Antibodies and bispecific antibodies targeting LAG-3 and their uses

Abstract

The present invention discloses 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 a CDR1 of an amino acid sequence as shown in SEQ ID NO:5, a CDR2 of an amino acid sequence as shown in SEQ ID NO:6, and / or a CDR3 of an amino acid sequence as shown in SEQ ID NO:7; the heavy chain variable region includes: a CDR1 of an amino acid sequence as shown in SEQ ID NO:8, a CDR2 of an amino acid sequence as shown in SEQ ID NO:9, and / or a CDR3 of an amino acid sequence as shown in SEQ ID NO:10. Also disclosed is a bispecific antibody targeting LAG-3 and its application. The above-mentioned LAG-3 binding protein and bispecific antibody can effectively block the binding of LAG-3 to MHC II and activate T cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an antibody and a bispecific antibody targeting LAG-3 and uses thereof. Background Art

[0002] Lymphocyte-activation gene 3 (LAG-3) belongs to the immunoglobulin superfamily and consists of three parts: the extracellular region, the transmembrane region, and the cytoplasmic region. The gene of LAG-3 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, exhausted T cells, tumor-infiltrating T cells, and regulatory T cells (Treg). After binding to the major histocompatibility complex 2 (MHC class II), the LAG-3 / MHC class II interaction leads to negative regulation of T cell proliferation, activation, and homeostasis. Inhibition of LAG-3 can relieve LAG3's inhibition of MHCII-peptide-T cell receptor antigen presentation and allow T cells to regain cytotoxicity, thereby enhancing the killing effect on tumors. At the same time, inhibition of LAG-3 can also reduce the function of regulatory T cells to suppress immune responses. Therefore, LAG-3 is considered to be a more attractive target than other immune checkpoint proteins.

[0003] LAG-3 is a target with relatively more clinical data and relatively certain drugability among the second-generation immune checkpoint targets. Antibody drugs targeting this target may become important anti-tumor drugs in the future. However, there are currently no drugs targeting LAG-3 on the market worldwide. By the end of 2018, there were a total of 30 drugs under clinical research. LAG-3 antibodies in the clinical research stage include GlaxoSmithKline's GSK2831781, Novartis's LAG525, Regeneron's REGN3767 and Tesaro's TSR-033. Most LAG-3 antibody drugs are committed to developing combination therapies with PD-1. Among them, the fastest-developed is Relatlimab, which is in the II / III clinical stage developed by BMS and Ono; IMP321 is in the Phase II clinical trial and LAG525 is in the Phase I / II clinical trial; there are 8 drugs in the Phase I clinical trial stage and 9 drugs in the preclinical trial stage. The main therapeutic areas of drugs targeting LAG-3 include cancer and autoimmune diseases. Bristol-Myers Squibb's Relatlimab (development code BMS-986016, originally developed by Medarex). MacroGenics' MGD013 is a PD-1 / LAG-3 bispecific antibody with a long half-life in serum. It has the potential to treat a variety of different cancers by simultaneously blocking the immunosuppression of these two immune checkpoint molecules.

[0004] However, there is still a lack of LAG-3-targeted antibodies with better thermal stability, better T cell activity, and effective blocking of LAG-3 binding to MHCII, better animal efficacy, and better PK in the clinic, as well as bispecific antibodies targeting LAG-3 with simple structure, stable molecules, and efficient process. Summary of the invention

[0005] In order to overcome the lack of more stable and active LAG-3-targeting antibodies and the lack of bispecific antibodies targeting LAG-3 with simple structure, stable molecules and efficient process in the art, an antibody targeting LAG-3 and a bispecific antibody and use thereof are provided. The bispecific antibody of the present invention is a sequence-based IgG like bispecific antibody (SBody). This patent will refer to this design as SBody.

[0006] The present invention provides an antibody targeting LAG-3 through optimized design and screening. The antibody has new light chain variable regions CDR1, CDR2 and CDR3 and heavy chain variable regions CDR1, CDR2 and CDR3, has good thermal stability and better T cell activity, can more effectively block the binding of LAG-3 to MHC II, and has better in vivo efficacy and PK. A bispecific antibody targeting LAG-3 is also provided, which can simultaneously target LAG-3 and another target; the new design can achieve the effect of one molecule targeting two specific targets at the same time, so that one molecule replaces the combination of two molecules, and even has the effect of synergistically treating tumors. The process, production cost, clinical trial, etc. of one molecule are more convenient and cost-effective than the combination of two separate molecules.

[0007] To solve the above technical problems, one of the technical solutions of the present invention is: a LAG-3 binding protein, comprising a light chain variable region and / or a heavy chain variable region; the light chain variable region comprises: a CDR1 with an amino acid sequence as shown in SEQ ID NO:5, a CDR2 with an amino acid sequence as shown in SEQ ID NO:6, and / or a CDR3 with an amino acid sequence as shown in SEQ ID NO:7; the heavy chain variable region comprises: a CDR1 with an amino acid sequence as shown in SEQ ID NO:8, a CDR2 with an amino acid sequence as shown in SEQ ID NO:9, and / or a CDR3 with an amino acid sequence as shown in SEQ ID NO:10.

[0008] The LAG-3 binding protein as described above, wherein 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;

[0009] And / or, 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.

[0010] In some preferred embodiments, for the LAG-3 binding protein as described above, 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; and / or 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 a substitution, deletion or addition of one or more amino acid residues in the original amino acid sequence, preferably having 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.

[0011] Preferably, 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 comprises the amino acid sequence shown in SEQ ID NO:23, and 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, and the heavy chain variable region comprises 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:24, and the heavy chain variable region comprises 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; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26, and the heavy chain variable region comprises 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:27, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: NO:31; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28, and the heavy chain variable region comprises 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:29, and the heavy chain variable region comprises 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:32; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28, and 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:28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34. NO:34; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:36;The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37. ;

[0012] In some preferred embodiments, the LAG-3 binding protein as described above is an antibody, Fab, Fab', F(ab') 2 , Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single region antibody, or monoclonal antibody or polyclonal antibody made from the above antibodies.

[0013] In some preferred specific embodiments, 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. Preferably, the human antibody light chain constant region is a κ chain or a λ chain, or the human antibody heavy chain constant region is hIgG1, hIgG2, hIgG4 or a mutation thereof; the mutation has one or more amino acid residues substituted, deleted or added to the original amino acid sequence, preferably having 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.

[0014] In some preferred embodiments, the light chain of the LAG-3 binding protein as described above comprises the amino acid sequence as shown in SEQ ID NO:38 or SEQ ID NO:39 or a mutation thereof; and / or the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:40 or SEQ ID NO:41 or a mutation thereof; the mutation has a substitution, deletion or addition of one or more amino acid residues in the original amino acid sequence, preferably having 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.

[0015] Preferably, the amino acid sequence of the light chain is as shown in SEQ ID NO:38; the amino acid sequence of the heavy chain is as shown in SEQ ID NO:40; or, the amino acid sequence of the light chain is as shown in SEQ ID NO:38; the amino acid sequence of the heavy chain is as shown in SEQ ID NO:41; or, the amino acid sequence of the light chain is as shown in SEQ ID NO:39; the amino acid sequence of the heavy chain is as shown in SEQ ID NO:40; or, the amino acid sequence of the light chain is as shown in SEQ ID NO:39; the amino acid sequence of the heavy chain is as shown in SEQ ID NO:41.

[0016] In order to solve the above technical problems, the second technical solution of the present invention is: a bispecific antibody targeting LAG-3, comprising a first protein functional region and a second protein functional region, characterized in that the first 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 respectively selected from immunoglobulin, scFv (single chain Fv, also known as single chain variable fragment), Fab, Fab' or F(ab') 2 , and only one of the first protein functional region and the second protein functional region is an immunoglobulin.

[0017] In order to design a bispecific antibody with a simple production process and retain effective activity, the bispecific antibody of the present invention is in the form of a structure similar to normal IgG. Specifically, a protein functional region of the light chain and / or heavy chain variable region that can target two targets is designed in its structure, and the two protein functional regions share the same heavy chain Fc region. Preferably, the antibody molecule of one target is connected to one end of the light chain or heavy chain of the complete antibody of another target in the form of one or more scFvs. This avoids the uneven expression product caused by the expression of different heavy chain Fc and / or different light chains, such as the co-expression of Knob-form Fc and Hole-form Fc, which will have uneven Fc-Fc pairing during its expression process, bringing a lot of inconvenience to the purification process; it can also avoid the influence of the cross design of the light and heavy chain partial regions on the structural activity, as well as the Fc mismatch phenomenon that occurs during the process. Through the design of one or more scFvs, the activity against specific targets can also be adjusted.

[0018] Therefore, in some preferred specific embodiments, the bispecific antibody targeting LAG-3 as described above, wherein the first protein functional region is an immunoglobulin, and the second protein functional region is one or more scFv; or, the first protein functional region is one or more scFv, and 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 antibody heavy chain constant region. Preferably, the human antibody light chain constant region is a κ chain or a λ chain, and the human antibody heavy chain constant region is hIgG1, hIgG2, hIgG4 or a mutation thereof; the mutation has one or more amino acid residues substituted, deleted or added to the original amino acid sequence, preferably having 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.

[0019] In some preferred embodiments, the bispecific antibody targeting LAG-3 as described above, wherein the scFv comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are connected by a linker, and the scFv is connected to the immunoglobulin by a linker, and the linker is preferably (Gly-Gly-Gly-Gly-Ser) w [Hereinafter abbreviated as (G 4 S) w ]; w is preferably an integer between 0 and 10, more preferably 1, 2, 3 or 4. The bispecific design summary (Formula 1) can be found in Table 10 of Example 12. In addition, the linker can also be selected from peptide segments conventionally used as linkers in the art.

[0020] In some preferred specific embodiments, the bispecific antibody targeting LAG-3 as described above, wherein the scFv is a light chain variable region-linker-heavy chain variable region structure, and the N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region is correspondingly 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, and the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region is correspondingly connected to the C-terminus or N-terminus of the immunoglobulin light chain and / or heavy chain through a linker.

[0021] In some preferred embodiments, the bispecific antibody targeting LAG-3 as described above, wherein the linker is (G 4 S) 3 , and / or, the number of the scFv is two, symmetrically connected to the immunoglobulin light chain and / or heavy chain. Preferably, 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 connected by (G 4 S) 3 Symmetrically connected to the N-termini of the two heavy chain variable regions of the immunoglobulin, or the N-termini of the light chain variable regions of the two scFvs, respectively through (G 4 S) 3 Symmetrically connected to the C-termini 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 C-termini of the light chain variable regions of the two scFvs are respectively connected by (G 4 S) 3 Symmetrically connected to the N-termini of the two light chain variable regions of the immunoglobulin, or the N-termini of the heavy chain variable regions of the two scFvs, respectively through (G 4 S) 3 Symmetrically connected to the C-termini of the two light chain variable regions of the immunoglobulin.

[0022] In some preferred embodiments, the bispecific antibody targeting LAG-3 as described above, wherein the second protein functional region targets PD-1. Preferably, the second protein functional region is an anti-PD-1 antibody. More preferably, the anti-PD-1 antibody is Nivolumab, Pembrolizumab or Ba08.

[0023] In some preferred embodiments, the bispecific antibody targeting LAG-3 as described above is selected from the following group:

[0024] (1) The first protein functional region is an immunoglobulin, which comprises the following light chain and heavy chain or a mutation thereof: wherein the amino acid sequence of the light chain is as shown in SEQ ID NO:38, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:40; the amino acid sequence of the light chain is as shown in SEQ ID NO:38, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:41; the amino acid sequence of the light chain is as shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:40; or, the amino acid sequence of the light chain is as shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:41; and / or,

[0025] 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 of positions 1 to 107 of SEQ ID NO:42, and the amino acid sequence of the heavy chain variable region of the scFv is as shown in the amino acid sequence of positions 1 to 113 of SEQ ID NO:43; or, the amino acid sequence of the light chain variable region of the scFv is as shown in the amino acid sequence of positions 1 to 111 of SEQ ID NO:44, and the amino acid sequence of the heavy chain variable region of the scFv is as shown in the amino acid sequence of positions 1 to 120 of SEQ ID NO:45;

[0026] The mutation has a substitution, deletion or addition of one or more amino acid residues in the original amino acid sequence, preferably having 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.;

[0027] Preferably, when the scFv is linked to the C-termini of the two heavy chains of the immunoglobulin, the C-termini of the heavy chains are mutated from K to A;

[0028] (2) The first protein functional region is scFv or a mutant thereof, wherein the amino acid sequence of the light chain variable region of the scFv is as shown in SEQ ID NOs: 23-29; and / or the amino acid sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NOs: 30-37; and / or,

[0029] The 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 amino acid sequence of the light chain of the immunoglobulin 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;

[0030] The mutation has a substitution, deletion or addition of one or more amino acid residues in the original amino acid sequence, preferably having 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.;

[0031] Preferably, when the scFv is linked to the C-termini of the two heavy chains of the immunoglobulin, the C-termini of the heavy chains are mutated from K to A.

