the VH and VL interfaces, a disulfide-stabilized multispecific antibody is obtained.
  • the multispecific antibody (preferably, a bispecific antibody) described in the present application comprises the amino acid sequence SEQ ID NO: 52 or a variant thereof, which has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 52; and/or the amino acid sequence SEQ ID NO: 53 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 53; and/or the amino acid sequence SEQ ID NO: 54 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 54.
  • the present application relates to a multispecific antibody (preferably a bispecific antibody) that can bind to preS1 and PDL1, which has the IgG-scFv structure as described above.
  • a multispecific antibody preferably a bispecific antibody
  • preS1 and PDL1 which has the IgG-scFv structure as described above.
  • the specific amino acid sequences of its heavy chain and light chain are shown in Table 8.
  • the present application relates to a multispecific antibody (preferably a bispecific antibody) that can bind to NKG2A and preS1, which has the IgG-scFv structure as described above.
  • a multispecific antibody preferably a bispecific antibody
  • the specific amino acid sequences of its heavy chain and light chain are shown in Table 9-1.
  • the IgG-(scFv)2 multispecific antibody structure (see, Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63) is a structure in which a scFv fragment is attached to each of the Fc chains of the two heavy chains of a full-length IgG antibody, forming an antigen-binding domain that can specifically bind to another different antigen, thereby achieving bispecificity.
  • the scFv is connected to the C-terminus of the Fc via a linker peptide (L).
  • L linker peptide
  • any multispecific antibody (preferably, a bispecific antibody) described herein has an IgG-(scFv)2 structure, which is a homodimer and a tetravalent multispecific antibody composed of two identical monomers.
  • Each monomer is composed of two polypeptide chains (hereinafter referred to as a heavy chain and a light chain), including two antigen-binding domains, one of which is a Fab and the other is an scFv.
  • the two antigen-binding domains bind to different antigens.
  • the IgG-(scFv)2 structure further includes two Fc regions, each comprising a CH2 and CH3 domain.
  • the scFv is linked to the C-terminus of the Fc via a linker peptide (L).
  • the multispecific antibody (preferably, a bispecific antibody) has an IgG-(scFv)2 structure, wherein two Fab antigen-binding domains specifically bind to a first antigen, and the other two scFv antigen-binding domains specifically bind to a second antigen.
  • the heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, the structure VH1 - CH1 - CH2 -CH3- LVH2 - L3 - VL2 .
  • the heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, the structure VH1 - CH1 - CH2 - CH3 - LVL2 -L3- VH2 .
  • the light chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, the structure VL1 - CL .
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to one antigen;
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to the other antigen;
  • L and L3 are connecting peptides;
  • CH1 is the CH1 domain of the heavy chain constant region;
  • CH2 is the CH2 domain of the heavy chain constant region;
  • CH3 is the CH3 domain of the heavy chain constant region;
  • CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) of a multispecific antibody (preferably, a bispecific antibody) that binds to the first antigen
  • VH2 -L3- VL2 or VL2 -L3- VH2 constitutes the antigen-binding domain (scFv) of the multispecific antibody (preferably, a bispecific antibody) that binds to the second antigen.
  • the leucine (L) at position 234 and the leucine (L) at position 235 of the hinge region of the heavy chain are replaced with alanine (A), and the proline (P) at position 331 is replaced with serine (S), forming the combined mutation LALAPS.
  • This combination of mutations can weaken the binding of the antibody Fc to the FcR receptors CD64, CD32A, CD16, and human complement component C1q, thereby weakening antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • the numbering is according to the EU index as in Kabat.
  • the present application relates to a multispecific antibody (preferably a bispecific antibody) that can bind to NKG2A and PDL1, which has the IgG-(scFv)2 structure as described above.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to NKG2A
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to PDL1
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) that specifically binds to NKG2A
  • VH2 -L3- VL2 or VL2 -L3- VH2 constitutes the antigen-binding domain (scFv) that specifically binds to PDL1.
  • the multispecific antibody (preferably, a bispecific antibody) can simultaneously bind to NKG2A and PDL1.
  • the antigen-binding domain scFv that specifically binds to PD-L1 includes genetically engineered cysteine mutations. By introducing two cysteine mutations at the VH and VL interfaces, a disulfide-stabilized multispecific antibody is obtained.
  • the multispecific antibody (preferably, a bispecific antibody) described in the present application comprises the amino acid sequence SEQ ID NO: 54 or a variant thereof, which has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 54; and/or the amino acid sequence SEQ ID NO: 58 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 58.
  • the present application relates to a multispecific antibody (preferably a bispecific antibody) that can bind to NKG2A and preS1, which has the aforementioned IgG-(scFv)2 structure.
  • a multispecific antibody preferably a bispecific antibody
  • the specific amino acid sequences of its heavy and light chains are shown in Table 9-2.
  • the multispecific antibody structure obtained by modification based on the bispecific antibody structure Bs4Ab is based on the Bs4Ab bispecific antibody structure (see the literature Bezabeh B, et al. Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in a tetravalent bispecific molecule with robust properties. MAbs. 2017 Feb/Mar; 9(2):240-256).
  • An scFv fragment is connected to the C-terminus of the Fc of one of the heavy chains to form an antigen-binding domain that can specifically bind to another different antigen, thereby achieving multispecificity.
  • a schematic diagram of the typical structure of the multispecific antibody is shown in Figure 1C.
  • any multispecific antibody described herein has a Bs4Ab-scFv structure, which is a heterodimer and a pentavalent multispecific antibody composed of two different monomers, wherein the first monomer is composed of two polypeptide chains (hereinafter referred to as the first heavy chain and the first light chain, respectively), including three antigen-binding domains, one of which is a Fab and the other two are scFv, each of which binds to a different antigen; the second monomer is composed of two polypeptide chains (hereinafter referred to as the second heavy chain and the second light chain, respectively), including two antigen-binding domains, one of which is a Fab and the other is an scFv, each of which binds to a different antigen.
  • the first monomer is composed of two polypeptide chains (hereinafter referred to as the first heavy chain and the first light chain, respectively), including three antigen-binding domains, one of which is a Fab and the other two are
  • the sequences of the first and second light chains are identical.
  • the Bs4Ab-scFv structure further includes two Fc regions, each comprising a CH2 and CH3 domain.
  • one of the scFv antigen binding domains is connected to the C-terminus of one of the Fcs via a connecting peptide (L), and the other two scFv antigen binding domains are respectively connected to the C-terminus of the heavy chain of the Fab binding domain and the N-terminus of one Fc via connecting peptides.
  • the multispecific antibody (preferably, a trispecific antibody) has a Bs4Ab-scFv structure, wherein the two Fab antigen-binding domains specifically bind to the first antigen, the two scFv antigen-binding domains connected to the Fab specifically bind to the second antigen, and the scFv antigen-binding domain connected to the C-terminus of the Fc specifically binds to the third antigen.
  • the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: VH1 - CH1 -LVH2- L3 - VL2 -LCH2- CH3 - LVH3 -L3- VL3 .
  • the first light chain of the multispecific antibody (preferably, a trispecific antibody) comprises , from N-terminus to C-terminus: VL1 - CL structure.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) of the multispecific antibody that binds to the first antigen
  • VH2 -L3- VL2 constitutes the antigen-binding domain (scFv) of the multispecific antibody that binds to the second antigen
  • VH3 -L3- VL3 constitutes the antigen-binding domain (scFv) of the multispecific antibody that binds to the third antigen.
  • the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: VH1 - CH1 - LVH2 -L3- VL2 .
  • the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: VL1 - CL structure.
  • the second heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: VH1 - CH1 - LVH2 -L3-VL2- LCH2 - CH3 .
  • the second light chain of the multispecific antibody comprises, from N-terminus to C-terminus: VL1 - CL structure.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to a first antigen; VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to a second antigen; L and L3 are connecting peptides; CH1 is the CH1 domain of the heavy chain constant region; CH2 is the CH2 domain of the heavy chain constant region; CH3 is the CH3 domain of the heavy chain constant region; and CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) of the multispecific antibody that binds to the first antigen
  • VH2 -L3- VL2 constitutes the antigen-binding domain (scFv) of the multispecific antibody that binds to the second antigen.
  • sequences of the first light chain and the second light chain are identical.
  • the leucine (L) at position 234 and the leucine (L) at position 235 of the hinge region of the heavy chain are replaced with alanine (A), and the proline (P) at position 331 is replaced with serine (S), forming the combined mutation LALAPS.
  • This combination of mutations can weaken the binding of the antibody Fc to the FcR receptors CD64, CD32A, CD16, and human complement component C1q, thereby weakening antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • the numbering is according to the EU index as in Kabat.
  • the scFv includes genetically engineered cysteine mutations, and a disulfide-stabilized multispecific antibody is obtained by introducing two cysteine mutations at the VH and VL interfaces.
  • the present application relates to a multispecific antibody (preferably a trispecific antibody) that specifically binds to NKG2A, PDL1, and preS1, which has the aforementioned Bs4Ab-scFv structure.
  • a multispecific antibody preferably a trispecific antibody
  • the specific amino acid sequences of its heavy and light chains are shown in Table 7.
  • the dual-variable domain immunoglobulin molecule DVD-Ig (Dual-variable domain-Ig) multispecific antibody structure (preferably, a bispecific antibody) (see, for example, Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules.
  • MAbs. 2009 Jul-Aug; 1(4): 339-47 is a structure in which the VL and VH domains of another antibody are connected to the N-termini of the VL and VH of a full-length IgG antibody, respectively.
  • the VH and VL of the two antibodies interact to form an antigen-binding domain, which can simultaneously bind to the corresponding antigens, thereby achieving multispecificity (preferably, bispecificity).
  • multispecificity preferably, bispecificity.
  • any multispecific antibody (preferably, a bispecific antibody) described herein has a DVD-IgG structure, which is a homodimer and a tetravalent multispecific antibody composed of two identical monomers, wherein each monomer is composed of two polypeptide chains (hereinafter referred to as a heavy chain and a light chain), including two antigen-binding domains, one of which is an Fv and the other is a Fab, each binding to a different antigen, and the two binding domains are connected in series by a connecting peptide (L).
  • the DVD-Ig structure further includes two Fc regions, each comprising a CH2 and CH3 domain.
  • the multispecific antibody (preferably, a bispecific antibody) has a DVD-Ig structure, wherein two Fab antigen-binding domains specifically bind to a first antigen, and the other two Fv antigen-binding domains specifically bind to a second antigen. In another preferred embodiment, wherein two Fv antigen-binding domains specifically bind to a first antigen, and the other two Fab antigen-binding domains specifically bind to a second antigen.
  • the heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises a VH1 - LVH2 - CH1 structure from N-terminus to C-terminus.
  • the light chain of the multispecific antibody comprises a VL1 - LVL2 - CL structure from N-terminus to C-terminus.
  • the heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the heavy chain of the multispecific antibody preferably, a bispecific antibody
  • the heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus, a VH1 - LVH2 - CH1 - CH2 - CH3 structure.
  • the light chain of the multispecific antibody comprises, from N-terminus to C-terminus, a VL1 - LVL2 - CL structure.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to one antigen; VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to another antigen; L is a connecting peptide; CH1 is the CH1 domain of the heavy chain constant region; CL is the light chain constant region.
  • VH1 and VL1 constitute one antigen-binding domain (Fv) of a multispecific antibody (preferably, a bispecific antibody); VH2 - CH1 and VL2 - CL constitute the other antigen-binding domain (Fab) of the multispecific antibody (preferably, a bispecific antibody).
  • the leucine (L) at position 234 and the leucine (L) at position 235 of the hinge region of the heavy chain are replaced with alanine (A), forming a combination mutation LALA.
  • A alanine
  • This combination of mutations can weaken the binding of the antibody Fc to the FcR receptors CD64, CD32A, CD16, and human complement component C1q, thereby weakening antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • the numbering is according to the EU index as in Kabat.
  • Hetero H a multispecific antibody (preferably, a bispecific antibody) structure of the CrossMab structure (see, for example, Klein C, et al. The use of CrossMAb technology for the generation of bi-and multispecific antibodies.
  • MAbs. 2016 Aug-Sep; 8(6): 1010-20 is a heterodimer based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, which can be the exchange of complete Fab domains (CrossMab Fab), or the exchange of only the variable region (CrossMab VH - VL ) or only the constant region (CrossMab CH1 - CL ) of the Fab domain, thereby ensuring the correct pairing between the light and heavy chains of the antibody.
  • a knobs-in-holes (KIH) structure can be designed in the Fc region (which comprises the CH2 and CH3 domains).
  • amino acid residues in the CH3 domain of one Fc region are replaced with amino acid residues with larger side chain volume to form a "knob”
  • amino acid residues in the CH3 domain of the other Fc region are replaced with amino acid residues with smaller side chain volume to form a "hole.”
  • two Cys residue mutations S354C on the "knob” side and Y349C on the "hole” side
  • the typical structure of this bispecific antibody is shown in Figure 1E.
  • the multispecific antibody (preferably, a bispecific antibody) has a Hetero H, CrossMab structure, in which one antigen binding domain specifically binds to a first antigen and the other antigen binding domain specifically binds to a second antigen.
  • the first heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises a VH1 - CH1 structure from N-terminus to C-terminus; and the first light chain comprises a VL1 - CL structure from N-terminus to C-terminus.
  • the first heavy chain further comprises an Fc comprising CH2 and CH3 domains. Therefore, in some embodiments, the first heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, a VH1 - CH1 - CH2 - CH3 structure; and the first light chain comprises, from N-terminus to C-terminus, a VL1 - CL structure.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to the first antigen
  • CH1 is the heavy chain constant region ( CH1 domain)
  • CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) that binds to the first antigen.
  • the second heavy chain of the multispecific antibody (preferably, bispecific antibody) comprises a VH2 - CL structure from N-terminus to C-terminus; in some embodiments, the second light chain of the multispecific antibody (preferably, bispecific antibody) comprises a VL2 - CH1 structure from N-terminus to C-terminus.
  • the second heavy chain further comprises an Fc comprising CH2 and CH3 domains. Therefore, in some embodiments, the second heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, a VH2 - CL - CH2 - CH3 structure. In some embodiments, the second light chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus, a VL2 - CH1 structure.
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to a second antigen
  • CH1 is the heavy chain constant region ( CH1 domain)
  • CL is the light chain constant region.
  • VH2 - CL and VL2 - CH1 comprise the antigen-binding domain (Fab) that binds to the second antigen.
  • the positions of CL and CH1 in the first or second monomer of the multispecific antibody are interchangeable.
  • the positions of VH1 and VL1 are interchangeable.
  • the positions of VH2 and VL2 are interchangeable.
  • an amino acid residue in the CH3 domain of the first heavy chain is replaced with an amino acid residue with a larger side chain volume to form a "knob”
  • an amino acid residue in the CH3 domain of the second heavy chain is replaced with an amino acid residue with a smaller side chain volume to form a "hole.”
  • an amino acid residue in the CH3 domain of the second heavy chain is replaced with an amino acid residue with a larger side chain volume to form a "knob”
  • an amino acid residue in the CH3 domain of the first heavy chain is replaced with an amino acid residue with a smaller side chain volume to form a "hole.”
  • the CH3 domain comprises, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.
  • the multispecific antibodies (preferably, bispecific antibodies) described herein have an scFv-Fab IgG structure, which is in the form of a heterodimer and comprises two monomers, referred to as a first monomer and a second monomer.
  • the first monomer is composed of two polypeptide chains (hereinafter referred to as a first heavy chain and a light chain) and comprises an antigen-binding domain (Fab) capable of binding to one antigen;
  • the second monomer is composed of one polypeptide chain (hereinafter referred to as a second heavy chain) and comprises an antigen-binding domain (scFv) capable of binding to another antigen.
  • the two monomers of the multispecific antibody (preferably, bispecific antibody) further comprise an Fc region comprising CH2 and CH3 domains.
  • the Fc regions of the two monomers further comprise amino acid substitutions that can promote the mutual binding of heterodimers.
  • the first heavy chain of any multispecific antibody (preferably, bispecific antibody) described herein comprises a VH1 - CH1 structure from N-terminus to C-terminus, and the light chain comprises a VL1 - CL structure.
  • the first heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the first heavy chain comprises, from N-terminus to C-terminus, a VH1 - CH1 - CH2 - CH3 structure, and the light chain comprises a VL1 - CL structure.
  • VH1 and VL1 are the heavy and light chain variable regions, respectively, that specifically bind to an antigen.
  • CH1 is the heavy chain constant region ( CH1 domain)
  • CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL comprise one antigen-binding domain (Fab).
  • the second heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises a VH2 -L3- VL2 structure from N-terminus to C-terminus. In other embodiments, the second heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises a VL2 -L3- VH2 structure from N-terminus to C-terminus.
  • the second heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the second heavy chain comprises the structure VH2 -L3- VL2 - CH2 - CH3 from N-terminus to C-terminus.
  • the second heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises the structure VL2 -L3- VH2 - CH2 - CH3 from N-terminus to C-terminus.
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to another antigen, and L3 is a connecting peptide.
  • VH2 -L3- VL2 or VL2 -L3- VH2 constitutes another antigen-binding domain (scFv).
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to NKG2A
  • VH1 - CH1 and VL1 - CL constitute an antigen-binding domain (Fab) that specifically binds to NKG2A
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to PDL1
  • VH2 -L3- VL2 or VL2 -L3- VH2 constitute an antigen-binding domain (scFv) that specifically binds to PDL1.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to PDL1, and VH1 - CH1 and VL1 - CL constitute an antigen-binding domain (Fab) that specifically binds to PDL1.
  • VH2 and VL2 are the heavy chain variable region and light chain variable region, respectively, that specifically bind to NKG2A, and VH2 -L3- VL2 or VL2 -L3- VH2 constitute an antigen-binding domain (scFv) that specifically binds to NKG2A.
  • one of the antigen binding domains specifically binds to NKG2A
  • the other antigen binding domain specifically binds to PDL1.
  • the multispecific antibody preferably a bispecific antibody
  • the scFv antigen-binding domain includes genetically engineered cysteine mutations, and disulfide-bond-stabilized multispecific antibodies (preferably, bispecific antibodies) are obtained by introducing two cysteine mutations at the VH and VL interfaces.
  • the Fc is derived from wild-type human IgG1.
  • the Fc in one monomer comprises, but is not limited to, the following amino acid substitutions relative to wild-type human IgG1: E357Q, and S364K; and the Fc in another monomer comprises, but is not limited to, the following amino acid substitutions relative to wild-type human IgG1: Q295E, L368D, K370S, N384D, Q418E, and N421D, wherein the numbering is according to the EU index as in Kabat.
  • the connecting peptide e.g., L3 connecting VH1 and VL1 in an exemplary scFv comprises the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 89).
  • a connecting peptide (or, may be referred to as a "linker”) can be used to connect the domains and/or structural regions of the heavy chain of a multispecific antibody (preferably, a bispecific antibody) into a continuous molecule.
  • the multispecific antibody (preferably, a bispecific antibody) may include additional linkers, such as a flexible linker connecting the variable heavy chain and light chain of an scFv.
  • the multispecific antibody (preferably, a bispecific antibody) may include additional linkers, such as a flexible linker connecting the variable heavy chain and light chain of an scFv and other linkers for connecting other binding units to the core structure of the multispecific antibody (preferably, a bispecific antibody).
  • the present invention relates to a polypeptide chain comprising at least 4 residues.
  • the position of a connecting peptide (or joint) is typically, hydrophilic, and the joints themselves rarely or do not form a secondary structure (joint site or flexible joint site).
  • at least 4 amino acid whose joints can be used to connect domains and/or districts close to each other. Longer joints can also be used.
  • the joint can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues.
  • the joint can be identical or different (for example, identical or different length and/or amino acid sequence).
  • the connecting peptide comprises or consists of a glycine-serine linker.
  • glycine-serine linker refers to a peptide consisting of glycine and serine residues.
  • Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly 4 Ser) n , where n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • a preferred glycine-serine linker is (Gly 4 Ser) 2 , i.e., GGGGSGGGGS (SEQ ID NO: 78).
  • a preferred glycine-serine linker is (Gly 4 Ser) 4 , i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79).
  • a preferred glycine-serine linker is (Gly 4 Ser) 3 , i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 80).
  • two or more glycine-serine linkers are connected in series in a connecting peptide.
  • the connecting peptide comprises at least a portion of a hinge region (e.g., from an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker such as ( G4S ) n .
  • a hinge region e.g., from an IgG1, IgG2, IgG3, or IgG4 molecule
  • a series of glycine-serine residues e.g., a glycine-serine linker such as ( G4S ) n .
  • L1 and/or L2 include a hinge portion and a linker portion, such as a linker portion comprising a glycine-serine linker. In other aspects, L1 and/or L2 include only a hinge portion or only a linker portion, such as a glycine-serine linker. In certain aspects, L1 and L2 include a glycine-serine linker. In certain aspects, the glycine-serine linker portions of L1 and L2 are the same length, while in other aspects, the glycine-serine linker portions of L1 and L2 are different in length.
  • a multispecific antibody preferably a bispecific antibody
  • the heavy chain and light chain of the scFv can be connected by a flexible linker.
  • a flexible linker generally does not include a hinge portion, but is a glycine-serine linker or other flexible linker.
  • the length and amino acid sequence of the flexible linker that interconnects the scFv domains can be selected and optimized.
  • the connecting peptide (L2) connecting the antigen-binding domain scFv to the Fc region in a multispecific antibody comprises the amino acid sequence GGGGSGGGGSEPKSDKTHTCPPCP (SEQ ID NO: 86), or GGGGSGGGGSCPPCP (SEQ ID NO: 87), or GGGGSGGGGSDKTHTCPPCP (SEQ ID NO: 88).
  • the connecting peptide (e.g., L) connecting the antigen-binding domain to the carboxyl-terminal CH3 of the Fc region in a multispecific antibody comprises the amino acid sequence GGGGSGGGGTGGGGS (SEQ ID NO: 90).
  • the multispecific antibody in addition to the connecting peptides connecting one antigen-binding domain to another antigen-binding domain or connecting one of the antigen-binding domains to the Fc (e.g., L1 and L2), may optionally include additional connecting peptides.
  • additional connecting peptides are independently selected.
  • the multispecific antibody preferably, bispecific antibody
  • This flexible connecting peptide may include a glycine-serine linker.
  • this linker does not include a hinge portion.
  • the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the amino acid sequence ASTKGP (SEQ ID NO: 81) or TVAAP (SEQ ID NO: 82).
  • the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79).
  • Exemplary antibody sequences are shown in Tables 2 to 9, where CDR numbering is based on the EU index in Kabat or IMGT. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining antibody light and heavy chain variable regions.
  • Antibodies comprising CDRs, VH, and/or VL sequences of antibodies or antigen-binding domains that specifically bind to NKG2A, antibodies or antigen-binding domains that specifically bind to PD-L1, and multispecific antibodies (preferably, bispecific antibodies) that specifically bind to NKG2A and PDL1 as described herein, but based on prediction algorithms other than those exemplified in the following tables, are also within the scope of this application.
  • the antibody or antigen-binding domain that specifically binds to PDL1 can be selected from the anti-PDL1 antibody or antigen-binding fragment described in patent application WO2024040212A2, and the antibody or antigen-binding domain that specifically binds to preS1 can be selected from the anti-HBV preS1 antibody or antigen-binding fragment described in patent application WO2023/066171A, all of which are incorporated into this application by reference.
  • Table 2-1 CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to NKG2A
  • Table 2-2 VH & VL sequences of exemplary antibodies or antigen-binding domains that specifically bind to NKG2A
  • Table 3-1 CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to PD-L1
  • Table 3-2 VH & VL sequences of exemplary antibodies or antigen-binding domains that specifically bind to PD-L1
  • Table 3-3 VH & VL cysteine variant sequences of exemplary antibodies or antigen-binding domains that specifically bind to PD-L1
  • Table 4-1 CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to HBV preS1
  • Table 4-2 VH & VL sequences of exemplary antibodies or antigen-binding domains that specifically bind to HBV preS1
  • Table 4-3 VH & VL cysteine variant sequences of exemplary antibodies or antigen-binding domains that specifically bind to HBV preS1
  • Table 6-1 Full-length heavy and light chain sequences of exemplary multispecific antibodies whose IgG-scFv structures specifically bind to NKG2A and PD-L1
  • Table 6-2 Full-length heavy and light chain sequences of exemplary multispecific antibodies with IgG-(scFv)2 structures that specifically bind to NKG2A and PD-L1
  • Table 7 Full-length heavy and light chain sequences of exemplary multispecific antibodies that specifically bind to NKG2A, PD-L1, and preS1, Bs4Ab-scFv structures
  • Table 8 Full-length heavy and light chain sequences of exemplary multispecific antibodies whose IgG-scFv structures specifically bind to PD-L1 and preS1
  • Table 9-1 Full-length heavy and light chain sequences of exemplary multispecific antibodies whose IgG-scFv structures specifically bind to NKG2A and preS1
  • Table 9-2 Full-length heavy and light chain sequences of exemplary multispecific antibodies whose IgG-(scFv)2 structure specifically binds to NKG2A and preS1
  • Binding affinity can be expressed as Kd, Koff, Kon or Ka.
  • Koff refers to the rate constant at which the antigen binding domain dissociates from the antigen binding domain/antigen complex, as determined by a kinetic selection device.
  • Kon refers to the association rate constant at which the antibody binds to the antigen to form the antigen binding domain/antigen complex.
  • the dissociation constant "Kd” used in this application refers to the dissociation constant during a specific antibody-antigen interaction, and refers to the antigen concentration required when the antigen occupies half of all antibody binding domains and reaches equilibrium in a solution of antibody molecules, which is equal to Koff/Kon. The determination of Kd assumes that all binding molecules are in solution.
  • the corresponding dissociation rate constant is expressed as EC50 , which is a good approximation of Kd.
  • the affinity binding constant Ka is the reciprocal of the dissociation constant Kd.
  • the equilibrium dissociation constant (Kd) can be used as an indicator of the affinity of the antigen-binding domain for the antigen.
  • Kd values obtained using these methods are expressed in units of M.
  • Antibodies that specifically bind to a target may have a Kd value of ⁇ 10-7 M, ⁇ 10-8 M, for example, ⁇ 10-9 M, ⁇ 10-10 M, ⁇ 10-11 M, ⁇ 10-12 M, or ⁇ 10-13 M.
  • the binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.
  • the antibody or antigen binding domain that specifically binds NKG2A specifically binds to the NKG2A target with a Kd value of 10-7 M to 10-13 M (e.g., 10-7 M to 10-13 M, 10-8 M to 10-13 M, 10-9 M to 10-13 M, or 10-10 M to 10-12 M).
  • the Kd value of binding between an antibody or antigen binding domain that specifically binds to NKG2A and NKG2A is 10-7 M to 10-13 M, 1 ⁇ 10-7 M to 5 ⁇ 10-13 M, 10-7 M to 10-12 M, 10-7 M to 10-11 M, 10-7 M to 10-10 M, 10-7 M to 10-9 M, 10-8 M to 10-13 M, 1 ⁇ 10-8 M to 5 ⁇ 10-13 M, 10-8 M to 10-12 M, 10-8 M to 10-11 M, 10-8 M to 10-10 M, 10-8 M to 10-9 M, 5 ⁇ 10-9 M to 1 ⁇ 10-13 M, 5 ⁇ 10-9 M to 1 ⁇ 10-12 M, 5 ⁇ 10-9 M to 1 ⁇ 10-11 M, 5 ⁇ 10-1 -9 M to 1 ⁇ 10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M, 10
  • the Kd value of binding between an antibody or antigen-binding domain that specifically binds to NKG2A and a non-target is higher than the Kd value of binding between the antibody or antigen-binding domain that specifically binds to NKG2A and the target, and in some embodiments cited herein, the binding affinity of an antibody or antigen-binding domain that specifically binds to NKG2A and the target (e.g., NKG2A) is higher than the binding affinity of the antibody or antigen-binding domain that specifically binds to NKG2A and the non-target.
  • the non-target is non-NKG2A.
  • the Kd value of the antibody or antigen binding domain that specifically binds NKG2A for binding to a non-NKG2A target is at least 10-fold, e.g., 10-10 2 -fold, 10 2 -10 3 -fold, 10 3 -10 4 -fold, 10 4 -10 5 -fold, 10 5 -10 6 -fold, 10 6 -10 7 -fold, 10 7 -10 8 -fold, 10 8 -10 9 -fold, 10 9 -10 10 -fold, 10 10 -10 11 -fold , 10 11 -10 12 -fold greater than the Kd of binding between the antibody or antigen binding domain that specifically binds NKG2A and the target NKG2A.
  • any multispecific antibody described herein comprises a first antigen-binding domain that specifically binds to NKG2A and a second antigen-binding domain that specifically binds to PDL1, wherein the first antigen-binding domain specifically binds to the NKG2A target with a Kd value of 10-7 M to 10-13 M ( e.g., 10-7 M to 10-13 M, 10-8 M to 10-13 M, 10-9 M to 10-13 M, or 10-10 M to 10-12 M).
  • a Kd value of 10-7 M to 10-13 M e.g., 10-7 M to 10-13 M, 10-8 M to 10-13 M, 10-9 M to 10-13 M, or 10-10 M to 10-12 M.
  • the Kd value for binding between the first antigen binding domain and NKG2A is 10-7 M to 10-13 M, 1 ⁇ 10-7 M to 5 ⁇ 10-13 M, 10-7 M to 10-12 M, 10-7 M to 10-11 M, 10-7 M to 10-10 M, 10-7 M to 10-9 M, 10-8 M to 10-13 M, 1 ⁇ 10-8 M to 5 ⁇ 10-13 M, 10-8 M to 10-12 M, 10-8 M to 10-11 M, 10-8 M to 10-10 M, 10-8 M to 10-9 M, 5 ⁇ 10-9 M to 1 ⁇ 10-13 M, 5 ⁇ 10-9 M to 1 ⁇ 10-12 M, 5 ⁇ 10-9 M to 1 ⁇ 10-11 M, 5 ⁇ 10-9 M to 1 ⁇ 10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M
  • the Kd value of the binding between the first antigen binding domain and NKG2A is 10 -7 M to 10 -13 M.
  • the second antigen binding domain specifically binds to the PDL1 target with a Kd value of 10 -7 M to 10 -13 M (e.g., 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, or 10 -10 M to 10 -12 M).
  • the Kd value for binding between the second antigen binding domain and PDL1 is 10-7 M to 10-13 M, 1 ⁇ 10-7 M to 5 ⁇ 10-13 M, 10-7 M to 10-12 M, 10-7 M to 10-11 M, 10-7 M to 10-10 M, 10-7 M to 10-9 M, 10-8 M to 10-13 M, 1 ⁇ 10-8 M to 5 ⁇ 10-13 M, 10-8 M to 10-12 M, 10-8 M to 10-11 M, 10-8 M to 10-10 M, 10-8 M to 10-9 M, 5 ⁇ 10-9 M to 1 ⁇ 10-13 M, 5 ⁇ 10-9 M to 1 ⁇ 10-12 M, 5 ⁇ 10-9 M to 1 ⁇ 10-11 M, 5 ⁇ 10-9 M to 1 ⁇ 10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -10 M to 1 ⁇ 10 -12 M,
  • any of the multispecific antibodies described herein comprises a first antigen-binding domain that specifically binds to NKG2A, and a second antigen-binding domain that specifically binds to PDL1.
  • the Kd value for binding between the first antigen-binding domain and a non-target is higher than the Kd value for binding between the first antigen-binding domain and the target, and in some embodiments cited herein, the binding affinity of the first antigen-binding domain to the target (e.g., NKG2A) is higher than the binding affinity of the first antigen-binding domain to the non-target.
  • the non-target is non-NKG2A.
  • the Kd value of the first antigen binding domain binding to the non-NKG2A target is at least 10 times greater than the Kd of the first antigen binding domain binding to the target NKG2A, for example, 10-10 2 times, 10 2 -10 3 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, or 10 11 -10 12 times.
  • the Kd value of the second antigen-binding domain binding to the non-PDL1 target is at least 10 times greater than the Kd of the binding between the second antigen-binding domain and PDL1, for example, 10-102 times , 102-103 times , 103-104 times , 104-105 times , 105-106 times, 106-107 times , 107-108 times , 108-109 times, 109-1010 times , 1010-1011 times , or 1011-1012 times .
  • the present application provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds to NKG2A and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1.
  • a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds NKG2A and (ii) an antibody or antigen-binding fragment that specifically binds PDL1, wherein: the antibody or antigen-binding fragment that specifically binds NKG2A comprises: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SNTMS (SEQ ID NO: 1); an HC-CDR2 comprising NINTGGNTYYANWAKG (SEQ ID NO: 2); and an HC-CDR3 comprising GSTIDSSGLSL (SEQ ID NO: 3); and a light chain variable region ( VL ), the VL comprising: a light chain complementarity determining region (LC-CDR) 1 comprising QASQNIGSDLA (SEQ ID NO: 4); an LC-CDR2 comprising LASTLAS (SEQ ID NO: 5); and an LC-C
  • a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds NKG2A and (ii) an antibody or antigen-binding fragment that specifically binds PDL1, the antibody or antigen-binding fragment that specifically binds PDL1 comprising: a heavy chain variable region ( VH ), the VH comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising GFTFGGFG (SEQ ID NO: 18); a HC-CDR2 comprising ITGDSSTI (SEQ ID NO: 19); and a HC-CDR3 comprising VRGPPGTWAY (SEQ ID NO: 20); and a light chain variable region ( VL ), the VL comprising: a light chain complementarity determining region (LC-CDR) 1 comprising ESVEFYGTTL (SEQ ID NO: 21); a LC-CDR2 comprising GAS (SEQ ID NO: 22); and a LC-CDR3 comprising
  • the antibody or antigen-binding fragment that specifically binds to NKG2A in the pharmaceutical composition comprises:
  • VH comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 13; and a VL comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 16; or
  • the antibody or antigen-binding fragment that specifically binds to PDL1 in the pharmaceutical composition comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 34, or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30 and 34; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 32 and 36, or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 32 and 36.
  • a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds to NKG2A and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1, wherein the antibody or antigen-binding fragment that specifically binds to NKG2A comprises: a VH comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 14; and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment that specifically binds to PDL1 comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 34 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 30 and
  • the present application provides nucleic acid molecules encoding antibodies or antigen-binding fragments that specifically bind to NKG2A, as well as multispecific antibodies (preferably, bispecific antibodies) that specifically bind to NKG2A and PDL1.
  • the present application provides a nucleic acid (or a group of nucleic acids) encoding an antibody or antigen-binding fragment that specifically binds to NKG2A, or a nucleic acid encoding a multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PDL1, including any of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies (preferably, bispecific antibodies) that specifically bind to NKG2A and PDL1 described herein.
  • a nucleic acid or a group of nucleic acids
  • a nucleic acid encoding an antibody or antigen-binding fragment that specifically binds to NKG2A
  • a multispecific antibody preferably, a bispecific antibody
  • the nucleic acid (or a group of nucleic acids) encoding the antibody, antigen-binding fragment, or multispecific antibody (preferably, a bispecific antibody) described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His-tag, an HA tag).
  • a polypeptide tag e.g., a protein purification tag, a His-tag, an HA tag
  • the present application also provides an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PDL1; or a nucleic acid molecule encoding the antibody or antigen-binding fragment; or an isolated host cell containing a vector carrying the nucleic acid molecule.
  • a multispecific antibody preferably, a bispecific antibody
  • variants include nucleotide sequences that hybridize with the nucleic acid sequence encoding the antibody or antigen-binding fragment or multispecific antibody (preferably, bispecific antibody) of the present application under at least moderate stringency hybridization conditions.
  • the present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.
  • a natural or synthetic nucleic acid encoding an antibody, antigen-binding fragment or bispecific antibody is inserted into a suitable expression vector so that the nucleic acid is operably linked to 5' and 3' regulatory elements, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), to express the antibody or antigen-binding fragment or multispecific antibody (preferably, a bispecific antibody).
  • the vector is suitable for replication and integration in eukaryotic host cells.
  • Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.
  • nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols.
  • Nucleic acid delivery methods are known in the art. For example, see U.S. Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.
  • the present application also provides gene therapy vectors.
  • Nucleic acids can be cloned into many types of vectors.
  • nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector can be provided to the cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as other virology or molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers (see, for example, WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.
  • Vectors derived from retroviruses are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells.
  • Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.
  • promoter elements such as enhancers, regulate the frequency of transcription initiation. These are typically located 30-110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are swapped or moved relative to one another. In the thymidine kinase (Tk) promoter, activity begins to decline only when the spacing between promoter elements increases to 50 bp.
  • Tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-1 ⁇ elongation factor 1 ⁇
  • constitutive promoters may also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.
  • this application should not be limited to the use of only constitutive promoters. Inducible promoters are also considered in this application.
  • an inducible promoter provides a molecular switch that can activate expression of an operably linked polynucleotide sequence when such expression is desired, and deactivate expression when such expression is not desired.
  • Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • expression of the antibody or antigen-binding fragment or multispecific antibody is inducible.
  • the nucleic acid sequence encoding the antibody or antigen-binding fragment or multispecific antibody is operably linked to an inducible promoter, including any inducible promoter described herein.
  • an inducible promoter provides a molecular switch that turns on the expression of an operably linked polynucleotide sequence when expression is desired and turns off expression when expression is not desired.
  • exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, D. M. et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R.
  • hormone-regulated elements e.g., see Mader, S. and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607
  • synthetic ligand-regulated elements see Spencer, D. M.
  • the inducible promoter system used to express antibodies or antigen-binding fragments or multispecific antibodies is the Tet system.
  • the inducible promoter system used to express antibodies or antigen-binding fragments or multispecific antibodies is the E. coli lac repression system.
  • an exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993).
  • the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites.
  • TetO Tet operator
  • TetR Tet repressor
  • the inducer releases TetR from TetO, thereby causing transcription to occur.
  • Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.
  • TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).
  • specific species e.g., prokaryotes
  • Tet-Off transcription is inactivated in the presence of Tc or Dox.
  • a tetracycline-regulated transcription activator protein tTA
  • TRE tetracycline-responsive promoter element
  • the TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus).
  • a promoter usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus.
  • rtTA is a fusion protein consisting of the TetR repressor and the VP16 transactivation domain.
  • rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.
  • lac repressor system of Escherichia coli (see Brown et al., Cell 49:603-612 (1987)).
  • the lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO).
  • lacO lac operator
  • lacR lac repressor
  • lacR lac repressor
  • lacR lacR
  • Expression of the target polynucleotide is induced by a suitable inducer, for example, isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector.
  • the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments.
  • Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.
  • Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences.
  • a reporter gene is a gene that is not present in or is not expressed by a recipient organism or tissue, and encodes a polypeptide whose expression is characterized by some properties that are easy to detect, such as enzymatic activity. After DNA is introduced into the recipient cells, the expression of the reporter gene is detected at the appropriate time.
  • Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared or obtained commercially by known techniques.
  • a construct with a minimum 5' flanking region that can show the highest expression level of a reporter gene is identified as a promoter.
  • This type of promoter region can be connected to a reporter gene and used to assess the ability of certain substances in regulating promoter-driven transcription.
  • a nucleic acid encoding any one of the antibodies or antigen-binding fragments or multispecific antibodies (preferably, bispecific antibodies) described herein is provided.
  • the nucleic acid includes one or more nucleic acid sequences encoding the heavy and light chains of antibodies or antigen-binding fragments or multispecific antibodies (preferably, bispecific antibodies).
  • each of the one or more nucleic acid sequences is contained in a separate vector.
  • at least some nucleic acid sequences are contained in the same vector.
  • all nucleic acid sequences are contained in the same vector.
  • the vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).
  • vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art.
  • expression vectors can be introduced into host cells by physical, chemical, or biological methods.
  • polynucleotides are introduced into host cells by calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).
  • the nucleic acid can be bound to a lipid.
  • the lipid-bound nucleic acid can be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule that binds to the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, suspended in lipids, contained in or mixed with micelles, or otherwise bound to lipids.
  • Lipid, lipid/DNA, or lipid/expression vector-related compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances that can be naturally occurring or synthetic.
  • lipids include fat droplets that naturally occur in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell.
  • Such experiments include, for example, "molecular biology” experiments familiar to those skilled in the art. For example, Southern and Northern blotting, RT-PCR and PCR; "biochemical” experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or by the experiments described in this application, all fall within the scope of this application.
  • the antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment that specifically binds to NKG2A) is a monoclonal antibody.
  • the antibody or antigen-binding fragment or multispecific antibody (preferably, a bispecific antibody) is derived from a monoclonal antibody.
  • the antibody, antigen-binding fragment or multispecific antibody (preferably, a bispecific antibody) comprises the VH and VL regions from a monoclonal antibody, or variants thereof.
  • the antibody, antigen-binding fragment or multispecific antibody (preferably, a bispecific antibody) further comprises the CH1 and CL regions from a monoclonal antibody, or variants thereof.
  • Monoclonal antibodies can be prepared using methods known in the art, such as hybridoma cell methods, yeast display, phage display, 293T cell display methods, or recombinant DNA methods.
  • exemplary yeast display and phage display methods are described in this application and in the following examples.
  • Multispecific antibodies preferably, bispecific antibodies
  • hamsters, mice or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent.
  • lymphocytes can be immunized in vitro.
  • the immunizing agent may include a polypeptide or fusion protein of the target protein.
  • PBLs peripheral blood lymphocytes
  • Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells.
  • Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used.
  • Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells.
  • suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells.
  • the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT)
  • hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.
  • the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium.
  • the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide.
  • the binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art.
  • the binding affinity of the monoclonal antibodies can be determined by, for example, Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107: 220 (1980).
  • the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.
  • DMEM modified Eagle medium
  • RPMI-1640 culture medium for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium.
  • hybridoma cells can be grown in mammalian ascites.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the antibody or antigen-binding fragment or multispecific antibody comprises a sequence selected from a clone of an antibody library (e.g., a phage library displaying scFv or Fab fragments).
  • the clone can be identified by screening a combinatorial library of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties.
  • the present invention further describes the present invention in Cerebral Biology 248:161-175(Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310(2004); Lee et al., J. Mol. Biol. 340(5):1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132(2004).
  • phage display methods the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then screened for phage that can bind to the antigen, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments in the form of scFv fragments or Fab fragments. Library phage from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridoma cells.
  • PCR polymerase chain reaction
  • natural libraries can be cloned to provide a single source of antibodies to a variety of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993).
  • natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.
  • the antibody or antigen-binding fragment or multispecific antibody is prepared by screening a library for an antigen-binding portion that can specifically bind to a target (e.g., NKG2A) through phage display.
  • the library can be a human scFv phage display library having at least 1 ⁇ 10 9 (e.g., at least 1 ⁇ 10 9 , 2.5 ⁇ 10 9 , 5 ⁇ 10 9 , 7.5 ⁇ 10 9 , 1 ⁇ 10 10 , 2.5 ⁇ 10 10 , 5 ⁇ 10 10, 7.5 ⁇ 10 10 , or 1 ⁇ 10 11 ) species of diversity of unique human antibody fragments.
  • the library is a human natural library constructed from DNA extracted from PMBCs and spleens of healthy subjects, comprising all human heavy and light chain subfamilies.
  • the library is a human natural library constructed from DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases.
  • the library is a semisynthetic human library in which the heavy chain CDR3 is completely random, with all amino acids (except cysteine) present at any given position with the same probability. (See, e.g., Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005).
  • the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids.
  • the library is a fully synthetic phage display library.
  • the library is a non-human phage display library.
  • Phage clones with high affinity for a target antigen can be screened by iterative binding of phage to the target antigen, which is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage.
  • a solid support e.g., beads for solution panning or mammalian cells for cell panning
  • the bound phage clones are then eluted and used to infect a suitable host cell, such as E. coli XL1-Blue, for expression and purification.
  • Phage clones that specifically bind to the target antigen can be enriched by multiple rounds of panning (e.g., 2, 3, 4, 5, 6, or more rounds), such as solution panning, cell panning, or a combination of both. Specific binding of the enriched phage clones to the target antigen can be detected by any method known in the art, including, for example, ELISA and FACS.
  • Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • the DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that can specifically bind to the genes encoding the light and heavy chains of mouse antibodies).
  • the hybridoma cells described above or the antigen-specific phage clones of this application can be used as the source of such DNA.
  • the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain monoclonal antibodies synthesized in recombinant host cells.
  • a host cell such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins.
  • the DNA can also be modified, for example, by replacing the homologous non-human sequence with the coding sequence for the human heavy and light chain constant structure and/or framework region (U.S. Patent No.
  • non-immunoglobulin polypeptides can replace the constant region of the antibody in the present application, or can replace one antigen binding site in the variable region of the antibody in the present application to form a chimeric bivalent antibody.
  • additional variable regions directed against different epitopes or antigens can be included to produce chimeric multispecific antibodies (preferably, bispecific antibodies).
  • the antibody can be a monovalent antibody.
  • Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of immunoglobulin light chains and modified heavy chains.
  • the heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains.
  • the relevant cysteine residues are replaced with other amino acid residues or deleted to prevent cross-linking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.
  • hybridoma cell lines to prepare multispecific antibodies (preferably, bispecific antibodies) refers to the fusion of two different hybridoma cell lines through cell fusion technology, and then identifying and isolating cells that can produce specific therapeutic antibodies (Kohler, G, et al. Continuous cultures of fused cells secreting antibodies of predefined specificity [J]. J Immunol., 2005, 174(5): 2453-2455). Because two hybridoma cells can produce two different light-heavy chains, and these light-heavy chains can be randomly combined, the multispecific antibodies (preferably, bispecific antibodies) prepared using this method have a high degree of randomness and low production efficiency.
  • KiH knock into hole
  • CrossMab are two common technologies currently used to improve the light-heavy chain pairing problem. KiH technology introduces an asymmetric mutation structure into the CH3 domain (a "knob” mutation refers to replacing a smaller residue with a larger amino acid residue in the CH3 domain, while a "hole” mutation refers to replacing a larger residue with a smaller amino acid residue).
  • the Fc region of engineered multispecific antibodies is more prone to heterodimerization than homodimerization due to steric hindrance (Ridgway J B, et al. “Knobs-into-holes” engineering of antibody CH3 domains for heavy chain heterodimerization [J]. Protein Eng. 1996, 9(7): 617-621). Introducing a Y349C mutation into the glycosylated CH3 domain can form disulfide bonds between glycosylated heavy chains and enhance the stability of KiH (Kuglstatter A, et al.
  • the charge effects of amino acid residues have also been used to enhance heterodimerization between the two heavy chains of multispecific antibodies (preferably, bispecific antibodies).
  • multispecific antibodies preferably, bispecific antibodies.
  • one chain is positively charged and the paired chain negatively charged, promoting heavy chain heterodimer formation through a pattern of like charges repelling and unlike charges attracting.
