CD3 specific binding protein as well as preparation method and application thereof

Abstract

The invention relates to a CD3 specific binding protein and a preparation method and application thereof in the field of biological medicine. The specific binding protein of the CD3 has a CD3 binding structural domain, and the CD3 binding structural domain has CDR1 to CDR3 of which the amino acid sequences are shown as SEQ ID NO: 3 to SEQ ID NO: 5. The CD3 specific binding protein provided by the invention has specific CDRs, can be bound with human and monkey CD3 epsilone delta dimer protein with high affinity, and has Jurkat cell binding activity.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to a specific binding protein of CD3, its preparation method and applications. Background Art

[0002] The immune system of the human body consists of two independent but interrelated systems: the cellular immune system and the humoral immune system. T cells are the main force of the cellular immune system and originate from pluripotent stem cells in the bone marrow (from the yolk sac and liver during the embryonic period). During the embryonic and neonatal periods of the human body, a part of the pluripotent stem cells or pre-T cells in the bone marrow migrate into the thymus, where they differentiate and mature under the induction of thymic hormones to become immunocompetent T cells.

[0003] Research has shown that the induction of T lymphocyte activation, proliferation, and differentiation into effector cells requires dual-signal stimulation. The first signal comes from the antigen peptide / MHC complex on the surface of antigen-presenting cells (APCs) and is transduced by the T cell receptor (TCR) complex. The second signal is the co-stimulatory signal, which is generated after the interaction between co-stimulatory molecules (such as CD28) on the surface of APCs and the corresponding receptors on T cells.

[0004] The TCR complex consists of TCR and CD3 molecules. Among them, TCR is composed of α and β chains, or γ and δ chains. The variable region parts of each chain protrude extracellularly and are responsible for the binding of antigen peptides, while the constant region is anchored inside the cell membrane, forming a disulfide bond between the two chains and having a very short intracellular domain. This intracellular domain has no signal transduction function. Therefore, the intracellular signaling pathway of T cells is entirely responsible for the CD3 protein, and CD3 is thus considered an essential molecule in the process of T cell activation.

[0005] The CD3 molecule is composed of one γ chain, one δ chain, two ε chains, and two ζ chains, forming three dimers εγ, εδ, and ζζ in the TCR / CD3 complex. Among them, ε, γ, and δ are all type I transmembrane proteins with extracellular functional domains similar to immunoglobulins. The intracellular domains of ε, γ, δ, and ζ contain a total of 10 immunoreceptor tyrosine-based activation motifs (ITAMs). The phosphorylation of ITAMs enables the CD3 chain to bind to the kinase ZAP70, which is very important for the T cell signaling pathway. Since there is a species-conserved epitope on the CD3ε chain, most CD3 antibodies target this epitope, such as the first anti-human CD3 monoclonal antibody muromonab-CD3 (OKT3) (Jones M et al., (1993) Journal of Immunology 150(12):5429 - 5435).

[0006] CD3-targeting antibodies can specifically recognize CD3 proteins on the cell surface and can be used for the detection of CD3-positive cells, the detection of CD3 proteins, and T cell activation. Based on the role of the CD3 molecule in stabilizing the TCR structure and transmitting activation signals, a series of monoclonal antibodies against the CD3 molecule have been developed to regulate the activation signal transduction of T cells, block or at least modulate the immune process, thereby treating inflammatory diseases and / or autoimmune diseases. For example, OKT3 has been used in the treatment / remission of organ transplantation and autoimmune diseases.

[0007] In addition, CD3 antibodies can also form bispecific molecules with functional groups targeting disease-related antigens (such as tumor-associated antigen TAA). By establishing a physical connection between T cells and disease-related antigens through bispecific CD3 antibodies, it bypasses the classical activation pathway in which T cells are activated only after the formation of a complex between MHC-antigen peptides and TCR and the binding of costimulatory molecules, leading to the activation of T cells and the killing of disease-related cells mediated by T cells. Through the killing function of T cells, the elimination of disease-related cells such as tumor cells can be achieved. For example, after a bispecific antibody targeting CD3 and tumor-associated antigen (TAA) brings T cells closer to tumor cells, the T cells are activated and release supramolecular attack particles (SMAP) containing more than 280 proteins to the tumor cells. Perforin and granzyme are carried in the core region of SMAP. Perforin can pierce the outer membrane of tumor cells, and granzyme will induce apoptosis of tumor cells.

[0008] Therefore, the research and development of CD3-targeting antibodies is of great importance. In view of this, the present application is specifically proposed. Summary of the Invention

[0009] One or more embodiments of the present application provide a specific binding protein of CD3, its preparation method, and applications.

[0010] One or more embodiments of the present application provide a specific binding protein of CD3. The specific binding protein of CD3 has a CD3 binding domain, and the CD3 binding domain has CDR1 to CDR3 with amino acid sequences shown in SEQ ID NO:3 to SEQ ID NO:5.