[0032] In some preferred embodiments, the bispecific antibody targeting LAG-3 as described above, wherein (i) the first protein functional region is scFv, the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 28, the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 31, and 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:42, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:43;

[0033] 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 connected by (G 4 S) 3 Symmetrically connected to the N-termini 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 N-termini of the heavy chain variable regions of the two scFvs are respectively connected by (G 4 S) 3The scFv is symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin, and the C-termini of the heavy chain are 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 connected by (G 4 S) 3 Symmetrically connected to the N-termini of the two light chains of the immunoglobulin; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the N-termini of the heavy chain variable regions of the two scFvs are respectively connected by (G 4 S) 3 symmetrically linked to the C-termini of the two light chains of the immunoglobulin; or,

[0034] (ii) the first protein functional region is scFv, the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 28, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 31, and 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;

[0035] 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 connected by linkers (G 4 S) 3 Symmetrically connected to the N-termini 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 N-termini of the heavy chain variable regions of the two scFvs are connected by linkers (G 4 S) 3 The scFv is symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin, and the C-termini of the heavy chain are 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 connected by linkers (G 4 S) 3 Symmetrically connected to the N-termini of the two light chains of the immunoglobulin; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the N-termini of the heavy chain variable regions of the two scFvs are connected by linkers (G 4 S) 3 symmetrically linked to the C-termini of the two light chains of the immunoglobulin; or,

[0036] (iii) the first protein functional region is scFv, the number of the scFv is a pair, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 36, and 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;

[0037] 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 connected by linkers (G 4 S) 3 Symmetrically connected to the N-termini 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 N-termini of the heavy chain variable regions of the two scFvs are connected by linkers (G 4 S) 3 It is symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin, and the C-termini of the heavy chains are mutated from K to A.

[0038] In some preferred embodiments, the bispecific antibody targeting LAG-3 as described above comprises the following amino acid sequence:

[0039] A light chain amino acid sequence as shown in SEQ ID NO:44, and an amino acid sequence containing a heavy chain as shown in SEQ ID NO:46; or, a light chain amino acid sequence as shown in SEQ ID NO:42, and an amino acid sequence containing a heavy chain as shown in SEQ ID NO:47; or, a light chain amino acid sequence as shown in SEQ ID NO:42, and an amino acid sequence containing a heavy chain as shown in SEQ ID NO:48; or, a light chain amino acid sequence as shown in SEQ ID NO:49, and a heavy chain amino acid sequence as shown in SEQ ID NO:43; or, a light chain amino acid sequence as shown in SEQ ID NO:50, and a heavy chain amino acid sequence as shown in SEQ ID NO:43.

[0040] Alternatively, the bispecific antibody of the present invention is a DVD-Ig (Dual-variable domain Ig) bispecific antibody, whose structure is to insert VL and VH of another antibody into the N-terminus of the light and heavy chains of a normal antibody, respectively, and to achieve dual functions by binding to two targets through two antibody variable regions. The design summary of the bispecific antibody (general formula 2) is shown in Table 12 of Example 12.

[0041] In a preferred embodiment, the bispecific antibody is composed of a sequence containing a light chain and a sequence containing a heavy chain. The bispecific antibody is selected from the following combinations: the sequence containing the light chain is Ab2317VL-(G 4 S) 3 -NivoVL-Lc (κ chain), containing the heavy chain sequence of Ab2317VH-(G 4 S) 3 -NivoVH-Hc(hIgG4); or, the sequence containing the light chain is PemVL-(G 4 S) 3 –Ab2317VL-Lc (κ chain), containing the heavy chain sequence of PemVH-(G 4 S) 3 –Ab2317VH-Hc(hIgG4); or, the sequence containing the light chain is NivoVL-(G 4 S) 3 –Ab2325VL-Lc (κ chain), containing the heavy chain sequence NivoVH-(G 4 S) 3 –Ab2325VH-Hc(hIgG1).

[0042] Alternatively, the bispecific antibody of the present invention comprises a first protein functional region and a second protein functional region, one of which is an immunoglobulin and the other is a Fab' or F(ab') 2 .

[0043] In a preferred embodiment, the first protein functional region is an immunoglobulin, and 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 The immunoglobulin is connected via a disulfide bond or a linker, wherein the linker is preferably a peptide segment or (Gly-Gly-Gly-Gly-Ser) that can be used as a linker conventionally in the art. w , the w is preferably an integer between 0 and 10, more preferably 1, 2, 3 or 4; the constant region of the immunoglobulin is preferably a human antibody constant region, the human antibody constant region preferably includes a human antibody light chain constant region and a human antibody heavy chain constant region, the human antibody light chain constant region is preferably a κ chain or a λ chain; the human antibody heavy chain constant region is preferably hIgG1, hIgG2 or hIgG4. When the Fab' or F(ab') 2 After the light chain and heavy chain are connected by a linker, because there is a linker between the light and heavy chains, it is no longer a Fab' or F(ab') in the strict sense.2 .

[0044] To solve the above technical problems, the third technical solution of the present invention is: an isolated nucleic acid encoding the LAG-3 binding protein as described above or the bispecific antibody targeting LAG-3 as described above.

[0045] To solve the above technical problems, the fourth technical solution of the present invention is: an expression vector comprising the isolated nucleic acid as described above.

[0046] To solve the above technical problems, the fifth technical solution of the present invention is: a host cell, which comprises the above expression vector. Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0047] To solve the above technical problems, the sixth technical solution of the present invention is: a method for preparing a LAG-3 binding protein or a bispecific antibody targeting LAG-3, which comprises culturing the host cell as described above, and obtaining the LAG-3 binding protein or the bispecific antibody targeting LAG-3 from the culture.

[0048] To solve the above technical problems, the seventh technical solution of the present invention is: an antibody-drug conjugate, which comprises a cytotoxic agent and the LAG-3 binding protein as described in one of the above technical solutions or the bispecific antibody targeting LAG-3 as described in the second technical solution.

[0049] In order to solve the above technical problems, the eighth technical solution of the present invention is: a pharmaceutical composition, which comprises the LAG-3 binding protein as described above, or the bispecific antibody targeting LAG-3 as described above, or the antibody-drug conjugate as described above.

[0050] To solve the above technical problems, the ninth technical solution of the present invention is: a drug kit combination, which comprises a drug kit A and a drug kit B; the drug kit A comprises the LAG-3 binding protein as described above, or the bispecific antibody targeting LAG-3 as described above, or the antibody-drug conjugate as described above, or the pharmaceutical composition as described above; the drug kit B comprises other drugs for treating cancer.

[0051] In order to solve the above technical problems, the tenth technical solution of the present invention is: use of the above-mentioned LAG-3 binding protein, bispecific antibody targeting LAG-3, antibody-drug conjugate, pharmaceutical composition and / or kit combination in the preparation of drugs for treating and / or preventing cancer.

[0052] Preferably, the cancer is selected from the group consisting of leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, urothelial carcinoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck squamous cell carcinoma, testicular cancer, endocrine gland cancer, adrenal cancer, pituitary cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, neural cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, uterine cancer, glioblastoma, medulloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome and sarcoma.

[0053] Thus, the present invention also provides a method for treating cancer, wherein the method is to use the LAG-3 binding protein, bispecific antibody targeting LAG-3, antibody-drug conjugate, pharmaceutical composition and / or kit combination as described above to treat patients suffering from the above cancer.

[0054] It should be understood that the terms "first" and "second" in the present invention have no practical meaning and are only used to distinguish the same terms. 2 When 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.

[0055] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0056] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0057] As used herein, 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]. In a general sense, the heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody, and the light chain refers to a polypeptide chain with a smaller molecular weight in an antibody. Light chains can be classified as κ and λ light chains. Heavy chains can generally be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains 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 consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one 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 (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability [called complementary determining regions (CDRs)], interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) corresponding to each heavy chain / light chain form the antibody binding site, respectively. The allocation of amino acids to each region or domain follows the definition of Kabat EA. Et al., Sequences of Proteins of Immunological Interest [National Institutes of Health, Bethesda, Md. (1987 and 1991)], or Chothia & Lesk 1987)]. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883. In particular, the heavy chain can also contain more than 3 CDRs, such as 6, 9 or 12. For example, in the bispecific antibody of the present invention, the heavy chain can be the N-terminus of the heavy chain of an IgG antibody connected to the ScFv of another antibody, in which case the heavy chain contains 9 CDRs.

[0058] In this article, unless the context clearly indicates, when referring to the term "antibody", it includes not only complete 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 to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "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 cleavage of complete antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab'), and Fab'. 2 , Fd, Fv, dAb and complementarity determining region (CDR) fragments, single-chain binding fragments (e.g., scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

[0059] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody; the term "Fab" refers to an antibody fragment consisting of the VL, VH, CL and CH1 (or CH) domains; the term "F(ab') 2 ” refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0060] In some cases, the antigen-binding fragment of an antibody is a single-chain binding fragment (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain [see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can 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 sequence or its variant. For example, a 4 S) 4 or (G 4 S) 3 linker, but variations thereof may also be used.

[0061] Antibody antigen-binding fragments (such as those described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0062] As used herein, the term "isolated" means obtained from the natural state by artificial means. 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 separated 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 separated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0063] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0064] As used herein, the term "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. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind to antigens at a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The antibody binds to the antigen with a dissociation equilibrium constant of 2 M or less, for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument. The affinity of the antibody and cell binding is determined, for example, using the KINEXA method on a KINEXA 400 instrument.

[0065] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0066] The reagents and raw materials used in the present invention are commercially available.

[0067] The positive and progressive effects of the present invention are:

[0068] (1) LAG-3 binding protein more effectively blocks the binding of LAG-3 to MHC II, thereby eliminating the negative regulation of signal transduction and antigen presentation between APC / T cells;

[0069] (2) The obtained humanized LAG-3 antibody exhibits good cell function activity, such as good activation of human T cells, human DC cell function and activity, animal efficacy, better PK characteristics, high expression yield, and thermal stability;

[0070] (3) Sequence-specific bispecific antibodies (SBody) have good dual-target activity, stability, high expression levels, a structure similar to normal IgG antibodies, and a simple purification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The functions and activities of the anti-human LAG-3 antibody mab23c of the present invention are as follows: a. the binding activity of mab23c and human LAG-3 (ELISA); b. the blocking activity of mab23c in preventing the binding of LAG-3 and Daudi cells.

[0072] Figure 2 It is a gel electrophoresis (PAGE) diagram used to evaluate the thermal stability of the anti-human LAG-3 humanized antibody Ab2317 of the present invention.

[0073] Figure 3 The functions and activities of the anti-human LAG-3 humanized antibody Ab2317 of the present invention are as follows: a. Ab2317 activates human T cell activity; b. Ab2317 activates human DC cells to stimulate human T cell activity (MLR assay).

[0074] Figure 4 This is an in vivo animal efficacy evaluation of the anti-human LAG-3 humanized antibody Ab2317 of the present invention.

[0075] Figure 5A The structure of the bispecific antibody (SBody) LB2373 of the present invention and related test data diagram, including a structural schematic diagram (c), and the binding activity test results of human LAG-3 (a), and the binding activity test results of human PD-1 (b).

[0076] Figure 5B The structure of the bispecific antibody (SBody) LB2374 of the present invention and related test data diagrams include a structural schematic diagram (c), and the binding activity test results of human LAG-3 (a), and the binding activity test results of human PD-1 (b).

[0077] Figure 5CThe structure of the bispecific antibody (SBody) LB2371 of the present invention and related test data diagram, including a structural schematic diagram (c), and the binding activity test results of human LAG-3 (a), and the binding activity test results of human PD-1 (b).

[0078] Figure 5D The structure of the bispecific antibody (SBody) LB2372 of the present invention and related test data diagram, including a structural schematic diagram (c), and the binding activity test results of human LAG-3 (a), and the binding activity test results of human PD-1 (b). DETAILED DESCRIPTION

[0079] The present invention is further described below in conjunction with the examples, but these examples are not intended to limit the scope of the present invention. The experimental methods in the examples of the present invention that do not specify specific conditions are usually carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify the specific source are conventional reagents purchased from the market.

[0080] Example 1: Cloning, expression and purification of antigens and antibodies

[0081] The antigens used in the present invention are purchased from the following different companies: Beijing Biopsies Biotechnology Co., Ltd. LAG-3-his (Cat. No.: LA3-H5222), cyno-LAG-3-mFc (Cat. No. LA3-C52A0) or Beijing Yiqiao Shenzhou Technology Co., Ltd. LAG-3-his (Cat. No.: 16498-H08H), LAG-3-hFc (Cat. No.: 16498-H05H), cyno-LAG-3-his (Cat. No. 90841-C08H), or expressed and purified by the present invention. The expressed human LAG-3 protein sequence is NCBI Reference Sequence: NP_002277.4, with a total length of 1-525 amino acids, of which 1-22 is a signal peptide; the extracellular (ECD) region is 23-422 amino acids; the first and second regions (domain #1 and #2, D12) of the extracellular (ECD) region 23-239 amino acids are D12-his, D12-hFc. The sequence of mouse LAG-3his tagged (mLAG-3-his) is from NCBI gi|148667361|gb|EDK99777.1, the extracellular region (ECD) is 23-406 amino acids. The sequence of Macaca LAG-3his tagged (cynoLAG-3-his) is NCBI No. NP_001271679.1, and its extracellular region (ECD) is 23-434 amino acids.

[0082] The antibodies used in the present invention, including the positive control antibody Ref (i.e., BMS-986016, sequence from WO2014008218A1, LAG3.5, #12 light chain, #14 heavy chain), and the 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.

[0083] The pTT5 vector (Biovector, Cat#: 102762) was used for expression. The recombinant protein, antibody light chain and heavy chain sequences were cloned into the pTT5 vector, expressed by transient transfection in HEK293E cells (Life Technologies Cat. No. 11625019), and then purified.

[0084] Specifically, 293 cells were cultured in Gibco FreeStyle 293 Expression Medium (Gibco, Cat#12338018). Before transient transfection, the cell concentration was adjusted to 6-8×10 5 cell / ml, 1% FBS (AusGene X FBS Excellent supplier: AusGeneX, China, Cat# FBSSA500-S), 37°C 8% CO 2 The cells were cultured on a shaker for 24 h and the survival rate was >95% and the cell density was 1.2×10 6 cell / ml.