  • Mutations K409D and D399K, K409D/K392D and D399K/E356K, or E356K/E357K/D399K and K370E/K409D/K439E in both chains can all enhance heterodimer formation to a certain extent (IGAWA T, et al. Methods for producing polypeptides by regulating polypeptides; association: US, 20100015133A1[P]. 2006). Combining the steric hindrance effect and charge effect of KiH is also one of the strategies to improve heterodimerization.
  • CrossMab technology is a new antibody pairing technology developed by Roche based on KiH technology. It is a multi-specific antibody (preferably a bispecific antibody) in which the light chain and heavy chain of one Fab are exchanged, while the other is not exchanged.
  • the exchanged light chain contains a fragment of a homologous heavy chain, which makes it impossible to pair with the unexchanged heavy chain, thereby ensuring the correct combination between the light chain and the heavy chain (Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies [J]. Proc Natl Acad Sci USA, 2011, 108 (27): 11187-11192).
  • the structure includes "CrossMab Fab", “CrossMab VH-VL " or "CrossMab CHI-CL " and other forms.
  • Antibody variable regions with the desired binding specificity can be fused to immunoglobulin constant regions.
  • the fusion is with an immunoglobulin heavy chain constant region, which includes at least a portion of the hinge, CH2 , and CH3 domains.
  • the CH1 domain of the heavy chain constant region, which contains the site necessary for light chain binding is present in at least one fusion.
  • DNA encoding the immunoglobulin heavy chain fusion, and if desired, DNA encoding the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism.
  • antibody variable regions directed against different antigenic epitopes or different antigens can be fused to immunoglobulin constant region sequences to generate chimeric multispecific antibodies (preferably, bispecific antibodies).
  • the antibody or antigen-binding fragment or multispecific antibody can be a humanized antibody or a fully human antibody.
  • the humanized form of a non-human (such as mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab', F(ab') 2 , scFv or other antigen-binding subsequences of an antibody), which generally includes a minimum sequence derived from a non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins, immunoglobulin chains or their fragments (receptor antibodies), wherein the residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues with desired specificity, affinity and performance, such as the CDRs of mice, rats or rabbits.
  • human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues.
  • Humanized antibodies can also include amino acid residues that are neither part of the receptor antibody nor in the CDR or framework region sequences introduced.
  • a humanized antibody comprises at least one, typically two variable regions, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin consensus sequences.
  • a humanized antibody typically contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are often referred to as "imported” residues, typically from the "imported” variable region.
  • humanization can be performed essentially according to the following method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences.
  • this "humanized" antibody portion U.S. Patent No.
  • the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.
  • Generating partially human antibodies is an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies upon immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production.
  • JH antibody heavy chain joining region
  • Fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced).
  • Human antibodies or human antibody portions can also be produced by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991).
  • the amino acid sequences of variants of the antibodies or antigen-binding fragments provided herein are also under consideration. For example, it may be necessary to improve the binding affinity and/or other biological activities of the antibody or antigen-binding fragment.
  • the amino acid sequence of the antigen-binding entity variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding entity or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antigen-binding entity.
  • the final construction can be completed by any combination of amino acid residue deletions, insertions, and substitutions to give it the desired characteristics. For example, antigen binding.
  • variants of antibodies or antigen-binding fragments having one or more amino acid substitutions are provided.
  • the target sites of the substitution mutations include hypervariable regions (HVRs) and framework regions (FRs).
  • Amino acid substitutions can be introduced into the target antibody to screen for products of desired activity, for example, improved affinity or activity.
  • the amino acid substitutions described herein are limited to the "exemplary substitutions" in Table 11 of the application.
  • the amino acid substitutions are limited to the "preferred substitutions" in Table 11 of the application.
  • Amino acids are divided into different categories based on the properties of their side chains:
  • Acidic amino acids Aspartic acid Asp, glutamic acid Glu;
  • Aromatic amino acids tryptophan Trp, tyrosine Tyr, phenylalanine Phe.
  • Non-conservative amino acid substitutions include substituting one class for another.
  • An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display.
  • affinity maturation techniques based on phage display.
  • one or more CDR residues are mutated, the variant antibody portion is displayed on phage, and variants with specific biological activity (e.g., based on RBC cell lysis inhibition assay or binding affinity) are screened.
  • Changes e.g., substitutions
  • Changes can be made in the HVRs region to obtain improved RBC lysis inhibition assay or antibody affinity. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol.
  • diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis).
  • a secondary library is then created. The library is screened to identify antibody variants with the desired affinity.
  • Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.
  • substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen.
  • conservative changes that do not substantially reduce binding affinity can be produced in HVRs (e.g., conservative substitutions provided herein). These changes may occur outside the HVR "hotspots" or SDRs regions.
  • each HVR is either unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
  • a useful method for identifying amino acid residues or regions in an antibody that can be targeted for mutation is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085.
  • target residues e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid
  • neutral or negatively charged amino acids e.g., alanine or glutamic acid
  • Further substitutions can be introduced at the amino acid position to demonstrate functional sensitivity of the position to the initial substitution.
  • contact sites between the antibody and antigen are identified by crystal structure of the antigen-antibody complex. These contact site residues and neighboring residues can be targeted or eliminated as substitution candidates.
  • the variants are screened to determine whether they have the desired properties.
  • Insertions of amino acid sequences include fusions at the amino and/or carboxyl termini ranging in length from one residue to polypeptides comprising 100 or more residues, and also include insertions of one or more amino acid residues within a sequence.
  • Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus.
  • Other insertion variants of antibody molecules include fusions of an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody molecule to the N- or C-terminus of the antibody molecule.
  • one or more amino acid modifications are introduced into the Fc region of an antibody or antigen-binding fragment described herein (e.g., a full-length antibody that specifically binds to NKG2A, a multispecific antibody that specifically binds to NKG2A and PDL1, or a fusion protein comprising the antibody or antigen-binding fragment or multispecific antibody), thereby generating an Fc variant.
  • the Fc variant has enhanced ADCC potency, typically associated with receptors that bind to Fc (FcRs).
  • the Fc variant has reduced ADCC potency.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • NK cells activated by antibodies.
  • NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the surface of target cells.
  • Fc receptors on the surface of NK cells are CD16 or Fc ⁇ RIII. Binding of Fc receptors to the Fc region of antibodies leads to activation of NK cells, release of cytolytic granules, and subsequent apoptosis of target cells.
  • Fc receptor (FcR) binding experiments can be performed to confirm that the antibody lacks Fc ⁇ R binding (and therefore may lack ADCC activity), but retains FcRn binding ability.
  • NK cells express only Fc ⁇ RIII, while monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch and Kinet Annu. Rev. Immunol. 9: 457-492 (1991), page 464.
  • Non-limiting examples of in vitro assessment of ADCC activity of target molecules are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)).
  • non-radioactive assays can be used (see, for example, the ACTI TM flow cytometry non-radioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and the CYTOTOX 96 TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in an animal model as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
  • a C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and, therefore, lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402.
  • a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).
  • Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Pat. No. 6,737,056).
  • These Fc variants include Fc variants with two or more substitutions at residues 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).
  • alterations in the Fc region result in altered (i.e., enhanced or decreased) opsonization, as described in Moore et al., MAbs. 2(2): 181–189 (2010).
  • a variant of an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1
  • a multispecific antibody e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1
  • FcRn Fc receptor
  • Antibodies with extended half-life and improved FcRn binding are described in US2005/0014934A1 (Hinton et al.). These antibodies comprise one or more amino acid substitutions in the Fc region that enhance binding of the Fc region to FcRn.
  • Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 in the Fc region, such as a substitution at residue 434 in the Fc region (U.S. Pat. No. 7,371,826).
  • antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1 (e.g., full-length antibodies that specifically bind to NKG2A, or bispecific antibodies that specifically bind to NKG2A and PDL1) comprising any one of the Fc variants described herein or a combination thereof.
  • the antibodies or antigen-binding fragments that specifically bind to NKG2A, or the multispecific antibodies that specifically bind to NKG2A and PDL1 are altered to increase or decrease the degree of glycosylation of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or the multispecific antibodies that specifically bind to NKG2A and PDL1.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1 comprises an Fc region
  • the sugars attached thereto may be modified.
  • Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically N-linked to the CH2 domain Asn297 of the Fc region, see, for example, Wright et al., TIBTECH 15:26-32 (1997).
  • Such oligosaccharides may comprise a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • sugars such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1 of the present application may be oligosaccharide-modified to generate antibody or antigen-binding fragment that specifically binds to NKG2A, or multispecific antibody that specifically binds to NKG2A and PDL1 variants having certain improved properties.
  • N-glycans attached to the CH2 domain of the Fc region are heterogeneous.
  • Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83).
  • a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum.
  • N-glycosylation of the Fc region is important for binding to Fc ⁇ Rs; however, non-fucosylated N-glycans enhance Fc binding to Fc ⁇ RIIIa.
  • Enhanced binding to Fc ⁇ RIIIa results in enhanced ADCC, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.
  • Fc-mediated cytotoxicity when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental.
  • the Fc fragment or CH2 domain is non-glycosylated.
  • glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.
  • variants of antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1 e.g., full-length antibodies that specifically bind to NKG2A, or bispecific antibodies that specifically bind to NKG2A and PDL1 are provided, comprising an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function.
  • the present application provides variants of antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1, which have reduced fucose relative to the same antibody or antigen-binding fragment that specifically binds to NKG2A, or multispecific antibody that specifically binds to NKG2A and PDL1 produced by wild-type CHO cells. That is, they are characterized by having a lower amount of fucose compared to antibodies produced by native CHO cells (e.g., CHO cells that produce native glycosylated forms, CHO cells containing a native FUT8 gene).
  • native CHO cells e.g., CHO cells that produce native glycosylated forms, CHO cells containing a native FUT8 gene.
  • the N-linked glycans of the antibody or antigen-binding fragment that specifically binds NKG2A, or the multispecific antibody that specifically binds NKG2A and PDL1 have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose.
  • the fucose content of the antibody or antigen-binding fragment that specifically binds NKG2A, or the multispecific antibody that specifically binds NKG2A and PDL1 may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%.
  • the N-linked glycans of the antibody or antigen-binding fragment that specifically binds NKG2A, or the multispecific antibody that specifically binds NKG2A and PDL1 do not contain fucose, i.e., the antibody or antigen-binding fragment that specifically binds NKG2A, or the multispecific antibody that specifically binds NKG2A and PDL1 is completely free of fucose, lacks fucose, or is defucosylated.
  • Fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all sugar structures (e.g., complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546.
  • Asn297 refers to the asparagine residue at position 297 in the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publication Nos.
  • Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US2003/0157108A1, Presta, L; and WO 2004/056312A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells with ⁇ -1,6-fucosyltransferase gene, FUT8 gene knockout (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003/085107).
  • Variants of antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1 e.g., full-length antibodies that specifically bind to NKG2A, or bispecific antibodies that specifically bind to NKG2A and PDL1
  • Such variants of antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1 may have reduced fucosylation and/or enhanced ADCC function.
  • Examples of such antibody variants are described in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006).
  • the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1) variant that specifically binds to NKG2A and PDL1 comprises an Fc region that can bind to Fc ⁇ RIII.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1) variant that specifically binds to NKG2A and PDL1 comprising an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or has enhanced ADCC activity in the presence of human effector cells compared to an otherwise identical antibody or antigen-binding fragment that specifically binds to NKG2A, or multispecific antibody (e.g., a full-length antibody that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1) that specifically binds to NKG2A and PDL1 having a human wild-type IgG1 Fc region.
  • the multispecific antibody e.g., a full-length antibody that specifically binds to NKG2A,
  • cysteine-engineered antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1), in which one or more amino acid residues are substituted with a cysteine residue.
  • the substituted residue occurs at an accessible site of the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1.
  • reactive sulfhydryl groups are located at accessible sites of an antibody or antigen-binding fragment that specifically binds NKG2A, or a multispecific antibody that specifically binds NKG2A and PDL1, which can be used to conjugate the antibody or antigen-binding fragment that specifically binds NKG2A, or a multispecific antibody that specifically binds NKG2A and PDL1 to other moieties, such as a drug moiety or a linker-drug moiety, to prepare an antibody or antigen-binding fragment that specifically binds NKG2A, or a multispecific antibody that specifically binds NKG2A and PDL1 immunoconjugate as further described herein.
  • Cysteine-engineered antibodies or antigen-binding fragments that specifically bind NKG2A, or multispecific antibodies that specifically bind NKG2A and PDL1 can be prepared, for example, as described in U.S. Pat. No. 7,521,541.
  • the multispecific antibodies specifically binding to NKG2A and PDL1 provided herein may be further modified to include other non-protein moieties known in the art and readily available. Suitable moieties for derivatizing multispecific antibodies that specifically bind to NKG2A and PDL1 include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • ethylene glycol/propylene glycol copolymers carboxymethyl cellulose
  • dextran polyvinyl alcohol
  • polyvinyl pyrrolidone poly
  • Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water.
  • the polymer can have any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the multispecific antibody that specifically binds NKG2A and PDL1 can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, whether the properties or functionality of the multispecific antibody that specifically binds NKG2A and PDL1 is to be improved, whether the multispecific antibody derivative that specifically binds NKG2A and PDL1 is intended for use in the treatment of a specific condition, etc.
  • compositions comprising any one of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A or a bispecific antibody that specifically binds to NKG2A and PDL1), a nucleic acid encoding the antibody or antigen-binding fragment, a vector comprising a nucleic acid encoding the antibody or antigen-binding fragment, or a host cell comprising the nucleic acid or vector described herein.
  • formulations comprising any one of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A or a bispecific antibody that specifically binds to NKG2A and PDL1), a nucleic acid encoding the antibody
  • Suitable preparations of antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1 can be obtained by mixing antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1, having the desired purity, with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), and prepared in the form of lyophilized preparations or liquid preparations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • Lyophilized formulations suitable for subcutaneous administration are described in WO97/04801. Such lyophilized formulations can be reconstituted into formulations with high protein concentrations by suitable diluents, and the reconstituted formulations can be administered to the individual to be treated in the present application by subcutaneous administration.
  • Cationic liposomes or liposomes can be used to deliver the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1, of the present application, to cells.
  • the formulations described herein may also contain one or more other active substances necessary for treating a specific condition, preferably substances with complementary activities and no adverse reactions to each other.
  • other active substances such as antibiotics.
  • the effective amount of the other active substances depends on the amount of the antibody or antigen-binding fragment that specifically binds NKG2A, or the multispecific antibody that specifically binds NKG2A and PDL1 in the formulation, the disease or condition, or the treatment modality, as well as other factors as described above.
  • These drugs are generally used in the same dosages and routes of administration as described herein, or at 1% to 99% of the currently used dosage.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 can also be embedded in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in coarse emulsions, respectively.
  • a sustained-release formulation can be prepared.
  • Rational strategies can be designed to stabilize antibodies or antigen-binding fragments that specifically bind to NKG2A, or multispecific antibodies that specifically bind to NKG2A and PDL1, based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be through intermolecular SS bonds formed by thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.
  • Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.
  • the present application provides a method for preventing or treating HBV infection in an individual, comprising administering to the individual an effective amount of any multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PD-L1 as described herein, or a composition comprising the same.
  • any multispecific antibody preferably, a bispecific antibody
  • the present application also provides the use of the above-mentioned multispecific antibody (preferably, a bispecific antibody) or a composition comprising the same in the preparation of a medicament for preventing or treating HBV infection.
  • the present application provides a method for preventing or treating HBV infection in an individual, comprising administering to the individual an effective amount of any multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PD-L1 as described herein, or a composition comprising the multispecific antibody, wherein the multispecific antibody comprises a first antigen-binding domain that specifically binds to NKG2A and a second antigen-binding domain that specifically binds to PDL1.
  • any multispecific antibody preferably, a bispecific antibody
  • the multispecific antibody comprises a first antigen-binding domain that specifically binds to NKG2A and a second antigen-binding domain that specifically binds to PDL1.
  • the present application provides use of any multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PD-L1 as described herein, or a composition comprising the multispecific antibody (preferably, a bispecific antibody) in the preparation of a medicament for preventing or treating hepatitis B virus infection, wherein the multispecific antibody comprises a first antigen-binding domain that specifically binds to NKG2A and a second antigen-binding domain that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody
  • the multispecific antibody comprises a first antigen-binding domain that specifically binds to NKG2A and a second antigen-binding domain that specifically binds to PDL1.
  • a method for preventing and/or treating HBV infection in an individual comprising administering to the individual an effective amount of a multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PDL1 as described herein, or a composition comprising the multispecific antibody (preferably, a bispecific antibody), wherein the method is more effective than administering an equivalent dose of an antibody that specifically binds to NKG2A or an equivalent dose of an antibody that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody
  • the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PDL1 as described herein, which enhances ADCC activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times more than the administration of an equivalent dose of an antibody that specifically binds to NKG2A or an equivalent dose of an antibody that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody
  • the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) described herein that specifically binds to NKG2A and PDL1, which enhances the neutralization activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times more compared to administering an equivalent dose of an antibody that specifically binds to NKG2A or an equivalent dose of an antibody that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody described herein that specifically binds to NKG2A and PDL1
  • the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) described herein that specifically binds to NKG2A and PDL1, which enhances ADCP activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more compared to administering an equivalent dose of an antibody that specifically binds to NKG2A or an equivalent dose of an antibody that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody described herein that specifically binds to NKG2A and PDL1
  • the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) that specifically binds to NKG2A and PDL1 as described herein, which reduces the HBsAg content in the patient's serum by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more compared to administering an equivalent dose of an antibody that specifically binds to NKG2A or an equivalent dose of an antibody that specifically binds to PDL1.
  • a multispecific antibody preferably, a bispecific antibody
  • a method for preventing or treating HBV infection in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein.
  • composition comprising (i) an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein for the preparation of a medicament for preventing or treating HBV infection.
  • a method for preventing or treating HBV infection in an individual comprising administering to the individual: (i) an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A as described herein is administered simultaneously with the antibody or antigen-binding fragment that specifically binds to PDL1.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and the antibody or antigen-binding fragment that specifically binds to PDL1 are administered sequentially.
  • an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein in the preparation of a medicament for preventing or treating HBV infection.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A as described herein is administered simultaneously with the antibody or antigen-binding fragment that specifically binds to PDL1.
  • the antibody or antigen-binding fragment that specifically binds to NKG2A as described herein is administered sequentially with the antibody or antigen-binding fragment that specifically binds to PDL1.
  • a method for preventing or treating HBV infection in an individual comprising administering to the individual an effective amount of (i) an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein, wherein the method is more effective than administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to NKG2A or an equivalent dose of an antibody or antigen-binding fragment that specifically binds to PDL1.
  • the method comprises administering an effective amount of (i) an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein, which method enhances ADCC activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more compared to administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to NKG2A or an equivalent amount of an antibody or antigen-binding fragment that specifically binds to PDL1.
  • the method comprises administering an effective amount of (i) an antibody or antigen-binding fragment that specifically binds to NKG2A as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to PDL1 as described herein, which method enhances the neutralization activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more compared to administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to NKG2A or an equivalent amount of an antibody or antigen-binding fragment that specifically binds to PDL1.
  • a product comprising a substance that can be used to prevent or treat HBV infection in an individual, or for delivering an antibody or antigen-binding fragment (an antibody that specifically binds to NKG2A) or a multispecific antibody (e.g., a bispecific antibody that specifically binds to NKG2A and PDL1), or a pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to NKG2A and an antibody or antigen-binding fragment that specifically binds to PDL1, to cells attached to pathogens expressing NKG2A or PDL1.
  • the product may include a container and a label or package insert on or accompanying the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the container can be made of a variety of materials, such as glass or plastic.
  • the container contains a composition that is effective for treating the disease or condition described herein and has a sterile port (e.g., the container can be an intravenous infusion bag or a vial with a lid that can be pierced by a hypodermic injection needle).
  • At least one active substance in the composition is the antibody or antigen-binding fragment or bispecific antibody described herein.
  • the label or package insert indicates the specific condition that the composition can be used to treat.
  • the label or package insert further includes instructions for administering the bispecific antibody or pharmaceutical composition to a patient. Combination therapies and kits comprising the combination therapies described herein are contemplated.
  • Package insert refers to instructions typically included in the commercial packaging of a therapeutic product, which includes indications, usage, dosage, administration, contraindications, and/or warning information related to the use of these therapeutic products.
  • the package insert indicates that the composition can be used to treat bacterial infections.
  • the package insert indicates that the composition can be used to treat HBV infection.
  • the product may further include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Green's solution, or glucose solution.
  • BWFI bacteriostatic water for injection
  • Other materials required from a commercial and user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.
  • kits that can be used for various purposes, such as for preventing or treating HBV infection in an individual, or for delivering an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1) to cells attached to pathogens expressing NKG2A or PDL1, optionally in combination with a product.
  • kits that can be used for various purposes, such as for preventing or treating HBV infection in an individual, or for delivering an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1) to cells attached to pathogen
  • kits of the present application include one or more containers containing an antibody or antigen-binding fragment that specifically binds to NKG2A, or a composition (or single-dose form and/or product) that specifically binds to NKG2A and PDL1, and in some embodiments, further contain another agent (e.g., an agent described herein) and/or instructions for use consistent with any of the methods described herein.
  • the kit may further include instructions for selecting an individual suitable for treatment.
  • the instructions for use included in the kits of the present application are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.
  • the kit comprises a composition comprising an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1).
  • a composition comprising an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1).
  • the kit comprises: a) a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 described herein, and b) at least one other agent in an effective amount that can enhance the effect (e.g., therapeutic effect, detection effect) of the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1.
  • a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 described herein
  • at least one other agent in an effective amount that can enhance the effect (e.g., therapeutic effect, detection effect) of the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1.
  • the kit comprises: a) a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 described herein, and b) instructions for administering the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody composition that specifically binds to NKG2A and PDL1 to an individual for treating HBV infection in the individual.
  • the kit includes: a) a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 described herein, and b) at least one other agent in an effective amount that can enhance the effect (e.g., therapeutic effect, detection effect) of the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1, and c) instructions for administering the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1, and the other agent to an individual for treating HBV infection in the individual.
  • a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 described herein
  • the antibody or antigen-binding fragment that specifically binds to NKG2A, or the multispecific antibody that specifically binds to NKG2A and PDL1, and the other agent can be present in separate containers or in the same container.
  • the kit can include one specific composition or two or more compositions, wherein one composition includes an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1, and the other composition includes another agent.
  • the kit comprises one (or a set of) nucleic acids encoding an antibody or antigen-binding fragment that specifically binds to NKG2A, or a multispecific antibody that specifically binds to NKG2A and PDL1 (e.g., a full-length antibody that specifically binds to NKG2A, or a bispecific antibody that specifically binds to NKG2A and PDL1).
  • NKG2A-CD94 heterodimers are expressed on the surface of NK cells, and their ligand, HLA-E tetramers, can bind to NKG2A on NK cells.
  • This assay measures the binding of HLA-E tetramers to NKG2A on the surface of NK cells (e.g., NKL cells or NK92 cells) to examine the ability of anti-NKG2A antibodies to block the binding of HLA-E to NKG2A.
  • NKL cells (Cat#BSCELL-0491, Shanghai Binsui Biotechnology Co., Ltd.) were resuspended and diluted in 1% BSA to a concentration of 1.0 ⁇ 106 cells /ml and seeded into 96-well plates at 100 ⁇ l/well. The plates were blocked at 4°C for 30 min.
  • the wells were incubated at 4°C in the dark for 1 h.
  • the cells were washed with 1 ⁇ PBS and fluorescence was detected by flow cytometry.
  • the antibody binding data were analyzed using FlowJo software, and the positive rate (%) was used to represent the proportion of positive cells with HLA-E fluorescent labeling.
  • Humanized anti-NKG2A antibodies R76-9 and R76-10 (human IgG4 format) can effectively block the binding of NKG2A on NKL cells to HLA-E tetramers, and their activity is better than that of the positive control antibody M15.
  • NKL cells expressing NKG2A on their cell surface with K562-E4 cells overexpressing the ligand HLA-E can activate NKG2A/HLA-E signaling, leading to intracellular ITIM phosphorylation, thereby inhibiting the cytotoxic activity of NKL cells.
  • anti-NKG2A antibodies promote the cytotoxic activity of NKL cells against K562-E4 target cells by neutralizing NKG2A expressed on NKL cells.
  • EuTDA cytotoxicity assay was used for detection.
  • the EuTDA cytotoxicity assay (Cat#AD0116, PerkinElmer) uses the fluorescent amplifying ligand BATDA to specifically label target cells.
  • BATDA rapidly enters cells and, upon hydrolysis, forms a hydrophilic chelate called TDA, which remains intracellular.
  • TDA hydrophilic chelate
  • BATDA is released and combines with the DELIFA Eu reagent to form a highly fluorescent, stable chelate called EuTDA, which is used to monitor target cell lysis.
  • K562-E4 stable cell line was constructed. Briefly, K562 cells were electroporated with the constructed plasmid pIRES2-SA-HLAE-EGFP and selectively cultured for several days in 1640 medium (10% FBS + 1% P.S. double-antibody) containing 400 ⁇ g/ml of the antibiotic G418. Cells expressing fluorescent proteins were then sorted by flow cytometry. The sorted monoclonal cells were plated in 24-well plates and cultured in 1640 medium containing G418 for 7-10 days. Afterwards, the cells were transferred to 75 cm cell culture flasks (Cat#430720, Corning) for expansion, thereby generating the K562-E4 stable cell line.
  • 1640 medium 10% FBS + 1% P.S. double-antibody
  • Short peptide stimulation of K562-E4 stably transfected cells Resuspend K562-E4 stably transfected cells in 3 ml of Opti-MEM containing 500 ⁇ g/ml of the short peptide VMAPRTVLL (synthesized by Nanjing GenScript) and incubate in a CO2 incubator at 26°C overnight (approximately 16 hours). Wash the cells with 1 ⁇ DPBS buffer. Add 1.5 ⁇ l of the fluorescence-enhancing ligand BATDA (Cat#AD0116, PerkinElmer) per 1 ml of cell suspension in the dark. Mix thoroughly and incubate in a CO2 incubator at 37°C for 30 minutes.
  • NKL cell treatment NKL cells were cultured in MyeloCult TM H5100 medium (Cat#05150, STEMCELL Technologies) supplemented with 1% anti-NKG2A antibody and 100 IU/ml IL-2 (Cat#589106, Biolegend). 100 ⁇ l of NKL cells at a density of 2 ⁇ 10 5 cells/ml were seeded into 96-well V-bottom plates (Cat#701201, NEST). 50 ⁇ l of serially diluted anti-NKG2A antibodies (8 ⁇ g/ml, 1.6 ⁇ g/ml, and 0.32 ⁇ g/ml) were added. Mon antibody (NOVO NORDISK) served as a positive control. Negative control wells were left untreated.
  • the plate was incubated at room temperature in the dark for 15 minutes with shaking.
  • the fluorescence value of each well was measured by a microplate reader.
  • the killing rate was calculated based on the fluorescence value of each well to measure the functional activity of the anti-NKG2 antibody.
  • the calculation formula is as follows:
  • the killing rate curve was drawn and the EC50 value was calculated.
  • the results are shown in Table 13.
  • the humanized anti-NKG2A antibody R76-10 (human IgG4 form) can effectively promote the killing of K562-E4 target cells by NKL cells, and its killing activity is significantly better than that of the positive control antibody Mon.
  • Example 2 Preparation and characterization of anti-NKG2A-PDL1 bispecific antibodies
  • VH and VL represent the heavy chain variable region and light chain variable region of the antibody, respectively;
  • CH represents the antibody heavy chain constant region, including the CH1 , CH2 , and CH3 domains;
  • scFv is a single-chain antibody formed by connecting the antibody VH and VL via a linker peptide;
  • IgG1 Fc represents the Fc region of an IgG1 subclass antibody, which includes the CH2 and CH3 domains;
  • CL represents the light chain constant region.
  • IgG-scFv bispecific antibodies are constructed by connecting an scFv fragment that binds to other antigens to the Fc terminus of one of the heavy chains of an IgG antibody to achieve bispecificity.
  • a knob-in-hole (KIH) structure was designed in the IgG1 Fc region, and two Cys residue mutations (S354C on the "knob” side and Y349C on the "hole” side) that form stabilizing disulfide bridges were introduced.
  • the KIH replaces the threonine (T) at position 366 of the CH3 region of one monomer with tryptophan (W), forming a "knob” structure.
  • the threonine (T) at position 366 is replaced with serine (S)
  • the leucine (L) at position 368 is replaced with alanine (A)
  • the tyrosine (Y) at position 407 is replaced with valine (V), forming a "hole” structure.
  • the combination mutation LALAPS is formed by replacing leucine (L) at positions 234 and 235 of the antibody hinge region with alanine (A), and proline (P) at position 331 with serine (S).
  • a disulfide-stabilized multispecific antibody was obtained by introducing two cysteine mutations at the VH and VL interfaces, namely, introducing a G44C mutation in the VH region of SBT451scFv and a Q100C mutation in the VL region of SBT451scFv; wherein the numbering is according to the EU index as in Kabat.
  • the schematic diagram of the IgG-scFv bispecific antibody structure used in this embodiment is shown in Figure 1A.
  • the anti-NKG2A-PDL1 bispecific antibody in this structure includes four polypeptide chains, namely the first heavy chain, the first light chain, the second heavy chain and the second light chain, wherein the sequence of the first light chain and the second light chain are the same, and their sequence composition is shown in Table 14.
  • the specific amino acid sequences of their full-length heavy chains and light chains are shown in Table 6-1.
  • the anti-PD-L1 antigen binding domain in the bispecific antibody is derived from the anti-PD-L1 antibody SBT451 (also known as Hum-6, see patent application WO2024040212A2, the entire content of which is incorporated herein in its entirety), and its amino acid sequence is shown in Tables 3-1, 3-2 and 3-3; the anti-NKG2A antigen binding domain is derived from the anti-NKG2A antibody R76-10.
  • the heavy chain variable region and light chain variable region sequences of the anti-NKG2A antibody and the heavy chain variable region and light chain variable region sequences of the anti-PD-L1 antibody were constructed into the eukaryotic expression vector pTTa1 by seamless cloning, obtaining two heavy chain expression vectors expressing the heavy chain and a light chain expression vector expressing the light chain, a total of three expression vectors.
  • bispecific antibodies Following the manufacturer's instructions, the three expression plasmids for the aforementioned bispecific antibodies were co-transfected into HEK293F cells. The transfected 293F cells were cultured at 37°C, 5% CO2 , and 120 rpm for 6 days. The cell culture fluids were collected separately. The antibodies were purified using Protein A resin (MabCap At 4FF 5ml prepacked column, Catalog No. SA023C15, Changzhou Tiandi Renhe Biotechnology Co., Ltd.).
  • Protein A resin MobCap At 4FF 5ml prepacked column, Catalog No. SA023C15, Changzhou Tiandi Renhe Biotechnology Co., Ltd.
  • the Protein A column was equilibrated with 6 column volumes of PBS buffer (containing 0.15M NaCl, pH 7.4) at a flow rate of 150 cm/h (flow rate of 5 ml/min).
  • the culture supernatant (adjusted to pH 7.2-7.4) was passed through the column at a flow rate of 150 cm/h (flow rate of 5 ml/min).
  • elution was performed using 6 column volumes of 0.1 M glycine buffer (containing 0.15 M NaCl, pH 3.2), the eluate was collected, the pH was adjusted to neutral, and the target protein solution was replaced with PBS buffer using an ultrafiltration concentrator.
  • Molecular sieve chromatography (HiLoad 16/600 superdex 200 pg, Cytiva) was then used to remove aggregate components in the antibody. Briefly, the molecular sieve chromatography column was equilibrated with 1 column volume of PBS buffer (containing 50 mM PBS and 0.15 M NaCl, pH 7.2) at a flow rate of 1 ml/min. A volume of no more than 5 ml of antibody sample was loaded, and then the molecular sieve chromatography column was rinsed with PBS buffer, and UV280 was detected to collect the components corresponding to the monomer peak. The solution was ultrafiltered into PBS using an ultrafiltration tube, and after ultrafiltration and concentration, the concentration of the target protein was determined using a BCA protein quantification kit.
  • PBS buffer containing 50 mM PBS and 0.15 M NaCl, pH 7.2
  • IgG-(scFv)2 bispecific antibodies see, Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63) achieve bispecificity by attaching scFv fragments that bind to other antigens to the Fc termini of the two heavy chains of an IgG antibody.
  • the combined mutation LALAPS was introduced into the Fc region, and two cysteine mutations were introduced at the VH and VL interfaces of the SBT451 scFv.
  • Example 2.1 See Example 2.1.
  • the schematic diagram of the bispecific antibody structure of IgG-(scFv) 2 used in the examples of the present application is shown in Figure 1B.
  • the bispecific antibody molecule of this structure is composed of four chains, namely two identical heavy chains and two identical light chains.
  • the anti-NKG2A antigen binding domain in this bispecific antibody is derived from R76-10
  • the anti-PD-L1 antigen binding domain is derived from SBT451.
  • Table 15 shows the composition of the bispecific antibody of the IgG-(scFv) 2 structure used in the examples, and the specific amino acid sequences of its full-length heavy and light chains are shown in Table 6-2.
  • Bispecific antibody construction process According to the operating instructions, a heavy chain expression vector (1) for expressing the heavy chain and a light chain expression vector (1) for expressing the light chain were constructed by seamless cloning, for a total of 2 expression vectors.
  • Table 15 Heavy and light chain composition of anti-NKG2A-PDL1 bispecific antibodies of IgG-(scFv)2 structure
  • an anti-NKG2A-PDL1 bispecific antibody for proof of concept was additionally constructed in this example for activity verification in mice.
  • a POC molecule with an IgG-scFv structure was constructed according to the steps described in 2.1; a POC molecule with an IgG-(scFv)2 structure was constructed according to the steps described in 2.2.
  • the anti-NKG2A antigen binding domain in the designed POC molecule adopts the sequence of the anti-mouse NKG2A antibody NKG2A.2 (refer to patent WO2020/102501A1, the entire contents of which are incorporated herein in their entirety); the anti-PD-L1 antigen binding domain adopts the sequence of the anti-mouse PD-L1 antibody S70 (refer to Clin Cancer Res (2020) 26(15):4154–4167), and a G44C mutation was introduced into the VH region of S70scFv and a Q100C mutation was introduced into the VL region to obtain a disulfide-stabilized multispecific antibody.
  • Table 16 shows the composition of the POC bispecific antibody used in the examples, and the specific amino acid sequences
  • the binding activity of the anti-NKG2A-PDL1 bispecific antibodies NP-2 and NP-1 to the huNKG2A-huCD94-musFc antigen was detected by ELISA binding assay, wherein the coated antigen concentration was 1 ⁇ g/ml, NKG2A monoclonal antibody R76-10 was used as a control molecule, and the other steps were carried out as described in Example 1.3.
  • the results are shown in Table 17.
  • the anti-NKG2A-PDL1 bispecific antibodies NP-2 and NP-1 can effectively bind to human NKG2A antigen, and the binding activity is comparable to that of the NKG2A monoclonal antibody R76-10.
  • An ELISA binding assay was used to detect the binding activity of the POC molecule corresponding to the anti-NKG2A-PDL1 bispecific antibody to the mouse NKG2A antigen.
  • the coated antigen was 1 ⁇ g/ml mouse NKG2A-CD94 heterodimer antigen (purchased from ACROBiosystems, catalog number #NC4-M5254), and the control antibody was the NKG2A monoclonal antibody NKG2A.2.
  • the other steps were performed as described in Example 1.3.
  • the results are shown in Table 18.
  • the POC molecules NP-2-POC and NP-1-POC corresponding to the anti-NKG2A-PDL1 bispecific antibody can effectively bind to mouse NKG2A antigen, and the binding activity is comparable to that of the NKG2A monoclonal antibody NKG2A.2.
  • Table 18 Binding activity of anti-NKG2A-PDL1 bispecific antibody POC molecules to NKG2A
  • the binding activity of the anti-NKG2A-PDL1 bispecific antibody to the human PD-L1 antigen was detected by ELISA binding assay, wherein the coating antigen was 1 ⁇ g/ml human PD-L1-Fc fusion protein (see patent application WO2024040212A2), the control antibody was PD-L1 monoclonal antibody SBT451, and the other steps were performed as described in Example 1.3.
  • the results are shown in Table 19.
  • the anti-NKG2A-PDL1 bispecific antibodies NP-2 and NP-1 can effectively bind to the human PD-L1 antigen, and the binding activity is comparable to that of the PD-L1 monoclonal antibody SBT451.
  • the binding activity of the POC molecule corresponding to the anti-NKG2A-PDL1 bispecific antibody to the mouse PD-L1 antigen was detected by ELISA binding assay, wherein the coated antigen was 1 ⁇ g/ml purified mouse PD-L1-Fc fusion protein, and the control antibody was PD-L1 monoclonal antibody S70.
  • the other steps were carried out as described in Example 1.3.
  • the results are shown in Table 20.
  • the POC molecule NP-2-POC corresponding to the exemplary anti-NKG2A-PDL1 bispecific antibody can effectively bind to the mouse PD-L1 antigen, and the binding activity is comparable to that of the PD-L1 monoclonal antibody S70.
  • Anti-NKG2A-PDL1 bispecific antibody blocks the binding of NKG2A to HLA-E tetramers on NKL cells
  • the blocking activity of the anti-NKG2A-PDL1 bispecific antibody against the binding of HLA-E tetramers to the NKL cell surface receptor NKG2A was detected by FACS.
  • the anti-NKG2A-PDL1 bispecific antibody was added at an initial concentration of 60 ⁇ g/ml and diluted 3-fold in a series of 11 dilutions.
  • the control antibody was the NKG2A monoclonal antibody R76-10. The other steps were performed as described in Example 1.4.
  • the results are shown in Table 21.
  • the anti-NKG2A-PDL1 bispecific antibodies NP-2 and NP-1 can effectively block the binding of NKG2A and HLA-E tetramers on NKL cells, and their activity is comparable to that of the NKG2A monoclonal antibody R76-10.
  • the DELFIA EuTDA cytotoxicity assay was used to detect the cytotoxicity of NKL cells against K562-E4 cells by the anti-NKG2A-PDL1 bispecific antibody.
  • the NKL cells were seeded at a density of 1 ⁇ 10 5 cells/ml, the initial concentration of the added bispecific antibody was 40 ⁇ g/ml, and 9 dilutions were performed in a 5-fold gradient.
  • the control antibody was the NKG2A monoclonal antibody R76-10. Other steps were performed as described in Example 1.5.
  • the interaction between PD-L1 and PD1 is detected using a HTF donor and HTF acceptor.
  • FRET fluorescence resonance energy transfer
  • This specific signal is proportional to the extent of the PD1/PD-L1 interaction. Therefore, compounds or antibodies that block the PD1/PD-L1 interaction will result in a decrease in the HTRF signal.
  • HTF donor anti-Tag1-Eu3 and HTF receptor anti-Tag2-XL665 10 ⁇ l of premixed HTF donor anti-Tag1-Eu3 and HTF receptor anti-Tag2-XL665 (Promega, 64ICP01PEG) were then added, the plate was blocked, and incubated at room temperature for 1 hour. Fluorescence was detected and the HTRF ratio was calculated. Binding curves were generated using Graphpad Prism to calculate the IC 50 value.
  • the results are shown in Table 23.
  • the anti-NKG2A-PDL1 bispecific antibodies NP-2 and NP-1 can effectively inhibit the interaction between PD1 and PDL1 proteins, and their blocking activity is better than that of the positive control antibody AM10-F2.
  • HBV-Tg HBV transgenic mouse model
  • the preparation process was as follows: a linearized DNA fragment 1.28 times the length of the HBV (type A, GenBank: AF305422.1) genome was injected into the pronuclei of C57BL/6NCrl mouse embryos to generate transgenic mice. Analysis of HBV DNA copy number in peripheral blood revealed that transgenic founder mice with a copy number of 107 to 108 IU/ml were retained.
  • the HBV-Tg mouse strain was established by mating hemizygous wild-type C57BL/6NCrl mice.
  • mice produce intact, infectious viral particles, and HBV replication levels are comparable to those in chronic hepatitis B patients.
  • high levels of HBsAg and HBeAg are also detected in peripheral blood. Since HBV antigens are continuously expressed during the embryonic stage, inducing immune tolerance in mice, these mice do not exhibit immune pathological changes similar to those of human hepatitis B.
  • mice were intraperitoneally injected with the anti-NKG2A-PDL1 bispecific antibody POC molecule NP-2-POC, the anti-PD-L1 monoclonal antibody S70, or a combination of the anti-NKG2A monoclonal antibody NKG2A.2 and the anti-PD-L1 monoclonal antibody S70. Dosing was performed twice weekly for four consecutive weeks. Blood samples were collected from the mice on the second day after administration, and HBsAg levels were measured using an HBsAg ELISA kit (Mike Biotech).
  • the Bs4Ab-scFv trispecific antibody structure is based on the Bs4Ab bispecific antibody structure (see the literature Bezabeh B, et al. Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties. MAbs. 2017 Feb/Mar; 9(2): 240-256).
  • a scFv fragment is connected to the C-terminus of one Fc to form an antigen-binding module that can specifically bind to another different antigen, thereby achieving trispecificity.
  • a knob-in-hole structure (KIH) was also designed in the Fc region.
  • two Cys residue mutations that form a stabilizing disulfide bridge were introduced, as well as the combined mutation LALAPS.
  • two cysteine mutations were introduced at the VH and VL interfaces of SBT451scFv. For details, see Example 2.1.
  • an anti-NKG2A-PDL1-preS1 trispecific antibody was additionally constructed for proof of concept (POC) to verify its activity in mice.
  • FIG. 1C A schematic diagram of the trispecific antibody structure of Bs4Ab-scFv used in the examples of the present application is shown in Figure 1C.
  • Table 24 shows the composition of the trispecific antibody of the Bs4Ab-scFv structure used in the examples, and the specific amino acid sequences of its full-length heavy and light chains are shown in Table 7.
  • the anti-NKG2A antigen binding domain in the trispecific antibody is derived from R76-9, R76-10 or NKG2A.2 (POC molecule)
  • the anti-PD-L1 antigen binding domain is derived from SBT451 or S70 (POC molecule)
  • the anti-preS1 antigen binding domain is derived from the anti-HBV preS1 antibody K127-9 (see patent publication number WO2023/066171A, the content of which is incorporated herein in its entirety), and its amino acid sequences are shown in Tables 4-1, 4-2 and 4-3.
  • Table 24 Heavy and light chain composition of the anti-NKG2A-PD-L1-preS1 trispecific antibody of Bs4Ab-scFv structure
  • the binding activity of the anti-NKG2A-PDL1-preS1 trispecific antibody to the huNKG2A-huCD94-musFc antigen was detected according to the steps described in Example 2.4; the binding activity of the anti-NKG2A-PDL1-preS1 trispecific antibody to the mouse NKG2A-CD94 antigen was detected according to the steps described in Example 2.5.
  • the results are shown in Table 25.
  • the anti-NKG2A-PDL1-preS1 trispecific antibody NPH-1 can effectively bind to the human NKG2A antigen, and its binding activity is comparable to that of the NKG2A monoclonal antibody R76-10.
  • the POC molecule NPH-1-POC corresponding to the above trispecific antibodies can also effectively bind to mouse NKG2A antigen (data not shown).
  • Table 25 Binding activity of anti-NKG2A-PDL1-preS1 trispecific antibodies to NKG2A antigen
  • the binding activity of the anti-NKG2A-PDL1-preS1 trispecific antibody to the human PD-L1 antigen was detected according to the steps described in Example 2.6; the binding activity of the anti-NKG2A-PDL1-preS1 trispecific antibody to the mouse PD-L1 antigen was detected according to the steps described in Example 2.7.
  • the results are shown in Table 26.
  • the anti-NKG2A-PDL1-preS1 trispecific antibody NPH-1 can effectively bind to the human PD-L1 antigen, and its binding activity is comparable to that of the PD-L1 monoclonal antibody SBT451.
  • the POC molecule NPH-1-POC corresponding to the above trispecific antibody can also effectively bind to the mouse PD-L1 antigen (data not shown).
  • An ELISA binding assay was used to detect the binding activity of the anti-NKG2A-PDL1-preS1 trispecific antibody to the HBV preS1 antigen, wherein the coating antigen was 1 ⁇ g/ml HBV-preS1-His fusion protein (see patent publication number WO2023/066171A), the control antibody was the anti-HBV preS1 monoclonal antibody K127-9, and the other steps were performed as described in Example 1.3.
  • the results are shown in Table 27.
  • the anti-NKG2A-PDL1-preS1 trispecific antibody NPH-1 can effectively bind to HBV preS1 antigen, and its binding activity is comparable to that of the anti-HBV preS1 monoclonal antibody K127-9.
  • the POC molecule NPH-1-POC corresponding to the above-mentioned trispecific antibodies can also effectively bind to HBV preS1 antigen (data not shown).
  • Anti-NKG2A-PDL1-preS1 trispecific antibody blocking the binding of NKG2A and HLA-E tetramers on NKL cells (cellular level)
  • the blocking activity of the anti-NKG2A-PDL1-preS1 trispecific antibody on the binding of HLA-E tetramer to the NKL cell surface receptor NKG2A was detected by FACS. The specific steps were carried out according to the description in Example 2.8.
  • the results are shown in Table 28.
  • the anti-NKG2A-PDL1-preS1 trispecific antibody NPH-1 can effectively block the binding of NKG2A and HLA-E tetramers on NKL cells, and its activity is comparable to that of the NKG2A monoclonal antibody R76-10.
  • the DELFIA EuTDA cytotoxicity assay was used to detect the cytotoxic activity of NKL cells in promoting the anti-NKG2A-PDL1-preS1 trispecific antibody against K562-E4 cells. The specific steps were carried out as described in Example 2.9.
  • the results are shown in Table 29.
  • the anti-NKG2A-PDL1-preS1 trispecific antibody NPH-1 can effectively promote the killing of K562-E4 target cells by NKL cells.

