CN120554509A - Antibodies or antigen-binding fragments thereof targeting CD3e/g, and their preparation and use - Google Patents

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof targeting CD3e/g, as well as their preparation and use. Disclosed herein are antibodies or antigen-binding fragments thereof targeting CD3e/g, comprising a VH and a VL, wherein the VH comprises a VH CDR1 as set forth in any of the amino acid sequences of SEQ ID NOs: 1-6, a VH CDR2 as set forth in any of the amino acid sequences of SEQ ID NOs: 7-15, and a VH CDR3 as set forth in any of the amino acid sequences of SEQ ID NOs: 16-22; and the VL comprises a VL CDR1 as set forth in any of the amino acid sequences of SEQ ID NOs: 23-27, a VL CDR2 as set forth in any of the amino acid sequences of SEQ ID NOs: 28 or 29, and a VL CDR3 as set forth in any of the amino acid sequences of SEQ ID NOs: 30-32. These antibodies are capable of binding to human or monkey CD3e/g and effectively activating the NFAT downstream signaling pathway in Jurkat cells.

Description

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

The application is a divisional application of an application patent with the application date of 2020, 9 and 27, the application number of 202011054853.5 and the application name of 'CD 3e/g targeting antibody or antigen binding fragment thereof, preparation and application thereof'.

Technical Field

The application relates to the field of biological medicine, in particular to an antibody targeting CD3e/g or an antigen binding fragment thereof, a preparation method and application thereof.

Background

Therapeutic antibodies, such as monoclonal antibodies, can be developed by a variety of techniques and pathways, including hybridoma techniques, phage display techniques, single lymphocyte gene cloning techniques, and the like. The preparation of monoclonal antibodies by hybridoma technology remains the mainstay of current methods for the preparation of therapeutic monoclonal antibodies.

Traditional hybridoma preparation techniques were established 40 years ago by Kohler AND MILSTEIN (Kohler AND MILSTEIN1975, nature 256:495), and are now widely used in the preparation and production of many related monoclonal antibodies for scientific research, diagnosis, treatment, and the like. The basic method, although extended to the present, has been varied, improved and innovated in many ways, including the use of different animal species such as transgenic animals, the introduction of electrofusion techniques, the use of highly efficient screening techniques such as ClonePix devices, etc., to make the use of hybridoma technology more diverse and more efficient. Monoclonal antibodies prepared from conventional animals such as mice and the like can be cloned by conventional molecular biology methods into antibody heavy chain variable region and light chain variable region genes, and 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, cabill et al) to greatly reduce immunogenicity in human use. Furthermore, the CDR structure of the variable region of the murine antibody can be grafted on the framework of the human antibody, so that the components of the murine antibody are reduced to below 5%, and the safety of the antibody in human body is greatly improved. This approach results in antibodies, known as humanized antibodies, and are currently the major products of the antibody drug market (U.S. Pat. No.5,225,539to 55, winter, and U.S. Pat. Nos.5,530,101;5,585,089;5,693,762 and 6,180,370to Queen et al).

Currently available antibodies to CD3e/g are OKT3, which is a trade name for CD3 monoclonal antibodies that inhibit acute rejection in organ transplantation, such as heart, kidney and liver, by blocking T cell function. T cell function usually returns to normal within one week after the end of treatment with OKT3, but the side effects of OKT3 at the time of initial use are also considerable, including OKT3 influenza-like syndrome, cytokine storm, etc. Furthermore OKT3 does not bind to cynomolgus derived CD3 (cyno CD3, CD 3), which makes it inconvenient to select primate cynomolgus in preclinical safety evaluation studies.

The difficulty in developing CD3e/g antibodies is that it is difficult to obtain a good immune response and serum titer of the transgenic mice of the antibodies, resulting in a small number of positive clones after fusion, and at present, CD3e/g antibodies have a function of Cross bioactivity with other CD3e/g antibodies except human sources. Conventional binding experiments such as cell flow cytometry (FACS) and enzyme-linked immunosorbent assay (ELISA) can screen for antibodies with affinity, but these antibodies do not necessarily affect subsequent signaling pathways and corresponding biological functions upon binding to CD3 e/g. Therefore, there is an urgent need in the art for CD3e/g antibodies that have better effects such as Cross bioactivity with CD3 from sources other than human CD3, without affecting subsequent signaling pathways and corresponding biological activities.

Disclosure of Invention

The invention aims to overcome the defects of the existing CD3e/g targeting antibody, and provides the CD3e/g targeting antibody or antigen binding fragment thereof, and a preparation method and application thereof. The antibody targeting CD3e/g or the antigen binding fragment thereof has high affinity, high biological activity and high diversity, and can be combined with human CD3e/g (hCD 3 e/g) and monkey CD3e/g (cCD 3 e/g), so that a disease model of primate macaque (cynomolgus, cyno) can be selected for pharmacological and toxicological experiments in preclinical safety evaluation research, and great convenience is brought to preclinical pharmacological and toxicological researches. In addition, the antibody or antigen binding fragment thereof targeting CD3e/g can effectively activate the NFAT downstream signal path of Jurkat cells, and is more beneficial to exerting subsequent biological activity.

The difficulty in developing CD3e/g antibodies is that it is difficult to obtain a good immune response and serum titer of the transgenic mice of the antibodies, resulting in a small number of positive clones after fusion, and the current CD3e/g antibodies have a Cross bioactive function with CD3e/g other than human sources, and do not affect subsequent signaling pathways and corresponding biological activities. Conventional binding experiments such as cell flow cytometry (FACS) and enzyme-linked immunosorbent assay (ELISA) can screen for antibodies with affinity, but these antibodies do not necessarily retain subsequent signaling pathways and corresponding biological functions after binding to CD3 e/g. The inventor adopts optimized hybridoma technology based on the current monoclonal antibody technology through a large number of experiments, establishes a detection method of related biological functions, and unexpectedly obtains the CD3e/g targeting antibody with high affinity, high biological activity and high diversity.

In order to solve the above technical problem, the first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting CD3e/g, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL),

Wherein the VH comprises a Complementarity Determining Region (CDR) or a mutation thereof, such as a VH CDR1 shown in any one of the amino acid sequences of SEQ ID NOS 1-6, a VH CDR2 shown in any one of the amino acid sequences of SEQ ID NOS 7-15, and/or a VH CDR3 shown in any one of the amino acid sequences of SEQ ID NOS 16-22;

Wherein the VL comprises a Complementarity Determining Region (CDR) or a mutation thereof, a VL CDR1 as shown in any one of the amino acid sequences of SEQ ID NOS.23-27, a VL CDR2 as shown in the amino acid sequence of SEQ ID NOS.28 or 29, and/or a VL CDR3 as shown in any one of the amino acid sequences of SEQ ID NOS.30-32.

