WO2020249041A1 - 靶向lag-3的抗体和双特异性抗体及其用途 - Google Patents

靶向lag-3的抗体和双特异性抗体及其用途 Download PDF

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WO2020249041A1
WO2020249041A1 PCT/CN2020/095577 CN2020095577W WO2020249041A1 WO 2020249041 A1 WO2020249041 A1 WO 2020249041A1 CN 2020095577 W CN2020095577 W CN 2020095577W WO 2020249041 A1 WO2020249041 A1 WO 2020249041A1
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amino acid
seq
acid sequence
heavy chain
chain variable
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刘佳建
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上海健信生物医药科技有限公司
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of biomedicine, in particular to an antibody and bispecific antibody targeting LAG-3 and uses thereof.
  • Lymphocyte-activation gene 3 (LAG-3, lymphocyte activation gene-3) belongs to the immunoglobulin superfamily and consists of three parts: extracellular domain, transmembrane domain and cytoplasmic domain.
  • the LAG-3 gene is located on chromosome 12 (12P13), which is similar to the location and structure of the CD4 molecule on the chromosome.
  • LAG-3 is expressed on activated T cells, depleted T cells, tumor infiltrating T cells, and regulatory T cells (Treg). After binding to major histocompatibility complex 2 (MHC class II), the LAG-3/MHC class II interaction leads to the negative regulation of T cell proliferation, activation and homeostasis.
  • MHC class II major histocompatibility complex 2
  • Inhibiting LAG-3 can relieve LAG3's inhibition of MHCII-polypeptide-T cell receptor antigen presentation, and allow T cells to regain cytotoxicity, thereby enhancing the tumor killing effect. At the same time, inhibiting LAG-3 can also reduce the function of regulating T cells to suppress immune response. Therefore, LAG-3 is considered to be a more attractive target than other immune checkpoint proteins.
  • LAG-3 is currently the second-generation target of immune checkpoints, with more clinical data and relatively definite druggability. Antibody drugs against this target may become important anti-tumor drugs in the future. However, there are currently no drugs on the market that target LAG-3. As of the end of 2018, there are a total of 30 drugs under clinical research.
  • the LAG-3 antibodies in the clinical research stage include GSK2831781 from GlaxoSmithKline, LAG525 from Novartis, REGN3767 from Regeneron and TSR-033 from Tesaro. LAG-3 antibody drugs are mostly devoted to the development of combination therapy with PD-1.
  • LAG targeting is provided.
  • the bispecific antibody of the present invention is a sequence-based IgG like bispecific antibody (SBody) with a similar structure. This patent will call this design SBody.
  • the present invention provides an antibody targeting LAG-3, which has new light chain variable regions CDR1, CDR2 and CDR3 and heavy chain variable regions CDR1, CDR2 and CDR3, and its thermal stability Good, T cell activity is better, it can more effectively block the binding of LAG-3 and MHC II, and better in vivo efficacy and PK characteristics. It also provides a bispecific antibody targeting LAG-3, which can target LAG-3 and another target at the same time; the new design can achieve the effect of one molecule on two specific targets at the same time, achieving one molecule instead of two The combination of individual molecules even has the effect of synergistic treatment of tumors. The process, production cost, and clinical trials of one molecule are more convenient and cheaper than the combination of two separate molecules.
  • a LAG-3 binding protein which includes a light chain variable region and/or a heavy chain variable region;
  • the light chain variable region includes: such as SEQ CDR1 of the amino acid sequence shown in ID NO: 5, CDR2 of the amino acid sequence shown in SEQ ID NO: 6, and/or CDR3 of the amino acid sequence shown in SEQ ID NO: 7;
  • the heavy chain is variable
  • the region includes: CDR1 of the amino acid sequence shown in SEQ ID NO: 8, CDR2 of the amino acid sequence shown in SEQ ID NO: 9, and/or CDR3 of the amino acid sequence shown in SEQ ID NO: 10.
  • the J gene region of the light chain variable region is selected from one of the following groups: hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, hJK3, hJK4 .1, hJK4.2, hJK5; preferably hJK4.1;
  • the following J gene region of the heavy chain variable region is selected from one of the following groups: hJh1, hJh2, hJh3.1, hJh3.2, hJh4.1, hJh4.2, hJh4.3, hJh5. 2. hJh6.1, hJh6.2, hJh6.3; preferably hJh4.1.
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 23-29 or a mutation thereof;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 30-37 or a mutation thereof;
  • the mutation has one or more amino acid residues in the original amino acid sequence Substitution, deletion or addition, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the original amino acid sequence, and the mutation maintains or improves The binding of the antibody to LAG-3.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region It comprises the amino acid sequence shown in SEQ ID NO: 23, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23
  • the amino acid sequence, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 31;
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 24, the heavy chain variable region Contains the amino acid sequence shown in SEQ ID NO: 31;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31 Amino acid sequence;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31;
  • the light chain variable region
  • the LAG-3 binding protein as described above is an antibody, Fab, Fab', F(ab') 2 , Fv, scFv, bispecific antibody, multispecific antibody, single Domain antibodies or single domain antibodies, or monoclonal antibodies or polyclonal antibodies prepared from the above antibodies.
  • the LAG-3 binding protein as described above is an immunoglobulin comprising a human antibody light chain constant region and a human antibody heavy chain constant region.
  • the human antibody light chain constant region is a kappa chain or a lambda chain, or the human antibody heavy chain constant region is hlgG1, hlgG2, hlgG4 or mutations thereof; the mutation has one or more mutations in the original amino acid sequence.
  • the substitution, deletion or addition of multiple amino acid residues preferably has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the original amino acid sequence, and said The mutation maintains or improves the binding of the antibody to LAG-3.
  • the light chain of the LAG-3 binding protein as described above comprises the amino acid sequence shown in SEQ ID NO: 38 or SEQ ID NO: 39 or a mutation thereof; and/or, its light chain
  • the chain contains the amino acid sequence shown in SEQ ID NO: 40 or SEQ ID NO: 41 or its mutation; the mutation has one or more amino acid residue substitutions, deletions or additions in the original amino acid sequence, preferably with the original amino acid sequence It has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity, and the mutation maintains or improves the binding of the antibody to LAG-3.
  • the amino acid sequence of the light chain is shown in SEQ ID NO: 38; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 40; or, the amino acid sequence of the light chain is shown in SEQ ID NO: 38
  • the amino acid sequence of the heavy chain is shown in SEQ ID NO: 41; or, the amino acid sequence of the light chain is shown in SEQ ID NO: 39; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 40
  • the amino acid sequence of the light chain is shown in SEQ ID NO: 39; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 41.
  • the second technical solution of the present invention is: a bispecific antibody targeting LAG-3, which includes a first protein functional region and a second protein functional region, characterized in that the first The protein functional region is the LAG-3 binding protein described in one of the technical solutions; the second protein functional region is a non-LAG-3 binding protein; preferably, the first protein functional region and the second protein functional region are selected separately From immunoglobulin, scFv (single chain Fv, also known as single chain variable fragment), Fab, Fab' or F(ab') 2 , and there is only one of the first protein functional region and the second protein functional region
  • the protein functional area is immunoglobulin.
  • the bispecific antibody of the present invention has a structure similar to that of normal IgG.
  • the structure of the bispecific antibody is designed to be capable of targeting two targets. / Or the protein functional region of the heavy chain variable region, the two protein functional regions share the same Fc region.
  • the antibody molecule of one target is linked to one end of the light chain or the heavy chain of a complete antibody of another target in the form of one or more scFv. In this way, it is avoided that the expression of different Fc and/or different light chains brings about inhomogeneous expression products.
  • the co-expression of Knob form of Fc and Hole form of Fc will result in heterogeneous Fc-Fc pairing during the expression process, which will cause purification
  • the process brings a lot of inconvenience; it can also avoid the influence of the cross design of the light chain and the heavy chain on the binding activity and the Fc mismatch phenomenon that occurs during the process.
  • the design of one or more scFvs the activity against specific targets can also be adjusted.
  • the bispecific antibody targeting LAG-3 as described above wherein the first protein functional domain is an immunoglobulin, and the second protein functional domain is one or Multiple scFv; or, the first protein functional region is one or more scFv, the second protein functional region is an immunoglobulin, and the constant region of the immunoglobulin includes a human antibody light chain constant region and a human Source antibody heavy chain constant region.
  • the constant region of the human antibody light chain is a kappa chain or a lambda chain
  • the constant region of the human antibody heavy chain is hIgG1, hIgG2, hIgG4 or a mutation thereof; the mutation has one or more amino acid residues in the original amino acid sequence.
  • substitution, deletion or addition of the group preferably has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the original amino acid sequence, and the mutation maintains or improves The binding of the antibody to antigens such as LAG-3, PD-1, etc.
  • the bispecific antibody targeting LAG-3 as described above wherein the scFv includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region is The light chain variable region is connected through a linker, and the scFv is connected to the immunoglobulin through a linker, and the linker is preferably (Gly-Gly-Gly-Gly-Ser) w [hereinafter abbreviated as (G 4 S ) w ]; Said w is preferably an integer between 0-10, more preferably 1, 2, 3 or 4.
  • the linker can also be selected from peptides conventionally used as linkers in the art.
  • the bispecific antibody targeting LAG-3 as described above, wherein the scFv has a light chain variable region-linker-heavy chain variable region structure, and its light chain can be The N-terminus of the variable region or the C-terminus of the heavy chain variable region is connected to the C-terminus or N-terminus of the immunoglobulin light chain and/or heavy chain through a linker; or the scFv is a heavy chain variable region- Linker-light chain variable region structure, the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region is connected to the C-terminus of the immunoglobulin light chain and/or heavy chain or N-terminal.
  • the scFv has a light chain variable region-linker-heavy chain variable region structure, and the C-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the immune system through (G 4 S) 3 respectively.
  • the N-terminals of the two heavy chain variable regions of the globulin, or the N-terminals of the light chain variable regions of the two scFvs are respectively symmetrically connected to the two heavy chains of the immunoglobulin through (G 4 S) 3 C-terminus; or, the scFv has a heavy chain variable region-linker-light chain variable region structure, and the C-termini of the light chain variable regions of two scFvs are symmetrically connected to each other through (G 4 S) 3
  • the N-terminals of the two light chain variable regions of the immunoglobulin, or the N-terminals of the heavy chain variable regions of the two scFvs are symmetrically connected to the two light-chain variable regions of the immunoglobulin respectively through (G 4 S) 3
  • the C-terminus of the chain variable region are respectively symmetrically connected to the two light-chain variable regions of the immunoglobulin respectively through (G 4 S) 3 The C-terminus of the chain variable region.
  • the second protein functional region is an anti-PD-1 antibody. More preferably, the anti-PD-1 antibody is Nivolumab or Pembrolizumab.
  • the bispecific antibody targeting LAG-3 as described above is selected from the following group:
  • the first protein functional region is an immunoglobulin, which includes the following light chain and heavy chain or mutations thereof: wherein the amino acid sequence of the light chain is shown in SEQ ID NO: 38, and the heavy chain
  • the amino acid sequence is shown in SEQ ID NO: 40; the amino acid sequence of the light chain is shown in SEQ ID NO: 38, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 41; the amino acid sequence of the light chain As shown in SEQ ID NO: 39, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 40; or, the amino acid sequence of the light chain is shown in SEQ ID NO: 39, and the amino acid sequence of the heavy chain As shown in SEQ ID NO: 41; and/or,
  • the second protein functional region is scFv or a mutation thereof, wherein the amino acid sequence of the light chain variable region of the scFv is as shown in the amino acid sequence 1-107 of SEQ ID NO: 42, and the heavy chain of the scFv
  • the amino acid sequence of the variable region is shown in the amino acid sequence 1-113 of SEQ ID NO: 43; or, the amino acid sequence of the light chain variable region of the scFv is shown in the amino acid sequence 1-111 of SEQ ID NO: 44
  • the amino acid sequence of the heavy chain variable region of the scFv is shown in the amino acid sequence 1-120 of SEQ ID NO: 45;
  • the mutation has one or more amino acid residue substitutions, deletions or additions in the original amino acid sequence, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and the original amino acid sequence. 99% sequence identity, and the mutation maintains or improves the binding of the antibody to antigens such as LAG-3, PD-1, etc.;
  • the C-terminus of the heavy chain is mutated from K to A;
  • the first protein functional region is scFv or a mutation thereof, wherein the amino acid sequence of the light chain variable region of the scFv is shown in SEQ ID NO: 23-29; and/or, the weight of the scFv The amino acid sequence of the chain variable region is shown in SEQ ID NO: 30-37; and/or,
  • amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO: 42, and the amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 43; or, the light chain of the immunoglobulin
  • amino acid sequence of is shown in SEQ ID NO: 44, and the amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 45;
  • the mutation has one or more amino acid residue substitutions, deletions or additions in the original amino acid sequence, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and the original amino acid sequence. 99% sequence identity, and the mutation maintains or improves the binding of the antibody to antigens such as LAG-3, PD-1, etc.;
  • the C-terminus of the heavy chain is mutated from K to A.
  • the bispecific antibody targeting LAG-3 as described above, wherein (i) the first protein functional region is scFv, and the amino acid sequence of the light chain variable region is as SEQ ID NO: 28, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, the linker is (G 4 S) 3 ; the second protein functional region is an immunoglobulin; The amino acid sequence of the light chain is shown in SEQ ID NO: 42, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 43;
  • the number of the scFv is two; the scFv is a light chain variable region-linker-heavy chain variable region structure, and the C-terminals of the heavy chain variable regions of the two scFvs respectively pass (G 4 S) 3 symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, two scFv heavy chain variable regions
  • the N-terminus of the immunoglobulin is symmetrically connected to the C-terminus of the two heavy chains of the immunoglobulin through (G 4 S) 3 , and the C-terminus of the heavy chain is mutated from K to A; or, the scFv is light Chain variable region-linker-heavy chain variable region structure, the C-terminals of the heavy chain variable regions of two scFvs are symmetrically connected to the two light chains of the immunoglobulin through (G 4 S) 3 respectively N-
  • the first protein functional region is scFv, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, so
  • the linker is (G 4 S) 3 ;
  • the second protein functional region is an immunoglobulin, the amino acid sequence of its light chain is shown in SEQ ID NO: 44, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 45 Shown
  • the number of the scFv is two; the scFv is a light chain variable region-linker-heavy chain variable region structure, and the C-terminus of the heavy chain variable regions of the two scFvs respectively pass through a linker (G 4 S) 3 is symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the heavy chains of the two scFv can be The N-terminus of the variable region is symmetrically connected to the C-terminus of the two heavy chains of the immunoglobulin through a linker (G 4 S) 3 , and the C-terminus of the heavy chain is mutated from K to A; or, The scFv is a light chain variable region-linker-heavy chain variable region structure, and the C-termini of the heavy chain variable regions of the two scFvs are respectively symmetrically connected to the immunoglob
  • the first protein functional region is scFv, the number of scFv is a pair, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, the linker is (G 4 S) 3 ;
  • the second protein functional region is an immunoglobulin, and the amino acid sequence of the light chain is shown in SEQ ID NO: 44, and the heavy chain The amino acid sequence is shown in SEQ ID NO: 45;
  • the number of the scFv is two; the scFv is a light chain variable region-linker-heavy chain variable region structure, and the C-terminus of the heavy chain variable regions of the two scFvs respectively pass through a linker (G 4 S) 3 is symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the heavy chains of the two scFv can be The N-terminus of the variable region is symmetrically connected to the C-terminus of the two heavy chains of the immunoglobulin through a linker (G 4 S) 3 , and the C-terminus of the heavy chain is mutated from K to A.
  • the bispecific antibody targeting LAG-3 as described above includes the following amino acid sequence:
  • the light chain amino acid sequence shown in SEQ ID NO: 44 such as the heavy chain-containing amino acid sequence shown in SEQ ID NO: 46; or, the light chain amino acid sequence shown in SEQ ID NO: 42, such as SEQ ID NO
  • the light chain-containing amino acid sequence shown in: 49 such as the heavy chain amino acid sequence shown in SEQ ID NO: 43; or, the light chain-containing amino acid sequence shown in SEQ ID NO: 50, such as SEQ ID NO: 43
  • the bispecific antibody of the present invention is a DVD-Ig (Dual-variable domain Ig) bispecific antibody, and its structure is that the VL and VL of another antibody are respectively connected to the N-terminus of the light and heavy chains of a normal antibody.
  • VH achieves dual functions by combining two antibody variable regions with dual targets. Please refer to Table 12 of Example 12 for a summary of the design of the bispecific antibody (General Formula 2).
  • the bispecific antibody is composed of a light chain-containing sequence and a heavy chain-containing sequence.
  • the bispecific antibody is selected from the following combinations: the light chain-containing sequence is Ab2317VL-(G 4 S) 3 -NivoVL-Lc ( ⁇ chain), and the heavy chain-containing sequence is Ab2317VH-(G 4 S) 3- NivoVH-Hc (hIgG4); or, the sequence containing the light chain is PemVL-(G 4 S) 3 -Ab2317VL-Lc ( ⁇ chain), and the sequence containing the heavy chain is PemVH-(G 4 S) 3 -Ab2317VH-Hc (hIgG4); Or, the sequence containing the light chain is NivoVL-(G 4 S) 3 -Ab2325VL-Lc ( ⁇ chain), and the sequence containing the heavy chain is NivoVH-(G 4 S) 3 -Ab2325VH-Hc(hIgG1) .
  • the bispecific antibody of the present invention includes a first protein functional region and a second protein functional region, one of which is an immunoglobulin, and the other protein is Fab' or F(ab' ) 2 .
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is Fab' or F(ab') 2 ; or the first protein functional region is Fab' or F(ab') 2 , the second protein functional region is an immunoglobulin; the Fab' or F(ab') 2 is connected to the immunoglobulin by a disulfide bond or a linker, the The linker is preferably a peptide fragment or (Gly-Gly-Gly-Gly-Ser) w which can be used as a linker conventional in the art.
  • the w is preferably an integer between 0-10, more preferably 1, 2, 3 or 4;
  • the constant region of the immunoglobulin is preferably a human antibody constant region, and the human antibody constant region preferably includes a human antibody light chain constant region and a human antibody heavy chain constant region, and the human antibody light chain constant region is preferably ⁇ Chain or lambda chain; the human antibody heavy chain constant region is preferably hIgG1, hIgG2 or hIgG4.
  • the third technical solution of the present invention is: an isolated nucleic acid encoding the above-mentioned LAG-3 binding protein or the above-mentioned LAG-3 bispecific antibody.
  • the fourth technical solution of the present invention is: an expression vector containing the isolated nucleic acid as described above.
  • the fifth technical solution of the present invention is: a host cell comprising the above-mentioned expression vector.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the sixth technical solution of the present invention is: a method for preparing LAG-3 binding protein or a bispecific antibody targeting LAG-3, which comprises culturing the host cell as described above, from the culture Obtain LAG-3 binding protein or bispecific antibody targeting LAG-3.
  • the seventh technical solution of the present invention is: an antibody drug conjugate comprising a cytotoxic agent, and the LAG-3 binding protein described in one of the above technical solutions or the second technical solution described above The described bispecific antibody targeting LAG-3.
  • the eighth of the technical solutions of the present invention is: a pharmaceutical composition comprising the LAG-3 binding protein as described above, or the bispecific antibody targeting LAG-3 as described above, or Antibody drug conjugate as described above.
  • the ninth technical solution of the present invention is: a kit combination comprising a kit A and a kit B; the kit A includes the LAG-3 binding protein as described above, or as described above The bispecific antibody targeting LAG-3, or the antibody-drug conjugate as described above, or the pharmaceutical composition as described above; the kit B contains other drugs for treating cancer.
  • ten of the technical solutions of the present invention are: the above-mentioned LAG-3 binding protein, LAG-3 targeting bispecific antibody, antibody drug conjugate, pharmaceutical composition and/or kit The application of the combination in the preparation of a medicine for treating and/or preventing cancer.
  • the cancer is selected from leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, urothelial cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell Squamous cell carcinoma, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, cholangiocarcinoma, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck squamous cell carcinoma, testicular cancer, endocrine adenocarcinoma , Adrenal gland cancer, pituitary gland cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, uterine cancer, glioblastoma, marrow The group consisting of blastoma, astromonide,
  • the present invention also provides a method for the treatment of cancer, which uses the above-mentioned LAG-3 binding protein, LAG-3 targeting bispecific antibody, antibody drug conjugate, and pharmaceutical composition And/or kits to treat patients suffering from the above-mentioned cancers.
  • first and second of the present invention have no actual meaning, and only distinguish the same terms.
  • a pair” and “two”, “two pairs” and “four” have the same meaning.
  • referring to the number of light chains or heavy chains or light chain variable regions or heavy chain variable regions “one” and “one”, “two” and “two” have the same meaning.
  • EC 50 refers to the concentration for 50% of maximal effect (concentration for 50% of maximal effect), which refers to the concentration that can cause 50% of the maximal effect.
  • the term "antibody” generally refers to an immunoglobulin consisting of two pairs of polypeptide chains [each pair has a light (L) chain and a heavy (H) chain].
