Antibodies or antigen-binding fragments thereof targeting CD3e / g, their preparation and uses

Abstract

The present invention discloses an antibody or an antigen-binding fragment thereof targeting CD3e / g, which comprises VH and VL. VH comprises VH CDR1 shown by any one of the amino acid sequences of SEQ ID NO: 1-6, VH CDR2 shown by any one of the amino acid sequences of SEQ ID NO: 7-15, and VH CDR3 shown by any one of the amino acid sequences of SEQ ID NO: 16-22; VL comprises VL CDR1 shown by any one of the amino acid sequences of SEQ ID NO: 23-27, VL CDR2 shown by the amino acid sequence of SEQ ID NO: 28 or 29, and VL CDR3 shown by any one of the amino acid sequences of SEQ ID NO: 30-32. It can bind to human or monkey CD3e / g and can effectively activate the NFAT downstream signaling pathway of Jurkat cells.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to an antibody or antigen-binding fragment thereof targeting CD3e / g, and its preparation method and application. Background Art

[0002] Therapeutic antibodies such as monoclonal antibodies can be developed by a variety of techniques and approaches, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The preparation of monoclonal antibodies by hybridoma technology remains the mainstream of current methods for preparing therapeutic monoclonal antibodies.

[0003] The traditional hybridoma preparation technology was established by Kohler and Milstein 40 years ago (Kohler and Milstein 1975, Nature 256:495), and is now widely used in the preparation and production of many related monoclonal antibodies in scientific research, diagnosis, treatment, etc. Although its basic method is still in use today, it has changed, improved and innovated in many aspects, including the use of different strains of animals such as transgenic animals, the introduction of electrofusion technology, the application of high-efficiency screening technology equipment such as ClonePix equipment, etc., making the application of hybridoma technology more diverse and efficient. Monoclonal antibodies prepared from conventional animals such as mice can clone the heavy chain variable region and light chain variable region genes of the antibody by conventional molecular biology methods. The variable region genes can be grafted onto human antibody constant region genes to form human-mouse chimeric antibodies (U.S. Pat. No. 4,816,567, Cabilly et al), so as to greatly reduce the immunogenicity when used in humans. Furthermore, the CDR domains of the murine antibody variable region can be grafted onto the human antibody framework, so that the murine antibody component is reduced to less than 5%, greatly increasing the safety of the antibody when used in humans. The antibody obtained by this approach is called a humanized antibody and is the main product in the current antibody drug market (U.S. Pat. Nos. 5,225,539 to 55, Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

[0004] The existing CD3e / g antibodies include OKT3, which is the trade name of a CD3 monoclonal antibody. It inhibits acute rejection in organ transplantation (such as heart, kidney, and liver) by blocking the function of T cells. After the treatment with OKT3 ends, the function of T cells usually returns to normal within a week. However, the side effects of OKT3 at the beginning of use are quite large, including OKT3 influenza-like syndrome, cytokine storm, etc. In addition, OKT3 does not bind to cynomolgus-derived CD3 (cyno CD3, cCD3), which brings inconvenience to the selection of the primate cynomolgus in preclinical safety evaluation studies.

[0005] The difficulty in developing CD3e / g antibodies lies in the fact that it is difficult to obtain a good immune response of antibody transgenic mice and serum titers, resulting in a small number of positive clones after fusion. Currently, there are few CD3e / g antibodies with Cross biological activity functions with other CD3e / g except human-derived ones. Conventional binding experiments such as flow cytometry (FACS) and enzyme-linked immunosorbent assay (ELISA) can screen out antibodies with affinity. However, after these antibodies bind to CD3e / g, they do not necessarily affect the subsequent signal pathways and corresponding biological functions. Therefore, there is an urgent need in the art for CD3e / g antibodies with better effects, such as having Cross biological activity functions with CD3 from other sources except human CD3 while not affecting the subsequent signal pathways and corresponding biological activities. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of current antibodies targeting CD3e / g, and provide an antibody targeting CD3e / g or its antigen-binding fragment, as well as its preparation method and application. The antibody targeting CD3e / g or its antigen-binding fragment of the present invention has high affinity, high biological activity, and high diversity; it can bind to human CD3e / g (hCD3e / g) and cynomolgus CD3e / g (cCD3e / g), so that in preclinical safety evaluation studies, a disease model of the primate cynomolgus can be selected for pharmacology and toxicology experiments, bringing great convenience to preclinical pharmacology, toxicology, and other studies. In addition, the antibody targeting CD3e / g or its antigen-binding fragment of the present invention can effectively activate the NFAT downstream signal pathway of Jurkat cells, which is more conducive to the subsequent biological activities.

[0007] The difficulty in developing CD3e / g antibodies lies in the difficulty of obtaining a good immune response and serum titer in antibody transgenic mice, resulting in a small number of positive clones after fusion. Currently, few CD3e / g antibodies have Cross biological activity functions with CD3e / g other than human sources, and do not affect subsequent signal pathways and corresponding biological activities. Conventional binding experiments such as flow cytometry (FACS) and enzyme-linked immunosorbent assay (ELISA) can screen out antibodies with affinity, but after these antibodies bind to CD3e / g, they may not necessarily maintain subsequent signal pathways and corresponding biological functions. Through a large number of experiments, based on the current monoclonal antibody technology, the present inventor adopted an optimized hybridoma technology and established a detection method for relevant biological functions, and unexpectedly obtained an antibody targeting CD3e / g with high affinity, high biological activity and high diversity.

[0008] To solve the above technical problems, in the first aspect of the present invention, there is provided an antibody targeting CD3e / g or an antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0009] Wherein, the VH comprises the following complementarity-determining regions (CDRs) or mutations thereof: VH CDR1 shown by any one of the amino acid sequences of SEQ ID NOs: 1-6; VH CDR2 shown by any one of the amino acid sequences of SEQ ID NOs: 7-15; and / or, VH CDR3 shown by any one of the amino acid sequences of SEQ ID NOs: 16-22.

[0010] Wherein, the VL comprises the following complementarity-determining regions (CDRs) or mutations thereof: VL CDR1 shown by any one of the amino acid sequences of SEQ ID NOs: 23-27; VL CDR2 shown by the amino acid sequence of SEQ ID NO: 28 or 29; and / or, VL CDR3 shown by any one of the amino acid sequences of SEQ ID NOs: 30-32.

[0011] Wherein the mutation is an insertion, deletion or substitution of 3, 2 or 1 amino acid in the amino acid sequence of the CDR.

[0012] In the present application, in phrases such as "having an insertion, deletion or substitution of 3, 2 or 1 amino acid", "amino acid mutation" means that compared with the original amino acid sequence, the sequence of the variant obtained after mutation has an amino acid mutation, including an insertion, deletion or substitution of an amino acid on the basis of the original amino acid sequence. An exemplary explanation is that the mutation of the CDR can include mutations of 3, 2 or 1 amino acid, and the same or different numbers of amino acid residues can be optionally selected for mutation among these CDRs. For example, it can be a mutation of 1 amino acid in CDR1, and no amino acid mutation in CDR2 and CDR3.

[0013] In the present application, the mutations may include mutations currently known to those skilled in the art. For example, during the production or application of an antibody, certain mutations may be made to the antibody. For example, mutations may be made to sites of potential post-translational modifications (PTMs), especially in the CDR regions, including mutations related to antibody aggregation, asparagine deamidation (sites such as NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}S / T) sensitive sites, and oxidation sensitive sites, etc.

[0014] Preferably, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:7, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:16; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:2, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:8, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:17; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:3, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:18; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:4, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:10, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:19; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:5, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:20; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:21; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:13, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:20; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:14, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:22; or, the amino acid sequence of the VH CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of the VH CDR2 is as shown in SEQ ID NO:15, and the amino acid sequence of the VH CDR3 is as shown in SEQ ID NO:20.

[0015] Preferably, the amino acid sequence of the VL CDR1 is as shown in SEQ ID NO: 23, the amino acid sequence of the VL CDR2 is as shown in SEQ ID NO: 28, and the amino acid sequence of the VL CDR3 is as shown in SEQ ID NO: 30; or, the amino acid sequence of the VL CDR1 is as shown in SEQ ID NO: 24, the amino acid sequence of the VL CDR2 is as shown in SEQ ID NO: 28, and the amino acid sequence of the VL CDR3 is as shown in SEQ ID NO: 30; or, the amino acid sequence of the VL CDR1 is as shown in SEQ ID NO: 24, the amino acid sequence of the VL CDR2 is as shown in SEQ ID NO: 28, and the amino acid sequence of the VL CDR3 is as shown in SEQ ID NO: 31; or, the amino acid sequence of the VL CDR1 is as shown in SEQ ID NO: 25, the amino acid sequence of the VL CDR2 is as shown in SEQ ID NO: 29, and the amino acid sequence of the VL CDR3 is as shown in SEQ ID NO: 32; or, the amino acid sequence of the VL CDR1 is as shown in SEQ ID NO: 26, the amino acid sequence of the VL CDR2 is as shown in SEQ ID NO: 29, and the amino acid sequence of the VL CDR3 is as shown in SEQ ID NO: 32; or, the amino acid sequence of the VL CDR1 is as shown in SEQ ID NO: 27, the amino acid sequence of the VL CDR2 is as shown in SEQ ID NO: 29, and the amino acid sequence of the VL CDR3 is as shown in SEQ ID NO: 32.