Wherein the mutation is a3, 2 or 1 amino acid insertion, deletion or substitution in the amino acid sequence of the CDR.

In the present application, "amino acid mutation" in "like" insertion, deletion or substitution with 3, 2 or 1 amino acids "means that the sequence of the variant obtained after mutation has an amino acid mutation compared with the original amino acid sequence, and includes an insertion, deletion or substitution of an amino acid on the basis of the original amino acid sequence. An exemplary interpretation is that mutations to a CDR may comprise 3, 2 or 1 amino acid mutations, and optionally the same or different numbers of amino acid residues may be selected between these CDRs for mutation, e.g., 1 amino acid mutation to CDR1, and no amino acid mutation to CDR2 and CDR 3.

In the present application, the mutations may include mutations which are currently known to those skilled in the art, for example, some mutations which may be performed on the antibody during the production or use of the antibody, for example, mutations at the site of post-transcriptional modification (Potentialpost-translational modifications, PTMs) which may exist, particularly in the CDR region, including aggregation, deamidation sensitivity (ASPARAGINE DEAMIDATION, site (NG, NS, NH, etc.), aspartic acid isomerism (DG, DP) sensitivity site, N glycosylation (N- { P } S/T) sensitivity site, oxidation sensitivity site, and the like.

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

Preferably, the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 28 and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 30, or the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 24, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 28 and the amino acid sequence of the VL 3 is shown as SEQ ID NO. 31, or the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 25, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 29 and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 32, or the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 26, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 29, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 32.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 1, 7 and 16, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 of the VL are shown in SEQ ID nos. 23, 28 and 30, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 2, 8 and 17, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 of the VL are shown in SEQ ID nos. 24, 28 and 30, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NOs 3, 9 and 18, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 of the VL are shown in SEQ ID NOs 24, 28 and 30, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 4, 10 and 19, respectively, and the amino acid sequences of the VL CDR1, VLCDR2 and VLCDR3 of the VL are shown in SEQ ID nos. 24, 28 and 31, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 5, 11 and 20, respectively, and the amino acid sequences of the VL CDR1, VLCDR2 and VLCDR3 of the VL are shown in SEQ ID nos. 25, 29 and 32, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NOS 6, 12 and 21, respectively, and the amino acid sequences of the VL CDR1, VLCDR2 and VLCDR3 are shown in SEQ ID NOS 26, 29 and 32, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 6, 13 and 20, respectively, and the amino acid sequences of the VL CDR1, VLCDR2 and VL CDR3 of the VL are shown in SEQ ID nos. 26, 29 and 32, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 6, 14 and 22, respectively, and the amino acid sequences of the VL CDR1, VLCDR2 and VLCDR3 of the VL are shown in SEQ ID nos. 26, 29 and 32, respectively.

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID nos. 6, 15 and 20, respectively, and the amino acid sequences of the VL CDR1, VLCDR2 and VLCDR3 of the VL are shown in SEQ ID nos. 27, 29 and 32, respectively.

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

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

In certain embodiments, the VH FWR comprises a VH FWR1 having an amino acid sequence as shown in any one of SEQ ID NO's 33-40 or mutations thereof, a VH FWR2 having an amino acid sequence as shown in any one of SEQ ID NO's 41-43 or mutations thereof, a VH FWR3 having an amino acid sequence as shown in any one of SEQ ID NO's 44-51 or mutations thereof, and/or a VH FWR4 having an amino acid sequence as shown in SEQ ID NO's 52 or 53 or mutations thereof.

In certain embodiments, the VL FWR comprises VL FWR1 having an amino acid sequence as shown in any one of SEQ ID NOS: 54-56 or mutations thereof, VL FWR2 having an amino acid sequence as shown in any one of SEQ ID NOS: 57 or 58 or mutations thereof, VL FWR3 having an amino acid sequence as shown in any one of SEQ ID NOS: 59-63 or mutations thereof, and/or VL FWR4 having an amino acid sequence as shown in any one of SEQ ID NOS: 64-66 or mutations thereof. Wherein said mutation is a 3, 2 or 1 amino acid insertion, deletion, substitution and repetition in the amino acid sequence of said VH FWR or VL FWR.

Preferably, the amino acid sequence of the VH is shown as any one of SEQ ID NO 67-75 or mutation thereof, and the nucleotide sequence of the VH is preferably shown as any one of SEQ ID NO 83-91 or mutation thereof.

Preferably, the amino acid sequence of the VL is shown in any one of SEQ ID NOS.76-82 or mutations thereof, and the nucleotide sequence thereof is preferably shown in any one of SEQ ID NOS.92-99 or mutations thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 74 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 76 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 75 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 77 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 67 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 77 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 73 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 78 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 69 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 81 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 72 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 79 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 71 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 80 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 68 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 80 or a mutation thereof.

In a preferred embodiment, the amino acid sequence of the VH is shown as SEQ ID NO. 70 or a mutation thereof, and the amino acid sequence of the VL is shown as SEQ ID NO. 82 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 84 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 92 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 91 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 93 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 90 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 93 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 85 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 94 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 86 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 95 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 83 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 97 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 89 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 98 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 87 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 96 or a mutation thereof.

In a preferred embodiment, the nucleotide sequence of the VH is shown as SEQ ID NO. 88 or a mutation thereof, and the nucleotide sequence of the VL is shown as SEQ ID NO. 99 or a mutation thereof.

The mutation is a deletion, substitution or addition of one or more amino acid residues on the amino acid sequence of the VH and/or VL, and the mutated 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, wherein 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.

In the present application, the amino acid sequences of the CDRs listed above are all shown according to the Kabat definition rules (the sequences shown according to the Kabat definition rules are also in the claims of the present application). However, it is well known to those skilled in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Kabat definition rules based on sequence variability (see, kabat et al, protein sequences in immunology, fifth edition, national institutes of health, besseda, maryland (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))、, chothia definition rules based on the location of structural loop regions (see, jmol Biol 273:927-48,1997) and AbM definition rules (trade-off between Kabat definition and Chothia methods), and the like.

It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the claimed scope of the present invention is based on the sequences shown by the Kabat definition rules, amino acid sequences corresponding to the definition rules according to other CDRs should also fall within the scope of the present invention.