  • a heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody
  • a light chain refers to a polypeptide chain with a smaller molecular weight in an antibody.
  • Light chains can be classified into kappa and lambda light chains.
  • Heavy chains can generally be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can also be subdivided into hyperdenaturation regions [called complementarity determining regions (CDR)], interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxy terminal.
  • the variable regions (VH and VL) corresponding to each heavy chain/light chain respectively form an antibody binding site.
  • the assignment of amino acids to each region or domain follows Kabat EA.Et al., Sequences of Proteins of Immunological Interest [National Institutes of Health, Bethesda, Md. (1987 and 1991)], or Chothia&Lesk 1987)].
  • the heavy chain may also include more than 3 CDRs, for example 6, 9 or 12 CDRs.
  • the heavy chain may be the N-terminus of the heavy chain of an IgG antibody connected to the ScFv of another antibody. In this case, the heavy chain contains 9 CDRs.
  • Antibody when referring to the term “antibody”, it includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • Antigen-binding fragment refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also This is called the "antigen binding portion”. See generally Fundamental Immunology, Ch. 7, Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes.
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of whole antibodies.
  • antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining region (CDR) fragments, single-chain binding fragments (e.g., scFv), chimeric antibodies, double An antibody (diabody) and such a polypeptide comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Fv means an antibody fragment consisting of the VL and VH domains of one arm of an antibody
  • Fab means an antibody fragment consisting of VL, VH, CL and CH1 (or CH) domains
  • F( ab') 2 means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region.
  • the antigen-binding fragment of the antibody is a single-chain binding fragment (e.g., scFv), in which the VL and VH domains pair to form a monovalent molecule by allowing them to be produced as a linker of a single polypeptide chain [see, for example, Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)].
  • scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated G 4 S amino acid sequences or variants thereof. For example, linkers having the amino acid sequence (G 4 S) 4 or (G 4 S) 3 can be used, but variants thereof can also be used.
  • isolated refers to artificially obtained from the natural state. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli, fungal cells such as yeast cells, such as S2 fruit fly cells or Sf9 Other insect cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli
  • fungal cells such as yeast cells
  • yeast cells such as S2 fruit fly cells or Sf9 Other insect cells
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • KD refers to the dissociation equilibrium constant (KD) of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • KD dissociation equilibrium constant
  • the antibody is less than about 10 -5 M, such as less than about 10 - 6 M, 10 -7 M , 10 -8 M, or 10 -10 M or less, a dissociation equilibrium constant antigen binding 10 -9 M, e.g. , As measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the reagents and raw materials used in the present invention are all commercially available.
  • LAG-3 binding protein more effectively blocks the binding of LAG-3 and MHC II, thereby removing the negative regulation of signal transduction and antigen presentation between APC/T cells;
  • the obtained humanized LAG-3 antibody shows good cell function activity, such as good activation of human T cell and human DC cell function and activity, animal drug efficacy, better PK characteristics, high expression yield, and Thermal stability
  • Sequence-specific bispecific antibodies have good activity against dual targets, are stable, have high expression levels, have structures similar to normal IgG antibodies, and have simple purification processes.
  • Figure 1 shows the function and activity of the anti-human LAG-3 antibody mab23c of the present invention: a. binding activity of mab23c and human LAG-3 (ELISA); b. mab23c blocking LAG-3 and Daudi cell blocking activity.
  • Figure 2 is a gel electrophoresis (PAGE) chart used to evaluate the thermal stability of the anti-human LAG-3 humanized antibody Ab2317 of the present invention.
  • Figure 3 shows the function and activity of the anti-human LAG-3 humanized antibody Ab2317 of the present invention: a. Ab2317 activates human T cell activity; b. Ab2317 activates human DC cells to stimulate human T cell activity (MLR assay).
  • Figure 4 is the evaluation of the efficacy of the anti-human LAG-3 humanized antibody Ab2317 in animals in vivo.
  • Figure 5A is a diagram of the structure of the bispecific antibody (SBody) LB2373 of the present invention and related detection data, including the schematic diagram (c), the detection result of the binding activity of human LAG-3 (a), and the detection of the binding activity of human PD-1 Result (b).
  • Figure 5B is a diagram of the structure of the bispecific antibody (SBody) LB2374 of the present invention and related detection data, including schematic diagram (c), and the detection result of binding activity of human LAG-3 (a), and detection of binding activity of human PD-1 Result (b).
  • Figure 5C is a diagram of the structure of the bispecific antibody (SBody) LB2371 of the present invention and related detection data, including the schematic diagram (c), and the detection result of the binding activity of human LAG-3 (a), and the detection of the binding activity of human PD-1 Result (b).
  • Figure 5D is a diagram of the structure of the bispecific antibody (SBody) LB2372 of the present invention and related detection data, including the schematic diagram (c), and the detection result of the binding activity of human LAG-3 (a), and the detection of the binding activity of human PD-1 Result (b).
  • the present invention is further described below in conjunction with examples, but these examples do not limit the scope of the present invention.
  • the experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • Reagents without specific sources are conventional reagents purchased on the market.
  • the antigen used in the present invention may be purchased from the following different companies: Beijing Biopsy Biotechnology Co., Ltd. LAG-3-his (article number: LA3-H5222), cyno-LAG-3-mFc (article number LA3-C52A0) or Beijing Yi Alice Shenzhou Technology Co., Ltd. LAG-3-his (article number: 16498-H08H), LAG-3-hFc (article number: 16498-H05H), cyno-LAG-3-his (article number 90841-C08H), or expressed and purified by the present invention get.
  • the expressed human LAG-3 protein sequence is NCBI Reference Sequence: NP_002277.4, full length 1-525 amino acids, of which 1-22 is the signal peptide; extracellular (ECD) region is 23-422 amino acids; extracellular (ECD) ) The first and second regions (domain#1and#2, D12) D12-his, D12-hFc of amino acids 23-239 in the region.
  • the sequence of mouse LAG-3his tagged (mLAG-3-his) comes from the 23-406 amino acids of the extracellular region (ECD) of NCBI gi
  • the sequence NCBI number of Macaca LAG-3his tagged (cynoLAG-3-his) is NP_001271679.1, and its extracellular domain (ECD) 23-434 amino acids.
  • the human PD-1 (hFc/histag) protein sequence is NCBI Reference Sequence: NP_005009.2, with a total length of 288 amino acids, of which positions 1-20 are signal peptides; ECD is amino acids 21-167.
  • the human PD-L1 (hFc/histag) protein sequence is NCBI Reference Sequence: NP_054862.1, with a total length of 290 amino acids, of which positions 1-18 are signal peptides; ECD is amino acids 19-239.
  • the antibodies used in the present invention include positive control antibody Ref (ie BMS-986016, sequence from WO2014008218A1, LAG3.5, #12 light chain, #14 heavy chain), and PD-1 antibody nivolumab (sequence from WO2013019906), Pembrolizumab (sequence Obtained from www.drugbank.ca, Accession Number: DB09037) are all expressed and purified by the present invention.
  • the pTT5 vector (Biovector, Cat#: 102762) used for expression.
  • the expressed recombinant protein, antibody light and heavy chain sequences were cloned into pTT5 vector, and transiently transfected into HEK293E cells (Life Technologies Cat. No. 11625019) for expression, and then purified.
  • 293 cells were expanded in Gibco FreeStyle 293 Expression Medium (Gibco, Cat#12338018) medium. Before the transient start, adjust the cell concentration to 6 ⁇ 8 ⁇ 10 5 cell/ml, 1% FBS (Aus Gene X FBS Excellent supplier: AusGeneX, China, Cat#FBSSA500-S), 37°C 8% CO 2 shaker After culturing for 24 hours, the survival rate was >95% by microscopic examination again, and the cell concentration was 1.2 ⁇ 10 6 cells/ml.
  • Gibco FreeStyle 293 Expression Medium Gibco, Cat#12338018
  • Purification of antibody or -Fc fusion protein Centrifuge the sample at high speed to remove impurities, and equilibrate the gravity column containing Protein A (Mabselect, GE Healthcare Life Science, Cat#71-5020-91AE) with PBS pH 7.4 ( ⁇ , Cat#F506606 -0001), wash 2-5 times the column volume. Pass the sample through the column. Wash the column with 5-10 column volumes of PBS (Biotech, Cat#B548117-0500). Then eluate the target protein with pH 3.5 0.1M acetic acid, then adjust to neutral with pH 8.0 Tris-HCl, measure the concentration with a microplate reader, aliquot and store for later use.
  • Protein A Manton, GE Healthcare Life Science, Cat#71-5020-91AE
  • PBS pH 7.4 ⁇ , Cat#F506606 -0001
  • Buffer replacement Centrifuge the eluted target protein through an ultrafiltration tube at 12000g for 10 minutes (Ultrafiltration tube Merck Millipore Cat#UFC500308), and add 1ml PBS to determine the concentration, aliquot and store for later use.
  • the hFc tag used in the present invention is all connected to the IgG1Fc region at the C-terminus, and the his tag is connected to 6 ⁇ his at the C-terminus.
  • Example 2 Construction of human LAG-3 highly expressing cell line (hLAG-3+ cells) and detection of binding activity (ELISA)
  • the human LAG-3 high expression cell line used in the present invention is completed through the company's stable cell line construction platform. The specific steps are as follows: On the first day of the experiment, 293T cells (Cell Bank Cat#GNHu17 of the Type Culture Collection Committee of the Chinese Academy of Sciences) were inoculated into two 6cm petri dishes, and the number of cells in each petri dish reached 7.5 ⁇ 10 5 .
  • each of the packaging plasmid (pGag-pol, pVSV-G and other BioVector plasmid vector strain cell gene collection center) and the plasmid pBabe-hLAG-3 cloned with human LAG-3 gene were added to OPTI-MEM (Thermofisher Scientific Cat# 31985070), make the final volume 200 ⁇ l, and prepare another 200 ⁇ l OPTI-MEM to add 36 ⁇ l transfection reagent fectin (Shanghai Yuanpei Biotechnology Co., Ltd. Cat#F210), mix the two evenly, leave at room temperature for 5 minutes, and then add the mixture (per dish Each 200 ⁇ l) was added dropwise to the cultured 293T cells.
  • OPTI-MEM Thermofisher Scientific Cat# 31985070
  • 36 ⁇ l transfection reagent fectin (Shanghai Yuanpei Biotechnology Co., Ltd. Cat#F210), mix the two evenly, leave at room temperature for 5 minutes, and then add
  • the 293T cell culture medium was changed to 4ml DMEM high glucose medium (Shanghai Yuanpei Biotechnology Co., Ltd./Yuanpei Bio: Cat#L130KJ).
  • CHO-K1 cells Cell Bank Cat#SCSP-507 of the Type Culture Collection Committee of the Chinese Academy of Sciences
  • the 293T cell supernatant was collected, filtered to the cultured CHO-K1 cells with a 0.45 ⁇ m filter membrane, and 10 ⁇ g/ml polybrene (Shanghai Yisheng Biotechnology Co., Ltd.
  • amino acid sequence NP_002277.4 of human LAG-3 (pBabe-hLAG-3) used in this example is a full sequence of amino acids 1-525, of which positions 1-22 are signal peptide sequences, that is, positions 23-525 are constructed by the present invention CHO-K1
  • LAG-3-his, LAG-3-D12-his, monkey LAG-3-his (cyno LAG-3-his) or mLAG-3-his and other recombinant proteins to 1 ⁇ g/ ml, 2 ⁇ g/ml (hLAG-3 D12-his), or 5 ⁇ g/ml (cyno LAG-3-his) concentration, add a 96-well microtiter plate (Corning, CLS3590-100EA) in a volume of 50 ⁇ l/well. Place in the 37°C incubator for 2 hours.
  • skimmed milk (bright skimmed milk powder) blocking solution diluted with PBS was added, and incubated in a 37°C incubator for 3 hours or 4°C overnight (16-18 hours) for sealing.
  • PBST buffer pH7.4 PBS containing 0.05% tweeen-20
  • 50 ⁇ l/well of supernatant (containing detection antibody) or 10 ⁇ g/ml starting, 5-fold gradient dilution to be tested Antibody incubate at 37°C for 1 hour
  • wash the plate 5 times with PBST add 50 ⁇ l/well 1:2500 diluted Anti-mouse or human HRP secondary antibody (Jackson Immuno Research, Cat#115-035-003 or 109-035-003) Incubate at 37°C for 1 hour.
  • the present invention uses human LAG-3 recombinant protein (prepared in Example 1) as the antigen, immunizes mice, screens fusion hybridomas, screens and optimizes from millions of hybridoma clones, and unexpectedly finds the binding activity of one strain to hLAG-3 Very good hybridoma cell line.
  • This cell line is further subcloned and screened to obtain a monoclonal cell line, and the murine antibody sequence obtained from the monoclonal cell line is subjected to computer modeling human design and screening to obtain an optimized humanized antibody.
  • the humanized antibody also maintains good binding activity to hLAG-3, and very surprisingly, the obtained humanized antibody also shows good cell function activity, animal efficacy, better PK characteristics, high expression yield and heat stability.
  • mice Female, 4 weeks old were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK ( ⁇ ) 2016-0011. After the mice are purchased, they are kept in a laboratory environment for 1 week, with day light/night dark cycle adjustment, temperature 20-25 °C; humidity 40-60%. Mice were divided into 3/group/cage.
  • the antigen prepared in Example 1 was used for immunization.
  • the adjuvant is Quickantibody-5w (Beijing Boaolong Immunology Technology Co., Ltd., KX0210041). The ratio of antigen to adjuvant is 1:1.
  • mice with high antibody titers and plateau titers in the serum were selected for spleen cell fusion, and the splenic lymphocytes With myeloma cells Sp2/0 cells ( CRL-8287 (TM )) was fused to obtain hybridoma cells and spread a 96-well plate.
  • Example 3 The ELISA method in Example 3 was used to screen the hybridoma cell lines in the 96-well plate to detect the binding activity of the antibody in the supernatant of the hybridoma cell lines to human LAG-3, select the clone with good activity, and select the above Use the method described in Example 4 to detect the secreted antibody to prevent the blocking activity of hLAG-3 and Daudi cells, preferably clones with good binding activity and blocking activity, and further limit dilution to preferably obtain monoclonal antibody cell lines, partial results See the table below.
  • Termination that is, the clones with poor blocking activity are terminated, and no subclonal screening will be performed;
  • ND No detection, that is, the terminated clones are not subsequently tested.
  • NA not adapted, that is, the subclone comes from the previous parent clone. Specifically in this table, the parent clone refers to clone 7B9 numbered #15.
  • Table 1 lists some of the screening data.
  • the data shows that in the initial screening of fusion hybridomas, clones with good binding activity and good blocking activity, such as #15 in the table, clone 7B9 (the higher the ELISA value, the better the binding activity. The lower the blocking activity, the Indicates that the blocking activity is better).
  • clone 7B9 was subjected to multiple limiting dilutions, 7-10 days after each dilution (subclone), after the clone proliferated, the binding activity and blocking activity of the antibody (supernatant) secreted by each subclone were re-tested by ELISA. .
  • the clones with poor blocking activity are discarded. For example, the numbers 1-14 in the above table are discarded, and subsequent subcloning and testing will not be performed.
  • Example 6 Extraction, analysis and identification of the murine anti-human LAG-3 antibody mab23 antibody sequence of the present invention
  • the process of extracting antibody sequences from monoclonal cell lines preferably obtained from hybridomas is a method commonly used by those skilled in the art. Specifically, the above-mentioned monoclonal cell lines were collected, and after expansion and culture, 1 ⁇ 10 6 cells were taken, and RNA was extracted with Trizol (Invitrogen, 15596-018) (according to the instructions of the kit), and the extracted RNA was reverse transcribed into cDNA, reverse transcription kit was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd., Cat#B532435. The cDNA obtained by reverse transcription is used as a template for PCR amplification. The amplified products were sequenced to obtain the base/coding sequences of the variable regions of the light and heavy chains of the mab23 antibody (as follows). The primers used refer to the manual TB326 Rev. C0308 published by Novagen.
  • the base sequence of the light chain variable region of the murine monoclonal antibody mab23 obtained from the preferred hybridoma cell line of the present invention (the underlined part is the coding sequence):
  • the base sequence of the heavy chain variable region of the murine monoclonal antibody mab23 obtained from the preferred hybridoma cell line of the present invention (the underlined part is the coding sequence):
  • amino acid sequences encoded by the base sequences of the light and heavy chain variable regions of the murine monoclonal antibody mab23 obtained in the present invention are the following SEQ ID NO: 3 and SEQ ID NO: 4.
  • amino acid sequence of the light chain variable region of the mouse monoclonal antibody mab23 obtained from the preferred hybridoma monoclonal cell line of the present invention is the amino acid sequence of the light chain variable region of the mouse monoclonal antibody mab23 obtained from the preferred hybridoma monoclonal cell line of the present invention:
  • amino acid sequence of the heavy chain variable region of the murine monoclonal antibody mab23 obtained in the preferred hybridoma cell monoclonal strain of the present invention:
  • the above-mentioned antibody light and heavy chain variable region sequences and IgG constant regions of different types such as human hIgG1, hIgG2, hIgG3, hIgG4, human light chain ⁇ , ⁇ type; mouse mIgG1, mIgG2, mIgG3, mouse light chain ⁇ , ⁇ Type and other recombinant expression and purification to obtain complete human-mouse chimeric antibody, mouse antibody.
  • the human heavy chain constant region as hIgG4 and human light chain ⁇ type as examples, the chimeric antibody mab23c was obtained according to the expression and purification method in Example 1.
  • the binding activity of mab23c and hLAG-3 was detected by the methods in Examples 3 and 4, and the prevention LAG-3 and Daudi cell binding activity (blocking activity), and parallel comparison with the control antibody (Ref) (see Figure 1 a and b).
  • the EC 50 and Emax of mab23c detected by ELISA are 0.41 nM and 1.7, respectively, which are better than the 0.64 nM and 1.4 of the control antibody (Ref).
  • the IC 50 of the present invention mab23c to prevent LAG3 and Daudi cell binding activity (blocking activity) is 2.3 nM, which is nearly 4 times better than the IC 50 (10.5 nM) detected by Ref under the same conditions ( Figure 1b). Blocking activity is directly related to the efficacy of the molecule as a therapeutic antibody. Therefore, the outstanding blocking activity of mab23c makes the antibody of the present invention have better and higher value as a candidate molecule for drug development.
  • Example 7 Humanization of the murine antibody mab23c of the present invention
  • the antibody mab23c (chimeric antibody) unexpectedly discovered in the present invention has a strong binding activity to the specific antigen hLAG-3, especially an unexpected blocking activity. It is shown that the antibody can be used for the development of monoclonal antibody drugs for tumor treatment targeting LAG-3, which has better value, such as possible better efficacy.
  • the present invention carries out humanized design and screening of murine mab23 antibody, as well as sequence optimization. The specific process is described as follows.
  • variable region of the murine anti-human hLAG-3 antibody mab23 of the present invention is marked/annotated as follows according to the various definition methods listed in the above table.
  • Light chain CDR1 RASQDIGSSLN (SEQ ID NO:5) Light chain CDR2 ATSSLDS(SEQ ID NO: 6) Light chain CDR3 LQYVTSPLT(SEQ ID NO: 7) Heavy chain CDR1 GYTFTDYEMH(SEQ ID NO: 8) Heavy chain CDR2 GIDPETEGIAYNQKFRG (SEQ ID NO: 9) Heavy chain CDR3 SNYYGGREAWFAY (SEQ ID NO: 10)
  • Antibody mab23 CDRs Light chain CDR1 RASQDIGSSLN(SEQ ID NO:5) Light chain CDR2 ATSSLDS(SEQ ID NO: 6) Light chain CDR3 LQYVTSPLT(SEQ ID NO: 7) Heavy chain CDR1 DYEMH(SEQ ID NO:11) Heavy chain CDR2 GIDPETEGIAYNQKFRG (SEQ ID NO: 9) Heavy chain CDR3 SNYYGGREAWFAY (SEQ ID NO: 10)
  • Anti-hLAG-3 (anti-hLAG-3) antibody mab23 of the present invention defines CDR sequence according to AbM
  • Antibody mab23 CDRs Light chain CDR1 RASQDIGSSLN(SEQ ID NO:5) Light chain CDR2 ATSSLDS(SEQ ID NO: 6) Light chain CDR3 LQYVTSPLT(SEQ ID NO: 7) Heavy chain CDR1 GYTFTDYEMH(SEQ ID NO: 8) Heavy chain CDR2 GIDPETEGIA(SEQ ID NO:12) Heavy chain CDR3 SNYYGGREAWFAY (SEQ ID NO: 10)
  • Anti-hLAG-3 (anti-hLAG-3) antibody mab23 of the present invention defines CDR sequence according to Chothia
  • Antibody mab23 CDRs Light chain CDR1 RASQDIGSSLN(SEQ ID NO:5) Light chain CDR2 ATSSLDS(SEQ ID NO: 6) Light chain CDR3 LQYVTSPLT(SEQ ID NO: 7) Heavy chain CDR1 GYTFTDY(SEQ ID NO:13) Heavy chain CDR2 DPETEG(SEQ ID NO:14) Heavy chain CDR3 SNYYGGREAWFAY (SEQ ID NO: 10)
  • Anti-hLAG-3 (anti-hLAG-3) antibody mab23 of the present invention defines CDR sequence according to Contact
  • Antibody mab23 CDRs Light chain CDR1 GSSLNWL(SEQ ID NO:15) Light chain CDR2 KRLIYATSSLD(SEQ ID NO:16) Light chain CDR3 LQYVTSPL(SEQ ID NO:17) Heavy chain CDR1 TDYEMH(SEQ ID NO:18) Heavy chain CDR2 WIGGIDPETEGIA(SEQ ID NO:19) Heavy chain CDR3 TNSNYYGGREAWFA(SEQ ID NO:20)
  • the murine antibody mab23 of the present invention After the above analysis, labeling, and definition of the CDR sequence of the murine antibody mab23 of the present invention, it is humanized according to the methods published in many documents in the field.