[0085] Prepare 300ml culture system cells, dissolve 150μg of heavy chain and light chain plasmids in 15ml Opti-MEM (Gibco, Cat#31985070) (if it is a recombinant protein, the amount of a single plasmid is 300μg), and sterilize by 0.22μm filtration. Take 15ml Opti-MEM and dissolve 600μl of 1mg / ml PEI (Polysciences, Inc, Cat#23966-2) and let it stand for 5min. Slowly add PEI to the plasmid, incubate at room temperature for 10min, and slowly drip the plasmid PEI mixed solution while shaking the culture bottle, 37℃ 8% CO 2 After 5 days of shaking culture, the samples were collected and the supernatant was taken for purification at 3300G for 10 min.

[0086] Purification of antibody or -Fc fusion protein: centrifuge the sample at high speed to remove impurities, balance the gravity column (Sangon Biotech, Cat#F506606-0001) containing Protein A (Mabselect, GE Healthcare Life Science, Cat#71-5020-91AE) with PBS pH 7.4, and rinse with 2-5 column volumes. Pass the sample through the column. Rinse the column with 5-10 column volumes of PBS (Sangon Biotech, Cat#B548117-0500). Elute the target protein with pH 3.5 0.1M acetic acid, and then adjust to neutral with Tris-HCl at pH 8.0. Measure the concentration with an ELISA reader, and aliquot and store for later use.

[0087] Purification of His Tagged protein: Centrifuge the sample at high speed to remove impurities. Equilibrate the nickel column (Ni smart beads 6FF Changzhou Tiandi Renhe Biotechnology Co., Ltd. Cat#SA036010): Equilibrate the nickel column with PBS pH7.4 solution containing 10mM imidazole and 0.5M NaCl, and rinse with 2-5 column volumes. Pass the sample supernatant through the column. Rinse the impurities: Rinse the column with PBS pH7.4 solution containing 10mM imidazole and 0.5M NaCl to remove non-specifically bound impurities, and collect the effluent. Elute the target protein with PBS pH7.4 containing 250mM imidazole and 0.5M NaCl. Buffer replacement: Centrifuge the eluted target protein through an ultrafiltration tube at 12000g for 10min (ultrafiltration tube Merck Millipore Cat#UFC500308), then add 1ml PBS, determine the concentration, aliquot, and store for later use.

[0088] Example 2: Construction of human LAG-3 high-expressing cell line (hLAG-3+ cells) and binding activity (ELISA) detection

[0089] The human LAG-3 high-expressing cell line used in the present invention was 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 (Cat#GNHu17 of the Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee) were inoculated into two 6 cm culture dishes, and the number of cells in each culture dish reached 7.5×10 5On the second day, 4 μg of the packaged plasmid (pGag-pol, pVSV-G and other BioVector plasmid vectors, bacterial strains and cell gene collection centers) and the plasmid pBabe-hLAG-3 cloned with the human LAG-3 gene were added to OPTI-MEM (Thermofisher Scientific Cat#31985070) to make the final volume 200 μl. Prepare another 200 μl of OPTI-MEM and add 36 μl of transfection reagent fectin (Shanghai Yuanpei Biotechnology Co., Ltd. Cat#F210). The two were mixed and placed at room temperature for 5 minutes, and then the mixture (200 μl per dish) was added dropwise to the cultured 293T cells. On the third day, the 293T cell culture medium was replaced with 4 ml of DMEM high-glucose medium (Shanghai Yuanpei Biotechnology Co., Ltd. / Yuanpei Biotechnology: Cat#L130KJ). On the 4th day, CHO-K1 cells (Cat# SCSP-507, Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee) were inoculated in a 10 cm culture dish to increase the cell number to 5 × 10 5 On the 5th day, the supernatant (virus) of 293T cells was collected and filtered through a 0.45 μm filter membrane to the cultured CHO-K1 cells. At the same time, 10 μg / ml polybrene (Shanghai Yisheng Biotechnology Co., Ltd. Cat#40804ES76) was added, mixed and placed in an incubator. After 3 to 4 hours, it was replaced with DMEM / F12 10% FBS medium (Source Culture Biology, Cat#L310KJ). On the 7th day, the CHO-K1 cells were passaged, and on the 8th day, 10 μg / ml puromycin was added to the passaged cells for screening (Source Culture Biology, Cat#S250J0). After 2-3 days, a large number of cells died, and the culture medium was replaced and continued until the cells stopped dying. The cells were amplified in large numbers, and monoclonal cell lines were screened, expanded, and frozen for seed preservation.

[0090] The amino acid sequence of human LAG-3 (pBabe-hLAG-3) used in this example is NP_002277.4, which is the entire sequence 1-525 amino acids, wherein positions 1-22 are the signal peptide sequence, i.e., positions 23-525 are the protein sequence expressed by the CHO-K1hLAG-3+ cell line constructed in the present invention.

[0091] hLAG-3+ cell binding activity (ELISA) detection:

[0092] The monoclonal cell line with high expression of human LAG-3 obtained in the above example was expanded and cultured at 1×10 5 / well of a 96-well plate, incubate overnight in a 37°C incubator, remove the supernatant, use 100μl / well of immunostaining fixative (Shanghai Biyuntian Biotechnology Co., Ltd. Cat#P0098), fix at room temperature for half an hour. Wash once with PBS (Source Culture Biology, Cat#B320), block with 230μl 5% milk at 37°C for 2 hours, and wash 3 times with PBST. Add 50μl, 10μg / ml, 5-fold gradient dilution of the sample to be tested to each well. Incubate at 37°C for 1 hour, then wash 5 times with PBST. Add Anti-human HRP (Jackson Immuno Research, Cat#109-035-003) 1:2500 50μl / well, incubate at 37°C for 1 hour, then wash 5 times with PBST, add 50μl TMB (Surmodic Cat#TTMB-1000-01) to each well for color development, and add 50μl / well 1M H 2 SO 4 The reaction was terminated, and the results were read using a microplate reader (MultiskanGO Thermo model 51119200), and data were analyzed using Graphpad prism 5.

[0093] Example 3: Anti-LAG-3 antibody and LAG-3 antigen binding assay (ELISA)

[0094] Dilute LAG-3-his, LAG-3-D12-his, monkey LAG-3-his (cyno LAG-3-his) or mLAG-3-his recombinant proteins to 1μg / ml, 2μg / ml (hLAG-3D12-his), or 5μg / ml (cyno LAG-3-his) with PBS buffer at pH 7.4, add 50μl / well to a 96-well ELISA plate (Corning, CLS3590-100EA), and place in a 37°C incubator for 2 hours. After discarding the liquid, add 230μl / well of 5% skim milk (Bright skim milk powder) blocking solution diluted with PBS, incubate in a 37°C incubator for 3 hours or place at 4°C overnight (16-18 hours) for blocking. Discard the blocking solution, wash the plate 5 times with PBST buffer (pH7.4PBS containing 0.05% tweeen-20), add 50μl / well supernatant (containing detection antibody) or 10μg / ml starting, 5-fold serial dilution of the test antibody, incubate at 37°C for 1 hour, wash the plate 5 times with PBST, add 50μl / well 1:2500 dilution of 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. After washing the plate 5 times with PBST, add 50μl / well TMB colorimetric substrate (KPL, 52-00-03), incubate at room temperature for 10-15min, add 50μl / well 1M H 2 SO 4 The reaction was terminated, and the absorbance was read at 450 nm using a MULTISKAN Go microplate reader (ThermoFisher, 51119200). The clones with high binding activity were selected according to the OD value, or the EC50 value (for antibodies with known concentrations) was calculated.

[0095] Example 4: Anti-LAG-3 antibody inhibits the binding activity of LAG-3 and Daudi cells

[0096] Daudi cell line (ATCC, CCL-213) was cultured and plated at 2x10 5 / well of a 96-well plate, centrifuge at 1600rpm for 10min, and fix with 100μl / well of immunostaining fixative (Shanghai Biotech Co., Ltd. Cat#P0098) at room temperature for half an hour. Wash once with PBS (Source Biotech, Cat#B320), block with 230μl 5% milk at 37℃ for 2 hours, and wash 3 times with PBST. Add 25μl, 100μg / ml, 3-fold gradient dilution of the sample to be tested and 25μl 2.5μg / ml bio-LAG3-mFc (Beijing Yiqiao Biotech Co., Ltd., 16498-H05H) to each well. Incubate at 37℃ for 1 hour, and then wash 5 times with PBST. Add 50 μl / well of 1:1000 diluted streptavidin-HRP secondary antibody (genscript, M00091), incubate at 37°C for 1 hour, wash 5 times with PBST, add 50 μl TMB (Surmodic Cat#TTMB-1000-01) to each well for color development, and add 50 μl / well 1M H 2 SO 4 The reaction was terminated, the results were read using a microplate reader (MultiskanGO Thermo model 51119200), and data were analyzed using Graphpad prism 5.

[0097] Example 5: Discovery of anti-human LAG-3 antibodies

[0098] The present invention uses human LAG-3 recombinant protein (prepared in Example 1) as an antigen, immunizes mice, screens fusion hybridomas, and screens and optimizes from millions of hybridoma clones, and unexpectedly finds a hybridoma cell line with very good binding activity to hLAG-3. The cell line is further subcloned and screened to obtain a monoclonal cell line, and the mouse antibody sequence obtained from the monoclonal cell line is screened by computer modeling and humanized design to obtain an optimized humanized antibody. The humanized antibody also maintains a good binding activity to hLAG-3, and surprisingly, the obtained humanized antibody also shows good cell function activity, animal efficacy, better PK characteristics, high expression yield and thermal stability.

[0099] Specifically, the experimental SJL mice, female, 4 weeks old, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., Animal Production License No.: SCXK (Beijing) 2016-0011. After the mice were purchased, they were kept in the laboratory environment for 1 week, with a light / dark cycle during the day and a temperature of 20-25°C; humidity of 40-60%. The mice were divided into 3 / group / cage. Immunization was performed with the antigen prepared in Example 1. The adjuvant was Quickantibody-5w (Beijing Biolong Immunotechnology Co., Ltd., KX0210041). The ratio of antigen to adjuvant was 1:1. 100μl / 10μg / mouse for the first immunization, 100μl / 10μg / mouse for the second, third, fourth, and fifth calf muscle injections. Three days before fusion, 100μl / 25μg / mouse for booster immunization. The immunization time is day 0, 14, 28, 46, 58, and 60 (boost immunization). On days 23, 50, and 58, the mouse serum antibody titers were detected by the ELISA method of Example 3, and mice with high antibody titers in the serum and at a plateau were selected for spleen cell fusion. Splenic lymphocytes were fused with myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells and plated in 96-well plates.

[0100] The hybridoma cell lines plated in the 96-well plate were initially screened by the ELISA method of Example 3, and the binding activity of the antibodies in the supernatant secreted by the hybridoma cell lines to human LAG-3 was detected. The clones with good activity were selected, and the supernatants were taken to detect the blocking activity of the secreted antibodies to hLAG-3 and Daudi cells by the method described in Example 4. The clones with good binding and blocking activities were preferred, and the monoclonal antibody cell lines obtained were further selected by limiting dilution. Some of the results are shown in the table below.

[0101] Table 1 Activity of monoclonal cells screened by hybridoma fusion

[0102]

[0103]

[0104] #: terminated, i.e., clones with poor blocking activity were terminated and no subclone screening was performed; ND: no detection, i.e., the terminated clones were not subsequently tested. ##: NA, not suitable, i.e., the subclones were derived from the previous mother clone. Specifically in this table, the mother clone refers to clone 7B9 numbered #15.

[0105] Table 1 lists some of the screening data. The data show that clones with good binding activity and blocking activity were selected in the initial screening of fusion hybridomas, such as clone #15 and clone 7B9 in the table (the higher the ELISA value, the better the binding activity. The lower the blocking activity value, the better the blocking activity). For clone #15 and clone 7B9, multiple limiting dilutions were performed. After 7-10 days of clonal proliferation after each dilution (subclone), the binding activity and blocking activity of the antibodies (supernatant) secreted by each subclone were retested by the ELISA method. Clones with poor blocking activity in the initial screening were discarded, such as clones 1-14 in the table above, which were discarded and no subsequent subcloning and testing were performed.

[0106] After multiple limiting dilutions of clone 7B9, number 15, it was unexpectedly found that the secreted supernatant of the screened monoclonal cell line 7B9G8G3G2D6 (number 23) maintained good binding activity and the best blocking activity (the lowest value under the same conditions, i.e. 0.8035). The antibody sequence was extracted from the monoclonal cell line number 23 to obtain the light and heavy chain sequences of the preferred mouse anti-human LAG-3 antibody mab23 of the present invention.

[0107] Example 6: Sequence extraction, analysis and identification of the mouse anti-human LAG-3 antibody mab23 of the present invention

[0108] The process of extracting antibody sequences from the monoclonal cell lines preferably obtained from hybridomas is a common method used by those skilled in the art. Specifically, the monoclonal cell lines are collected, amplified and cultured, and 1 x 10 6 10 cells, RNA was extracted using Trizol (Invitrogen, 15596-018) (according to the kit instructions), and the extracted RNA was reverse transcribed into cDNA. The reverse transcription kit was purchased from Sangon Biotech (Shanghai) Co., Ltd., Cat#B532435. PCR amplification was performed using the cDNA obtained by reverse transcription as a template. The amplified products were sequenced to obtain the base / coding sequences of the light and heavy chain variable regions of the mab23 antibody (as follows). The primers used refer to the manual TB326Rev.C0308 published by Novagen.