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Abstract

涉及特异性地结合NKG2A和PD-L1的多特异性抗体或抗原结合片段，及其制备方法和用途。

Description

特异性结合NKG2A抗原和PD-L1抗原的多特异性抗体及其应用
以下提交的序列表的内容通过整体引用并入本申请中：文本名称：NKG2A-PDL1.xml，记录日期：2025.3.12，大小：96KB)。
技术领域
本申请涉及特异性结合NKG2A和PD-L1的多特异性抗体，以及包含特异性结合NKG2A和PD-L1的多特异性抗体的药物组合物，及其制备方法和用途，包括用其预防和治疗HBV感染或与HBV感染相关的疾病的方法。
背景技术
NKG2A，是自然杀伤细胞受体家族2(NKG2)的成员之一，又被称为自然杀伤细胞凝集素样受体C1(killer cell lectin like receptor C1,KLRC1)或CD159a，是一类II型跨膜蛋白。NKG2家族还包括NKG2B/C/D/E/F，其中NKG2A是NKG2受体家族的抑制性受体成员,是自然杀伤(natural killer,NK)细胞表面优先表达的跨膜蛋白之一。
NKG2A主要表达于NK细胞表面，还可以表达在部分T细胞表面。在人的NK细胞中，该蛋白与共同表达在NK细胞表面的CD94分子由二硫键连接形成异质二聚体复合物NKG2A-CD94后，被靶细胞上非经典的主要组织相容性复合体I类分子(major histocompatibility complex class I,MHC I)人类白细胞抗原α链E(HLA-E)识别。位于NKG2A蛋白N-端的NK细胞胞质域含有2个免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motifs,ITIMs)，可将抑制信号传递到NK细胞内，诱导级联的抑制信号，从而抑制NK的细胞毒活性和细胞因子的分泌。这种机制是NK细胞中天然自我识别/耐受的一部分。除了调控NK细胞功能，NKG2A还可以调控CD8+T细胞的活化过程，也可以在CD8+T细胞与靶细胞的相互作用中发挥抑制CD8+T细胞的功能。
在一些病毒，例如乙肝病毒(HBV)感染病人体内，T细胞免疫功能不足已被明确证实(Ye B,Liu X,Li X,et al.Cell Death Dis 2015；6:e1694)。此外，NK细胞功能受损，会导致其抗病毒功能出现缺陷(Stelma F,de Niet A,Tempelmans Plat-Sinnige MJ,et al..J Infect Dis 2015；212:1042–51.)。有研究表明，HBV感染能够增加小鼠和人类NK细胞上NKG2A抑制性受体的水平，并降低清除HBV的能力(Li F,et al.GASTROENTEROLOGY 2013；144:392–401)。
PD-L1分子程序性死亡配体1(PDL1；又被称为CD274或B7-H1)，同样是一种免疫检查点抑制性分子，组成性地表达于免疫细胞表面，例如T细胞、B细胞、巨噬细胞和树突状细胞(DCs)，并且也表达于多种非造血细胞，包括血管内皮、胰岛和胎盘合体滋养层细胞(Keir等，2008)。此外，PD-L1也可以由肿瘤细胞表达，作为逃避抗肿瘤反应的“适应性免疫机制”(Ohaegbulam KC等，2015；Anand V R Kornepati.等，Nat Rev Cancer,2022Mar；22(3):174-189)，并且可被任何炎症刺激上调(Yamazaki等，2002)。当浸润的肿瘤反应性细胞毒性T淋巴细胞(CTL)分泌的促炎细胞因子诱导肿瘤微环境中PD-L1表达时，PD-L1与细胞毒性T淋巴细胞(CTL)上的PD-1结合可下调抗肿瘤免疫。因此，阻断PD-L1/PD-1通路是恢复和增强抗肿瘤免疫应答的理想治疗方法。在临床前模型中，抗PD-1Ab和抗PD-L1Ab通过增强肿瘤抗原特异性T细胞应答(包括细胞因子产生、存活、细胞运动和糖酵解)，在各种肿瘤模型中(尤其是使用免疫原性肿瘤的模型)显示出抗肿瘤作用(Chang等2015；Zinselmeyer等，2013；Okazaki等，2013)。除了在肿瘤方面的作用，PD-L1抗原也高表达于HBV病毒感染的病人体内，通过PD-1/PD-L1的相互作用引起T细胞耗竭，从而导致持续的HBV感染(Mol Immunol.2008；45(4):963-70；Cell Death Dis.2015Mar 19；6:e1694)。目前已开发的PD-L1抗体如envafolimab(ASC-22,Alphamab Oncology)显示在治疗HBV感染方面取得了一定的效果，但针对HBV感染安全且有效的功能性治愈仍在探索中。
最新的研究报道，NKG2A与PD-1在头颈癌及黑色素瘤浸润性的CD8+T细胞上共表达，同时阻断NKG2A/HLA-E以及PD-1/PD-L1两条信号通路有很强的协同抗肿瘤作用(Cell.2018；175:1-13,Cell.2018；175:1744-1755)。另外有在研的靶向NKG2A和PDL1的双特异性抗体药物XB-628(Exelixis公司)，目前尚处于临床前阶段。目前已有报道的针对NKG2A和PDL1靶点的多特异性抗体数量有限，同时针对HBV感染的有效治愈仍然亟待解决，因此本领域仍然需要活性更高、效果更好的靶向NKG2A和PDL1的多特异性抗体以及利用该多抗有效治疗HBV感染相关疾病。
本申请提及的所有出版物、专利、专利申请和已公开的专利申请中披露的内容，以引用方式全部并入本申请中。
申请概述
一方面，本申请提供了特异性结合NKG2A和PD-L1的多特异性抗体(例如，双特异性抗体)，以及包含上述多特异性抗体(例如，双特异性抗体)的药物组合物。在另一些实施例中，本申请还提供使用上述多特异性抗体(例如，双特异性抗体)或其药物组合物预防和/或治疗HBV感染相关疾病的方法。在另一些实施例中，本申请还提供上述多特异性抗体(例如，双特异性抗体)或其药物组合物在制备用于预防和/或治疗HBV感染的药物中的用途。
在一些实施例中，本申请提供了一种结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)，其包含特异性结合NKG2A的第一抗原结合域，和特异性结合PD-L1的第二抗原结合域，其中，所述特异性结合NKG2A的第一抗原结合域包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含SNTMS(SEQ ID NO:1)；HC-CDR2，其包含NINTGGNTYYANWAKG(SEQ ID NO:2)；和HC-CDR3，其包含GSTIDSSGLSL(SEQ ID NO:3)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含QASQNIGSDLA(SEQ ID NO:4)；LC-CDR2，其包含LASTLAS(SEQ ID NO:5)；和LC-CDR3，其包含QQSWSSSNVDNV(SEQ ID NO:6)；以及，所述特异性结合PD-L1的第二抗原结合域包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含GFTFGGFG(SEQ ID NO:18)；HC-CDR2，其包含ITGDSSTI(SEQ ID NO:19)；和HC-CDR3，其包含VRGPPGTWAY(SEQ ID NO:20)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含ESVEFYGTTL(SEQ ID NO:21)；LC-CDR2，其包含GAS(SEQ ID NO:22)；和LC-CDR3，其包含QQIRKVPWT(SEQ ID NO:23)。
在一些实施例中，根据本申请所述的任一多特异性抗体(例如，双特异性抗体)，其包含特异性结合NKG2A的第一抗原结合域，和特异性结合PD-L1的第二抗原结合域，其中特异性结合NKG2A的第一抗原结合域包含：(a)VH，其包含氨基酸序列SEQ ID NO:13或其变体，所述变体与氨基酸序列SEQ ID NO:13具有至少80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少80％序列同一性；或(b)VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少80％序列同一性；以及，所述特异性结合PDL1的第二抗原结合域包含：VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少80％序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少80％序列同一性。
在一些实施例中，本申请所述的多特异性抗体(例如，双特异性抗体)包含Fc。在另一些实施例中，本申请所述的Fc选自IgGl、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD的Fc。在一些实施例中，所述Fc包含Fc变体。在一些实施例中，所述Fc是糖基化的。在一些实施例中，所述Fc是去糖基化的。在一些实施例中，所述Fc是岩藻糖基化减少的或是无岩藻糖基化。在一些实施例中，所述Fc变体包括在位点297上的取代。在一些实施例中，位点297上的取代是297Q。在一些实施例中，所述可变的Fc区包括在位点234、235、239、282、289、297、312、324、330、331、335、337、339、356、359、361、383、384、398、400、440、422和442中的一个或多个位点上的取代，根据EU索引编号。
在一些实施例中，所述多特异性抗体(例如，双特异性抗体)的结构选自IgG-scFv、IgG-(scFv)2、Bs4Ab-scFv、DVD-Ig、Hetero H,CrossMab或scFv-Fab IgG等结构形式。
在一些实施例中，所述多特异性抗体(例如，双特异性抗体)具有IgG-scFv结构。在一些实施例中，所述多特异性抗体(例如，双特异性抗体)包含四条多肽链：其中，
一条多肽链从N端到C端包含VH1-CH1-CH2-CH3-L-VH2-L3-VL2结构，其中VH1是特异性结合NKG2A的重链可变区；VH2是特异性结合PD-L1的重链可变区；VL2是特异性结合PD-L1的轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；
两条多肽链从N端到C端包含VL1-CL，其中VL1是特异性结合NKG2A的轻链可变区，CL是轻链恒定区；以及
一条多肽链从N端到C端包含VH1-CH1，其中VH1是特异性结合NKG2A抗原的重链可变区；CH1是重链恒定区CH1结构域；优选地，该多肽链进一步包含Fc，该Fc包含CH2和CH3结构域；
其中，VH1-CH1和VL1-CL组成可特异性结合NKG2A的抗原结合域(Fab)，VH2-L3-VL2组成可特异性结合PD-L1的抗原结合域(scFv)。
在一些实施例中，本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)包含：氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有80％序列同一性；和/或氨基酸序列SEQ ID NO:53或其变体，所述变体与氨基酸序列SEQ ID NO:53具有至少80％序列同一性；和/或氨基酸序列SEQ ID NO:54或其变体，所述变体与氨基酸序列SEQ ID NO:54具有至少80％序列同一性。
在一些实施例中，所述多特异性抗体(例如，双特异性抗体)具有IgG-(scFv)2结构。在一些实施例中，所述多特异性抗体(例如，双特异性抗体)包含四条多肽链：其中，
两条多肽链从N端到C端包含VH1-CH1-CH2-CH3-L-VH2-L3-VL2结构，其中VH1是特异性结合NKG2A的重链可变区；VH2是特异性结合PD-L1的重链可变区；VL2是特异性结合PD-L1的轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；以及
另外两条多肽链从N端到C端包含VL1-CL结构，其中VL1是特异性结合NKG2A的轻链可变区，CL是轻链恒定区。
其中VH1-CH1和VL1-CL组成特异性结合NKG2A的抗原结合域(Fab)，VH2-L3-VL2组成可特异性结合PD-L1的抗原结合域(scFv)。
在一些实施例中，本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)包含：氨基酸序列SEQ ID NO:54或其变体，所述变体与氨基酸序列SEQ ID NO:54具有80％序列同一性；和/或氨基酸序列SEQ ID NO:58或其变体，所述变体与氨基酸序列SEQ ID NO:58具有至少80％序列同一性。
在一些实施例中，提供一种治疗所需个体疾病或病症的方法，包括向所述个体施用有效量的如上所述的任一种多特异性抗体(例如，双特异性抗体)或包含其的药物组合物。在一些实施例中，提供如上所述的任一种多特异性抗体(例如，双特异性抗体)在制备用于治疗所需个体疾病或病症的药物组合物中的用途。在一些实施例中，提供如上所述的任一种多特异性抗体(例如，双特异性抗体)或包含其的药物组合物在制备用于治疗疾病或病症的药物中的用途。在一些实施例中，所述疾病或病症包括HBV感染或与HBV感染相关的疾病或病症。在一些实施例中，所述疾病或病症包括乙型肝炎、肝功能衰竭、肝硬化或肝癌。
一方面，本申请提供了在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的本申请所述的多特异性抗体(例如，双特异性抗体)和/或任一药物组合物。
在一些实施例中，根据本申请所述的任一方法，所述疾病或病症包含HBV感染或与HBV感染相关的疾病。在一些实施例中，所述的疾病或病症包括乙型肝炎、肝功能衰竭、肝硬化或肝癌。
在一些实施例中，本申请提供了一种分离的核酸分子，其编码如上所述的任一多特异性抗体(例如，双特异性抗体)。在一些实施例中，提供了一种载体，其包含如上所述的任一核酸分子。在一些实施例中，提供了一种宿主细胞，其包含如上所述的任一多特异性抗体(例如，双特异性抗体)、任一核酸分子或任一载体。在一些实施例中，提供了一种制备特异性结合NKG2A和PD-L1的多特异性抗体(例如，双特异性抗体)的方法，包括：a)在有效表达特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)的条件下培养如上所述的任一宿主细胞；并且b)在宿主细胞中获得所表达的多特异性抗体(例如，双特异性抗体)。
还提供了包含如上所述的任一种多特异性抗体(例如，双特异性抗体)、核酸分子、载体、或宿主细胞的药物组合物、试剂盒和制品。
附图说明
图1A所示为IgG-scFv多特异性抗体的示例性结构示意图；图1B所示为IgG-(scFv)2多特异性抗体的示例性结构示意图；图1C所示为Bs4Ab-scFv多特异性抗体的示例性结构示意图；图1D所示为DVD-Ig(Dual-variable domain-Ig)多特异性抗体的示例性结构示意图；图1E所示为Hetero H,CrossMab多特异性抗体的示例性结构示意图；图1F所示为scFv-Fab IgG多特异性抗体的示例性结构示意图。
本申请的详细描述
一方面，本申请提供特异性结合NKG2A的抗体或抗原结合片段。在另一方面，本申请提供特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)。
另一方面，本申请还提供了包含特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)的药物组合物。
另一方面，本申请还提供一种治疗所需个体疾病或病症的方法，包括向所述个体施用有效量的特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)或包含其的药物组合物。
另一方面，本申请提供特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)或包含其的药物组合物在制备用于预防或治疗HBV感染的药物中的用途。
通过293T细胞展示库筛选、抗体人源化、亲和力成熟以及适当设计的生物化学及生物学实验的组合，鉴定出特异性结合人NKG2A的抗体或抗原结合片段。通过杂交瘤筛选、人源化以及适当设计的生物化学及生物学实验的组合，鉴定出特异性结合人PD-L1的抗体或抗原结合片段(参见WO2024040212A2)。同时制备了特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)。当以多特异性抗体(例如，双特异性抗体)的方式用于预防和/或治疗相关疾病时能够达到累积或协同作用。
本申请同时还提供编码特异性结合NKG2A和PDL1的多特异性抗体(例如，双特异性抗体)的核酸，包含上述核酸的载体及宿主细胞，以及制备上述多特异性抗体的方法。
定义
如本申请所述，“治疗(treatment)”或“治疗(treating)”是一种获得有益的或期望的结果的方法，包括临床结果。鉴于本申请的目的，所述有益的或期望的临床结果，包括但不限于以下一种或多种：缓解由疾病引起的一种或多种症状，减轻疾病程度，稳定疾病(例如，预防或延迟疾病恶化)，预防或延迟疾病的扩散(例如，转移)，预防或延迟疾病复发，延迟或减缓疾病进展，改善疾病状态，缓解疾病(部分或全部)，减少治疗疾病所需的一种或多种其他药物的剂量，延迟疾病进展，改善或提高生活质量，增加体重，和/或延长生存期。同时，“治疗”还包括疾病病理结果的减少(例如，对HBV病毒感染而言，病毒载量、肝损伤程度)。本申请的方法考虑了这些治疗的任何一个或多个方面。
术语“预防(prevent)”以及类似的词，如“预防(prevented)”、“预防(preventing)”、“预防(prevention)”或“预防(prophylactic)”等，表示一种预防、抑制或减少疾病或病症(如HBV感染)发生或复发可能性的方法。它还指延缓一种疾病或病症的发生或复发，或延缓一种疾病或病症的症状的发生或复发。正如在此所使用的，“预防(prevention)”和类似的词还包括在疾病或病症发生或复发之前减轻其强度、影响、症状和/或负担。正如在此所使用的，“预防(prevention)”和类似的词还包括减少疾病或病症发生或复发的风险和易感性，例如HBV感染。
抗体或抗原结合片段如本申请所述，术语“抗体”是广义的，包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、单特异性、多特异性抗体(如双特异性抗体)、全长抗体及其抗原结合片段，只要它们表现出所需的抗原结合活性。全长抗体包括两条重链和两条轻链。轻链和重链的可变区负责抗原的结合。两条链中的可变区通常包括3个高变的环，被称为互补决定区(CDRs)，轻链(LC)CDRs包括LC-CDR1、LC-CDR2和LC-CDR3，重链(HC)CDRs包括HC-CDR1、HC-CDR2和HC-CDR3。本申请所披露的抗体或抗原结合片段的CDR边界可通过Kabat,Chothia或Al-Lazikani惯例来定义或识别(Al-Lazikani 1997；Chothia 1985；Chothia 1987；Chothia 1989；Kabat 1987；Kabat 1991)。重链或轻链的3个CDR区插入到被称为框架区(FRs)的侧翼区段之间，所述框架区比CDR区具有更高的保守性，并形成支撑高变环的支架。重链和轻链的恒定区并不参与抗原结合，但展示出多种效应功能。抗体是基于它们重链恒定区的氨基酸序列进行分类的。抗体的五种主要类别或同种型是IgA、IgD、IgE、IgG和IgM，其特征在于分别具有α、δ、ε、γ和μ型重链。几种主要的抗体类别被分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)。
如本申请所述，术语“抗原结合片段”包括一种抗体片段，包括，例如，双链抗体(diabody)、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、多特异性dsFv(dsFv-dsFv’)、二硫键稳定的双链抗体(ds双链抗体)、单链抗体(scFv)、scFv二聚体(二价双链抗体)，由包含一个或多个CDRs的抗体片段组成的多特异性抗体、单域抗体、纳米抗体、域抗体、二价域抗体或者能够与抗原结合但不包含完整抗体结构的任何其他抗体片段。其中如本申请所述的Fab(fragment antigen-binding)，其是包含抗体的VL结构域、VH结构域、CL结构域、以及CH1结构域的单价片段。抗原结合片段还包括包含上述抗体片段的融合蛋白。抗原结合片段能够与亲本抗体或亲本抗体片段(如亲本scFv)结合相同的抗原。在一些实施例中，抗原结合片段可能包括来自特定人抗体的一个或多个CDRs，该CDRs被移植到来自一个或多个不同人抗体的框架区。
如本申请所述，术语“多特异性抗体”是指在一个分子中对至少两个不同抗原或表位具有结合特异性的抗体分子(例如，双特异性抗体、三特异性抗体)。优选地，多特异性抗体是双特异性抗体。如本申请所述，术语“双特异性抗体”是指在一个分子中对两个不同抗原或表位具有结合特异性的抗体分子。在本申请的一些优选的实施方案中，所述多特异性抗体(例如的，双特异性抗体)的结构可在完整抗体的结构基础上进行改造而获得。多特异性抗体(例如，双特异性抗体)的生产过程包括完整分子的设计、每个结构域的核苷酸序列的合成和克隆、哺乳动物细胞的表达和最终产品的纯化。示例性的多特异性抗体(例如，双特异性抗体)的结构是本领域所已知的，例如，IgG-scFv结构、DVD-Ig结构、Bs4Ab结构、Hetero H,CrossMab结构、IgG-(scFv)2结构或scFv-Fab IgG结构等(例如，参见综述文献Labrijn AF,et al.Nat Rev Drug Discov.2019Aug；18(8):585-608)。
如本申请所述，术语“抗原结合域”是指抗原结合分子中与抗原特异性结合的部分。更具体地说，术语“抗原结合域”指的是抗体的一部分，该部分包括一个与部分或全部抗原特异性结合并与之互补的区域。如果是大抗原，抗原结合分子可能只结合抗原的一个特定部分，这个部分被称为抗原表位。例如，抗原结合域可以由一个或多个可变区(也称作可变域)提供。优选地，所述抗原结合域包括抗体轻链可变区(VL)和抗体重链可变区(VH)。一方面，抗原结合域能够结合其抗原并阻断或部分阻断所述抗原的功能。特异性结合NKG2A抗原或PDL1抗原的抗原结合域包括本申请进一步定义的抗体及抗原结合片段。
如本申请所述，术语“表位”是指抗体或抗体部分结合的抗原上特定的原子或氨基酸组。如果两种抗体或抗体部分表现出与某抗原竞争性结合，则它们可能结合抗原上相同表位。
如本申请所述，当第一抗体在等摩尔浓度下抑制第二抗体与NKG2A抗原靶标结合至少50％(例如至少55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或99％)时，第一抗体与第二抗体“竞争”结合NKG2A抗原靶标，反之亦然。PCT出版物WO 03/48731描述了基于交叉竞争的高通量抗体“表位归类”(binning)方法。
如本申请所述，术语“特异性(地)结合”、“特异性地识别”或“对…来说是特异性的”是指可测量的和可再现的相互作用，例如靶标与抗体的结合可以确定在异质分子群，包括生物分子中存在该靶标。例如，抗体能够特异性地识别某靶标(可以是表位)是指，与其它靶标结合相比，该抗体与该靶标的结合具有更高的亲和力，亲合力，更容易和/或更持久。在一些实施例中，特异性地识别抗原的抗体与抗原的一个或多个抗原决定簇反应，其结合亲和力是其与其它靶标结合亲和力的至少10倍。
如本申请所述，一种“分离的”抗体是指一种抗体，其(1)与天然存在的蛋白无关，(2)不含相同来源的其他蛋白，(3)由不同种属的细胞所表达，或(4)自然界中不存在。
如本申请所述，术语“分离的核酸”，是指基因组、cDNA或合成来源的核酸或其组合。根据其来源，所述“分离的核酸”(1)与自然界中发现的“分离的核酸”中的全部或部分多核苷酸无关，(2)可与自然状态下不与之相连的多核苷酸可操作性地连接，或(3)在自然界中不作为较长序列的一部分而存在。
如本申请所述，术语“CDR”或“互补决定区”是指重链和轻链多肽的可变区内发现的非连续抗原结合位点。在文献Kabat et al.,J.Biol.Chem.252:6609-6616(1977)；Kabat et al.,U.S.Dept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991)；Chothia et al.,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997)；MacCallum et al.,J.Mol.Biol.262:732-745(1996)；Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.et al.,Dev.Comp.Immunol.,27:55-77(2003)；和Honegger and Plückthun,J.Mol.Biol.,309:657-670(2001)中已经描述这些特殊的区域，其中当彼此之间互相比较时，这些定义包括氨基酸残基的重合或子集。然而，采用任何一种定义方式来指示抗体或移植抗体或其变体的CDR，均包括在本申请所定义和使用的术语范围之内。表1中列了由上述引用的各篇参考文献所定义的CDR所包括的氨基酸残基的位置，以示比较。CDR预测的算法和结合界面在本领域是已知的，包括，例如Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008)；Ehrenmann F.et al.,Nucleic Acids Res.,38:D301-D307(2010)；和Adolf-Bryfogle J.et al.,Nucleic Acids Res.,43:D432-D438(2015)中均有描述。本段中所引用的参考文献的内容以其整体引用并入本申请中，以用于本申请和可能包含在本申请中的一个或多个权利要求中。
表1:CDR定义