[0016] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 1, 7, and 16, respectively; the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO: 23, 28, and 30, respectively.

[0017] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 2, 8, and 17, respectively; the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO: 24, 28, and 30, respectively.

[0018] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown as SEQ ID NO:3, 9, and 18 respectively; the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown as SEQ ID NO:24, 28, and 30 respectively.

[0019] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown as SEQ ID NO:4, 10, and 19 respectively; the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown as SEQ ID NO:24, 28, and 31 respectively.

[0020] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown as SEQ ID NO:5, 11, and 20 respectively; the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown as SEQ ID NO:25, 29, and 32 respectively.

[0021] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown as SEQ ID NO:6, 12, and 21 respectively; the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown as SEQ ID NO:26, 29, and 32 respectively.

[0022] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown as SEQ ID NO:6, 13, and 20 respectively; the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown as SEQ ID NO:26, 29, and 32 respectively.

[0023] In a preferred embodiment, in the antibody or its antigen-binding fragment, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown as SEQ ID NO:6, 14, and 22 respectively; the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown as SEQ ID NO:26, 29, and 32 respectively.

[0024] In a preferred embodiment, in the antibody or its antigen-binding fragment, the VH contains VH CDR1, VH CDR2, and VH CDR3 with amino acid sequences as shown in SEQ ID NO:6, 15, and 20 respectively; the VL contains VL CDR1, VL CDR2, and VL CDR3 with amino acid sequences as shown in SEQ ID NO:27, 29, and 32 respectively.

[0025] Preferably, the heavy chain variable region (VH) further includes a heavy chain variable region framework region (VH FWR), preferably the heavy chain variable region framework region of a human antibody or a murine antibody.

[0026] Preferably, the light chain variable region (VL) further includes a light chain variable region framework region (VL FWR), preferably the light chain variable region framework region of a human antibody or a murine antibody.

[0027] In certain embodiments, the VH FWR includes the following VH FWR: VH FWR1 with an amino acid sequence as shown in any of SEQ ID NO:33 - 40 or its mutants, VH FWR2 with an amino acid sequence as shown in any of SEQ ID NO:41 - 43 or its mutants, VH FWR3 with an amino acid sequence as shown in any of SEQ ID NO:44 - 51 or its mutants, and / or VH FWR4 with an amino acid sequence as shown in SEQ ID NO:52 or 53 or its mutants.

[0028] In certain embodiments, the VL FWR includes the following VL FWR: VL FWR1 with an amino acid sequence as shown in any of SEQ ID NO:54 - 56 or its mutants, VL FWR2 with an amino acid sequence as shown in SEQ ID NO:57 or 58 or its mutants, VL FWR3 with an amino acid sequence as shown in any of SEQ ID NO:59 - 63 or its mutants, and / or VL FWR4 with an amino acid sequence as shown in any of SEQ ID NO:64 - 66 or its mutants. The mutations are insertions, deletions, substitutions, and duplications of 3, 2, or 1 amino acids in the amino acid sequence of the VH FWR or VL FWR.

[0029] Preferably, the amino acid sequence of the VH is as shown in any of SEQ ID NO:67 - 75 or its mutants, and its nucleotide sequence is preferably as shown in any of SEQ ID NO:83 - 91 or its mutants.

[0030] Preferably, the amino acid sequence of the VL is as shown in any of SEQ ID NO:76 - 82 or its mutants, and its nucleotide sequence is preferably as shown in any of SEQ ID NO:92 - 99 or its mutants.

[0031] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:74 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:76 or its mutation.

[0032] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:75 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:77 or its mutation.

[0033] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:67 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:77 or its mutation.

[0034] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:73 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:78 or its mutation.

[0035] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:69 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:81 or its mutation.

[0036] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:72 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:79 or its mutation.

[0037] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:71 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:80 or its mutation.

[0038] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:68 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:80 or its mutation.

[0039] In a preferred embodiment, the amino acid sequence of the VH is as shown in SEQ ID NO:70 or its mutation, and the amino acid sequence of the VL is as shown in SEQ ID NO:82 or its mutation.

[0040] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO:84 or its mutation, and the nucleotide sequence of the VL is as shown in SEQ ID NO:92 or its mutation.

[0041] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 91 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 93 or its mutants.

[0042] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 90 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 93 or its mutants.

[0043] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 85 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 94 or its mutants.

[0044] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 86 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 95 or its mutants.

[0045] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 83 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 97 or its mutants.

[0046] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 89 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 98 or its mutants.

[0047] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 87 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 96 or its mutants.

[0048] In a preferred embodiment, the nucleotide sequence of the VH is as shown in SEQ ID NO: 88 or its mutants, and the nucleotide sequence of the VL is as shown in SEQ ID NO: 99 or its mutants.

[0049] The above mutations are deletions, substitutions or additions of one or more amino acid residues in the amino acid sequence of the VH and / or VL, and the mutant amino acid sequence has at least 85% sequence identity with the amino acid sequence of the VH and / or VL, and maintains or improves the binding of the antibody to CD3e / g; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, 98% sequence identity, and most preferably at least 99% sequence identity.

[0050] In the present application, the amino acid sequences of the above-listed CDRs are all shown according to the Kabat definition rule (the sequences in the claims of the present application are also shown according to the Kabat definition rule). However, as is well known to those skilled in the art, there are various methods in the art to define the CDRs of antibodies, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991); “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989)), the Chothia definition rule based on the position of structural loop regions (see J Mol Biol 273:927-48, 1997), and the AbM definition rule (a compromise between the Kabat definition and the Chothia method), etc. The methods and techniques for identifying the variable regions within an antibody molecule and the CDRs within the amino acid sequences of the antibody variable regions are well known in the art and can be used to identify the CDRs within the specific antibody variable region amino acid sequences disclosed herein.

[0051] Those skilled in the art should understand that, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or its region (such as the variable region) should be understood to cover the complementary determining regions defined by any of the above-known schemes described in the present invention. Although the scope claimed in the present invention is based on the sequences shown according to the Kabat definition rule, the amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.

[0052] Preferably, the antibody or its antigen-binding fragment targeting CD3e / g further comprises an antibody heavy chain constant region and / or an antibody light chain constant region. For example, the antibody heavy chain constant region is a murine-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region; the antibody light chain constant region is a murine-derived light chain antibody constant region or a human-derived antibody light chain constant region. More preferably, the antibody heavy chain constant region is a human-derived antibody heavy chain constant region, such as the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 antibodies; the antibody light chain constant region is a human-derived antibody light chain κ or λ chain constant region.

[0053] Preferably, the antibody or antigen-binding fragment thereof targeting CD3e / g is a full-length antibody, Fab, Fab’, F(ab’)2, Fv, scFv, bispecific antibody, multispecific antibody, single-domain antibody, single-region antibody or any other antibody that retains part of the ability of an antibody to specifically bind an antigen, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies. When it includes a framework region of a human antibody variable region, it is generally a humanized antibody.

[0054] The full-length antibody mentioned above can be a conventional full-length antibody in the art, which includes a heavy-chain variable region, a light-chain variable region, a heavy-chain constant region and a light-chain constant region, such as IgG1, IgG2a, IgG2b or IgG2c, etc.

[0055] The single-chain antibody (scFv) mentioned above can be a conventional single-chain antibody in the art, which includes a heavy-chain variable region, a light-chain variable region and a short peptide of 15-20 amino acids.

[0056] The single-domain antibody mentioned above can be a conventional single-domain antibody in the art, which includes a heavy-chain variable region and a heavy-chain constant region.

[0057] The single-region antibody mentioned above can be a conventional single-region antibody in the art, which only includes a heavy-chain variable region.

[0058] To solve the above technical problems, the second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof targeting CD3e / g as described in the first aspect of the present invention.

[0059] The preparation method of the nucleic acid is a conventional preparation method in the art. For example, it may include the following steps: obtaining a nucleic acid molecule encoding the above antibody through gene cloning technology, or obtaining a nucleic acid molecule encoding the above antibody through the method of artificial total sequence synthesis.

[0060] Those skilled in the art know that the base sequence of the amino acid sequence encoding the above antibody can be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a homolog of the polynucleotide. The homolog of the polynucleotide in the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the antibody sequence within the range of maintaining the antibody activity.

[0061] To solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector containing the isolated nucleic acid as described in the second aspect of the present invention.

[0062] The recombinant expression vector can be obtained by a conventional method in the art, that is: constructed by ligating the nucleic acid molecule described in the present application to various expression vectors. The expression vectors mentioned above are various conventional vectors in the art, as long as they can accommodate the aforementioned nucleic acid molecule.