  • the mouse antibody sequence is compared with the human antibody germline database (v-base) to find out the human antibody light and heavy chain germlines with high homology.
  • v-base human antibody germline database
  • computer modeling and simulation of antibody structure may affect and
  • the antigen binding sites, key sites and combinations of back mutations are selected to screen out humanized antibody molecules with optimal activity.
  • Back mutation is also called reverse mutation (reverse mutation), that is, the specific amino acid residue of the humanized antibody is mutated into the amino acid residue at the corresponding position of the original source antibody.
  • human antibody germlines with good homology to mab23 light chain include IGKV1-16*01, IGKV1-17*01, IGKV1-39*01, IGKV1-NL1* 01, IGKV1/OR-2*01, IGKV1/OR-3*01, IGKV1/OR-4*01, IGKV1/OR10-1*01, IGKV1/OR2-1*01, IGKV1/OR2-2*01, etc.
  • the human antibody germline light chain IGKV1-39*01 is preferred.
  • hJK4.1 is preferably used for mab23 light chain humanized human antibody germline J region for humanized design, screening and sequence optimization.
  • human antibody germlines with good homology to mab23 heavy chain include IGHV1-69*02, IGHV1-69*04, IGHV1-69*06, IGHV1-69*08, IGHV1 -69*09, IGHV1-69*10, IGHV1-69*14, IGHV1-69*17, IGHV1-18*01, IGHV1-18*03, etc.
  • the human germline heavy chain IGHV1-18*01 sequence is preferably used for humanization of the antibody of the present invention.
  • hJh4.1 is preferably used in the mouse-derived antibody mab23 heavy chain humanized human antibody germline J region for humanization design , Screening and sequence optimization.
  • the antibody of the present invention is transplanted with the CDR region of mab23 (see the definition of CDR above) to the selected humanized light and heavy chain human antibody germline template, and then recombined with the IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the murine antibody, reverse mutations are performed on the embedded residues, residues that directly interact with the CDR region, and residues that have an important impact on the conformation of VL and VH, and screen these mutations and The combination of mutations can see the effect on antibody activity, and optimize the chemically unstable amino acid residues in the CDR region to obtain an antibody molecule sequence optimized for structure and activity, which completes the humanization of the murine antibody of the present invention.
  • Human antibody light chain constant region ⁇ chain SEQ ID NO: 21; human IgG4 heavy chain constant region: SEQ ID NO: 22
  • variable region of the humanized light chain of the anti-human LAG-3 antibody mab23 of the present invention is variable region of the humanized light chain of the anti-human LAG-3 antibody mab23 of the present invention.
  • LG2312 SEQ ID NO: 23; LG2313: SEQ ID NO: 24; LG2314: SEQ ID NO: 25; LG2315: SEQ ID NO: 26, that is
  • LG2316 SEQ ID NO: 27; LG2317: SEQ ID NO: 28, that is
  • LG2318 SEQ ID NO: 29.
  • LG2342 SEQ ID NO: 30, that is
  • LG2343 SEQ ID NO: 31, that is
  • LG2344 SEQ ID NO: 32; LG2345: SEQ ID NO: 33; LG2346: SEQ ID NO: 34; LG2347: SEQ ID NO: 35; LG2348: SEQ ID NO: 36; LG2349: SEQ ID NO: 37.
  • the humanized sequence of the light chain of the murine antibody mab23 of the present invention contains different back mutations, and the number of back mutation sites can be 10 or more, preferably 0-10, such as the sequence listed above.
  • These arbitrary sequences are combined with the constant region sequence of the human antibody light chain constant region kappa chain or lambda chain to obtain the light chain sequence of the humanized antibody of the present invention, such as the kappa type light chain constant region for the light chain of the present invention, as listed above.
  • the heavy chain variable region used for humanization also has a different number of back mutations, and the number of back mutation sites can be 10 or more, preferably 0-10, such as the heavy chain variable region sequences listed above.
  • heavy chain variable region sequences with different numbers of back mutations are recombined with optional human IgG1, 2, 3, and 4 chain constant region sequences to obtain the humanized antibody heavy chain sequence of the present invention.
  • the heavy chain of the present invention uses hIgG4 as a constant
  • the region sequence is illustrated as an example.
  • the mab23 antibody of the present invention partially optimizes the humanized antibody light and heavy chain sequence, expression amount (expression and purification using the method of Example 1 of the present invention and detection of antibody production) and activity evaluation (the ELISA detection method of Example 3 of the present invention, of Example 5)
  • the results of blocking activity detection method are as follows.
  • Table 8 The partial sequence of the humanized antibody of the mab23 antibody of the present invention (take human kappa light chain, hIgG4 heavy chain constant region as an example)
  • the humanized antibody molecule obtained from the murine antibody mab23 sequence of the present invention retains the binding activity to hLAG-3. More preferably, many molecules recover the same binding activity as the murine antibody mab23c, among which Ab2315, The binding activity of Ab2317, Ab2322, Ab2325 and other antibodies is no different from mab23c. That is, the humanized preferred antibody of the present invention retains the binding activity of the previous murine antibody. In addition, except for antibodies with very weak binding activity (NB), most of the humanized antibodies have good blocking activity and are close to mab23c, such as Ab2314, Ab2316, Ab2317, Ab2318, Ab2320, Ab2325 and so on.
  • NB very weak binding activity
  • amino acid sequences (including constant regions) of light and heavy chains of humanized antibodies in the part of Table 8 are as follows.
  • Heavy chain same as Ab2317 heavy chain (SEQ ID NO: 40)
  • Light chain same as Ab2317 light chain (SEQ ID NO: 39)
  • Example 8 Comprehensive evaluation of humanized anti-human LAG-3 antibody of the present invention, preferably antibody binding activity
  • the Biacore method is as follows: use Biacore T200, GE Healthcare instrument to determine the affinity of the antibody of the present invention and human LAG-3. Using pH7.4 running buffer HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20), first couple Protein A (Thermo Pierce, Cat#21181) to the biosensing chip CM5 (Cat.
  • the mouse-derived antibody of the present invention is humanized preferably antibodies (Ab2317, Ab2325, etc.) and human LAG-3-his (monomer), human LAG3-hFc (dimer), human LAG-3 extracellular loop 1 and loop 2 (hLAG-3-D12) his form (monomer), namely hLAG-3-D12-his (monomer) and hLAG-3-D12-hFc (dimer) form, hLAG3+ cells are all very good Combination.
  • Bicore test results show that the affinities of Ab2317 and Ab2325 are both below 0.2nM, which is close to the affinity of the control molecule.
  • This specific blocking activity is related to the efficacy of the antibody for clinical treatment of tumor patients. The blocking activity is good and the efficacy is expected to be better.
  • another unexpected advantage of the antibody of the present invention is that its expression yield is very high.
  • the amount achieved is more than 100mg/L
  • the expression amount of the control antibody Ref is only 60mg/L. L.
  • the average expression yield of multiple comparisons, Ab2317 is more than 60% higher than the control antibody Ref. This shows that the expression level of the antibody of the present invention is better than that of the control molecule (this difference in expression level is related to the sequence), which brings advantages for the antibody of the present invention to be used in the later process development to increase the antibody yield.
  • the results of electrophoresis showed that the concentration of the antibody Ab2317 of the present invention remained unchanged at 45°C for 7 days and 14 days, regardless of whether it was a non-denatured or denatured sample, indicating that there was no price reduction.
  • the control sample Ref showed a decrease in samples (less on the gel). For example, the samples at 14 days (lanes 11 and 12) were significantly less than the 0 days, indicating significant degradation. This result well illustrates the excellent stability characteristics of the antibody Ab2317 of the present invention. This feature brings great convenience and advantages to its development as a drug, especially the development of formulations.
  • the humanized antibodies of the present invention can all behave as cynomolgus LAG-3 (cynomolgus, cyno-LAG3-hFc, purchased from Beijing Biopsy Biotechnology Co., Ltd., catalog number) LA3-C5252) binding, the binding activity was 40.2nM, 44.5nM, 35.3nM, respectively. This binding activity is more than 200 times weaker than that of human LAG-3. This is consistent with the result of Biacore detecting the affinity of the antibody of the present invention with cyno-LAG3-hFc.
  • the affinity of Ref is 836nM (more than 4000 times weaker than the affinity of the same human LAG-3); Ab2317 and Ab2325 did not detect the binding to cyno-LAG-3-hFc in Biacore. In addition, none of the above antibodies bind to murine LAG-3.
  • the present invention is represented by the antibody LG2317, and cell function evaluation is carried out through the following experiments.
  • Example 9 Evaluation of the functional activity of human T cells activated by anti-LAG-3 antibodies
  • the cell culture plate was incubated in a 37°C, 5% CO 2 incubator for 3 days, the cell culture plate was taken out, centrifuged at 3000 rpm for 10 min, and 80 ⁇ l of supernatant was taken out of each well for human IL-2 detection.
  • the IL-2 ELISA test is performed in accordance with the instructions of the kit (Shenzhen Xinbosheng Biotechnology Co., Ltd., cat: EHC003.96), and the steps are as follows:
  • a Dilute the taken-out cell culture supernatant by 25 times (diluted in different experiments) and add it to the ELISA plate (100 ⁇ l/well); the standard is diluted with the specimen universal diluent to a different concentration gradient: 1000pg/ml , 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, add 100 ⁇ l to each well; add sample general diluent to blank wells.
  • Result analysis Calculate the IL-2 value, and compare it with the blank control, and convert it into an increase percentage (%) to evaluate the sample's activation of human T cell activity.
  • T cells from different donors experiment showed Ab2317 antibody of the invention is preferably activated human T cells releasing activity EC IL-2 is 50 0.34 ⁇ g / ml. Under the same conditions, the EC 50 of the control antibody Ref was 1.4 ⁇ g/ml. That is, the antibody of the present invention is at least 3 times better than the control Ref on the T cell activity.
  • Fig. 3 a shows one of the representative data.
  • Example 10 Activity detection of anti-LAG-3 antibody of the present invention alone and in combination with PD-1 antibody in mixed lymphocyte reaction (MLR assay)
  • the mixed lymphocyte reaction (MLR assay) method is used to detect the secretion of INF- ⁇ (INFg) to evaluate the activity of the series of antibodies of the present invention in activating human T cells. That is, the human blood cell PBMC (isolated from peripheral blood donated by healthy volunteers) isolated from the present invention induces dendritic cells (DC), and the obtained DC is used to stimulate T cells from different volunteers.
  • MLR assay mixed lymphocyte reaction
  • dendritic cell culture On the first day of the experiment, inoculate PBMC in a 6-well plate with RPMI 1640 medium, 2ml per well, 1 ⁇ 10 6 cells/ml, 37°C, 5% CO 2 incubator 2 After hours, gently aspirate the suspended cells and add 2ml complete RPMI 1640 medium (containing 10% FBS, 100ng/ml GM-CSF, Peprotech, Cat#:300-03, and 100ng/ml IL-4 to the adherent cells). , Peprotech, Cat#:200-04). After culturing for 2 days, add 1ml fresh complete RPMI 1640 medium to each well.
  • DC stimulated T cell (MLR) assay Use 10ng/ml anti-CD3 antibody (Miltenyl Biotec, Cat#: 130-093-387) to coat 96-well cell culture plates at 100 ⁇ l/well, incubate at 37°C for 2 hours, Wash again with PBS. Collect the above-mentioned DC cells on the 7th day of culture, centrifuge, resuspend in 10% FBS RPMI 1640 medium, count, make 5 ⁇ 10 4 cells/ml, add the above-mentioned anti-CD3 coated 96 cells at 90 ⁇ l/well -In the orifice plate.
  • MLR DC stimulated T cell
  • the sample to be tested was prepared in proportion with PBS, including control antibody (Ref) and Ab2317, and PD-1 antibody (obtained by expression and purification of the present invention) was added to the above 96-well plate.
  • Ref or Ab2317+PD-1 antibody each 10 ⁇ l/well.
  • the final concentration of PD-1 antibody in 200 ⁇ l system is 0.5 ⁇ g/ml.
  • the concentration of the antibody to be tested (Ref or Ab2317) in the 200 ⁇ l system is the required concentration gradient.
  • Control group 90 ⁇ l PBMC cells+90 ⁇ l DC+20 ⁇ l PBS. After the cell culture plate was incubated in a 37°C, 5% CO 2 incubator for 4 days, the cell culture plate was taken out, centrifuged at 3000 rpm for 10 min, and 150 ⁇ l of supernatant was taken from each well for human INF- ⁇ detection.
  • INF- ⁇ value (pg) is calculated by the standard curve formula to evaluate the activity of the antibody of the present invention.
  • Example 11 Evaluation of in vivo efficacy of LAG-3 antibody of the present invention
  • the human PD-1 and LAG-3 double transgenic mice (C57BL/6-hPD1/hLAG3) are used to establish an animal drug efficacy model to evaluate the drug efficacy of the LAG-3 antibody of the present invention in animals.
  • Double transgenic mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number: SCXK ( ⁇ )2018-0008.
  • MC38 cells purchased from the Cell Institute of the Chinese Academy of Sciences
  • 10% fetal bovine serum (Shanghai Bosheng Biotechnology Co., Ltd., item number: BS-0002-500)
  • 1% HEPES Thermo Fisher Technology Co., Ltd., item number: 15630080
  • DMEM/high sugar medium (Shanghai Yuanpei Biotechnology Co., Ltd., article number: L110KJ)
  • continuously cultured in a 37°C cell incubator containing 5% CO 2 C57BL/6-hPD1/hLAG3 female mice, 6 weeks old, 5 mice/cage, housed in an SPF environment, temperature 20-25°C; humidity 40%-60%, free eating and water, changing the litter regularly.
  • MC38 cells grow to the logarithmic growth phase (the confluence rate is 80%-90%), they are digested with 0.25% trypsin, the cells are collected, and the cells are washed twice with serum-free DMEM/high glucose medium, and then with serum-free Resuspend the DMEM/high glucose medium, count, and mix Matrigel (BD Medical Devices (Shanghai) Co., Ltd., catalog number: 354234) and DMEM/high glucose medium at a ratio of 1:1, and adjust the cell concentration to 1 ⁇ 10 7 cells/ml were used for inoculation. Inoculate 100 ⁇ l (10 6 cells) of MC38 cell suspension subcutaneously on the right ribs of mice, and select tumor cells to grow to a size of about 100-150 mm 3 and randomly group them into groups, 5 in each group.
  • Matrigel BD Medical Devices (Shanghai) Co., Ltd., catalog number: 354234
  • the sample to be tested is prepared with PBS and is sterile.
  • the Blank group is PBS.
  • the PD-1 antibody (cloned and expressed according to the published sequence of Keytruda) was used as a single drug control group.
  • PD-1 antibody + Ref is the combination control group.
  • PD-1 antibody + Ab2317 is the test group of combination medication.
  • the mode of administration is intraperitoneal injection. PD-1 antibody is administered alone or in combination, and the dose is 10 ⁇ g/100 ⁇ l/head. In the combined administration group, the dose of Ref and Ab2317 was 120 ⁇ g/100 ⁇ l/head.
  • the frequency of administration is 2 times a week.
  • the day of each injection sample is day 0. Measure body weight, tumor volume, and record data before each administration. This experiment is given 4 times.
  • Table 9b is the statistics of tumor volume on day 0 and day 23. The results showed that on the 23rd day, compared with the PBS group, the efficacy (inhibition rate) of the PD-1 antibody + antibody Ab2317 combination group of the present invention was 103%, that is, 100% inhibited tumor growth and the tumor shrank from the beginning. The efficacy (inhibition rate) of PD-1 antibody alone is 63%, and the efficacy (inhibition rate) of PD-1 antibody + Ref combination group is 80%.
  • the present invention has carried out a variety of bispecific antibody designs.
  • the general formula of the designed bispecific antibody is as follows.
  • the light chain-containing sequence means that in addition to the light chain sequence, the sequence can also include scFv linked to the light chain sequence;
  • the heavy chain-containing sequence means that the sequence can include, in addition to the heavy chain sequence, ScFv linked to heavy chain sequence.
  • T1 represents the first protein functional region for target 1 (such as LAG3)
  • T2 represents the second protein functional region for target 2 (non-LAG3)
  • T1 (scFv) represents the scFv sequence against target 1 antibody
  • T2 (scFv) represents the scFv sequence against target 2.
  • n1, n2, n3, and n4 are natural numbers respectively, which can be 0, 1, 2, 3, etc., in specific embodiments of the present invention Among them, at least one of n1, n2, n3, and n4 has a value of 1, and the rest are 0.
  • VL stands for the variable region sequence of the antibody light chain against target 1 or 2
  • VH stands for the variable region sequence of the antibody heavy chain against target 1 or 2.
  • Lc represents the constant region sequence of the light chain ( ⁇ or ⁇ ), preferably human light chain constant region sequence
  • Hc represents the heavy chain, including the constant region sequence of IgG1, IgG2, IgG3, IgG4, etc. (abbreviated as Hc-IgG1, Hc- IgG2, Hc-IgG3, Hc-IgG4), preferably human heavy chain constant region sequence (Hc-hIgG).
  • Hc-hIgG human heavy chain constant region sequence
  • T1 is immunoglobulin and T2 is scFv; in scheme 2, T2 is immunoglobulin, and T1 is scFv; scFv targets the same target; in schemes 3 and 4, the scFv at both ends Aimed at two different targets.
  • the N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region is connected to the immune system through a linker.
  • the C-terminus or N-terminus of the globulin light chain and/or heavy chain; or the scFv is the heavy chain variable region-linker-light chain variable region, and the N-terminus of the heavy chain variable region or the light chain variable region
  • the C-terminus is correspondingly connected to the C-terminus or N-terminus of the immunoglobulin light chain and/or heavy chain through a linker.
  • scFv is a light chain variable region-linker-heavy chain variable region
  • its connection mode is that the C-terminus of the light chain variable region is connected to the linker, and the linker is then connected to the heavy chain
  • the N-terminus of the variable region is connected, thereby exposing the N-terminus of the light chain variable region of the scFv and the C-terminus of the heavy chain variable region, so that it can be connected to the light chain and/or heavy chain of the immunoglobulin through a linker.
  • the C-terminus of the variable region of the heavy chain of scFv is preferably connected to the N-terminus of the immunoglobulin heavy chain through a linker;
  • the scFv is a heavy chain variable region-linker-light chain variable region
  • its connection mode is that the N-terminus of the light chain variable region is connected to the linker, and the linker is connected to the heavy chain variable region.
  • the C-terminus is connected, thereby exposing the C-terminus of the scFv light chain variable region and the N-terminus of the heavy chain variable region, so that it can be connected to the light chain and/or heavy chain of the immunoglobulin through a linker.
  • the C-terminus of the light chain variable region of the scFv when it is connected to the light chain of an immunoglobulin, in some specific embodiments, it is preferable to use the C-terminus of the light chain variable region of the scFv to be connected to the N-terminus of the immunoglobulin heavy chain; when it is connected In the case of the heavy chain of an immunoglobulin, in some specific embodiments, it is preferable to connect the N-terminus of the heavy chain variable region of the scFv to the C-terminus of the immunoglobulin heavy chain.
  • the linker is (Gly-Gly-Gly-Gly-Ser) w [abbreviated as (G 4 S) w ].
  • the w is preferably an integer between 0-10.
  • the linker is (G 4 S) 3 , and/or the number of the scFv is a pair, which is symmetrically connected to the immunoglobulin light chain and/or heavy chain.
  • the antibody of the present invention can also be used in a manner different from Formula 1, for example, Formula 2 below.
  • Table 11 DVD format bispecific design based on the anti-LAG3 antibody of the present invention (general formula 2)
  • T1 and T2 stand for target 1 (for example, LAG3) and target 2 (non-LAG3), respectively.
  • a light chain-containing sequence means that in addition to the normal and complete light chain sequence, the sequence also includes another light chain variable region sequence.
  • a sequence containing a heavy chain means that the sequence includes another heavy chain variable region sequence in addition to the normal and complete heavy chain sequence. The variable region of the light chain and the complete light chain, and the variable region of the heavy chain and the complete heavy chain are connected by a linker.