1氨基酸残基编号参照上述Kabat et al.中的命名方法
2氨基酸残基编号参照上述Chothia et al.中的命名方法
3氨基酸残基编号参照上述MacCallum et al.中的命名方法
4氨基酸残基编号参照上述Lefranc et al.中的命名方法
5氨基酸残基编号参照上述Honegger and Plückthun中的命名方法
术语“嵌合抗体”是指重链和/或轻链的一部分与来自特定种属或属于特定抗体种类或亚类的抗体中的相应序列一致或具有同源性，而这个(些)链的剩余部分与来自另一种属或属于其它抗体种类或亚类的抗体中的相应序列一致或具有同源性的抗体，以及此类抗体的片段，只要其具有本申请中的生物学活性(见U.S.Patent No.4,816,567；and Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。
“Fv”是包含完整抗原识别及结合位点的最小抗体片段。该片段是由一个重链可变区和一个轻链可变区紧密非共价连接形成的二聚体。通过这两个域的折叠衍生出6个高变环(轻链和重链中各3个环)，所述高变环为抗体提供了用于结合抗原的氨基酸残基，并且赋予抗体与抗原结合的特异性。然而，即使单个可变区(或Fv片段的一半，其仅包含对抗原具有特异性的3个CDRs)也具有识别和结合抗原的能力，尽管其亲和力低于完整的结合位点。
“单链Fv”，也可简写成“sFv”或“scFv”，是包含被连接成单一多肽链的VH和VL抗体域的抗体片段。在一些实施例中，scFv多肽进一步包括VH和VL域之间的连接多肽，该连接多肽使得scFv形成抗原结合的理想结构。关于scFv的概述，见Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,Springer-Verlag,New York,pp.269-315(1994)。
术语“双链抗体(diabodies)”是指，在VH和VL之间采用短接头(例如5～10个残基)构建scFv片段(见上段内容)制备而成的一种小抗体片段，这样就使得可变区在链间而不是链内进行配对，产生一个双价片段，即具有两个抗原结合位点的片段。多特异性的双链抗体是两个“交叉”scFv片段的异二聚体，其中两个抗体的VH和VL域位于不同的多肽链上。在EP 404,097；WO 93/11161；Hollinger et al.,Proc.Natl.Acad.Sci.USA,90:6444-6448(1993)中全面描述了双链抗体。
非人源(如啮齿类)抗体的“人源化”形式是嵌合抗体，其包括最少的来自非人源抗体的序列。大多数情况下，人源化抗体是人源免疫球蛋白(受体抗体)，其中受体抗体的高变区(HVR)残基被来自非人源种属例如小鼠、大鼠、兔或非人类灵长类动物的且具有理想的抗体特异性，亲和力和性能的高变区残基所取代(供体抗体)。在某些情况下，人源免疫球蛋白框架区(FR)中的残基被相应的非人源残基所取代。另外，人源化抗体可以包括在受体抗体或供体抗体中均不存在的残基。这些修饰能够进一步改善抗体的性能。通常，人源化抗体会包含基本上至少一个，通常两个可变区，其中所有或基本上所有的高变环均与非人免疫球蛋白的高变环相对应，以及所有或基本上所有的框架区均是人免疫球蛋白序列。人源抗体任选地也还包括免疫球蛋白恒定区(Fc)的至少一部分，通常是人免疫球蛋白的恒定区。具体细节可以参考Jones et al.,Nature 321:522-525(1986)；Riechmann et al.,Nature 332:323-329(1988)；和Presta,Curr.Op.Struct.Biol.2:593-596(1992)。
本申请所鉴定的多肽和抗体序列的“氨基酸序列同源性百分比(％)”或“同源性”或“同一性”被定义为：候选序列与待比较多肽序列中相同氨基酸残基所占的百分比。可以通过本领域技术范围内的多种比对方式来确定氨基酸序列同源性或同一性百分比，例如，使用如BLAST、BLAST-2、ALIGN、Megalign(DNASTAR)、或MUSCLE软件等可公开获得的计算机软件。本领域技术人员可以确定用于测量比对的合适的参数，包括在所比较序列的全长上实现最大化比对所需的任何算法。然而，为了本申请的目的，氨基酸序列同源性或同一性百分比数值是使用序列比对电脑程序MUSCLE(Edgar,R.C.,Nucleic Acids Research 32(5):1792-1797,2004；Edgar,R.C.,BMC Bioinformatics 5(1):113,2004)生成的。
术语“Fc(fragment crystallizable)”或“Fc区”，是指包含完整抗体恒定区的多肽，不包含CH1结构域，在某些情况下包含部分铰链，无论是单体形式还是多聚体形式。天然Fc的原始免疫球蛋白来源优选地为人类来源，并且可以是任何免疫球蛋白，例如，IgG1、IgG2、IgG3或IgG4。天然Fc由单体多肽组成，单体多肽可以通过共价(即二硫键)和非共价缔合连接成二聚体或多聚体形式。免疫球蛋白的Fc区一般包含重链恒定区的CH2结构域和CH3结构域，且任选包含CH4结构域。
在一些实施例中，Fc二聚体中的两个Fc单体中的每一个包含促进两个单体异源二聚体化的氨基酸取代。在一些实施例中，Fc单体的异源二聚体化可以通过在两个Fc单体中引入不同但相容的取代诸如“knob-into-hole”残基对来促进。“knob-into-hole”技术也公开于美国专利公布第8,216,805号中。在一些实施例中，一个Fc单体包含knob突变T366W，并且另一个Fc单体包含hole突变T366S、L358A和Y407V。在一些实施例中，引入形成稳定化的二硫桥的两个Cys残基(S354C在“knob”侧，并且Y349C在“hole”侧)。
术语“Fc受体”或“FcR”用于描述结合抗体Fc区的受体。在一些实施例中，本申请所述的FcR是结合IgG抗体(一种γ受体)的FcR，包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和可变剪接形式。FcγRII受体包括FcγRIIA(激活受体)和FcγRIIB(抑制受体)，它们具有相似的氨基酸序列，主要在细胞质结构域有所不同。激活受体FcγRIIA的胞质结构域中含有免疫受体酪氨酸活化基序(ITAM)。抑制受体FcγRIIB的胞质结构域中含有免疫受体酪氨酸抑制基序(ITIM)(见M.inAnnu.Rev.Immunol.15:203-234(1997))。所述术语还包括同种异型，例如FcγRIIIA同种异型:FcγRIIIA-Phe158、FcγRIIIA-Val158、FcγRIIA-R131和/或FcγRIIA-H131。在Ravetch and Kinet,Annu.Rev.Immunol 9:457-92(1991)和Capel et al.,Immunomethods 4:25-34(1994)；and de Haas et al.,J.Lab.Clin.Med.126:330-41(1995)中对FcRs进行了描述。本申请中术语FcR涵盖其他类型的FcRs，包括将来鉴定的FcRs。术语FcR同时还包括新生儿受体FcRn，其负责向新生儿转移母体IgGs(Guyer et al.,J.Immunol.117:587(1976)and Kim etal.,J.Immunol.24:249(1994))。
术语“FcRn”指新生儿Fc受体(FcRn)。FcRn与主要组织相容性复合体(MHC)在结构上相似，由α链非共价结合到β2微球蛋白上组成。新生儿Fc受体FcRn的多种功能在Ghetie and Ward(2000)Annu.Rev.Immunol.18,739-766.中进行了描述。FcRn在免疫球蛋白IgGs从母体向新生儿的被动转运和调控血清IgG水平中起到重要作用。FcRn作为一种救助受体，可以在细胞内和细胞间以完整的形式结合和运输胞吞化的IgG，并使它们免于经受默认的降解途径。
人IgG重链恒定区的“CH1结构域”通常从118位氨基酸延伸到215位氨基酸(EU编号系统)。
“铰链区”通常被定义为从人IgG1的216位Glu延伸到230位Pro(Burton,Molec.Immunol.22:161-206(1985))。通过将形成重链间二硫键的第一个和最后一个半胱氨酸残基置于与IgG1相同位置后，可以使得其他IgG亚型的铰链区与IgG1序列比对。
人IgG Fc区的“CH2结构域”通常从231位氨基酸延伸到340位氨基酸。CH2结构域的独特之处在于，它不会与另一个区域紧密配对。而是在完整的天然IgG分子的两个CH2结构域之间插入了两条N端连接的支链糖链。据推测，糖类可能作为域与域间配对的替代，有助于保持CH2结构域稳定。Burton,Molec Immunol.22:161-206(1985)。
“CH3”结构域包括在Fc区内从C末端残基延伸到CH2结构域(从341位氨基酸到抗体序列的C末端，通常为IgG的第446或447位氨基酸残基)。
“功能性Fc片段”具有天然Fc区序列所具有的“效应功能”。示例性的“效应功能”包括C1q结合；补体依赖的细胞毒作用(CDC)；Fc受体结合；抗体依赖的细胞介导的细胞毒作用(ADCC)；吞噬作用；细胞表面受体的下调(如B细胞受体；BCR)等。这类效应功能通常需要Fc区与结合结构域(如抗体可变区)结合，并且可以使用本领域公知的多种实验方法进行评估。
具有“改变的”FcR结合亲和力或ADCC活性的IgG Fc变体的抗体，与亲本多肽或包含天然Fc序列的多肽相比，其FcR结合活性和/或ADCC活性增强或减弱。表现出与FcR“结合增强”的Fc变体与亲本多肽或包含天然IgG Fc序列的多肽相比，其与至少一种FcR具有更高的结合亲和力(例如更低的表观Kd或IC50值)。在一些实施例中，与亲本多肽相比，结合能力增强3倍，例如5、10、25、50、60、100、150、200，甚至高达500倍或结合力提高25％到1000％。表现出与FcR“结合降低”的Fc变体，与亲本多肽相比，其与至少一种FcR具有更低的亲和力(例如更高的表观Kd或IC50值)。与亲本多肽相比，其结合能力下降40％或更多。
“抗体依赖的细胞介导的细胞毒作用”或“ADCC”是一种细胞毒性形式，指分泌型的Ig与存在于某些细胞毒性细胞(例如自然杀伤细胞(NK)、中性粒细胞和巨噬细胞)上的Fc受体(FcRs)结合，使这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后使用细胞毒素杀死靶细胞。抗体“武装”细胞毒性细胞并且是这种杀伤所必需的。介导ADCC的主要细胞类型中，NK细胞只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。在Ravetch and Kinet,Annu.Rev.Immunol 9:457-92(1991)第464页的Table 3中总结了在造血细胞上FcR的表达。评估目标分子的ADCC活性，可以进行体外ADCC实验，在美国专利No.5,500,362或5,821,337中进行了描述。适用于此类实验的效应细胞包括外周血单个核细胞(PBMC)和自然杀伤细胞(NK)。可选地，或者此外，目标分子的ADCC活性也可以在体内进行评估，例如在如Clynes et al.PNAS(USA)95:652-656(1998)中所公开的动物模型中进行了描述。
包含Fc变体的多肽与包含野生型IgG Fc多肽或亲本多肽相比，在人体效应细胞存在下表现出“增强的ADCC活性”或能够更有效的介导ADCC效应，所述包含Fc变体的多肽在实验时与包含野生型IgG Fc多肽(或亲本多肽)数量上基本相同时，无论在体外或体内均能更有效的介导ADCC。通常采用本领域已知的任何体外ADCC实验方法来鉴定此类变体，例如用于鉴定ADCC活性的实验或方法，例如在动物模型中等。在一些实施例中，此类变体与野生型Fc(或亲代多肽)相比，介导ADCC的效率提高5到100倍，例如25到50倍。
“补体依赖的细胞毒作用”或“CDC”是指在补体存在的情况下裂解靶细胞。经典的补体途径的激活是由补体系统第一组分(C1q)与结合同源抗原的抗体(具有适宜结构的亚类)相结合而启动的。为了评估补体激活，可以进行CDC实验，如Gazzano-Santoro et al.,J.Immunol.Methods202:163(1996)中所描述的。在美国专利No.6,194,551B1和WO99/51642中描述了具有改变的Fc区氨基酸序列并增加或降低的C1q结合能力的多肽变体。这些专利出版物的内容通过引用明确地并入本申请中。另见Idusogie et al.J.Immunol.164:4178-4184(2000)。
除非另有说明，一种“编码氨基酸序列的核苷酸序列”包括相互之间互为简并形式且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质或RNA的核苷酸序列也可包括内含子，例如编码蛋白质的核苷酸序列在某些形式中包含内含子。
术语“可操作性地连接”是指调控序列与异源核苷酸序列之间的功能性连接，从而使后者表达。例如，当第一个核苷酸序列与第二个核苷酸序列处于功能性关系时，第一个核苷酸序列与第二个核苷酸序列为可操作性地连接。例如，如果启动子影响编码序列的转录或表达，该启动子与编码序列为可操作性地连接。通常，可操作性连接的DNA序列是连续的，并且在必要时，可以在同一个阅读框中连接两个蛋白质编码区。
“同源”是指两个多肽之间或两个核酸分子之间的序列相似性或序列同源性。如果两个比较序列的同一位置为相同的碱基或氨基酸单体亚基时，例如两个DNA分子的同一位置均为腺嘌呤，则这两个DNA分子在该位置是同源的。两个序列间的同源百分比是指两个序列中共有的匹配或同源位置的数量与位置总数之比再乘以100所得函数。例如，两个序列中如果10个位置中有6个位置是相匹配或同源的，则这两个序列的同源性为60％。举例来说，DNA序列ATTGCC和TATGGC具有50％的同源性。通常来说，在比对两个序列时，以得到最大同源性为目的来进行对比。
本申请所公开的抗体(包括多特异性抗体)或组合物的“有效量”是指足以实现特定目的的量。“有效量”可以凭经验和通过已知的与所述目的相关的方法确定。
术语“治疗有效量”是指本申请所述抗体(包括多特异性抗体)或组合物能够有效治疗个体的疾病或者症状的用量。即足以减轻或改善疾病或其一种或多种症状的严重程度和/或持续时间的量；预防疾病发展，引起病症消退，预防与疾病相关的一种或多种症状的复发、发展、发作或进展，检测疾病，或增强/改善另一疗法(例如预防剂或治疗剂)的预防或治疗效果的量。在HBV感染的情况中，本申请公开的抗体或组合物的治疗有效量能够有效预防或治疗由于HBV感染造成的病情的结合分子的用量。本申请公开的抗体或组合物在某种程度上能够阻止和/或杀死现有的感染HBV的细胞，它可以是细胞抑制性的或细胞毒性的。在一些实施例中，治疗有效量是指能够延长患者生存期的用量。在一些实施例中，治疗有效量是指能够改善患者无进展生存期的用量。
如本申请所用的，“药学上可接受的”或“药理学上相容的”是指无生物学活性或者其它不期望性质的材料，例如该材料能够加入到给予患者的药物组合物中，而不会引起显著的不良生物反应，或者，不与组合物中包含的任何其它组分以有害的方式相互作用。药学上可接受的载体或赋形剂优选满足毒理学或制造检测的所需标准和/或包含在美国食品和药品管理局编制的非活性成分指南中。
本申请中描述的本申请的实施例应理解为包含“由……组成”和/或“基本上由……组成”的实施例。
本申请中提及“约”为一个数值或参数，包含(和描述)针对该值或参数本身的变体。例如，涉及“约X”的描述，包括“X”的描述。
如本申请所用的，提及“不是(not)”一个数值或参数，通常表示并描述“除了(other than)”某一数值或参数之外。例如，该方法不能用于治疗X型感染，意味着该方法通常用于治疗除X型感染之外的其他类型。
除非上下文另有明确说明，本申请和所述权利要求中所采用的单数形式“一”，“一个”和“该”包括复数对象。
特异性结合NKG2A或PDL1的抗体或抗原结合域
本申请提供了特异性结合NKG2A或PDL1的抗体或抗原结合域，其包括，但不限于，人源化抗体，嵌合抗体，小鼠抗体，人抗体，以及本申请所述的包含重链和/或轻链CDRs的抗体分子。一方面，所述抗体或抗原结合域是与NKG2A或PDL1结合的分离的抗体或抗原结合域。预期的特异性结合NKG2A或PDL1的抗体或抗原结合域包括，特异性结合NKG2A或PDL1的全长抗体(如全长IgG1、IgG2或IgG4)的全部或片段，特异性结合NKG2A或PDL1的单链抗体，多特异性(如双特异性)结合NKG2A和PDL1的抗体，特异性结合NKG2A或PDL1的免疫偶联物，以及诸如此类。在一些实施例中，特异性结合NKG2A或PDL1的抗体或抗原结合域是Fab、Fab’、F(ab)’2、Fab’-SH、单链抗体(scFv)、Fv片段、dAb、Fd、纳米抗体或双链抗体(diabody)。在一些实施例中，特异性结合NKG2A或PDL1的抗体或抗原结合域是指抗体或抗原结合域与NKG2A或PDL1结合的亲和力至少是与非靶标结合亲和力的10倍以上(包括例如10、102、103、104、105、106、或107倍)。在一些实施例中，非靶标是指不是NKG2A或PDL1的抗原。
结合亲和力可通过本领域已知的方法来测定，如ELISA，荧光激活细胞分选(FACS)分析或放射免疫沉淀分析(RIA)。Kd值可以通过本领域已知的方法来测定，如表面等离子共振(SPR)技术或生物层干涉(BLI)技术。
尽管本申请广泛地讨论了包含人序列的特异性结合NKG2A或PDL1的抗体或抗原结合域(例如，包含人CDR序列的人重链和轻链可变区)，但同时也考虑了非人抗体。在一些实施例中，非人抗体包括本申请所述的特异性结合NKG2A或PDL1的抗体或抗原结合域的人CDR序列和非人框架区序列，在一些实施例中，非人框架区序列包括任何的用于使用如本申请所述的一种或多种人CDR序列产生重链和/或轻链可变区的序列，包括例如哺乳动物，例如小鼠、大鼠、兔子、猪、牛(例如，牛、公牛、水牛)、鹿、绵羊、山羊、鸡、猫、狗、雪貂、灵长类(例如，狨猴，猕猴)等。在一些实施例中，非人特异性结合NKG2A或PDL1的抗体或抗原结合域包括将一种或多种本申请所述的人CDR序列移植到非人框架区中(例如，鼠或鸡的框架区序列)所产生的特异性结合NKG2A或PDL1的抗体或抗原结合域。
特异性结合NKG2A的抗体或抗原结合域
一方面，本申请提供了特异性结合NKG2A的抗体或抗原结合域。
在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合域与NKG2A抗原结合。在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合域是NKG2A特异性的，并且没有种属交叉反应或不与其它类型的非NKG2A发生交叉反应。在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合域与其它类型的非NKG2A发生交叉反应。
在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合域包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含SNTMS(SEQ ID NO:1)；HC-CDR2，其包含NINTGGNTYYANWAKG(SEQ ID NO:2)；和HC-CDR3，其包含GSTIDSSGLSL(SEQ ID NO:3)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含QASQNIGSDLA(SEQ ID NO:4)；LC-CDR2，其包含LASTLAS(SEQ ID NO:5)；和LC-CDR3，其包含QQSWSSSNVDNV(SEQ ID NO:6)。
在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合域包含：
(i)VH，其包含氨基酸序列SEQ ID NO:13或其变体，所述变体与氨基酸序列SEQ ID NO:13具有至少80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少80％序列同一性；或
(ii)VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少80％序列同一性。
特异性结合PDL1的抗体或抗原结合域
一方面，本申请提供了特异性结合PDL1的抗体或抗原结合域。
在一些实施例中，所述特异性结合PDL1的抗体或抗原结合域与PDL1抗原结合。在一些实施例中，所述特异性结合PDL1的抗体或抗原结合域是PDL1特异性的，并且没有种属交叉反应或不与其它类型的非PDL1发生交叉反应。在一些实施例中，所述特异性结合PDL1的抗体或抗原结合域与其它类型的非PDL1发生交叉反应。
在一些实施例中，所述特异性结合PDL1的抗体或抗原结合域包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含GFTFGGFG(SEQ ID NO:18)；HC-CDR2，其包含ITGDSSTI(SEQ ID NO:19)；和HC-CDR3，其包含VRGPPGTWAY(SEQ ID NO:20)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含ESVEFYGTTL(SEQ ID NO:21)；LC-CDR2，其包含GAS(SEQ ID NO:22)；和LC-CDR3，其包含QQIRKVPWT(SEQ ID NO:23)。
在一些实施例中，所述特异性结合PDL1的抗体或抗原结合域包含：VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少80％序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少80％序列同一性。
特异性结合preS1的抗体或抗原结合域
一方面，本申请提供了特异性结合preS1的抗体或抗原结合域。所述抗体的具体氨基酸序列如表4-1、4-2和4-3所示。其中根据Kabat定义方式进行CDR编号。
关于前述的特异性结合NKG2A或PDL1的抗体或抗原结合域，在一些实施例中，如前所述的氨基酸取代限于本申请表11中所示的“示例性取代”。在一些实施例中，氨基酸取代限于本申请表10中所示的“优选取代”。
在一些实施例中，本申请提供能够与上述任一种特异性结合NKG2A或PDL1的抗体竞争性地结合NKG2A或PDL1的抗体或抗原结合域。在一些实施例中，本申请提供与上述任一种特异性结合NKG2A或PDL1的抗体或抗原结合域竞争性地结合相同表位的抗体或抗原结合域。
在一些实施例中，可以利用竞争实验来鉴定与本申请所述的特异性结合NKG2A或PDL1的抗体或抗原结合域竞争性结合NKG2A或PDL1的单克隆抗体或抗原结合域。竞争实验可以通过识别相同的或空间上重叠的表位或者通过一个抗体竞争性抑制另一抗体与抗原结合来确定两个抗体是否结合相同的表位。在某些实施例中，这种竞争性抗体与本申请所述的抗体结合相同的表位。一些示例性的竞争实验包括，但不限于如Harlow and Lane(1988)Antibodies:A Laboratory Manual ch.14(Cold Spring Harbor Laboratory，Cold Spring Harbor,N.Y.)中所提到的常规实验。用于解析抗体结合的表位的详细示例性方法如Morris(1996)"Epitope Mapping Protocols,"in Methods in Molecular Biology vol.66(Humana Press,Totowa,N.J.)中所述。在一些实施例中，如果每种抗体阻断另一种抗体结合的50％或更多，则称其结合相同的表位。在一些实施例中，与本申请所述的特异性结合NKG2A或PDL1的抗体竞争的抗体是嵌合抗体、人源化抗体或全人抗体。
在一些实施例中，所述特异性结合PDL1的抗体或抗原结合域可以选自专利申请WO2024040212A2中所述的特异性结合PDL1的抗体或抗原结合片段。示例性特异性结合PDL1的抗体或抗原结合域的序列如表3-1、表3-2和表3-3所示，其中根据Kabat(S70)或IMGT(SBT451)定义方式进行CDR编号。
在一些实施例中，所述特异性结合preS1的抗体或抗原结合域可以选自专利申请WO2023/066171A中所述的特异性结合preS1的抗体或抗原结合片段。示例性特异性结合preS1的抗体或抗原结合域的序列如表4-1、表4-2和表4-3所示，其中根据Kabat定义方式进行CDR编号。
示例性特异性结合NKG2A的抗体或抗原结合域的序列如表2-1和表2-2所示，其中根据Kabat定义方式进行CDR编号。
本领域技术人员将认识到有多种已知算法来预测CDR的位置以及界定抗体轻、重链可变区。包含如本申请所述抗体的CDRs、包含如本申请所述抗体的CDRs、VH和/或VL序列，但基于预测算法而非下表中所示例的抗体或抗原结合域也在本申请的范围内
多特异性抗体
结合NKG2A和PDL1的多特异性抗体
一方面，本申请提供了一种多特异性抗体(优选的，双特异性抗体)，其包含特异性结合NKG2A的第一抗原结合域，和特异性结合PDL1的第二抗原结合域。
在一些实施例中，所述特异性结合NKG2A的第一抗原结合域包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含SNTMS(SEQ ID NO:1)；HC-CDR2，其包含NINTGGNTYYANWAKG(SEQ ID NO:2)；和HC-CDR3，其包含GSTIDSSGLSL(SEQ ID NO:3)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含QASQNIGSDLA(SEQ ID NO:4)；LC-CDR2，其包含LASTLAS(SEQ ID NO:5)；和LC-CDR3，其包含QQSWSSSNVDNV(SEQ ID NO:6)。
在一些实施例中，所述特异性结合PDL1的第二抗原结合域包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含GFTFGGFG(SEQ ID NO:18)；HC-CDR2，其包含ITGDSSTI(SEQ ID NO:19)；和HC-CDR3，其包含VRGPPGTWAY(SEQ ID NO:20)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含ESVEFYGTTL(SEQ ID NO:21)；LC-CDR2，其包含GAS(SEQ ID NO:22)；和LC-CDR3，其包含QQIRKVPWT(SEQ ID NO:23)。
在一些实施例中，所述特异性结合NKG2A的第一抗原结合域包含：
(a)VH，其包含氨基酸序列SEQ ID NO:13或其变体，所述变体与氨基酸序列SEQ ID NO:13具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；或
(b)VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性。
在一些实施例中，所述特异性结合PDL1的第二抗原结合域包含：VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性。
在一些实施例中，本申请提供了一种多特异性抗体(优选的，双特异性抗体)，其包括特异性结合NKG2A的第一抗原结合域，和特异性结合PDL1的第二抗原结合域，所述第一抗原结合域包含：VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少约80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少约80％序列同一性；以及其中所述第二抗原结合域包含：VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少约80％序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少约80％序列同一性。
特异性结合NKG2A、PDL1和preS1的多特异性抗体
另一方面，本申请提供了一种多特异性抗体(优选的，三特异性抗体)，其包含特异性结合NKG2A的第一抗原结合域，特异性结合PDL1的第二抗原结合域和特异性结合preS1的第三抗原结合域。
特异性结合preS1和PDL1的多特异性抗体
另一方面，本申请提供了一种多特异性抗体(优选的，双特异性抗体)，其包含特异性结合PDL1的第一抗原结合域，和特异性结合preS1的第二抗原结合域。
特异性结合NKG2A和preS1的多特异性抗体
另一方面，本申请提供了一种多特异性抗体(优选的，双特异性抗体)，其包含特异性结合NKG2A的第一抗原结合域，和特异性结合preS1的第二抗原结合域。
多特异性抗体的结构
如前所述，本申请提供了一种多特异性抗体(优选的，双特异性抗体)，其包括特异性结合NKG2A的第一抗原结合域，和特异性结合PDL1的第二抗原结合域。在一些优选的实施方案中，所述的多特异性抗体(优选的，双特异性抗体)的结构选自IgG-scFv、IgG-(scFv)2、Bs4Ab-scFv、DVD-Ig、Hetero H,CrossMab或scFv-Fab IgG。
IgG-scFv结构的多特异性抗体
在一些实施例中，IgG-scFv多特异性抗体结构(优选的，双特异性抗体)，是在一个IgG抗体其中一条重链的Fc上连接一个scFv片段，构成能够特异性结合另外一个不同抗原的抗原结合域，以此实现多特异性(优选的，双特异性)结合。优选的，所述scFv通过连接肽(L)连接至该Fc的C末端。此外，为促进异源二聚体的相互结合，可以在Fc区(其包含CH2和CH3结构域)设计杵臼结构(knobs-in-holes，KIH)，即，其中一个Fc中的CH3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基，形成“knob”，以及，另一个Fc中的CH3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基，形成“hole”；同时，还可以引入可形成稳定化二硫桥的两个Cys残基突变(S354C在“knob”侧，并且Y349C在“hole”侧)。该多特异性抗体的典型结构的示意图如图1A所示。
在一些实施例中，根据本申请所述的任一多特异性抗体(优选的，双特异性抗体)，其具有IgG-scFv结构，该结构为异源二聚体，是由两个不同的单体组成的三价多特异性抗体，其中，一个单体由两条多肽链(下文分别称之为第一重链和第一轻链)组成，其包含两个抗原结合域，其中一个抗原结合域为Fab，另一个抗原结合域为scFv，分别结合不同的抗原；另外一个单体由两条多肽链(下文分别称之为第二重链和第二轻链)组成，其包含一个抗原结合域(Fab)。在一些实施例中，第一轻链和第二轻链的序列相同。
在一些优选实施例中，其中所述多特异性抗体(优选的，双特异性抗体)包含两个Fc，其包含CH2和CH3结构域，两个Fc中可进一步包含能够促进异源二聚体相互结合的氨基酸替换。。
在本申请的一个优选实施方案中，所述多特异性抗体(优选的，双特异性抗体)具有IgG-scFv结构，其中，两个Fab抗原结合域特异性地与第一种抗原结合，以及另外一个scFv抗原结合域特异性地与第二种抗原结合。
在一些实施例中，本申请所述的任一多特异性抗体(优选的，双特异性抗体)的第一重链从N端到C端包含：VH1-CH1-CH2-CH3-L-VH2-L3-VL2结构。在另一些实施例中，多特异性抗体(优选的，双特异性抗体)的第一重链从N端到C端包含：VH1-CH1-CH2-CH3-L-VL2-L3-VH2结构。在一些实施例中，多特异性抗体(优选的，双特异性抗体)的第一轻链从N端到C端包含：VL1-CL结构。
其中VH1和VL1是特异性结合其中第一种抗原的重链可变区和轻链可变区；VH2和VL2是特异性结合第二种抗原的重链可变区和轻链可变区；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；CL是轻链恒定区；L和L3是连接肽。其中，VH1-CH1和VL1-CL组成多特异性抗体(优选的，双特异性抗体)的结合第一种抗原的抗原结合域(Fab)，VH2-L3-VL2或VL2-L3-VH2组成多特异性抗体(优选的，双特异性抗体)的结合第二种抗原的抗原结合域(scFv)。
在一些实施例中，本申请所述的任一多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包含：VH1-CH1。在一些实施例中，多特异性抗体(优选的，双特异性抗体)的第二轻链从N端到C端包含：VL1-CL结构。
在一些实施例中，第二重链进一步包含Fc，该Fc包含CH2和CH3结构域。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包含：VH1-CH1-CH2-CH3结构；在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第二轻链从N端到C端包含：VL1-CL结构。
其中VH1和VL1分别是结合第一种抗原的重链可变区和轻链可变区，CH1是重链恒定区CH1结构域，CH2是重链恒定区CH2结构域，CH3是重链恒定区CH3结构域，CL是轻链恒定区。其中VH1-CH1和VL1-CL组成结合第一种抗原的抗原结合域(Fab)。
在一些实施例中，第一轻链和第二轻链的序列相同。
在一些实施例中，第一重链的CH3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”，和第二重链的CH3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”。在另一些实施例中，第二重链的CH3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”，和第一重链的CH3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”。在一些实施例中，所述CH3结构域包含，但不限于如下氨基酸置换：S354C，T366W，Y349C，T366S，L368A和Y407V，其中所述编号依照如Kabat的EU索引。
在一些实施例中，所述重链的铰链区的第234位的亮氨酸(L)以及第235位的亮氨酸(L)替换为丙氨酸(A)，第331位的脯氨酸(P)替换为丝氨酸(S)，形成组合突变LALAPS。该突变组合可以减弱抗体Fc与FcR受体CD64、CD32A、CD16以及人补体成分C1q的结合，从而削弱抗体依赖细胞介导的细胞毒性作用(ADCC)及补体依赖的细胞毒性作用(CDC)。其中所述编号依照如Kabat的EU索引。
NKG2A和PDL1
本申请涉及可结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)，其具有如前所述的IgG-scFv结构。
在本申请的一些优选的实施方案中，VH1和VL1分别是特异性结合NKG2A的重链可变区和轻链可变区,VH2和VL2分别是特异性结合PDL1的重链可变区和轻链可变区。VH1-CH1和VL1-CL组成特异性结合NKG2A的抗原结合域(Fab)，VH2-L3-VL2或VL2-L3-VH2组成特异性结合PDL1的抗原结合域(scFv)。
在本申请的一些实施例中，所述多特异性抗体(优选的，双特异性抗体)可以同时结合NKG2A和PDL1。在一些实施例中，特异性结合PD-L1的抗原结合域scFv包括基因工程半胱氨酸突变，通过在VH和VL界面引入两个半胱氨酸突变，获得了二硫键稳定型的多特异性抗体。
在一些实施例中，根据本申请所述的多特异性抗体(优选的，双特异性抗体)，其包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；和/或氨基酸序列SEQ ID NO:53或其变体，所述变体与氨基酸序列SEQ ID NO:53具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；和/或氨基酸序列SEQ ID NO:54或其变体，所述变体与氨基酸序列SEQ ID NO:54具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性。
PreS1和PDL1
本申请涉及可结合preS1和PDL1的多特异性抗体(优选的，双特异性抗体)，其具有如前所述的IgG-scFv结构。其重链和轻链的具体氨基酸序列如表8所示。
NKG2A和preS1
本申请涉及可结合NKG2A和preS1的多特异性抗体(优选的，双特异性抗体)，其具有如前所述的IgG-scFv结构。其重链和轻链的具体氨基酸序列如表9-1所示。
IgG-(scFv)2结构的多特异性抗体
在一些实施例中，IgG-(scFv)2多特异性抗体结构(可参见，Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.1997Feb；15(2):159-63)，是在全长IgG抗体的两条重链的Fc上均分别连接一个scFv片段，构成能够特异性结合另外一个不同抗原的抗原结合域，以此实现双特异性。优选的，所述scFv通过连接肽(L)连接至该Fc的C末端。该多特异性抗体的典型结构的示意图如图1B所示。
在一些实施例中，根据本申请所述的任一多特异性抗体(优选的，双特异性抗体)，其具有IgG-(scFv)2结构，该结构为同源二聚体，是由两个相同的单体组成的四价多特异性抗体。其中，每个单体均由两条多肽链(下文分别称之为重链和轻链)组成，包括两个抗原结合域，其中一个抗原结合域为Fab，另一个抗原结合域为scFv，优选的，该两个抗原结合域分别结合不同的抗原。在一些优选实施例中，所述IgG-(scFv)2结构还包括两个Fc区域，所述Fc区域包含CH2和CH3结构域。在一些实施例中，所述scFv通过连接肽(L)连接至Fc的C末端。
在本申请的一个优选实施方案中，所述多特异性抗体(优选的，双特异性抗体)具有IgG-(scFv)2结构，其中，两个Fab抗原结合域特异性地与第一种抗原结合，以及另外两个scFv抗原结合域特异性地与第二种抗原结合。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的重链从N端到C端包含：VH1-CH1-CH2-CH3-L-VH2-L3-VL2结构。在另一些实施例中，多特异性抗体(优选的，双特异性抗体)的重链从N端到C端包含：VH1-CH1-CH2-CH3-L-VL2-L3-VH2结构。在一些实施例中，多特异性抗体(优选的，双特异性抗体)的轻链从N端到C端包含：VL1-CL结构。
其中，VH1和VL1分别是特异性结合其中一个抗原的重链可变区和轻链可变区；VH2和VL2分别是特异性结合另一个抗原的重链可变区和轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；以及CL是轻链恒定区。其中，VH1-CH1和VL1-CL组成多特异性抗体(优选的，双特异性抗体)的结合第一种抗原的抗原结合域(Fab)，VH2-L3-VL2或VL2-L3-VH2组成多特异性抗体(优选的，双特异性抗体)的结合第二种抗原的抗原结合域(scFv)。
在一些实施例中，所述重链的铰链区的第234位的亮氨酸(L)以及第235位的亮氨酸(L)替换为丙氨酸(A)，第331位的脯氨酸(P)替换为丝氨酸(S)，形成组合突变LALAPS。该突变组合可以减弱抗体Fc与FcR受体CD64、CD32A、CD16以及人补体成分C1q的结合，从而削弱抗体依赖细胞介导的细胞毒性作用(ADCC)及补体依赖的细胞毒性作用(CDC)。其中所述编号依照如Kabat的EU索引。
NKG2A和PDL1
本申请涉及可结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)，其具有如前所述的IgG-(scFv)2结构。
在本申请的一些优选的实施方案中，VH1和VL1分别是特异性结合NKG2A的重链可变区和轻链可变区,VH2和VL2分别是特异性结合PDL1的重链可变区和轻链可变区，VH1-CH1和VL1-CL组成特异性结合NKG2A的抗原结合域(Fab)，VH2-L3-VL2或VL2-L3-VH2组成特异性结合PDL1的抗原结合域(scFv)。
在本申请的一些实施例中，所述多特异性抗体(优选的，双特异性抗体)可以同时结合NKG2A和PDL1。在一些实施例中，特异性结合PD-L1的抗原结合域scFv包括基因工程半胱氨酸突变，通过在VH和VL界面引入两个半胱氨酸突变，获得了二硫键稳定型的多特异性抗体。
在一些实施例中，根据本申请所述的多特异性抗体(优选的，双特异性抗体)，其包含氨基酸序列SEQ ID NO:54或其变体，所述变体与氨基酸序列SEQ ID NO:54具有约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；和/或氨基酸序列SEQ ID NO:58或其变体，所述变体与氨基酸序列SEQ ID NO:58具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性。
NKG2A和preS1
本申请涉及可结合NKG2A和preS1的多特异性抗体(优选的，双特异性抗体)，其具有如前所述的IgG-(scFv)2结构。其重链和轻链的具体氨基酸序列如表9-2所示。
Bs4Ab-scFv结构的多特异性抗体
以双特异性抗体结构Bs4Ab为基础进行改造获得的多特异性抗体结构(本申请称之为Bs4Ab-scFv结构，优选的，三特异性抗体)，是在Bs4Ab双特异性抗体结构(可参见文献Bezabeh B,et al.Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.MAbs.2017Feb/Mar；9(2):240-256)基础上，在其中一条重链的Fc的C端连接一个scFv片段，构成能够特异性结合另外一个不同抗原的抗原结合域，以此实现多特异性。该多特异性抗体的典型结构的示意图如图1C所示。
在一些实施例中，根据本申请所述的任一多特异性抗体，其具有Bs4Ab-scFv结构，该结构为异源二聚体，是由两个不同的单体组成的五价多特异性抗体，其中，第一单体由两条多肽链(下文分别称之为第一重链和第一轻链)组成，包括三个抗原结合域，其中一个抗原结合域为Fab，另外两个抗原结合域为scFv，三个抗原结合域分别结合不同的抗原；第二单体由两条多肽链(下文分别称之为第二重链和第二轻链)组成，包括两个抗原结合域，其中一个抗原结合域为Fab，另外一个抗原结合域为scFv，分别结合不同的抗原。在一些实施例中，第一轻链和第二轻链的序列相同。在一些优选实施例中，所述Bs4Ab-scFv结构还进一步包括两个Fc区域，所述Fc区域包含CH2和CH3结构域。在一些实施例中，所述其中一个scFv抗原结合域通过连接肽(L)连接至其中一个Fc的C末端，另外两个scFv抗原结合域均通过连接肽分别连接Fab结合域重链的C端和一个Fc的N端。
在本申请的一些实施例中，所述多特异性抗体(优选的，三特异性抗体)具有Bs4Ab-scFv结构，其中，两个Fab抗原结合域特异性地与第一种抗原结合，与Fab连接的两个scFv抗原结合域特异性地与第二种抗原结合，以及与Fc的C端连接的scFv抗原结合域特异性地与第三种抗原结合。
在一些实施例中，所述多特异性抗体(优选的，三特异性抗体)的第一重链从N端到C端包含：VH1-CH1-L-VH2-L3-VL2-L-CH2-CH3-L-VH3-L3-VL3。在一些实施例中，多特异性抗体(优选的，三特异性抗体)的第一轻链从N端到C端包含：VL1-CL结构。
其中，VH1和VL1分别是特异性结合第一抗原的重链可变区和轻链可变区；VH2和VL2分别是特异性结合第二抗原的重链可变区和轻链可变区；VH3和VL3分别是特异性结合第三抗原的重链可变区和轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；以及CL是轻链恒定区。其中，VH1-CH1和VL1-CL组成多特异性抗体的结合第一抗原的抗原结合域(Fab)，VH2-L3-VL2组成多特异性抗体的结合第二抗原的抗原结合域(scFv)，VH3-L3-VL3组成多特异性抗体的结合第三抗原的抗原结合域(scFv)。
在一些实施例中，所述多特异性抗体(优选的，三特异性抗体)的第二重链从N端到C端包含：VH1-CH1-L-VH2-L3-VL2。在一些实施例中，多特异性抗体(优选的，三特异性抗体)的第二轻链从N端到C端包含：VL1-CL结构。
在一些实施例中，第二重链进一步包含Fc，该Fc包含CH2和CH3结构域。
在一些实施例中，所述多特异性抗体(优选的，三特异性抗体)的第二重链从N端到C端包含：VH1-CH1-L-VH2-L3-VL2-L-CH2-CH3。在一些实施例中，多特异性抗体的第二轻链从N端到C端包含：VL1-CL结构。
其中，VH1和VL1分别是特异性结合第一抗原的重链可变区和轻链可变区；VH2和VL2分别是特异性结合第二抗原的重链可变区和轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；以及CL是轻链恒定区。其中，VH1-CH1和VL1-CL组成多特异性抗体的结合第一抗原的抗原结合域(Fab)，VH2-L3-VL2组成多特异性抗体的结合第二抗原的抗原结合域(scFv)。
在一些实施例中，第一轻链和第二轻链的序列相同。
在一些实施例中，所述重链的铰链区的第234位的亮氨酸(L)以及第235位的亮氨酸(L)替换为丙氨酸(A)，第331位的脯氨酸(P)替换为丝氨酸(S)，形成组合突变LALAPS。该突变组合可以减弱抗体Fc与FcR受体CD64、CD32A、CD16以及人补体成分C1q的结合，从而削弱抗体依赖细胞介导的细胞毒性作用(ADCC)及补体依赖的细胞毒性作用(CDC)。其中所述编号依照如Kabat的EU索引。
在一些实施例中，所述scFv包括基因工程半胱氨酸突变，通过在VH和VL界面引入两个半胱氨酸突变，获得了二硫键稳定型的多特异性抗体。
NKG2A、PDL1和preS1
本申请涉及特异性结合NKG2A、PDL1和preS1的多特异性抗体(优选的，三特异性抗体)，其具有如前所述的Bs4Ab-scFv结构。其重链和轻链的具体氨基酸序列如表7所示。
DVD-Ig结构的多特异性抗体
双重可变区免疫球蛋白分子DVD-Ig(Dual-variable domain-Ig)多特异性抗体结构(优选的，双特异性抗体)(可参见，例如，Wu C,et al.Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin(DVD-Ig)molecules.MAbs.2009Jul-Aug；1(4):339-47)，是在全长IgG抗体的VL和VH的N末端分别连接另外一个抗体的VL和VH结构域，通过两个抗体的VH与VL相互作用形成抗原结合域，能够同时与相对应的抗原结合，以此实现多特异性(优选的，双特异性)。该多特异性抗体的典型结构的示意图如图1D所示。
在一些实施例中，根据本申请所述的任一多特异性抗体(优选的，双特异性抗体)，其具有DVD-IgG结构，该结构为同源二聚体，是由两个相同的单体组成的四价多特异性抗体，其中，每个单体均由两条多肽链(下文分别称之为重链和轻链)组成，包括两个抗原结合域，其中一个抗原结合域为Fv，另一个抗原结合域为Fab，分别结合不同的抗原，且上述两个结合域通过连接肽(L)串联连接。在一些优选实施例中，所述DVD-Ig结构还进一步包括两个Fc区域，所述Fc区域包含CH2和CH3结构域。
在本申请的一个优选实施方案中，所述多特异性抗体(优选的，双特异性抗体)具有DVD-Ig结构，其中，两个Fab抗原结合域特异性地与第一种抗原结合，以及另外两个Fv抗原结合域特异性地与第二种抗原结合。在另外一个优选实施方案中，其中两个Fv抗原结合域特异性地与第一种抗原结合，以及另外两个Fab抗原结合域特异性地与第二种抗原结合。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的重链从N端到C端包含：VH1-L-VH2-CH1结构。在一些实施例中，所述多特异性抗体的轻链从N端到C端包含：VL1-L-VL2-CL结构。
在一些优选实施例中，其中所述重链进一步包含Fc，该Fc包含CH2和CH3结构域。因此，在这些实施例中，所述多特异性抗体(优选的，双特异性抗体)的重链从N端到C端包含：VH1-L-VH2-CH1-CH2-CH3结构。在一些实施例中，所述多特异性抗体的轻链从N端到C端包含：VL1-L-VL2-CL结构。
其中，VH1和VL1分别是特异性结合一个抗原的重链可变区和轻链可变区；VH2和VL2分别是特异性结合另一个抗原的重链可变区和轻链可变区；L是连接肽；CH1是重链恒定区CH1结构域；CL是轻链恒定区。其中，VH1和VL1组成多特异性抗体(优选的，双特异性抗体)的一个抗原结合域(Fv)；VH2-CH1和VL2-CL组成多特异性抗体(优选的，双特异性抗体)的另一个抗原结合域(Fab)。
在一些实施例中，所述重链的铰链区的第234位的亮氨酸(L)以及第235位的亮氨酸(L)替换为丙氨酸(A)，形成组合突变LALA。该突变组合可以减弱抗体Fc与FcR受体CD64、CD32A、CD16以及人补体成分C1q的结合，从而削弱抗体依赖细胞介导的细胞毒性作用(ADCC)及补体依赖的细胞毒性作用(CDC)。其中所述编号依照如Kabat的EU索引。
Hetero H,CrossMab结构的多特异性抗体
Hetero H,CrossMab结构的多特异性抗体(优选的，双特异性抗体)结构(可参见，例如，Klein C,et al.The use of CrossMAb technology for the generation of bi-and multispecific antibodies.MAbs.2016Aug-Sep；8(6):1010-20)，是一个异源二聚体，其基于双特异性IgG抗体的一个Fab臂内抗体域的交换，可以是完整的Fab域的交换(CrossMab Fab)，或者是Fab域中仅可变区的交换(CrossMab VH-VL)或仅恒定区的交换(CrossMab CH1-CL)，从而确保抗体轻、重链间的正确配对。此外，为促进异源二聚体的相互结合，可以在Fc区(其包含CH2和CH3结构域)设计杵臼结构(knobs-in-holes，KIH)，其中一个Fc中的CH3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基，形成“knob”，以及，另一个Fc中的CH3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基，形成“hole”；同时，还可以引入可形成稳定化二硫桥的两个Cys残基突变(S354C在“knob”侧，并且Y349C在“hole”侧)。该双特异性抗体的典型结构如图1E所示。
在一些实施例中，根据本申请所述的任一多特异性抗体(优选的，双特异性抗体)，其具有Hetero H,CrossMab结构。该结构是由两个单体组成的二价双特异性抗体。其中第一单体由两条多肽链(下称为第一重链和第一轻链)组成，包含一个抗原结合域(Fab)；第二单体也由两条多肽链(下称为第二重链和第二轻链)组成，包含另外一个抗原结合域(Fab)。在一些实施例中，第一单体和/或第二单体的Fab中，轻链恒定区(CL)和重链恒定区CH1结构域(CH1)的位置可相互替换；或重链可变区(VH)和轻链可变区(VL)的位置可相互替换；或轻链恒定区(CL)和重链恒定区CH1结构域(CH1)以及重链可变区(VH)和轻链可变区(VL)可同时相互替换，以确保轻、重链之间的正确配对。在一些实施例中，其中所述多特异性抗体(优选的，双特异性抗体)进一步包含两个Fc，其包含CH2和CH3结构域。
在本申请的一个优选实施方案中，所述多特异性抗体(优选的，双特异性抗体)具有Hetero H,CrossMab结构，其中一个抗原结合域特异性地与第一种抗原结合，另外一个抗原结合域特异性地与第二种抗原结合。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第一重链从N端到C端包括：VH1-CH1结构；第一轻链从N端到C端包括：VL1-CL结构。
在一些实施例中，第一重链还包含Fc，该Fc包含CH2和CH3结构域。因此，在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第一重链从N端到C端包括：VH1-CH1-CH2-CH3结构；第一轻链从N端到C端包括：VL1-CL结构。
其中，VH1和VL1分别是特异性结合第一种抗原的重链可变区和轻链可变区，CH1是重链恒定区CH1结构域，CL是轻链恒定区。其中VH1-CH1和VL1-CL组成结合第一种抗原的抗原结合域(Fab)。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包括：VH2-CL结构；在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第二轻链从N端到C端包括：VL2-CH1结构。
在一些实施例中，第二重链进一步包含Fc，该Fc包含CH2和CH3结构域。因此，在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包括：VH2-CL-CH2-CH3结构。在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的第二轻链从N端到C端包括：VL2-CH1结构。
其中，VH2和VL2分别是特异性结合第二种抗原的重链可变区和轻链可变区，CH1是重链恒定区CH1结构域，CL是轻链恒定区。其中VH2-CL和VL2-CH1组成结合第二种抗原的抗原结合域(Fab)。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)第一单体或第二单体中CL和CH1的位置相互替换。在一些实施例中，VH1和VL1的位置可相互替换。在另一些实施例中，VH2和VL2的位置可相互替换。
在一些实施例中，第一重链的CH3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”，和第二重链的CH3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”。在另一些实施例中，第二重链的CH3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”，和第一重链的CH3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”。在一些实施例中，所述CH3结构域包含，但不限于如下氨基酸置换：S354C，T366W，Y349C，T366S，L368A和Y407V，其中所述编号依照如Kabat的EU索引。
scFv-Fab IgG结构的多特异性抗体
scFv-Fab IgG结构的多特异性抗体为异二聚体抗体，其是在IgG抗体结构的基础上，将其中一个Fab臂被替换成结合不同抗原的scFv结构，以此实现抗体的多特异性(优选的，双特异性)。在本申请的一个优选实施方案中，该多特异性抗体的典型结构如图1F所示。
在一些实施例中，本申请所述的多特异性抗体(优选的，双特异性抗体)具有scFv-Fab IgG结构，其为异二聚体形式，包含两个单体，分别称为第一单体和第二单体。其中，所述第一单体由两条多肽链(下称为第一重链和轻链)组成，包含能够结合一个抗原的抗原结合域(Fab)；所述第二单体由一条多肽链(下称为第二重链)组成，包含能够结合另一个抗原的抗原结合域(scFv)。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)的两个单体还进一步包含Fc区域，其包含CH2和CH3结构域。上述两个单体中的Fc中进一步包含氨基酸替换，能够促进异源二聚体的相互结合。
在一些实施例中，本申请所述的任一多特异性抗体(优选的，双特异性抗体)的第一重链从N端到C端包含：VH1-CH1结构。轻链包含VL1-CL结构。
在一些实施例中，所述第一重链进一步包含Fc，该Fc包含CH2和CH3结构域。
在一些实施例中，第一重链从N端到C端包含：VH1-CH1-CH2-CH3结构，轻链包含VL1-CL结构。
其中，VH1和VL1分别是特异性结合其中一个抗原的重链可变区和轻链可变区，CH1是重链恒定区CH1结构域，CL是轻链恒定区。VH1-CH1和VL1-CL组成其中一个抗原结合域(Fab)。
在一些实施例中，本申请所述的任一多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包含：VH2-L3-VL2结构。在另外一些实施例中，本申请所述的任一多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包含：VL2-L3-VH2结构。
在一些实施例中，所述第二重链进一步包含Fc，该Fc包含CH2和CH3结构域。
在一些实施例中，第二重链从N端到C端包含：VH2-L3-VL2-CH2-CH3结构。在另外一些实施例中，本申请所述的任一多特异性抗体(优选的，双特异性抗体)的第二重链从N端到C端包含：VL2-L3-VH2-CH2-CH3结构。
其中VH2和VL2分别是特异性结合另一个抗原的重链可变区和轻链可变区，L3是连接肽。VH2-L3-VL2或VL2-L3-VH2组成另一个抗原结合域(scFv)。
在一些实施例中，VH1和VL1分别是特异性结合NKG2A的重链可变区和轻链可变区，VH1-CH1和VL1-CL组成特异性结合NKG2A的抗原结合域(Fab)。VH2和VL2分别是特异性结合PDL1的重链可变区和轻链可变区，VH2-L3-VL2或VL2-L3-VH2组成特异性结合PDL1的抗原结合域(scFv)。
在一些实施例中，VH1和VL1分别是特异性结合PDL1的重链可变区和轻链可变区，VH1-CH1和VL1-CL组成特异性结合PDL1的抗原结合域(Fab)。VH2和VL2分别是特异性结合NKG2A的重链可变区和轻链可变区，VH2-L3-VL2或VL2-L3-VH2组成特异性结合NKG2A的抗原结合域(scFv)。
在一些实施例中，所述其中一个抗原结合域(Fab或scFv)特异性地与NKG2A结合，以及另一个抗原结合域(scFv或Fab)特异性地与PDL1结合。在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)可以同时结合NKG2A和PDL1。
在一些实施例中，scFv抗原结合域包括基因工程半胱氨酸突变，通过在VH和VL界面引入两个半胱氨酸突变，获得了二硫键稳定型的多特异性抗体(优选的，双特异性抗体)。
在一些实施例中，所述Fc来源于人野生型IgG1。在另一个实施例中，一个单体中的Fc相对于人野生型IgG1包含，但不限于如下氨基酸置换E：357Q、和S364K；以及，另一个单体中的Fc相对于人野生型IgG1包含，但不限于如下氨基酸置换：Q295E、L368D、K370S、N384D、Q418E和N421D，其中所述编号依照如Kabat的EU索引。在一些实施例中，示例性连接scFv中VH1与VL1的连接肽(例如L3)包含序列GKPGSGKPGSGKPGSGKPGS(SEQ ID NO：89)。
连接肽
一方面，连接肽(或者，可称之为“接头”)可用于将多特异性抗体(优选的，双特异性抗体)的重链的结构域和/或结构区连接成一个连续的分子。所述多特异性抗体(优选的，双特异性抗体)可能包括额外的接头，例如连接scFv的可变重链和轻链的柔性接头。在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)可能包括额外的接头，例如连接scFv的可变重链和轻链的柔性接头以及用于将其他结合单元与多特异性抗体(优选的，双特异性抗体)的核心结构连接的其他接头。
连接肽(或接头)的一个典型的、非限制性的例子是包含至少4个残基的多肽链。这类接头的部位可能是柔性的、亲水的，并且它们自己很少或不会形成二级结构(接头部位或柔性接头部位)。在分子组装完成后，至少4个氨基酸的接头可以用于连接彼此靠近的结构域和/或区。也可以使用较长的接头。在一些实施例中，接头可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45、50、100、125、150、175或200个残基。当使用多个接头连接分子的各个部分时，接头可以是相同的或不同的(例如，相同或不同的长度和/或氨基酸序列)。
在某些方面，所述连接肽包含或由甘氨酸-丝氨酸接头组成。如本申请所述，术语“甘氨酸-丝氨酸接头”指由甘氨酸和丝氨酸残基组成的肽。示例性的甘氨酸-丝氨酸接头包括通式为(Gly4Ser)n的的氨基酸序列，其中n是正整数(例如，1、2、3、4、5、6、7、8、9或10)。一个优选的甘氨酸-丝氨酸接头为(Gly4Ser)2，即GGGGSGGGGS(SEQ ID NO：78)。一个优选的甘氨酸-丝氨酸接头为(Gly4Ser)4，即GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：79)。一个优选的甘氨酸-丝氨酸接头为(Gly4Ser)3，即GGGGSGGGGSGGGGS(SEQ ID NO：80)。在其它方面，两个或两个以上的甘氨酸-丝氨酸接头串联在一个连接肽中。在某些方面，所述连接肽包括铰链区的至少一部分(例如，来源于IgGl、IgG2、IgG3或IgG4分子)和一系列的甘氨酸-丝氨酸残基(例如，甘氨酸-丝氨酸接头，例如(G4S)n。
在一些实施例中，L1和/或L2包括铰链部分和接头部分，例如包括甘氨酸-丝氨酸接头的接头部分。在其它方面，L1和/或L2仅包括铰链部分或仅包括接头部分，例如甘氨酸-丝氨酸接头。在某些方面，L1和L2包括甘氨酸-丝氨酸接头。在某些方面，L1和L2的甘氨酸-丝氨酸接头部分的长度相同，而在其它方面，LI和L2的甘氨酸-丝氨酸接头部分的长度不同。当多特异性抗体(优选的，双特异性抗体)包含scFv时，scFv的重链和轻链可以通过柔性接头连接。在一些实施例中，这种柔性接头通常不包括铰链部分，而是一种甘氨酸-丝氨酸接头或其它柔性接头。可以选择和优化相互连接scFv结构域的柔性接头的长度和氨基酸序列。
在一些实施例中，连接肽(例如L1和/或L2)包含甘氨酸-丝氨酸或全部为甘氨酸的接头以及铰链区域的一部分或经修饰的一部分。在某些方面，多特异性抗体(优选的，双特异性抗体)中连接其中一个抗原结合域(如Fab或scFv)和另一个抗原结合域(如scFv或Fab)的连接肽(L1)包含氨基酸序列EPKSDKTGGGGSGGGGS(SEQ ID NO：83)或EPKSCGKTGGGGSGGGGS(SEQ ID NO：84)或EPKSCGGGGSGGGGS(SEQ ID NO：85)。在某些方面，多特异性抗体(优选的，双特异性抗体)中连接结抗原结合域scFv与Fc的连接肽(L2)包含氨基酸序列GGGGSGGGGSEPKSDKTHTCPPCP(SEQ ID NO：86)或GGGGSGGGGSCPPCP(SEQ ID NO：87)或GGGGSGGGGSDKTHTCPPCP(SEQ ID NO：88)。在一些实施例中，多特异性抗体(优选的，双特异性抗体)中连接抗原结合域与Fc区羧基末端CH3的连接肽(例如L)包含氨基酸序列GGGGSGGGGTGGGGS(SEQ ID NO：90)。
在一些实施例中，所述多特异性抗体(优选的，双特异性抗体)除了将一个抗原结合域与另一个抗原结合域连接的连接肽或将其中一个抗原结合域与Fc连接的连接肽(例如L1和L2)，多特异性抗体(优选的，双特异性抗体)都可以选择性地包含额外的连接肽。这些额外的连接肽的长度和序列是独立选择的。例如，多特异性抗体(优选的，双特异性抗体)还可以包括一个柔性连接肽(L3)，其连接抗原结合域scFv中的可变重链和轻链(VHSCFV和VLSCFV)。这种柔性的连接肽可以包括甘氨酸-丝氨酸接头。通常，这种接头不包括铰链部分。在一些实施例中，连接scFv的可变重链和轻链的柔性连接肽(L3)包含氨基酸序列ASTKGP(SEQ ID NO：81)或TVAAP(SEQ ID NO：82)。在一些实施例中，连接scFv的可变重链和轻链的柔性连接肽(L3)包含序列GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：79)。
示例性抗体序列如表2至表9所示，其中CDR编号依据Kabat或IMGT中的EU索引。本领域技术人员将认识到有多种已知算法来预测CDR的位置以及界定抗体轻、重链可变区。包含如本申请所述特异性结合NKG2A的抗体或抗原结合域，特异性结合PD-L1的抗体或抗原结合域，和特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)的CDRs、VH和/或VL序列，但基于预测算法而非下表中所示例的抗体也在本申请的范围内。
所述特异性结合PDL1的抗体或抗原结合域可以选自专利申请WO2024040212A2中所述的抗PDL1的抗体或抗原结合片段，所述特异性结合preS1的抗体或抗原结合域可以选自专利申请WO2023/066171A中所述的抗HBV preS1的抗体或抗原结合片段，以引用的方式均并入本申请中。
表2-1：示例性特异性结合NKG2A的抗体或抗原结合域的CDR序列
表2-2：示例性特异性结合NKG2A的抗体或抗原结合域的VH&VL序列