[0063] Preferably, the expression vector comprises an eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

[0064] To solve the above technical problems, a fourth aspect of the present invention provides a transformant, which comprises the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention.

[0065] The preparation method of the transformant can be a conventional preparation method in the art, for example: the above recombinant expression vector is transformed into a host cell to obtain it. The host cell of the transformant is various conventional host cells in the art, as long as it can satisfy that the above recombinant expression vector can stably replicate by itself, and the carried nucleic acid can be effectively expressed. Preferably, the host cell is a prokaryotic cell and / or an eukaryotic cell. The prokaryotic cell is preferably an E.coli cell such as TG1, BL21 (expressing single-chain antibody or Fab antibody), and the eukaryotic cell is preferably a HEK293 cell or a CHO cell (expressing full-length IgG antibody). Transforming the aforementioned recombinant expression plasmid into a host cell can obtain the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0066] To solve the above technical problems, a fifth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting CD3e / g, which comprises culturing the transformant as described in the fourth aspect of the present invention, and obtaining the antibody or the antigen-binding fragment thereof targeting CD3e / g from the culture.

[0067] To solve the above technical problems, a sixth aspect of the present invention provides a chimeric antigen receptor, which comprises the antibody or an antigen-binding fragment thereof targeting CD3e / g as described in the first aspect of the present invention.

[0068] To solve the above technical problems, a seventh aspect of the present invention provides a genetically modified cell, which comprises the antibody or an antigen-binding fragment thereof targeting CD3e / g as described in the first aspect of the present invention; the genetically modified cell is preferably an eukaryotic cell, more preferably an isolated human cell, and further more preferably an immune cell such as a T cell, or an NK cell such as the NK92 cell line.

[0069] To solve the above technical problems, an eighth aspect of the present invention provides an antibody-drug conjugate, which comprises a cytotoxic agent and the antibody or an antigen-binding fragment thereof targeting CD3e / g as described in the first aspect of the present invention.

[0070] To solve the above technical problems, a ninth aspect of the present invention provides a pharmaceutical composition, which comprises an antibody targeting CD3e / g or an antigen-binding fragment thereof as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0071] Preferably, the pharmaceutical composition further comprises other anti-tumor antibodies as active ingredients.

[0072] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0073] The pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, and preferably includes pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above antibody or its antigen-binding fragment and / or the above antibody-drug conjugate, etc., and 0.01-99.99% of the pharmaceutical carrier, and the percentages are by mass of the pharmaceutical composition.

[0074] The administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc. The pharmaceutical composition is various conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, that is, it can be an aqueous solution, a non-aqueous solution or a suspension, and more preferably tablets, capsules, granules, injections or infusions, etc. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally or intramuscularly. Preferably, the pharmaceutical composition can also be administered as an aerosol or a coarse spray, that is, intranasally; or, intrathecally, intramedullary or intraventricularly. More preferably, the pharmaceutical composition can also be administered transdermally, percutaneously, topically, enterally, vaginally, sublingually or rectally. The pharmaceutical composition of the present invention can be made into various dosage forms as needed, and the physician can determine the dosage beneficial to the patient according to factors such as the type, age, weight and general disease condition of the patient, and the administration method. The administration method can be, for example, injection or other treatment methods.

[0075] The dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition required to achieve the desired diagnostic or therapeutic result. The administration regimen can also be a single injection or multiple injections, or adjusted. The selected dosage level and regimen are reasonably adjusted depending on various factors including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the administration route, the combination with other drugs or treatments, the disease or disorder to be detected and / or treated, and the health status and previous medical history of the subject to be treated.

[0076] The therapeutically effective dose of the pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine the appropriate range of administration concentrations and routes. Subsequently, they can be used to determine the useful doses and routes of administration in humans. Generally, the determination and adjustment of the effective amount or dose to be administered, and the evaluation of when and how such adjustments are to be made, are known to those skilled in the art.

[0077] For combination therapies, the above-mentioned antibodies or their antigen-binding fragments, the above-mentioned antibody-drug conjugates, etc., and / or additional therapeutic or diagnostic agents can each be used as a single agent within any time frame suitable for performing the intended treatment or diagnosis. Thus, these single agents can be administered substantially simultaneously (i.e., as a single formulation or within a few minutes or hours) or sequentially in succession.

[0078] For additional guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see works such as Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co., Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, etc.

[0079] To solve the above technical problems, the tenth aspect of the present invention provides an application of an antibody targeting CD3e / g or its antigen-binding fragment as described in the first aspect of the present invention, a chimeric antigen receptor as described in the sixth aspect of the present invention, a genetically modified cell as described in the seventh aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, and a pharmaceutical composition as described in the ninth aspect of the present invention in the preparation of a drug for diagnosing, preventing, and / or treating tumors.

[0080] Preferably, the tumors include, but are not limited to, colorectal cancer, lung cancer, breast cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, pancreatic cancer, and / or lymphoma, and the lymphoma is, for example, adult T-cell leukemia lymphoma (ATLL), acute myeloid lymphoma (AML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), pediatric acute lymphoblastic lymphoma (B-ALL), anaplastic large cell lymphoma (ALCL), natural killer cell lymphoma, and / or peripheral T-cell lymphoma (PTCL), etc.

[0081] To solve the above technical problems, the present invention also provides a kit, comprising: the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the chimeric antigen receptor as described in the sixth aspect of the present invention, the genetically modified cell as described in the seventh aspect of the present invention, the antibody-drug conjugate as described in the eighth aspect of the present invention, the pharmaceutical composition as described in the ninth aspect of the present invention; preferably further comprising a device for administering the antibody or its antigen-binding fragment or the antibody-drug conjugate or the pharmaceutical composition; more preferably further comprising an instruction manual, etc.

[0082] To solve the above technical problems, the present invention also provides a method for detecting CD3e / g in a sample, which comprises detecting using the anti-CD3e / g antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0083] To solve the above technical problems, the present invention also provides the use of the above-mentioned anti-CD3e / g antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned genetically modified cell, the above-mentioned antibody-drug conjugate, and / or the above-mentioned pharmaceutical composition in the diagnosis, prevention, and / or treatment of cancer. Preferably, the cancer is as described in the tenth aspect of the present invention.

[0084] To solve the above technical problems, the present invention also provides a set of medicine boxes, which comprises medicine box A and medicine box B. The medicine box A contains the above-mentioned anti-CD3e / g antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned genetically modified cell, the above-mentioned antibody-drug conjugate, the above-mentioned pharmaceutical composition, and the medicine box B is other anti-cancer (tumor) antibodies or a pharmaceutical composition containing the other anti-cancer (tumor) antibodies. The medicine box A and the medicine box B can be used simultaneously, or medicine box A can be used first and then medicine box B, or medicine box B can be used first and then medicine box A, which can be determined according to the actual needs in specific applications.

[0085] In this application, unless otherwise specified, the scientific and technical terms used in this application have the meanings commonly understood by those skilled in the art. And the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used in this application are all conventional steps widely used in the corresponding fields. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0086] In the present application, the term "variable" generally refers to the fact that certain parts of the sequence of the variable domain of an antibody vary strongly, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the entire variable region of the antibody. It is concentrated in three segments in the variable regions of the light and heavy chains, which are called complementarity-determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts in the variable domain are called framework regions (FWRs). The variable domains of natural heavy and light chains each contain four FWR regions, mostly adopting a β-sheet configuration, connected by three CDRs, forming loop connections and, in some cases, part of the β-sheet structure. The CDRs in each chain are brought close together by the FWR regions and, together with the CDRs from the other chain, form the antigen-binding site of the antibody. The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity of the antibody.

[0087] The three-letter and single-letter codes for amino acids used in the present application are as known to those skilled in the art or as described in J. Biol. Chem, 243, p3558 (1968).

[0088] As used in the present application, the term "comprising" or "including" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements, and depending on the context, also includes the case of "consisting of...".

[0089] The term "antibody" as described in the present application may include immunoglobulins, which are four-peptide chain structures composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulin heavy chains are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains according to the differences in their constant regions. Each of the five classes of Ig can have κ chains or λ chains.

[0090] In the present application, the variable region of the antibody light chain as described in the present application may further contain a light chain constant region, and the light chain constant region contains human κ, λ chains or variants thereof. In the present application, the variable region of the antibody heavy chain as described in the present application may further contain a heavy chain constant region, and the heavy chain constant region contains human IgG1, 2, 3, 4 or variants thereof.

[0091] Within the light and heavy chains, the variable and constant regions are joined 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 the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly and are the variable regions (V regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions (C regions). The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FWRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FWR regions, and the order from the amino-terminus to the carboxyl-terminus is: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The three CDR regions of the light chain refer to VL CDR1, VL CDR2, and VL CDR3; the three CDR regions of the heavy chain refer to VH CDR1, VH CDR2, and VH CDR3.