Preferably, the CD3 e/g-targeting antibody or antigen binding fragment thereof 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 mouse-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and the antibody light chain constant region is a mouse-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 antibody heavy chain constant region, such as a human IgG1, igG2, igG3, or IgG4 antibody heavy chain constant region, and the antibody light chain constant region is a human antibody light chain kappa or lambda chain constant region.

Preferably, the CD3 e/g-targeting antibody or antigen binding fragment thereof is a full length antibody, fab ', F (ab ') 2, fv, scFv, bispecific antibody, multispecific antibody, single domain antibody, or any other antibody that retains the antibody's partial ability to specifically bind to an antigen, or a monoclonal or polyclonal antibody made from the above antibodies. Or when comprising framework regions of human antibody variable regions, are typically humanized antibodies.

The full length antibody may be a full length antibody conventional in the art comprising 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, and the like.

The single chain antibody (scFv) may be a conventional single chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

The single domain antibody may be a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

The single region antibody may be a conventional single region antibody in the art, which includes only the heavy chain variable region.

In order to solve the above technical problem, the second aspect of the present invention provides an isolated nucleic acid encoding the CD3e/g targeting antibody or antigen binding fragment thereof according to the first aspect of the present invention.

The preparation method of the nucleic acid is a preparation method conventional in the art, and may include, for example, a step of obtaining a nucleic acid molecule encoding the above antibody by a gene cloning technique or a step of obtaining a nucleic acid molecule encoding the above antibody by a method of artificial total sequence synthesis.

It is known to those skilled in the art that a nucleotide sequence encoding the amino acid sequence of the above antibody may be appropriately introduced into a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide. Homologs of the polynucleotides of the invention may be prepared by substitution, deletion, or addition of one or more bases of the gene encoding the antibody sequence within a range that retains antibody activity.

In order to solve the above technical problem, the third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid according to the second aspect of the present invention.

The recombinant expression vector can be obtained by a conventional method in the art, namely, the recombinant expression vector is constructed by connecting the nucleic acid molecule of the application to various expression vectors. The expression vector is a variety of vectors conventional in the art, as long as it can accommodate the aforementioned nucleic acid molecule.

Preferably, the expression vector comprises a eukaryotic expression vector and/or a prokaryotic expression vector.

In order to solve the above technical problem, the fourth aspect of the present invention provides a transformant comprising the isolated nucleic acid according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention.

The transformant can be prepared by methods conventional in the art, for example, by transforming the above recombinant expression vector into a host cell. The host cell of the transformant is a variety of host cells conventional in the art, as long as it is capable of stably autonomously replicating the above recombinant expression vector and the nucleic acid carried thereby can be efficiently expressed. Preferably, the host cell is a prokaryotic cell, preferably an e.coli cell such as TG1, BL21 (expressing a single chain antibody or Fab antibody), and/or a eukaryotic cell, preferably a HEK293 cell or CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell, so that the preferred recombinant expression transformant of the invention can be obtained. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.

In order to solve the above technical problems, a fifth aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof targeting CD3e/g, comprising culturing the transformant according to the fourth aspect of the present invention, and obtaining the antibody or the antigen-binding fragment thereof targeting CD3e/g from the culture.

In order to solve the above technical problem, a sixth aspect of the present invention provides a chimeric antigen receptor comprising the CD3 e/g-targeting antibody or antigen binding fragment thereof according to the first aspect of the present invention.

In order to solve the above technical problem, the seventh aspect of the present invention provides a genetically modified cell comprising the CD3e/g targeting antibody or antigen binding fragment thereof according to the first aspect of the present invention, preferably a eukaryotic cell, more preferably an isolated human cell, even more preferably an immune cell such as a T cell, or an NK cell such as an NK92 cell line.

In order to solve the above technical problem, the eighth aspect of the present invention provides an antibody drug conjugate comprising a cytotoxic agent and the CD3e/g targeting antibody or antigen binding fragment thereof according to the first aspect of the present invention.

In order to solve the above technical problem, the ninth aspect of the present invention provides a pharmaceutical composition comprising the CD3 e/g-targeting antibody or antigen binding fragment thereof according to the first aspect of the present invention, and a pharmaceutically acceptable carrier.

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

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

The pharmaceutically acceptable carrier may be any carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the antibody or antigen binding fragment thereof and/or antibody drug conjugate and the like, and 0.01-99.99% of a medicinal carrier, wherein the percentages are mass percentages of the pharmaceutical composition.

The route of administration of the pharmaceutical composition according to the invention is preferably parenteral, injectable or oral.

The administration by injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, i.e. in the form of an aqueous solution, non-aqueous solution or suspension, more preferably in the form of tablets, capsules, granules, injections or infusions, etc. More preferably via intravascular, subcutaneous, intraperitoneal or intramuscular administration. Preferably, the pharmaceutical composition may also be administered as an aerosol or a coarse spray, i.e. nasally, or intrathecally, intramedullary or intraventricular. More preferably, the pharmaceutical composition may also be administered transdermally, topically, enterally, intravaginally, sublingually or rectally. The pharmaceutical composition of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by the physician according to the type, age, weight and general disease condition of the patient, the administration mode, etc. The administration may be, for example, injection or other therapeutic means.

The dosage level of the pharmaceutical composition of the present invention may be adjusted depending on the amount of the composition that achieves the desired diagnostic or therapeutic result. The administration regimen may also be single or multiple injections, or may be modified. The dosage level and regimen selected is reasonably adjusted depending on various factors including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the route of administration, the combination with other drugs or treatments, the disease or condition to be detected and/or treated, and the health and prior medical history of the subject to be treated.

The therapeutically effective dose for the pharmaceutical composition of the invention may be estimated initially 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 concentration ranges and routes of administration. And can then be used to determine useful dosages and routes of administration in humans. In general, the determination and adjustment of the administration of an effective amount or dose, and the assessment of when and how such adjustment is made, are known to those skilled in the art.

For combination therapy, the above-described antibodies or antigen-binding fragments thereof, the above-described antibody drug conjugates, and the like, and/or additional therapeutic or diagnostic agents, may each be used as a single agent, within any time frame suitable for performing the intended therapy or diagnosis. Thus, these single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially and continuously.

Additional guidance regarding formulations, dosages, administration regimens, and measurable therapeutic results is provided by Berkow et al (2000) The Merck Manual of Medical Information (Merck medical information handbook) and Merck&Co.Inc.,Whitehouse Station,New Jersey;Ebadi(1998)CRC Desk Reference of Clinical Pharmacology( clinical pharmacology handbook), among others.