表3-1：示例性特异性结合PD-L1的抗体或抗原结合域的CDR序列
表3-2：示例性特异性结合PD-L1的抗体或抗原结合域的VH&VL序列
表3-3：示例性特异性结合PD-L1的抗体或抗原结合域的VH&VL半胱氨酸变体序列

表4-1：示例性特异性结合HBV preS1的抗体或抗原结合域的CDR序列
表4-2：示例性特异性结合HBV preS1的抗体或抗原结合域的VH&VL序列
表4-3：示例性特异性结合HBV preS1的抗体或抗原结合域的VH&VL半胱氨酸变体序列
表5：示例性抗体恒定区序列

表6-1：IgG-scFv结构特异性结合NKG2A和PD-L1的示例性多特异性抗体的全长重链和轻链序列

表6-2：IgG-(scFv)2结构特异性结合NKG2A和PD-L1的示例性多特异性抗体的全长重链和轻链序列
表7：Bs4Ab-scFv结构特异性结合NKG2A、PD-L1和preS1的示例性多特异性抗体的全长重链和轻链序列
表8：IgG-scFv结构特异性结合PD-L1和preS1的示例性多特异性抗体的全长重链和轻链序列
表9-1：IgG-scFv结构特异性结合NKG2A和preS1的示例性多特异性抗体的全长重链和轻链序列
表9-2：IgG-(scFv)2结构特异性结合NKG2A和preS1的示例性多特异性抗体的全长重链和轻链序列