[0092] The term "human antibody" includes antibodies having variable and constant regions with human germline immunoglobulin sequences. The human antibodies of the present application may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto a human framework sequence (i.e., "humanized antibodies").

[0093] As used herein, the term "specificity" with respect to an antibody means an antibody that recognizes a specific antigen but substantially does not recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind that antigen from one or more species. However, this interspecies cross-reactivity by itself does not change the classification of the antibody according to specificity. In another example, an antibody that specifically binds an antigen may also bind different allelic forms of that antigen. However, this cross-reactivity by itself does not change the classification of the antibody according to specificity. In some cases, the terms "specific" or "specifically binds" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody generally recognizes and binds to a specific protein structure, rather than to the protein. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0094] As used in this application, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificities of the parental antibody. Generally, when activities are expressed on a molar basis, antibody fragments retain at least 10% of the parental binding activity. Preferably, antibody fragments retain at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more of the parental antibody's binding affinity for the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0095] As used herein, the term "chimeric antigen receptor" or "CAR" refers to: a polypeptide comprising an extracellular domain (extracellular binding domain) capable of binding an antigen, a hinge domain, a transmembrane domain (transmembrane region), and a cytoplasmic signaling domain (i.e., intracellular signaling domain). The hinge domain can be considered as a part for providing flexibility to the extracellular antigen-binding region. The intracellular signaling domain refers to a protein that transmits information into the cell through a defined signaling pathway by generating second messengers to regulate cell activity, or a protein that functions as an effector in response to such messengers, generating signals that can promote the immune effector function of cells expressing the CAR (e.g., CAR T cells). The intracellular signaling domain contains a signaling domain and may also include a co-stimulatory intracellular domain derived from a co-stimulatory molecule.

[0096] "Identity" and "mutation" refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when two sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparison is made when the maximum percentage of identity is obtained by aligning the two sequences.

[0097] The terms "polypeptide", "peptide" and "protein" (if single-stranded) are used interchangeably herein. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably.

[0098] As used herein, the term "vector" is a composition comprising an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0099] As used in this application, the terms "cell" and "cell line" are used interchangeably, and all such names include progeny. The term "host cell" refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0100] The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polyamine-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.

[0101] As used in this application, the term "immune cell" refers to a cell that can initiate an immune response, and "immune cell" and its other grammatical forms can refer to immune cells from any source. "Immune cells" include, for example, white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells)). The term "immune cell" can also be human or non-human. For example, immune cells can be from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or can be derived from cell lines, such as NK cell lines prepared by EBV virus infection, NK cells induced from embryonic stem cells and iPSCs, and the NK92 cell line, etc.

[0102] As used in this application, the term "T cell" refers to a class of lymphocytes that mature in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes (such as B cells) by the presence of T cell receptors on the cell surface. "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Tregs) and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells and neutrophils, which are capable of mediating cytotoxic reactions. As used herein, the term "NK cell" refers to a class of lymphocytes that originate in the bone marrow and play an important role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells or other stressed cells, even in the absence of antibodies and major histocompatibility complex on the cell surface.

[0103] "Optional", "any one", "any", or "any item" means that the subsequently described event or circumstance may but does not have to occur, and this description includes the occasions where the event or circumstance occurs or does not occur. For example, "optionally comprising a variable region of an antibody heavy chain" means that a variable region of an antibody heavy chain of a specific sequence may but does not have to be present. As used in the present invention, "a" and "an" are used in the present invention to refer to one or more than one grammatical object. Unless the context clearly indicates otherwise, the term "or" is used in the present invention to mean the term "and / or" and is used interchangeably therewith. "About" and "approximately" should generally mean the acceptable degree of error of the measured quantity in view of the nature or accuracy of the measurement. Exemplary degrees of error are generally within 10% thereof and more generally within 5% thereof. The methods and compositions disclosed in the present invention cover such polypeptides and nucleic acids that have a specified sequence, a variant sequence, or a sequence that is substantially the same or similar thereto, for example, a sequence that is at least 85%, 90%, 95%, 99% or more identical to the specified sequence. In the case of an amino acid sequence, the term "substantially the same" is used in the present invention to refer to a first amino acid sequence.

[0104] As used herein, the term EC50 refers to the concentration for 50% of maximal effect, i.e., the concentration that can cause 50% of the maximal effect.

[0105] Those skilled in the art should understand that products containing the anti-CD3E / G antibodies or antigen-binding fragments thereof described in the present invention, such as CAR-T, TCR-T, bispecific antibodies, multispecific antibodies, ADCs, etc., should fall within the scope of protection of the present invention.

[0106] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0107] The reagents and raw materials used in the present invention are all commercially available.

[0108] The positive and progressive effects of the present invention are as follows:

[0109] The anti-CD3e / g antibodies or antigen-binding fragments thereof described in the present invention have high affinity, high biological activity, and high diversity. They can bind to human CD3e / g (hCD3e / g) and cynomolgus monkey CD3e / g (cCD3e / g), so that in preclinical safety assessment studies, a disease model of the primate cynomolgus monkey (cynomolgus, cyno) can be selected for pharmacology and toxicology experiments, which brings great convenience to preclinical pharmacology, toxicology and other studies. In addition, the anti-CD3e / g antibodies or antigen-binding fragments thereof described in the present invention can effectively activate the NFAT downstream signaling pathway of Jurkat cells, which is more conducive to the subsequent biological activity. Brief Description of the Drawings

[0110] Figure 1 It is a result graph of ELISA for detecting the serum titer of the immunogen in mouse serum.

[0111] Figure 2 It is a result graph of ELISA for detecting the binding of CD3e / g antibody to hCD3e / g.

[0112] Figure 3 It is a result graph of ELISA for detecting the binding of CD3e / g antibody to cyno CD3e / g.

[0113] Figure 4 It is a result graph of FACS for detecting the binding of CD3e / g antibody to human CD3 on the surface of Jurkat and primary CD3-positive T cells.

[0114] Figure 5 It is a result graph of NF-kB reporter gene assay for detecting the activation of NFAT signaling pathway by CD3 antibody. Detailed Description of the Invention

[0115] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0116] Example 1 Preparation of CD3E / G Antibody

[0117] (I) Preparation of Immunogen

[0118] CD3e / g is a soluble ligand. We designed the product of CD3e / g protein and human constant region - CD3e / g recombinant protein as the immunogen. The specific sequence of CD3e / g protein is shown in Table 1.

[0119] Table 1 Immunogen Sequence

[0120]

[0121] (II) Preparation of Hybridoma Cells and Antibody Screening

[0122] The above immunogen was used to immunize SJL mice aged 6 - 8 weeks (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.), and the mice were raised under SPF conditions. At the first immunization, the immunogen was emulsified with Freund's complete adjuvant and 0.25 ml was injected into the tail vein end, that is, 50 μg of immunogen was injected into each mouse. At the booster immunization, the immunogen was emulsified with Freund's incomplete adjuvant and 0.25 ml was injected into the tail vein end, that is, 50 μg of immunogen was injected into each mouse. The interval between the first immunization and the first booster immunization was 2 weeks, and the interval between each subsequent booster immunization was 3 weeks. Blood was collected 1 week after each booster immunization, and the antibody titer and specificity of the immunogen in the serum were detected by ELISA (the experimental steps were the same as those in Example 3A, only the samples were different). The results were as Figure 1 and Table 2 showed. From Figure 1 and Table 2, it can be seen that the sera of the mice immunized with the CD3e / g recombinant protein had different degrees of binding to the immunogen, showing an antigen - antibody reaction, and the highest dilution was about one thousand. The blank control was 1% (w / w) BSA, the batch refers to the sera of the mice on the seventh day after the second booster immunization, and the data in the table are OD450nm values.

[0123] Table 2 Antibody titers of sera of mice immunized with CD3e / g recombinant protein detected by ELISA

[0124]

[0125] Before the completion of the immunization procedure, 100 μg of CD3e / g recombinant protein was intraperitoneally injected into each selected mouse at the last immunization. The mice were sacrificed 3 days later, and spleen cells were collected. NH4OH was added to a final concentration of 1% (w / w) to lyse the red blood cells mixed in the spleen cells, and a spleen cell suspension was obtained. The cells were washed 3 times by centrifugation at 1000 revolutions per minute with DMEM basal medium, and then mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC) at a ratio of 5:1 by the number of live cells. Cell fusion was carried out using a high - efficiency electrofusion method (see METHODS IN ENZYMOLOGY, VOL.220). The fused cells were diluted into DMEM medium containing 20% fetal bovine serum and 1×HAT, and the percentages are mass percentages. Then, they were added to a 96 - well cell culture plate at a density of 1×10 5 / 200 μl per well and placed in an incubator with 5% CO2 at 37 °C, and the percentage is volume percentage. The fused cells were diluted into DMEM medium containing 20% fetal bovine serum and 1×HAT, and the percentages are mass percentages. Then, they were added to a 96 - well cell culture plate at a density of 1×10 5Add 200 μL per well to a 96-well cell culture plate and place it in an incubator with 5% CO2 at 37 °C, where the percentage is by volume. After 14 days, screen the supernatant of the cell fusion plate with an ELISA (microporous plate protein detection method) plate coated with antigen CD3e / g. Amplify the positive clones with a ratio greater than 2 in the ELISA result to a 24-well plate and expand the culture in DMEM (invitrogen) containing 10% (w / w) HT fetal bovine serum at 37 °C and 5% (v / v) CO2. After 3 days of culture, centrifuge the culture medium in the 24-well plate for expansion, collect the supernatant, and perform antibody subtype analysis on the supernatant. Use ELISA and FACS to determine the binding activity to the antigen (for the detection methods of binding activity, please refer to Example 3A and Example 3B respectively).