[0011] In some embodiments of the present application, the CD3 binding domain has FR1 with an amino acid sequence shown in SEQ ID NO:6.

[0012] In some embodiments of the present application, the CD3 binding domain has FR2 with an amino acid sequence shown in SEQ ID NO:7.

[0013] In some embodiments of the present application, the CD3 binding domain has FR3 with the amino acid sequence shown in SEQ ID NO:8.

[0014] In some embodiments of the present application, the CD3 binding domain has FR4 with the amino acid sequence shown in SEQ ID NO:9.

[0015] In some embodiments of the present application, the amino acid sequence of the CD3 binding domain is as shown in SEQ ID NO:2.

[0016] In some embodiments of the present application, the specific binding protein of CD3 is a fusion expression protein of the CD3 binding domain and the Fc region.

[0017] One or more embodiments of the present application provide a nucleic acid molecule encoding the specific binding protein.

[0018] In some embodiments of the present application, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1.

[0019] One or more embodiments of the present application provide a vector comprising the nucleic acid molecule.

[0020] One or more embodiments of the present application provide a cell comprising the nucleic acid molecule or the vector.

[0021] In some embodiments of the present application, the cell comprises mammalian cells.

[0022] One or more embodiments of the present application provide a method for preparing the cell, the preparation method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0023] One or more embodiments of the present application provide a method for producing the specific binding protein of CD3, the production method comprising: culturing the cell, and isolating the specific binding protein of CD3 from the resulting culture supernatant.

[0024] One or more embodiments of the present application provide a CD3 detection product comprising the specific binding protein of CD3.

[0025] One or more embodiments of the present application provide a method for detecting CD3 in a sample to be tested, the detection method using the specific binding protein of CD3 as a detection antibody to determine the presence of CD3 in the sample to be tested through an immunobinding reaction.

[0026] One or more embodiments of the present application provide a drug, which comprises the specific binding protein of CD3 as described above.

[0027] One or more embodiments of the present application provide a treatment method, which comprises administering an effective dose of the specific binding protein of CD3 or the drug to a subject.

[0028] Details of one or more embodiments of the present application are set forth in the following description. Other features, objects, and advantages of the present application will become apparent from the specification and the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more fully understand the present application and its beneficial effects, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 Is the antigen binding curve of CR17-63G5;

[0031] Figure 2 Is the determination result of the binding activity of CR17-63G5 at the cellular level;

[0032] Figure 3 Is the determination result of the affinity of CR17-63G5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present application will be further described in detail below in conjunction with the drawings, embodiments, and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing the embodiments and examples and are not intended to limit the present application.

[0035] Term

[0036] Unless otherwise specified or there is a contradiction, the terms or phrases used in this document have the following meanings:

[0037] The selection range of the terms "and / or", "or / and", and "and / or" used in this document includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution connected by "logical AND").

[0038] In this application, the terms "multiple", "diverse", "multiple times", "multiple elements", etc., unless otherwise specified, refer to a quantity greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0039] The "combinations thereof", "any combinations thereof", "any combination modes thereof", etc. used in this document include all suitable combination modes of any two or any two or more of the listed items.

[0040] In this document, the "suitable" in "suitable combination modes", "suitable modes", "any suitable modes", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0041] In this document, "preferred", "better", "more preferable", "it is advisable" are only used to describe the implementation manners or embodiments with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0042] In this application, "further", "even further", "especially", etc. are used for descriptive purposes and represent differences in content, but should not be understood as a limitation on the protection scope of this application.

[0043] In this application, "optionally", "optional", and "option" mean optional, that is, any one of the two alternative options of "yes" or "no". If "optional" appears multiple times in a technical solution, without special instructions, and without contradictions or mutual constraints, each "optional" is independent of each other.

[0044] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0045] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0046] In this application, regarding a numerical interval (i.e., a numerical range), without special instructions, the optional numerical values are considered continuous within the above numerical interval and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Without special instructions, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0047] The temperature parameter in this application, without special limitations, allows both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0048] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) represents volume percentage, and %(w / v) represents mass-volume percentage.

[0049] In the first aspect of the embodiments of the present application, a specific binding protein of CD3 is provided. The specific binding protein of CD3 has a CD3 binding domain, and the CD3 binding domain has CDR1 to CDR3 with amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 5.

[0050] The term "CD3" refers to cluster of differentiation 3, which includes γ chain, δ chain, ε chain, ζ chain, etc. The term "CD3ε" refers to the ε chain of CD3. This term includes variants, homologs, orthologs and paralogs. For example, an antibody specific for human CD3 (such as CD3ε) can cross-react with CD3 (such as CD3ε) protein of another species such as monkey in some cases. In other embodiments, an antibody specific for human CD3 (such as CD3ε) protein can be completely specific for human CD3 (such as CD3ε) protein without cross-reacting with CD3 (such as CD3ε) proteins of other species or other types of proteins, or can cross-react with CD3 (such as CD3ε) proteins of some other species but not all other species.