In order to solve the technical problem, the tenth aspect of the present invention provides the use of the CD3e/g targeting antibody or antigen binding fragment thereof according to the first aspect of the present invention, the chimeric antigen receptor according to the sixth aspect of the present invention, the genetically modified cell according to the seventh aspect of the present invention, the antibody drug conjugate according to the eighth aspect of the present invention, and the pharmaceutical composition according to the ninth aspect of the present invention for preparing a medicament for diagnosing, preventing and/or treating tumor.

Preferably, the tumor includes, but is not limited to, colorectal cancer, lung cancer, breast cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, pancreatic cancer, and/or lymphoma, such as adult T-cell leukemia lymphoma (ATLL), acute Myelogenous 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), and the like.

In order to solve the technical problem, the invention also provides a kit which comprises the antibody or the antigen binding fragment thereof according to the first aspect of the invention, the chimeric antigen receptor according to the sixth aspect of the invention, the genetically modified cell according to the seventh aspect of the invention, the antibody drug conjugate according to the eighth aspect of the invention and the drug composition according to the ninth aspect of the invention, and preferably further comprises a device for applying the antibody or the antigen binding fragment thereof or the antibody drug conjugate or the drug composition, further preferably further comprises a use instruction and the like.

In order to solve the technical problem, the invention also provides a method for detecting CD3e/g in a sample, which comprises detecting by using the antibody or antigen binding fragment thereof targeting CD3e/g according to the first aspect of the invention.

In order to solve the technical problems, the invention also provides application of the CD3e/g targeting antibody or antigen binding fragment thereof, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate and/or the pharmaceutical composition in diagnosis, prevention and/or treatment of cancers. Preferably, the cancer is as described in the tenth aspect of the invention.

In order to solve the above technical problems, the present invention further provides a kit comprising a kit a and a kit B, wherein the kit a comprises the CD3e/g targeting antibody or antigen binding fragment thereof, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate, and the pharmaceutical composition, and the kit B is another anti-cancer (tumor) antibody or a pharmaceutical composition comprising the other anti-cancer (tumor) antibody. The medicine box A and the medicine box B can be used simultaneously, the medicine box A can be used first and then the medicine box B can be used, the medicine box B can be used first and then the medicine box A can be used according to the actual requirements in specific application.

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used in the present application are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

In the present application, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domain of an antibody vary strongly, which results in the binding and specificity of various specific antibodies for their specific antigens. However, variability is not evenly distributed throughout the variable regions of antibodies. It focuses on three segments in the light and heavy chain variable regions, known as Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts of the variable domain are called the Framework (FWR). The variable domains of the natural heavy and light chains each comprise four FWR regions, mostly in a β -sheet configuration, connected by three CDRs, forming a loop connection, and in some cases forming part of a β -sheet structure. The CDRs in each chain are in close proximity by the FWR region and together with CDRs from the other chain form the antigen binding site of the antibody, and the constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity.

The three-letter and one-letter codes for amino acids used in the present application are known to the person skilled in the art or are described in J.biol. Chem,243, p3558 (1968).

As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements, but also include the case of "consisting of.

The term "antibody" as used herein may include immunoglobulins which are four peptide chain structures made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further divided into different subclasses, e.g., igG can be divided into IgG1, igG2, igG3 and IgG4, depending on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

In the present application, the antibody light chain variable region of the present application may further comprise a light chain constant region comprising a kappa, lambda chain of human origin or variants thereof. In the present application, the antibody heavy chain variable region of the present application may further comprise a heavy chain constant region comprising human IgG1, 2, 3, 4 or variants thereof.

Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises 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 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody may mediate the binding of an 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 (C1 q). The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region), and the remaining amino acid sequences near the C-terminus are relatively stable, being the constant region (C region). The variable region includes 3 hypervariable regions (HVRs) and 4 framework regions (FWRs) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of 3 CDR regions and 4 FWR regions, which are arranged in the order of FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4 from the amino-terminus to the carboxy-terminus. The 3 CDR regions of the light chain refer to VL CDR1, VL CDR2 and VL CDR3 and the 3 CDR regions of the heavy chain refer to VH CDR1, VH CDR2 and VH CDR3.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the application may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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 human framework sequences (i.e., a "humanized antibody").

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

In the present application, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally comprise at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, fv fragments, linear antibodies (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.

The term "chimeric antigen receptor" or "CAR" as used herein refers to a polypeptide comprising an extracellular domain capable of binding an antigen (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region), and a cytoplasmic signaling domain (i.e., an intracellular signaling domain). The hinge domain may be considered to be part of a mechanism for providing flexibility to the extracellular antigen-binding region. Intracellular signaling domain refers to a protein that transmits information into a cell via a defined signaling pathway by generating a second messenger to modulate cellular activity, or by functioning as an effector corresponding to such a messenger, to generate a signal that can promote immune effector function of a cell of a CAR (e.g., a CART cell). The intracellular signaling domain comprises a signaling domain and may also include a costimulatory intracellular domain derived from a costimulatory molecule.

"Identity", "mutation" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is 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 percent identity between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the maximum percent identity.

The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably in the present application. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably.

The term "vector" as used herein is a composition comprising an isolated nucleic acid and useful for delivering 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 autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

The expressions "cell", "cell line" as used herein are used interchangeably and all such designations include offspring. The term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, a prokaryotic cell such as E.coli, a fungal cell such as a yeast cell, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK 293 cell, or human cell.

The term "transfection" refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics (biolistics).

The term "immune cell" refers to a cell that can elicit an immune response, and "immune cell" and grammatical variations thereof can refer to any source of immune cells. The term "immune cells" includes, for example, white blood cells (leukocytes) derived from Hematopoietic Stem Cells (HSCs) produced in 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 cells" may also be human or non-human.

As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and differ from other lymphocytes (e.g., B cells) in 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 gamma-delta T cells. "cytotoxic cells" include CD8+ T cells, natural Killer (NK) cells and neutrophils, which are capable of mediating a cytotoxic response. As used herein, the term "NK cells" refers to a class of lymphocytes that originate from 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 complexes on the cell surface.