[0126] According to the screening results of the 24-well plate, select the hybridoma cells with an OD value > 2 in the ELISA experiment as the qualified positive clones. Select the qualified hybridoma cells and perform subcloning in a 96-well plate by the limiting dilution method, and culture them in DMEM medium containing 10% (w / w) FBS (purchased from invitrogen) at 37 °C and 5% (v / v) CO2. Ten days after subcloning, perform preliminary screening with ELISA, and select individual positive monoclonal clones and amplify them to a 24-well plate for continuous culture. After 3 days, determine the antigen-binding positivity with ELISA and evaluate the biological activity with the VEGFR receptor ligand binding experiment (the evaluation criterion is an OD value > 2 in the ELISA experiment).

[0127] According to the sample detection results of the 24-well plate, select the optimal clone, and expand the culture of the optimal clone in DMEM medium containing 10% (w / w) FBS (purchased from invitrogen) at 37 °C and 5% (v / v) CO2, and store it in liquid nitrogen to obtain the hybridoma cells of the present invention, which can be used for subsequent antibody production and purification.

[0128] Example 2 Production and Purification of Lead Antibody

[0129] The antibody concentration produced by hybridoma cells is relatively low, only about 1 - 10 μg / mL, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the medium and the components of fetal bovine serum contained in the medium interfere with many biological activity analysis methods to varying degrees. Therefore, small-scale (1 - 5 mg) antibody production and purification are required.

[0130] The hybridoma cells obtained in Example 1 were inoculated into a T-75 cell culture flask and passaged for 3 generations by acclimation using a production medium (Hybridoma serum free medium, purchased from Invitrogen). When their growth state was good, the cells were inoculated into a cell culture spinner flask. 500 mL of the production medium was added to each 2-L spinner flask, and the inoculation cell density was 1.0×10 5 / mL. The bottle cap was tightened, and the spinner flask was placed on a spinner in an incubator at 37°C, with a rotation speed of 3 revolutions per minute. After continuous rotational culture for 14 days, the cell culture fluid was collected, the cells were removed by filtration, and the culture supernatant was filtered through a 0.45-μm filter membrane until it was clear. The clear culture supernatant could be immediately purified or stored frozen at -30°C.

[0131] The monoclonal antibody in the clear culture supernatant (300 mL) of the hybridoma cells was purified using a 2-mL protein A column (purchased from GE Healthcare). The protein A column was first equilibrated with an equilibration buffer (PBS phosphate buffer, pH 7.2), and then the clear culture supernatant was loaded onto the protein A column, controlling the flow rate at 3 mL / min. After loading, the protein A column was washed with the equilibration buffer, and the volume of the equilibration buffer was 4 times the bed volume of the protein A column. The CD3E / G antibody bound to the protein A column was eluted with an elution buffer (0.1 M glycine hydrochloride buffer, pH 2.5), and the elution was monitored using an ultraviolet detector (A280 ultraviolet absorption peak). The eluted antibody was collected, 10% 1.0 M Tris-HCl buffer was added to neutralize the pH (the percentage is by volume), and then it was immediately dialyzed overnight against PBS phosphate buffer. The solution was changed once the next day and dialysis continued for 3 hours. The dialyzed CD3e / g antibody was collected and sterile filtered using a 0.22-μm filter, and stored aseptically to obtain the purified CD3e / g antibody.

[0132] The purified CD3e / g antibody was subjected to protein concentration (A280 / 1.4), purity, and other detection and analysis, and the results are shown in Table 3.

[0133] Table 3 Detection and analysis of the purified CD3e / g antibody

[0134]

[0135] Example 3 Verification of the lead antibody

[0136] A. Detection of antigen-antibody binding sites by enzyme-linked immunosorbent assay (ELISA)

[0137] The purified CD3e / g antibody obtained in Example 2 was subjected to a binding reaction with the CD3e / g protein (the sequence is shown in Table 1).

[0138] Dilute human CD3e / g protein or cynomolgus CD3e / g protein (sequences are shown in Table 1) with PBS to a final concentration of 5.0 μg / mL, and then add 100 μL per well to a 96-well ELISA plate. Seal it with plastic wrap and incubate overnight at 4°C. The next day, wash the plate twice with wash buffer [PBS + 0.01% (v / v) Tween 20], and add blocking buffer [PBS + 0.01% (v / v) Tween 20 + 1% (w / w) BSA] to block at room temperature for 2 hours. Pour out the blocking buffer, and add 100 μL of the purified CD3e / g antibody obtained in Example 2 per well. After incubating at 37°C for 2 hours, wash the plate 3 times with wash buffer [PBS + 0.01% (v / v) Tween 20]. Add HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma), and after incubating at 37°C for 2 hours, wash the plate 3 times with wash buffer [PBS + 0.01% (v / v) Tween 20]. Add 100 μL of TMB substrate per well, and after incubating at room temperature for 30 minutes, add 100 μL of stop solution (1.0 N HCl) per well. Read the A450nm value with an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device), Figure 2 and Table 4 show the result graph of ELISA detecting the binding of CD3e / g antibody to hCD3e / g, Figure 3 and Table 5 show the result graph of ELISA detecting the binding of CD3e / g antibody to cynoCD3e / g, where the IgG control is mouse IgG, and the data in the table are OD450nm values. Among them, HIT3a is purchased from BD pharmigen, Lot number is 555336; SP34 is purchased from BD pharmigen, Lot number is 551916. One kind of OKT3 is purchased from Biolegend, and the other is self-made by WiseChemistry, and the sequences of its heavy and light chains are shown in SEQ ID NO:100 and SEQ ID NO:101 respectively.

[0139] From Figure 2 and 3 Tables 4 and 5, it can be seen that the antibody of the present invention can bind to human CD3 (hCD3e / g), and the binding affinity of the antibody of the present invention to human CD3e / g is better than that of the existing similar antibody OKT3. At the same time, the antibody of the present invention can bind to cynomolgus CD3 (cyno CD3e / g), while the existing similar antibody OKT3 does not have the binding affinity to cyno CD3e / g or has poor binding.

[0140] Table 4 ELISA detection of the binding of CD3e / g antibody to hCD3e / g

[0141]

[0142]

[0143] Table 5 Binding of anti-CD3e / g antibody to cCD3e / g detected by ELISA

[0144]

[0145] B. Detection of antigen-antibody binding sites by flow cytometry (FACS)

[0146] The purified anti-CD3e / g antibody obtained in Example 2 was subjected to a binding reaction with cell surface CD3e / g (the sequence is shown in Table 1).

[0147] In the present invention, two types of cells endogenously expressing CD3 were used for the binding experiment. One is Jurkat, a human T cell lymphoma, and the other is CD3-positive T cells isolated from human peripheral monocytes. These two types of cells were expanded in a T-75 cell culture flask to 3×10 6 / ml. The culture medium was aspirated, and the cells were washed twice with PBS buffer (purchased from Invitrogen). After cell counting, the cells were diluted with PBS buffer to 2×10 6 cells per milliliter. 1% goat serum blocking solution was added, and the percentage is the mass percentage. The cells were incubated on ice for 30 minutes and then centrifuged and washed twice with PBS buffer. The collected cells were suspended in FACS buffer (PBS + 1% BSA, the percentage is the mass percentage) to 2×10 6 cells / mL. 100 μL of the cells were added to each well of a 96-well FACS reaction plate, and 100 μL of the purified anti-CD3 antibody test sample obtained in Example 2 was added to each well. The cells were incubated on ice for 2 hours. The cells were centrifuged and washed twice with FACS buffer, 100 μL of fluorescent (Alexa 488) labeled secondary antibody (purchased from Invitrogen) was added to each well, and the cells were incubated on ice for 1 hour. The cells were centrifuged and washed three times with FACS buffer, and then the cells were suspended in 100 μL of FACS buffer. The results were detected and analyzed by FACS (FACS Calibur, purchased from BD). The results are as Figure 4 shown, where the IgG control is mouse IgG (mIgG).

[0148] The results showed that these antibodies could bind to human CD3 on the surface of Jurkat and primary CD3-positive T cells.