  • the PD1 antibody sequence includes the antibody sequence that has been published, including the anti-PD-1 antibody Nivolumab/Opdivo (Nivo for short), Pembrolizumab/ Keytruda (Pem for short).
  • Nivolumab/Opdivo Navo for short
  • Pembrolizumab/ Keytruda Pem for short
  • sequences such as Nivolumab (Nivo) and Pembrolizumab (Pem) can also be found from public sources such as www.drugbank.ca. The expression numbers and sequences of these individual antibodies are shown in the table below.
  • Nivolumab and Pembrolizumab can be obtained from public information such as www.drugbank.ca.
  • the present invention numbers them as follows:
  • NivoVL-Lc ( ⁇ chain) SEQ ID NO: 42; NivoVH-Hc (hIgG4): SEQ ID NO: 43;
  • PemVL-Lc ( ⁇ chain): SEQ ID NO: 44; PemVH-Hc (hIgG4): SEQ ID NO: 45;
  • Ab835VL is SEQ ID NO: 58 in application number 201810917684.X; Ab385VH is SEQ ID NO: 59 in application number 201810917684.X.
  • Example 13 Design and activity evaluation of bispecific antibodies against LAG-3 and PD-1 dual targets
  • the present invention designs bispecific antibodies with different sequence structures for the two targets of LAG-3 and PD1, as shown in the following table.
  • ⁇ chain indicates that the light chain is the constant region of the ⁇ type light chain of human IgG.
  • the SBody designed by the present invention that introduces scFv into the C-terminus of the heavy chain all mutate the last K to A.
  • the above-mentioned bispecific antibodies were cloned, expressed, and purified respectively, and the binding activities of these designed bispecific molecules with human LAG-3 and PD-1 were tested by the methods in the foregoing examples.
  • the results are as follows table.
  • sequences based on specific activity exhibit unexpected differences in molecular design, which is similar to the conventional structure of IgG molecules, the present invention is referred to as S equence-based IgG like bispecific anti body format (SBody), i.e., sequence similarity specific IgG structure Bispecific antibodies.
  • SBody bispecific anti body format
  • the affinity of the bispecific antibody of the present invention was analyzed by Biacore (the method is the same as that in Example 8).
  • PD-1 expressed in the present invention
  • a pH 7.4 PBS buffer a pH 7.4 PBS buffer
  • a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 50 ⁇ l/well. Incubate for 2 hours at °C. After discarding the liquid, add 200 ⁇ l/well of 5% skim milk (Shanghai Shenggong Bioengineering Co., Ltd., A600669-0250) blocking solution diluted with PBS, and place overnight (16-18 hours) at 4°C for blocking.
  • skim milk Shanghai Shenggong Bioengineering Co., Ltd., A600669-0250
  • the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), and the bispecific antibody to be tested (10 ⁇ g/ml) was added to start, and the plate was diluted 5-fold with 1% BSA.
  • PBST buffer pH7.4PBS containing 0.05% tween-20
  • LAG3-his was diluted to 1 ⁇ g/ml with pH7.4 PBS buffer, added to a 96-well microtiter plate at a volume of 50 ⁇ l/well, and incubated at 37°C for 2 hours. After the liquid was discarded, 200 ⁇ l/well of 5% skimmed milk blocking solution diluted with PBS was added, and it was placed overnight (16-18 hours) at 4°C for blocking.
  • PBST buffer pH 7.4 PBS containing 0.05% tween-20
  • PBST buffer pH 7.4 PBS containing 0.05% tween-20
  • the bispecific antibody SBody of the present invention can further bind to the other target after binding to one of the dual targets, that is, it can bind to two targets at the same time.
  • IC 50 change factor that is, the ratio of the IC 50 of the bispecific antibody and the corresponding monoclonal antibody (control antibody).
  • the larger the ratio the more the functional activity of the designed bispecific antibody against a single target is weakened.
  • the ratio of 2 indicates that the functional activity of the designed bispecific antibody against the target is weakened compared with the corresponding monoclonal antibody. 1 times.
  • a ratio within 2 is the experimental error range, that is, the activity is not affected.
  • ND The molecule has no detectable activity to prevent LAG3 from binding to Daudi cells.
  • the bispecific antibody (SBody) designed in the present invention retains the activity of preventing the binding of PD-1 and PD-L1, and only LB2373 prevents the binding activity of PD-1/PD-L1 is slightly weaker (change factor 3.78 ).
  • LB2379, LB2380, LB2371 and LB2372 prevented LAG-3 and Daudi cells from weakening the binding activity more (8.16, 3.78, 7.92 and 5.63 times respectively), while LB2373 and LB2374 almost did not weaken (0.82, 1.98 times respectively).
  • the antibody of the present invention was prepared at 1 mg/ml in PBS (pH 7.4), incubated at 37°C for 5 days (d5), 10 days (d10) and stored at -80°C for 60 Days’ samples were compared for activity and stability was evaluated. The results are shown in the table below.
  • the design of the present invention targets LAG3 and PD1 bispecific antibodies (SBody) in terms of expression yields that differ greatly.
  • SBody LAG3 and PD1 bispecific antibodies
  • the expression levels of LB2373, LB2374, LB2379, and LB2380 are relatively high.
  • the expression level of SBody designed with Ab835 as scFv is much lower than that of the same designed Ab2317 scFv, which is more than 65 times lower, such as LB2374 vs LB234; and more than 140 times lower, such as LB2373 vs LB211.
  • LB2379 contains light chain amino acid sequence: SEQ ID NO: 49; heavy chain amino acid sequence: SEQ ID NO: 43; LB2380 contains light chain amino acid sequence: SEQ ID NO: 50; heavy chain amino acid sequence: SEQ ID NO: 43.
  • Example 14 PK evaluation of LAG-3 antibody of the present invention
  • mice of C57BL/6cnc strain purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Zhejiang) 2018-0001), female, 8 weeks old, 6 mice, about 20g, breeding environment : SPF level. Temperature 20-25°C; humidity 40-60%.
  • C57BL/6cnc mice were reared in a laboratory environment for 3 days and then randomly divided into groups, with 3 mice in each group.
  • the drug to be tested (the LAG-3 antibody Ab2317 and Ref of the present invention) was injected subcutaneously into the back of mice with an injection volume of 200 ⁇ l. The dosage is 20mg/kg/head.
  • the test sample was taken from the orbit of the mouse after injection. Time points are 0.5, 1, 2, 4, 7, 24, 31, 48, 56, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408 , 432, 456, 480, 504 hours. Centrifuge the blood sample, take the supernatant, and store at -20°C for testing. After collecting blood samples, ELISA was used to detect the content of blood samples at various time points. Use Excel to analyze the PK data and calculate the T1/2 of the drug to be tested. The results are shown in the table below.
  • Example 15 PK evaluation of the bispecific antibody (SBody) designed for the dual target of LAG3 and PD-1 of the present invention
  • mice of C57BL/6cnc strain purchasedd from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Zhejiang) 2018-0001
  • human PD-1 and human LAG-3 double transgenic CB7BL/6 Mice (abbreviated as C57BL/6-DKI, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number: SCXK ( ⁇ )2018- 0008) female, 8 weeks old, 6 each, about 20g, breeding environment: SPF level. Temperature 20-25°C, humidity 40-60%. After raising these two strains of mice in a laboratory environment for 3 days, the mice in good condition were randomly divided into groups with 3 mice in each group.
  • the test drug Ab2374 was injected subcutaneously into the back of mice with an injection volume of 200 ⁇ l. The dosage is 20mg/kg/head.
  • Blood was taken from the orbit after injection of mice. Time points are 0, 0.5, 1, 2, 6, 24, 31, 48, 56, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408 , 432 hours. Centrifuge the blood sample, take the supernatant, and store at -20°C for testing. After collecting blood samples, ELISA was used to detect the content of blood samples at various time points. specifically:
  • PD-1 detection method Pave a 96-well plate with PD-1 protein (corning, catalog number 3590). The milk is sealed for 2 hours, and the plate is washed for later use. The standard curve was prepared with Ab2374, the serum to be tested was diluted in proportion and added to the processed 96-well plate. After reacting at 37°C for 1 hour, the plate was washed, and the diluted enzyme-labeled antibody Peroxidase AffiniPure Goat Anti-Human IgG (Jackson, catalog number 109-035-003) was added. After half an hour, the plate was washed, TMB (Surmodic, article number TTMB-1000-01) was added for color development, and terminated with sulfuric acid, and the OD450 was measured by a microplate reader.
  • TMB Purmodic, article number TTMB-1000-01
  • PD-1/LAG-3 (sandwich ELISA) detection method Pave a 96-well plate (corning, catalog number 3590) with PD-1 protein. The milk is sealed for 2 hours, and the plate is washed for later use. The standard curve was prepared with Ab2374, the serum to be tested was diluted in proportion and added to the processed 96-well plate. After reacting at 37°C for 1 hour, the plate was washed, and a fixed concentration of biotinylated LAG-3 protein was added. After reacting at 37°C for 1 hour, the plate was washed, and the diluted enzyme-labeled antibody streptavidin-HRP (genscript, catalog number M00091) was added. After half an hour, the plate was washed, TMB (Surmodic, article number TTMB-1000-01) was added for color development, and terminated with sulfuric acid, and the OD450 was measured by a microplate reader.
  • TMB Purmodic, article number TTMB-1000-01
  • Sandwich ELISA detects the PK characteristics of LAG-3 molecules after binding to PD-1.
  • the results in Table 20 show that, unexpectedly, the PK characteristics (Cmax, T 1/2 ) detected by the sandwich ELISA of the bispecific antibody of the present invention in C57BL/6 mice are almost the same as those obtained by PD-1 detection alone. It shows that the bispecific antibody of the present invention is stable in mice, and there is no scFv (LAG-3) shedding. Moreover, the PK characteristics are similar to those of Ab2317 antibody alone (Table 18).
  • the antibody of the present invention has a specific target binding in hPD-1/hLAG-3 double transgenic mice (C57BL/6-DKI), and Cmax and T 1/2 are reduced.
  • Example 1 of the present invention the above-mentioned DVD-designed bispecific antibodies were cloned, expressed, and purified, respectively.
  • Gel electrophoresis (PAGE) results showed that the light and heavy chains of these antibodies are prone to interlinker breakage.
  • one target antibody is connected to the N or C-terminus of the light chain or heavy chain of another target antibody, and the sequence and design of the optimized bispecific antibody can be obtained by screening, which can avoid/reduce the linker break ( See the previous example), and retain the binding activity to the dual target, the in vitro functional activity, and the in vivo efficacy against the dual target.
  • the preferred bispecific antibody (called SBody in the present invention) is not only stable, but also because of its similar structure to normal IgG, the purification process is simple, which provides great convenience for the process and purification in the later development process.

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Abstract

本发明公开了一种LAG-3结合蛋白，其包括轻链可变区和/或重链可变区；所述轻链可变区包含如SEQ ID NO:5所示的氨基酸序列的CDR1，如SEQ ID NO:6所示的氨基酸序列的CDR2，和/或，如SEQ ID NO:7所示的氨基酸序列的CDR3；所述重链可变区包含：如SEQ ID NO:8所示的氨基酸序列的CDR1，如SEQ ID NO:9所示的氨基酸序列的CDR2，和/或，如SEQ ID NO:10所示的氨基酸序列的CDR3。还公开了一种靶向LAG-3的双特异性抗体及其应用。上述LAG-3结合蛋白和双特异性抗体能有效地阻断LAG-3与MHC II的结合，并激活T细胞。

Description

靶向LAG-3的抗体和双特异性抗体及其用途
本申请要求申请日为2019年6月13日的中国专利申请号201910510292.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，具体涉及一种靶向LAG-3的抗体和双特异性抗体及其用途。