"Optional," "any," or "any" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1 antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence may be, but is not required to be, present. As used herein, "a" and "an" are used in this disclosure to refer to one or more than one grammar object. The term "or" is used in this disclosure to mean and is used interchangeably with the term "and/or" unless the context clearly dictates otherwise. "about" and "approximately" shall generally mean an acceptable degree of error in the measured quantity in view of the nature or accuracy of the measurement. Exemplary error levels are generally within 10% thereof and more generally within 5% thereof. The methods and compositions disclosed herein encompass polypeptides and nucleic acids having a specified sequence, variant sequence, or sequence substantially identical or similar thereto, e.g., a sequence that is at least 85%, 90%, 95%, 99% or more identical to the specified sequence. In the case of amino acid sequences, the term "substantially identical" is used in the present invention to refer to the first amino acid sequence.

As used herein, the term EC50 refers to the half maximal effect concentration (concentration for 50%of maximal effect), i.e., the concentration that causes 50% of the maximal effect.

It will be appreciated by those skilled in the art that products comprising antibodies or antigen binding fragments thereof, such as CAR-T, TCR-T, diabodies, polyclonal antibodies, ADCs, etc., that target CD3E/G as described herein are intended to fall within the scope of the present invention.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

The CD3e/g targeting antibody or antigen binding fragment thereof has high affinity, high biological activity and high diversity. The kit can be combined with human CD3e/g (hCD 3 e/g) and monkey CD3e/g (cCD 3 e/g), so that a disease model of primate macaque (cyno) can be selected for pharmacological and toxicological experiments in preclinical safety evaluation research, and great convenience is brought to preclinical pharmacological and toxicological researches. In addition, the antibody or antigen binding fragment thereof targeting CD3e/g can effectively activate the NFAT downstream signal path of Jurkat cells, and is more beneficial to exerting subsequent biological activity.

Drawings

FIG. 1 is a graph showing the results of ELISA detection of serum titers against immunogens in mouse serum.

FIG. 2 is a graph showing the results of ELISA detection of binding of CD3e/g antibody to hCD3 e/g.

FIG. 3 is a graph showing the results of ELISA detection of CD3e/g antibody binding to cyno CD3 e/g.

FIG. 4 is a graph showing the results of FACS detection of binding of CD3e/g antibody to human CD3 on the surface of Jurkat and primary CD 3-positive T cells.

FIG. 5 is a graph showing the results of NF-kB reporter gene assay to detect activation of the NFAT signaling pathway by CD3 antibodies.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of CD3E/G antibodies

Preparation of (one) immunogens

CD3e/g is a soluble ligand, we designed the CD3e/g protein and human constant region product-CD 3e/g recombinant protein (CD 3e/g recombinant protein) as immunogen, CD3e/g protein specific sequence is shown in Table 1.

TABLE 1 immunogen sequences

Preparation of (II) hybridoma cells and antibody screening

The immunogen is adopted to immunize SJL mice (purchased from Shanghai Laek) with 6-8 weeks old antibodies, and the mice are bred under SPF conditions. At the time of primary immunization, the immunogen was emulsified with Freund's complete adjuvant and injected into the tail vein end with 0.25 ml, i.e., 50. Mu.g of immunogen per mouse. At boost, the immunogen was emulsified with Freund's incomplete adjuvant and injected into the tail vein end with 0.25 ml, i.e., 50. Mu.g of immunogen per mouse. The primary and primary boost were separated by 2 weeks, followed by 3 weeks between each boost. Blood was collected 1 week after each boost, and the serum was assayed for antibody titer and specificity by ELISA (experimental procedure was different from example 3A, only samples), and the results are shown in FIG. 1 and Table 2. As can be seen from FIGS. 1 and 2, the serum of mice immunized with CD3e/g recombinant protein had different degrees of binding to the immunogen, exhibiting an antigen-antibody response, with the highest dilution being around one thousand. Wherein the blank is 1% (w/w) BSA, wherein the batch refers to mouse serum from the seventh day after the second booster immunization, and the data in the table are OD450nm values.

TABLE 2ELISA detection of serum antibody titers of mice after CD3e/g recombinant protein immunization

Before the immunization step was completed, each mouse was selected and was finally immunized with 100. Mu.g of CD3e/g recombinant protein intraperitoneally, and after 3 days, the mice were sacrificed and spleen cells were collected. NH ₄ OH was added to a final concentration of 1% (w/w), and the spleen cells were lysed for the presence of the red blood cells that had been mixed with the spleen cells, to obtain a spleen cell suspension. Cells were washed 3 times per minute Zhong Lixin with DMEM basal medium 1000 and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 5:1 viable cell number, and cell fused 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, 1 xhat, the percentages being mass percentages. Then 1X 10 ⁵/200 microliters per well was added to a 96 well cell culture plate and placed in a 5% CO ₂, 37℃incubator, the percentages being by volume. The fused cells were diluted into DMEM medium containing 20% fetal bovine serum, 1 xhat, the percentages being mass percentages. Then 1X 10 ⁵/200 microliters per well was added to a 96 well cell culture plate and placed in a 5% CO ₂, 37℃incubator, the percentages being by volume. After 14 days, the cell fusion plate supernatants were screened with ELISA (microplate protein assay) plates coated with antigen CD3e/g, positive clones with a ratio of > 2 in ELISA results were amplified to 24 well plates and expanded in the presence of 10% (w/w) HT fetal bovine serum DMEM (invitrogen) at 37℃and 5% (v/v) CO ₂. After 3 days of incubation, the culture broth from the expansion culture in 24-well plates was centrifuged, the supernatant was collected, and the supernatant was subjected to antibody subtype analysis to determine the binding activity to the antigen by ELISA and FACS (for detection of binding activity see example 3A and example 3B, respectively).

According to the 24-well plate screening result, hybridoma cells with OD value of >2 in ELISA experiments are selected as positive clones meeting the conditions, subcloning is carried out on the hybridoma cells meeting the conditions in a 96-well plate by a limiting dilution method, and the hybridoma cells are cultured in DMEM medium (purchased from Invitrogen) containing 10% (w/w) FBS at 37 ℃ and 5% (v/v) CO ₂. The primary screening was performed by ELISA 10 days after subcloning, and single positive monoclonal amplifications were selected to 24 well plates for further culture. After 3 days, antigen binding positivity was determined by ELISA and bioactivity was assessed by VEGFR receptor ligand binding experiments (evaluation criteria were OD >2 in ELISA experiments).

According to the detection result of the 24-well plate sample, the optimal clone is selected, and the optimal clone is subjected to expansion culture in DMEM medium (purchased from invitrogen) containing 10% (w/w) FBS at 37 ℃ and 5% (v/v) CO ₂, so that the hybridoma cell is obtained after freezing in liquid nitrogen, and can be used for subsequent antibody production and purification.