[0149] C. Detection of activation of the NFAT signaling pathway by anti-CD3 antibody using the NF-κB reporter gene assay

[0150] The plasmid pGL4.30 [luc2P / NFAT-RE / Hygro] (purchased from Promega) was transfected into Jurkat cells and cultured in RPMI1640 medium containing hygromycin and 10% fetal bovine serum to screen for the Jurkat cell line stably expressing NFAT RE-luciferase (hereinafter referred to as Jurkat-NFAT Luc).

[0151] A 96-well plate (purchased from Perkin Elmer) was pre-coated with 10 μg / ml variable region fragment of goat anti-mouse antibody and incubated overnight at 4°C. It was washed three times with PBS before use. The diluted antibody was added to the 96-well plate and incubated at 37°C for 30 minutes. Then, 1×10 5 / well of Jurkat-NFAT Luc was added and co-incubated for 5 hours, and the luciferase content was measured using the One-Glo Luciferase Assay System (purchased from Promega).

[0152] The results were as Figure 5 shown, and the said antibody could significantly activate the NFAT signaling pathway, and its activation ability was comparable to that of OKT3.

[0153] Example 4 Determination of Variable Region Amino Acid Sequence

[0154] Total RNA isolation: After the supernatant obtained from the subclonal culture in Example 1 was tested for antigen binding (i.e., after the verification and activity determination in Examples 2 to 3), 5×10 7 hybridoma cells were collected by centrifugation, 1 mL of Trizol was added, mixed well and transferred to a 1.5 mL centrifuge tube, and left standing at room temperature for 5 minutes; 0.2 mL of chloroform was added, shaken for 15 seconds, left standing for 2 minutes, and then centrifuged at 4°C, 12,000 g for 5 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube; 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed. After leaving standing at room temperature for 10 minutes, it was centrifuged at 4°C, 12,000 g for 15 minutes, and the supernatant was discarded; 1 mL of 75% ethanol (the percentage is volume percentage) was added to gently wash the precipitate. After centrifugation at 4°C, 12,000 g for 5 minutes, the supernatant was discarded, the precipitate was air-dried, and dissolved in DEPC-treated H2O (promoted by a 55°C water bath for 10 minutes) to obtain total RNA.

[0155] Reverse Transcription and PCR: Take 1 μg of total RNA, prepare a 20 μl reaction system, add reverse transcriptase and react at 42 °C for 60 minutes, and then react at 7 °C for 10 minutes to terminate the reaction. Prepare a 50 μl PCR system, including 1 μl of cDNA, 25 pmol of each primer, 1 μl of DNA polymerase and the corresponding buffer system, 250 μmol of dNTPs; set the PCR program, pre-denature at 95 °C for 3 minutes, denature at 95 °C for 30 seconds, anneal at 55 °C for 30 seconds, extend at 72 °C for 35 seconds, and then extend at 72 °C for an additional 5 minutes after 35 cycles to obtain the PCR product. The kit used for reverse transcription is PrimeScript RT Master Mix, purchased from Takara, catalog number RR036; the kit used for PCR includes Q5 Ultra-Fidelity Enzyme, purchased from NEB, catalog number M0492.

[0156] Cloning and Sequencing: Take 5 μl of the PCR product for agarose gel electrophoresis detection, and purify the positive samples detected using a column recovery kit. The recovery kit is Gel&PCR Clean-up, purchased from MACHEREY-NAGEL, catalog number 740609. Perform a ligation reaction: 50 ng of the sample, 50 ng of the T vector, 0.5 μl of ligase, 1 μl of buffer, and a reaction system of 10 μl. React at 16 °C for half an hour to obtain the ligation product. The kit for ligation is T4 DNA Ligase, purchased from NEB, catalog number M0402; take 5 μl of the ligation product and add it to 100 μl of competent cells (Ecos 101 competent cells, purchased from Yeastern, catalog number FYE607), incubate on ice for 5 minutes, then heat shock in a 42 °C water bath for 1 minute, put it back on ice for 1 minute, and then add 650 μl of antibiotic-free SOC medium. Resuscitate at 37 °C on a shaker at a speed of 200 RPM for 30 minutes. Take out 200 μl and spread it on an LB solid medium containing antibiotics and incubate overnight at 37 °C in an incubator; the next day, prepare a 30 μl PCR system using primers M13F and M13R on the T vector for colony PCR. Dip a pipette tip into the colony and pipette in the PCR reaction system, and aspirate 0.5 μl and spot it on another LB solid culture dish containing 100 nM ampicillin to preserve the strain; after the PCR reaction is completed, take out 5 μl for agarose gel electrophoresis detection, and sequence and analyze the positive samples [see Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)]. The sequencing results are shown in Table 6-1, Table 6-2, and Table 7.

[0157] Table 6-1 CD3e / g Antibody Protein CDR Sequence Numbers (Taking Kabat Definition Rules as an Example)

[0158]

[0159] Table 6-2 CD3e / g Antibody Protein CDR Sequence Numbers (Taking Kabat Definition Rules as an Example)

[0160]

[0161]

[0162] The following Table 7 shows the optional framework region sequence combinations of the present invention.

[0163] Table 7 Framework Region Sequence Combinations (Taking Kabat Definition Rules as an Example)

[0164]

[0165] The light chain variable region and heavy chain variable region of the antibody obtained by the present invention are shown in the following Table 8.

[0166] Table 8 CD3E / G Antibody Gene (DNA) Sequence Numbers

[0167]

[0168]

[0169] After obtaining the light and heavy chain variable domain sequences encoding the antibody molecule, conventional recombinant DNA techniques can be used to fuse and express the light and heavy chain variable domain sequences with the corresponding murine or human antibody light and heavy chain constant domain sequences to obtain a recombinant antibody molecule.