背景技术
Lymphocyte-activation gene 3(LAG-3，淋巴细胞活化基因-3)属于免疫球蛋白超家族，由胞外区、跨膜区和胞质区3个部分组成。LAG-3的基因定位于12号染色体(12P13)，与CD4分子在染色体上的定位和结构相似。LAG-3在活化的T细胞、耗竭的T细胞、肿瘤浸润性T细胞和调节性T细胞(Treg)上表达。在与主要组织相容性复合物2(MHC II类)结合后，LAG-3/MHC II类相互作用导致T细胞增殖、活化以及体内平衡的负调节。抑制LAG-3能够解除LAG3对MHCII-多肽-T细胞受体抗原递呈的抑制，并让T细胞重新获得细胞毒性，从而增强对肿瘤的杀伤效果。同时抑制LAG-3还能够降低调节T细胞抑制免疫反应的功能。因此，LAG-3被认为是一个比其它免疫检查点蛋白更吸引人的靶点。
LAG-3是目前免疫检查点二代靶点中，临床数据较多、成药性相对确定的靶点，针对该靶点的抗体药物将来有可能成为重要的抗肿瘤药物。然而，目前以LAG-3为靶点全球尚无药物上市。至2018年年末，临床在研的药物共计30个，处于临床研究阶段的LAG-3抗体包括葛兰素史克的GSK2831781、诺华的LAG525、再生元的REGN3767和Tesaro的TSR-033。LAG-3抗体药物大多致力于开发与PD-1的联合疗法。其中研发进展最快的是BMS和小野公司开发处于II/III临床阶段的Relatlimab；处于II期临床IMP321和I/II期临床的LAG525；处于I期临床试验阶段有8个药物，处于临床前试验阶段有9个药物，以LAG-3为靶点的药物主要治疗领域包括癌症和自身免疫性疾病。百时美施贵宝的Relatlimab(研发代码为BMS-986016，最初由Medarex开发)。MacroGenics的MGD013是PD-1/LAG-3双特异性抗体，在血清中的半衰期很长，通过同时阻断这两个免疫检查点分子的免疫抑制，它具有治疗多种不同癌症的潜力。
然而，目前临床上仍缺乏热稳定性更好、T细胞活性更好并且有效阻断LAG-3与 MHC II结合，动物药效更好、PK更好的靶向LAG-3的抗体，以及结构简单，分子稳定，工艺简单针对LAG-3的双特异性抗体。
发明内容
为了克服本领域缺乏更稳定、活性更好的靶向LAG-3的抗体和缺乏结构简单、分子稳定和工艺简单的靶向LAG-3的双特异性抗体的缺陷，提供了一种靶向LAG-3的抗体和双特异性抗体及用途。本发明的双特异性抗体为序列特异的IgG结构类似的双特异性抗体(Sequence-based IgG like bispecific antibody,SBody)，本专利将称这种设计称为SBody。
本发明通过优化设计和筛选，提供一种靶向LAG-3的抗体，该抗体具有新的轻链可变区CDR1、CDR2和CDR3及重链可变区CDR1、CDR2和CDR3，其热稳定性好、T细胞活性更好，能更有效阻断LAG-3与MHC II结合，更好的体内药效和PK特性。还提供一种靶向LAG-3的双特异性抗体，其能同时靶向LAG-3和另外一个靶点；新的设计能实现一个分子同时针对两个特异靶点作用，达到一个分子代替两个分子联合，甚至有协同治疗肿瘤的效果。一个分子的工艺，生产成本，临床试验等要比两个单独的分子联合具有更便捷，成本更低等优势。
为解决上述技术问题，本发明的技术方案之一为：一种LAG-3结合蛋白，其包括轻链可变区和/或重链可变区；所述轻链可变区包含：如SEQ ID NO:5所示的氨基酸序列的CDR1，如SEQ ID NO:6所示的氨基酸序列的CDR2，和/或，如SEQ ID NO:7所示的氨基酸序列的CDR3；所述重链可变区包含：如SEQ ID NO:8所示的氨基酸序列的CDR1，如SEQ ID NO:9所示的氨基酸序列的CDR2，和/或，如SEQ ID NO:10所示的氨基酸序列的CDR3。
如上所述的LAG-3结合蛋白，所述轻链可变区的J基因区选自以下组的一种：hJK1、hJK2.1、hJK2.2、hJK2.3、hJK2.4、hJK3、hJK4.1、hJK4.2、hJK5；优选hJK4.1；
和/或，所述重链可变区的以下J基因区选自以下组的一种：hJh1、hJh2、hJh3.1、hJh3.2、hJh4.1、hJh4.2、hJh4.3、hJh5.2、hJh6.1、hJh6.2、hJh6.3；优选hJh4.1。
在一些优选的具体实施例中，如上所述的LAG-3结合蛋白，所述轻链可变区包含如SEQ ID NO:3、SEQ ID NO:23-29或其突变所示的氨基酸序列；和/或，所述重链可变区包含如SEQ ID NO:4、SEQ ID NO:30-37或其突变所示的氨基酸序列；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性，并且所述突变保持或改善了所述抗体对LAG-3的结合。
优选地，所述轻链可变区包含如SEQ ID NO:3所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:4所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:23所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:30所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:23所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:24所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:26所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:27所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:29所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:32所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:26所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:32所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:30所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:33所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:34所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:36所示的氨基酸序列；所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:37所示的氨基酸序列。
在一些优选的具体实施例中，如上所述的LAG-3结合蛋白，其是抗体、Fab、Fab’、F(ab’) 2、Fv、scFv、双特异性抗体、多特异性抗体、单域抗体或单区抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。
在一些优选的具体实施例中，如上所述的LAG-3结合蛋白，其为包括人源抗体轻链恒定区和人源抗体重链恒定区的免疫球蛋白。优选地，所述人源抗体轻链恒定区为κ链或者λ链，或，所述人源抗体重链恒定区为hIgG1、hIgG2、hIgG4或其突变；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列 具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性，并且所述突变保持或改善了所述抗体对LAG-3的结合。
在一些优选的具体实施例中，如上所述的LAG-3结合蛋白，其轻链包含如SEQ ID NO:38或SEQ ID NO:39所示的氨基酸序列或其突变；和/或，其重链包含如SEQ ID NO:40或SEQ ID NO:41所示的氨基酸序列或其突变；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性，并且所述突变保持或改善了所述抗体对LAG-3的结合。
优选地，所述轻链的氨基酸序列如SEQ ID NO:38所示；所述重链的氨基酸序列如SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:38所示；所述重链的氨基酸序列如SEQ ID NO:41所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示；所述重链的氨基酸序列如SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示；所述重链的氨基酸序列如SEQ ID NO:41所示。
为解决上述技术问题，本发明的技术方案之二为：一种靶向LAG-3的双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其特征在于，所述第一蛋白功能区为技术方案之一所述的LAG-3结合蛋白；所述第二蛋白功能区为非LAG-3结合蛋白；优选地，所述第一蛋白功能区和第二蛋白功能区分别选自免疫球蛋白、scFv(single chain Fv，也称为单链可变片段)、Fab、Fab’或F(ab’) 2，且所述第一蛋白功能区和第二蛋白功能区中只有一个蛋白功能区为免疫球蛋白。
为了设计生产工艺简单且保留有效活性的双特异性抗体，本发明的双特异性抗体的形式为类似正常IgG的结构，具体地，在其结构上设计能够靶向两个靶点的轻链和/或者重链可变区的蛋白功能区，两个蛋白功能区共享相同的Fc区域。较佳地，将一个靶点的抗体分子以一个或者多个scFv的形式，连到另一靶点完整抗体的轻链或重链的一端。这样既避免表达不同Fc和/或不同轻链带来表达产物的不均一，例如Knob形式的Fc和Hole形式的Fc共表达，其表达过程中会有不均一的Fc-Fc配对形式，给纯化工艺带来很多不便利；也可以避免轻、重链部分区域交换(cross)设计可能对结合活性的影响，以及工艺过程中出现的Fc错配现象。通过一个或者多个scFv设计，还可以调节针对特定靶点的活性。
因此，在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为一个或多个scFv；或，所述第一蛋白功能区为一个或多个scFv，所述第二蛋白功能区为免疫球蛋白，所述免疫球蛋 白的恒定区包括人源抗体轻链恒定区和人源抗体重链恒定区。优选地，所述人抗体轻链恒定区为κ链或者λ链，所述人抗体重链恒定区为hIgG1、hIgG2、hIgG4或其突变；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性，并且所述突变保持或改善了所述抗体对抗原例如LAG-3、PD-1等的结合。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其中，所述scFv包括重链可变区与轻链可变区，所述重链可变区与轻链可变区通过连接子连接，所述scFv通过连接子与所述免疫球蛋白连接，所述连接子优选为(Gly-Gly-Gly-Gly-Ser) w[以下简写为(G 4S) w]；所述的w优选为0～10之间的整数，更优选为1、2、3或者4。所述双特异设计概括(通式1)请参见实施例12之表10。此外，所述连接子还可以选自本领域常规的用作连接子的肽段。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其中，所述scFv为轻链可变区-连接子-重链可变区结构，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区结构，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其中，所述连接子为(G 4S) 3，和/或，所述scFv的数量为两个，且两个scFv对称地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。优选地，所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链可变区的N末端，或两个scFv的轻链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的轻链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链可变区的N末端，或两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链可变区的C末端。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其中，所述第二蛋白功能区靶向PD-1。优选地，所述第二蛋白功能区为抗PD-1抗体。更优选地，所述抗PD-1抗体为Nivolumab或Pembrolizumab。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其选自以下组：
(1)所述第一蛋白功能区为免疫球蛋白，其包含以下轻链和重链或其突变：其中， 所述轻链的氨基酸序列如SEQ ID NO:38所示，所述重链的氨基酸序列如SEQ ID NO:40所示；所述轻链的氨基酸序列如SEQ ID NO:38所示，所述重链的氨基酸序列如SEQ ID NO:41所示；所述轻链的氨基酸序列如SEQ ID NO:39所示，所述重链的氨基酸序列如SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示，所述重链的氨基酸序列如SEQ ID NO:41所示；和/或，
所述第二蛋白功能区为scFv或其突变，其中，所述scFv的轻链可变区的氨基酸序列如SEQ ID NO:42的第1-107位氨基酸序列所示，所述scFv的重链可变区的氨基酸序列如SEQ ID NO:43的第1-113位氨基酸序列所示；或，所述scFv的轻链可变区的氨基酸序列如SEQ ID NO:44的第1-111位氨基酸序列所示，所述scFv的重链可变区的氨基酸序列如SEQ ID NO:45的第1-120位氨基酸序列所示；
所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性，并且所述突变保持或改善了所述抗体对抗原例如LAG-3、PD-1等的结合；
优选地，当所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端由K突变为A；
(2)所述第一蛋白功能区为scFv或其突变，其中，所述scFv的轻链可变区的氨基酸序列如SEQ ID NO:23-29所示；和/或，所述scFv的重链可变区的氨基酸序列如SEQ ID NO:30-37所示；和/或，
所述免疫球蛋白的轻链的氨基酸序列如SEQ ID NO:42所示，所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:43所示；或，所述免疫球蛋白的轻链的氨基酸序列如SEQ ID NO:44所示，所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:45所示；
所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性，并且所述突变保持或改善了所述抗体对抗原例如LAG-3、PD-1等的结合；
优选地，当所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端由K突变为A。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其中，(i)所述第一蛋白功能区为scFv，其轻链可变区的氨基酸序列如SEQ ID NO:28所示，其重链可变区的氨基酸序列如SEQ ID NO:31所示，所述连接子为(G 4S) 3；所述第二蛋白功能区为免疫球蛋白；其轻链的氨基酸序列如SEQ ID NO:42所示，其重链的氨基酸序列如SEQ ID NO:43所示；
其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端，且所述重链的C末端由K突变为A；或，所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的C末端；或，
(ii)所述第一蛋白功能区为scFv，其轻链可变区的氨基酸序列如SEQ ID NO:28所示，其重链可变区的氨基酸序列如SEQ ID NO:31所示，所述连接子为(G 4S) 3；所述第二蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:44所示，其重链的氨基酸序列如SEQ ID NO:45所示；
其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过连接子(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过连接子(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端，且所述重链的C末端由K突变为A；或，所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过连接子(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过连接子(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的C末端；或，
(iii)所述第一蛋白功能区为scFv，所述scFv数量为一对，其轻链可变区的氨基酸序列如SEQ ID NO:28所示，其重链可变区的氨基酸序列如SEQ ID NO:36所示，所述连接子为(G 4S) 3；所述第二蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:44所示，其重链的氨基酸序列如SEQ ID NO:45所示；
其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过连接子(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过连接子(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端，且所述重链的C末端由K突变为A。
在一些优选的具体实施例中，如上所述的靶向LAG-3的双特异性抗体，其包括以下 氨基酸序列：
如SEQ ID NO:44所示的轻链氨基酸序列，如SEQ ID NO:46所示的含重链的氨基酸序列；或，如SEQ ID NO:42所示的轻链氨基酸序列，如SEQ ID NO:47所示的含重链的氨基酸序列；或，如SEQ ID NO:42所示的轻链氨基酸序列，如SEQ ID NO:48所示的含重链的氨基酸序列；或，如SEQ ID NO:49所示的含轻链的氨基酸序列，如SEQ ID NO:43所示的重链氨基酸序列；或，如SEQ ID NO:50所示的含轻链的氨基酸序列，如SEQ ID NO:43所示的重链氨基酸序列。
或者，本发明所述的双特异性抗体为DVD-Ig(Dual-variable domain Ig)双特异性抗体，其结构是在正常抗体轻、重链的N末端分别再接入另一个抗体的VL和VH，通过两个抗体可变区结合双靶点实现双功能。所述双特异性抗体的设计概括(通式2)请参见实施例12之表12。
在一优选的具体实施例中，所述的双特异性抗体由含轻链的序列和含重链的序列组成。所述的双特异性抗体选自以下组合：含轻链的序列为Ab2317VL-(G 4S) 3-NivoVL-Lc(κ链)，含重链的序列为Ab2317VH-(G 4S) 3-NivoVH-Hc(hIgG4)；或，含轻链的序列为PemVL-(G 4S) 3–Ab2317VL-Lc(κ链)，含重链的序列为PemVH-(G 4S) 3–Ab2317VH-Hc(hIgG4)；或，含轻链的序列为NivoVL-(G 4S) 3–Ab2325VL-Lc(κ链)，含重链的序列为NivoVH-(G 4S) 3–Ab2325VH-Hc(hIgG1)。
或者，本发明所述的双特异性抗体为包括第一蛋白功能区和第二蛋白功能区，其中之一的蛋白功能区为免疫球蛋白，另一蛋白功能区为Fab’或F(ab’) 2。
在一优选的具体实施例中，所述的第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为Fab’或F(ab’) 2；或者所述的第一蛋白功能区为Fab’或F(ab’) 2，所述第二蛋白功能区为免疫球蛋白；所述Fab’或F(ab’) 2与所述免疫球蛋白通过二硫键或连接子连接，所述连接子优选本领域常规的可作为连接子的肽段或(Gly-Gly-Gly-Gly-Ser) w，所述w优选为0～10之间的整数，更优选为1、2、3或者4；所述免疫球蛋白的恒定区较佳地为人抗体恒定区，所述人抗体恒定区优选包括人抗体轻链恒定区和人抗体重链恒定区，所述人抗体轻链恒定区优选κ链或者λ链；所述人抗体重链恒定区优选hIgG1、hIgG2或者hIgG4。当所述Fab’或F(ab’) 2的轻链与重链之间通过连接子连接后，因为轻重链之间有连接子，其已不是严格意义上的Fab’或F(ab’) 2。
为解决上述技术问题，本发明的技术方案之三为：一种分离的核酸，其编码如上所述的LAG-3结合蛋白或如上所述的靶向LAG-3的双特异性抗体。
为解决上述技术问题，本发明的技术方案之四为：一种包含如上所述的分离的核酸 的表达载体。
为解决上述技术问题，本发明的技术方案之五为：一种宿主细胞，其包含如上所述的表达载体。优选地，所述宿主细胞是原核细胞或真核细胞。
为解决上述技术问题，本发明的技术方案之六为：一种LAG-3结合蛋白或靶向LAG-3的双特异性抗体的制备方法，其包含培养如上所述的宿主细胞，从培养物中获得LAG-3结合蛋白或靶向LAG-3的双特异性抗体。
为解决上述技术问题，本发明的技术方案之七为：一种抗体药物偶联物，其包含细胞毒性剂，以及如上技术方案之一所述的LAG-3结合蛋白或如上技术方案之二所述的靶向LAG-3的双特异性抗体。
为解决上述技术问题，本发明的技术方案之八为：一种药物组合物，其包含如上所述的LAG-3结合蛋白，或如上所述的靶向LAG-3的双特异性抗体，或如上所述的抗体药物偶联物。
为解决上述技术问题，本发明的技术方案之九为：一种药盒组合，其包含药盒A和药盒B；所述药盒A包括如上所述的LAG-3结合蛋白，或如上所述的靶向LAG-3的双特异性抗体，或如上所述的抗体药物偶联物，或如上所述的药物组合物；所述药盒B包含其它治疗癌症的药物。
为解决上述技术问题，本发明的技术方案之十为：如上所述的LAG-3结合蛋白、靶向LAG-3的双特异性抗体、抗体药物偶联物、药物组合物和/或药盒组合在制备治疗和/或预防癌症的药物中的应用。
优选地，所述的癌症选自由白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、膀胱癌、尿路上皮癌、非小细胞肺癌、肺腺癌、肺鳞状细胞癌、支气管癌、骨癌、前列腺癌、胰腺癌、胃癌、肝细胞癌、胆囊癌、胆管癌、食管癌、肾细胞癌、甲状腺癌、头颈鳞状细胞癌、睾丸癌、内分泌腺癌、肾上腺癌、脑垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽癌、宫颈癌、子宫癌、成胶质细胞瘤、髓母细胞瘤、星形细胞瘤、神经胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤，黑素瘤、骨髓增生异常综合征和肉瘤组成的群组。
由此，本发明还提供了一种癌症的治疗方法，所述方法为使用如上所述的LAG-3结合蛋白、靶向LAG-3的双特异性抗体、抗体药物偶联物、药物组合物和/或药盒组合来治疗患有上述癌症的患者。
应了解，本发明“第一”、“第二”均无实际意义，仅为区分相同的术语。在提及scFv或细胞因子或细胞因子受体或Fab’或F(ab’) 2的数量时，“一对”和“两个”、“两对”和 “四个”具有相同的含义。在提及轻链或重链或轻链可变区或重链可变区的数量时，“一个”和“一条”、“两个”和“两条”具有相同的含义。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”，通常是指由两对多肽链[每对具有一条轻(L)链和一条重(H)链]组成的免疫球蛋白。从一般意义上，重链可以理解为抗体中分子量较大的多肽链，轻链是指抗体中分子量较小的多肽链。轻链可分类为κ和λ轻链。重链通常可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域[称为互补决定区(CDR)]，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末排列的3个CDR和4个FR组成。各重链/轻链对应的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat EA.Et al.,Sequences of Proteins of Immunological Interest[National Institutes of Health,Bethesda,Md.(1987and 1991)]，或Chothia&Lesk 1987)].Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:877-883的定义。特别地，重链还可以包含3个以上CDR，例如6、9或12个。例如在本发明的双特异性抗体中，重链可以是IgG抗体的重链的N端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见Fundamental Immunology,Ch.7，Paul, W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab’、F(ab’) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链结合片段(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段；术语“Fab”意指由VL、VH、CL和CH1(或者CH)结构域组成的抗体片段；术语“F(ab’) 2”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链结合片段(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见例如Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)]。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的G 4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G 4S) 4或(G 4S) 3接头，但也可使用其变体。