[0109] The base sequence of the light chain variable region of the mouse monoclonal antibody mab23 obtained in the preferred hybridoma cell line of the present invention (the underlined part is the coding sequence):

[0110] gatgaggacccctgctcagattcttgggatcttgttgctcttgtttccaggtaccagatgt gacattc agatgatccagtctccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcgggcaagtcagga cattggtagtagtttaaactggcttcagcaggaaccagatggaactatcaaacgcctgatctacgccacatccagt ttagattctggtgtccccaaaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttg agtctgaagattttgtagactattactgtctacaatatgttatacttctccgctcacgttcggtgctgggaccaagct ggagctgaaa cgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttctgaacaactctaccccagagacatcaattccctg (SEQ ID NO: 1)

[0111] The base sequence of the heavy chain variable region of the mouse monoclonal antibody mab23 obtained in the preferred hybridoma cell line of the present invention (the underlined part is the coding sequence):

[0112] gatgaaatggcagctgggtttttctctcttcctcctgtcagtaattgcaggtgtccaatcc caggttcaa ctgcagcagtctggggctgagctggtgaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacat ttactgactatgaaatgcactgggtgaaacagacacctgtgcatggcctggaatggattggaggtattgatcctga aactgaaggcattgcctataatcagaagttcaggggcaaggccatactgactgcagacaaatcctccatcacagcc tacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaaactccaattactacggtggaa gggaggcctggtttgcttactggggccaagggactctggtcactgtctctgga gccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatacatagccattactcaaa (SEQ ID NO: 2)

[0113] The amino acid sequences encoded by the base sequences of the light and heavy chain variable regions of the above-mentioned mouse monoclonal antibody mab23 obtained in the present invention are as follows: SEQ ID NO: 3 and SEQ ID NO: 4. The amino acid sequence of the light chain variable region of the mouse monoclonal antibody mab23 obtained in the preferred hybridoma monoclonal cell line of the present invention is:

[0114] DIQMIQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYVTSPLTFGAGTKLELK(SEQ ID NO:3)

[0115] The amino acid sequence of the heavy chain variable region of the mouse monoclonal antibody mab23 obtained in the preferred hybridoma cell monoclonal strain of the present invention is:

[0116] QVQLQQSGAELVRPGASVTLSKASGYTFTDYEMHWVKQTPVHGLEWIGGIDPETEGIAYNQKFRGKAILTADKSSITAYMELRSLTSEDSAVYYCTNSNYYGGREAWFAYWGQGTLVTVSG(SEQ ID NO:4)

[0117] The above antibody light and heavy chain variable region sequences and different types of IgG constant regions, such as human hIgG1, hIgG2, hIgG3, hIgG4, human light chain κ, λ type; mouse mIgG1, mIgG2, mIgG3, mouse light chain κ, λ type, etc., were recombinantly expressed and purified to obtain complete human-mouse chimeric antibodies and mouse antibodies. Taking the human heavy chain constant region as hIgG4 and human light chain κ type as an example, the chimeric antibody mab23c was obtained by the expression and purification method of Example 1. The binding activity of mab23c and hLAG-3 and the blocking activity of LAG-3 and Daudi cells were detected by the methods of Examples 3 and 4, and compared with the control antibody (Ref) in parallel (see Figure 1 a and b).

[0118] The results showed that mab23c of the present invention was different from the control antibody (Ref). The EC50 and Emax of mab23c detected by ELISA were 0.41nM and 1.7, respectively, which were better than 0.64nM and 1.4 of the control antibody (Ref). It was particularly surprising that the IC50 of mab23c of the present invention in blocking the binding activity of LAG3 and Daudi cells was 2.3nM, which was 1.04nM higher than the IC50 detected by Ref under the same conditions. 50 (10.5nM) is nearly 4 times better ( Figure 1 b) Good blocking activity is directly correlated with the pharmacodynamic effect 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.

[0119] Example 7: Humanization of the murine antibody mab23c of the present invention

[0120] The antibody mab23c (chimeric antibody) unexpectedly discovered in the present invention has strong binding activity with the specific antigen hLAG-3, especially unexpectedly very good blocking activity. It shows that the antibody can be used for the development of monoclonal antibody drugs for tumor treatment targeting LAG-3 targets with better value, such as possible better efficacy. In order to avoid the risks of immunogenicity and other aspects in the process of drug development, the present invention has carried out humanized design and screening of the mouse source mab23 antibody, as well as sequence optimization. The specific process is described as follows.

[0121] There are many different methods for defining antibody CDRs in the art, and these CDR labeling methods can be summarized in the following table.

[0122] Table 2 Summary of different methods for defining antibody CDRs in this field*

[0123] Loop CCG Definition Kabat Definition AbM Definition Chothia Definition Contact definition Light chain CDR1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36 Light chain CDR2 L50-L56 L50-L56 L50-L56 L50-L56 L45-L55 Light chain CDR3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96 Heavy chain CDR1 H26-H35 H31-H35 H26-H35 H26-H32 H30-H35 Heavy chain CDR2 H50-H65 H50-H65 H50-H58 H52-H56 H47-H58 Heavy chain CDR3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101

[0124] *For more information, please refer to the website: http: / / www.bioinf.org.uk / abs / #cdrdef

[0125] The variable regions of the murine anti-human hLAG-3 antibody mab23 of the present invention are labeled / annotated as follows according to the various definition methods listed in the above reference table.

[0126] Table 3 CDR sequences of the anti-hLAG-3 antibody mab23 of the present invention according to CCG definition

[0127] Antibody mab23CDRs 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)

[0128] Table 4 CDR sequences of the anti-hLAG-3 antibody mab23 of the present invention according to Kabat definition

[0129]

[0130]

[0131] Table 5 CDR sequences of the anti-hLAG-3 antibody mab23 of the present invention according to AbM definition

[0132] Antibody mab23CDRs 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)

[0133] Table 6 CDR sequences of the anti-hLAG-3 antibody mab23 of the present invention according to Chothia definition

[0134] Antibody mab23CDRs 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)

[0135] Table 7 CDR sequences of the anti-hLAG-3 antibody mab23 of the present invention defined by Contact

[0136]

[0137]

[0138] After the above analysis, labeling and definition of the CDR sequence of the mouse antibody mab23 of the present invention, humanization is performed as disclosed in many documents in the art. The mouse antibody sequence is compared with the human antibody germline database (v-base) to find the human antibody light and heavy chain germlines with high homology. On this basis, computer modeling is performed to simulate the sites in the antibody structure that may affect the binding to the antigen, and the key mutation sites and combinations are reversed to screen out the humanized antibody molecules with preferred activity.

[0139] Specifically, through sequence homology comparison analysis, it was found that human antibody germlines with good homology to the 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. After further comparison and analysis, the human antibody germline light chain IGKV1-39*01 was preferred. Sequence comparison revealed that the J gene region of the mab23 light chain had high homology with human antibody germline hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, hJK3, hJK4.1, hJK4.2, and hJK5. After further comparison and analysis, hJK4.1 was selected for the mab23 light chain humanized human antibody germline J region for humanized design, screening, and sequence optimization.

[0140] By sequence homology comparison analysis, it was found that human antibody germlines with good homology to the heavy chain of mab23 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. After further comparison and analysis, the human germline heavy chain IGHV1-18*01 sequence was preferably used for the humanization of the antibody of the present invention. Sequence comparison revealed high homology with the heavy chain J gene region of mab23 and human antibody germline heavy chain J genes hJh1, hJh2, hJh3.1, hJh3.2, hJh4.1, hJh4.2, hJh4.3, hJh5.2, hJh6.1, hJh6.2, and hJh6.3. After further comparison and analysis, hJh4.1 was preferably used for the humanized human antibody germline J region of the heavy chain of the murine antibody mab23 of the present invention for humanized design, screening, and sequence optimization.

[0141] The antibody of the present invention is transplanted with the CDR region of mab23 (see the definition of CDR above) onto 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 mouse antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of VL and VH are back-mutated, and these mutations and mutation combinations are screened to see the effect on the antibody activity, and the chemically unstable amino acid residues in the CDR region are optimized to obtain an antibody molecule sequence with optimized structure, activity, etc., that is, the humanization of the mouse antibody of the present invention is completed.

[0142] The following is an explanation based on the specific sequence of mab23, taking the hIgG4 heavy chain and κ light chain (sequence as follows) as an example.

[0143] Human antibody light chain constant region κ chain: SEQ ID NO: 21; human IgG4 heavy chain constant region: SEQ ID NO: 22

[0144] The preferred sequence of the humanized light chain variable region of the anti-human LAG-3 antibody mab23 of the present invention is:

[0145] LG2312: SEQ ID NO:23; LG2313: SEQ ID NO:24;

[0146] LG2314: SEQ ID NO:25; LG2315: SEQ ID NO:26, that is

[0147] DIQMTQSPSSSLSASVGDRVTITCRASQDIGSSLNWLQQKPGKAIKRLIYATSSLDSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVTSPLTFGGGTKVEIK

[0148] LG2316: SEQ ID NO: 27

[0149] LG2317: SEQ ID NO: 28, i.e.

[0150] DIQMTQSPSSSLSASVGDRVTITCRASQDIGSSLNWYQQKPGKAPKRLIYATSSLDSGVPSRFSGSRSGSDFTLTISSLQPEDFATYYCLQYVTSPLTFGGGTKVEIK

[0151] LG2318: SEQ ID NO:29.

[0152] The preferred sequence of the humanized heavy chain variable region of the anti-human LAG-3 antibody mab23 of the present invention is:

[0153] LG2342: SEQ ID NO: 30, i.e.

[0154] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGGIDPETEGIAYNQKFRGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARSNYYGGREAWFAYWGQGTLVTVSS

[0155] LG2343: SEQ ID NO: 31, i.e.

[0156] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVKQAPGQGLEWIGGIDPETEGIAYNQKFRGRATLTADKSTSTAYMELRSLRSDDTAVYYCTNSNYYGGREAWFAYWGQGTLVTVSS

[0157] LG2344: SEQ ID NO:32; LG2345: SEQ ID NO:33;

[0158] LG2346: SEQ ID NO:34; LG2347: SEQ ID NO:35;

[0159] LG2348: SEQ ID NO:36; LG2349: SEQ ID NO:37.

[0160] The humanized sequence of the light chain of the mouse 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, as listed above. These arbitrary sequences are combined with the constant region sequence of the κ chain or λ chain of the human antibody light chain constant region to obtain the light chain sequence of the humanized antibody of the present invention, such as the κ type light chain constant region of the present invention, as listed above. Similarly, the heavy chain variable region used for humanization also has different numbers 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. These heavy chain variable region sequences containing different numbers of back mutations are recombined with the optional human IgG1, 2, 3, and 4 chain constant region sequences to obtain the heavy chain sequence of the humanized antibody of the present invention, such as the heavy chain of the present invention using hIgG4 as a constant region sequence as an example to illustrate.

[0161] The light and heavy chain sequences of the humanized antibody mab23 antibody of the present invention are partially optimized, and the expression amount (expression, purification and antibody yield detection using the method of Example 1 of the present invention) and activity evaluation (ELISA detection method of Example 3 of the present invention, blocking activity detection method of Example 5) results are shown in the following table.

[0162] Table 8 Partial sequence of humanized antibody of mab23 antibody of the present invention (taking human κ type light chain and hIgG4 heavy chain constant region as examples)

[0163]

[0164] *: NB, no binding. The binding curve shows that only weak binding can be seen at 100nM. #: Not detected. Because the binding activity is very weak, no blocking activity is detected.

[0165] The above results show that the above humanized antibody molecules obtained from the sequence of the mouse antibody mab23 of the present invention retain the binding activity to hLAG-3. More preferably, many molecules recover the same binding activity as the mouse antibody mab23c, among which the binding activity of antibodies such as Ab2315, Ab2317, Ab2322, and Ab2325 is no different from that of ab23c. That is, the humanized preferred antibodies of the present invention retain the binding activity of the previous mouse antibodies. In addition, except for antibodies with very weak binding activity (NB), most humanized antibodies have good blocking activity, which is close to that of ab23c, such as Ab2314, Ab2316, Ab2317, Ab2318, Ab2320, and Ab2325.

[0166] Table 8 shows the amino acid sequences of the light and heavy chains (including constant regions) of some preferred humanized antibodies.

[0167] Humanized Ab2315 antibody amino acid sequence:

[0168] Light chain:

[0169] DIQMTQSPSSSLSASVGDRVTITCRASQDIGSSLNWLQQKPGKAIKRLIYATSSLDSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVTSPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:38)

[0170] Heavy chain: same as Ab2317 heavy chain (SEQ ID NO: 40)

[0171] Humanized Ab2317 antibody amino acid sequence:

[0172] Light chain:

[0173] DIQMTQSPSSSLSASVGDRVTITCRASQDIGSSLNWYQQKPGKAPKRLIYATSSLDSGVPSRFSGSRSGSDFTLTISSLQPEDFATYYCLQYVTSPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:39)

[0174] Heavy Chain:

[0175] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVKQAPGQGLEWIGGIDPETEGIAYNQKFRGRATLTADKSTSTAYMELRSLRSDDTAVYYCTNSNYYGGREAWFAYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:40)

[0176] Humanized Ab2325 antibody amino acid sequence:

[0177] Light chain: same as Ab2317 light chain (SEQ ID NO: 39)

[0178] Heavy Chain:

[0179] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVKQAPGQGLEWMGGIDPETEGIAYNQKFRGRATMTTDTSTAYMELRSLRSDDTAVYYCANSNYYGGREAWFAYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:41)

[0180] Example 8: Comprehensive evaluation of binding activity of humanized anti-human LAG-3 antibodies of the present invention, preferably antibodies

[0181] In order to further evaluate the binding activity of the humanized antibodies of the present invention to LAG-3, the above-mentioned preferred humanized preferred antibody Ab2317 was taken as an example to evaluate its binding activity to LAG3 of different forms and species, and its affinity was evaluated by the Baicore method. The results are shown in Table 9.