[0170] It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. SEQUENCE LISTING <110> Shanghai RAAS Chemical Co., Ltd. <120> Antibody Targeting CD3e / g or Its Antigen-Binding Fragment, Preparation and Application <130> P20013309C <160> 101 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 (mAb001 / 003) <400> 1 Asn Tyr Tyr Met His 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 (mAb002) <400> 2 Thr Phe Gly Met Gly Val Gly 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 (mAb004 / 006) <400> 3 Asn Tyr Trp Met His 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 (mAb007) <400> 4 Asp Asn Tyr Ile His 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 (mAb008) <400> 5 Asp Tyr Thr Val Asn 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 (mAb009-012) <400> 6 Asp Tyr Thr Ile His 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb001 / 003) <400> 7 Trp Thr Tyr Pro Gly Asn Asn Asn Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb002) <400> 8 His Ile Trp Trp Asp Asp Glu Tyr Tyr Asn Pro Val Leu Lys Ser 1 5 10 15 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb004 / 006) <400> 9 Tyr Ile Asn Pro Ser Ser Gly Tyr Ile Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb007) <400> 10 Trp Ile Tyr Pro Gly Ser Val Asn Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb008) <400> 11 Tyr Ile Asn Pro Phe Asn Ser Tyr Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb009) <400> 12 Tyr Ile Asn Pro Phe Ser Asp Tyr Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb010) <400> 13 Tyr Ile Asn Pro Tyr Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb011) <400> 14 Tyr Ile Asn Pro Phe Asn Asp Tyr Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 (mAb012) <400> 15 Tyr Ile Asn Pro Tyr Ser Asp Tyr Thr Lys Tyr Asn Gln Arg Phe Lys 1 5 10 15 Asp <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb001 / 003) <400> 16 Asp Gly Tyr Gly Tyr Tyr Phe Phe Asp Tyr 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb002) <400> 17 Ile Val Arg Tyr Gly Ser Ser Leu Asp Tyr 1 5 10 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb004 / 006) <400> 18 Asp Asn Ser Gly Gln Tyr Tyr Phe Asp Tyr 1 5 10 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb007) <400> 19 Asp Ile Ser Arg Tyr Tyr Phe Asp Tyr 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb008 / 010 / 012) <400> 20 Ser Val Ser Thr Tyr 1 5 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb009) <400> 21 Ser Val Ser Ile Tyr 1 5 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 (mAb011) <400> 22 Ser Val Ser Leu Tyr 1 5 <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 (mAb001 / 003) <400> 23 Lys Ser Ser Gln Ser Leu Leu Asn Asn Arg Thr Arg Lys Asn Tyr Leu 1 5 10 15 Ala <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 (mAb002 / 004 / 006 / 007) <400> 24 Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu 1 5 10 15 Ala <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 (mAb008) <400> 25 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 (mAb009 - 011) <400> 26 Lys Ser Ser Gln Ser Leu Leu Asp Gly Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 (mAb012) <400> 27 Lys Ser Ser Gln Ser Leu Leu Asp Val Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 (mAb001-004 / 006 / 007) <400> 28 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 (mAb008-012) <400> 29 Leu Val Ser Lys Leu Asn Ser 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 (mAb001-004 / 006) <400> 30 Lys Gln Ser Tyr Thr Leu Arg Thr 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 (mAb007) <400> 31 Lys Gln Ser Phe Ile Leu Arg Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 (mAb008-012) <400> 32 Trp Gln Gly Thr His Phe Pro Arg Thr 1 5 <210> 33 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Ala Ala Leu Ala Glu Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile 20 25 30 <210> 34 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 34 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 35 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 35 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 36 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 36 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 37 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 37 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Thr Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 38 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 38 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr 20 25 30 <210> 39 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 39 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 <210> 40 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FWR1 <400> 40 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr 20 25 30 <210> 41 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> VH FWR2 <400> 41 Trp Ile Arg Gln Pro Ser Gly Lys Ser Leu Glu Trp Leu Ala 1 5 10 <210> 42 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> VH FWR2 <400> 42 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Gly Ile Gly 1 5 10 <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> VH FWR2 <400> 43 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 44 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 44 Lys Ala Thr Leu Thr Ala Asp Lys Pro Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 45 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 45 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Ile Gln 1 5 10 15 Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Asn 20 25 30 <210> 46 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 46 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys Ala Asn 20 25 30 <210> 47 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 47 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Asn 20 25 30 <210> 48 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 48 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Asn 20 25 30 <210> 49 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 49 Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 50 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 50 Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Thr Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Ser Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg 20 25 30 <210> 51 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FWR3 <400> 51 Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu Lys 1 5 10 15 Ile Ala Thr Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 20 25 30 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH FWR4 <400> 52 Trp Gly Gln Gly Ile Ile Leu Thr Val Ser Ser 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH FWR4 <400> 53 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 54 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VL FWR1 <400> 54 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys 20 <210> 55 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VL FWR1 <400> 55 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Arg Val Thr Met Asn Cys 20 <210> 56 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VL FWR1 <400> 56 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 <210> 57 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> VL FWR2 <400> 57 Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Gln 1 5 10 15 <210> 58 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> VL FWR2 <400> 58 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 59 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VL FWR3 <400> 59 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Phe Tyr Cys 20 25 30 <210> 60 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VL FWR3 <400> 60 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys 20 25 30 <210> 61 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VL FWR3 <400> 61 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 20 25 30 <210> 62 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VL FWR3 <400> 62 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys 20 25 30 <210> 63 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VL FWR3 <400> 63 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys 20 25 30 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VL FWR4 <400> 64 Phe Gly Gly Gly Thr Lys Leu Ala Ile Lys 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VL FWR4 <400> 65 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VL FWR4 <400> 66 Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 1 5 10 <210> 67 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb004 / 006) <400> 67 Gln Val Gln Leu Gln Gln Ser Gly Ala Ala Leu Ala Glu Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asn Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Gly Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ile Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asn Ser Gly Gln Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 68 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb011) <400> 68 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Phe Asn Asp Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ser Val Ser Leu Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 69 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb008) <400> 69 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Thr Val Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Phe Asn Ser Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Asn Ser Val Ser Thr Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 70 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb012) <400> 70 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Ser Asp Tyr Thr Lys Tyr Asn Gln Arg Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ser Val Ser Thr Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 71 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb010) <400> 71 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ser Val Ser Thr Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 72 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb009) <400> 72 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Thr Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Phe Ser Asp Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Ile Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ser Val Ser Ile Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 73 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb007) <400> 73 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr Asp Asn 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Val Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ser Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Ser Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 74 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb001 / 003) <400> 74 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Thr Tyr Pro Gly Asn Asn Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Gly Tyr Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ile Ile Leu Thr Val Ser Ser 115 <210> 75 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH (mAb002) <400> 75 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Phe 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Ser Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Glu Tyr Tyr Asn Pro Val Leu 50 55 60 Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe 65 70 75 80 Leu Lys Ile Ala Thr Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ile Val Arg Tyr Gly Ser Ser Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 76 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb001 / 003) <400> 76 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Asn 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Ala Ile Lys 100 105 110 <210> 77 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb002 / 004 / 006) <400> 77 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 78 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb007) <400> 78 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Phe Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Ala Ile Lys 100 105 110 <210> 79 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb009) <400> 79 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Gly 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Gln Leu Val Ser Lys Leu Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 80 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb010 / 011) <400> 80 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Gly 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Gln Leu Val Ser Lys Leu Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 81 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb008) <400> 81 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Gln Leu Val Ser Lys Leu Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Phe Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 82 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL (mAb012) <400> 82 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Gln Leu Val Ser Lys Leu Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 83 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb009) <400> 83 caggtccaac tgcagcagtc tggggctgaa ctgacaagac ctggtgcctc agtgaagatg 60 tcctgcaagg cttctggcta cacctttact gactacacga tacactgggt caaacagaga 120 cctggacagg gtctggaatg gattggatac attaatcctt tcagtgatta tactaagtac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atacaactga acagcctgac atctgaggac tctgcagtct attactgtgc aaactccgtt 300 agtatttatt ggggccaagg caccactctc acagtctcct ca 342 <210> 84 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb001 / 003) <400> 84 caggtccagc tgcagcagtc tggacctgaa ctggtgaagc ctggggcttc agtgaagata 60 tcctgcaagg cttctgggta cagcttcacg aactactata tgcactgggt gaagcagagg 120 cctggacagg gactggagtg gattggatgg acttatcctg gaaataataa tattaagtac 180 aatgagaagt tcaagggcaa ggccacactg acggcagaca catcttccag tactgcctac 240 atgcagctca gcagcctaac atctgaggac tctgcggtct attactgtgc aagagatggg 300 tatggttact acttctttga ctactggggc caaggcatta ttctcacagt ctcctca 357 <210> 85 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb007) <400> 85 caggtccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatt 60 tcctgcaagg cttctggctc caccttcaca gacaactata tacactgggt gaagcagagg 120 cctggacagg gacttgagtg gattggatgg atttatcctg gaagtgttaa tattaagtat 180 aatgagaagt tcaaggacaa ggccacactg acggcagaca catcctccac cactgcctac 240 atgcagctca gcagcctctc atctgaggac tctgcgatct attactgtgc aagagacatc 300 tcaagatatt actttgacta ctggggccaa ggcaccactc tcacagtctc ctca 354 <210> 86 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb008) <400> 86 caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggtgcctc agtgaagatg 60 tcctgcaaga cttctggcta cacctttact gactacacgg tgaactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaatcctt tcaatagtta tactaaatac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atgcaactga gcagcctgac atctgaggac tctgcagtct atttctgtgc aaactccgtt 300 agtacttatt ggggccaagg cacaactctc acagtctcct ca 342 <210> 87 <211> 262 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb011) <400> 87 cacttttact gactacacga tacactgggt caaacagaga cctggacagg gtctggaatg 60 gattggatac attaatcctt tcaatgatta tactaagtac aatcagaagt tcaaggacaa 120 ggccacattg actgcagaca aatcctccag cacagcctac atgcaactga gcagtttgac 180 atctgaggac tctgcagtct attactgtgc aaactccgtt agtctttatt ggggccaagg 240 caccactctc acagtctctt ca 262 <210> 88 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb012) <400> 88 caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggtgcctc agtgaggatg 60 caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggtgcctc agtgaggatg 60 tcctgcaagg cttctggcta cacctttact gactacacga ttcactgggt aaaacagagg 120 tcctgcaagg cttctggcta cacctttact gactacacga ttcactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaatcctt acagtgatta tactaagtac 180 cctggacagg gtctggaatg gattggatac attaatcctt acagtggtta tactaagtac 180 aatcagaggt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctat 240 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 ttgcaactga gcagcctgac atctgacgac tctgcagtct attactgtgc aaactccgtt 300 ttgcaactga gcagcctgac atctgacgac tctgcagtct attactgtgc aaactccgtt 300 agtacctact ggggccaagg caccactctc acagtctcct ca 342 agtacctact ggggccaagg caccactctc acagtctcct ca 342 <210> 89<210> 89 <211> 342<211> 342 <212> DNA<212> DNA <213> Artificial Sequence<213> Artificial Sequence <220> <220> <223> VH (mAb010) <223> VH (mAb010) <400> 89 <400> 89 caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggtgcctc agtgaggatg 60 caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggtgcctc agtgaggatg 60 tcctgcaagg cttctggcta cacctttact gactacacga ttcactgggt aaaacagagg 120 tcctgcaagg cttctggcta cacctttact gactacacga ttcactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaatcctt acagtggtta tactaagtac 180 cctggacagg gtctggaatg gattggatac attaatcctt acagtggtta tactaagtac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aaactccgtt 300 agtacctact ggggccaagg caccactctc acagtctcct ca 342 <210> 90 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb004 / 006) <400> 90 caggtccagc tgcagcagtc tggggctgca ctggcagaac ctggggcctc agtgaagctg 60 tcctgcaagg cctctggcta cacctttatt aactactgga tgcactgggt aaaacagagg 120 cctggacagg gtctggaagg gattggatac attaatccta gcagtggtta tattaagtac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aaccctccag tacagcctac 240 atgcagttga gcagcctgac atatgaggac tctgcagtct attactgtgc aagagacaac 300 tcaggccagt actactttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 91 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> VH (mAb002) <400> 91 caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60 acttgttctt tctctgggtt ttcactgacc acttttggta tgggtgtcgg ctggattcgt 120 cagccttcag ggaagagtct ggaatggctg gcacacattt ggtgggatga tgaatactat 180 aatccagtcc tgaagagtcg gctcacaatc tccaaggata cctccaaaaa ccaggtattc 240 ctcaagatcg ccactgtaga cactgcagat actgccacat actactgtgc tcgaatagtg 300 cggtacggta gtagccttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 92 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb001 / 003) <400> 92 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60 atgagctgca aatccagtca gagtctgctc aacaatagaa cccgaaagaa ctacttggct 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggctctg ggacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca atttattact gcaagcaatc ttatactctt 300 cggaccttcg gtggaggcac caagctggca atcaaa 336 <210> 93 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb002 / 004 / 006) <400> 93 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60 atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttatactctt 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 94 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb007) <400> 94 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gagggtcact 60 atgaactgca aatccagtca gagtctcctc aacagtagaa cccgaaagaa ctacttggct 120 tggtaccagc agaagccagg gcagtctcct aaactactga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttttattctt 300 cggacgttcg gtggaggcac caagctggca atcaaa 336 <210> 95 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb008) <400> 95 gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatcc agctggtgtc taaactgaac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggaatt ttttattgct ggcaaggtac acattttcct 300 cggacgttcg gtggaggcac caagttggaa atcaaa 336 <210> 96 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb011) <400> 96 gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgca agtcaagtca gagcctctta gatggtgatg gaaagacata tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatcc agctggtgtc taaactgaac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattattgtt ggcaaggcac acattttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 97 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb009) <400> 97 gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60 gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgta agtcaagtca gagcctctta gatggtgatg gaaagacata tttgaattgg 120 atctcttgta agtcaagtca gagcctctta gatggtgatg gaaagacata tttgaattgg 120 ttattacaga ggccaggcca gtctccaaag cgcctaatcc agctggtgtc taaactgaac 180 ttattacaga ggccaggcca gtctccaaag cgcctaatcc agctggtgtc taaactgaac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggaatt tattattgtt ggcaaggcac acattttcct 300 agcagagtgg aggctgagga tttgggaatt tattattgtt ggcaaggcac acattttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 98<210> 98 <211> 336<211> 336 <212> DNA<212> DNA <213> Artificial Sequence<213> Artificial Sequence <220> <220> <223> VL (mAb010) <223> VL (mAb010) <400> 98 <400> 98 gatgttgtga tgacccagac tccgctcact ttgtcggtta ccattggaca accagcctcc 60 gatgttgtga tgacccagac tccgctcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgca agtcaagtca gagcctctta gatggtgatg gaaagacata tttgaattgg 120 atctcttgca agtcaagtca gagcctctta gatggtgatg gaaagacata tttgaattgg 120 ttgttacaga ggccaggcca gtcgccaaag cgcctaatcc agctggtgtc taaactgaac 180 ttgttacaga ggccaggcca gtcgccaaag cgcctaatcc agctggtgtc taaactgaac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 99 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL (mAb012) <400> 99 gatgttgtga tgacccagac tccgctcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgca agtcaagtca gagcctctta gatgttgatg gaaagacata tttgaattgg 120 ttgttacaga ggccaggcca gtcgccaaag cgcctaatcc agctggtgtc taaactgaat 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgaaga tttgggagtt tattattgct ggcaaggtac acattttcct 300 cggacgttcg gtggaggcac caggctggaa atcaaa 336 <210> 100 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> OKT3-VH <400> 100 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 101 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> OKT3-VL <400> 101 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn 100 105