可使用本领域技术人员已知的常规技术(例如重组DNA技术或酶促或化学断裂法)从给定的抗体获得抗体的抗原结合片段(例如上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌等原核细胞，如酵母细胞等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“KD”是指特定抗体一抗原相互作用的解离平衡常数(KD)，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体一抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 - 6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数结合抗原，例如，如使用表 面等离子体共振术(SPR)在BIACORE仪中测定的。例如用KINEXA方法在KINEXA 400仪器上检测到的抗体和细胞结合的亲和力。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
(1)LAG-3结合蛋白更加有效阻断LAG-3与MHC II的结合，从而解除负调控APC/T细胞之间的信号转导与抗原递呈；
(2)所得人源化LAG-3抗体显示很好的细胞功能活性，例如很好地激活人T细胞、人DC细胞功能与活性，动物药效，更好的PK特征，高表达产量，和热稳定性；
(3)序列特异的双特异性抗体(SBody)针对双靶活性好，稳定，表达量高，结构类似正常IgG抗体，纯化工艺简单。
附图说明
图1为本发明抗人LAG-3抗体mab23c的功能与活性：a.mab23c和人LAG-3的结合活性(ELISA)；b.mab23c阻止LAG-3和Daudi细胞结合(blocking)活性。
图2为凝胶电泳(PAGE)图，用于评价本发明抗人LAG-3人源化抗体Ab2317热稳定性。
图3为本发明抗人LAG-3人源化抗体Ab2317的功能与活性：a.Ab2317激活人T细胞活性；b.Ab2317激活人DC细胞刺激人T细胞活性(MLR assay)。
图4为本发明抗人LAG-3人源化抗体Ab2317动物体内药效评估。
图5A为本发明双特异性抗体(SBody)LB2373结构及相关检测数据图，包括结构示意图(c)，和人LAG-3的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图5B为本发明双特异性抗体(SBody)LB2374结构及相关检测数据图，包括结构示意图(c)，和人LAG-3的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图5C为本发明双特异性抗体(SBody)LB2371结构及相关检测数据图，包括结构示意图(c)，和人LAG-3的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图5D为本发明双特异性抗体(SBody)LB2372结构及相关检测数据图，包括结构示意图(c)，和人LAG-3的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
具体实施方式
以下结合实施例进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：抗原、抗体的克隆、表达和纯化
本发明所用的抗原或购自如下不同公司：北京百普赛斯生物科技有限公司LAG-3-his(货号：LA3-H5222)，cyno-LAG-3-mFc(货号LA3-C52A0)或北京义翘神州科技有限公司LAG-3-his(货号：16498-H08H)，LAG-3-hFc(货号：16498-H05H)，cyno-LAG-3-his(货号90841-C08H)，或由本发明表达纯化得到。表达的人LAG-3蛋白序列为NCBI Reference Sequence:NP_002277.4，全长1-525氨基酸，其中1-22位为信号肽；胞外(ECD)区域为23-422位氨基酸；胞外(ECD)区域23-239位氨基酸的第一，第二区域(domain#1and#2，D12)D12-his,D12-hFc。鼠LAG-3his tagged(mLAG-3-his)的序列来自NCBI gi|148667361|gb|EDK99777.1的胞外区(ECD)23-406位氨基酸。Macaca LAG-3his tagged(cynoLAG-3-his)的序列NCBI号NP_001271679.1，其胞外区(ECD)23-434位氨基酸。
人PD-1(hFc/his tag)蛋白序列为NCBI Reference Sequence:NP_005009.2，全长288个氨基酸，其中第1-20位为信号肽；ECD为第21-167位氨基酸。
人PD-L1(hFc/his tag)蛋白序列为NCBI Reference Sequence:NP_054862.1，全长290个氨基酸，其中第1-18位为信号肽；ECD为第19-239位氨基酸。
本发明所用抗体，包括阳性对照抗体Ref(即BMS-986016，序列来自WO2014008218A1，LAG3.5，#12轻链，#14重链)，和PD-1抗体nivolumab(序列来自WO2013019906)，Pembrolizumab(序列从www.drugbank.ca得到，Accession Number：DB09037)均由本发明表达纯化。
表达所用pTT5载体(Biovector，Cat#:102762)。将所表达的重组蛋白，抗体轻、重链序列克隆到pTT5载体上，经瞬时转染HEK293E细胞(Life Technologies Cat.No.11625019)表达，后纯化得到。
具体地，293细胞在Gibco FreeStyle 293 Expression Medium(Gibco，Cat#12338018)培养基扩培。瞬转开始之前，调节细胞浓度至6～8×10 5cell/ml，1％FBS(Aus Gene X FBS Excellent供应商：AusGeneX，China，Cat#FBSSA500-S)，37℃8％CO 2摇床培养24h，再次镜检存活率>95％，细胞浓度在1.2×10 6cells/ml。
准备300ml培养体系细胞，15ml Opti-MEM(Gibco，Cat#31985070)溶入重链、轻链质粒各150μg(如果是重组蛋白，单个质粒用量为300μg)，0.22μm过滤除菌。再取 15ml Opti-MEM溶入1mg/ml PEI(Polysciences,Inc,Cat#23966-2)600μl后静置5min。把PEI缓慢加入质粒中，室温孵育10min，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，37℃8％CO 2摇床培养5天收样，3300g 10min取上清进行纯化。
抗体或-Fc融合蛋白纯化：将样品高速离心去除杂质，用PBS pH 7.4平衡含有Protein A(Mabselect，GE Healthcare Life Science，Cat#71-5020-91AE)的重力柱(生工生物，Cat#F506606-0001)，2-5倍柱体积冲洗。将样品过柱。用5-10倍柱体积的PBS(生工生物，Cat#B548117-0500)冲洗柱子。再用pH 3.5 0.1M乙酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，酶标仪测定浓度，分装、储存备用。
His Tagged蛋白纯化：将样品高速离心去除杂质。平衡镍柱(Ni smart beads 6FF常州天地人和生物科技有限公司Cat#SA036010)：用含有10mM咪唑0.5M NaCl的PBS pH7.4溶液平衡镍柱，2-5倍柱体积冲洗。将样品上清过柱。漂洗杂蛋白：使用含有10mM咪唑0.5M NaCl的PBS pH7.4溶液冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。用含有250mM咪唑0.5M NaCl的PBS pH7.4洗脱目的蛋白。buffer置换：将洗脱的目的蛋白过超滤管12000g 10min离心(超滤管Merck Millipore Cat#UFC500308)，再补加1ml PBS，测定浓度，分装、储存备用。
本发明所用hFc tag均是在C端连接IgG1Fc区域，his tag为在C端连接6×his。
实施例2：人LAG-3高表达细胞株(hLAG-3+细胞)构建及结合活性(ELISA)检测
本发明所用的人LAG-3高表达细胞株通过公司稳定细胞株构建平台完成。具体步骤如下：实验开始第1天，将293T细胞(中国科学院典型培养物保藏委员会细胞库Cat#GNHu17)接种于两个6cm培养皿，每个培养皿里的细胞数达到7.5×10 5。第2天将包裹质粒(pGag-pol,pVSV-G等BioVector质粒载体菌种细胞基因保藏中心)和克隆有人LAG-3基因的质粒pBabe-hLAG-3各4μg加入OPTI-MEM(Thermofisher Scientific Cat#31985070)，使最终体积为200μl，另准备200μl OPTI-MEM加入36μl转染试剂fectin(上海源培生物科技股份有限公司Cat#F210)，二者混匀，室温放置5min，然后将混合物(每皿各200μl)滴加入培养好的293T细胞。第3天将293T细胞培养液换为4ml DMEM高糖培养基(上海源培生物科技股份有限公司/源培生物：Cat#L130KJ)。第4天将CHO-K1细胞(中国科学院典型培养物保藏委员会细胞库Cat#SCSP-507)接种于10cm培养皿，使细胞数达到5×10 5。第5天收集293T细胞上清(病毒)，用0.45μm滤膜过滤至培养好的CHO-K1细胞，同时加入10μg/ml polybrene(上海翊圣生物科技有限公司Cat#40804ES76)，混匀后放置培养箱，3～4h后换成DMEM/F12 10％FBS培养基(源培生 物，Cat#L310KJ)。第7天将CHO-K1细胞传代，第8天传代的细胞开始加入10μg/ml puromycin进行筛选(源培生物，Cat#S250J0)。2-3天细胞大量死亡，更换培养基继续培养，直到细胞不再死亡时，细胞大量扩增，筛选单克隆细胞株，扩培，冻存保种。
本实施例所用的人LAG-3(pBabe-hLAG-3)的氨基酸序列NP_002277.4之全序列1-525氨基酸，其中1-22位为信号肽序列，即第23-525位为本发明构建CHO-K1 hLAG-3+细胞系所表达的蛋白序列。
hLAG-3+细胞结合活性(ELISA)检测：
将上述实施例得到的细胞人LAG-3高表达的单克隆细胞株扩培后，按1×10 5/孔铺96孔板，37℃培养箱过夜孵育后去除上清，用免疫染色固定液(上海碧云天生物技术有限公司Cat#P0098)100μl/孔，室温固定半小时。PBS(源培生物，Cat#B320)洗一遍后230μl 5％牛奶37℃封闭2小时，PBST洗3遍。每孔加入50μl，10μg/ml，5倍梯度稀释的待测样品。37℃孵育1小时，后PBST洗5遍。加Anti-human HRP(Jackson Immuno Research，Cat#109-035-003)1:2500 50μl/孔37℃孵育1小时，后PBST洗5遍，每孔加入50μl TMB(Surmodic Cat#TTMB-1000-01)显色，加入50μl/孔1M H 2SO 4终止反应。酶标仪(MultiskanGO Thermo型号51119200)读数，Graphpad prism 5进行数据分析。
实施例3：抗LAG-3抗体和LAG-3抗原结合实验(ELISA)
用pH7.4的PBS缓冲液将LAG-3-his，LAG-3-D12-his，猴LAG-3-his(cyno LAG-3-his)或者mLAG-3-his等重组蛋白稀释至1μg/ml，2μg/ml(hLAG-3 D12-his),或者5μg/ml(cyno LAG-3-his)浓度，以50μl/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液230μl/孔，37℃孵育箱孵育3小时或4℃放置过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(PH7.4 PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔上清(含检测抗体)或10μg/ml起始，5倍梯度稀释的待测抗体，37℃孵育1小时，PBST洗板5次，加入50μl/孔1:2500稀释的Anti-mouse或human HRP二抗(Jackson Immuno Research，Cat#115-035-003或109-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，室温孵育10-15min，加入50μl/孔1M H 2SO 4终止反应，用MULTISKAN GO酶标仪(ThermoFisher,51119200)在450nm处读取吸收值，根据OD值挑选结合活性高的克隆或者计算EC 50值(对浓度已知的抗体)。
实施例4：抗LAG-3抗体阻止LAG-3和Daudi细胞结合活性实验
将Daudi细胞株(ATCC,CCL-213)扩培后，按2x10 5/孔铺96孔板，1600rpm离心10min后，用免疫染色固定液(上海碧云天生物技术有限公司Cat#P0098)100μl/孔室温固定半小时。PBS(源培生物，Cat#B320)洗一遍后230μl 5％牛奶37℃封闭2小时，PBST洗3遍。每孔加入25μl，100μg/ml，3倍梯度稀释的待测样品和25μl 2.5μg/ml的bio-LAG3-mFc(北京义翘生物科技有限公司，16498-H05H)。37℃孵育1小时，后PBST洗5遍。加入50μl/孔1:1000稀释的streptavidin-HRP二抗(genscript，M00091)，37℃孵育1小时，后PBST洗5遍，每孔加入50μl TMB(Surmodic Cat#TTMB-1000-01)显色，加入50μl/孔1M H 2SO 4终止反应。酶标仪(MultiskanGO Thermo型号51119200)读数，Graphpad prism 5进行数据分析。
实施例5：抗人LAG-3抗体的发现
本发明用人LAG-3重组蛋白(实施例1准备)作为抗原，免疫小鼠、筛选融合杂交瘤，从数百万株杂交瘤克隆中筛选、优化，意外地发现一株和hLAG-3结合活性非常好的杂交瘤细胞株。对该细胞株进一步亚克隆筛选得到单克隆细胞株，从单克隆细胞株中得到的鼠源抗体序列，经计算机建模人源设计筛选后得到优化人源化抗体。人源化抗体同样保持和hLAG-3很好的结合活性，并且非常意外地，所得人源化抗体还显示很好的细胞功能活性、动物药效、更好的PK特征、高表达产量和热稳定性。
具体地，实验用SJL小鼠，雌性，4周龄购自北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2016-0011。小鼠购进后，实验室环境饲养1周，白天光/夜晚暗周期调节，温度20-25℃；湿度40-60％。小鼠分成3只/组/笼。用实施例1准备的抗原进行免疫。佐剂为Quickantibody-5w(北京博奥龙免疫技术有限公，KX0210041)。抗原与佐剂比例为1:1。100μl/10μg/只首免，100μl/10μg/只二免、三免，四免，五免小腿肌肉注射。融合前3天，100μl/25μg/只加强免疫。免疫时间为第0、14、28、46、58天和60天(加强免疫)。于第23、50、58天，用上述实施例3的ELISA方法检测小鼠血清抗体滴度，选择血清中抗体滴度高并且滴度处于平台期的小鼠进行脾细胞融合，将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2020095577-appb-000001
CRL-8287 TM)进行融合得到杂交瘤细胞铺96孔板。
用实施例3的ELISA方法对所铺96孔板杂交瘤细胞株进行初次筛选，检测杂交瘤细胞株分泌上清中的抗体和人LAG-3的结合活性，选择活性好的克隆，取其上清用实施例4所述方法检测所分泌的抗体阻止hLAG-3和Daudi细胞的结合(Blocking)活性，优选结合活性和Blocking活性好的克隆，进一步有限稀释优选所得单克隆抗体细胞株，部分结果见下表。
表1杂交瘤融合筛选单克隆细胞活性
Figure PCTCN2020095577-appb-000002
Figure PCTCN2020095577-appb-000003
#：终止，即blocking活性不好的克隆终止，不进行亚克隆筛选；ND:没有检测，即终止的克隆没有后续检测。##：NA，不适应，即亚克隆从之前的母克隆而来。具体到本表中，母克隆指编号#15的克隆7B9。
表1列出了部分筛选数据。数据显示，在融合杂交瘤初始筛选结合活性好，且blocking活性也很好的克隆，比如表中编号#15，克隆7B9(ELISA数值越高，表明结合活性越好。Blocking活性数值越低，则表明blocking活性越好)。对#15，克隆7B9进行多次有限稀释，每一次稀释(亚克隆)后7-10天待克隆增殖后，用ELISA方法重新检测各个亚克隆所分泌抗体(上清)的结合活性和blocking活性。初始筛选中blocking活性不好的克隆均丢弃，例如上表中的编号1-14均丢弃，不再做后续亚克隆和检测。
编号15的克隆7B9经过多次有限稀释后，意外发现，筛选得到的单克隆细胞株7B9G8G3G2D6(编号23)所分泌上清保持了较好的结合活性和最好的Blocking活性(同等条件相检测所得数值最低,即0.8035)。从编号23的单克隆细胞株中提取抗体序列，得到本发明优选鼠源抗人LAG-3的抗体mab23轻、重链序列。
实施例6：本发明鼠源抗人LAG-3抗体mab23抗体序列提取、分析鉴定
从杂交瘤优选得到的单克隆细胞株中提取抗体序列过程为本领域技术人员常用的方法。具体地，收集上述单克隆细胞株，扩增培养后，取1x 10 6个细胞，用Trizol(Invitrogen，15596-018)提取RNA(按照试剂盒说明书步骤)，将提取的RNA并反转录成cDNA，反 转录试剂盒购自生工生物技术(上海)股份有限公司，Cat#B532435。以反转录得到的cDNA为模板，进行PCR扩增。扩增产物测序，分别得到mab23抗体轻、重链可变区碱基/编码序列(如下)。所用引物参阅Novagen发表的手册TB326 Rev.C0308。
本发明优选的杂交瘤细胞株中获得的鼠源单克隆抗体mab23轻链可变区碱基序列(划线部分为编码序列)：
Figure PCTCN2020095577-appb-000004
本发明优选的杂交瘤细胞株中获得的鼠源单克隆抗体mab23重链可变区碱基序列(划线部分为编码序列)：
Figure PCTCN2020095577-appb-000005
本发明所得到的上述鼠源单克隆抗体mab23轻、重链可变区的碱基序列所编码的氨基酸序列为如下SEQ ID NO:3和SEQ ID NO:4。本发明优选的杂交瘤单克隆细胞株中获得的鼠源单克隆抗体mab23轻链可变区氨基酸序列：
Figure PCTCN2020095577-appb-000006
本发明优选的杂交瘤细胞单克隆株中获得的鼠源单克隆抗体mab23重链可变区氨基酸序列:
Figure PCTCN2020095577-appb-000007
将上述抗体轻、重链可变区序列和IgG不同型的恒定区，例如人hIgG1、hIgG2、 hIgG3、hIgG4，人轻链κ、λ型；鼠mIgG1、mIgG2、mIgG3，鼠轻链κ、λ型等重组表达纯化得到完整的人鼠嵌合抗体，鼠抗体。以人重链恒定区为hIgG4、人轻链κ型为例，按实施例1表达纯化方法得到嵌合抗体mab23c，用实施例3、4之方法检测了mab23c和hLAG-3的结合活性、阻止LAG-3和Daudi细胞结合活(blocking活性)，并和对照抗体(Ref)平行比较(参见图1的a与b)。
结果表明，本发明mab23c和对照抗体(Ref)不同，ELISA检测到mab23c的EC 50和Emax分别为0.41nM和1.7，比对照抗体(Ref)的0.64nM和1.4要好。特别意外的是，本发明mab23c阻止LAG3和Daudi细胞结合活性(blocking活性)IC 50为2.3nM，这比同条件下Ref检测到的IC 50(10.5nM)好近4倍(图1b)。Blocking活性好直接和该分子作为治疗抗体的药效效果正相关。因此mab23c突出的blocking活性使本发明抗体作为药物开发的候选分子具有更好更高的价值。
实施例7:本发明鼠源抗体mab23c人源化
本发明意外发现的抗体mab23c(嵌合抗体)具有和特异抗原hLAG-3强结合活性，特别是意想不到的阻断(blocking)活性。显示所述抗体可用于针对LAG-3靶点的肿瘤治疗单克隆抗体药物开发具有更好价值，比如可能更好的药效。为了避免用于药物开发过程中的免疫原性等方面的风险，本发明对鼠源mab23抗体进行了人源化设计和筛选，以及序列优化。具体过程描述如下。
抗体的CDR定义本领域还有多种不同的方法，这些标记CDR方法可总结如下表。
表2本领域抗体CDR定义不同方法汇总*
Loop CCG定义 Kabat定义 AbM定义 Chothia定义 Contact定义
轻链CDR1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36
轻链CDR2 L50-L56 L50-L56 L50-L56 L50-L56 L45-L55
轻链CDR3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96
重链CDR1 H26-H35 H31-H35 H26-H35 H26-H32 H30-H35
重链CDR2 H50-H65 H50-H65 H50-H58 H52-H56 H47-H58
重链CDR3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101
*更多信息可以参阅网站：http://www.bioinf.org.uk/abs/#cdrdef
本发明鼠源抗人hLAG-3抗体mab23可变区按上述照表所列各种定义方法，对其CDR序列标记/注释如下。
表3本发明抗hLAG-3(anti-hLAG-3)抗体mab23按CCG定义的CDR序列
抗体 mab23 CDRs
轻链CDR1 RASQDIGSSLN(SEQ ID NO:5)
轻链CDR2 ATSSLDS(SEQ ID NO:6)
轻链CDR3 LQYVTSPLT(SEQ ID NO:7)
重链CDR1 GYTFTDYEMH(SEQ ID NO:8)
重链CDR2 GIDPETEGIAYNQKFRG(SEQ ID NO:9)
重链CDR3 SNYYGGREAWFAY(SEQ ID NO:10)
表4本发明抗hLAG-3(anti-hLAG-3)抗体mab23按Kabat定义的CDR序列
抗体 mab23 CDRs
轻链CDR1 RASQDIGSSLN(SEQ ID NO:5)
轻链CDR2 ATSSLDS(SEQ ID NO:6)
轻链CDR3 LQYVTSPLT(SEQ ID NO:7)
重链CDR1 DYEMH(SEQ ID NO:11)
重链CDR2 GIDPETEGIAYNQKFRG(SEQ ID NO:9)
重链CDR3 SNYYGGREAWFAY(SEQ ID NO:10)
表5本发明抗hLAG-3(anti-hLAG-3)抗体mab23按AbM定义CDR序列
抗体 mab23 CDRs
轻链CDR1 RASQDIGSSLN(SEQ ID NO:5)
轻链CDR2 ATSSLDS(SEQ ID NO:6)
轻链CDR3 LQYVTSPLT(SEQ ID NO:7)
重链CDR1 GYTFTDYEMH(SEQ ID NO:8)
重链CDR2 GIDPETEGIA(SEQ ID NO:12)
重链CDR3 SNYYGGREAWFAY(SEQ ID NO:10)
表6本发明抗hLAG-3(anti-hLAG-3)抗体mab23按Chothia定义CDR序列
抗体 mab23 CDRs
轻链CDR1 RASQDIGSSLN(SEQ ID NO:5)
轻链CDR2 ATSSLDS(SEQ ID NO:6)
轻链CDR3 LQYVTSPLT(SEQ ID NO:7)
重链CDR1 GYTFTDY(SEQ ID NO:13)
重链CDR2 DPETEG(SEQ ID NO:14)
重链CDR3 SNYYGGREAWFAY(SEQ ID NO:10)
表7本发明抗hLAG-3(anti-hLAG-3)抗体mab23按Contact定义CDR序列
抗体 mab23 CDRs
轻链CDR1 GSSLNWL(SEQ ID NO:15)
轻链CDR2 KRLIYATSSLD(SEQ ID NO:16)
轻链CDR3 LQYVTSPL(SEQ ID NO:17)
重链CDR1 TDYEMH(SEQ ID NO:18)
重链CDR2 WIGGIDPETEGIA(SEQ ID NO:19)
重链CDR3 TNSNYYGGREAWFA(SEQ ID NO:20)
对本发明鼠源抗体mab23的CDR序列做上述分析、标记、定义后，如本领域许多文献公示的方法进行人源化。将鼠源抗体序列和人抗体种系数据库(v-base)比较，找出同源性高的人抗体轻、重链种系，在此基础上，计算机建模，模拟抗体结构中可能影响和抗原结合的位点，回复突变关键位点和组合，筛选出活性优选的人源化抗体分子。回复突变(back mutation)又称反向突变(reverse mutation)，即将人源化抗体的特定的氨基酸残基突变成原始来源抗体对应位置的氨基酸残基。
具体地，通过序列同源性比较分析，发现和mab23轻链同源性比较好的人抗体种系包含有IGKV1-16*01、IGKV1-17*01、IGKV1-39*01、IGKV1-NL1*01、IGKV1/OR-2*01，IGKV1/OR-3*01、IGKV1/OR-4*01、IGKV1/OR10-1*01、IGKV1/OR2-1*01、IGKV1/OR2-2*01等。进一步比较、分析，优选人抗体种系轻链IGKV1-39*01。序列比对发现mab23轻链的J基因区和人抗体种系hJK1、hJK2.1、hJK2.2、hJK2.3、hJK2.4、hJK3、hJK4.1、hJK4.2、hJK5同源性高，进一步比较、分析，优选hJK4.1用于mab23轻链人源化人抗体 种系J区，进行人源化设计、筛选和序列优化。
通过序列同源性比较分析，发现和mab23重链同源性比较好的人抗体种系包含有IGHV1-69*02、IGHV1-69*04、IGHV1-69*06、IGHV1-69*08、IGHV1-69*09、IGHV1-69*10、IGHV1-69*14、IGHV1-69*17、IGHV1-18*01、IGHV1-18*03等。进一步比较、分析，优选人种系重链IGHV1-18*01序列用于本发明抗体人源化。序列比对发现和mab23的重链J基因区和人抗体种系重链J基因hJh1、hJh2、hJh3.1、hJh3.2、hJh4.1、hJh4.2、hJh4.3、hJh5.2、hJh6.1、hJh6.2、hJh6.3等同源性高，进一步比较、分析，优选hJh4.1用于本发明鼠源抗体mab23重链人源化人抗体种系J区，进行人源化设计、筛选和序列优化。