[0182] The Biacore method is as follows: The affinity of the antibody of the present invention to human LAG-3 was determined using a Biacore T200, GE Healthcare instrument. Protein A (Thermo Pierce, Cat#21181) was first coupled to a biosensor chip CM5 (Cat.#BR-1005-30, GE) using a running buffer HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20) at pH 7.4, and the chip was activated with freshly prepared 50mM NHS (N-hydroxysuccinimide) and 200mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), and then 10μg / ml Protein A prepared in pH 4.0 10mM NaAC was injected. The concentration of the antibody to be tested was 5 μg / ml, the concentration gradient of the antigen LAG-3-his was 0 nM, 1.875 nM, 3.75 nM, 7.5 nM, 15 nM and 30 nM, the flow rate was 30 μl / min, the binding time was 180 seconds, and the dissociation time was 300 seconds. After the experiment, the chip was washed with 10 mM Glycine-HCl, pH 1.5, 30 μl / min, 30 seconds. The experimental data were fitted with the 1:1 Langmuir model using Biacore T200 evaluation version 3.0 (GE) software to obtain the affinity value KD.

[0183] Table 9 Comprehensive evaluation of the binding activity (EC50, nM) and affinity (Biacore) of the antibodies of the present invention to LAG-3

[0184]

[0185] The above results show that the preferred humanized murine antibodies of the present invention (Ab2317, Ab2325, etc.) have good binding to human LAG-3-his (monomer), human LAG3-hFc (dimer), his form (monomer) of human LAG-3 extracellular loop 1 and loop 2 (hLAG-3-D12), i.e. hLAG-3-D12-his (monomer) and hLAG-3-D12-hFc (dimer), and hLAG3+ cells.

[0186] Bicore test results showed that the affinities of Ab2317 and Ab2325 were both below 0.2 nM, close to the affinity of the control molecules.

[0187] Compared with the control positive antibody (Ref), Ab2317 and Ab2325 have the advantage of good blocking activity (parallel assay comparison results show that the blocking activity of ab2317 is at least 2 times better than that of Ref, 2.7nM vs 8.2nM). This is consistent with the blocking activity result of mab23c being better than that of Ref ( Figure 1 , b) are consistent. In addition, the above data show that the antibody of the present invention binds to the 1st and 2nd regions of hLAG3 (hLAG-3-D12), indicating that the blocking activity of the antibody of the present invention is to prevent the binding of the D12 region of hLAG3 to MHCII (daudi cells). It is difficult to screen out antibodies with such specific blocking activity, especially those with very good blocking activity, which is more than 2 times better than the blocking activity of the current clinical molecule (Ref) in this field, and it is even more difficult and unpredictable. This specific blocking activity is related to the efficacy of the antibody in the clinical treatment of tumor patients. The better the blocking activity, the better the expected efficacy.

[0188] In addition, another unexpected advantage of the antibody of the present invention, such as Ab2317, is that its expression yield is very high, reaching more than 100 mg / L under the conditions of the present invention, while the expression of the control antibody Ref under parallel and equal conditions is only 60 mg / L. The average expression yield of multiple comparisons, Ab2317 is more than 60% higher than that of the control antibody Ref. This shows that the expression of the antibody of the present invention is better than that of the control molecule (this difference in expression is related to the sequence), which brings advantages for the antibody of the present invention to be used in later process development to increase the antibody yield.

[0189] The antibody Ab2317 of the present invention was prepared into a PBS solution at a concentration of 1 mg / L and stored at 45°C for 0, 7 and 14 days. The stability of the samples was evaluated by gel electrophoresis. Figure 2 , from left to right, M is the molecular weight marker. Lanes 1, 2; 3, 4; 5, 6 are the non-denaturing and denaturing electrophoresis of Ab2317 after being stored at 45°C for 0 days, 7 days, and 14 days, respectively. Lanes 7, 8; 9, 10; 11, 12 are the non-denaturing and denaturing electrophoresis of Ref after being stored at 45°C for 0 days, 7 days, and 14 days under parallel conditions, respectively. The electrophoresis results show that the concentration of the antibody Ab2317 of the present invention has not changed after being stored at 45°C for 7 days and 14 days, whether it is a non-denatured or denatured sample, indicating that there is no price reduction. The control sample Ref shows a reduction in the sample (less on the gel), for example, the sample at 14 days (lanes 11, 12) is significantly less than that at 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.

[0190] The humanized antibodies of the present invention, such as Ab2317 and Ab2325, can bind to cynomolgus monkey LAG-3 (cynomolgus, cyno-LAG3-hFc, purchased from Beijing Biopsies Biotechnology Co., Ltd., catalog number LA3-C5252) like the control antibody (Ref), with binding activities of 40.2nM, 44.5nM, and 35.3nM, respectively. This binding activity is more than 200 times weaker than the binding to human LAG-3. This is consistent with the results of Biacore detection of the affinity of the antibodies of the present invention to cyno-LAG3-hFc. The affinity of Ref is 836nM (more than 4000 times weaker than the affinity to human LAG-3); Ab2317 and Ab2325 were not detected to bind to cyno-LAG-3-hFc in Biacore. In addition, none of the above antibodies bind to mouse LAG-3.

[0191] The present invention is represented by antibody LG2317, and cell function evaluation is performed by the following experiments.

[0192] Example 9: Evaluation of the functional activity of anti-LAG-3 antibodies in activating human T cells

[0193] On the day of the experiment, human PBMCs (isolated from peripheral blood donated by healthy volunteers) were collected, resuspended in RPMI1640 medium containing 10% FBS and counted, and the cell density was adjusted to 1×10 6 cell / ml, add to 96-well plate, 85μl / well, and put into incubator. Prepare superantigen (SEB, purchased from Beijing Kangbo Bening Technology Co., Ltd.) with culture medium, make the initial concentration 2μg / ml, add 5μl to each well (make the final concentration 100ng / ml). Prepare the samples to be tested in proportion with culture medium, including negative antibody and control antibody. Add 10μl to each well, so that the concentration of the antibody to be tested in the 100μl system is the required concentration gradient. Place the cell culture plate at 37℃, 5% CO 2 After incubation in the incubator for 3 days, the cell culture plate was removed and centrifuged at 3000 rpm for 10 min. 80 μl of supernatant was removed from each well for detection of human IL-2.

[0194] IL-2 ELISA test was performed according to the instructions of the kit (Shenzhen Xinbosheng Biotechnology Co., Ltd., cat: EHC003.96), and the steps are as follows:

[0195] a. Dilute the cell culture supernatant by 25 times (the dilution factor varies in different experiments) and add it to the ELISA plate (100μl / well); dilute the standard with universal specimen diluent to different concentration gradients: 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.25pg / ml, 15.625pg / ml, add 100μl to each well; add universal specimen diluent to the blank wells.

[0196] b. Seal the reaction wells with sealing tape and incubate at 37°C for 90 minutes.

[0197] c. Wash the plate 5 times, 3 minutes each time, add 100 μl of biotin antibody working solution to each well, add biotinylated antibody diluent to the blank wells, seal the reaction wells with new sealing tape, and incubate at 37°C for 60 minutes.

[0198] d. Wash the plate 5 times, 3 minutes each time, add 100 μl of enzyme binding working solution to each well, add enzyme binding diluent to the blank wells, seal the reaction wells with new sealing tape, and incubate at 37°C in the dark for 30 minutes.

[0199] e. Wash the plate 5 times, 3 minutes each time, add 100 μl of chromogenic substrate TMB to each well, incubate at 37°C in the dark for 15 minutes

[0200] f. Add stop solution, 100 μl / well, mix well, and read OD450 with a microplate reader within 3 minutes.

[0201] g. Result analysis: Calculate the IL-2 value and convert it into an increase percentage (%) compared with the blank control to evaluate the human T cell activation activity of the sample.

[0202] The results showed that the EC50 of the preferred antibody Ab2317 of the present invention for activating human T cells to release IL-2 was 0.34 μg / ml. 50 The activity of the antibody of the present invention on T cells is at least 3 times better than that of the control Ref. Figure 3 The a display represents one of the data.

[0203] Example 10 Activity detection of the anti-LAG-3 antibody of the present invention alone and in combination with PD-1 antibody in mixed lymphocyte reaction (MLRassay)

[0204] The activity of the series of antibodies of the present invention in activating human T cells was evaluated by detecting the secretion of INF-γ (INFg) by mixed lymphocyte reaction (MLR assay). That is, dendritic cells (DC) were induced from human blood cells PBMC (isolated from peripheral blood donated by healthy volunteers) separated by the present invention, and the obtained DC were used to stimulate T cells from different volunteers. Specifically, dendritic cell (DC) culture: on the first day of the experiment, PBMC were inoculated in 6-well plates with RPMI 1640 medium, 2 ml per well, 1×10 6 / ml, 37°C, 5% CO 2 After 2 hours of incubation in the incubator, the suspended cells were gently aspirated, and 2 ml of complete RPMI 1640 medium (containing 10% FBS, 100 ng / ml GM-CSF, Peprotech, Cat#: 300-03, and 100 ng / ml IL-4, Peprotech, Cat#: 200-04) was added to the adherent cells. After 2 days of continuous culture, 1 ml of fresh complete RPMI 1640 medium was added to each well. On the 5th day, 3 μl of 100 μg / ml TNF-α was added to each well to a final concentration of 100 ng / ml (TNF-α was purchased from Peprotech, Cat#: AF-300-01A), and culture was continued for 2 days. The resulting dendritic cells (DC) were used in the following experiments.

[0205] DC stimulated T cells (MLR) assay: 10 ng / ml anti-CD3 antibody (Miltenyl Biotec, Cat#: 130-093-387) was used to coat 96-well cell culture plates at 100 μl / well, incubated at 37°C for 2 hours, and washed once with PBS. DC cells cultured on day 7 were collected, centrifuged, resuspended in RPMI 1640 medium containing 10% FBS, counted, and prepared into 5×10 4 cell / ml, and add 90 μl / well to the 96-well plate coated with anti-CD3. PBMC cells from different volunteers were collected, counted, and prepared into 5×10 5cell / ml, add 90μl / well to the 96-well plate coated with the above anti-CD3 and plated with DC cells. Prepare the test samples in proportion with PBS, including the control antibody (Ref) and Ab2317, and PD-1 antibody (expressed and purified by the present invention) and add them to the above 96-well plate. 10μl / well of Ref or Ab2317+PD-1 antibody. The final concentration of PD-1 antibody in the 200μl system is 0.5μg / ml. The concentration of the antibody to be tested (Ref or Ab2317) in the 200μl system is the required concentration gradient. Control group = 90μl PBMC cells + 90μl DC + 20μl PBS. The cell culture plate is placed at 37°C, 5% CO 2 After incubation in the incubator for 4 days, the cell culture plate was removed and centrifuged at 3000 rpm for 10 min. 150 μl of supernatant was removed from each well for detection of human INF-γ.

[0206] INF-γ ELISA test was performed according to the instructions of the kit (Shanghai Xinaosheng Biotechnology Co., Ltd. cat: EHC102g.96), and the steps are as follows:

[0207] h. Add the removed cell culture supernatant to the ELISA plate (100μl / well); dilute the standard into different concentration gradients with universal specimen diluent: 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.25pg / ml, 15.625pg / ml, add 100μl to each well; add universal specimen diluent to the blank well.

[0208] i. Seal the reaction wells with sealing tape and incubate at 37°C for 90 minutes.

[0209] j. Wash the plate 5 times, 3 minutes each time, add 100 μl of biotin antibody working solution to each well, add biotinylated antibody diluent to the blank wells, seal the reaction wells with new sealing tape, and incubate at 37°C for 60 minutes.

[0210] k. Wash the plate 5 times, 3 minutes each time, add 100 μl of enzyme binding working solution to each well, add enzyme binding diluent to the blank wells, seal the reaction wells with new sealing tape, and incubate at 37°C in the dark for 30 minutes.

[0211] l. Wash the plate 5 times, 3 minutes each time, add 100 μl of chromogenic substrate TMB to each well, incubate at 37°C in the dark for 15 minutes

[0212] m. Add stop solution, 100 μl / well, mix well, and read OD450 with a microplate reader within 3 minutes.

[0213] n. Result analysis: The INF-γ value (pg) was calculated using the standard curve formula to evaluate the activity of the antibody of the present invention.

[0214] Figure 3 b The results show that the EC50 of the antibody Ab2317 of the present invention for activating DC to stimulate T cells (used in combination with the PD-1 antibody Keytruda) is 0.21 nM, which is more than twice as strong as Ref (EC50 is 0.61 nM) under the same conditions.

[0215] Example 11: In vivo efficacy evaluation of the LAG-3 antibody of the present invention

[0216] The animal efficacy model was established using human PD-1 and LAG-3 double transgenic mice (C57BL / 6-hPD1 / hLAG3) to evaluate the in vivo efficacy of the LAG-3 antibody of the present invention. Double transgenic mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number: SCXK (Su) 2018-0008.

[0217] MC38 cells (purchased from the Institute of Cell Biology, Chinese Academy of Sciences) were cultured in DMEM / high glucose medium (Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No.: L110KJ) containing 10% fetal bovine serum (Shanghai Bosheng Biotechnology Co., Ltd., Catalog No.: BS-0002-500) and 1% HEPES (Thermo Fisher Scientific, Inc., Catalog No.: 15630080) in an atmosphere containing 5% CO. 2 C57BL / 6-hPD1 / hLAG3 female mice, 6 weeks old, were housed in an SPF environment, 5 mice / cage, at a temperature of 20-25°C and a humidity of 40%-60%, with free access to food and water, and litter was changed regularly.