EXAMPLE 2 production and purification of lead antibodies

The concentration of antibody produced by hybridoma cells is relatively low, about only 1-10 μg/ml, and the concentration varies greatly. And various proteins produced by cell culture in the culture medium and the fetal bovine serum components contained in the culture medium interfere with many biological activity analysis methods to different extents, so that small-scale (1-5 mg) antibody production and purification are required.

The hybridoma cells obtained in example 1 were inoculated into a T-75 cell culture flask and acclimatized with a production medium (Hybridoma serum free medium, available from Invitrogen company) for passage 3 generations. And inoculating the cell culture roller bottle after the growth state is good. 500 ml of production medium was added to each 2 l flask and the inoculated cell density was 1.0X10 ⁵/ml. The bottle cap was closed and the roller bottle was placed on a roller bottle machine in a 37 ℃ incubator at 3 revolutions per minute. After 14 days of continuous rotation culture, the cell culture broth was collected, filtered to remove cells, and filtered through a 0.45 μm filter until the culture supernatant was clear. The clarified culture supernatant may be immediately purified or frozen at-30 ℃.

Monoclonal antibodies in the culture supernatant (300 mL) of clarified hybridoma cells were purified using a 2mL protein a column (purchased from GE HEALTHCARE). The protein A column was equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2) and the clarified culture supernatant was then applied to the protein A column at a flow rate of 3 mL/min. After the sample is loaded, the protein A column is washed by using an equilibrium buffer solution, and the volume of the equilibrium buffer solution is 4 times of the volume of the column bed of the protein A column. The CD3E/G antibodies bound to the protein A column were eluted with an eluent (0.1M glycine HCl buffer, pH 2.5) and the elution was monitored by UV detector (A280 UV absorption peak). The eluted antibody was collected, neutralized to pH by adding 10%1.0M Tris-HCl buffer, the percentage being volume percentage, and then immediately dialyzed with PBS phosphate buffer overnight, changing the solution 1 time the following day and continuing the dialysis for 3 hours. Collecting CD3e/g antibody after dialysis, performing aseptic filtration by using a filter with the size of 0.22 microns, and performing aseptic preservation to obtain the purified CD3e/g antibody.

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

TABLE 3 detection assay for purified CD3e/g antibodies

EXAMPLE 3 detection of lead antibodies

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

The purified CD3e/g antibody obtained in example 2 was subjected to binding reaction with CD3e/g protein (see Table 1 for sequence).

Human CD3e/g protein or monkey CD3e/g protein (see Table 1 for sequences) was diluted to a final concentration of 5.0. Mu.g/mL with PBS and then added to a 96-well ELISA plate at 100. Mu.l per well. Incubation was performed overnight at 4℃with plastic film, and the next day plates were washed 2 times with plate wash [ PBS+0.01% (v/v) Tween20], blocking solution [ PBS+0.01% (v/v) Tween20+1% (w/w) BSA ] was added and blocked for 2 hours at room temperature. The blocking solution was removed and 100. Mu.l of purified CD3e/g antibody obtained in example 2 was added to each well. After incubation for 2 hours at 37 ℃, the plates were washed 3 times with plate wash [ PBS+0.01% (v/v) Tween20 ]. HRP (horseradish peroxidase) -labeled secondary antibody (purchased from Sigma) was added and after incubation at 37 ℃ for 2 hours, the plates were washed 3 times with plate wash [ pbs+0.01% (v/v) Tween20 ]. 100. Mu.l of TMB substrate per well was added and after incubation for 30 minutes at room temperature, 100. Mu.l of stop solution (1.0N HCl) per well was added. The A450nm values were read with ELISA plate reader (SpectraMax 384plus, available from Molecular Device), FIG. 2 and Table 4 show graphs of the results of ELISA detection of CD3e/g antibodies binding to hCD3e/g, FIG. 3 and Table 5 show graphs of the results of ELISA detection of CD3e/g antibodies binding to cyno CD3e/g, where the IgG control is mouse IgG and the data in the tables are OD450nm values. Wherein HIT3a is purchased from BD pharmigen, lot No. 555336, and SP34 is purchased from BD pharmigen, lot No. 551916.OKT3 was purchased from Biolegend and was made by Dairy Chemie, and the heavy and light chains were sequenced as shown in SEQ ID NO. 100 and SEQ ID NO. 101, respectively.

As can be seen from fig. 2 and 3, tables 4 and 5, the antibodies of the present invention can bind to human CD3 (hCD 3 e/g), and the binding affinity of the antibodies of the present invention to human CD3e/g is better than that of the existing cognate antibody OKT 3. Meanwhile, the antibody of the invention can be combined with monkey-derived CD3 (cyno CD3 e/g), and the existing similar antibody OKT3 does not have the binding affinity or poor binding with monkey CD3 e/g.

TABLE 4ELISA detection of binding of CD3e/g antibodies to hCD3e/g

TABLE 5ELISA detection of binding of CD3e/g antibodies to cCD3e/g

B. detection of antigen-antibody binding sites by flow cytometry (FACS)

The purified CD3e/g antibody obtained in example 2 was subjected to binding reaction with cell surface CD3e/g (see Table 1 for sequences).

The present invention uses two cells endogenously expressing CD3 for binding experiments. One was Jurkat, a human T cell lymphoma, and the other was CD3 positive T cells isolated from human peripheral mononuclear cells, both cells were expanded to 3 x 10 ⁶/ml in T-75 cell flasks, medium was drained, washed 2 times with PBS buffer (available from Invitrogen), cells were diluted to 2 x 10 ⁶ cells per ml with PBS buffer after cell counting, 1% goat serum blocking solution was added, the percentages were mass percentages, incubated for 30 minutes on ice, and then washed 2 times with PBS buffer by centrifugation. The collected cells were suspended to 2X 10 ⁶ cells/mL with FACS buffer (PBS+1% BSA, the percentages being by mass), added to a 96-well FACS reaction plate at 100. Mu.l per well, 100. Mu.l per well of the purified CD3 antibody test sample obtained in example 2 was added, and incubated on ice for 2 hours. The cells were washed 2 times with FACS buffer, and 100 μl of fluorescent (Alexa 488) -labeled secondary antibody (available from Invitrogen) per well was added and incubated on ice for 1 hour. The cells were centrifuged 3 times with FACS buffer, then suspended with 100. Mu.L of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD Corp.). The results are shown in FIG. 4, where the IgG control was murine IgG (mIgG).

The results indicate that these antibodies bind to human CD3 on the surface of Jurkat and primary CD3 positive T cells.