将本发明抗体以mab23的CDR区(见上述CDR之定义)移植到所选择的人源化轻、重链人抗体种系模板上，再与IgG轻、重链恒定区重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，筛选这些突变以及突变组合，看对抗体活性的影响，并对CDR区化学不稳定氨基酸残基优化，得到结构、活性等优化的抗体分子序列，即完成本发明鼠源抗体的人源化。
以下结合mab23的具体序列，以hIgG4重链，κ型轻链(序列如下)为例进行说明。
人抗体轻链恒定区κ链：SEQ ID NO:21；人IgG4的重链恒定区：SEQ ID NO:22
本发明抗人LAG-3抗体mab23人源化轻链可变区优选序列：
LG2312：SEQ ID NO:23；LG2313：SEQ ID NO:24；LG2314：SEQ ID NO:25；LG2315：SEQ ID NO:26，即
Figure PCTCN2020095577-appb-000008
LG2316：SEQ ID NO:27；LG2317：SEQ ID NO:28，即
Figure PCTCN2020095577-appb-000009
LG2318：SEQ ID NO:29。
本发明抗人LAG-3抗体mab23人源化重链可变区优选序列：
LG2342：SEQ ID NO:30，即
Figure PCTCN2020095577-appb-000010
LG2343：SEQ ID NO:31，即
Figure PCTCN2020095577-appb-000011
LG2344：SEQ ID NO:32；LG2345：SEQ ID NO:33；LG2346：SEQ ID NO:34；LG2347：SEQ ID NO:35；LG2348：SEQ ID NO:36；LG2349：SEQ ID NO:37。
本发明鼠源抗体mab23轻链的人源化序列中含有不同的回复突变，回复突变位点数目可以是10个或更多，优选0-10个，如上述所列序列。这些任意序列与人抗体轻链恒定区κ链或λ链的恒定区序列组合得到本发明人源化抗体的轻链序列,比如本发明轻链用κ型轻链恒定区，如上所列序列。同样，人源化所用重链可变区也有不同数目的回复突变，回复突变位点数目可以是10个或更多，优选0-10个，如上述所列重链可变区序列。这些含不同数量回复突变的重链可变区序列，同任选人IgG1、2、3、4链恒定区序列重组得到本发明人源化抗体重链序列，比如本发明重链用hIgG4作为恒定区序列为例子加以说明。
本发明mab23抗体部分优化人源化抗体轻重链序列，表达量(用本发明实施例1的方法表达纯化并检测抗体产量)和活性评估(本发明实施例3之ELISA检测方法，实施例5之blocking活性检测方法)结果如下表。
表8本发明mab23抗体人源化抗体部分序列(以人κ型轻链,hIgG4重链恒定区为例子)
Figure PCTCN2020095577-appb-000012
Figure PCTCN2020095577-appb-000013
*：NB,没有结合。结合曲线显示在100nM才能看到微弱的结合。#：没有检测。因结合活性很弱，未检测到blocking活性。
上述结果表明，由本发明鼠源抗体mab23序列得到的上述人源化抗体分子保留了和hLAG-3的结合活性，更佳地，很多分子恢复到和鼠源抗体mab23c同样的结合活性，其中Ab2315、Ab2317、Ab2322、Ab2325等抗体的结合活性，和mab23c没有差别。即本发明人源化优选抗体保留了之前鼠源抗体的结合活性。此外，除了结合活性很弱(NB)的抗体之外，大部分人源化抗体blocking活性较好，和mab23c接近，比如Ab2314、Ab2316、Ab2317、Ab2318、Ab2320、Ab2325等。
表8部分优选人源化抗体的轻、重链氨基酸(包括恒定区)序列如下。
人源化Ab2315抗体氨基酸序列：
轻链：
Figure PCTCN2020095577-appb-000014
重链：同Ab2317重链(SEQ ID NO:40)
人源化Ab2317抗体氨基酸序列：
轻链：
Figure PCTCN2020095577-appb-000015
重链：
Figure PCTCN2020095577-appb-000016
人源化Ab2325抗体氨基酸序列：
轻链：同Ab2317轻链(SEQ ID NO:39)
重链：
Figure PCTCN2020095577-appb-000017
实施例8：本发明人源化抗人LAG-3抗体，优选抗体结合活性综合评价
为了进一步评估本发明人源化抗体和LAG-3结合活性，以上述优选人源化优选抗体Ab2317为例，评价其和不同形式，不同种属LAG3结合活性，以及Baicore方法评估其亲和力，结果见表9。
Biacore方法如下：用Biacore T200，GE Healthcare仪器测定本发明抗体和人LAG-3的亲和力。用pH7.4的运行缓冲液HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA和0.05％的P20)，先将Protein A(Thermo Pierce,Cat#21181)偶联到生物传感芯片CM5(Cat.#BR-1005-30，GE)上,将芯片用新配制的50mM NHS(N-hydroxysuccinimide)和200mM EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)激活,然后 注入pH4.0 10mM NaAC配制的10μg/ml的Protein A。待测抗浓度为5μg/ml，抗原LAG-3-his浓度梯度0nM、1.875nM、3.75nM、7.5nM、15nM和30nM，流速30μl/分钟，结合时间180秒，解离时间300秒。实验后，用10mM Glycine-HCl，pH 1.5，30μl/min,30s清洗芯片。实验数据用Biacore T200evaluation version 3.0(GE)软件以1:1Langmuir模型进行拟合，得出亲和力数值KD。
表9本发明抗体和LAG-3结合活性(EC 50，nM)、亲和力(Biacore)综合评价
Figure PCTCN2020095577-appb-000018
上述结果表明本发明鼠源抗体人源化优选抗体(Ab2317、Ab2325等)和人LAG-3-his(单体)、人LAG3-hFc(二聚体)、人LAG-3细胞外loop 1和loop 2(hLAG-3-D12)的his形式(单体)，即hLAG-3-D12-his(单体)以及hLAG-3-D12-hFc(二聚体)形式、hLAG3+细胞都有很好的结合。
Bicore检测结果表明，Ab2317和Ab2325的亲和力均在0.2nM以下，和对照分子亲和力接近。
Ab2317、Ab2325和对照阳性抗体(Ref)相比，突出的优点是其blocking活性好(平行assay比较结果，ab2317的blocking活性至少比Ref好2倍以上，2.7nM vs 8.2nM)，这和mab23c优于Ref的blocking活性结果(图1，b)一致。不仅如此，上述数据表明，本发明抗体结合hLAG3的第1、2区域(hLAG-3-D12)，说明本发明抗体的blocking活性是阻止hLAG3的D12区域和MHCII(daudi细胞)的结合。筛选出具有这样特异blocking活性的抗体是很难获取的事情，特别是blocking活性很好，比目前本领域临床上的分子(Ref)blocking活性还好2倍以上就更加困难，是不可预料的事情。这一特异blocking活性和该抗体用于临床治疗肿瘤患者药效相关，blocking活性好，预期药效会更好。
此外本发明抗体，例如Ab2317的另一意想不到的优点是其表达产量很高，在本发明条件下达到量在100mg/L以上，而平行、同等条件下，对照抗体Ref的表达量仅达60mg/L。多次比对的平均表达产量，Ab2317比对照抗体Ref高60％以上。这说明本发明抗体表达量优于对照分子(这种表达量的不同是和序列相关的)，这为本发明抗体用于后期工艺开发提高抗体产量带来了优势。
将本发明抗体Ab2317配制成1mg/L浓度，PBS溶液，45℃保存0天、7天、14天后，用凝胶电泳对样品进行稳定性评价。结果见图2，左起，M为分子量标记。泳道1、2；3、4；5、6分别为Ab2317在45℃保存0天、7天、14天后的样品非变性和变性电泳。泳道7、8；9、10；11、12分别为平行条件下Ref在45℃保存0天、7天、14天后的样品非变性和变性电泳。电泳结果表明，本发明抗体Ab2317在45℃保存7天、14天后不管是非变性还是变性的样品，浓度都没有改变，说明没有任何降价。而对照样品Ref则显示样品减少(凝胶上变少)，比如14天(泳道11、12)样品比0天明显变少了，说明有显著降解。该结果很好说明本发明抗体Ab2317优良的稳定性特点。这一特点为其作为药物开发，特别是制剂处方开发带来非常好的便利和优势。
本发明人源化抗体例如Ab2317、Ab2325和对照抗体(Ref)一样，均能与食蟹猴LAG-3(cynomolgus，cyno-LAG3-hFc，购自北京百普赛斯生物科技有限公司，目录号LA3-C5252)结合，结合活性分别为40.2nM、44.5nM、35.3nM。这一结合活性与人LAG-3的结合相比，结合活性弱200倍以上。这和Biacore检测本发明抗体同cyno-LAG3-hFc亲和力结果一致。Ref亲和力为836nM(比同人LAG-3亲和力弱了4000倍以上)；Ab2317、Ab2325则在Biacore未检测到和cyno-LAG-3-hFc的结合。此外，上述抗体也都不和鼠LAG-3结合。
本发明以抗体LG2317为代表，通过以下实验进行细胞功能评价。
实施例9：抗LAG-3抗体激活人T细胞功能活性评价
实验当天，收集人PBMC(由健康志愿者捐献外周血液中分离)，用含10％FBS的RPMI 1640培养基重悬细胞并计数，调整细胞密度为1×10 6cells/ml，加入96孔板，85μl/孔，放入培养箱。用培养基配制超抗原(SEB，购自北京康博贝宁科技有限公司)，使初始浓度为2μg/ml，每孔加入5μl(使终浓度为100ng/ml)。用培养基按比例配制待测样品，包括阴性抗体，对照抗体。每孔加入10μl，使待测抗体在100μl体系中的浓度为需要的浓度梯度。细胞培养板置于37℃、5％CO 2培养箱孵育3天后，取出细胞培养板，3000rpm离心10min，每孔取出80μl上清进行人IL-2的检测。
IL-2 ELISA检测按照试剂盒(深圳欣博盛生物科技有限公司，cat:EHC003.96)说明 书进行操作，步骤如下:
a.将取出的细胞培养上清以25倍(不同次实验稀释倍数有不同)稀释后加入酶标板中(100μl/孔)；标准品用标本通用稀释液稀释成不同浓度梯度：1000pg/ml、500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.25pg/ml、15.625pg/ml，每孔加100μl；空白孔加标本通用稀释液。
b.封板胶纸封住反应孔，37℃，孵育90分钟。
c.洗板5次，每次3分钟，每孔加入100μl生物素抗体工作液，空白孔加生物素化抗体稀释液，新封板胶纸封住反应孔，37℃，孵育60分钟。
d.洗板5次，每次3分钟，每孔加入100μl酶结合工作液，空白孔加酶结合稀释液，新封板胶纸封住反应孔，37℃，避光孵育30分钟。
e.洗板5次，每次3分钟，每孔加入100μl显色底物TMB，37℃，避光孵育15分钟
f.加入终止液，100μl/孔，混匀后，3分钟内，酶标仪读OD450。
g.结果分析：计算IL-2值，并和空白对照相比，换算成增加百分比(％)来评估样品激活人T细胞活性。
结果表明，不同供体T细胞实验均显示本发明优选抗体Ab2317激活人T细胞释放IL-2的活性EC 50为0.34μg/ml。同条件下比对实验对照抗体Ref的EC 50为1.4μg/ml。即本发明抗体比对照Ref在T细胞上的活性至少优3倍以上。图3的a显示代表数据之一。
实施例10本发明抗LAG-3抗体单独和联合PD-1抗体在混合淋巴细胞反应(MLR assay)中的活性检测
用混合淋巴细胞反应(mixed lymphocyte reaction,MLR assay)检测INF-γ(INFg)分泌的方法来评估本发明系列抗体在激活人T细胞的活性。即由本发明分离的人血细胞PBMC(由健康志愿者捐献外周血液中分离)诱导得到树突细胞(DC)，用得到的DC刺激来自不同志愿者的T细胞。具体地，树突细胞培养：在实验第1天，用RPMI 1640培养基，接种PBMC于6孔板，每孔2ml，1×10 6cells/ml，37℃、5％CO 2培养箱培养2小时后，轻轻吸出悬浮细胞，向贴壁细胞中加入2ml完全RPMI 1640培养基(包含10％FBS，100ng/ml GM-CSF，Peprotech，Cat#:300-03，和100ng/ml IL-4，Peprotech，Cat#:200-04)，继续培养2天后，每孔补加1ml新鲜的完全RPMI 1640培养基。第5天，每孔补加3μl 100μg/ml的TNF-α，使终浓度为100ng/ml(TNF-α购自Peprotech，Cat#:AF-300-01A)，继续培养2天，所得树突细胞(DC)用于如下实验。
DC刺激T细胞(MLR)assay：用10ng/ml anti-CD3抗体(Miltenyl Biotec，Cat#: 130-093-387)，按100μl/孔包被96孔细胞培养板，37℃孵育2小时，用PBS洗一遍。收集上述培养第7天的DC细胞，离心，重悬于10％FBS的RPMI 1640培养基，计数，配成5×10 4cells/ml，按90μl/孔加入上述anti-CD3包被好的96-孔板中。取来自不同志愿者的PBMC细胞，计数，配成5×10 5cells/ml，按90μl/孔加入上述anti-CD3包被并铺有DC细胞的96-孔板中。用PBS按比例配制待测样品，包括对照抗体(Ref)和Ab2317，PD-1抗体(本发明表达纯化得到)加入上述96孔板中。Ref或者Ab2317+PD-1抗体各10μl/孔。PD-1抗体在200μl体系中终浓度为0.5μg/ml。待测抗体(Ref或Ab2317)在200μl体系中的浓度为需要的浓度梯度。对照组＝90μl PBMC细胞+90μl DC+20μl PBS。细胞培养板置于37℃，5％CO 2培养箱孵育4天后，取出细胞培养板，3000rpm离心10min，每孔取出150μl上清进行人INF-γ的检测。
INF-γELISA检测，按照试剂盒(上海欣奥盛生物科技有限公司cat:EHC102g.96)说明书进行操作，步骤如下：
h.将取出的细胞培养上清加入酶标板中(100μl/孔)；标准品用标本通用稀释液稀释成不同浓度梯度：1000pg/ml、500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.25pg/ml、15.625pg/ml，每孔加100μl；空白孔加标本通用稀释液。
i.封板胶纸封住反应孔，37℃，孵育90分钟。
j.洗板5次，每次3分钟，每孔加入100μl生物素抗体工作液，空白孔加生物素化抗体稀释液，新封板胶纸封住反应孔，37℃，孵育60分钟。
k.洗板5次，每次3分钟，每孔加入100μl酶结合工作液，空白孔加酶结合稀释液，新封板胶纸封住反应孔，37℃，避光孵育30分钟。
l.洗板5次，每次3分钟，每孔加入100μl显色底物TMB，37℃，避光孵育15分钟
m.加入终止液，100μl/孔，混匀后，3分钟内，酶标仪读OD450。
n.结果分析：通过标准曲线公式，计算INF-γ值(pg)，来评估本发明抗体的活性。
图3b结果表明，本发明抗体Ab2317激活DC刺激T细胞活性(和PD-1抗体Keytruda联用)EC 50为0.21nM比同样条件下Ref(EC 50为0.61nM)强2倍以上。
实施例11:本发明LAG-3抗体体内药效评价
用人PD-1和LAG-3双转基因小鼠(C57BL/6-hPD1/hLAG3)建立动物药效模型，评价本发明LAG-3抗体动物体内药效。双转基因小鼠购自江苏集萃药康生物科技有限公司，生产许可证编号：SCXK(苏)2018-0008。
MC38细胞(购自中科院细胞所)培养于含10％胎牛血清(上海博升生物科技有限公 司，货号：BS-0002-500)，1％HEPES(赛默飞世尔科技有限公司，货号：15630080)的DMEM/高糖培养基中(上海源培生物科技股份有限公司，货号：L110KJ)，在含5％CO 2的37℃的细胞培养箱中连续培养。C57BL/6-hPD1/hLAG3雌性小鼠，6周龄，5只/笼饲养于SPF级环境，温度20-25℃；湿度40％-60％，自由进食进水，定期更换垫料。
待MC38细胞长至对数生长期(汇合率在80％-90％)时，用0.25％胰酶消化，收集细胞，并用无血清的DMEM/高糖培养基洗涤细胞两次，后用无血清的DMEM/高糖培养基重悬，计数，用Matrigel(碧迪医疗器械(上海)有限公司，货号：354234)与DMEM/高糖培养基按1:1的比例混合，调整细胞浓度为1×10 7细胞/ml用于接种。接种MC38细胞悬液100μl(10 6个)于小鼠右肋部皮下，挑选肿瘤细胞长至体积约100-150mm 3大小后随机分组，每组5只。
待测样品用PBS配制，无菌。Blank组为PBS。PD-1抗体(据Keytruda公开序列克隆表达)为单独用药对照组。PD-1抗体+Ref为联合用药对照组。PD-1抗体+Ab2317为联合用药待测组。给药方式为腹腔注射。PD-1抗体单独或联合给药，剂量均为10μg/100μl/只。联合给药组中，Ref和Ab2317药剂量为120μg/100μl/只。给药频率2次/周。各注射样品当天为第0天。每次给药前测量体重，肿瘤体积，记录数据。本次实验给药4次。
肿瘤大小计算公式：肿瘤体积TV(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)；相对肿瘤增长率(T/C％)＝100％*(T-T0)/(C-C0)；抑瘤率(TGI)＝(1-T/C)*100％；其中T0、T分别为样品组实验开始时及实验结束时的肿瘤体积；C0、C分别为对照组实验开始时及实验结束时的肿瘤体积。
结果见图4和表9b。
表9b本发明LAG-3抗体体内药效评价
Figure PCTCN2020095577-appb-000019
图4结果表明，本发明抗体和PD-1抗体联用在第13天开始，抑瘤率达到了100％，并且肿瘤比开始时候缩小，保持到实验结束。明显优于Ref+PD-1抗体组，和PD-1单独抗体组。
表9b是第0天和第23天肿瘤体积的统计结果。结果表明，第23天，和PBS组比较，PD-1抗体+本发明抗体Ab2317联合组的药效(抑制率)为103％，即100％抑制了肿瘤生长并且肿瘤比开始时候缩小了。优于PD-1抗体单独药效(抑制率)为63％，也优于PD-1抗体+Ref联合组的药效(抑制率为)80％。
统计分析(T test)结果表明，PD-1抗体单独药效和PBS组P值为0.06，未达到统计学显著差异水平。PD-1抗体+Ref联用药效和PBS组P值为0.01，达到显著差异水平(标记为*)。意想不到的是，PD-1抗体+本发明抗体Ab2317联用药效和PBS组P值为0.001，达到极显著差异水平(标记为**)。更加意想不到的是，PD-1抗体+本发明抗体Ab2317联用药效和PD-1抗体+Ref联用组统计分析，P值为0.04，达到显著差异水平(P<0.05，标记为*)。
本实验各组实验期间，动物体重都没有明显的变化，表明本发明抗体没有显著的毒性作用。
实施例12:针对LAG-3靶点的双特异性抗体设计
用上述发现的抗LAG-3抗体，本发明进行了多种双特异性抗体的设计。所设计的双特异性抗体的通式如下。
表10基于本发明抗LAG-3抗体进行的双特异设计(通式1)
Figure PCTCN2020095577-appb-000020
表10及本文中，含轻链的序列指该序列除了包括轻链序列以外，还可以包括与轻链序列连接的scFv；含重链的序列指该序列除了包括重链序列以外，还可以包括与重链序 列连接的scFv。其中，T1代表针对靶点1(比如LAG3)的第一蛋白功能区，T2代表针对靶点2(非LAG3)的第二蛋白功能区。T1(scFv)代表针对靶点1抗体的scFv序列；T2(scFv)代表针对靶点2的scFv序列。
(scFv) n1、(scFv) n2、(scFv) n3、(scFv) n4中的n1、n2、n3、n4分别为自然数，可以是0、1、2、3等，在本发明的具体实施例中，n1、n2、n3，n4中其中至少1个数值为1，其余为0。VL，代表针对靶点1或者2的抗体轻链可变区序列；VH，代表针对靶点1或者2的抗体重链可变区序列。Lc，代表轻链(κ或者λ)的恒定区序列，优选人轻链恒定区序列；Hc代表重链，包括IgG1、IgG2、IgG3、IgG4等的恒定区序列(缩写为Hc-IgG1、Hc-IgG2、Hc-IgG3、Hc-IgG4)，优选人的重链恒定区序列(Hc-hIgG)。重链恒定区C末端连接scFv或其它蛋白序列的时候，其C末端末位氨基酸K可以突变，优选突变为A。由此，在方案1中，T1为免疫球蛋白，T2为scFv；在方案2中，T2为免疫球蛋白，T1为scFv；scFv针对的靶点相同；在方案3、4中，两端的scFv针对两个不同的靶点。
当表10中所述scFv为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。
需说明的是，当上述scFv为轻链可变区-连接子-重链可变区时，其连接方式为轻链可变区的C末端与连接子连接，所述连接子再与重链可变区的N末端连接，从而将scFv轻链可变区的N末端和重链可变区的C末端暴露出来，使其可以通过连接子与免疫球蛋白的轻链和或重链连接。在本发明中，当其连接免疫球蛋白的轻链时，在一些具体的实施例中优选地使用scFv的重链可变区的C末端通过连接子与免疫球蛋白重链的N末端连接；当其连接免疫球蛋白的重链时，在一些具体的实施例中优选地使用scFv的轻链可变区的N末端与免疫球蛋白重链的C末端连接。
当所述scFv为重链可变区-连接子-轻链可变区时，其连接方式为轻链可变区的N末端与连接子连接，所述连接子再与重链可变区的C末端连接，从而将scFv轻链可变区的C末端和重链可变区的N末端暴露出来，使其可以通过连接子与免疫球蛋白的轻链和/或重链连接。在此情况下，当其连接免疫球蛋白的轻链时，在一些具体的实施例中优选地使用scFv的轻链可变区的C末端与免疫球蛋白重链的N末端连接；当其连接免疫球蛋白的重链时，在一些具体的实施例中优选地使用scFv的重链可变区的N末端与免疫球蛋 白重链的C末端连接。
所述连接子为(Gly-Gly-Gly-Gly-Ser) w[缩写为(G 4S) w]所述的w优选为0～10之间的整数。优选地，所述连接子为(G 4S) 3，和/或，所述scFv的数量为一对，对称地连接在所述的免疫球蛋白轻链和/或重链。
此外，本发明抗体也可用不同于通式1的方式，例如如下通式2。
表11基于本发明抗LAG3抗体进行DVD形式的双特异设计(通式2)
Figure PCTCN2020095577-appb-000021
表11中，T1、T2分别代表针对靶点1(例如LAG3)和靶点2(非LAG3)。含轻链的序列指该序列除了包括正常完整的轻链序列以外，还包括另一个轻链可变区序列。含重链的序列指该序列除了包括正常完整的重链序列以外，还包括另一个重链可变区序列。轻链可变区和完整的轻链之间、重链可变区和完整的重链之间通过连接子(Linker)连接。
上述双特异设计涉及的各个靶点抗体序列，除了本发明所述anti-LAG3抗体序列外，PD1抗体序列包括已经公开的抗体序列，包括抗PD-1抗体Nivolumab/Opdivo(简称Nivo)、Pembrolizumab/Keytruda(简称Pem)。除了专利中公开的序列外，Nivolumab(Nivo)、Pembrolizumab(Pem)等序列也都能从www.drugbank.ca等公开资源查到。这些单独抗体表达编号和序列见下表。
表12本发明克隆表达的单克隆抗体编号、轻重链序列及说明
Figure PCTCN2020095577-appb-000022
其中Nivolumab和Pembrolizumab的序列可以从www.drugbank.ca等公开信息得到，本发明将其编号如下所示：
NivoVL-Lc(κ链)：SEQ ID NO:42；NivoVH-Hc(hIgG4)：SEQ ID NO:43；
PemVL-Lc(κ链)：SEQ ID NO:44；PemVH-Hc(hIgG4)：SEQ ID NO:45；
Ab835VL为申请号201810917684.X中的SEQ ID NO:58；Ab385VH为申请号201810917684.X中的SEQ ID NO:59。
实施例13针对LAG-3和PD-1双靶点的双特异性抗体设计和活性评价
本发明针对LAG-3和PD1两个靶点设计了不同序列结构的双特异性抗体，见下表。
表13针对LAG-3和PD1双靶点设计的双特异性抗体
Figure PCTCN2020095577-appb-000023
Figure PCTCN2020095577-appb-000024
*:κ链表示轻链为人IgG的κ型轻链恒定区。#：IgG4的C末端连接连接子的时候，其最末端氨基酸K突变为A。本发明申请中，在重链C末端引入scFv的本发明所设计的SBody，均将最末端K突变为A。
按本发明克隆表达纯化方法，分别克隆、表达、纯化上述双特异性抗体，用前述实施例中的方法分别检测这些设计的双特异分子和人LAG-3以及PD-1的结合活性，结果如下表。
表14针对LAG-3和PD-1双靶点设计的双特异性抗体的结合活性评价
Figure PCTCN2020095577-appb-000025
Figure PCTCN2020095577-appb-000026
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体的结合活性EC 50。*：同样实验条件下，双特异性抗体和对应的单克隆抗体的结合活性EC 50的比值。比值越大，说明所设计的双特抗体对单靶点的结合力减弱越多，比如比值为2，则说明所设计的双特异性抗体对靶点结合活性和对应的单克隆抗体相比减弱了1倍。比值在2以内(实验误差范围)，说明结合活性没有受到影响。
上表中结果表明，LB2373 vs LB2371表明同样的本发明Ab2317 scFv和不同的PD-1抗体以同样的结构(scFv均在PD-1抗体重链的N末端)设计的双特异性抗体，LB2373对PD-1的结合活性影响大(EC 50改变倍数为2.92，见图5A、5C)，对LAG3的结合活性则基本没有影响。而LB2371对PD-1和LAG3的活性均没有影响(图5C)。比较LB2373 vs LB2374、LB2371 vs LB2372，数据表明(见图5A、5B、5C、5D)，同样的Ab2317 scFv序列和同样的PD-1抗体序列设计成的双特异性抗体，Ab2317 scFv位置不同(N末端vs C末端)，对PD-1和LAG3的结合活性也不同。LB2371和双靶点的结合活性最好。LB2383 vs LB2384显示Ab2325 scFv在Nivo重链N末端(LB2383)活性优于C末端(LB2384)。
比较LB2371-LB2374和LB211、LB152、LB234、LB203的活性，发现在LAG-3抗 体scFv的不同(Ab2317 scFv vs Ab835 scFv)的前提下，同样的PD-1抗(Nivo or Pem)的同样结构(重链的N-末端，或C-末端)，由Ab2317 scFv所设计的SBody比Ab835 scFv设计的SBody活性要好很多。特别意想不到的是，LB2374和LB234为同样的SBody设计结构，PD-1抗体序列(Nivo)也相同，只是Ab2317 scFv vs Ab835 scFv的不同。结果LB2317几乎很好保留了对LAG-3和PD-1的结合活性，而LB234几乎失去了和LAG-3的结合活性(减弱了175倍)。根据这些数据，非常意外地发现，本发明抗体Ab2317 scFv设计的SBody的活性是序列特异的。