表10：示例性连接肽(或接头)序列
结合亲和力
结合亲和力可以采用Kd、Koff、Kon或Ka来表示。如本申请所用，术语“Koff”是指抗原结合域从抗原结合域/抗原复合物中解离的速率常数，通过动力学选择装置测定。术语“Kon”是指抗体与抗原结合形成抗原结合域/抗原复合物的结合速率常数。本申请所用的解离常数“Kd”是指特定抗体抗原相互作用时的解离常数，是指在抗体分子溶液中，抗原占据所有抗体结合域的一半并且达到平衡时所需的抗原浓度，等于Koff/Kon。Kd的测定假设所有的结合分子均在溶液中。抗原结合域与细胞壁连接的情况，例如在酵母表达系统中，相应的解离速率常数采用EC50来表示，其是Kd的一个良好的近似值。亲和结合常数Ka是解离常数Kd的倒数。
平衡解离常数(Kd)可以作为抗原结合域部分与抗原亲和力的指标。例如，可以通过Scatchard方法使用标记有各种标记物的抗体，和Biacore仪器(由Amersham Biosciences制造)进行简单分析，根据用户手册或附带试剂盒，通过表面等离子体共振来分析生物分子间的相互作用。使用这些方法得到的Kd值，用单位M来表示。与靶标特异性结合的抗体可能具有，≤10-7M、≤10-8M、例如≤10-9M、≤10-10M、≤10-11M、≤10-12M或≤10-13M的Kd值。
抗体的结合特异性可以通过本领域已知的方法进行实验测定。这些方法包括，但不限于Western blots、ELISA-、RIA-、ECL-、IRMA-、EIA-、BIAcore测试和肽扫描等。
在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合域特异性结合NKG2A靶标，其Kd值为10-7M至10-13M(例如10-7M至10-13M、10-8M至10-13M、10-9M至10-13M或10-10M至10-12M)。因此，在一些实施例中，特异性结合NKG2A的抗体或抗原结合域与NKG2A之间结合的Kd值为10-7M至10-13M、1×10-7M至5×10-13M、10-7M至10-12M、10-7M至10-11M、10-7M至10-10M、10-7M至10-9M、10-8M至10-13M、1×10-8M至5×10-13M、10-8M至10-12M、10-8M至10-11M、10-8M至10-10M、10-8M至10-9M、5×10-9M至1×10-13M、5×10-9M至1×10-12M、5×10-9M至1×10-11M、5×10-9M至1×10-10M、10-9M至10-13M、10-9M至10-12M、10-9M至10-11M、10-9M至10-10M、5×10-10M至1×10-13M、5×10-10M至1×10-12M、5×10-10M至1×10-11M、10- 10M至10-13M、1×10-10M至5×10-13M、1×10-10M至1×10-12M、1×10-10M至5×10-12M、1×10-10M至1×10-11M、10-11M至10-13M、1×10-11M至5×10-13M、10-11M至10-12M、10-12M至10-13M。在一些实施例中，特异性结合NKG2A的抗体或抗原结合域与NKG2A之间结合的Kd值为10-7M至10-13M。
在一些实施例中，特异性结合NKG2A的抗体或抗原结合域与非靶标之间结合的Kd值高于特异性结合NKG2A的抗体或抗原结合域与靶标的Kd值，并且本申请中引用的一些实施例中，特异性结合NKG2A的抗体或抗原结合域与靶标(例如，NKG2A)的结合亲和力高于特异性结合NKG2A的抗体或抗原结合域与非靶标的结合亲和力。一些实施例中，非靶标是指非NKG2A。在一些实施例中，特异性结合NKG2A的抗体或抗原结合域与非NKG2A靶标结合的Kd值是特异性结合NKG2A的抗体或抗原结合域与靶标NKG2A之间结合的Kd的至少10倍，例如10-102倍、102-103倍、103-104倍、104-105倍、105-106倍、106-107倍、107-108倍、108-109倍、109-1010倍、1010-1011倍、1011-1012倍。
在一些实施例中，本申请中任一所述的多特异性抗体包含特异性结合NKG2A的第一抗原结合域，和特异性结合PDL1的第二抗原结合域。其中第一抗原结合域特异性结合NKG2A靶标，其Kd值为10-7M至10-13M(例如10-7M至10-13M、10-8M至10-13M、10-9M至10-13M或10-10M至10- 12M)。因此，在一些实施例中，第一抗原结合域与NKG2A之间结合的Kd值为10-7M至10-13M、1×10-7M至5×10-13M、10-7M至10-12M、10-7M至10-11M、10-7M至10-10M、10-7M至10-9M、10-8M至10-13M、1×10-8M至5×10-13M、10-8M至10-12M、10-8M至10-11M、10-8M至10-10M、10-8M至10-9M、5×10-9M至1×10-13M、5×10-9M至1×10-12M、5×10-9M至1×10-11M、5×10-9M至1×10-10M、10-9M至10-13M、10-9M至10-12M、10-9M至10-11M、10-9M至10-10M、5×10-10M至1×10-13M、5×10-10M至1×10-12M、5×10-10M至1×10-11M、10-10M至10-13M、1×10-10M至5×10-13M、1×10-10M至1×10-12M、1×10-10M至5×10-12M、1×10-10M至1×10-11M、10-11M至10- 13M、1×10-11M至5×10-13M、10-11M至10-12M、10-12M至10-13M。在一些实施例中，第一抗原结合域与NKG2A之间结合的Kd值为10-7M至10-13M。其中第二抗原结合域特异性结合PDL1靶标，其Kd值为10-7M至10-13M(例如10-7M至10-13M、10-8M至10-13M、10-9M至10-13M或10-10M至10-12M)。因此，在一些实施例中，第二抗原结合域与PDL1之间结合的Kd值为10-7M至10-13M、1×10-7M至5×10-13M、10-7M至10-12M、10-7M至10-11M、10-7M至10-10M、10-7M至10-9M、10-8M至10-13M、1×10-8M至5×10-13M、10-8M至10-12M、10-8M至10-11M、10-8M至10-10M、10-8M至10-9M、5×10-9M至1×10-13M、5×10-9M至1×10-12M、5×10-9M至1×10-11M、5×10-9M至1×10-10M、10-9M至10-13M、10-9M至10-12M、10-9M至10-11M、10-9M至10-10M、5×10-10M至1×10-13M、5×10-10M至1×10-12M、5×10-10M至1×10-11M、10-10M至10-13M、1×10-10M至5×10-13M、1×10-10M至1×10-12M、1×10-10M至5×10-12M、1×10-10M至1×10-11M、10-11M至10-13M、1×10-11M至5×10-13M、10-11M至10-12M、10-12M至10-13M。在一些实施例中，第二抗原结合域与PDL1之间结合的Kd值为10-7M至10-13M。
在一些实施例中，本申请中任一所述的多特异性抗体包含特异性结合NKG2A的第一抗原结合域，和特异性结合PDL1的第二抗原结合域。其中第一抗原结合域与非靶标之间结合的Kd值高于第一抗原结合域与靶标的Kd值，并且本申请中引用的一些实施例中，第一抗原结合域与靶标(例如，NKG2A)的结合亲和力高于第一抗原结合域与非靶标的结合亲和力。一些实施例中，非靶标是指非NKG2A。在一些实施例中第一抗原结合域与非NKG2A靶标结合的Kd值是第一抗原结合域与靶标NKG2A之间结合的Kd的至少10倍，例如10-102倍、102-103倍、103-104倍、104-105倍、105-106倍、106-107倍、107-108倍、108-109倍、109-1010倍、1010-1011倍、1011-1012倍。其中第二抗原结合域与非靶标之间结合的Kd值高于第二抗原结合域与靶标的Kd值，并且本申请中引用的一些实施例中，第二抗原结合域与靶标(例如，PDL1)的结合亲和力高于第二抗原结合域与非靶标的结合亲和力。一些实施例中，非靶标是指非PDL1。在一些实施例中第二抗原结合域与非PDL1靶标结合的Kd值是第二抗原结合域与PDL1之间结合的Kd的至少10倍，例如10-102倍、102-103倍、103-104倍、104-105倍、105-106倍、106-107倍、107-108倍、108-109倍、109-1010倍、1010-1011倍、1011-1012倍。
抗体组合物
一方面，本申请提供了一种药物组合物，包括：(i)特异性结合NKG2A的抗体或抗原结合片段和(ii)特异性结合PDL1的抗体或抗原结合片段。
在一些实施例中，提供了一种药物组合物，包括：(i)特异性结合NKG2A的抗体或抗原结合片段和(ii)特异性结合PDL1的抗体或抗原结合片段，其中：特异性结合NKG2A的抗体或抗原结合片段包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含SNTMS(SEQ ID NO:1)；HC-CDR2，其包含NINTGGNTYYANWAKG(SEQ ID NO:2)；和HC-CDR3，其包含GSTIDSSGLSL(SEQ ID NO:3)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含QASQNIGSDLA(SEQ ID NO:4)；LC-CDR2，其包含LASTLAS(SEQ ID NO:5)；和LC-CDR3，其包含QQSWSSSNVDNV(SEQ ID NO:6)。
在一些实施例中，提供了一种药物组合物，包括：(i)特异性结合NKG2A的抗体或抗原结合片段和(ii)特异性结合PDL1的抗体或抗原结合片段，特异性结合PDL1的抗体或抗原结合片段包含：重链可变区(VH)，所述VH包含：重链互补决定区(HC-CDR)1，其包含GFTFGGFG(SEQ ID NO:18)；HC-CDR2，其包含ITGDSSTI(SEQ ID NO:19)；和HC-CDR3，其包含VRGPPGTWAY(SEQ ID NO:20)；以及轻链可变区(VL)，所述VL包含：轻链互补决定区(LC-CDR)1，其包含ESVEFYGTTL(SEQ ID NO:21)；LC-CDR2，其包含GAS(SEQ ID NO:22)；和LC-CDR3，其包含QQIRKVPWT(SEQ ID NO:23)。
在一些实施例中，所述药物组合物中特异性结合NKG2A的抗体或抗原结合片段包含：
(i)VH，其包含氨基酸序列SEQ ID NO:13或其变体，所述变体与氨基酸序列SEQ ID NO:13具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；或
(ii)VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性。
在一些实施例中，所述药物组合物中特异性结合PDL1的抗体或抗原结合片段包含：VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同一性。
在一些实施例中，提供了一种药物组合物，包括：(i)特异性结合NKG2A的抗体或抗原结合片段和(ii)特异性结合PDL1的抗体或抗原结合片段，其中特异性结合NKG2A的抗体或抗原结合片段包含：VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少约80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少约80％序列同一性；以及，其中特异性结合PDL1的抗体或抗原结合片段包含：VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少约80％序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少约80％序列同一性。
核酸、载体及抗体的制备
核酸和载体
本申请提供了特异性结合编码特异性结合NKG2A的抗体或抗原结合片段、以及特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)的核酸分子也被考虑在内。
在一些实施例中，本申请提供一种(或一组)编码特异性结合NKG2A的抗体或抗原结合片段的核酸，或编码特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)的核酸，包括本申请所述的任一种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)。在一些实施例中，本申请所述的抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的核酸(或一组核酸)还可以包括编码多肽标签的核酸序列(例如蛋白纯化标签，His-标签、HA标签)。
同时本申请还提供了包含特异性结合NKG2A的抗体或抗原结合片段，或特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)；或包含编码所述抗体或抗原结合片段的核酸分子；或包含携带所述核酸分子的载体的分离的宿主细胞。
本申请还包括这些核酸序列的变体。例如，变体包括至少在中等严格杂交条件下与编码本申请的抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的核酸序列杂交的核苷酸序列。
本申请同时还提供可将本申请中核酸序列插入到其中的载体。
简言之，将编码抗体、抗原结合片段或双特异性抗体的天然或合成的核酸插入到合适的表达载体中，使得核酸可操作性的连接到5’和3’端调控元件，例如包括启动子(例如淋巴细胞特异性启动子)和3’非翻译区(UTR)，可表达抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)。所述载体可适用于在真核宿主细胞中复制和整合。典型的克隆与表达载体包含调控目标核酸序列的表达的转录和翻译终止子、起始序列和启动子。
本申请所述的核酸也可以通过使用标准的基因递送方案，用于核酸免疫和基因治疗。核酸递送方法是本领域已知的。例如参见U.S.Pat.Nos.5,399,346、5,580,859、5,589,466，通过引用其全部内容并入本申请。在一些实施例中，本申请还提供基因治疗载体。
可以将核酸克隆到许多类型的载体中。例如，可以将核酸克隆到载体中，所述载体包括，但不限于，质粒、噬菌粒、噬菌体衍生物、动物病毒和柯斯质粒。特别感兴趣的载体包括表达载体、复制载体、探针生成载体和测序载体。
此外，表达载体可以以病毒载体的形式提供给细胞。病毒载体技术是本领域熟知的，并且描述于例如Green and Sambrook(2013,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)，以及其它病毒学或分子生物学手册中。可用作载体的病毒包括，但不限于，逆转录病毒、腺病毒、腺相关病毒、疱疹病毒和慢病毒。通常，合适的载体包括一个在至少一种生物体中起作用的复制起点、启动子序列、方便的限制性内切酶位点以及一个或多个选择标记物(参见例如，WO 01/96584；WO 01/29058；和U.S.Pat.No.6,326,193)。
已经开发了许多基于病毒的系统，用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以应用本领域已知的技术，将选择的基因插入载体中并包装在逆转录病毒颗粒中。然后分离重组病毒，在体内或体外递送至受试者的细胞中。许多逆转录病毒系统在本领域中是已知的。在一些实施例中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一些实施例中，使用慢病毒载体。衍生自逆转录病毒的载体，例如慢病毒，是实现长期基因转移的合适工具，因为它们使得转基因长期稳定的整合以及在子代细胞中繁殖。慢病毒载体相对于衍生自肿瘤的逆转录病毒例如小鼠白血病病毒具有额外的优势，因为它们可以转导非分裂细胞，例如肝细胞。同时，其还具有低免疫原性的额外优势。
其它的启动子元件，例如，增强子，调控转录起始频率。通常它们位于起始位点上游30-110bp处，虽然最近发现很多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔通常是灵活的，所以当元件彼此之间位置互换或移动时仍保持启动子的功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔增加到50bp，活性才会开始下降。
合适启动子的一个示例是即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是一个很强的组成型启动子序列，可以驱动任何与其可操作性连接的多核苷酸序列高水平表达。合适启动子的另一个示例是延伸因子1α(EF-1α)启动子。然而，也可以使用其它组成型启动子，包括但不限于，猿猴病毒40(SV40)早期启动子、小鼠乳腺肿瘤病毒(MMTV)、人免病缺陷病毒长末端重复序列(HIV-LTR)启动子、MoMuLV启动子、禽类白血病病毒启动子、Epstein-Barr病毒即刻早期启动子、劳斯氏肉瘤病毒启动子以及人类基因启动子，例如包括但不限于，肌动蛋白启动子、肌球蛋白启动子、血红蛋白启动子和肌酸激酶启动子。此外，不应将本申请局限在仅使用组成型启动子。诱导型启动子也是本申请考虑的部分。诱导型启动子的使用提供了一种分子开关，当需要这种表达时，能启动其与之可操作性连接的多核苷酸序列表达，当不需要时，则关闭表达。诱导型启动子，包含但不局限于，金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
在一些实施例中，所述抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的表达是可诱导的。在一些实施例中，编码所述抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的核酸序列可操作的连接到诱导型启动子上，包括本申请所述的任一诱导型启动子。
诱导型启动子
诱导型启动子的使用提供了一种分子开关，当需要表达时，可启动与之可操作性连接的多核苷酸序列表达，而在不需要表达时，则关闭表达。真核细胞中适用的示例性诱导型启动子，包括但不限于，激素调节元件(例如，参见Mader,S.and White,J.H.(1993)Proc.Natl.Acad.Sci.USA 90:5603-5607)合成配体调节元件(参见Spencer,D.M.et al(1993)Science 262:1019-1024)以及电离辐射调控元件(参见Manome,Y.et al.(1993)Biochemistry 32:10607-10613；Datta,R.et al.(1992)Proc.Natl.Acad.Sci.USA 89:1014-10153)。其它适用于体内或体外哺乳动物系统的示例性诱导型启动子参见Gingrich et al.(1998)Annual Rev.Neurosci 21:377-405。在一些实施例中，用于表达抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的诱导型启动子系统为Tet系统。在一些实施例中，表达抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的诱导型启动子系统为大肠杆菌lac抑制系统。
本申请所采用的一个示例性诱导型启动子系统为Tet系统。该系统是基于Gossen等(1993)描述的Tet系统。在一个示例性实施例中，目标多核苷酸由包含一个或多个Tet操纵子(TetO)位点的启动子控制。在非激活状态，Tet阻遏物(TetR)与TetO位点结合并抑制启动子的转录。在激活状态，例如，在存在诱导剂如四环素(Tc)、无水四环素、多西环素(Dox)或其活性类似物的情况下，诱导剂会使TetR从TetO上释放，从而导致转录发生。多西环素是四环素抗生素家族中的一员，其化学名为1-二甲氨基-2,4a,5,7-五羟基-11-甲基-4,6-二氧基-1,4a,11,11a,12,12a-六氢四烯-3-甲酰胺。
在一个实施例中，TetR经密码子优化适用于在哺乳动物细胞中表达，例如小鼠或人类细胞。由于遗传密码的简并性，大多数氨基酸由不止一个密码子编码，从而使得给定核酸的序列具有大量的变体，而其编码的氨基酸序列没有任何改变。然而，许多生物体在密码子使用方面存在差异，也称为“密码子偏好”(即，给定氨基酸使用特定密码子的偏好)。密码子偏好通常与特定密码子的优势tRNA种类的存在有关，反过来又提高了mRNA翻译的效率。因此可以通过密码子优化来定制源自特定物种的编码序列(例如，原核生物)，以提高其在不同物种(例如，真核生物)中的表达。
Tet系统的其它具体变体，包括以下的“Tet-Off”和“Tet-On”系统。在Tet-off系统中，转录在Tc或Dox存在下是失活的。在该系统中，由TetR与单纯疱疹病毒VP16强转录激活结构域融合组成的四环素调控的转录激活蛋白(tTA)，在四环素反应启动子元件(TRE)转录控制下调控靶核酸的表达。TRE元件由TetO序列串联与启动子(通常是来源于人巨细胞病毒即刻早期启动子的最小启动子序列)融合组成。在不存在Tc或Dox的情况下，tTA结合TRE并激活靶基因的转录。在存在Tc或Dox的情况下，tTA不能结合TRE，靶基因不能表达。
相反，在Tet-On系统中，转录在Tc或Dox存在下是激活的。Tet-On系统是基于反向四环素调控的转录激活因子rtTA。与tTA一样，rtTA是由TetR阻遏物与VP16反式激活域组成的融合蛋白。然而，TetR的DNA结合区中4个氨基酸的变化改变了rtTA的结合特性，使其在存在Dox的情况下只能识别靶转基因TRE上的tetO序列。所以在Tet-On系统中，只有在存在Dox的情况下，rtTA才能激活TRE调控的靶基因的转录。
另一种诱导型启动子系统是大肠杆菌的lac阻遏物系统(参见Brown et al.,Cell 49:603-612(1987))。Lac阻遏物系统通过调控与包含lac操纵子(lacO)的启动子可操作性连接的目标多核苷酸的转录发挥功能。Lac阻遏物(lacR)与LacO结合，进而阻止目标多核苷酸的转录。通过合适的诱导剂来诱导目标多核苷酸的表达，例如，异丙基-β-D硫代半乳糖吡喃苷(IPTG)。
为了评估多肽或其部分的表达，待导入细胞的表达载体还可包含选择标记基因或报告基因或二者都有，以便于从病毒载体转染或感染的细胞群体中识别和选择表达细胞。在其他方面，选择标记可以携带在单独的DNA片段上并在共转染实验中使用。选择标记基因或报告基因都可侧接于合适的调控序列，使其在宿主细胞中能够表达。有用的选择标记包括，例如，抗生素耐药基因，如neo以及类似基因。
报告基因可用于鉴定潜在的转染细胞和评价调控序列的功能。通常，报告基因是不存在于受体生物体或组织中或不由受体生物体或组织表达的基因，其编码一种多肽，其表达表现为一些易于检测的特性，例如酶活性。当DNA导入受体细胞后，在合适的时间检测报告基因的表达。合适的报告基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌碱性磷酸酶或绿色荧光蛋白的基因合适的表达系统是公知的，可以通过已知的技术制备或通过商业途径获得。通常，把具有能够显示报告基因最高表达水平的最小5'侧翼区的构建体认定为启动子。此类启动子区可以与报告基因连接，并用于评估某些物质在调节启动子驱动的转录中能力。
在一些实施例中，提供编码本申请所述的任一种抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)的核酸。在一些实施例中，所述核酸包括编码抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)重链和轻链的一个或多个核酸序列。在一些实施例中，所述一个或多个核酸序列中的每一个包含在单独的载体中。在一些实施例中，至少有一些核酸序列包含在同一载体中。在一些实施例中，所有核酸序列包含在同一载体中。载体可以选自，例如，哺乳动物表达载体和病毒载体(如源自逆转录病毒、腺病毒、腺相关病毒、疱疹病毒和慢病毒的载体)。
将基因导入细胞并表达的方法在本领域是已知的。在涉及表达载体的上下文中，通过本领域的任何方法载体可以很容易地导入宿主细胞中，如哺乳动物细胞、细菌、酵母或昆虫细胞。例如表达载体可以通过物理、化学或生物方法导入宿主细胞。
将多核苷酸导入宿主细胞的物理方法包括磷酸钙沉淀、脂质体转染、基因枪法、显微注射、电穿孔法以及诸如此类。制备包含载体和/或外源核酸的细胞的方法在本领域是熟知的。参见例如Green and Sambrook(2013,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。在一些实施例中，通过磷酸钙转染法将多核苷酸导入宿主细胞。
将目标多核苷酸导入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已成为将基因插入哺乳动物细胞，例如人类细胞中的最广泛使用的方法。其它病毒载体可以源自慢病毒、痘病毒、单纯疱疹病毒1型、腺病毒和腺相关病毒等。参见如U.S.Pat.Nos.5,350,674和5,585,362。
将多核苷酸导入宿主细胞的化学方法包括胶体分散系统，例如高分子复合物、纳米胶囊、微球、磁珠和以脂质为基础的系统，其包括水包油乳剂、胶团、混合胶团和脂质体。一种在体内和体外被用作递送载体的示例性胶体系统是脂质体(例如，人工膜囊)。
在另一方面，所述核酸可以与脂质结合。与脂质结合的核酸可被包裹进脂质体的水性内部，散布在脂质体的脂质双层内，通过与脂质体和寡核苷酸结合的连接分子连接在脂质体，包埋在脂质体中，与脂质体形成复合物，分散在含有脂质的溶液中，与脂质混合，与脂质结合，悬浮在脂质中，包含在胶束中或与胶束混合，或以其它方式与脂质结合。脂质、脂质/DNA或脂质/表达载体相关的组合物在溶液中不限于任何特定结构。例如，它们可能以双分子层结构、以胶束或以“塌陷”结构存在。它们也可以简单的分散在溶液中，可能形成大小或形状不均匀的聚集体。脂质是脂肪物质，可以是天然存在的或是合成的脂质。例如，脂质包括天然存在于细胞质中的脂肪滴，以及含有长链脂肪烃及其衍生物的一类化合物，例如脂肪酸、醇、胺、氨基醇和醛。
无论采用何种方法将外源核酸导入宿主细胞中或以其他方式将细胞暴露于本申请的抑制剂中，为了确认重组DNA序列存在于宿主细胞中，可以进行多种实验。这类实验包括例如本领域技术人员熟知的“分子生物学”实验。例如Southern和Northern blotting，RT-PCR和PCR；“生物化学”实验，例如检测某一特定多肽是否存在或不存在，例如通过免疫学方法(ELISAs和Western blots)或者通过本申请所述的实验来进行鉴定均落入本申请范围内。
抗体或抗原结合片段和多特异性抗体的制备
在一些实施例中，所述抗体或抗原结合片段(例如，特异性结合NKG2A的抗体或抗原结合片段)是单克隆抗体。在一些实施例中，所述抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)源于单克隆抗体。在一些实施例中，所述抗体、抗原结合片段或多特异性抗体(优选的，双特异性抗体)包括来自单克隆抗体的VH和VL，或者其变体。在一些实施例中，所述，所述抗体、抗原结合片段或多特异性抗体(优选的，双特异性抗体)进一步包括来自单克隆抗体的CH1和CL区域，或者其变体。单克隆抗体可以应用例如本领域已知的方法制备，包括杂交瘤细胞法、酵母展示、噬菌体展示、293T细胞展示方法或应用重组DNA法。此外，示例性的酵母展示和噬菌体展示法在本申请及以下的实施例中进行了描述。多特异性抗体(优选的，双特异性抗体)可以应用例如本领域已知的方法制备，包括化学偶联、杂交瘤法和基因工程法等。
在杂交瘤细胞法中，通常用免疫剂免疫仓鼠、小鼠或其他适合的宿主动物，以引发产生或能够产生与免疫剂特异性结合的抗体的淋巴细胞。或者，可以在体外免疫淋巴细胞。免疫剂可包括目标蛋白的多肽或融合蛋白。通常，如果需要人源细胞，采用外周血淋巴细胞(PBLs)，而如果需要非人哺乳动物来源细胞，则会使用脾细胞或淋巴结细胞。使用适当的融合剂将淋巴细胞与永生细胞系进行融合，例如聚乙二醇，以形成杂交瘤细胞。永生细胞系通常是转化的哺乳动物细胞，尤其是啮齿类、牛科和人源的骨髓瘤细胞。通常使用大鼠或小鼠骨髓瘤细胞系。杂交瘤细胞可以在合适的培养基中进行培养，所述培养基优选含有一种或多种抑制未融合永生细胞生长或存活的物质。例如，如果亲本细胞缺乏次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，则杂交瘤细胞培养基通常包括次黄嘌呤、氨蝶呤和胸苷(HAT培养基)，该培养基能阻止HGPRT缺陷细胞生长。
在一些实施例中，永生化细胞系有效融合，通过所选择的抗体生产细胞保证抗体高水平稳定表达，并且对某些培养基敏感，例如HAT培养基。在一些实施例中，永生细胞系是小鼠骨髓瘤细胞系，可以从例如，加利福尼亚圣地亚哥的索尔克细胞保藏中心和弗吉尼亚马纳萨斯的美国典型培养物保藏中心获得。同时还描述了人骨髓瘤和鼠-人杂交骨髓瘤细胞系用于制备人源单克隆抗体。
然后可以测定培养杂交瘤细胞的培养基中是否存在针对多肽的单克隆抗体。由杂交瘤细胞产生的单克隆抗体的结合特异性可以通过免疫沉淀法或体外结合实验确定，如放射性免疫测定法(RIA)或酶联免疫吸附法(ELISA)。此类技术或分析方法在本领域是已知的。单克隆抗体的结合亲和力可以通过例如Munson and Pollard,Anal.Biochem.,107:220(1980)中所述的斯卡查德(Scatchard)分析确定。
在鉴定出所需的杂交瘤细胞后，可以通过有限稀释法对目标克隆进行亚克隆，并通过标准方法进行培养。基于此目的适合的培养基包括，例如改良Eagle培养基(DMEM)和RPMI-1640培养基。或者，杂交瘤细胞可以在哺乳动物体内以腹水的形式生长。
亚克隆分泌的单克隆抗体可以通过常规免疫球蛋白纯化方法从培养基或腹水中分离或纯化，例如蛋白A-琼脂糖凝胶、羟基磷灰石色谱层析、凝胶电泳、透析或亲和层析。
在一些实施例中，根据本申请所述的任一抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)，所述抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)包含选自抗体文库(例如展示scFv或Fab片段的噬菌体文库)的克隆的序列。所述克隆可以通过筛选具有所需活性的抗体片段组合文库的方法进行鉴定。例如，本领域已知多种方法用于产生噬菌体展示文库以及筛选这些文库来获得所需结合特性的抗体。这些方法在例如Hoogenboom et al.,Methods in Molecular Biology 178:1-37(O'Brien et al.,ed.,Human Press,Totowa,N.J.,2001)中进行了综述，并且在例如McCafferty et al.,Nature 348:552-554；Clackson et al.,Nature 352:624-628(1991)；Marks et al.,J.Mol.Biol.222:581-597(1992)；Marks and Bradbury,Methods in Molecular Biology 248:161-175(Lo,ed.,Human Press,Totowa,N.J.,2003)；Sidhu et al.,J.Mol.Biol.338(2):299-310(2004)；Lee et al.,J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004)；and Lee et al.,J.Immunol.Methods 284(1-2):119-132(2004)中进行了进一步描述。
在某些噬菌体展示方法中，通过聚合酶链式反应(PCR)分别克隆VH和VL基因的所有组成成分，并在噬菌体文库中随机重组，然后筛选能够结合抗原的噬菌体，如Winter et al.,Ann.Rev.Immunol.,12:433-455(1994)中所述。噬菌体通常以scFv片段或以Fab片段形式展示抗体片段。免疫来源的文库噬菌体提供针对免疫原的高亲和力抗体而不需要构建杂交瘤细胞。或者，可以克隆天然库(例如来自人)，来提供针对多种非自身抗原和自身抗原的单一抗体来源，而不需任何免疫，如Griffiths et al.,EMBO J,12:725-734(1993)中所述。最后，天然文库也可以通过克隆来自干细胞的非重排V-gene片段，并使用包含随机序列的PCR引物编码CDR3高变区并且在体外完成重排的方法进行制备，如Hoogenboom and Winter,J.Mol.Biol.,227:381-388(1992)中所述。描述人抗体噬菌体文库的专利出版物包括，例如U.S.Pat.No.5,750,373、和US Patent Publication Nos.2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936和2009/0002360。
通过噬菌体展示筛选文库中能够特异性结合靶标(例如，NKG2A)的抗原结合部分的方法来制备所述的抗体或抗原结合片段或多特异性抗体(优选的，双特异性抗体)。该文库可以是人scFv噬菌体展示文库，具有至少1×109(例如至少1×109、2.5×109、5×109、7.5×109、1×1010、2.5×1010、5×1010、7.5×1010或1×1011)种多样性的独特的人抗体片段。在一些实施例中，所述文库是人天然文库，通过从健康受试者的PMBCs和脾脏中提取的DNA构建，包含所有人重链和轻链亚家族。在一些实施例中，所述文库是人天然文库，通过从各种疾病患者体内分离的PMBCs中提取的DNA构建，例如自身免疫病的患者、癌症患者和感染性疾病的患者。在一些实施例中，所述文库是半合成的人文库，其中重链CDR3完全是随机的，所有氨基酸(除了半胱氨酸)以相同的概率存在于任何给定的位置。(参见，例如，Hoet,R.M.et al.,Nat.Biotechnol.23(3):344-348,2005)。在一些实施例中，半合成的人文库的重链CDR3长度在5到24个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24个)氨基酸之间。在一些实施例中，所述文库是全合成的噬菌体展示文库。在一些实施例中，所述文库是非人噬菌体展示文库。
对靶标抗原(例如，NKG2A)具有高亲和力的噬菌体克隆可以通过噬菌体与靶标抗原的迭代结合进行筛选，所述靶标抗原与固相支持物结合(例如用于溶液淘选的珠子或用于细胞淘选的哺乳动物细胞)，接下来去除未结合的噬菌体，并洗脱特异性结合噬菌体。随后，洗脱结合的噬菌体克隆并用于感染合适的宿主细胞，例如E.coli XL1-Blue，进行表达和纯化。可以通过多轮淘选(例如，2、3、4、5、6或更多轮)，例如溶液淘选、细胞淘选或两者结合以富集特异性结合靶标抗原的噬菌体克隆。富集的噬菌体克隆与靶标抗原的特异性结合可以通过本领域已知的任何方法进行检测，包括例如ELISA和FACS。
单克隆抗体也可以通过重组DNA方法进行制备，例如U.S.Patent No.4，816,567中所述。编码本申请中所述单克隆抗体的DNA可以通过常规方法(例如通过能特异性结合编码鼠源抗体轻链和重链基因的寡聚核苷酸探针)轻易的分离和测序。如上所述的杂交瘤细胞或本申请的抗原特异性噬菌体克隆可以作为这种DNA的来源。分离后，可将DNA置于表达载体中，然后该载体转染入宿主细胞，例如猿猴COS细胞、中华仓鼠卵巢癌(CHO)细胞或不产生免疫球蛋白的骨髓瘤细胞中，获得在重组宿主细胞中合成的单克隆抗体。所述DNA也可以被修饰，例如用人类重链和轻链恒定结构和/或框架区域的编码序列代替同源的非人类序列(美国专利号4,816,567；Morrison etal.，同上)，或者将非免疫球蛋白多肽的全部或部分编码序列共价连接到免疫球蛋白编码序列上。这种非免疫球蛋白多肽可以取代本申请中抗体的恒定区，或可以取代本申请中抗体可变区中的一个抗原结合位点，形成嵌合的二价抗体。在一些实施例中，针对不同表位或抗原的额外的可变区可以被包括进来以产生嵌合的多特异性抗体(优选的，双特异性抗体)。
所述抗体可以是单价抗体。制备单价抗体的方法是本领域已知的。例如，一种涉及免疫球蛋白轻链和修饰重链的重组表达方法。通常在Fc区的任意位置截短重链，以阻止重链相互交联。或者，相关的半胱氨酸残基被其它氨基酸残基取代或被缺失以防止交联。
体外方法也适用于制备单价抗体。消化抗体产生抗体片段，特别是Fab片段，可以使用任何本领域已知的方法完成。
化学偶联法化学偶联是最早应用于制备多特异性抗体(优选的，双特异性抗体)的技术。1985年，Brennan首次利用两个单克隆抗体G1片段的化学结合来制备多特异性抗体(优选的，双特异性抗体)(Brennan M,et al.Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments[J].Science,1985,229(4708):81-83)。化学偶联法主要有两种方式，一是将两种单抗抗体或其衍生物直接偶联形成多特异性抗体(优选的，双特异性抗体)，二是先通过各种理化方式先将两种单抗解离成游离的轻链和重链，再将这些轻链和重链重新组合。化学偶联法的优点是快速，操作简便，回收率高，但也容易破坏抗体的抗原结合域，影响抗体活性，且容易形成多聚体。
利用杂交瘤细胞系来制备多特异性抗体(优选的，双特异性抗体)，是指通过细胞融合技术，将两种不同的杂交瘤细胞系进行融合，再对可产生具有特定治疗性抗体的细胞进行鉴定和分离(Kohler，G，et al.Continuous cultures of fused cells secreting antibody of predefined specificity[J].J Immunol.,2005,174(5):2453-2455)。因为两种杂交瘤细胞可以产生两种不同的轻-重链，而这些轻-重链可以随机组合，故用此方法制备的多特异性抗体(优选的，双特异性抗体)具有较大的随机性，制备效率低。
基因工程技术目前也被用于制备多种多特异性抗体(Roland E K.Antibody-cytokine fusion proteins[J].Arch Biochem Biophys.,2012,526(2):194-205)。利用基因工程来编辑重组抗体，可以通过多种方式限制两对轻－重链结合的选择性来解决随机组合的问题。KiH(Knob into hole)和CrossMab是目前被应用于改善轻-重链配对问题的两种常用技术。KiH技术，即在CH3结构域引入不对称的突变结构(“knob”突变指在CH3结构域中，用一个大的氨基酸残基替换一个较小的残基，而“hole”突变指使用小的氨基酸残基来替换较大的残基)。经过改造的多特异性抗体(优选的，双特异性抗体)的Fc区由于空间位阻的影响更倾向于发生异二聚化而不是同二聚化(Ridgway J B，et al.“Knobs-into-holes”engineering of antibody CH3 domains for heavy chain heterodimerization[J].Protein Eng.1996，9(7):617-621)。而在糖基化的CH3结构域中引入一个Y349C突变，可以使糖基化重链间形成二硫键，增强KiH的稳定性(Kuglstatter A,et al.Structural differences between glycosylated,disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J].Protein Eng Des Sel.,2017,30(9):649-656)。
除了空间位阻效应外，氨基酸残基的电荷效应也被用于提高多特异性抗体(优选的，双特异性抗体)两条重链之间的异源二聚化。通过结构模拟和分子设计改造，使一个链中产生“正电”，配对链中产生“负电”，通过同种电荷相斥、异种电荷相吸的模式促进重链异二聚体形成。两条链中分别包括突变K409D和D399K，K409D/K392D和D399K/E356K，或者E356K/E357K/D399K和K370E/K409D/K439E等模式，均可在一定程度上提高异二聚体形成(IGAWA T,et al.Methods for producing polypeptides by regulating polypeptide；association:US,20100015133A1[P].2006)。将KiH空间位阻效应和电荷效应相结合，也是提高异源二聚的策略之一。
CrossMab技术是Roche公司基于KiH技术基础上开发的一种新的抗体配对技术，通过将多特异性抗体(优选的，双特异性抗体)中一条Fab的轻链与重链进行结构域交换，而另一条不交换。交换后的轻链中会含有一部分同源重链的片段，使其不能与未经交换的重链配对，从而确保了轻链与重链之间的正确组合(Schaefer W,et al.Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies[J].Proc Natl Acad Sci USA，2011,108(27):11187-11192)，该结构包括“CrossMab Fab”，“CrossMabVH-VL”或“CrossMabCH1-CL”等形式。
具有所需结合特异性(抗体-抗原结合位点)的抗体可变区可以与免疫球蛋白恒定区融合。优选与免疫球蛋白重链恒定区进行融合，其包括至少部分铰链，CH2和CH3结构域。在一些实施例中，包含轻链结合必要位点的重链恒定区CH1结构域至少出现在一种融合体中。编码免疫球蛋白重链融合体的DNA，如果需要，还可以包括编码免疫球蛋白轻链的DNA，被插入进独立的表达载体中，并共转染至合适的宿主生物中。在一些实施例中，针对不同抗原表位或不同抗原的抗体可变区可与免疫球蛋白恒定区序列融合以产生嵌合的多特异性抗体(优选的，双特异性抗体)。
全人和人源化抗体
所述抗体或抗原结合片段或多特异性抗体可以是人源化抗体或全人抗体。非人(如小鼠)抗体部分的人源化形式是嵌合的免疫球蛋白、免疫球蛋白链或其片段(例如Fv、Fab、Fab’、F(ab’)2、scFv或抗体的其他抗原结合子序列)，其通常包括最少的源于非人免疫球蛋白的序列。人源化抗体包括人免疫球蛋白、免疫球蛋白链或其片段(受体抗体)，其中受体CDR的残基被具有所需特异性、亲和力和性能的非人源(供体抗体)CDR残基取代，例如小鼠、大鼠或兔子的CDR。在一些实施例中，人免疫球蛋白Fv框架区残基被相应的非人源残基取代。人源化抗体还可以包含既不属于受体抗体也不在引入的CDR或框架区序列中的氨基酸残基。通常，人源化抗体包含至少一个，通常两个可变区，其中全部或基本上全部CDR区对应于非人免疫球蛋白的CDR区，全部或基本上全部框架区是人免疫球蛋白共有序列。
通常，人源化抗体含有一个或多个从非人源引入的氨基酸残基。那些非人源氨基酸残基通常被称为“移入”残基，通常来自“移入”可变区。根据一些实施例，人源化基本上可以按照Winter和其同事的如下方法进行(Jones et al.,Nature,321:522-525(1986)；Riechmann et al.,Nature,332:323-327(1988)；Verhoeyen et al.,Science,239:1534-1536(1988))，通过用啮齿动物CDRs或CDR序列取代人源抗体的相应序列。因此，这种“人源化”抗体部分(U.S.Patent No.4,816,567)，其基本上少于完整的人源抗体，其可变区已被来自非人源的相应序列所取代。在实际中，人源化抗体部分是典型的人源抗体部分，其中一些CDR残基和可能的一些框架区残基被来自啮齿类抗体中类似位点的残基所取代。
产生人抗体部分是人源化的一种替代方式。例如，目前可以制备在免疫后能够产生完整的全人抗体文库而不产生内源性免疫球蛋白的转基因动物(例如，小鼠)。例如，已有报道，嵌合和种系突变小鼠中抗体重链连接区(JH)基因的纯合子缺失，完全抑制了内源性抗体的产生。将人种系免疫球蛋白基因阵列转移到这种种系突变小鼠体内，可在抗原刺激下产生全人抗体，参见，例如akobovits et al.,PNAS USA,90:2551(1993)；Jakobovits et al.,Nature,362:255-258(1993)；Bruggemann et al.,Year in Immunol.,7:33(1993)；U.S.Patent Nos.5,545,806,5,569,825,5,591,669；5,545,807；和WO 97/17852。或者，可以通过将人类免疫球蛋白基因座引入转基因动物中(例如内源性免疫球蛋白基因已经被部分或全部沉默的小鼠)来制备全人抗体。抗原刺激后，可以发现全人抗体的产生在各个方面都与其在人类中的产生非常相似，包括基因重排、组装和抗体文库。这种方法在例如U.S.Patent Nos.5,545,807；5,545,806；5,569,825；5,625,126；5,633,425；and 5,661,016,and Marks et al.,Bio/Technology,10:779-783(1992)；Lonberg et al.,Nature,368:856-859(1994)；Morrison,Nature,368:812-813(1994)；Fishwild et al.,Nature Biotechnology,14:845-851(1996)；Neuberger,Nature Biotechnology,14:826(1996)；Lonberg and Huszar,Intern.Rev.Immunol.,13:65-93(1995)中进行了描述。
人抗体或人抗体部分也以通过体外活化B细胞(见U.S.Patents 5,567,610和5,229,275)或通过使用本领域已知的各种技术来产生，包括噬菌体展示文库。Hoogenboom and Winter,J.Mol.Biol.,227:381(1991)；Marks et al.,J.Mol.Biol.,222:581(1991).Cole et al.和Boerner et al.等人的技术也可以用于制备全人单克隆抗体。见Cole et al.,Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,p.77(1985)and Boerner et al.,J.Immunol.,147(1):86-95(1991)。
抗体或抗原结合片段的变体
在一些实施例中，本申请提供的抗体或抗原结合片段(例如，特异性结合NKG2A的抗体，或特异性结合NKG2A和PDL1的多特异性抗体)的变体的氨基酸序列也在考虑中。例如，可能需要改善抗体或抗原结合片段的结合亲和力和/或其它生物学活性。抗原结合实体变体的氨基酸序列可以通过在编码抗原结合实体的核苷酸序列中引入适当的修饰或通过肽合成来制备。此类修饰包括例如，抗原结合实体氨基酸序列中残基的缺失和/或插入和/或取代。可以通过氨基酸残基缺失、插入和取代的任一组合来完成最终的构建，使其具有所需的特征。例如，抗原结合性。
在一些实施例中，提供具有一个或多个氨基酸取代的抗体或抗原结合片段的变体。取代突变的目标位点包括高变区(HVRs)和框架区(FRs)。可以在目标抗体中引入氨基酸取代，筛选所需活性的产物，例如，改善的亲和力或活性。在一些实施例中，本申请所述的氨基酸取代仅限于本申请表11中的“示例性取代”。在一些实施例中，所述氨基酸取代仅限于本申请表11中的“优选取代”。
保守取代如下表11所示。
表11：保守取代
根据侧链性质将氨基酸分为不同类别：
a、疏水氨基酸：去甲亮氨酸Norleucine、蛋氨酸Met、丙氨酸Ala、缬氨酸Val、亮氨酸Leu、异亮氨酸Ile；
b、中性亲水性氨基酸：半胱氨酸Cys、丝氨酸Ser、苏氨酸Thr、天冬酰胺Asn、谷氨酰胺Gln；
c、酸性氨基酸：天冬氨酸Asp、谷氨酸Glu；
d、碱性氨基酸：组氨酸His、赖氨酸Lys、精氨酸Arg；
e、含有影响链方向的氨基酸：甘氨酸Gly、脯氨酸Pro；
f、芳香族氨基酸：色氨酸Trp、酪氨酸Tyr、苯丙氨酸Phe。
非保守氨基酸的取代包含将以上一种类别取代为另一种类别。
一种示例性的取代变体是亲和力成熟的抗体，可采用例如以噬菌体展示为基础的亲和力成熟技术而方便地产生。简言之，将一个或多个CDR残基进行突变，变体抗体部分展示在噬菌体上，并筛选具有特定生物活性(例如，基于RBC细胞裂解抑制实验或结合亲和力)的变体。可以在HVRs区进行改变(例如，取代)来获得改善的基于RBC裂解抑制实验或抗体亲和力。可以在HVR的“热点区”产生改变，即在体细胞成熟过程中发生高频突变的密码子编码的残基(参见，例如Chowdhury,Methods Mol.Biol.207:179-196(2008))，和/或在特异的决定性残基(SDRs)，检测所得变体VH和VL的结合亲和力。从二级文库中构建和重新选择亲和力成熟的方法已经在一些文献中进行描述，例如，Hoogenboom et al.in Methods in Molecular Biology 178:1-37(O'Brien et al.,ed.,Human Press,Totowa,NJ,(2001))。
在一些亲和力成熟的实施例中，通过多种方法中的任一种(例如易错PCR，链改组或寡核苷酸定向突变)，将多样性引入选择的用于亲和力成熟的可变基因中。然后创建二级文库。对该文库进行筛选，鉴定出具有所需亲和力的抗体变体。另一种引入多样性的方法包括HVR介导的方式，其中几个HVR残基(例如，一次4-6个残基)被随机化。涉及抗原结合的HVR残基被特异性地识别，例如，采用丙氨酸扫描诱变或建模。通常CDR-H3和CDR-L3区域尤其是重点靶标。
在一些实施例中，取代、插入或缺失可能发生在一个或多个HVRs内，只要这种改变基本上不降低抗体结合抗原的能力。例如，可以在HVRs中产生基本上不降低结合亲和力的保守性改变(例如，本文中提供的保守性取代)。这些改变可能发生在HVR“热点区”或SDRs区域之外。在一些实施例中上文提供的变体VH和VL序列，每一个HVR或者是未发生改变，或者包含不超过1个、2个或3个氨基酸取代。
一种有用的可以鉴定出抗体中能被靶向性突变的氨基酸残基或区域的方法称为“丙氨酸扫描突变”，如Cunningham and Wells(1989)Science,244:1081-1085中所述。在该方法中，一个或一组目标残基(例如，带电残基如精氨酸、天冬氨酸、组氨酸、赖氨酸和谷氨酸)被中性或带负电荷氨基酸(例如，丙氨酸或谷氨酸)取代，以此来确定抗体与抗原相互作用是否受到影响。可以在氨基酸的位置进一步引入取代，来证明该位置对初始取代具有功能敏感性。或者/另外，通过抗原-抗体复合物的晶体结构来鉴定抗体和抗原之间的接触位点。这些接触位点残基和邻近残基可作为取代候选物而被靶向或消除。筛选变体，确定它们是否具有所需要的性质。
氨基酸序列的插入，包括在氨基端和/或羧基末端的融合，长度范围从1个残基到包含100个或更多个残基的多肽，还包括在序列内插入1个或多个氨基酸残基。末端插入的例子包括N末端具有甲硫氨酰残基的抗体。抗体分子的其它插入变体，包括在抗体分子N-末端或C-末端融合一个酶(例如，ADEPT)或增加抗体血清半衰期的多肽。
Fc变体
在一些实施例中，将一个或多个氨基酸修饰引入本申请所述的抗体或抗原结合片段(例如，特异性结合NKG2A的全长抗体、特异性结合NKG2A和PDL1的多特异性抗体，或包含所述抗体或抗原结合片段或多特异性抗体的融合蛋白)的Fc区，从而产生Fc变体。在一些实施例中，Fc变体具有增强的ADCC效能，通常与结合Fc的受体(FcRs)有关。在一些实施例中，Fc变体具有降低的ADCC效能。有很多关于Fc序列的改变或突变影响其效能的例子，例如，WO 00/42072和Shields et al.J Biol.Chem.9(2):6591-6604(2001)描述了与FcRs的结合增强或减弱的抗体变体。这些出版物的内容通过引用并入本申请。
抗体依赖的细胞介导的细胞毒作用(ADCC)是治疗性抗体针对肿瘤细胞的作用机制。ADCC是细胞介导的免疫防御，当靶细胞膜表面的抗原被特异性抗原结合部分(例如，特异性结合NKG2A的抗体、或特异性结合NKG2A和PDL1的多特异性抗体)结合，免疫系统的效应细胞主动裂解靶细胞(例如，感染的细胞)。通常ADCC效应涉及由抗体激活的NK细胞。NK细胞表达Fc受体CD16。该受体识别并结合与靶细胞表面相结合的抗体分子的Fc部分。NK细胞表面最常见的Fc受体为CD16或FcγRIII。Fc受体与抗体Fc区的结合导致NK细胞的活化，细胞裂解颗粒的释放，及随后靶细胞的凋亡。
在一些实施例中，本申请还提供特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体变体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体的变体)其包含具有一种或多种效应功能的Fc区，这使得它成为一个理想的申请候选抗体，在特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的体内半衰期是重要的，但某些效应功能(如CDC和ADCC)是不必要或有害的。通过进行体外和/或体内的细胞毒性试验，来确认CDC和/或ADCC活性的降低/消除。例如，可以进行Fc受体(FcR)结合实验来确认抗体缺乏FcγR结合(因此可能缺乏ADCC活性)，但是保留FcRn结合能力。介导ADCC的主要细胞中，NK细胞仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch and Kinet Annu.Rev.Immunol.9:457-492(1991)第464页的表3中总结了FcR在造血细胞上的表达。在体外评估目标分子的ADCC活性的非限制性实例在U.S.Pat.No.5,500,362中进行了描述(参见例如Hellstrom,I.et al.Proc.Nat'l Acad.Sci.USA 83:7059-7063(1986))and Hellstrom,I et al.,Proc.Nat'l Acad.Sci.USA 82:1499-1502(1985)；U.S.Pat.No.5,821,337(see Bruggemann,M.et al.,J.Exp.Med.166:1351-1361(1987))。或者，可以采用非放射性检测方法(参见，例如ACTITM流式细胞术非放射性细胞毒性检测(CellTechnology,Inc.Mountain View,Calif.)和CYTOTOX 96TM非放射性细胞毒性检测(Promega,Madison,Wis.))。此类检测实验采用的效应细胞包括外周血单核细胞(PBMC)和自然杀伤细胞(NK)。或者，另外地，目标分子的ADCC活性在体内进行检测，例如，在动物模型中，如Clynes et al.Proc.Nat'l Acad.Sci.USA 95:652-656(1998)中所述。同时还可以进行C1q结合试验来确认抗体不能与C1q结合，从而缺乏CDC活性。参见，例如WO2006/029879和WO 2005/100402中C1q和C3c结合ELISA。为了评估补体激活情况，可进行CDC检测(参见，例如Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)；Cragg,M.S.et al.,Blood 101:1045-1052(2003)；和Cragg,M.S.and M.J.Glennie,Blood 103:2738-2743(2004))。使用本领域已知的方法来测定FcRn结合和体内清除/半衰期(参见，例如，Petkova,S.B.et al.,Int'l.Immunol.18(12):1759-1769(2006))。
具有降低的效应功能的抗体，包括在Fc区残基238、265、269、270、297、327和329位进行一个或多个残基的取代(U.S.Pat.No.6,737,056)。这些Fc变体包括在265、269、270、297和327位进行两个或多个残基的取代的Fc变体，包括被称为“DANA”的Fc变体，其在265和297位残基取代为丙氨酸(U.S.Pat.No.7,332,581)。
这类与FcRs结合能力提高或降低的抗体变体已有描述(参见例如U.S.Pat.No.6,737,056；WO 2004/056312,和Shields et al.,J.Biol.Chem.9(2):6591-6604(2001))。
在一些实施例中，Fc区的改变导致调理作用的改变(即增强或减弱)，参见Moore et al.,MAbs.2(2):181–189(2010)中所述。
在一些实施例中，提供一种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体，其包含具有一个或多个氨基酸取代的Fc变体，能够延长半衰期和/或增强与Fc受体(FcRn)的结合。具有延长半衰期和改善FcRn结合的抗体在US2005/0014934A1(Hinton等)中有所描述。这些抗体Fc区包含一个或多个氨基酸取代，增强了Fc区与FcRn的结合。这些Fc变体在Fc区包含238、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424或434位的残基中的一个或多个取代，例如Fc区434位残基的取代(U.S.Pat.No.7,371,826)。
同时参见Duncan&Winter,Nature 322:738-40(1988)；U.S.Pat.No.5,648,260；U.S.Pat.No.5,624,821和WO 94/29351中提供其它Fc变体的例子。
同时考虑了包括本申请所述的任一种Fc变体或其组合的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)。
糖基化变体
在一些实施例中，对本申请所提供的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)进行改变，以增加或降低特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体糖基化的程度。通过改变特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体或其多肽部分的氨基酸序列以此来增加或去除一个或多个糖基化位点，可以方便地实现添加或删除特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体上的糖基化位点。
其中特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体包含Fc区，可以改变与其连接的糖。由哺乳动物细胞产生的天然抗体通常包含分支的双触角寡糖，该寡糖通常通过N-连接与Fc区CH2结构域Asn297连接，参见例如Wright et al.，TIBTECH 15:26-32(1997)。所述寡糖可包含多种糖类，例如甘露糖、N-乙酰氨基葡萄糖苷(GlcNAc)、半乳糖和唾液酸，以及与双触角寡糖结构“茎”部的GlcNAc相连接的海藻糖。在一些实施例中，可对本申请的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体进行寡糖修饰，从而产生具有某些改进特性的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体变体。
与Fc区的CH2结构域连接的N-聚糖是异质的。CHO细胞中产生的抗体或Fc融合蛋白通过岩藻糖基转移酶活性被岩藻糖基化，参见Shoji-Hosaka et al.,J.Biochem.2006,140:777-83。通常，可以在人血清中检测出一小部分天然存在的非岩藻糖基化IgGs。Fc区的N-糖基化对于其与FcγR结合很重要；而非岩藻糖基化的N-聚糖增强了Fc与FcγRIIIa的结合能力。与FcγRIIIa结合能力增强使得ADCC效应增强，这在需要细胞毒性的某些抗体治疗应用中是有利的。
在一些实施例中，当不需要Fc介导的细胞毒作用时，增强的效应功能可能是有害的。在一些实施例中，Fc片段或CH2结构域是非糖基化的。在一些实施例中，通过对CH2结构域中的N-糖基化位点进行突变以阻止其糖基化。
在一些实施例中，提供特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体，其包含Fc区，其中连接于Fc区的糖类结构具有减少的岩藻糖或缺乏岩藻糖，这可能会增强ADCC功能。具体地，本申请提供特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体变体，其相对于野生型CHO细胞产生的相同的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体具有减少的岩藻糖。也就是说，它们的特征在于，与天然CHO细胞(例如，产生天然糖基化形式的CHO细胞，含有天然FUT8基因的CHO细胞)产生的抗体相比，具有更少量的岩藻糖。在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的N-连接聚糖具有少于50％、40％、30％、20％、10％或5％的岩藻糖。例如，该变特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的岩藻糖含量可能是1％-80％、1％-65％、5％-65％或20％-40％。在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的N-连接聚糖不包含岩藻糖，即，其中特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体完全不含岩藻糖，或没有岩藻糖或是去岩藻糖基化。岩藻糖的含量是通过计算连接到Asn297上的糖链内岩藻糖平均含量相对于通过MALDI-TOF质谱测量的所有连接在Asn297上的糖结构(如复合、杂交或甘露糖结构)的总量来确定的，如WO 2008/077546所述。Asn297是指位于Fc区297位的天冬酰胺残基(EU Fc区残基编号体系)。然而，由于抗体的微小序列变化，Asn297也可位于297位的上游或下游±3个氨基酸，即在294和300位之间。这些岩藻糖基化变体可能具有增强的ADCC功能。参见例如US Patent Publication Nos.US2003/0157108(Presta,L.),US 2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。与“去岩藻糖基化”或“岩藻糖缺乏”的抗体变体相关的出版物的实例，包括US2003/0157108；WO 2000/61739；WO 2001/29246；US2003/0115614；US 2002/0164328；US2004/0093621；US2004/0132140；US2004/0110704；US2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586；WO 2005/035778；WO2005/053742；WO2002/031140；Okazaki et al.J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004)。能够产生去岩藻糖基化抗体的细胞系包括缺乏蛋白岩藻糖基化功能的Lec13 CHO细胞(Ripka et al.Arch.Biochem.Biophys.249:533-545(1986)；US Pat Appl No US2003/0157108A1,Presta,L；和WO 2004/056312A1,Adams et al.,尤其是实施例11)，和基因敲除细胞系，例如α-1,6-岩藻糖基转移酶基因，FUT8基因敲除的CHO细胞(参见Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004)；Kanda,Y.et al.,Biotechnol.Bioeng.,94(4):680-688(2006)；和WO2003/085107)。
特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体进一步提供二等分寡糖，例如，其中连接于特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体Fc区的双触角寡糖被GlcNAc等分。这种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体可能具有减少的岩藻糖基化和/或增强的ADCC功能。这类抗体变体的实例在WO 2003/011878(Jean-Mairet et al.)；U.S.Pat.No.6,602,684(Umana et al.)；US2005/0123546(Umana et al.),和Ferrara et al.,Biotechnology and Bioengineering,93(5):851-861(2006)中有所描述。还提供特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体，其在与Fc区连接的寡糖中具有至少一个半乳糖残基。这类特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体变体可能具有增强的CDC功能。这类变体在例如WO 1997/30087(Patel et al.)；WO 1998/58964(Raju,S.)；和WO 1999/22764(Raju,S.)中有所描述。
在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体包含能与FcγRIII相结合的Fc区。在一些实施例中，包含Fc区的所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)变体在人效应细胞(例如T细胞)存在下具有ADCC活性，或者与具有人野生型IgG1 Fc区的其他相同特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的多特异性抗体)相比，在人效应细胞存在下，具有增强的ADCC活性
半胱氨酸工程变体
在一些实施例中，需要制备半胱氨酸工程化的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)，在该抗体中一个或多个氨基酸残基被半胱氨酸残基取代。在一些实施例中，取代残基出现在特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的可及位点。通过用半胱氨酸取代那些残基，具有活性的巯基基团位于特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的可及位点，可以用于将该特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体与其它部分偶联，例如药物部分或接头-药物部分，来制备如本申请中进一步描述的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体免疫偶联物。半胱氨酸工程化的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如，特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)可以按照例如U.S.Pat.No.7,521,541所述进行制备。
衍生物
在一些实施例中，本申请所提供的特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A和PDL1的双特异性抗体)可进一步修饰以包含本领域已知并且容易获得的其它非蛋白部分。适用于衍生化特异性结合NKG2A和PDL1的多特异性抗体的部分包括但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、右旋糖酐、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二氧戊烷、聚-1,3,6-三氧杂环已烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、右旋糖酐或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、环氧丙烷/环氧乙烷共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇及其混合物。聚乙二醇丙醛由于其在水中的稳定性，在制造中具有优势。聚合物可以具有任意分子量，可以是支链或非支链的。连接在特异性结合NKG2A和PDL1的多特异性抗体上的聚合物数量可以变化，并且如果连接多于一个多聚物，它们可以是相同的或不同的分子。通常，用于衍生化的聚合物的数量和/或类型可基于以下考虑因素来确定，包括但不限于，需要改进特异性结合NKG2A和PDL1的多特异性抗体的特性或功能，特异性结合NKG2A和PDL1的多特异性抗体衍生物是否用于特定条件下的治疗等。
药物组合物
本申请还提供包含任一种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体或特异性结合NKG2A和PDL1的双特异性抗体)、编码抗体或抗原结合片段的核酸、包含编码抗体或抗原结合片段的核酸的载体或者包含本申请所述的核酸或载体的宿主细胞的组合物(例如药物组合物，在这里也称为制剂)。在一些实施例中，提供一种药物组合物，包含本申请所述的任一种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体和药学上可接受的载体。
可通过混合具有所需纯度的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体与任选的药学上可接受的载体、赋形剂或稳定剂(Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980))获得合适的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体制剂，制备成冻干制剂或液体制剂形式。可接受的载体、赋形剂或稳定剂在所用剂量和浓度下对接受者无毒，包括缓冲剂如：磷酸盐、柠檬酸和其它有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；六甲基氯化铵；苯扎氯铵；苄索氯铵；苯酚；丁醇或苄醇；对羟基苯甲酸烷基酯，如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇和间甲酚)；低分子量(少于10个残基)多肽；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂如EDTA；糖类，如蔗糖、甘露醇、海藻糖或山梨糖醇；成盐反离子如钠；金属复合物(如锌-蛋白复合物)；和/或非离子表面活性剂如TWEENTM，PLURONICSTM或聚乙二醇(PEG)；示例性制剂如WO98/56418中所述，并通过引用明确并入本申请。适合皮下给药的冻干制剂在WO97/04801中有所描述。这类冻干制剂可通过合适的稀释剂重构成高蛋白浓度的制剂，并且重构的制剂可以通过皮下给药的方式给予本申请中待治疗个体。阳离子脂质体或脂质体可以用于将本申请中的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体递送至细胞。
本申请所述的制剂除包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体或特异性结合NKG2A和PDL1的双特异性抗体)之外，还可以包含一种或多种治疗特定病症所必要的其它活性物质，优选具有活性互补且彼此无不良反应的物质。例如，除了特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体之外，可能需要进一步包含其它具有治疗活性的物质，例如，抗生素类药物。这些分子以对预期目的有效的量组合存在。其它活性物质的有效量取决于制剂中的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的含量，疾病或病症或治疗方式等，以及如上所述的其它因素。这些药物通常以与本申请描述的相同剂量和给药途径使用，或者以目前应用剂量的1％至99％使用。
所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体或特异性结合NKG2A和PDL1的双特异性抗体)也可以包埋在例如通过凝聚技术和界面聚合制备的微胶囊中，例如分别在胶体药物递送系统(例如，脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊)中或粗乳液中的羟甲基纤维素或明胶-微胶囊和聚(甲基丙烯酸甲酯)微胶囊。可以制备缓释制剂。
可以制备特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体或特异性结合NKG2A和PDL1的双特异性抗体)的缓释制剂。缓释制剂的适合的实例包括含有抗体(或其片段)的固体疏水聚合物半透性基质，这些基质是成型制品的形式，例如，薄膜或微胶囊。缓释基质的实例包括聚酯、水凝胶(例如，聚(2-羟乙基甲基丙烯酸酯)或聚(乙烯醇))、聚乳酸(U.S.Pat.No.3,773,919)，L-谷氨酸和L-谷氨酸乙酯共聚物，不可降解的乙烯-醋酸乙烯酯，可降解的乳酸-乙醇酸共聚物如LUPRON DEPOTTM(由乳酸-乙醇酸共聚物和醋酸亮丙瑞林组成的可注射微球)以及聚-D(-)-3-羟基丁酸。虽然诸如乙烯-醋酸乙烯酯和乳酸-乙醇酸之类的聚合物可以使分子的释放超过100天，某些水凝胶可以在更短的时间内释放蛋白质。当包封的抗体在体内长时间停留时，它们会因暴露于37℃的潮湿环境中发生变性或聚集，可能导致生物活性的丧失或免疫原性的改变。可以根据相应的机制，设计合理的策略来稳定特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体。例如，如果发现聚集机制是通过硫代二硫化物交换形成分子间S-S键，则可以通过修饰巯基残基、在酸性溶液中冻干、控制含水量、使用适当的添加剂、以及开发特定的聚合物基质组合物来实现稳定化。
在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体或特异性结合NKG2A和PDL1的双特异性抗体)配制在含有柠檬酸盐、氯化钠、乙酸盐、琥珀酸盐、甘氨酸、聚山梨酯80(吐温80)或上述任何组合的缓冲液中。在一些实施例中，特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体配制在pH值介于4-9之间的缓冲液中。
用于体内给药的制剂必须是无菌的。这可以通过例如应用无菌过滤膜过滤而容易地实现。
预防或治疗HBV感染疾病的方法
一方面，本申请提供了一种预防或治疗个体中HBV感染的方法，所述方法包括向个体施用有效量的包含本申请所述的任一特异性结合NKG2A和PD-L1的多特异性抗体(优选的，双特异性抗体)或包含其的组合物。在一些实施例中，本申请还提供了上述多特异性抗体(优选的，双特异性抗体)或包含其的组合物在制备用于预防或治疗HBV感染的药物中的用途。
另一方面，本申请还提供了一种预防或治疗个体中HBV感染的方法，所述方法包括向个体施用有效量的本申请所述的(i)特异性结合NKG2A的抗体或抗原结合片段和(ii)特异性结合PDL1的抗体或抗原结合片段；或者向个体施用包含上述(i)特异性结合NKG2A的抗体或抗原结合片段和(ii)特异性结合PDL1的抗体或抗原结合片段的组合物。在一些实施例中，本申请还提供了上述抗体或组合物在制备用于预防或治疗HBV感染的药物中的用途。
与HBV感染相关的疾病和/或病症包括但不限于乙型肝炎、肝功能衰竭、肝硬化或肝癌。在一些实施例中，预防或治疗HBV感染的方法降低了HBV感染导致的死亡率。
抗NKG2A-PDL1多特异性抗体预防或治疗HBV感染疾病的方法
在一些实施例中，本申请提供了一种预防或治疗个体中HBV感染的方法，所述方法包括向个体施用有效量的本申请所述的任一特异性结合NKG2A和PD-L1的多特异性抗体(优选的，双特异性抗体)，或包含所述多特异性抗体的组合物，其中所述多特异性抗体包含特异性结合NKG2A的第一抗原结合域和特异性结合PDL1的第二抗原结合域。
在一些实施例中，本申请提供了本申请所述的任一特异性结合NKG2A和PD-L1的多特异性抗体(优选的，双特异性抗体)，或包含所述多特异性抗体(优选的，双特异性抗体)的组合物在制备用于预防或治疗乙型肝炎病毒感染的药物中的用途，其中所述多特异性抗体包含特异性结合NKG2A的第一抗原结合域和特异性结合PDL1的第二抗原结合域。
在一些实施例中，提供了一种预防和/或治疗个体中HBV感染的方法，所述方法包括向个体施用有效量的本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体),或者包含所述多特异性抗体(优选的，双特异性抗体)的组合物，该方法比施用等价剂量的特异性结合NKG2A的抗体或等价剂量的特异性结合PDL1的抗体更有效。
在一些实施例中，所述方法包括施用有效量的本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)，该方法与施用等价剂量的特异性结合NKG2A的抗体或等价剂量的特异性结合PDL1的抗体相比，增强针对HBV感染细胞的ADCC活性至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、、2倍、5倍、10倍、20倍、50倍或100倍以上。
在一些实施例中，所述方法包括施用有效量的本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)，该方法与施用等价剂量的特异性结合NKG2A的抗体或等价剂量的特异性结合PDL1的抗体相比，增强对HBV感染细胞的中和活性至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
在一些实施例中，所述方法包括施用有效量的本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)，该方法与施用等价剂量的特异性结合NKG2A的抗体或等价剂量的特异性结合PDL1的抗体相比，增强针对HBV感染细胞的ADCP活性至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
在一些实施例中，所述方法包括施用有效量的本申请所述的特异性结合NKG2A和PDL1的多特异性抗体(优选的，双特异性抗体)，该方法与施用等价剂量的特异性结合NKG2A的抗体或等价剂量的特异性结合PDL1的抗体相比，降低病人血清中HBsAg含量至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
包含抗NKG2A抗体和抗PDL1抗体的组合物用于预防或治疗HBV感染疾病的方法
在一些实施例中，提供了一种预防或治疗个体中HBV感染的方法，所述方法包括向个体施用有效量的药物组合物，其包括：(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段。
在一些实施例中，提供了包含(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段的组合物在制备用于预防或治疗HBV感染的药物中的用途。
在一些实施例中，提供了一种预防或治疗个体中HBV感染的方法，所述方法包括向个体施用：(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段。在一些实施例中，本申请所述的特异性结合NKG2A的抗体或抗原结合片段与特异性结合PDL1的抗体或抗原结合片段同时施用。在一些实施例中，本申请所述的特异性结合NKG2A的抗体或抗原结合片段与特异性结合PDL1的抗体或抗原结合片段顺序施用。
在一些实施例中，提供了(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段在制备预防或治疗HBV感染的药物中的用途。在一些实施例中，本申请所述的特异性结合NKG2A的抗体或抗原结合片段与特异性结合PDL1的抗体或抗原结合片段同时施用。在一些实施例中，本申请所述的特异性结合NKG2A的抗体或抗原结合片段与特异性结合PDL1的抗体或抗原结合片段顺序施用。
在一些实施例中，所述方法包括施用药物组合物，所述组合物包括：本申请所述的特异性结合NKG2A的抗体或抗原结合片段和本申请所述的特异性结合PDL1的抗体或抗原结合片段，其中特异性结合NKG2A的抗体或抗原结合片段和特异性结合PDL1的抗体或抗原结合片段的摩尔比约为5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4或1:5。在一些实施例中，所述特异性结合NKG2A的抗体或抗原结合片段和特异性结合PDL1的抗体或抗原结合片段的摩尔比约为2:1或1:1。
在一些实施例中，提供了预防或治疗个体中HBV感染的方法，所述方法包括向个体施用有效量的(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段，该方法比施用等价剂量的特异性结合NKG2A的抗体或抗原结合片段或等价剂量的特异性结合PDL1的抗体或抗原结合片段更有效。
在一些实施例中，所述方法包括施用有效量的(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段，该方法与施用等价剂量的特异性结合NKG2A的抗体或抗原结合片段或等价量的特异性结合PDL1的抗体或抗原结合片段相比，增强针对HBV感染细胞的ADCC活性至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
在一些实施例中，所述方法包括施用有效量的(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段，该方法与施用等价剂量的特异性结合NKG2A的抗体或抗原结合片段或等价量的特异性结合PDL1的抗体或抗原结合片段相比，增强对HBV感染细胞的中和活性至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
在一些实施例中，所述方法包括施用有效量的(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段，该方法与施用等价剂量的特异性结合NKG2A的抗体或抗原结合片段或等价量的特异性结合PDL1的抗体或抗原结合片段相比，增强针对HBV感染细胞的ADCP活性至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
在一些实施例中，所述方法包括施用有效量的(i)本申请所述的特异性结合NKG2A的抗体或抗原结合片段和(ii)本申请所述的特异性结合PDL1的抗体或抗原结合片段，该方法与施用等价剂量的特异性结合NKG2A的抗体或抗原结合片段或等价量的特异性结合PDL1的抗体或抗原结合片段相比，降低病人血清中HBsAg含量至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
制品及试剂盒
一方面，在本申请的一些实施例中，提供一种制品，所述制品包含一种物质，所述物质能够用于预防或治疗个体中的HBV感染，或者用于递送抗体或抗原结合片段(特异性结合NKG2A的抗体)或多特异性抗体(例如特异性结合NKG2A和PDL1的双特异性抗体)，或者包含特异性结合NKG2A的抗体或抗原结合片段和特异性结合PDL1的抗体或抗原结合片段的药物组合物到被表达NKG2A或PDL1的病原菌附着的细胞中。所述制品可以包括一种容器以及在容器上或随该容器附带的标签或包装说明书。合适的容器包括，例如瓶子、小瓶、注射器等。容器可以由多种材料制成，例如玻璃或塑料。通常，该容器内装有能够有效治疗本申请所述疾病或病症的组合物，并且具有一个无菌端口(例如该容器可以是一个静脉输液袋或是一个具有皮下注射针头可刺穿盖子的小瓶)。组合物中的至少一种活性物质即为本申请所述的抗体或抗原结合片段或双特异性抗体。标签或包装说明书标示了该组合物可以用于治疗的特定病症。标签或包装说明书进一步包含给患者施用双特异性抗体或药物组合物的说明书。包括本申请所述的包含联合治疗的制品和试剂盒均在考虑范围之内。
包装说明书是指通常包含在治疗产品的商业包装内的说明书，其包含关于与这些治疗产品使用有关的适应症、用法、剂量、施用、禁忌症和/或警告信息。在一些实施例中，包装说明书标明该组合物可以用于治疗细菌感染。在一些实施例中，包装说明书标明该组合物可用于治疗HBV感染。此外，所述制品还可以包括第二容器，其包含药学上可接受的缓冲液，例如抑菌性注射用水(BWFI)、磷酸盐缓冲液、格林氏溶液或葡萄糖溶液。还可以包括从商业和用户角度而言所需的其他材料，包括其他缓冲液、稀释液、过滤器、针头和注射器。
同时还提供可用于各种目的的试剂盒，例如用于预防或治疗个体中HBV感染，或者用于递送特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)到被表达NKG2A或PDL1的病原菌附着的细胞中，任选与制品组合。本申请的试剂盒包括一个或多个容器，其包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的组合物(或单剂量形式和/或制品)，并且在一些实施例中，进一步包含另一种药剂(例如本申请所述的药剂)和/或与本申请所述任一方法相一致的使用说明书。该试剂盒可进一步包括选择适合治疗个体的描述。本申请中试剂盒中所附带的使用说明书通常是标签或包装说明书上的书面说明(例如包含在试剂盒内的纸页)，机器可读的说明(例如，磁性或光学储存光盘上的说明)也是可以接受的。
例如，在一些实施例中，试剂盒包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)的组合物。在一些实施例中，试剂盒包括：a)包含本申请所述的任一种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的组合物，和b)至少一种有效量的其它药剂，其能够增强特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的效果(如治疗效果、检测效果)。在一些实施例中，试剂盒包括：a)包含本申请所述的任一种特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的组合物，和b)向个体施用特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体组合物用于治疗个体中HBV感染的使用说明书。在一些实施例中，试剂盒包括：a)包含本申请所述的任一种特异性结合NKG2A抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的组合物，和b)至少一种有效量的其它药剂，其能够增强特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的效果(如治疗效果、检测效果)和c)向个体施用特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体组合物和其它物质用于治疗个体中HBV感染的使用说明书。所述特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体和其他物质可以存在于独立的容器或同一个容器中。例如，该试剂盒可以包括一种特定组合物或两种或更多种组合物，其中一种组合物包括特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体，另一种组合物包括另一种药剂。
在一些实施例中，试剂盒包含一种(或一组)编码特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)的核酸。在一些实施例中，试剂盒包含：a)一种(或一组)编码特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的核酸，和b)一种表达核酸(或一组核酸)的宿主细胞。在一些实施例中，试剂盒包含：a)一种(或一组)编码特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的核酸，和b)使用说明书，适用于：i)在宿主细胞中表达特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体，ii)制备包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的双特异性抗体的组合物，和iii)向个体施用包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的组合物来预防或治疗个体中HBV感染。在一些实施例中，试剂盒包括：a)一种(或一组)编码特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的核酸，b)一种表达核酸(或一组核酸)的宿主细胞，和c)使用说明书，适用于：i)在宿主细胞中表达特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体，ii)制备包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的组合物，和iii)向个体施用包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体的组合物来预防或治疗个体中HBV感染。
本申请所述的试剂盒以合适的形式进行包装。合适的包装包括，但不限于，小瓶、瓶子、广口瓶、软包装(例如密封的聚酯薄膜或塑料袋)等。试剂盒可以任选地提供其它的组分，例如缓冲液和说明信息。本申请因此还提供制品，其包括小瓶(例如密封的小瓶)、瓶子、广口瓶、软包装等等。
关于包含特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体组合物的使用说明书，通常包括一些信息，诸如，剂量，给药周期和给药途径。容器可以是单位剂量的，大包装的(如，多剂量包装)或亚单位剂量的。例如，提供一种包含足够剂量的如本申请所述的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体(例如特异性结合NKG2A的全长抗体、或特异性结合NKG2A和PDL1的双特异性抗体)的试剂盒以对个体进行长期有效的治疗，例如一周、8天、9天、10天、11天、12天、13天、2周、3周、4周、6周，8周，3个月、4个月、5个月、7个月、8个月、9个月或更长时间。试剂盒还可包含多单位剂量的特异性结合NKG2A的抗体或抗原结合片段、或特异性结合NKG2A和PDL1的多特异性抗体、药物组合物和使用说明书，并且以足够在药房中储存和使用的量进行包装，例如，医院药房和复合药房。
本领域的技术人员将认识到在本申请的范围和宗旨内可能的若干实施例。现在将通过参考以下非限制性实施例来更详细地描述本申请。以下实施例进一步阐明本申请，但不应解释为以任何方式进行限制其范围。
具体实施方式
实施例1：筛选、制备并表征抗NKG2A抗体
1.1.制备重组的NKG2A抗原
通过亚克隆将编码人NKG2A(huNKG2A)蛋白胞外区(94aa-233aa)、人CD94(huCD94)蛋白胞外区(32aa-179aa)的核酸序列分别构建到带有人IgG Fc(huFc)或小鼠IgG Fc(musFc)的表达载体中，并按照已建立的标准分子生物学方法制备上述表达载体质粒。之后将上述表达载体质粒共转染HEK293F细胞，培养后收集细胞培养液。按照制造商的操作说明，采用MabCap At4FF预装柱纯化具有Fc标签的重组蛋白，获得huNKG2A-huCD94-Fc异源二聚体蛋白。
1.2.筛选、制备全长的抗NKG2A抗体
使用huNKG2A-huCD94异源二聚体抗原免疫新西兰兔，收集免疫后的兔血清，ELISA检测上述血清中的总IgG滴度。几轮免疫后，使用兔脾脏构建293T细胞展示库。采用流式细胞仪分选出结合人NKG2A-musFc的阳性单细胞。筛选后进一步通过ELISA法筛选出与人NKG2A结合且不与NKG2C结合的阳性克隆。对筛选后的阳性克隆进行测序，获得候选先导抗体ST14-R76的序列。之后对先导抗体ST14-R76进行人源化，并优化CDR区化学不稳定的氨基酸残基，从而获得一系列人源化分子。选择其中的hum-ST14-R76-9(本文中简称为R76-9)、hum-ST14-R76-10(本文中简称为R76-10)抗体进行后续的验证和进一步开发，其具体氨基酸序列如表2-1和表2-2所示。
1.3.ELISA法检测抗NKG2A抗体的结合活性
将浓度为2μg/ml的huNKG2A-huCD94-musFc抗原分别包被到3590酶标板，100μl/孔，4℃孵育过夜，0.5％ PBST洗板3次；随后加入梯度稀释(初始浓度为200μg/ml，6倍梯度稀释，共8个浓度)的待检测抗NKG2A抗体或阳性对照抗体M15(上海怀越)，100μl/孔，以及100μl BSA封闭液，混匀后37℃孵育1h；弃去酶标板中液体，用0.5％ PBST洗板3次；随后加入100μl/孔的1：4000稀释(1％ BSA)的山羊抗人kappa-AP二抗(Cat#2060-04，SouthernBiotech)，37℃孵育1h；弃去酶标板中液体，用0.5％PBST清洗3次，甩干残余液体；最后每孔加入100μl PNPP，37℃显色15min，测定OD405，并通过PRISM软件生成结合曲线，分析各候选抗体与抗原人NKG2A的亲和力，计算EC50值。
结果如表12所示，人源化抗NKG2A抗体(人IgG4形式)R76-9、R76-10均能够有效结合人NKG2A，其结合活性与阳性对照抗体M15相当。
表12：人源化抗NKG2A抗体与人NKG2A抗原的结合活性
1.4.FACS检测抗NKG2A抗体阻断NKG2A与HLA-E四聚体的结合试验
NKG2A-CD94异源二聚体在NK细胞表面表达，其配体HLA-E四聚体可以结合NK细胞上的NKG2A。本实验通过检测HLA-E四聚体与NK细胞(例如NKL细胞或NK92细胞)表面NKG2A的结合，检测抗NKG2A抗体阻断HLA-E与NKG2A结合的活性。
简言之，用1％的BSA重悬稀释NKL细胞(Cat#BSCELL-0491，上海宾穗生物科技有限公司)至浓度为1.0×106个/ml，并接种至96孔板，100μl/孔，于4℃下封闭30min；随后加入梯度稀释(初始浓度为20μg/ml，4倍梯度稀释，共8个梯度)的待测抗NKG2A抗体，50μl/孔，以及50μl链霉亲和素-藻红蛋白(SA-PE)缀合的HLA-E四聚体(Cat#TS-ME01-1，MBL)稀释液(包含1.2μl原液和98.8μl PBS)，其中M15抗体(上海怀越)作为阳性对照，空白对照组补加100μl的1％BSA；各孔吸打混匀后于4℃避光孵育1h，1×PBS清洗细胞后用流式细胞仪检测荧光。用FlowJo软件分析抗体结合数据，以阳性率(％)表示带有HLA-E荧光标记的阳性细胞比例。
结果如表13所示，人源化抗NKG2A抗体R76-9、R76-10(人IgG4形式)能够有效地阻断NKL细胞NKG2A与HLA-E四聚体的结合，其活性优于阳性对照抗体M15。
表13：人源化抗NKG2A抗体阻断NKG2A与HLA-E四聚体结合的活性
1.5.DELFIA EuTDA细胞毒实验检测人源化抗NKG2A抗体促进NKL细胞对K562-E4细胞的杀伤活性
将细胞表面表达NKG2A的NKL细胞和过表达配体HLA-E的K562-E4细胞系共培养后，可以激活NKG2A/HLA-E信号，导致细胞内ITIM磷酸化，从而抑制NKL细胞的杀伤活性。在本实验中，抗NKG2A抗体通过中和表达于NKL细胞的NKG2A，从而促进NKL细胞对K562-E4靶细胞的杀伤活性，并利用EuTDA细胞毒法来检测。EuTDA细胞毒法(Cat#AD0116，珀金埃尔默)是利用荧光放大配体BATDA特异性标记靶细胞。BATDA能迅速进入细胞，并在水解作用下形成亲水的TDA留在细胞内，并在靶细胞裂解下释放，和DELIFA Eu试剂相结合形成强荧光、稳定的螯合物EuTDA，用于检测靶细胞的裂解情况。
首先构建K562-E4稳转细胞系。简言之，将构建的质粒pIRES2-SA-HLAE-EGFP电转染K562细胞，在含有400μg/ml抗生素G418的1640培养基(含10％FBS+1％ P.S.双抗)中选择性培养数天，随后用流式细胞仪分选出有荧光表达的细胞，并将分选出的单克隆细胞置于24孔板，在含有G418抗生素的1640培养基中培养7～10天，之后转入75cm细胞培养瓶(Cat#430720，Corning)中扩大培养，从而得到K562-E4稳转细胞系。
短肽刺激处理K562-E4稳转细胞：将K562-E4稳转细胞重悬于3ml含有500μg/ml短肽VMAPRTVLL(南京金斯瑞合成)的Opti-MEM，置于CO2培养箱中26℃孵育过夜(约16h)；用1×DPBS缓冲液清洗细胞，在避光条件下，每1ml细胞悬液加入1.5μl荧光增强配体BATDA(Cat#AD0116，PerkinElmer)，混匀后置于CO2培养箱中37℃孵育30min；随后1300rpm离心5min去上清，重悬于1ml 1×DPBS，并转移至新的15ml离心管，重复3次，以达到充分清洗培养基内残余BATDA的目的。K562-E4细胞孵育BATDA的同时准备NKL细胞。
NKL细胞的处理：NKL细胞培养于MyeloCultTMH5100培养基(Cat#05150，STEMCELL Technologies)，其中补加1％双抗和100IU/ml的IL-2(Cat#589106，Biolegend)。将100μl密度为2×105个/ml的NKL细胞接种至96孔V底板(Cat#701201，NEST)，加入50μl梯度稀释的待测抗NKG2A抗体(8μg/ml，1.6μg/ml，0.32μg/ml)，其中Mon抗体(NOVO NORDISK)作为阳性对照，阴性对照孔不加抗体，吸打混匀后置于CO2培养箱孵育15min。随后加入50μl/孔上述BATDA处理的K562-E4细胞(1×104细胞/孔)，混匀后1000rpm离心5min，置于37℃CO2培养箱孵育1h；500g离心5min后，各孔内吸取20μl上清转移至96孔平底透明板(由DELFIA EuTDA细胞毒法试剂盒提供)内，每孔加入200μl Europium Solution(Cat#AD0116，PerkinElmer)，室温避光震荡孵育15min，酶标仪检测各孔的荧光值。通过各孔荧光值计算杀伤率，以衡量抗NKG2抗体的功能活性，计算公式如下：
根据杀伤率绘制曲线并计算EC50值。
结果如表13所示，人源化抗NKG2A抗体R76-10(人IgG4形式)能够有效地促进NKL细胞对K562-E4靶细胞的杀伤，其杀伤活性显著优于阳性对照抗体Mon。
表13：人源化抗人NKG2A抗体促进NKL细胞对K562-E4细胞的杀伤活性
实施例2：抗NKG2A-PDL1双特异性抗体的制备和表征
下述序列设计中:VH和VL分别代表抗体的重链可变区和轻链可变区；CH代表抗体重链恒定区，包括CH1、CH2和CH3结构域；scFv是由抗体的VH和VL通过连接肽(Linker)连接而成的单链抗体；IgG1 Fc代表IgG1亚类抗体的Fc区域，其包含CH2和CH3结构域；CL代表轻链恒定区。
2.1.IgG-scFv结构的双特异性抗体的构建
序列设计：IgG-scFv结构双特异性抗体，是在一个IgG抗体的其中一条重链的Fc末端连接结合其他抗原的scFv片段，以此实现双特异性。
本实施例中所使用的该结构的双特异性抗体中，在IgG1 Fc区设计了杵臼结构(knobs-in-holes，KIH)，同时引入形成稳定化二硫桥的两个Cys残基突变(S354C在“knob”侧，并且Y349C在“hole”侧)。所述KIH即将一个单体的CH3区366位苏氨酸(T)替换为色氨酸(W)形成“杵”(knobs)结构，并将配对的另一个单体的CH3区366位苏氨酸(T)替换为丝氨酸(S)，368位亮氨酸(L)替换为丙氨酸(A)，407位酪氨酸(Y)替换为缬氨酸(V)形成“臼”(holes)结构。另外将抗体铰链区的第234位的亮氨酸(L)、第235位的亮氨酸(L)替换为丙氨酸(A)、第331位的脯氨酸(P)替换为丝氨酸(S)，形成组合突变LALAPS。另外，通过在VH和VL界面引入两个半胱氨酸突变，从而获得了二硫键稳定型的多特异性抗体，即在SBT451scFv的VH区中引入G44C突变，以及在SBT451scFv的VL区中引入Q100C突变；其中所述编号依照如Kabat的EU索引。
本实施例中所使用的IgG-scFv双特异性抗体结构的示意图如图1A所示。该结构中的抗NKG2A-PDL1双特异性抗体共包括四条多肽链，分别称为第一重链、第一轻链、第二重链和第二轻链，其中，第一轻链和第二轻链的序列相同，其序列组成如表14所示，其全长重链和轻链的具体氨基酸序列如表6-1所示。其中，双特异性抗体中的抗PD-L1抗原结合域来自于抗PD-L1抗体SBT451(又称为Hum-6，参见专利申请WO2024040212A2，其全部内容以其整体并入本文之中)，其氨基酸序列如表3-1、3-2和3-3所示；抗NKG2A抗原结合域来自于抗NKG2A抗体R76-10。
双特异性抗体的构建过程：按照操作说明书，通过无缝克隆的方式将抗NKG2A抗体的重链可变区、轻链可变区序列，抗PD-L1抗体的重链可变区、轻链可变区序列构建入真核表达载体pTTa1中，获得分别表达重链的重链表达载体(2个)，以及表达轻链的轻链表达载体，共计3个表达载体。
双特异性抗体的表达和纯化：按照操作说明书，将上述双特异性抗体的3个表达质粒共转染HEK293F细胞，并将转染后的293F细胞在37℃、5％ CO2、120rpm条件下培养6天。分别收集细胞培养液。采用蛋白A树脂(MabCap At 4FF 5ml预装柱，货号为SA023C15，常州天地人和生物科技有限公司)纯化上述抗体。具体操作如下：首先采用6倍柱体积PBS缓冲液(包含0.15M NaCl，pH7.4)以150cm/h(流速5ml/min)的流速平衡蛋白A柱。培养液上清(调节pH至7.2-7.4)以150cm/h(流速5ml/min)流速流穿柱子。进一步平衡该柱后，采用6倍柱体积的0.1M甘氨酸缓冲液(含0.15M NaCl，pH3.2)洗脱，收集洗脱液，调节pH值至中性。利用超滤浓缩管将目的蛋白溶液置换于PBS缓冲液中。
然后采用分子筛层析(HiLoad 16/600superdex 200pg，cytiva)去除抗体中的聚集体成分。简言之，采用1倍柱体积的PBS缓冲液(包含50mM PBS和0.15M NaCl，pH7.2)平衡分子筛层析柱，流速1ml/min。将体积不大于5ml的抗体样品上样，接着采用PBS缓冲液冲洗分子筛层析柱，并检测UV280，收集单体峰对应的组分。采用超滤管超滤换液至PBS中，超滤浓缩后采用BCA蛋白定量试剂盒测定目的蛋白浓度。
表14：IgG-scFv结构的抗NKG2A-PDL1双特异性抗体的重链和轻链组成
2.2.IgG-(scFv)2结构的双特异性抗体的构建
序列设计：IgG-(scFv)2结构双特异性抗体(可参见，Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.1997Feb；15(2):159-63)，是在一个IgG抗体的两条重链的Fc末端连接结合其他抗原的scFv片段，以此实现双特异性。对于本申请实施例中使用的IgG-(scFv)2结构的双特异性抗体，在Fc区引入组合突变LALAPS，另外在SBT451scFv的VH和VL界面引入两个半胱氨酸突变，具体详见实施例2.1部分。
本申请实施例中所使用的IgG-(scFv)2的双特异性抗体结构示意图如图1B所示，该结构的双特异性抗体分子由四条链组成，即两条相同的重链和两条相同的轻链。其中，该双特异性抗体中的抗NKG2A抗原结合域来源于R76-10，抗PD-L1抗原结合域来源于SBT451。表15为实施例中所使用的IgG-(scFv)2结构的双特异性抗体的组成，表6-2中示出了其全长重链和轻链的具体氨基酸序列。
双特异性抗体的构建过程：按照操作说明书，通过无缝克隆的方式构建分别表达重链的重链表达载体(1个)，以及分别表达轻链的轻链表达载体(1个)，共计2个表达载体。
双特异性抗体的表达纯化：具体的操作步骤如实施例2.1中所述。
表15：IgG-(scFv)2结构的抗NKG2A-PDL1双特异性抗体的重链和轻链组成
2.3.用于概念验证的双特异性抗体的构建
此外，由于上述的抗NKG2A抗体结合域与小鼠NKG2A抗原、以及上述的抗PD-L1抗体结合域与小鼠PD-L1抗原均没有交叉结合活性，因此，为了在小鼠模型中验证本申请的抗NKG2A-PDL1双特异性抗体的作用，本实施例中额外构建了用于概念验证(Proof of Concepts,POC)的抗NKG2A-PDL1双特异性抗体，用于在小鼠体内的活性验证。
序列设计：按照2.1中所述的步骤构建具有IgG-scFv结构的POC分子；按照2.2中所述的步骤构建具有IgG-(scFv)2结构的POC分子。其中，设计的POC分子中抗NKG2A抗原结合域采用抗鼠NKG2A抗体NKG2A.2的序列(参考专利WO2020/102501A1，其全部内容以其整体并入本文之中)；抗PD-L1抗原结合域是采用抗鼠PD-L1抗体S70的序列(参考Clin Cancer Res(2020)26(15):4154–4167)，并在S70scFv的VH区中引入G44C突变，VL区中引入Q100C突变，以获得二硫键稳定型的多特异性抗体。表16为实施例中所使用的POC双特异性抗体的组成，表6-1和表6-2中示出了其重链和轻链的具体氨基酸序列。
POC双特异性抗体的表达纯化：具体的操作步骤如实施例2.1中所述。
表16：抗NKG2A-PDL1双特异性抗体POC分子的重链和轻链组成
2.4.抗NKG2A-PDL1双特异性抗体与NKG2A的结合活性检测
采用ELISA结合试验检测抗NKG2A-PDL1双特异性抗体NP-2、NP-1与huNKG2A-huCD94-musFc抗原的结合活性，其中包被的抗原浓度为1μg/ml，NKG2A单抗R76-10作为对照分子，其他步骤按照实施例1.3中所述进行。
结果如表17所示，抗NKG2A-PDL1双特异性抗体NP-2、NP-1能够有效结合人NKG2A抗原，且结合活性与NKG2A单抗R76-10相当。
表17：抗NKG2A-PDL1双特异性抗体与NKG2A的结合活性