[0218] When MC38 cells reached the logarithmic growth phase (confluence rate was 80%-90%), they were digested with 0.25% trypsin, collected, and washed twice with serum-free DMEM / high glucose medium. Then, the cells were resuspended with serum-free DMEM / high glucose medium, counted, and Matrigel (Bio-Dixon Medical Devices (Shanghai) Co., Ltd., Catalog No.: 354234) was mixed with DMEM / high glucose medium in a ratio of 1:1 to adjust the cell concentration to 1×10 7 Cells / ml were used for inoculation. 100 μl (10 6 The tumor cells were selected and grown to a volume of about 100-150 mm. 3 The mice were randomly divided into groups with 5 in each group.

[0219] The samples to be tested were prepared with PBS and were sterile. The Blank group was PBS. The PD-1 antibody (cloned and expressed according to the Keytruda public sequence) was the control group for single medication. The PD-1 antibody + Ref was the control group for combined medication. The PD-1 antibody + Ab2317 was the combined medication test group. The administration method was intraperitoneal injection. The PD-1 antibody was administered alone or in combination, with a dose of 10μg / 100μl / mouse. In the combined administration group, the doses of Ref and Ab2317 were 120μg / 100μl / mouse. The frequency of administration was 2 times / week. The day of each sample injection was day 0. Measure body weight and tumor volume before each administration, and record the data. The experiment was administered 4 times.

[0220] Tumor size calculation formula: tumor volume TV (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ); relative tumor growth rate (T / C%) = 100%*(T-T0) / (C-C0); tumor inhibition rate (TGI) = (1-T / C)*100%; T0 and T are the tumor volumes at the beginning and end of the experiment for the sample group, respectively; C0 and C are the tumor volumes at the beginning and end of the experiment for the control group, respectively.

[0221] Results Figure 4 and Table 9b.

[0222] Table 9b In vivo efficacy evaluation of the LAG-3 antibody of the present invention

[0223]

[0224] Figure 4 The results showed that the combination of the antibody of the present invention and the PD-1 antibody had a tumor inhibition rate of 100% starting on the 13th day, and the tumor was smaller than at the beginning and maintained until the end of the experiment, which was significantly better than the Ref+PD-1 antibody group and the PD-1 antibody group alone.

[0225] Table 9b is the statistical results of tumor volume on day 0 and day 23. The results show that on day 23, compared with the PBS group, the efficacy (inhibition rate) of the PD-1 antibody + Ab2317 combination group of the present invention was 103%, that is, 100% inhibition of tumor growth and the tumor was smaller than at the beginning. It is better than the efficacy (inhibition rate) of 63% of the PD-1 antibody alone, and also better than the efficacy (inhibition rate) of 80% of the PD-1 antibody + Ref combination group.

[0226] The results of statistical analysis (T test) showed that the P value of the efficacy of PD-1 antibody alone and the PBS group was 0.06, which did not reach the level of statistically significant difference. The P value of the efficacy of PD-1 antibody + Ref combined with the PBS group was 0.01, reaching the level of significant difference (marked with *). Unexpectedly, the P value of the efficacy of PD-1 antibody + Ab2317 of the present invention combined with the PBS group was 0.001, reaching the level of extremely significant difference (marked with **). Even more unexpectedly, the statistical analysis of the efficacy of the combination of PD-1 antibody + Ab2317 of the present invention and the PD-1 antibody + Ref combined group showed a P value of 0.04, reaching the level of significant difference (P<0.05, marked with *).

[0227] During the experimental period of each group in this experiment, there was no significant change in the body weight of the animals, indicating that the antibody of the present invention had no significant toxic effect.

[0228] Example 12: Design of bispecific antibodies targeting LAG-3

[0229] Using the anti-LAG-3 antibodies discovered above, the present invention designs a variety of bispecific antibodies. The general formula of the designed bispecific antibodies is as follows.

[0230] Table 10 Bispecific design based on the anti-LAG-3 antibody of the present invention (General Formula 1)

[0231]

[0232] In Table 10 and herein, a sequence containing a light chain means that the sequence may include a scFv linked to the light chain sequence in addition to the light chain sequence; a sequence containing a heavy chain means that the sequence may include a scFv linked to the heavy chain sequence in addition to the heavy chain sequence. Among them, T1 represents the first protein functional region for target 1 (such as LAG3), and T2 represents the second protein functional region for target 2 (non-LAG3). T1 (scFv) represents the scFv sequence of an antibody for target 1; T2 (scFv) represents the scFv sequence for target 2.

[0233] (scFv) n1 , (scFv) n2 , (scFv) n3 , (scFv) n4Where n1, n2, n3, n4 are natural numbers, which can be 0, 1, 2, 3, etc. In a specific embodiment of the present invention, at least one of n1, n2, n3, and n4 is 1, and the rest are 0. VL represents the variable region sequence of the antibody light chain for target 1 or 2; VH represents the variable region sequence of the antibody heavy chain for target 1 or 2. Lc represents the constant region sequence of the light chain (κ or λ), preferably the human light chain constant region sequence; Hc represents the heavy chain, including the constant region sequences of IgG1, IgG2, IgG3, IgG4, etc. (abbreviated as Hc-IgG1, Hc-IgG2, Hc-IgG3, Hc-IgG4), preferably the human heavy chain constant region sequence (Hc-hIgG). When the C-terminus of the heavy chain constant region is connected to scFv or other protein sequences, the last amino acid K at its C-terminus can be mutated, preferably mutated to A. Therefore, in scheme 1, T1 is an immunoglobulin and T2 is a scFv; in scheme 2, T2 is an immunoglobulin and T1 is a scFv; the scFvs target the same target; in schemes 3 and 4, the scFvs at both ends target two different targets.

[0234] When the scFv in Table 10 is a light chain variable region-linker-heavy chain variable region, the N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region is correspondingly 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, the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region is correspondingly connected to the C-terminus or N-terminus of the immunoglobulin light chain and / or heavy chain through a linker.

[0235] It should be noted that when the above-mentioned scFv is a light chain variable region-linker-heavy chain variable region, the connection method is that the C-terminus of the light chain variable region is connected to the linker, and the linker is further connected to the N-terminus of the heavy chain variable region, 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 the linker. In the present invention, when it is connected to the light chain of the immunoglobulin, in some specific embodiments, it is preferred to use the C-terminus of the heavy chain variable region of the scFv to connect to the N-terminus of the heavy chain of the immunoglobulin through the linker; when it is connected to the heavy chain of the immunoglobulin, in some specific embodiments, it is preferred to use the N-terminus of the light chain variable region of the scFv to connect to the C-terminus of the heavy chain of the immunoglobulin.

[0236] When the scFv is a heavy chain variable region-linker-light chain variable region, the connection method is that the N-terminus of the light chain variable region is connected to the linker, and the linker is then connected to the C-terminus of the heavy chain variable region, 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 the linker. In this case, when it is connected to the light chain of the immunoglobulin, in some specific embodiments, it is preferred to use the C-terminus of the light chain variable region of the scFv to connect to the N-terminus of the heavy chain of the immunoglobulin; when it is connected to the heavy chain of the immunoglobulin, in some specific embodiments, it is preferred to use the N-terminus of the heavy chain variable region of the scFv to connect to the C-terminus of the heavy chain of the immunoglobulin.

[0237] The linker is (Gly-Gly-Gly-Gly-Ser) w [Abbreviated as (G 4 S) w ]W is preferably an integer between 0 and 10. Preferably, the linker is (G 4 S) 3 , and / or, the number of the scFv is a pair, symmetrically connected to the immunoglobulin light chain and / or heavy chain.

[0238] In addition, the antibody of the present invention can also be used in a manner different from Formula 1, such as the following Formula 2.

[0239] Table 11 Bispecific design of DVD format based on the anti-LAG3 antibody of the present invention (General Formula 2)

[0240]

[0241] In Table 11, T1 and T2 represent targets 1 (e.g., LAG3) and target 2 (non-LAG3), respectively. A sequence containing a light chain means that the sequence includes another light chain variable region sequence in addition to a normal complete light chain sequence. A sequence containing a heavy chain means that the sequence includes another heavy chain variable region sequence in addition to a normal complete heavy chain sequence. The light chain variable region and the complete light chain, and the heavy chain variable region and the complete heavy chain are connected by a linker.

[0242] The target antibody sequences involved in the above-mentioned bispecific design, in addition to the anti-LAG3 antibody sequence described in the present invention, include the disclosed antibody sequences of PD1 antibody, including the anti-PD-1 antibody Nivolumab / Opdivo (Nivo for short) and Pembrolizumab / Keytruda (Pem for short). In addition to the sequences disclosed in the patent, the sequences of Nivolumab (Nivo) and Pembrolizumab (Pem) can also be found in public resources such as www.drugbank.ca. The expression numbers and sequences of these individual antibodies are shown in the table below.

[0243] Table 12 Number, light and heavy chain sequences and description of monoclonal antibodies cloned and expressed by the present invention

[0244]

[0245]

[0246] The sequences of Nivolumab and Pembrolizumab can be obtained from public information such as www.drugbank.ca, and the present invention numbered them as follows:

[0247] NivoVL-Lc (κ chain): SEQ ID NO: 42; NivoVH-Hc (hIgG4): SEQ ID NO: 43;

[0248] PemVL-Lc (κ chain): SEQ ID NO: 44; PemVH-Hc (hIgG4): SEQ ID NO: 45;

[0249] Ab835VL is SEQ ID NO: 58 in application number 201810917684.X; Ab385VH is SEQ ID NO: 59 in application number 201810917684.X.

[0250] Example 13 Design and activity evaluation of bispecific antibodies targeting LAG-3 and PD-1

[0251] The present invention designs bispecific antibodies with different sequence structures for the two targets of LAG-3 and PD1, as shown in the table below.

[0252] Table 13 Bispecific antibodies designed for LAG-3 and PD1 dual targets

[0253]

[0254]

[0255] *: κ chain indicates that the light chain is the κ type light chain constant region of human IgG. #: When the C-terminus of IgG4 is connected to the linker, its terminal amino acid K is mutated to A. In the present application, the SBody designed by the present invention that introduces scFv at the C-terminus of the heavy chain has the terminal K mutated to A.

[0256] According to the cloning, expression and purification method of the present invention, 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 detected respectively using the methods of the aforementioned embodiments. The results are shown in the following table.

[0257] Table 14 Evaluation of binding activity of bispecific antibodies designed for dual targets of LAG-3 and PD-1

[0258]

[0259]

[0260] #: The value in brackets is the binding activity EC of the monoclonal antibody corresponding to the same target under the same experimental conditions. 50 *: The binding activity EC of the bispecific antibody and the corresponding monoclonal antibody under the same experimental conditions. 50 The larger the ratio, the more the designed bispecific antibody's binding force to the single target is weakened. For example, if the ratio is 2, it means that the designed bispecific antibody's binding activity to the target is weakened by 1 times compared with the corresponding monoclonal antibody. If the ratio is within 2 (experimental error range), it means that the binding activity is not affected.

[0261] The results in the above table show that LB2373 vs LB2371 show that the same Ab2317scFv of the present invention and different PD-1 antibodies are designed with the same structure (scFv is at the N-terminus of the PD-1 antibody heavy chain), and LB2373 has a greater effect on the binding activity of PD-1 (EC50 change fold is 2.92, see Figure 5A , 5C ), but had little effect on the binding activity of LAG3. LB2371 had no effect on the activity of PD-1 and LAG3 ( Figure 5C ). Comparing LB2373 vs LB2374, LB2371 vs LB2372, the data show (see Figure 5A , 5B, 5C, 5D), the same Ab2317scFv sequence and the same PD-1 antibody sequence were designed into bispecific antibodies. The Ab2317scFv had different positions (N-terminus vs C-terminus), and the binding activity to PD-1 and LAG3 was also different. LB2371 had the best binding activity to both targets. LB2383 vs LB2384 showed that Ab2325scFv was more active at the N-terminus (LB2383) than the C-terminus (LB2384) of the Nivo heavy chain.

[0262] Comparing the activities of LB2371-LB2374 and LB211, LB152, LB234, and LB203, it was found that under the premise of different LAG-3 antibody scFvs (Ab2317scFv vs Ab835scFv), the same structure (N-terminus or C-terminus of the heavy chain) of the same PD-1 antibody (Nivo or Pem), the SBody designed by Ab2317scFv has much better activity than the SBody designed by Ab835scFv. What is particularly unexpected is that LB2374 and LB234 have the same SBody design structure and the PD-1 antibody sequence (Nivo) is also the same, but the difference is Ab2317scFv vs Ab835scFv. As a result, LB2317 almost retains the binding activity to LAG-3 and PD-1, while LB234 almost loses the binding activity to LAG-3 (reduced by 175 times). Based on these data, it was found very unexpectedly that the activity of the SBody designed from the antibody Ab2317scFv of the present invention is sequence-specific.

[0263] Comparing the binding data of LB2379-LB2382 and LB202, LB201, LB214, and LB216 against dual targets (PD-1 and LAG-3), in the same structure, the LAG-3 antibody sequence (scFv) is different, and the activity of LB2379-LB2382 is better than that of the corresponding LB202, LB201, LB214, and LB216.

[0264] These molecules designed based on sequence specificity and showing unexpected differences in activity have a molecular structure similar to conventional IgG, which is referred to in the present invention as Sequence-based IgG like bispecific antibody format (SBody), i.e., bispecific antibodies with sequence-specific IgG-like structures.

[0265] Taking LB2374 as a representative, the affinity of the bispecific antibody of the present invention was analyzed by Biacore (the method is the same as Example 8). The affinity data of LB2374 for LAG-3 are: ka (1 / Ms) = 2.21E+6; kd (1 / s) = 4.13E-4; KD (M) = 1.87E-10 (very close to the affinity of Ab2317 of 1.68E-10 in Table 9). The affinity data for PD-1 are: ka (1 / Ms) = 2.098E+5; kd (1 / s) = 1.41E-3; KD (M) = 6.71E-9 (this is close to the affinity of Nivo alone, 8.59E-9). The data show that the bispecific antibody LB2374 of the present invention retains the same affinity (KD) for dual targets.