比较LB2379-LB2382和LB202、LB201、LB214、LB216针对双靶点(PD-1和LAG-3)的结合数据，在同样结构中，LAG-3抗体序列(scFv)不同，LB2379-LB2382活性优于对应的LB202、LB201、LB214、LB216。
这些基于序列特异而表现出意料之外活性差别的分子设计，其分子结构类似常规IgG，本发明称之为 Sequence-based IgG like bispecific anti body format(SBody)，即序列特异的IgG结构相似的双特异性抗体。
以LB2374为代表，用Biacore(方法同实施例子8)对本发明双特异性抗体亲和力进行分析。LB2374对LAG-3亲和力数据为：ka(1/Ms)＝2.21E+6；kd(1/s)＝4.13E-4；KD(M)＝1.87E-10(和表9数据Ab2317的亲和力1.68E-10非常接近)。对PD-1的亲和力数据为：ka(1/Ms)＝2.098E+5；kd(1/s)＝1.41E-3；KD(M)＝6.71E-9(这和Nivo单独测得的亲和力8.59E-9接近)。该数据表明，本发明双特异性抗体LB2374保留了对双靶点同样的亲和力(KD)。
为了评估本发明设计的双特异性抗体(SBody)对两个靶点同时结合活性，用双夹心ELISA对LB2374、LB2371活性进行了评估。
具体地，用pH7.4的PBS缓冲液将PD-1(本发明表达)稀释至1μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于37℃孵育2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(上海生工生物工程有限公司，A600669-0250)封闭液200μl/孔，4℃放置过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.05％tween-20)洗板5次后，加入待测双特异性抗体(10μg/ml)起始，用1％BSA 5倍连续稀释，50μl/孔，37℃孵育1小时，PBST洗板5次后，加入50μl/孔1μg/ml的Bio-LAG3-his(ACROBio systems，TM-H5229，biotin标记)，37℃孵育1小时，PBST洗板5次后，加入50μl/孔1:1000稀释的streptavidin-HRP二抗(南京金斯瑞生物科技有限公司，M00091)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，室温孵育5-10min，加入50μl/孔1M H 2SO 4终止 反应，用MULTISKAN GO酶标仪(ThermoFisher，51119200)在450nm处读取吸收值，根据OD值计算EC 50。
用pH7.4的PBS缓冲液将LAG3-his稀释至1μg/ml，以50μl/孔的体积加入96孔酶标板中，于37℃孵育2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶封闭液200μl/孔，4℃放置过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.05％tween-20)洗板5次后，加入待测双特异性抗体样品(10μg/ml起始)，用1％BSA 5倍连续稀释，50μl/孔，37℃孵育1小时，PBST洗板5次后，加入50μl/孔10μg/ml的Bio-PD-1-his(本发明表达后，Biotin标记)，37℃孵育1小时，PBST洗板5次后，加入50μl/孔1：1000稀释的streptavidin-HRP二抗，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物，室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用MULTISKAN GO酶标仪在450nm处读取吸收值，根据OD值计算EC 50。
结果见下表。
表14a针对LAG-3和PD-1双靶点设计的双特异性抗体双夹心ELISA
Figure PCTCN2020095577-appb-000027
上述结果表明，本发明双特异性抗体SBody在结合双靶点其中之一以后，能进一步结合另一靶点，即其能同时结合两个靶点。
将上述保留了双靶点活性的SBody分别针对两个靶点进行功能(阻止抗原和受体结合实验)评估，结果见下表。
表15针对LAG3和PD-1双靶点设计的双特异性抗体功能活性评价
Figure PCTCN2020095577-appb-000028
Figure PCTCN2020095577-appb-000029
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体阻断抗原和配体结合活性IC 50。*：IC 50改变倍数，即双特异性抗体和对应的单克隆抗体(对照抗体)的IC 50的比值。比值越大，说明所设计的双特抗体对单靶点的功能活性减弱越多，比如比值为2，则说明所设计的双特异性抗体对靶点功能活性和对应的单克隆抗体相比减弱了1倍。比值在2以内为实验误差范围，即活性没有受到影响。ND：该分子没有检测到阻止LAG3和Daudi细胞结合活性。
上述功能活性结果表明，本发明设计的双特异性抗体(SBody)保留了阻止PD-1和PD-L1的结合的活性，仅LB2373阻止PD-1/PD-L1结合活性稍弱(改变倍数3.78)。LB2379、LB2380、LB2371和LB2372阻止LAG-3和Daudi细胞的结合活性减弱比较多(分别为8.16、3.78、7.92和5.63倍)，而LB2373、LB2374几乎没有减弱(分别为0.82、1.98倍)。这也表明，在同样结构下，PD-1抗体序列的不同(LB2373、LB2374为Pem；LB2371、LB2372为Nivo)对功能活性影响显著不同，即本发明设计的SBody是序列特异的。综合对两个靶点功能活性结果，LB2374最优，其对两个靶点的功能活性(阻止抗原与受体结合)影响最小。
为评估本发明双特异性抗体稳定性，将本发明抗体配置成1mg/ml在PBS(pH 7.4)中，37℃孵育5天(d5)、10天(d10)的样品和-80℃保存60天的样品进行活性比较，评估稳定性。结果见下表。
表16针对LAG3和PD-1双靶点设计的双特异性抗体(SBody)稳定性评价
Figure PCTCN2020095577-appb-000030
#：为该样品在-80℃条件下保存后取出，在相同条件下检测到的的活性值；*：相对于-80℃保存样品活性(括号内数值)改变倍数。d5、d10分别代表37℃孵育第5天、第10天的样品。
上述结果表明，本发明设计双特异分子LB2371、LB2374在37℃保存5天、10天，对双靶点的结合活性和-80℃保存60天的样品的结合活性一样，即结合活性没有改变 (EC 50差别均在2倍以内)。说明这些分子稳定。此外，这些样品凝胶电泳(PAGE)分析，变性和非变性条件下均没有发现降解现象。凝胶电泳结果同样说明这些双特异分子结构稳定。
表17针对LAG3和PD-1双靶点设计的双特异性抗体表达水平评价
Figure PCTCN2020095577-appb-000031
上述结果表明，本发明设计针对LAG3和PD1双特抗体(SBody)表达产量差别很大。非常意外地，LB2373、LB2374、LB2379、LB2380表达量均比较高。以Ab835为scFv设计的SBody的表达量比同样设计的Ab2317 scFv的SBody表达量低很多，低65倍以上的，比如LB2374 vs LB234；低140倍以上，比如LB2373 vs LB211。
这些数据表明，本发明抗LAG3抗体Ab2317和PD1抗体设计的双特异性抗体SBody意想不到的效果不仅是其活性、功能、稳定性，而且表达量也是序列特异设计相关的。
LB2371轻链氨基酸序列：SEQ ID NO:44；含重链的氨基酸序列：SEQ ID NO:46；LB2373轻链氨基酸序列：SEQ ID NO:42；含重链的氨基酸序列：SEQ ID NO:47；
LB2374轻链氨基酸序列：SEQ ID NO:42；LB2374含重链的氨基酸序列：SEQ ID NO:48
Figure PCTCN2020095577-appb-000032
Figure PCTCN2020095577-appb-000033
LB2379含轻链的氨基酸序列：SEQ ID NO:49；重链氨基酸序列：SEQ ID NO:43；LB2380含轻链的氨基酸序列：SEQ ID NO:50；重链氨基酸序列：SEQ ID NO:43。
实施例14:本发明LAG-3抗体PK评价
实验用C57BL/6cnc品系小鼠(购自浙江维通利华实验动物技术有限公司，生产许可证编号：SCXK(浙)2018-0001)，雌性，8周龄，6只，20g左右，饲养环境：SPF级。温度20-25℃；湿度40-60％。将C57BL/6cnc小鼠实验室环境饲养3天后随机分组，每组3只。将待测药物(本发明LAG-3抗体Ab2317和Ref)背部皮下注射小鼠，注射体积200μl。给药剂量为20mg/kg/只。
检测样品在注射小鼠后眼眶取血。时间点为0.5、1、2、4、7、24、31、48、56、72、96、120、144、168、192、216、240、264、288、312、336、360、384、408、432、456、480、504小时。所取血样离心，取上清，-20度保存，待测。等收集完血液样本后，用ELISA检测各个时间点血液中样品的含量。用Excel来分析PK数据并计算待测药物的T1/2，结果见下表。
表18本发明抗体Ab2317 PK评价
Figure PCTCN2020095577-appb-000034
上述结果表明本发明抗体Ab2317显示更好的PK特征，包括更长的T1/2(Ab2317 T1/2为202小时，Ref为188.7小时)，更高的Cmax等。
实施例15:本发明针对LAG3和PD-1双靶点设计的双特异性抗体(SBody)PK评价
实验用C57BL/6cnc品系小鼠(购自浙江维通利华实验动物技术有限公司，生产许可证编号：SCXK(浙)2018-0001)与人PD-1和人LAG-3双转基因CB7BL/6小鼠(简写C57BL/6-DKI，购自江苏集萃药康生物科技有限公司，生产许可证编号：SCXK(苏)2018- 0008)雌性，8周龄，各6只，20g左右，饲养环境：SPF级。温度20-25℃，湿度40-60％。将这两品系小鼠实验室环境饲养3天后，各选状态良好的小鼠随机分组，每组3只。将待测药物Ab2374背部皮下注射小鼠，注射体积200μl。给药剂量为20mg/kg/只。
注射小鼠后眼眶取血。时间点为0，0.5、1、2、6、24、31、48、56、72、96、120、144、168、192、216、240、264、288、312、336、360、384、408、432小时。所取血样离心，取上清，-20℃保存，待测。等收集完血液样本后，用ELISA检测各个时间点血液中样品的含量。具体地：
PD-1检测方法：用PD-1蛋白铺96孔板(corning，货号3590)。牛奶封闭2小时，洗板后待用。用Ab2374配制标准曲线，待测血清按比例稀释，加入处理好的96孔板。37℃反应1小时后洗板，加入稀释后酶标记抗体Peroxidase AffiniPure Goat Anti-Human IgG(Jackson，货号109-035-003)。半小时后洗板，加入TMB(Surmodic，货号TTMB-1000-01)显色，并用硫酸终止，酶标仪测定OD450。
PD-1/LAG-3(夹心ELISA)检测方法：用PD-1蛋白铺96孔板(corning，货号3590)。牛奶封闭2小时，洗板后待用。用Ab2374配制标准曲线，待测血清按比例稀释，加入处理好的96孔板。37℃反应1小时后洗板，加入固定浓度的生物素化LAG-3蛋白，37℃反应1小时后洗板，加入稀释后的酶标记抗体streptavidin-HRP(genscript，货号M00091)。半小时后洗板，加入TMB(Surmodic，货号TTMB-1000-01)显色，并用硫酸终止，酶标仪测定OD450。
用Excel来分析PK数据并计算待测药物的T1/2，结果见下表。
表19本发明双特异性抗体Ab2374 PK评价(PD-1检测结果)
Figure PCTCN2020095577-appb-000035
表20本发明抗体Ab2374 PK评价(PD-1/LAG-3检测结果)
Figure PCTCN2020095577-appb-000036
Figure PCTCN2020095577-appb-000037
夹心ELISA检测的是结合PD-1后再结合LAG-3分子的PK特征。表20结果表明，意外地，本发明双特异性抗体在C57BL/6小鼠体内夹心ELISA检测到的PK特征(Cmax、T 1/2)和单独检测PD-1得到特征几乎一致。说明本发明双特异性抗体在小鼠体内稳定，没有发生scFv(LAG-3)脱落的情况。而且该PK特征和单独Ab2317抗体PK表征(表18)也接近。此外，本发明抗体在hPD-1/hLAG-3双转基因小鼠(C57BL/6-DKI)因为有特异的靶点结合，Cmax、T 1/2均有减少，意外地，用夹心ELISA检测到的PK和单独PD-1检测到的PK特征一致，说明本发明双特异性抗体在有特异靶点结合的小鼠体内同样稳定，没有scFv脱落情况。
按本发明实施例1的方法分别克隆表达、纯化上述DVD设计双特异性抗体，凝胶电泳(PAGE)结果表明，这些抗体轻、重链均易出现linker间断裂现象。而以scFv的形式将一个靶点抗体连接在另一个靶点抗体的轻链或重链的N或C末端，通过筛选得到序列和设计优化的双特异性抗体，可以避免/减少linker间断裂(见前述实施例)，并且保留对双靶点的结合活性，体外功能活性，以及针对双靶点的体内药效。优选双特异性抗体(本发明称之为SBody)不仅稳定，而且因其类似正常IgG结构，纯化工艺简单，这为后期开发过程中的工艺、纯化都提供了极大便捷。

Claims (20)

  1. 一种LAG-3结合蛋白，其包括轻链可变区和/或重链可变区；所述轻链可变区包含：如SEQ ID NO:5所示的氨基酸序列的CDR1，如SEQ ID NO:6所示的氨基酸序列的CDR2，和/或，如SEQ ID NO:7所示的氨基酸序列的CDR3；所述重链可变区包含：如SEQ ID NO:8所示的氨基酸序列的CDR1，如SEQ ID NO:9所示的氨基酸序列的CDR2，和/或，如SEQ ID NO:10所示的氨基酸序列的CDR3。
  2. 如权利要求1所述的LAG-3结合蛋白，所述轻链可变区包含如SEQ ID NO:3、SEQ ID NO:23-29或其突变所示的氨基酸序列；和/或，所述重链可变区包含如SEQ ID NO:4、SEQ ID NO:30-37或其突变所示的氨基酸序列；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少99％的序列同一性，并且所述突变保持或改善了所述抗体对LAG-3的结合；
    优选地，所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:27所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:29所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:26所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:32所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:36所示的氨基酸序列。
  3. 如权利要求1或2所述的LAG-3结合蛋白，其是抗体、Fab、Fab’、F(ab’) 2、Fv、scFv、双特异性抗体、多特异性抗体、单域抗体或单区抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。
  4. 如权利要求3所述的LAG-3结合蛋白，其包括人源抗体轻链恒定区和人源抗体重链恒定区；优选地，所述人源抗体轻链恒定区为κ链或者λ链，和/或，所述人源抗体重链恒定区为hIgG1、hIgG2、hIgG4或其突变。
  5. 如权利要求4所述的LAG-3结合蛋白，其轻链包含如SEQ ID NO:38或SEQ ID NO:39所示的氨基酸序列或其突变，和/或，其重链包含如SEQ ID NO:40或SEQ ID NO:41所示的氨基酸序列或其突变；
    优选地，所述轻链的氨基酸序列如SEQ ID NO:38所示；所述重链的氨基酸序列如 SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:38所示；所述重链的氨基酸序列如SEQ ID NO:41所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示；所述重链的氨基酸序列如SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示；所述重链的氨基酸序列如SEQ ID NO:41所示。
  6. 一种靶向LAG-3的双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中，所述第一蛋白功能区为如权利要求1-5中任一项所述的LAG-3结合蛋白；所述第二蛋白功能区为非LAG-3结合蛋白；优选地，所述第一蛋白功能区和第二蛋白功能区分别选自免疫球蛋白、scFv、Fab、Fab’或F(ab’) 2，且所述第一蛋白功能区和第二蛋白功能区中只有一个蛋白功能区为免疫球蛋白。
  7. 如权利要求6所述的靶向LAG-3的双特异性抗体，其特征在于，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为一个或多个scFv；或，所述第一蛋白功能区为一个或多个scFv，所述第二蛋白功能区为免疫球蛋白，所述免疫球蛋白的恒定区包括人源抗体轻链恒定区和人源抗体重链恒定区；优选地，所述人抗体轻链恒定区为κ链或者λ链，所述人抗体重链恒定区为hIgG1、hIgG2、hIgG4或其突变。
  8. 如权利要求6或7所述的靶向LAG-3的双特异性抗体，其中，所述scFv包括重链可变区与轻链可变区，所述重链可变区与轻链可变区通过连接子连接；所述scFv通过连接子与所述免疫球蛋白连接，所述连接子优选(G 4S) w；所述w优选为0～10之间的整数，更优选为1、2、3或者4。
  9. 根据权利要求6-8中任一项所述的靶向LAG-3的双特异性抗体，其中，所述scFv为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。
  10. 根据权利要求6-9中任一项所述的靶向LAG-3的双特异性抗体，其中，所述连接子为(G 4S) 3，和/或，所述scFv的数量为两个，且两个scFv对称地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；
    优选地，所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链可变区的N末端，或两个scFv的轻链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的轻链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链可变区的N末端， 或两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链可变区的C末端。
  11. 如权利要求6-10中任一项所述的靶向LAG-3的双特异性抗体，其中，所述第二蛋白功能区靶向PD-1；优选地，所述第二蛋白功能区为抗PD-1抗体；更优选地，所述抗PD-1抗体为Nivolumab或Pembrolizumab。
  12. 根据权利要求6-11中任一项所述的靶向LAG-3的双特异性抗体，其选自以下组：
    (1)所述第一蛋白功能区为免疫球蛋白，其包含以下轻链和重链或其突变：所述轻链的氨基酸序列如SEQ ID NO:38所示，所述重链的氨基酸序列如SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:38所示，所述重链的氨基酸序列如SEQ ID NO:41所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示，所述重链的氨基酸序列如SEQ ID NO:40所示；或，所述轻链的氨基酸序列如SEQ ID NO:39所示，所述重链的氨基酸序列如SEQ ID NO:41所示；优选地，当所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端由K突变为A；和/或，
    所述第二蛋白功能区为scFv或其突变，其中，所述scFv的轻链可变区的氨基酸序列如SEQ ID NO:42的第1-107位氨基酸序列所示，所述scFv的重链可变区的氨基酸序列如SEQ ID NO:43的第1-113位氨基酸序列所示；或，所述scFv的轻链可变区的氨基酸序列如SEQ ID NO:44的第1-111位氨基酸序列所示，所述scFv的重链可变区的氨基酸序列如SEQ ID NO:45的第1-120位氨基酸序列所示；
    (2)所述第一蛋白功能区为scFv或其突变，其中，所述scFv的轻链可变区选自如SEQ ID NO:3、SEQ ID NO:23-29所示的氨基酸序列；和/或，所述scFv的重链可变区选自如SEQ ID NO:4、SEQ ID NO:30-37所示的氨基酸序列；和/或，
    所述第二蛋白功能区为免疫球蛋白，其包含以下轻链和重链或其突变：所述轻链的氨基酸序列如SEQ ID NO:42所示；所述重链的氨基酸序列如SEQ ID NO:43所示；或，所述轻链的氨基酸序列如SEQ ID NO:44所示；所述重链的氨基酸序列如SEQ ID NO:45所示；优选地，当所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端由K突变为A；
    所述突变优选与原氨基酸序列具有至少99％的序列同一性，并且保持或改善了所述抗体的功能。
  13. 如权利要求6-12中任一项所述的靶向LAG-3的双特异性抗体，其选自以下组：
    (i)所述第一蛋白功能区为scFv，其轻链可变区的氨基酸序列如SEQ ID NO:28所 示，其重链可变区的氨基酸序列如SEQ ID NO:31所示，所述连接子为(G 4S) 3；所述第二蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:42所示，其重链的氨基酸序列如SEQ ID NO:43所示；
    其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的N末端，且所述C末端由K突变为A；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端；或，所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的C末端；或，
    (ii)所述第一蛋白功能区为scFv，其轻链可变区的氨基酸序列如SEQ ID NO:28所示，其重链可变区的氨基酸序列如SEQ ID NO:31所示，所述连接子为(G 4S) 3；所述第二蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:44所示，所述重链的氨基酸序列如SEQ ID NO:45所示；
    其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端，且所述C末端由K突变为A；或，所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条轻链的C末端；或，
    (iii)所述第一蛋白功能区为scFv，其轻链可变区的氨基酸序列如SEQ ID NO:28所示，其重链可变区的氨基酸序列如SEQ ID NO:36所示，所述连接子为(G 4S) 3；所述第二蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:44所示，其重链的氨基酸序列如SEQ ID NO:45所示；
    其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的N末端；或，所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3对称地连接在所述免疫球蛋白的两条重链的C末端，且所 述C末端由K突变为A；
    优选地，所述靶向LAG-3的双特异性抗体包括以下氨基酸序列：
    如SEQ ID NO:44所示的轻链氨基酸序列，如SEQ ID NO:46所示的含重链的氨基酸序列；或，如SEQ ID NO:42所示的轻链氨基酸序列，如SEQ ID NO:47所示的含重链的氨基酸序列；或，如SEQ ID NO:42所示的轻链氨基酸序列，如SEQ ID NO:48所示的含重链的氨基酸序列；或，如SEQ ID NO:49所示的含轻链的氨基酸序列，如SEQ ID NO:43所示的重链氨基酸序列；或，如SEQ ID NO:50所示的含轻链的氨基酸序列，如SEQ ID NO:43所示的重链氨基酸序列。
  14. 一种分离的核酸，其编码如权利要求1-5中的任一项所述的LAG-3结合蛋白或如权利要求6-13中任一项所述的靶向LAG-3的双特异性抗体。
  15. 一种包含根据权利要求14所述的分离的核酸的表达载体。
  16. 一种宿主细胞，其包含根据权利要求15所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞。
  17. 一种LAG-3结合蛋白或靶向LAG-3的双特异性抗体的制备方法，其包含培养如权利要求16中所述的宿主细胞，从培养物中获得LAG-3结合蛋白或靶向LAG-3的双特异性抗体。
  18. 一种抗体药物偶联物，其包含细胞毒性剂，以及如权利要求1-5任一项中所述的LAG-3结合蛋白或如权利要求6-13任一项所述的靶向LAG-3的双特异性抗体。
  19. 一种药物组合物，其包含如权利要求1-5任一项所述的LAG-3结合蛋白，或如权利要求6-13中任一项所述的靶向LAG-3的双特异性抗体，或如权利要求18所述的抗体药物偶联物。
  20. 如权利要求1-5中任一项所述的LAG-3结合蛋白、权利要求6-13中任一项所述的靶向LAG-3的双特异性抗体、权利要求18中所述的抗体药物偶联物和/或权利要求19中所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。
PCT/CN2020/095577 2019-06-13 2020-06-11 靶向lag-3的抗体和双特异性抗体及其用途 WO2020249041A1 (zh)

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