2.5.抗NKG2A-PDL1双抗POC分子与NKG2A的结合活性检测
采用ELISA结合试验检测抗NKG2A-PDL1双特异性抗体对应的POC分子与小鼠NKG2A抗原的结合活性，其中包被的抗原为1μg/ml的小鼠NKG2A-CD94异二聚体抗原(购自ACROBiosystems，货号#NC4-M5254)，对照抗体为NKG2A单抗NKG2A.2，其他步骤按照实施例1.3中所述进行。
结果如表18所示，抗NKG2A-PDL1双特异性抗体对应的POC分子NP-2-POC、NP-1-POC能够有效地结合小鼠NKG2A抗原，且结合活性与NKG2A单抗NKG2A.2相当。
表18：抗NKG2A-PDL1双特异性抗体POC分子与NKG2A的结合活性
2.6.抗NKG2A-PDL1双特异性抗体与PD-L1的结合活性检测
采用ELISA结合试验检测抗NKG2A-PDL1双特异性抗体与人PD-L1抗原的结合活性，其中包被抗原为1μg/ml的人PD-L1-Fc融合蛋白(参见专利申请WO2024040212A2)，对照抗体为PD-L1单抗SBT451，其他步骤按照实施例1.3中所述进行。
结果如表19所示，抗NKG2A-PDL1双特异性抗体NP-2、NP-1能够有效结合人PD-L1抗原，且结合活性与PD-L1单抗SBT451相当。
表19：抗NKG2A-PDL1双特异性抗体与PDL1的结合活性
2.7.抗NKG2A-PDL1双抗POC分子与PD-L1的结合活性检测
采用ELISA结合试验检测抗NKG2A-PDL1双特异性抗体对应的POC分子与小鼠PD-L1抗原的结合活性，其中包被的抗原为1μg/ml的纯化的小鼠PD-L1-Fc融合蛋白，对照抗体为PD-L1单抗S70，其他步骤按照实施例1.3中所述进行。
结果如表20所示，示例性的抗NKG2A-PDL1双特异性抗体对应的POC分子NP-2-POC能够有效地结合小鼠PD-L1抗原，且结合活性与PD-L1单抗S70相当。
表20：抗NKG2A-PDL1双特异性抗体POC分子与PDL1的结合活性
2.8.抗NKG2A-PDL1双特异性抗体阻断NKL细胞NKG2A与HLA-E四聚体的结合试验
采用FACS方法检测抗NKG2A-PDL1双特异性抗体对HLA-E四聚体与NKL细胞表面受体NKG2A结合的阻断活性，其中加入的抗NKG2A-PDL1双特异性抗体的初始浓度为60μg/ml，3倍梯度稀释，共11个梯度，对照抗体为NKG2A单抗R76-10，其他步骤按照实施例1.4中所述进行。
结果如表21所示，抗NKG2A-PDL1双特异性抗体NP-2、NP-1均能够有效地阻断NKL细胞上NKG2A与HLA-E四聚体的结合，其活性与NKG2A单抗R76-10相当。
表21：抗NKG2A-PDL1双特异性抗体对NKG2A-HLAE结合的阻断活性
2.9.DELFIA EuTDA细胞毒实验检测抗NKG2A-PDL1双特异性抗体促进NKL细胞对K562-E4细胞的杀伤活性
采用DELFIA EuTDA细胞毒法检测抗NKG2A-PDL1双特异性抗体促进NKL细胞对K562-E4细胞的杀伤活性，其中接种的NKL细胞密度为1×105个/ml，加入的双特异性抗体的初始浓度为40μg/ml，5倍梯度稀释，共9个梯度，对照抗体为NKG2A单抗R76-10，其他步骤按照实施例1.5中所述进行。
结果如表22所示，抗NKG2A-PDL1双特异性抗体NP-2、NP-1均能够有效地促进NKL细胞对K562-E4靶细胞的杀伤，其杀伤活性与NKG2A单抗R76-10相当。
表22：抗NKG2A-PDL1双特异性抗体促进NKL细胞对K562-E4细胞的杀伤活性
2.10.ELISA检测抗NKG2A-PDL1双特异性抗体对PD1-PDL1信号通路的阻断活性
采用HTF供体和HTF受体检测PD-L1和PD1之间的相互作用。当供体和受体由于PD-L1和PD1结合而接近时，供体的激发触发了向受体的荧光共振能量转移(FRET)，而受体又在665nm处特异性发射。这个特定的信号与PD1/PD-L1相互作用的程度成正比。因此，阻断PD1/PD-L1相互作用的化合物或抗体会导致HTRF信号的减少。
简言之，在96孔板(Corning,3903)中加入2μl的待测抗NKG2A-PDL1双特异性抗体或阳性对照抗体AM10-F2(来自迈威公司的抗NKG2A-PDL1双特异性抗体，可参见专利CN113583127A)，4μl Tag1-PD-L1蛋白以及4μl Tag2-PD1蛋白(Promega,64ICP01PEG)，室温静置15分钟；然后加入10μl预混的HTF供体anti-Tag1-Eu3和HTF受体anti-Tag2-XL665(Promega,64ICP01PEG)，封闭板子室温静置1小时；检测荧光计算HTRF Ratio，并通过Graphpad Prism生成结合曲线，计算IC50值。
结果如表23所示，抗NKG2A-PDL1双特异性抗体NP-2、NP-1均能够有效地抑制PD1-PDL1蛋白的相互作用，其阻断活性优于阳性对照抗体AM10-F2。
表23：抗NKG2A-PDL1双特异性抗体对PD1-PDL1信号通路的阻断活性
2.11.抗NKG2A-PDL1双特异性抗体在HBV转基因小鼠模型中的药效学研究
HBV转基因小鼠模型(HBV-Tg)由维通利华公司制备，用于抗NKG2A-PDL1抗体抑制HBV复制的体内药效学评价。制备过程如下，将1.28倍HBV(A型，GenBank:AF305422.1)基因组长度的DNA线性化片段注入C57BL/6NCrl小鼠胚胎原核，获得转基因阳性小鼠。通过外周血HBV DNA拷贝数分析，保留了拷贝数达到107～108IU/ml的转基因首建鼠。按照半合子配野生C57BL/6NCrl小鼠的方式建立HBV-Tg小鼠品系。该小鼠体内可产生完整的、具有感染性的病毒颗粒，HBV复制水平与慢性乙型肝炎患者相当。除高滴度的HBV DNA，外周血中也可检测到高含量HBsAg、HBeAg。由于HBV抗原在胚胎阶段就持续表达，诱导小鼠产生免疫耐受，所以该小鼠不会表现出类似于人类乙型肝炎的免疫病理变化。
通过腹腔注射向上述模型小鼠分别施用抗NKG2A-PDL1双特异性抗体的POC分子NP-2-POC，抗PD-L1单抗S70或抗NKG2A单抗NKG2A.2和抗PD-L1单抗S70的组合。每周给药2次，连续给药4周。给药后的第二天采集小鼠血液样品，采用HBsAg Elisa试剂盒(迈克生物)检测血液中HBsAg含量。
初步的结果显示，抗NKG2A-PDL1双特异性抗体NP-2-POC能够降低小鼠体内的HBsAg含量，且其活性优于抗NKG2A单抗NKG2A.2和抗PD-L1单抗S70的组合使用的活性(数据未显示)。实施例3：抗NKG2A-PDL1-preS1三特异性抗体的制备和表征
3.1.Bs4Ab-scFv结构的三特异性抗体的构建
序列设计：Bs4Ab-scFv三特异性抗体结构，是在Bs4Ab双特异性抗体结构(可参见文献Bezabeh B,et al.Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.MAbs.2017Feb/Mar；9(2):240-256)基础上，在其中一个Fc的C端连接一个scFv片段，构成能够特异性结合另外一个不同抗原的抗原结合模块，以此实现三特异性。对于本申请实施例中使用的Bs4Ab-scFv结构的三特异性抗体，在Fc区也设计了杵臼结构(knobs-in-holes，KIH)，同时引入形成稳定化二硫桥的两个Cys残基突变，以及引入组合突变LALAPS，另外在SBT451scFv的VH和VL界面引入两个半胱氨酸突变，具体详见实施例2.1部分。
此外，本实施例中额外构建了用于概念验证(Proof of Concepts,POC)的抗NKG2A-PDL1-preS1三特异性抗体，用于在小鼠体内的活性验证。
本申请实施例中所使用的Bs4Ab-scFv的三特异性抗体结构示意图如图1C所示。表24为实施例中所使用的Bs4Ab-scFv结构的三特异性抗体的组成，表7中示出了其全长重链和轻链的具体氨基酸序列。其中，三特异性抗体中的抗NKG2A抗原结合域来源于R76-9，R76-10或NKG2A.2(POC分子)，抗PD-L1抗原结合域来源于SBT451或S70(POC分子)，抗preS1抗原结合域来源于抗HBV preS1抗体K127-9(参见专利公开号WO2023/066171A，该专利申请的内容以其整体并入到本文之中)，其氨基酸序列如表4-1、4-2和4-3所示。
三特异性抗体的构建过程：对于各个三特异性抗体分子，按照操作说明书，通过无缝克隆的方式构建分别表达重链的重链表达载体(2个)，以及表达轻链的轻链表达载体(1个)，共计3个表达载体。
三特异性抗体的表达纯化：具体的操作步骤如实施例2.1中所述。
表24：Bs4Ab-scFv结构的抗NKG2A-PD-L1-preS1三特异性抗体的重链和轻链组成