[0051] The terms "specific binding", "selective binding", "selectively bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, an antibody binds with an affinity (KD) of about less than 10-8M, such as about less than 10-9M, 10-10M, 10-11M, 10-12M or less.

[0052] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Generally, the antibodies of the present disclosure bind to TrKA or its epitope with a dissociation equilibrium constant (KD) of less than about 10-7M, such as less than about 10-8M or 10-9M. For example, in the present disclosure, the affinity of an antibody for a cell surface antigen is determined by the FACS method to obtain the KD value.

[0053] The specific binding protein of CD3 provided by the embodiments of the present application has specific CDRs, can bind to human and monkey CD3epsilon&delta dimer proteins with high affinity and has Jurkat cell binding activity.

[0054] The specific binding protein of CD3 of the present application may have the above CDRs, or a derivative fragment having the above CDRs. The derivative fragment is formed by amino acid substitution at no more than 6 sites ("conservative modification" or "conservative substitution or replacement") relative to its corresponding CDR, and retains the biological activity consistent with its corresponding complementarity determining region. For example, the derivative fragment may be substituted at 1, 2, 3, 4, 5 or 6 sites relative to its corresponding complementarity determining region, and one amino acid may be replaced by another amino acid, or one amino acid may be replaced by multiple (such as 2) amino acids.

[0055] Among the CDRs provided in the present application, a derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, or 3 amino acids in the amino acid sequence of the antibody of the present application with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0056] Table A

[0057] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0058] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.), such that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide basically does not change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). Additionally, the replacement of amino acids with similar structure or function is unlikely to destroy the biological activity.

[0059] The specific binding protein of CD3 provided in the present application can be an antibody, an antigen-binding fragment of an antibody, or a small modular immunopharmaceutical. Further, the binding protein is a monoclonal antibody, F(ab’)2 fragment, Fab’ fragment, Fab fragment, Fv fragment, scFv fragment, bispecific antibody, multispecific antibody, minibody, chelate recombinant antibody, intrabody, nanobody, binding domain immunoglobulin fusion protein, or small modular immunopharmaceutical.

[0060] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α, or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, or IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region 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 3 domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (termed complementarity-determining regions, abbreviated as CDRs) separated by relatively conserved regions (termed framework regions, abbreviated as FRs). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site(s) / portion(s). The distribution of amino acids in the various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of the present disclosure, the Kabat system numbering definition is used for the determination of CDR sequences.

[0061] The term "monoclonal antibody" or "mAb" or "monoclonal antibody composition" refers to an antibody molecule preparation composed of a single molecule. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0062] The term "bispecific" or "bifunctional" molecule refers to a molecule that specifically binds to two target molecules, or two different epitopes on the same target molecule. Bispecific molecules include bispecific antibodies that specifically bind CD3 and a disease-related antigen in the present application. In contrast, a "monospecific" molecule refers to a molecule that specifically binds to a single target molecule, particularly to one epitope on a single target molecule, such as a monoclonal antibody that binds CD3 in the present application.

[0063] "Nanobody" is generally defined as in WO 2008 / 020079 or WO 2009 / 138519, and in a specific aspect generally refers to VHH, humanized VHH or camelized VH (such as camelized human VH), or generally refers to sequence-optimized VHH (optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression). "Nanobody" is obtained by genetic engineering methods and mainly has three categories. The first category is the heavy chain variable region obtained from HCAb of camelids, which is a single folding unit and retains the complete antigen-binding activity, and is the smallest natural antibody fragment. The second category is the heavy chain variable region obtained from IgNAR of cartilaginous fish such as sharks, which is represented by VNAR. The third category is the heavy or light chain variable region obtained from human or murine monoclonal antibodies, which retains the antigen-binding activity, but the affinity and solubility are greatly reduced. In one example, the specific binding protein of CD3 provided in the embodiments of the present application can be a nanobody. Nanobody is the VHH domain of heavy chain antibodies found in the sera of camelids and other animals. Its molecular weight is as low as 12-15 kDa, only 1 / 10 of that of traditional antibodies, and it is the smallest functional single-domain antibody that can stably bind to antigens currently available. Due to its small molecular mass, strong tissue penetration, high specific affinity, ability to recognize crevice epitopes, low immunogenicity, high water solubility, strong stability, simple production, easy expression and easy engineering modification, etc. Nanobodies can be used as affinity capture reagents, biosensors, in vivo imaging tracers, etc., and have broad application prospects in the fields of scientific research, substance detection, disease diagnosis and treatment.

[0064] In some of these examples, the CD3 binding domain has an FR1 with the amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 6.

[0065] In some of these examples, the CD3 binding domain has an FR2 with the amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 7.