C. NF-kB reporter gene experiment to detect activation of NFAT signaling pathway by CD3 antibody

PGL4.30[ Luc2P/NFAT-RE/Hygro ] plasmid (purchased from Promega) was transfected into Jurkat cells, cultured in RPMI1640 medium containing hygromycin (hygromycin) and 10% fetal bovine serum, and screened to obtain Jurkat cell lines stably expressing NFAT RE-luciferases (referred to herein as Jurkat-NFAT Luc).

96-Well plates (purchased from PERKIN ELMER) were pre-coated with 10ug/ml goat anti-mouse antibody variable region fragment and incubated overnight at 4 ℃. The cells were washed three times with PBS before use. Diluted antibodies were added to 96-well plates and incubated at 37 ℃ for 30 minutes. Then, jurkat-NFAT Luc (1X 10 ⁵/well) was added and incubated for 5 hours, and the luciferase content was determined using One-Glo luciferase assay system (from Promega).

The results are shown in fig. 5, where the antibody significantly activated the NFAT signaling pathway, with an activation capacity comparable to OKT 3.

Example 4 determination of variable region amino acid sequence

Total RNA isolation, namely, after antigen binding is detected on the supernatant obtained by subcloning culture in example 1 (namely, after detection and activity measurement in examples 2-3), 5X 10 ⁷ hybridoma cells are collected through centrifugation, 1mL of Trizol is added and mixed uniformly and transferred into a 1.5mL centrifuge tube, standing still for 5 minutes at room temperature, 0.2mL of chloroform is added, shaking is carried out for 15 seconds, standing still for 2 minutes, then 4 ℃ and 12000g of chloroform are added, transferring the supernatant into a new 1.5mL centrifuge tube, 0.5mL of isopropanol is added, liquid in the tube is mixed lightly, standing still for 10 minutes at room temperature and then centrifuging for 15 minutes at 4 ℃, the supernatant is discarded, 1mL of 75% ethanol (the percentage is the volume percentage) is added, precipitation is washed lightly, 4 ℃ and 12000g of the supernatant is centrifuged for 5 minutes, the precipitate is dried, and H2O treated by DEPC is added and dissolved (the solvent is promoted by a water bath at 55 ℃ for 10 minutes), so that total RNA is obtained.

Reverse transcription and PCR 1. Mu.g of total RNA was taken, 20. Mu.l of the system was prepared, and after addition of reverse transcriptase, the reaction was stopped at 42℃for 60 minutes and at 7℃for 10 minutes. A50. Mu.l PCR system was configured, comprising 1. Mu. lcDNA, 25pmol of each primer, 1. Mu.l DNA polymerase and the appropriate buffer system, 250. Mu. Mol dNTPs, and a PCR program was set, pre-denatured at 95℃for 3 minutes, denatured at 95℃for 30 seconds, annealed at 55℃for 30 seconds, extended at 72℃for 35 seconds, and extended for an additional 5 minutes at 72℃after 35 cycles to obtain the PCR product. Wherein the kit used for reverse transcription is PRIMESCRIPT RT MASTER Mix, purchased from Takara under the accession number RR036, and the kit used for PCR comprises Q5 super fidelity enzyme, purchased from NEB under the accession number M0492.

Cloning and sequencing, namely taking 5 mu l of PCR product to carry out agarose gel electrophoresis detection, purifying a positive detection sample by using a column recovery kit, wherein the recovery kit isGel & PCR Clean-up, available from MACHEREY-NAGEL, cat# 740609. 50ng of sample, 50ng of T carrier, 0.5 μl of ligase, 1 μl of buffer, 10 μl of reaction system, and half an hour at 16 ℃ to obtain ligation product, wherein the ligation kit is T4 DNA ligase, purchased from NEB under the trade name M0402, 5 μl of ligation product is added to 100 μl of competent cells (Ecos 101competent cells, purchased from Yeastern under the trade name FYE 607) and subjected to ice bath for 5 minutes, then heat shock for 1 minute in a 42 ℃ water bath, after 1 minute after being put back on ice, 650 μl of antibiotic-free SOC medium is added, resuscitated at a speed of 200RPM for 30 minutes on 37 ℃,200 μl of LB solid medium containing antibiotics is taken out, incubated overnight in a 37 ℃ incubator, on the next day, 30 μl of PCR system is configured by using primers M13F and M13R on the T carrier, colony PCR is performed by dipping the colony in the PCR reaction by a pipette gun, 0.5 μl of colony is sucked out from the PCR system, and the other 100 μl of solid medium is subjected to the PCR system, and the colony PCR system is subjected to the electrophoresis for 3 μl of PCR system, and the PCR system is subjected to the electrophoresis for 3 to the test of 5-6 nM solid strain, and the positive test results of the PCR table are shown in the table 1-6 nM, and the PCR table is carried out, and the positive test results of the PCR table are obtained after the PCR table is carried out.

TABLE 6-1CD3e/g antibody protein CDR sequence numbering (based on the kabat definition rules as an example)

TABLE 6-2CD3e/g antibody protein CDR sequence numbering (based on the kabat definition rules as an example)

Table 7 below shows alternative framework region sequence combinations of the present invention.

TABLE 7 combinations of framework regions sequences (exemplified by kabat definition rules)

The light chain variable region and the heavy chain variable region of the antibodies obtained in the present invention are shown in table 8 below.

TABLE 8CD3E/G antibody Gene (DNA) sequence numbering

After obtaining the light and heavy chain variable domain sequences encoding the antibody molecules, conventional recombinant DNA techniques may be employed to fusion express the light and heavy chain variable domain sequences and the corresponding murine or human antibody light and heavy chain constant domain sequences to obtain recombinant antibody molecules.

It will be appreciated that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the application, and that such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

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),

Wherein the VH comprises the following Complementarity Determining Regions (CDRs):

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 10, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 19;

the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 6, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 13, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 20;

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO.1, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 7, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 16;

the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 2, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 8, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 17;

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 3, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 9, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 18;

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 20;

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 6, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 12, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 21;

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 6, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 22, or,

The amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 6, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 15, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 20;

And/or, the VL comprises the following Complementarity Determining Regions (CDRs):

the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 24, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 28, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 31;

the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 26, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 29, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 32;

the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 28, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 30;

the amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 24, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 28, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 30;

The amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 25, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 29, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 32, or

The amino acid sequence of the VL CDR1 is shown as SEQ ID NO. 27, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 29, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 32;

preferably, in the antibody or antigen binding fragment thereof,

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 4, 10 and 19, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 24, 28 and 31, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 are respectively shown in SEQ ID NO. 6, 13 and 20, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO. 26, 29 and 32, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 1, 7 and 16, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 23, 28 and 30, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 2, 8 and 17, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 24, 28 and 30, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 3, 9 and 18, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 24, 28 and 30, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 are respectively shown in SEQ ID NO. 5, 11 and 20, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO. 25, 29 and 32, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 6, 12 and 21, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 26, 29 and 32, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 are respectively shown in SEQ ID NO. 6, 14 and 22, the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are respectively shown in SEQ ID NO. 26, 29 and 32, or

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 are shown as SEQ ID NO. 6, 15 and 20 respectively, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are shown as SEQ ID NO. 27, 29 and 32 respectively.