[0266] In order to evaluate the simultaneous binding activity of the bispecific antibody (SBody) designed in the present invention to two targets, the activities of LB2374 and LB2371 were evaluated using double sandwich ELISA.

[0267] Specifically, PD-1 (expressed in the present invention) was diluted to a concentration of 1 μg / ml with a PBS buffer at pH 7.4, added to a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 50 μl / well, and incubated at 37° C. for 2 hours. After discarding the liquid, 200 μl / well of a blocking solution of 5% skim milk (Shanghai Sangon Biotechnology Co., Ltd., A600669-0250) diluted with PBS was added, and the plate was placed at 4° C. overnight (16-18 hours) for blocking. The blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), and then the bispecific antibody to be tested (10 μg / ml) was added initially, and serially diluted 5-fold with 1% BSA, 50 μl / well, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well 1 μg / ml Bio-LAG3-his (ACROBiosystems, TM-H5229, biotin labeling) was added, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well 1:1000 diluted streptavidin-HRP secondary antibody (Nanjing GenScript Biotechnology Co., Ltd., M00091) was added and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well TMB colorimetric substrate (KPL, 52-00-03) was added and incubated at room temperature for 5-10 min. 50 μl / well 1M H 2 SO 4 The reaction was terminated, and the absorbance was read at 450 nm using a MULTISKAN Go microplate reader (ThermoFisher, 51119200), and the EC50 was calculated based on the OD value.

[0268] LAG3-his was diluted to 1 μg / ml with PBS buffer at pH 7.4, added to a 96-well ELISA plate at a volume of 50 μl / well, and incubated at 37°C for 2 hours. After discarding the liquid, 200 μl / well of 5% skim milk blocking solution diluted with PBS was added and placed at 4°C overnight (16-18 hours) for blocking. The blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and then the bispecific antibody sample to be tested (starting at 10 μg / ml) was added, and 1% BSA was used for 5-fold serial dilution, 50 μl / well, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well 10 μg / ml Bio-PD-1-his (expressed in the present invention, Biotin labeled) was added, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well 1:1000 diluted streptavidin-HRP secondary antibody was added, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well TMB colorimetric substrate was added, and incubated at room temperature for 5-10 minutes, and 50 μl / well 1M H 2 SO 4 The reaction was terminated, the absorbance was read at 450 nm using a MULTISKAN Go microplate reader, and the EC50 was calculated based on the OD value.

[0269] The results are shown in the table below.

[0270] Table 14a Dual-specific antibody double sandwich ELISA designed for LAG-3 and PD-1 dual targets

[0271]

[0272] The above results indicate that 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.

[0273] The SBody that retained dual-target activity was evaluated for function (antigen and receptor binding blocking experiments) against the two targets, and the results are shown in the table below.

[0274] Table 15 Evaluation of functional activity of bispecific antibodies designed for dual targets of LAG3 and PD-1

[0275]

[0276]

[0277] #: The value in the brackets is the IC50 of the monoclonal antibody corresponding to the same target under the same experimental conditions to block the antigen and ligand binding activity. *: IC50 change multiple, that is, the ratio of the IC50 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 the single target is weakened. For example, if the ratio is 2, it means that the functional activity of the designed bispecific antibody against the target is weakened by 1 times compared with the corresponding monoclonal antibody. The ratio within 2 is within the experimental error range, that is, the activity is not affected. ND: The molecule has no detected activity to prevent LAG3 and Daudi cell binding.

[0278] The above functional activity results show that the bispecific antibody (SBody) designed by the present invention retains the activity of preventing the binding of PD-1 and PD-L1, and only LB2373 has a slightly weaker activity in preventing the binding of PD-1 / PD-L1 (change factor 3.78). The binding activity of LB2379, LB2380, LB2371 and LB2372 in preventing LAG-3 and Daudi cells is greatly weakened (8.16, 3.78, 7.92 and 5.63 times, respectively), while LB2373 and LB2374 are almost not weakened (0.82 and 1.98 times, respectively). This also shows that under the same structure, the different PD-1 antibody sequences (LB2373 and LB2374 are Pem; LB2371 and LB2372 are Nivo) have significantly different effects on functional activity, that is, the SBody designed by the present invention is sequence-specific. Comprehensively considering the functional activity results of the two targets, LB2374 is the best, with the least impact on the functional activity of the two targets (preventing antigen from binding to the receptor).

[0279] To evaluate the stability of the bispecific antibody of the present invention, the antibody of the present invention was prepared into 1 mg / ml in PBS (pH 7.4), and the activity of the samples incubated at 37°C for 5 days (d5) and 10 days (d10) and the samples stored at -80°C for 60 days were compared to evaluate the stability. The results are shown in the table below.

[0280] Table 16 Stability evaluation of bispecific antibodies (SBody) designed for dual targets of LAG3 and PD-1

[0281]

[0282] #: The activity value of the sample taken out after being stored at -80℃ and detected under the same conditions; *: The change factor relative to the activity of the sample stored at -80℃ (the value in brackets). d5 and d10 represent the samples on the 5th and 10th days of incubation at 37℃, respectively.

[0283] The above results show that the bispecific molecules LB2371 and LB2374 designed by the present invention are stored at 37°C for 5 days and 10 days, and the binding activity to the dual targets is the same as that of the samples stored at -80°C for 60 days, that is, the binding activity has not changed (EC50 difference is within 2 times). This shows that these molecules are stable. In addition, these samples were analyzed by polyacrylamide gel electrophoresis (PAGE), and no degradation was found under denaturing and non-denaturing conditions. This also shows that the structures of these bispecific molecules are stable.

[0284] Table 17 Evaluation of expression levels of bispecific antibodies designed for dual targets of LAG3 and PD-1

[0285]

[0286] The above results show that the expression yields of the bispecific antibodies (SBody) designed by the present invention for LAG3 and PD1 vary greatly. Surprisingly, the expression levels of LB2373, LB2374, LB2379, and LB2380 are all relatively high. The expression level of the SBody designed with Ab835 as scFv is much lower than that of the SBody with the same design of Ab2317scFv, more than 65 times lower, such as LB2374 vs LB234; more than 140 times lower, such as LB2373 vs LB211.

[0287] These data indicate that the unexpected effects of the bispecific antibody SBody designed from the anti-LAG3 antibody Ab2317 and the PD1 antibody of the present invention are not only its activity, function, stability, but also the expression level, which is related to the sequence-specific design.

[0288] LB2371 light chain amino acid sequence: SEQ ID NO: 44; heavy chain amino acid sequence: SEQ ID NO: 46;

[0289] LB2373 light chain amino acid sequence: SEQ ID NO: 42; heavy chain amino acid sequence: SEQ ID NO: 47;

[0290] LB2374 light chain amino acid sequence: SEQ ID NO: 42

[0291] LB2374 heavy chain amino acid sequence: SEQ ID NO: 48

[0292] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGAGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVKQAPGQGLEWIGGIDPETEGIAYNQKFRGRATLTADKSTSTAYMELRSLRSDDTAVYYCTNS NYYGGREAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDIGSSLNWYQQKPGKAPKRLIYATSSLDSGVPSRFSGSRSGSDFTLTISSLQPEDFATYYCLQYVTSPLTFGGGTKVEIK

[0293] LB2379 contains the amino acid sequence of the light chain: SEQ ID NO: 49; the amino acid sequence of the heavy chain: SEQ ID NO: 43;

[0294] LB2380 contains the light chain amino acid sequence: SEQ ID NO:50; the heavy chain amino acid sequence: SEQ ID NO:43.

[0295] Example 14: PK evaluation of LAG-3 antibodies of the present invention

[0296] The experiment used C57BL / 6cnc mice (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, and the breeding environment was SPF level. Temperature 20-25°C; humidity 40-60%. C57BL / 6cnc mice were randomly divided into groups after being raised in the laboratory environment for 3 days, with 3 mice in each group. The drug to be tested (LAG-3 antibody Ab2317 and Ref of the present invention) was subcutaneously injected into the back of the mice, with an injection volume of 200 μl. The dosage was 20 mg / kg / mouse.

[0297] Blood samples were collected from the eye sockets of mice after injection. The time points were 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, and 504 hours. The blood samples were centrifuged, the supernatant was taken, and stored at -20 degrees for testing. After the blood samples were collected, the content of the samples in the blood at each time point was detected by ELISA. Excel was used to analyze the PK data and calculate the T1 / 2 of the drug to be tested. The results are shown in the table below.

[0298] Table 18 PK evaluation of antibody Ab2317 of the present invention

[0299]

[0300] The above results show that the antibody Ab2317 of the present invention shows better PK characteristics, including longer T1 / 2 (Ab2317 T1 / 2 is 202 hours, Ref is 188.7 hours), better Cmax and the like.

[0301] Example 15: PK evaluation of the bispecific antibody (SBody) designed for dual targets of LAG3 and PD-1 of the present invention

[0302] The experiment used C57BL / 6cnc mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Zhejiang) 2018-0001) and 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 (Su) 2018-0008) female, 8 weeks old, 6 each, about 20g, breeding environment: SPF level. Temperature 20-25°C, humidity 40-60%. After the two strains of mice were raised in the laboratory environment for 3 days, mice in good condition were randomly divided into groups, with 3 mice in each group. The drug Ab2374 to be tested was injected subcutaneously into the back of the mice, with an injection volume of 200μl. The dose was 20mg / kg / mouse.

[0303] Blood was collected from the eye sockets of mice after injection. The time points were 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, and 432 hours. The blood samples were centrifuged, the supernatant was collected, and stored at -20°C for testing. After the blood samples were collected, the content of the samples in the blood at each time point was detected by ELISA. Specifically:

[0304] PD-1 detection method: 96-well plates (corning, catalog number 3590) were coated with PD-1 protein. The plates were blocked with milk for 2 hours, and then washed and set aside for use. The standard curve was prepared with Ab2374, and the serum to be tested was diluted in proportion and added to the treated 96-well plates. After reacting at 37°C for 1 hour, the plates were 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 plates were washed, TMB (Surmodic, catalog number TTMB-1000-01) was added for color development, and sulfuric acid was used to terminate the reaction. The OD450 was measured by an enzyme reader.

[0305] PD-1 / LAG-3 (sandwich ELISA) detection method: 96-well plates (corning, catalog number 3590) were coated with PD-1 protein. Milk was used to block for 2 hours, and the plates were washed and set aside for use. The standard curve was prepared with Ab2374, and the serum to be tested was diluted in proportion and added to the treated 96-well plates. After reacting at 37°C for 1 hour, the plates were washed, and a fixed concentration of biotinylated LAG-3 protein was added. After reacting at 37°C for 1 hour, the plates were washed, and the diluted enzyme-labeled antibody streptavidin-HRP (genscript, catalog number M00091) was added. After half an hour, the plates were washed, TMB (Surmodic, catalog number TTMB-1000-01) was added for color development, and sulfuric acid was used to terminate the reaction, and the OD450 was measured by an enzyme reader.

[0306] Excel was used to analyze the PK data and calculate the T1 / 2 of the drug to be tested. The results are shown in the table below.

[0307] Table 19 PK evaluation of the bispecific antibody Ab2374 of the present invention (PD-1 detection results)

[0308]

[0309] Table 20 PK evaluation of antibody Ab2374 of the present invention (PD-1 / LAG-3 detection results)

[0310]

[0311]

[0312] The sandwich ELISA detected the PK characteristics of binding to PD-1 and then to LAG-3 molecules. The results in Table 20 show that, unexpectedly, the PK characteristics (Cmax, T ... 1 / 2 ) and the characteristics obtained by detecting PD-1 alone are almost the same. This 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 also close to the PK characteristics of the Ab2317 antibody alone (Table 18). In addition, the antibody of the present invention has a specific target binding in hPD-1 / hLAG-3 double transgenic mice (C57BL / 6-DKI), and Cmax, T 1 / 2 Surprisingly, the PK detected by sandwich ELISA was consistent with the PK characteristics detected by PD-1 alone, indicating that the bispecific antibody of the present invention is also stable in mice with specific target binding and there is no scFv shedding.