[0066] In some of these examples, the CD3 binding domain has FR3 with the amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO:8.

[0067] In some of these examples, the CD3 binding domain has FR4 with the amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO:9.

[0068] In some of these examples, the amino acid sequence of the CD3 binding domain is as shown in SEQ ID NO:2 or an amino acid sequence having at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO:2.

[0069] "Identity" of amino acid sequences refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence when the amino acid sequences are aligned (introducing gaps if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, the alignment can be achieved in a variety of ways within the scope of those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequences being compared.

[0070] In some embodiments of the present application, the specific binding protein of CD3 is a fusion expression protein of the CD3 binding domain and the Fc region.

[0071] "Fc region" or "Fc" refers to the C-terminal region of an antibody heavy chain, which contains at least a portion of the hinge region, CH2 domain, and CH3 domain, and mediates the binding of the antibody to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (e.g., C1q), including native sequence Fc regions and variant Fc regions. Generally, the human IgG heavy chain Fc region is the segment from the amino acid residue at its Cys226 or Pro230 position to the carboxyl terminus, although its boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc can also refer to this isolated region, or in the case of a protein polypeptide containing Fc, such as a "binding protein containing an Fc region", also referred to as an "Fc fusion protein" (e.g., an antibody or an immunoadhesin). In the present application, native sequence Fc regions include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In IgG, IgA, and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0072] In the embodiments of the present application, there is no particular limitation on the source of the Fc region, and there is no particular limitation on the species source, and each independently can be, but is not limited to: mouse, human, bovine, equine, porcine, ovine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, or goose. In one example, the Fc region is derived from a human.

[0073] In the second aspect of the embodiments of the present application, there is provided a nucleic acid molecule encoding the specific binding protein described above.

[0074] In some embodiments of the present application, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequence has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO:1.

[0075] The nucleic acid molecule of the present application mainly refers to an isolated nucleic acid molecule. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffer, or salt, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds described herein.

[0076] In the third aspect of the embodiments of the present application, a vector is provided, and the vector includes the nucleic acid molecule described above.

[0077] The term "vector", which may also be referred to as "nucleic acid construct", refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes effectively linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology usually exist in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

[0078] In the fourth aspect of the embodiments of the present application, a cell is provided, and the cell includes the nucleic acid molecule or the vector described above.

[0079] The term "cell", also known as "host cell", refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plant or animal cells. Bacteria that are easily transformable include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0080] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in DNA content. This includes mutant progeny having the same function or biological activity as screened in the original transformed cells. Where different names are meant, it is clear from the context.

[0081] In a fifth aspect of the embodiments of the present application, there is provided a method for preparing the cell as described above, the preparation method including the step of introducing the nucleic acid molecule or the vector into a target cell.

[0082] In one example, the introduction is by transfection.

[0083] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0084] In a sixth aspect of the embodiments of the present application, there is provided a method for producing the specific binding protein of CD3 as described above, the production method including: culturing the cell as described above, and isolating the specific binding protein of CD3 from the resulting culture supernatant.

[0085] The present application does not particularly limit the source of the specific binding protein of CD3. In some examples, the specific binding protein of CD3 of the present application is screened from a phage display library.

[0086] The term "phage display library" refers to a "library" of phages on the surface of which exogenous peptides or proteins are expressed. The foreign peptide or polypeptide is displayed on the outer surface of the phage capsid. The foreign peptide can be displayed as a recombinant fusion protein incorporated as part of a phage coat protein; a recombinant fusion protein that is not normally a phage coat protein but is capable of becoming incorporated into the outer surface of the capsid; or a protein or peptide that becomes covalently or non-covalently linked to said protein. This is achieved by inserting an exogenous nucleic acid sequence into a nucleic acid that can be packaged into a phage particle. The exogenous nucleic acid sequence is, for example, inserted into the coding sequence of a phage coat protein gene. If the foreign sequence is cloned in-frame, the protein it encodes will be expressed as part of the coat protein. Thus, a library of nucleic acid sequences, such as a library of antibody lineages prepared from gene segments encoding an entire B cell lineage of one or more individuals, can be inserted into phages in this way to produce a "phage library". When those peptides and proteins representing the peptides and proteins encoded by the nucleic acid library are displayed by the phages, a "peptide display library" is produced. Although a variety of phages are used in the construction of the library, filamentous phages are commonly used (Dunn (1996) Curr. Opin. Biotechnol. 7:547 - 553). See, for example, the following description of phage display libraries.

[0087] In some of these examples, the specific binding protein of CD3 of the present application is obtained by biosynthesis, such as culturing the cells. Those skilled in the art can understand that, in the case of knowing the amino acid sequence of the specific binding protein of CD3, it can also be obtained by other means such as solid-phase synthesis.

[0088] In the seventh aspect of the embodiments of the present application, a CD3 detection product is provided, and the detection product includes the specific binding protein of CD3 described above.