2. The CD3 e/g-targeting antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region (VH) further comprises a heavy chain variable region framework region (VH FWR), and/or the light chain variable region (VL) further comprises a light chain variable region framework region (VL FWR);

Preferably, the VH FWR is a heavy chain variable region framework region of a human or murine antibody, and the VL FWR is a light chain variable region framework region of a human or murine antibody;

More preferably, the VH FWR comprises a VH FWR1 with an amino acid sequence shown as any one of SEQ ID NO 33-40, a VH FWR2 with an amino acid sequence shown as any one of SEQ ID NO 41-43, a VH FWR3 with an amino acid sequence shown as any one of SEQ ID NO 44-51, and a VH FWR4 with an amino acid sequence shown as any one of SEQ ID NO 52 or 53, wherein the VL FWR comprises a VL FWR1 with an amino acid sequence shown as any one of SEQ ID NO 54-56, a VL FWR2 with an amino acid sequence shown as any one of SEQ ID NO 57 or 58, a VL FWR3 with an amino acid sequence shown as any one of SEQ ID NO 59-63, and a VL FWR4 with an amino acid sequence shown as any one of SEQ ID NO 64-66.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the VH is shown in any one of SEQ ID NOS.67-75, preferably in any one of SEQ ID NOS.83-91, and/or the amino acid sequence of the VL is shown in any one of SEQ ID NOS.76-82, preferably in any one of SEQ ID NOS.92-99;

preferably:

The amino acid sequence of the VH is shown as SEQ ID NO. 74 and the amino acid sequence of the VL is shown as SEQ ID NO. 76, or the amino acid sequence of the VH is shown as SEQ ID NO. 75 and the amino acid sequence of the VL is shown as SEQ ID NO. 77, or the amino acid sequence of the VH is shown as SEQ ID NO. 67 and the amino acid sequence of the VL is shown as SEQ ID NO. 77, or the amino acid sequence of the VH is shown as SEQ ID NO. 73 and the amino acid sequence of the VL is shown as SEQ ID NO. 78, or the amino acid sequence of the VH is shown as SEQ ID NO. 69 and the amino acid sequence of the VL is shown as SEQ ID NO. 81, or the amino acid sequence of the VH is shown as SEQ ID NO. 72 and the amino acid sequence of the VL is shown as SEQ ID NO. 79, or the amino acid sequence of the VL is shown as SEQ ID NO. 71 and the amino acid sequence of the VL is shown as SEQ ID NO. 80, or the amino acid sequence of the VL is shown as SEQ ID NO. 68 and the amino acid sequence of the VL is shown as SEQ ID NO. 80;

More preferably:

the nucleotide sequence of the VH is shown as SEQ ID NO. 84 and the nucleotide sequence of the VL is shown as SEQ ID NO. 92, or the nucleotide sequence of the VH is shown as SEQ ID NO. 91 and the nucleotide sequence of the VL is shown as SEQ ID NO. 93, or the nucleotide sequence of the VH is shown as SEQ ID NO. 90 and the nucleotide sequence of the VL is shown as SEQ ID NO. 93, or the nucleotide sequence of the VH is shown as SEQ ID NO. 85 and the nucleotide sequence of the VL is shown as SEQ ID NO. 94, or the nucleotide sequence of the VH is shown as SEQ ID NO. 86 and the nucleotide sequence of the VL is shown as SEQ ID NO. 95, or the nucleotide sequence of the VH is shown as SEQ ID NO. 83 and the nucleotide sequence of the VL is shown as SEQ ID NO. 97, or the nucleotide sequence of the VL is shown as SEQ ID NO. 89 and the nucleotide sequence of the VL is shown as SEQ ID NO. 98, or the nucleotide sequence of the VL is shown as SEQ ID NO. 88 and the nucleotide sequence of the VL is shown as SEQ ID NO. 88.

4. The CD3 e/g-targeting antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the CD3 e/g-targeting antibody or antigen-binding fragment thereof further comprises an antibody heavy chain constant region and an antibody light chain constant region;

Preferably, the antibody heavy chain constant region is a mouse-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and the antibody light chain constant region is a mouse-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 antibody heavy chain constant region, preferably a human IgG1, igG2, igG3, or IgG4 antibody heavy chain constant region, and the antibody light chain constant region is a human antibody light chain kappa or lambda chain constant region.

5. The CD3 e/g-targeting antibody or antigen-binding fragment thereof according to any one of claims 1-3, which is a full length antibody, fab ', F (ab') ₂, fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or any other antibody that retains the partial ability of the antibody to specifically bind to an antigen, or a monoclonal or polyclonal antibody made from the above antibody.

6. An isolated nucleic acid encoding the CD3 e/g-targeting antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. A recombinant expression vector comprising the isolated nucleic acid of claim 6, preferably said expression vector comprises a eukaryotic expression vector and/or a prokaryotic expression vector.

8. A transformant comprising the isolated nucleic acid of claim 6 or the recombinant expression vector of claim 7, preferably the host cell of the transformant is a prokaryotic cell, preferably an e.coli cell such as TG1, BL21, and/or a eukaryotic cell, preferably a HEK293 cell or CHO cell.

9. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof targeting CD3e/g according to any one of claims 1-5, and a pharmaceutically acceptable carrier, preferably the pharmaceutical composition further comprises other anti-tumor antibodies as active ingredient.

10. Use of an antibody or antigen-binding fragment thereof targeting CD3e/g according to any one of claims 1-5 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor;

preferably:

The tumors include colorectal cancer, lung cancer, breast cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, pancreatic cancer and/or lymphomas such as adult T-cell leukemia lymphomas, acute myelomas, diffuse large B-cell lymphomas, follicular lymphomas, pediatric acute lymphoblastic lymphomas, anaplastic large cell lymphomas, natural killer cell lymphomas and/or peripheral T-cell lymphomas.