Claims

1. An antibody or antigen-binding fragment thereof targeting CD3e / g, characterized in that, It comprises a heavy chain variable region (VH) and a light chain variable region (VL). Among them, the VH comprises the following complementary determining regions (CDRs): VH CDR1 shown by the amino acid sequence of SEQ ID NO:5; VH CDR2 shown by the amino acid sequence of SEQ ID NO:11; and VH CDR3 shown by the amino acid sequence of SEQ ID NO:

20. The VL comprises the following complementary determining regions (CDRs): VL CDR1 shown by the amino acid sequence of SEQ ID NO:25; VL CDR2 shown by the amino acid sequence of SEQ ID NO:29; and VL CDR3 shown by the amino acid sequence of SEQ ID NO:

32.

2. The anti-CD3e / g antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region (VH) further includes a heavy chain variable region framework region (VH FWR), and the light chain variable region (VL) further includes a light chain variable region framework region (VL FWR).

3. The antibody or antigen-binding fragment thereof targeting CD3e / g according to claim 2, wherein The VH FWR is a heavy chain variable region framework region of a human antibody or a murine antibody, and the VL FWR is a light chain variable region framework region of a human antibody or a murine antibody.

4. The antibody or antigen-binding fragment thereof targeting CD3e / g according to claim 2, characterized in that, The VH FWR comprises the following VH FWRs: VH FWR1 with an amino acid sequence shown by any one of SEQ ID NO:33 - 40, VH FWR2 with an amino acid sequence shown by any one of SEQ ID NO:41 - 43, VH FWR3 with an amino acid sequence shown by any one of SEQ ID NO:44 - 51, and VH FWR4 with an amino acid sequence shown by SEQ ID NO:52 or 53; the VL FWR comprises the following VL FWRs: VL FWR1 with an amino acid sequence shown by any one of SEQ ID NO:54 - 56, VL FWR2 with an amino acid sequence shown by SEQ ID NO:57 or 58, VL FWR3 with an amino acid sequence shown by any one of SEQ ID NO:59 - 63, and VL FWR4 with an amino acid sequence shown by any one of SEQ ID NO:64 - 66.

5. The anti-CD3e / g antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The amino acid sequence of the VH is shown by SEQ ID NO:69, and the amino acid sequence of the VL is shown by SEQ ID NO:

81.

6. The anti-CD3e / g antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein The nucleotide sequence of the VH is shown by SEQ ID NO:86, and the nucleotide sequence of the VL is shown by SEQ ID NO:

95.

7. The antibody or antigen-binding fragment thereof targeting CD3e / g according to any one of claims 1-4, characterized in that, The antibody or its antigen-binding fragment targeting CD3e / g further includes an antibody heavy chain constant region and an antibody light chain constant region.

8. The antibody or antigen-binding fragment thereof targeting CD3e / g according to claim 7, characterized in that, The antibody heavy chain constant region is a murine antibody heavy chain constant region or a human antibody heavy chain constant region; the antibody light chain constant region is a murine antibody light chain constant region or a human antibody light chain constant region.

9. The antibody or antigen-binding fragment thereof targeting CD3e / g according to claim 7, wherein The antibody heavy chain constant region is a human IgG1, IgG2, IgG3 or IgG4 antibody heavy chain constant region; the antibody light chain constant region is a human antibody light chain κ or λ chain constant region.

10. The anti-CD3e / g antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, It is a full-length antibody, Fab, Fab’, F(ab’)2, Fv, or a monoclonal antibody prepared from the above antibodies.

11. The anti-CD3e / g antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, It is a scFv.

12. An isolated nucleic acid encoding an anti-CD3e / g antibody or antigen-binding fragment thereof as claimed in any one of claims 1-11.

13. A recombinant expression vector comprising the isolated nucleic acid as claimed in claim 12.

14. The recombinant expression vector as claimed in claim 13, wherein the recombinant expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

15. A transformant comprising the isolated nucleic acid as claimed in claim 12 or the recombinant expression vector as claimed in claim 13 or 14.

16. The transformant as claimed in claim 15, wherein the host cell of the transformant is a prokaryotic cell and / or a eukaryotic cell.

17. The transformant as claimed in claim 16, wherein the prokaryotic cell is an E. coli cell and the eukaryotic cell is a HEK293 cell or a CHO cell.

18. The transformant as claimed in claim 17, wherein the E. coli cell is TG1 or BL21.

19. A method for preparing an anti-CD3e / g antibody or antigen-binding fragment thereof, comprising culturing the transformant as claimed in any one of claims 15-18 and obtaining the anti-CD3e / g antibody or antigen-binding fragment thereof from the culture.

20. A genetically modified cell, characterized in that, It comprises an anti-CD3e / g antibody or antigen-binding fragment thereof as claimed in any one of claims 1-11.

21. The genetically modified cell as claimed in claim 20, wherein the genetically modified cell is a eukaryotic cell.

22. The genetically modified cell as claimed in claim 20, wherein the genetically modified cell is an isolated human cell.

23. The genetically modified cell as claimed in claim 20, wherein the genetically modified cell is an immune cell.

24. The genetically modified cell as claimed in claim 23, wherein the immune cell is a T cell or an NK cell.

25. The genetically modified cell as claimed in claim 24, wherein the NK cell is the NK92 cell line.

26. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an anti-CD3e / g antibody or antigen-binding fragment thereof as claimed in any one of claims 1-11 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition as claimed in claim 26, wherein the pharmaceutical composition further comprises other anti-tumor antibodies as active ingredients.

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