[0089] The embodiments of the present application do not make a particular limitation on the type of the detection product, such as a diagnostic reagent, a test strip, a detection plate or a kit. In the detection product, the specific binding protein of CD3 competitively binds to CD3 in the test sample.

[0090] When the term "competition" is used in the context of antigen-binding proteins that compete for the same epitope, it means competition between the antigen-binding proteins, which is determined by the following assay: in the assay, the antigen-binding protein to be detected (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., the TrKA antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such assays including: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA with I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct-labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Generally, the assay involves using a purified antigen that binds to a solid surface or cell that is loaded with either the unlabeled test antigen-binding protein or the labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label that binds to the solid surface or cell in the presence of the test antigen-binding protein. Generally, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope adjacent to the binding epitope of the reference antigen-binding protein that is sufficiently close such that the two epitopes sterically hinder binding to occur. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Generally, when the competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more of the reference antigen-binding protein to the common antigen.In some cases, the binding is inhibited by at least 80 - 85%, 85 - 90%, 90 - 95%, 95 - 97% or 97% or more.

[0091] In an eighth aspect of the embodiments of the present application, a method for detecting CD3 in a sample to be tested is provided. The detection method uses the specific binding protein of CD3 as a detection antibody to determine the presence of CD3 in the sample to be tested through an immunobinding reaction.

[0092] The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen (for which the antibody is specific). The strength or affinity of the immunobinding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunobinding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rates of antigen - binding site / antigen complex formation and dissociation.

[0093] The present application relates to methods for immuno - detecting or assaying a target antigen (such as CD3), reagents for immuno - detecting or assaying a target antigen (such as CD3), methods for immuno - detecting or assaying cells expressing a target antigen (such as CD3), and diagnostic agents for diagnosing diseases associated with target - antigen (such as CD3) - positive cells, which contain an antibody or antibody fragment that specifically recognizes the target antigen (such as CD3) and binds to the amino acid sequence of the extracellular region or its three - dimensional structure as an active ingredient.

[0094] In the present application, the method for detecting or assaying the amount of a target antigen (such as CD3) can be any known method. For example, it includes immuno - detection or assay methods. Immuno - detection or assay methods are methods for detecting or assaying the amount of an antibody or an antigen using a labeled antigen or antibody. Examples of immuno - detection or assay methods include radioimmunoassay (RIA) using a radioactive substance - labeled immuno - antibody, enzyme - linked immunosorbent assay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, Western blotting, physicochemical methods, etc.

[0095] To detect cells expressing a polypeptide, known immuno - detection methods can be used, and preferably immunoprecipitation, fluorescence cell staining, immunohistochemical staining, etc. can be used. In addition, fluorescence antibody staining methods using the FMAT8100HTS system (Applied Biosystem) can be used.

[0096] In the application, there is no particular limitation on the sample to be tested for detecting or assaying a target antigen (such as CD3), as long as it has the possibility of containing cells expressing the target antigen (such as CD3), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0097] Depending on the required detection method, the test product may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assay methods, such as a second antibody labeled to recognize the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label, etc.

[0098] In a ninth aspect of the embodiments of the present application, there is provided a drug, which includes the specific binding protein of CD3 described above.

[0099] In a tenth aspect of the embodiments of the present application, there is provided a treatment method, which includes administering an effective dose of the specific binding protein of CD3 described above or the drug to a subject.

[0100] When "administering", "giving", and "treating" are applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "giving", and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of cells includes the contact of a reagent with the cells, as well as the contact of the reagent with a fluid, where the fluid contacts the cells. "Administering", "giving", and "treating" also mean the in vitro and ex vivo treatment of cells by a reagent, diagnostic, binding composition, or by another cell. When "treating" is applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, preventive or prophylactic measures, research, and diagnostic applications.

[0101] "Treatment" means administering to a patient a therapeutic agent, either internally or externally, such as a composition comprising any one of the antibodies or antigen-binding fragments of the present disclosure, to a patient having one or more disease symptoms, wherein the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to alleviate one or more disease symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any particular disease symptom (also referred to as "therapeutically effective amount") can vary depending on various factors, such as the disease state, age and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare person to evaluate the severity or progression of the symptom. Although the embodiments of the present disclosure (such as treatment methods or articles) may not be effective in alleviating each target disease symptom, it should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test method known in the art, such as Student's t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0102] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcomes. For prophylactic use, beneficial or desired outcomes include eliminating or reducing the risk, alleviating the severity, or delaying the onset of a disease condition, including the biochemical, histological, and / or behavioral symptoms of the disease condition, its complications, and intermediate pathological phenotypes presented during the development of the disease condition. For therapeutic applications, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various disease conditions related to the target antigens of the present application or improving one or more symptoms of the disease condition, reducing the dosage of other agents required to treat the disease condition, enhancing the efficacy of another agent, and / or delaying the progression of the disease condition related to the target antigens of the present disclosure in the patient.

