WO2025190332A1 - Anticorps multi-spécifique se liant de manière spécifique à l'antigène nkg2a et à l'antigène pd-l1, et utilisation d'un anticorps multi-spécifique - Google Patents

Anticorps multi-spécifique se liant de manière spécifique à l'antigène nkg2a et à l'antigène pd-l1, et utilisation d'un anticorps multi-spécifique

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
nkg2a
amino acid
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/082284
Other languages
English (en)
Chinese (zh)
Inventor
韩荣荣
李忠
唐巧朋
张思
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Solobio Genetechnology Co Ltd
Original Assignee
Beijing Solobio Genetechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Solobio Genetechnology Co Ltd filed Critical Beijing Solobio Genetechnology Co Ltd
Publication of WO2025190332A1 publication Critical patent/WO2025190332A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un anticorps multi-spécifique ou un fragment de liaison à l'antigène qui se lie spécifiquement à NKG2A et PD-L1, et un procédé de préparation de l'anticorps multi-spécifique et son utilisation.
PCT/CN2025/082284 2024-03-14 2025-03-13 Anticorps multi-spécifique se liant de manière spécifique à l'antigène nkg2a et à l'antigène pd-l1, et utilisation d'un anticorps multi-spécifique Pending WO2025190332A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2024/081645 2024-03-14
CN2024081645 2024-03-14

Publications (1)

Publication Number Publication Date
WO2025190332A1 true WO2025190332A1 (fr) 2025-09-18

Family

ID=97062862

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2025/082284 Pending WO2025190332A1 (fr) 2024-03-14 2025-03-13 Anticorps multi-spécifique se liant de manière spécifique à l'antigène nkg2a et à l'antigène pd-l1, et utilisation d'un anticorps multi-spécifique

Country Status (1)

Country Link
WO (1) WO2025190332A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583127A (zh) * 2020-04-30 2021-11-02 迈威(上海)生物科技股份有限公司 一种靶向nkg2a和pd-l1的双特异性抗体及应用
CN116368154A (zh) * 2020-10-08 2023-06-30 阿菲姆德股份有限公司 三特异性结合剂
CN117157107A (zh) * 2021-02-08 2023-12-01 西纳福克斯股份有限公司 多功能抗体
WO2024040212A2 (fr) * 2022-08-19 2024-02-22 Staidson Biopharma Inc. Anticorps reconnaissant spécifiquement le ligand 1 de mort cellulaire programmée 1 et utilisations associées
WO2024056010A1 (fr) * 2022-09-16 2024-03-21 北京三诺佳邑生物技术有限责任公司 Anticorps reconnaissant spécifiquement nkg2a et utilisation associée

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583127A (zh) * 2020-04-30 2021-11-02 迈威(上海)生物科技股份有限公司 一种靶向nkg2a和pd-l1的双特异性抗体及应用
CN116368154A (zh) * 2020-10-08 2023-06-30 阿菲姆德股份有限公司 三特异性结合剂
CN117157107A (zh) * 2021-02-08 2023-12-01 西纳福克斯股份有限公司 多功能抗体
WO2024040212A2 (fr) * 2022-08-19 2024-02-22 Staidson Biopharma Inc. Anticorps reconnaissant spécifiquement le ligand 1 de mort cellulaire programmée 1 et utilisations associées
WO2024056010A1 (fr) * 2022-09-16 2024-03-21 北京三诺佳邑生物技术有限责任公司 Anticorps reconnaissant spécifiquement nkg2a et utilisation associée

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAN SEN, ZENG HAN, JIN KAIFENG, SHAO FEI, LIU ZHAOPEI, CHANG YUAN, WANG YIWEI, ZHU YU, WANG ZEWEI, XU LE, XU JIEJIE: "NKG2A and PD-L1 expression panel predicts clinical benefits from adjuvant chemotherapy and PD-L1 blockade in muscle-invasive bladder cancer", JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 10, no. 5, 4 April 2022 (2022-04-04), GB , pages 1 - 9, XP093354796, ISSN: 2051-1426, DOI: 10.1136/jitc-2022-004569 *

Similar Documents

Publication Publication Date Title
WO2024056010A1 (fr) Anticorps reconnaissant spécifiquement nkg2a et utilisation associée
JP2023011887A (ja) 顆粒球－マクロファージコロニー刺激因子受容体αを特異的に認識する抗体及びその使用
US20260015423A1 (en) Antibodies specifically recognizing programmed cell death1 ligand 1 and uses thereof
CN118812719A (zh) 特异性结合HBV pre-S1抗原和CD3抗原的多特异性抗体及其应用
JP7755725B2 (ja) Ｆｃｒｎを特異的に認識する抗体およびその使用
TW202321291A (zh) 特異性結合乙型肝炎病毒表面抗原ｐｒｅ—Ｓ１的抗體及其應用
WO2025190332A1 (fr) Anticorps multi-spécifique se liant de manière spécifique à l'antigène nkg2a et à l'antigène pd-l1, et utilisation d'un anticorps multi-spécifique
CN118103397A (zh) 特异性识别tnfr2的抗体及其用途
WO2025167631A1 (fr) Anticorps multi-spécifique se liant de manière spécifique à l'antigène pré-s1 du vhb, à l'antigène pd-l1 et à l'antigène cd3 et son utilisation
WO2025002221A1 (fr) Anticorps multi-spécifiques comprenant des domaines de liaison à l'antigène reconnaissant de manière spécifique c5ar1 et gm-csfr alpha
EP4606886A1 (fr) Combinaison d'anticorps se liant spécifiquement à trail ou fasl, et anticorps bispécifique
US20260035464A1 (en) Antibodies specifically recognizing b- and t-lymphocyte attenuator (btla) and uses thereof
US20250326828A1 (en) Antibodies Specifically Recognizing Gdf15 and Uses Thereof
WO2025195443A1 (fr) Anticorps multi-spécifique se liant de manière spécifique à light et à c5a, composition et utilisation associées
CN120699159A (zh) 特异性结合LIGHT和GM-CSFRα的多特异性抗体以及组合物及其应用
WO2025195445A1 (fr) Anticorps multi-spécifique se liant de manière spécifique à light et tslp, composition et utilisation associées
CN120682370A (zh) 特异性结合LIGHT和IL-4Rα的多特异性抗体以及组合物及其应用
WO2024251199A1 (fr) Anticorps se liant de manière spécifique à masp3, et anticorps multispécifique se liant de manière spécifique à masp3 et masp2
WO2025040163A1 (fr) Anticorps isolé et son utilisation
WO2023030501A1 (fr) Anticorps capable de reconnaître de manière spécifique fcrn et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25769712

Country of ref document: EP

Kind code of ref document: A1