3.2.抗NKG2A-PDL1-preS1三特异性抗体与NKG2A的结合活性检测
按照实施例2.4部分所述步骤检测抗NKG2A-PDL1-preS1三特异性抗体与huNKG2A-huCD94-musFc抗原的结合活性；按照实施例2.5部分所述步骤检测抗NKG2A-PDL1-preS1三特异性抗体与小鼠NKG2A-CD94抗原的结合活性。
结果如表25所示，抗NKG2A-PDL1-preS1三特异性抗体NPH-1能够有效结合人NKG2A抗原，且结合活性与NKG2A单抗R76-10相当。
上述三特异性抗体对应的POC分子NPH-1-POC也能有效地结合小鼠NKG2A抗原(数据未显示)。
表25：抗NKG2A-PDL1-preS1三特异性抗体与NKG2A抗原的结合活性
3.3.抗NKG2A-PDL1-preS1三特异性抗体与PDL1的结合活性检测
按照实施例2.6部分所述步骤检测抗NKG2A-PDL1-preS1三特异性抗体与人PD-L1抗原的结合活性；按照实施例2.7部分所述步骤检测抗NKG2A-PDL1-preS1三特异性抗体与小鼠PD-L1抗原的结合活性。
结果如表26所示，抗NKG2A-PDL1-preS1三特异性抗体NPH-1能够有效结合人PD-L1抗原，且结合活性与PD-L1单抗SBT451相当。
上述三特异性抗体对应的POC分子NPH-1-POC也能有效地结合小鼠PD-L1抗原(数据未显示)。
表26：抗NKG2A-PDL1-preS1三特异性抗体与PDL1抗原的结合活性
3.4.抗NKG2A-PDL1-preS1三特异性抗体与HBV preS1抗原的结合活性检测
采用ELISA结合试验检测抗NKG2A-PDL1-preS1三特异性抗体与HBV preS1抗原的结合活性，其中包被抗原为1μg/ml的HBV-preS1-His融合蛋白(参见专利公开号WO2023/066171A)，对照抗体为抗HBV preS1单抗K127-9，其他步骤按照实施例1.3中所述进行。
结果如表27所示，抗NKG2A-PDL1-preS1三特异性抗体NPH-1能够有效结合HBV preS1抗原，且结合活性与抗HBV preS1单抗K127-9相当。
上述三特异性抗体对应的POC分子NPH-1-POC也能有效地结合HBV preS1抗原(数据未显示)。
表27：抗NKG2A-PDL1-preS1三特异性抗体与preS1抗原的结合活性
3.5.抗NKG2A-PDL1-preS1三特异性抗体阻断NKL细胞NKG2A与HLA-E四聚体的结合试验(细胞水平)
采用FACS方法检测抗NKG2A-PDL1-preS1三特异性抗体对HLA-E四聚体与NKL细胞表面受体NKG2A结合的阻断活性，具体步骤按照实施例2.8部分所述进行。
结果如表28所示，抗NKG2A-PDL1-preS1三特异性抗体NPH-1能够有效地阻断NKL细胞NKG2A与HLA-E四聚体的结合，其活性与NKG2A单抗R76-10相当。
表28：抗NKG2A-PDL1-preS1三特异性抗体对NKG2A-HLAE结合的阻断活性
3.6.DELFIA EuTDA细胞毒实验检测抗NKG2A-PDL1-preS1三特异性抗体促进NKL细胞对K562-E4细胞的杀伤活性
采用DELFIA EuTDA细胞毒法检测抗NKG2A-PDL1-preS1三特异性抗体促进NKL细胞对K562-E4细胞的杀伤活性，具体步骤按照实施例2.9部分所述进行。
结果如表29所示，抗NKG2A-PDL1-preS1三特异性抗体NPH-1能够有效地促进NKL细胞对K562-E4靶细胞的杀伤。
表29：抗NKG2A-PDL1-preS1三特异性抗体促进NKL细胞对K562-E4细胞的杀伤活性

Claims (15)

  1. 一种多特异性抗体，包括特异性结合NKG2A的第一抗原结合域和特异性结合PD-L1的第二抗原结合域，其中：
    所述特异性结合NKG2A的第一抗原结合域包含：
    VH，所述VH包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:3；以及VL，所述VL包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:4，LC-CDR2，其包含氨基酸序列SEQ ID NO:5，和LC-CDR3，其包含氨基酸序列SEQ ID NO:6；
    以及，所述特异性结合PD-L1的第二抗原结合域包含：
    VH，所述VH包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:18，HC-CDR2，其包含氨基酸序列SEQ ID NO:19，和HC-CDR3，其包含氨基酸序列SEQ ID NO:20；以及VL，所述VL包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:22，和LC-CDR3，其包含氨基酸序列SEQ ID NO:23。
  2. 根据权利要求1中所述的多特异性抗体，其中：
    所述特异性结合NKG2A的第一抗原结合域包含：
    (a)VH，其包含氨基酸序列SEQ ID NO:13或其变体，所述变体与氨基酸序列SEQ ID NO:13具有至少80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少80％序列同一性；或
    (b)VH，其包含氨基酸序列SEQ ID NO:14或其变体，所述变体与氨基酸序列SEQ ID NO:14具有至少80％序列同一性；以及VL，其包含氨基酸序列SEQ ID NO:16或其变体，所述变体与氨基酸序列SEQ ID NO:16具有至少80％序列同一性；
    以及，所述特异性结合PD-L1的第二抗原结合域包含：
    VH，其包含SEQ ID NO:30和34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:30和34中任一所示的氨基酸序列具有至少80％序列同一性；以及VL，其包含SEQ ID NO:32和36中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NO:32和36中任一所示的氨基酸序列具有至少80％序列同一性。
  3. 根据权利要求1-2中任一项所述的多特异性抗体，其结构选自IgG-scFv、IgG-(scFv)2、Bs4Ab-scFv、DVD-Ig、Hetero H,CrossMab或scFv-Fab IgG。
  4. 根据权利要求1-3中任一项所述的多特异性抗体，其结构为IgG-scFv，其包含四条多肽链：
    其中，一条多肽链从N端到C端包含VH1-CH1-CH2-CH3-L-VH2-L3-VL2结构，其中VH1是特异性结合NKG2A的重链可变区；VH2是特异性结合PD-L1的重链可变区；VL2是特异性结合PD-L1的轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；
    两条多肽链从N端到C端包含VL1-CL，其中VL1是特异性结合NKG2A的轻链可变区，CL是轻链恒定区；以及
    一条多肽链从N端到C端包含VH1-CH1，其中VH1是特异性结合NKG2A的重链可变区；CH1是重链恒定区CH1结构域；优选地，该多肽链进一步包含Fc，该Fc包含CH2和CH3结构域。
  5. 根据权利要求4中所述的多特异性抗体，其包含：
    氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有80％序列同一性；和/或
    氨基酸序列SEQ ID NO:53或其变体，所述变体与氨基酸序列SEQ ID NO:53具有至少80％序列同一性；和/或
    氨基酸序列SEQ ID NO:54或其变体，所述变体与氨基酸序列SEQ ID NO:54具有至少80％序列同一性。
  6. 根据权利要求1-3中任一项所述的多特异性抗体，其结构为IgG-(scFv)2，其包含四条多肽链：
    其中，两条多肽链从N端到C端包含VH1-CH1-CH2-CH3-L-VH2-L3-VL2结构，其中VH1是特异性结合NKG2A的重链可变区；VH2是特异性结合PD-L1的重链可变区；VL2是特异性结合PD-L1的轻链可变区；L和L3是连接肽；CH1是重链恒定区CH1结构域；CH2是重链恒定区CH2结构域；CH3是重链恒定区CH3结构域；以及
    另外两条多肽链从N端到C端包含VL1-CL结构，其中VL1是特异性结合NKG2A的轻链可变区，CL是轻链恒定区。
  7. 根据权利要求6中所述的多特异性抗体，其包含：
    氨基酸序列SEQ ID NO:54或其变体，所述变体与氨基酸序列SEQ ID NO:54具有80％序列同一性；和/或
    氨基酸序列SEQ ID NO:58或其变体，所述变体与氨基酸序列SEQ ID NO:58具有至少80％序列同一性。
  8. 一种分离的编码权利要求1-7中任一项所述的多特异性抗体的核酸分子。
  9. 一种包含权利要求8中所述的核酸分子的载体。
  10. 一种分离的宿主细胞，其包含权利要求1-7中任一项所述的多特异性抗体、权利要求8中所述的核酸分子或权利要求9中所述的载体。
  11. 一种制备权利要求1-7中任一项所述的多特异性抗体的方法，其包含：
    a)在能有效表达抗体的条件下培养权利要求10中所述的宿主细胞；和
    b)从宿主细胞中获得表达的抗体。
  12. 一种药物组合物，包含权利要求1-7中任一项所述的多特异性抗体、权利要求8中所述的核酸分子、权利要求9中所述的载体、权利要求10中所述的分离的宿主细胞或由权利要求11中所述方法制备得到的多特异性抗体，以及药学上可接受的载体或辅料。
  13. 权利要求1-7中任一项所述的特异性结合NKG2A抗原和PD-L1抗原的多特异性抗体、权利要求8中所述的核酸分子、权利要求9中所述的载体、权利要求10中所述的宿主细胞、由权利要求11中所述方法制备得到的多特异性抗体、或权利要求12中所述的药物组合物在制备治疗有此需求的个体疾病或病症的药物中的用途。
  14. 根据权利要求13中所述的用途，其中所述的疾病或病症包含HBV感染或与HBV感染相关的疾病。
  15. 根据权利要求14中所述的用途，其中所述的疾病或病症包括乙型肝炎、肝功能衰竭、肝硬化或肝癌。
PCT/CN2025/082284 2024-03-14 2025-03-13 特异性结合nkg2a抗原和pd-l1抗原的多特异性抗体及其应用 Pending WO2025190332A1 (zh)

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