[0103] The diseases to be treated are typically CDR3-mediated diseases, such as autoimmune diseases (type 1 diabetes, multiple sclerosis, psoriasis, etc.), prevention of transplant rejection in the field of organ transplantation, and inflammatory bowel diseases (Crohn's disease, ulcerative colitis, etc.).

[0104] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions indicated in the following examples, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0105] In the following specific embodiments, regarding the measurement parameters of raw material components, if there is no special instruction, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0106] Example 1

[0107] 1. Immunize camels with CD3-related protein

[0108] Use Human CD3 epsilon&CD3 delta Heterodimer Protein,His Tag, purchased from ACRO BIOSYSTEMS, product number CDD-H52W1 and Human CD3 epsilon protein purchased from ACRO BIOSYSTEMS, product number CDE-H5223 to immunize camels. For the first immunization, use complete Freund's adjuvant to mix and emulsify with the antigen protein (0.2 mg for each single immunization dose) at a mass ratio of 1:1, and inject subcutaneously at multiple points. Every 2 weeks, boost with Freund's incomplete adjuvant emulsified with the two antigen proteins (0.2 mg each) at a mass ratio of 1:1, and immunize a total of 4 times. Draw blood after immunization to separate antiserum, use the immunogenic protein to coat the ELISA plate, and determine the titer of the antiserum by ELISA method. Among them, the total IgG titer is determined using Goat anti-camelid IgG H&L (HRP) (abcam, product number ab112786), and the heavy chain antibody titer is determined using Anti-VHH, Rabbit antibody(HRP)(AlpVHHs, product number 077-201-005).

[0109] After 4 immunizations, the total serum titer of the animal reaches more than 1.0E5, and the heavy chain antibody titer > 2.5E4, then stop immunization.

[0110] 2. Construction of an immunized camel nanobody phage display library

[0111] An outsourcing company for immunization draws peripheral blood from camels to isolate PBMC (peripheral blood mononuclear cells). Use HiPure Total RNA Mini Kit (Magen, product number R4111-02) to extract PBMC Total RNA, and use The IIIFirst-Strand Synthesis System for RT-PCR (Invitrogen, catalog number 18080-051) kit was used for reverse transcription into cDNA. The gene fragment from the signal peptide to CH2 was amplified from the cDNA using the nested PCR method. After recovering the product of about 700 bp in size, it was used as a template, and a set of forward and reverse primers (with SfiI restriction site sequences) matching the camelid heavy chain antibody variable region genes FR1 and FR4 were used for secondary amplification to obtain the heavy chain antibody variable region fragment VHH. The VHH was inserted into the phage display pComb3XSS vector using conventional molecular biological techniques, and the ER2738 electrotransformation competent cells (BIOSEARCH, catalog number 60522-2) were electrotransformed to construct a library. The gradient diluted bacterial library was coated on the antibiotic-containing plate, and the number of clones was counted to calculate the library transformant capacity. The bacterial library was expanded to the logarithmic growth phase, and helper phage M13KO7 was added for superinfection. After adding the corresponding antibiotics and culturing overnight, the culture supernatant was collected, and the nanoantibody phage display library was obtained by the PEG / NaCl precipitation method. The host was infected with the gradient diluted phage library and then coated with antibiotic-containing plates, and the phage library titer was calculated by counting the number of clones.

[0112] The library capacity of the nanoantibody library obtained in this example is greater than 1.0×10 9 .

[0113] 3. Screening of Nanobody Phage Display Library

[0114] 3.1 Liquid phase panning

[0115] Block the phage library with > 1.0E+12 cfu and streptavidin-conjugated magnetic beads (BeaverBeads Mag Streptavidin, 2μm, 10mg / mL, Beaver Biotech, Cat.22305-1) using SuperBlock PBS Blocking Buffer (Thermo, Cat. No. 37580) respectively, and incubate at room temperature using a turning mixer. Add Biotin-labeled antigen CD3E&D (ACRO BIOSYSTEMS, Cat. No. CDD-H82W6), or Biotin-labeled antigen CD3E (ACRO BIOSYSTEMS, Cat. No. CDE-H82E1), or Biotin-labeled CD3E polypeptide-BSA (synthesized and conjugated by Jitai Peptide Industry) to the magnetic beads and incubate for 0.5 h. After rinsing with PBST, add the blocked phage library and incubate at room temperature for 1 h. After rinsing with PBST, separate the magnetic beads, elute the phage sub-library using 1mg / mL Trypsin and infect the host, culture and amplify for the next round of panning, and at the same time sample the sub-library infected with the host for dilution plating to count colonies. After 2 - 3 rounds of panning, pick monoclonal colonies, culture in deep well plates, add M13KO7 helper phage (purchased from NEB, Cat. No. N0315S, and amplified by oneself later) to infect, package monoclonal recombinant phages, collect the supernatant and screen for positive clones that can bind to the antigen by ELISA method, and sequence the positive clones to obtain positive single sequences.