[0313] According to the method of Example 1 of the present invention, the above-mentioned DVD-designed bispecific antibodies were cloned, expressed, and purified, and the gel electrophoresis (PAGE) results showed that the light and heavy chains of these antibodies were prone to linker breakage. By connecting one target antibody to the N or C terminus of the light chain or heavy chain of another target antibody in the form of scFv, a bispecific antibody with optimized sequence and design is obtained by screening, which can avoid / reduce linker breakage (see the above examples), and retain the binding activity to the dual targets, in vitro functional activity, and in vivo efficacy against the dual targets. The preferred bispecific antibody (referred to as SBody in the present invention) is not only stable, but also simple in purification process because of its similarity to conventional IgG structure, which provides great convenience for the process and purification in the later development process. SEQUENCE LISTING <110> Shanghai Jianxin Biopharmaceutical Technology Co., Ltd. <120> Antibodies and bispecific antibodies targeting LAG-3 and their uses <130> P19011013C <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Mouse MAb23 light chain variable region bases <400> 1 gatgaggacc cctgctcaga ttcttgggat cttgttgctc ttgtttccag gtaccagatg 60 tgacattcag atgatccagt ctccatcctc cttatctgcc tctctgggag aaagagtcag 120 tctcacttgt cgggcaagtc aggacattgg tagtagttta aactggcttc agcaggaacc 180 agatggaact atcaaacgcc tgatctacgc cacatccagt ttagattctg gtgtccccaa 240 aaggttcagt ggcagtaggt ctgggtcaga ttattctctc accatcagca gccttgagtc 300 tgaagatttt gtagactatt actgtctaca atatgttact tctccgctca cgttcggtgc 360 tgggaccaag ctggagctga aacgggctga tgctgcacca actgtatcca tcttcccacc 420 atccagtgag cagttaacat ctggaggtgc ctcagtcgtg tgcttctgaa caactctacc 480 ccagagacat caattccctg 500 <210> 2 <211> 539 <212> DNA <213> Artificial Sequence <220> <223> Base sequence of murine mab23 heavy chain variable region <400> 2 gatgaaatgg cagctgggtt tttctcttcc tcctgtcagt aattgcaggt gtccaatccc 60 aggttcaact gcagcagtct ggggctgagc tggtgaggcc tggggcttca gtgacgctgt 120 cctgcaaggc ttcgggctac acatttactg actatgaaat gcactgggtg aaacagacac ctgtgcatgg cctggaatgg attggaggta ttgatcctga aactgaaggc attgcctata atcagaagtt caggggcaag gccatactga ctgcagacaa atcctccatc acagcctaca tggagctccg cagcctgaca tctgaggact ctgccgtcta ttactgtaca aactccaatt actacggtgg aagggaggcc tggtttgctt actggggcca agggactctg gtcactgtct 420 480. ctggagccaa aacgacaccc cctctgtct atccactggc ccctggatct gctgcccaaa ctaactccat ggtgaccctg ggatgcctgg tcaagggcta tacatagcca ttactcaaa <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Free mab23 license plates <400> 3 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 4 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Amino acids of the variable region of the murine mab23 heavy chain <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ile Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Gly 115 120 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 5 Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn 1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 <400> 6 Ala Thr Ser Ser Leu Asp Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 <400> 7 Leu Gln Tyr Val Thr Ser Pro Leu Thr 1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 8 Gly Tyr Thr Phe Thr Asp Tyr Glu Met His 1 5 10 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 <400> 9 Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe Arg 1 5 10 15 Gly <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 10 Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr 1 5 10 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1, Kabat definition <400> 11 Asp Tyr Glu Met His 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2, AbM definition <400> 12 Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1, Chothia definition <400> 13 Gly Tyr Thr Phe Thr Asp Tyr 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2, Chothia definition <400> 14 Asp Pro Glu Thr Glu Gly 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1, Contact definition <400> 15 Gly Ser Ser Leu Asn Trp Leu 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2, Contact definition <400> 16 Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp 1 5 10 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3, Contact definition <400> 17 Leu Gln Tyr Val Thr Ser Pro Leu 1 5 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1, Contact definition <400> 18 Thr Asp Tyr Glu Met His 1 5 <210> 19 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2, Contact definition <400> 19 Trp Ile Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala 1 5 10 <210> 20 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3, Contact definition <400> 20 Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala 1 5 10 <210> twenty one <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human antibody light chain constant region kappa chain <400> twenty one Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 22 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Constant region of the heavy chain of human IgG4 <400> 22 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2312 <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2313 <400> 24 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 25 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2314 <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2315 <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2316 <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2317 <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region, LG2318 <400> 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2342 <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 31 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2343 <400> 31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2344 <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Thr Thr Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 33 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2345 <400> 33 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2346 <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2347 <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2348 <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region, LG2349 <400> 37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain, Ab2315 <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 39 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain, Ab2317 / Ab2325 <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 40 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain, Ab2315 / Ab2317 <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 41 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain, Ab2325 <400> 41 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 42 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Nivolumab light chain <400> 42 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 43 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> Nivolumab heavy chain <400> 43 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 44 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Pembrolizumab light chain <400> 44 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 45 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Pembrolizumab heavy chain <400> 45 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 46 <211> 706 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of heavy chain-containing LB2371 <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys 145 150 155 160 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Gly Ile Asp Pro Glu 165 170 175 Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu 195 200 205 Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Thr Asn Ser Asn Tyr Tyr 210 215 220 Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys 260 265 270 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 275 280 285 Thr Asn Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 290 295 300 Glu Trp Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn 305 310 315 320 Glu Lys Phe Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr 325 330 335 Thr Ala Tyr Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr 355 360 365 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 370 375 380 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 385 390 395 400 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 405 410 415 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 420 425 430 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 435 440 445 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 450 455 460 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 465 470 475 480 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 515 520 525 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 545 550 555 560 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 610 615 620 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 660 665 670 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 675 680 685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 690 695 700 Gly Lys 705 <210> 47 <211> 699 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain of LB2373 <400> 47 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys 145 150 155 160 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Gly Ile Asp Pro Glu 165 170 175 Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu 195 200 205 Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Thr Asn Ser Asn Tyr Tyr 210 215 220 Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 260 265 270 Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe 275 280 285 Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 290 295 300 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala 305 310 315 320 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 325 330 335 Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val 355 360 365 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 370 375 380 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 385 390 395 400 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 405 410 415 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 420 425 430 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 435 440 445 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 450 455 460 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 595 600 605 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 690 695 <210> 48 <211> 699 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain of LB2374 <400> 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala 450 455 460 Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 465 470 475 480 Gly Tyr Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Ala Pro 485 490 495 Gly Gln Gly Leu Glu Trp Ile Gly Gly Ile Asp Pro Glu Thr Glu Gly 500 505 510 Ile Ala Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Leu Thr Ala Asp 515 520 525 Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp 530 535 540 Asp Thr Ala Val Tyr Tyr Cys Thr Asn Ser Asn Tyr Tyr Gly Gly Arg 545 550 555 560 Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 565 570 575 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 595 600 605 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 610 615 620 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 625 630 635 640 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 645 650 655 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 660 665 670 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 675 680 685 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 690 695 <210> 49 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of LB2379 <400> 49 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys 145 150 155 160 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Gly Ile Asp Pro Glu 165 170 175 Thr Glu Gly Ile Ala Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu 195 200 205 Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Thr Asn Ser Asn Tyr Tyr 210 215 220 Gly Gly Arg Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 260 265 270 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 275 280 285 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 290 295 300 Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg 305 310 315 320 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 325 330 335 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn 340 345 350 Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 355 360 365 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 370 375 380 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 385 390 395 400 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 405 410 415 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 420 425 430 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 435 440 445 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 450 455 460 Thr Lys Ser Phe Asn Arg Gly Glu Cys 465 470 <210> 50 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> LB2380 contains the light chain amino acid sequence <400> 50 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 225 230 235 240 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 245 250 255 Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Ala Pro Gly Gln 260 265 270 Gly Leu Glu Trp Ile Gly Gly Ile Asp Pro Glu Thr Glu Gly Ile Ala 275 280 285 Tyr Asn Gln Lys Phe Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser 290 295 300 Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Thr Asn Ser Asn Tyr Tyr Gly Gly Arg Glu Ala 325 330 335 Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 340 345 350 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 355 360 365 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 370 375 380 Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn 385 390 395 400 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala 405 410 415 Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg 420 425 430 Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 435 440 445 Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Thr Ser Pro Leu Thr Phe 450 455 460 Gly Gly Gly Thr Lys Val Glu Ile Lys 465 470

Claims

1. An anti-LAG-3 antibody, comprising a light chain variable region and a heavy chain variable region; the light chain variable region comprises: a CDR1 with an amino acid sequence as shown in SEQ ID NO: 5, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 6, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 7; the heavy chain variable region comprises: a CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:

10.

2. The anti-LAG-3 antibody of claim 1, wherein 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; and / or 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; wherein the mutation has a substitution, deletion or addition of one or more amino acid residues in the original amino acid sequence, has at least 99% sequence identity with the original amino acid sequence, and the mutation maintains or improves the binding of the anti-LAG-3 antibody to LAG-3.

3. The anti-LAG-3 antibody of claim 2, wherein 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; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

36.

4. The anti-LAG-3 antibody of any one of claims 1 to 3, which is a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv.

5. The anti-LAG-3 antibody according to claim 4, 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 κ chain or a λ chain, and / or the human antibody heavy chain constant region is hIgG1, hIgG2, or hIgG4.

6. The anti-LAG-3 antibody of claim 5, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 38 or SEQ ID NO: 39, and / or the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 40 or SEQ ID NO:

41.

7. The anti-LAG-3 antibody of claim 6, wherein the amino acid sequence of the light chain is as shown in SEQ ID NO: 38; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 40; or, the amino acid sequence of the light chain is as shown in SEQ ID NO: 38; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 41; or, the amino acid sequence of the light chain is as shown in SEQ ID NO: 39; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 40; or, the amino acid sequence of the light chain is as shown in SEQ ID NO: 39; the amino acid sequence of the heavy chain is as shown in SEQ ID NO:

41.

8. A bispecific antibody targeting LAG-3, comprising a first protein functional region and a second protein functional region, wherein: The first protein functional region is the anti-LAG-3 antibody according to any one of claims 1 to 7; the second protein functional region is an anti-PD-1 antibody, and the anti-PD-1 antibody is Nivolumab or Pembrolizumab; The first protein functional region is two scFvs; 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 symmetrically connected to the N-termini of the two heavy chain variable regions of the second protein functional region through (G4S)3, or the N-termini of the light chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two heavy chains of the second protein functional region through (G4S)3; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the C-termini of the light chain variable regions of the two scFvs are symmetrically connected to the N-termini of the two light chain variable regions of the second protein functional region through (G4S)3, or the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two light chain variable regions of the second protein functional region through (G4S)3.

9. The bispecific antibody targeting LAG-3 according to claim 8, which is selected from the following: The amino acid sequence of the light chain variable region of the scFv 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; or, the amino acid sequence of the light chain variable region of the scFv 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; and / or, The second protein functional region comprises the following light chain and heavy chain or mutations thereof: the amino acid sequence of the light chain is as shown in SEQ ID NO: 42; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 43; or, the amino acid sequence of the light chain is as shown in SEQ ID NO: 44; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 45; when the scFv is connected to the C-termini of the two heavy chains of the immunoglobulin, the C-terminus of the heavy chain mutates from K to A.

10. The bispecific antibody targeting LAG-3 according to claim 8 or 9, which is selected from the following group: (i) the amino acid sequence of the light chain variable region of the first protein functional region is as shown in SEQ ID NO: 28, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 31, and the linker is (G4S)3; the amino acid sequence of the light chain of the second protein functional region is as shown in SEQ ID NO: 42, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 43; in, The scFv is a light chain variable region-linker-heavy chain variable region structure, the C-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the N-termini of the two heavy chains of the immunoglobulin through (G4S)3, and the C-termini are mutated from K to A; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin through (G4S)3; or, the scFv is a light chain variable region-linker-heavy chain variable region structure, the C-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the N-termini of the two light chains of the immunoglobulin through (G4S)3; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two light chains of the immunoglobulin through (G4S)3; or, (ii) the amino acid sequence of the light chain variable region of the first protein functional region is as shown in SEQ ID NO: 28, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 31, and the linker is (G4S)3; the amino acid sequence of the light chain of the second protein functional region is as shown in SEQ ID NO: 44, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 45; Wherein, 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 symmetrically connected to the N-termini of the two heavy chains of the immunoglobulin through (G4S)3; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin through (G4S)3, and the C-termini are 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 symmetrically connected to the N-termini of the two light chains of the immunoglobulin through (G4S)3; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two light chains of the immunoglobulin through (G4S)3; or, (iii) the amino acid sequence of the light chain variable region of the first protein functional region is as shown in SEQ ID NO: 28, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 36, and the linker is (G4S)3; the amino acid sequence of the light chain of the second protein functional region is as shown in SEQ ID NO: 44, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 45; Wherein, 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 symmetrically connected to the N-termini of the two heavy chains of the immunoglobulin through (G4S)3; or, the scFv is a heavy chain variable region-linker-light chain variable region structure, and the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin through (G4S)3, and the C-termini are mutated from K to A.

11. The bispecific antibody targeting LAG-3 according to claim 10, wherein the bispecific antibody comprises the following amino acid sequence: A light chain amino acid sequence as shown in SEQ ID NO: 44, an amino acid sequence containing a heavy chain as shown in SEQ ID NO: 46; or, a light chain amino acid sequence as shown in SEQ ID NO: 42, an amino acid sequence containing a heavy chain as shown in SEQ ID NO: 47; or, a light chain amino acid sequence as shown in SEQ ID NO: 42, an amino acid sequence containing a heavy chain as shown in SEQ ID NO: 48; or, a light chain amino acid sequence as shown in SEQ ID NO: 49, an amino acid sequence containing a heavy chain as shown in SEQ ID NO: 43; or, a light chain amino acid sequence as shown in SEQ ID NO: 50, an amino acid sequence containing a heavy chain as shown in SEQ ID NO:

43.

12. An isolated nucleic acid encoding the anti-LAG-3 antibody of any one of claims 1 to 7 or the bispecific antibody targeting LAG-3 of any one of claims 8 to 11.

13. An expression vector comprising the isolated nucleic acid according to claim 12. A host cell comprising the expression vector according to claim 13 .

15. A method for preparing an anti-LAG-3 antibody or a bispecific antibody targeting LAG-3, comprising culturing the host cell as claimed in claim 14, and obtaining the anti-LAG-3 antibody or the bispecific antibody targeting LAG-3 from the culture.

16. A pharmaceutical composition comprising the anti-LAG-3 antibody according to any one of claims 1 to 7, or the bispecific antibody targeting LAG-3 according to any one of claims 8 to 11.

17. Use of the anti-LAG-3 antibody according to any one of claims 1 to 7, the bispecific antibody targeting LAG-3 according to any one of claims 8 to 11, and / or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating and / or preventing cancer; The cancer is selected from the group consisting of melanoma, colorectal cancer, non-small cell lung cancer, renal cell carcinoma, follicular lymphoma, head and neck squamous cell carcinoma, breast cancer, diffuse large B-cell lymphoma, fibrosarcoma, epithelial ovarian cancer, chronic lymphocytic leukemia.

Images