[0116] 3.2 Cell panning

[0117] Block the phage library with > 1.0E+12 cfu using a turning mixer at room temperature for 1 h with 1% BSA. Take 1E7 Jurkat cells (Novizan, Cat. No.: DD1302-01) in the logarithmic growth phase, centrifuge and rinse once with PBS, resuspend the cells in 1% BSA solution, incubate with turning at 4°C for 1 h, add the blocked phage library, and incubate with turning at 4°C for 30 min. Centrifuge at 1000g for 5 min to remove the supernatant, then add 10 mL of 1% BSA and wash 3 times. Elute the phage sub-library using 1mg / mL Trypsin (Sigma Aldrich, Cat. No. T8802 50MG) and infect the host, culture and amplify for the next round of panning, and at the same time sample the sub-library infected with the host for dilution plating to count colonies. After 3 rounds of panning, pick monoclonal colonies, culture in deep well plates, add M13KO7 helper phage (purchased from NEB, Cat. No. N0315S, and amplified by oneself later) to infect, package monoclonal recombinant phages, collect the supernatant and screen for positive clones that can bind to the immunogen by ELISA method and perform gene sequencing.

[0118] 4. Nanobody preparation

[0119] Candidate nanobody clones were selected, the VHH DNA fragment encoding the nanobody was amplified by PCR, and subcloned into the expression vector pcDNA3.4 pre-containing the human IgG1Fc gene. The vector was transfected into mammalian cells CHO for recombinant protein expression, and the cell culture supernatant was collected and the expressed fusion nanobody was purified using a Protein A affinity chromatography column.

[0120] SEQ ID NO:1 (Native DNA sequence, amplified from PBMC cDNA):

[0121] GAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAAAATCTCCTGTGAAGCCTCCGGATACAACAGTATTCTTTTTATGGCGTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGTTATGCATACTGGTGGAATTAGTCCATATTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAAGTCCAAGAACACGGTGTATCTGCAATTGAATAGCCTGGAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGCCCTGCGGAGCCGCTTGAAGTCCTATACCGTTCTCGACCCCCGGAAGTATAACGTCTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCA.

[0122] SEQ ID NO:2 (CR17-63G5 nanobody amino acid sequence):

[0123] EVQLQESGGGSVQAGGSLKISCEAS GYNSILF MAWFRQAPGKEREGVAV MHTGGISP YYADSVKGR FTISQDKSKNTVYLQLNSLEPEDTAMYYC AAALRSRLKSYTVLDPRKYNV WGQGTLVTVSS.

[0124] SEQ ID NO:3 (CDR1 sequence, IMGT numbering): GYNSILF.

[0125] SEQ ID NO:4 (CDR2 sequence, IMGT numbering): MHTGGISP.

[0126] SEQ ID NO:5 (CDR3 sequence, IMGT numbering): AAALRSRLKSYTVLDPRKYNV.

[0127] SEQ ID NO:6 (FR1, IMGT numbering): EVQLQESGGGSVQAGGSLKISCEAS.

[0128] SEQ ID NO:7 (FR2, IMGT numbering): MAWFRQAPGKEREGVAV.

[0129] SEQ ID NO:8 (FR3, IMGT numbering):

[0130] YYADSVKGRFTISQDKSKNTVYLQLNSLEPEDTAMYYC.

[0131] SEQ ID NO:9 (FR4): WGQGTLVTVSS.

[0132] 5. Determination of the binding activity of candidate antibody proteins at the protein level

[0133] Use Human CD3 epsilon&CD3 delta Heterodimer Protein, His Tag (ACROBIOSYSTEMS, catalog number CDD-H52W1) and CD3E N-terminal polypeptide-BSA (synthesized by Jitai Peptide Industry), 0.5 μg / mL, 100 μL / well to coat an ELISA plate (Thermo, catalog number 442404) overnight. After washing with PBST, add 2% BSA to block at room temperature for 1 h. Dilute the candidate antibody 4-fold with 1% BSA, add it to the blocked and rinsed ELISA plate, and incubate at room temperature for 1 h. After washing with PBST, add rabbit anti-human IgG H&L (HRP) (abcam, catalog number ab6759) diluted with 1% BSA and incubate at room temperature for 1 h. After washing with PBST, add TMB chromogenic solution (Sino Biological, catalog number: SEKCR01-15) for color development. After termination, read the OD450 value with an ELISA reader and plot the curve.

[0134] The results of the binding curves of the candidate antibodies measured in this example are as Figure 1 shown. Clone CR17-63G5 was obtained, which has binding activity to Human CD3 epsilon&CD3 delta Heterodimer Protein and can bind to the CD3E N-terminal polypeptide.

[0135] 6. Determination of the binding activity of candidate nanobodies at the cellular level

[0136] Take NFAT Jurkat cells in the logarithmic growth phase, centrifuge the cell suspension at 300 g for 5 min, resuspend with 4% FBS, and adjust the cell density to 2.0E+6 cfu / mL for standby. Dilute the nanobody to be tested with 4% FBS, starting from 800 nM, and perform 3-fold serial dilution for a total of 8 gradients. After mixing the cell suspension, add 50 μL / well to a 96-well U-shaped plate, add 50 μL of the diluted nanobody to each well, incubate at 4°C for 1 h, and wash the 96-well plate 3 times with PBS. Dilute the secondary antibody APC-labelled anti-human IgGFc (Jackson ImmunoResearch, catalog number: 109-135-170) with 4% FBS at a ratio of 1:1000, add it to the washed cells, 100 μL per well, resuspend and incubate at 4°C for 40 min. Wash the 96-well plate 3 times with PBS. Gently resuspend the cells with 100 μL / well of 4% FBS, and then use a FACS detector (BD Lyric) to detect the cell fluorescence.

[0137] The results are as Figure 2 shown, and the candidate nanobody clone CR17-63G5 can bind to Jurkat cells.

[0138] 7. Affinity determination of CD3 nanobody

[0139] Use a Biacore 8k instrument to determine the affinity of the nanobody Fc fusion protein. Capture the candidate antibody through a Protein A chip, and determine its affinity for antigens Human CD3 epsilon&CD3delta Heterodimer Protein, His Tag (ACRO BIOSYSTEMS, catalog number CDD-H52W1) and cynomolgus CD3 epsilon&CD3 delta Heterodimer Protein, His Tag (ACRO BIOSYSTEMS, catalog number CDD-C52W4) at 100, 50, 25, 12.5, 6.25, 3.125, 0 nM. Set the binding time to 120 s, the dissociation time to 480 s, and the regeneration time with Glycine pH1.5 to 30 s.

[0140] In this application, the gene sequence of the nanobody ANb27-M249-4M-905 with the amino acid sequence shown in SEQ ID NO:10 was synthesized (i.e., codon-optimized for the amino acid sequence of SEQ ID NO:10), cloned into the same expression vector as in "4. Preparation of nanobody", transfected and expressed, and the purified nanobody was named 905Fc, and its affinity was determined under the same conditions as in this example.

[0141] Amino acid sequence of SEQ ID NO:10 (ANb27-M249-4M-905):

[0142] EVQLVESGGGLVQAGGSLRLTCAASGRGISNYHMGWFRQAPGKEREIVAAITSTGARTYYVDSVKG RFTISRDNPKNTLYLEMNSLKPEDTAMYYCVANSVDDSYWGQGTQVTVSS。

[0143] The results are as Figure 3 shown in Table 1. CR17-63G5 has high affinity for both human and monkey CD3 epsilon&CD3 delta HeterodimerProtein, with KD values of 6.23E-10 M and 9.57E-10 M respectively, and 905Fc has no binding activity.

[0144] Table 1

[0145]

[0146] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0147] The above-described examples only represent several embodiments of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be determined by the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A CD3-specific binding protein, characterized in that: The CD3-specific binding protein has a CD3-binding domain, and the CD3-binding domain has CDR1 to CDR3 with amino acid sequences as shown in SEQ ID NO:3 to SEQ ID NO:

5.

2. The CD3-specific binding protein according to claim 1, characterized in that: The specific binding protein satisfies one or more of the following conditions (1) to (2): (1) the amino acid sequence of the CD3 binding domain is shown in SEQ ID NO: 2; and, (2) The CD3-specific binding protein is a fusion protein of the CD3 binding domain and the Fc region.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the specific binding protein according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:

1.

5. A carrier, characterized in that The vector comprises the nucleic acid molecule according to any one of claims 3 to 4.

6. A cell, characterized in that The cell comprises the nucleic acid molecule of any one of claims 3 to 4 or the vector of claim 5 .

7. The cell according to claim 6, characterized in that The cells include mammalian cells.

8. A method for preparing a cell according to any one of claims 6 to 7, characterized in that: The preparation method comprises the step of introducing the nucleic acid molecule or the vector into target cells.

9. A method for producing a CD3-specific binding protein according to any one of claims 1 to 2, characterized in that: The production method comprises: Cultivating the cell of claim 6, and The CD3-specific binding protein is isolated from the obtained culture supernatant.

10. A CD3 detection product, characterized in that: The detection product comprises the CD3-specific binding protein according to any one of claims 1 to 2.

11. A method for detecting CD3 in a sample to be tested, characterized in that: The detection method uses the CD3-specific binding protein of any one of claims 1 to 2 as a detection antibody to determine whether the sample to be tested contains CD3 through an immune binding reaction.

12. A drug, characterized in that The drug comprises the CD3-specific binding protein according to any one of claims 1 to 2.

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