Nanobody or antigen-binding fragment thereof specifically binding to CD3 / TCR complex, and use thereof

Abstract

The present invention relates to the field of biotechnology. Provided are a nanobody or an antigen-binding fragment thereof specifically binding to a CD3 / TCR complex, and the use thereof. The nanobody or the antigen-binding fragment thereof comprises HCDRs as defined in the heavy chain variable region as shown in any one of amino acid sequences of SEQ ID NOs: 1-15.

Description

Nanobodies or antigen-binding fragments thereof specifically binding to CD3 / TCR complex and uses thereof

[0001] Priority Information

[0002] This application claims priority to and the benefit of Chinese Patent Application No. 202410744852.5, filed on June 7, 2024, and is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, and in particular to a nanobody or antigen-binding fragment thereof specifically binding to CD3 / TCR complex and uses thereof. BACKGROUND

[0004] CD3 (Cluster of Differentiation 3) is a multimeric protein complex present on the surface of T cells, and is one of the key molecules in the immune system. CD3 is composed of four different polypeptide chains, including epsilon (ε), gamma (γ), delta (δ) and zeta (ζ), among which CD3ε is essential for T cell activation and T cell development. These subunits can assemble into three pairs of heterodimers (εγ, εδ, ζζ) to form the CD3 complex. The CD3 complex, as a T cell co-receptor, non-covalently binds with the TCR (T Cell Receptor) to form a TCR-CD3 complex containing 8 peptide chains, which is involved in T cell antigen recognition, signal transduction and regulation of T cell development.

[0005] The cytoplasmic segment of CD3ε, CD3γ and CD3δ contains a single ITAM, while the cytoplasmic domain of CD3ζ subunit contains three ITAMs, for a total of ten ITAMs, making the complex very sensitive to antigen binding. The main function of CD3 molecules is to transduce the activation signal generated by TCR recognition of antigens. It is connected to the T cell antigen receptor through a salt bridge and is involved in T cell signal transduction. CD3 antibodies can specifically recognize CD3 molecules on the surface of T cells, causing cross-linking of the TCR-CD3 complex of T cells, directly generating an activation signal, leading to T cell activation and proliferation. Current research shows that CD3 antibodies play an important role in tumor therapy.

[0006] Anti-CD3 agonist antibodies are difficult to develop due to their complex extracellular structure. SP34 is the first widely used anti-CD3 agonist antibody, which can cross-react with cynomolgus monkey CD3. Based on the parent SP34 antibody, a large number of derivatives have been developed, however, it is a great challenge to transform the SP34 antibody or its derivatives into scFv form, and there is a great risk in the CMC production development stage, which seriously limits the application of SP34. Another widely used anti-CD3 antibody is OKT3, but it does not cross-react with cynomolgus monkey CD3, and its clinical application is hindered due to the lack of a suitable preclinical test model.

[0007] Nanobodies, also known as VHH (variable domain of heavy chain of heavy-chain antibody), are a kind of natural light chain-deleted antibodies existing in the peripheral blood of Camelidae (such as camels, llamas and alpacas). It has the advantages of stable physicochemical properties, easy large-scale production, rapid screening through display library, low immunogenicity, small molecular weight, strong tissue penetration ability, and rapid blood clearance. The bispecific antibody TCE (T Cell Engager) formed by CD3 nanobodies and tumor-associated antigens (TAA) is a new type of immunotherapy drug. One arm of TCE targets TAA, and the other arm mainly targets the CD3 / TCR complex. Its main role is to guide T cells to the location of tumor cells by binding to CD3 or TCR antigens on the surface of T cells, thereby activating T cells to enable them to specifically kill tumor cells, thereby improving the effect of immunotherapy. Therefore, it is crucial to develop a nanobody targeting CD3 / TCR.

[0008] In view of the above, the present application is proposed. SUMMARY

[0009] The purpose of the present application is to provide a nanobody or antigen-binding fragment thereof that can specifically bind to CD3 and its application.

[0010] To solve the above technical problems, the present application adopts the following technical solutions:

[0011] In a first aspect, the present application provides a nanobody or antigen-binding fragment thereof that specifically binds to a CD3 / TCR complex. Specifically as follows:

[0012] In one aspect, a nanobody or antigen-binding fragment thereof that specifically binds to a CD3 / TCR complex is provided, which comprises HCDRs defined in the heavy chain variable region as shown in any one of SEQ ID NO: 1-15.

[0013] In another aspect, there is provided a Nanobody or an antigen-binding fragment thereof that specifically binds to a CD3 / TCR complex, comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region;

[0014] the complementarity determining region HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58;

[0015] the complementarity determining region HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59;

[0016] the complementarity determining region HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60.

[0017] In another aspect, there is provided a Nanobody or an antigen-binding fragment thereof that specifically binds to a CD3 / TCR complex, comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region;

[0018] the amino acid sequence of the complementarity determining region HCDR1 comprises D or N;

[0019] the amino acid sequence of the complementarity determining region HCDR2 comprises: S-X1-X2-G-X3, wherein X1 is T, I or Y, X2 is S, G or D, and X3 is S, A or T;

[0020] the amino acid sequence of the complementarity determining region HCDR3 comprises: L-X4, or AGFTG, wherein X4 is G or H.

[0021] In a second aspect, the present application provides a biomaterial comprising any one of (i) to (vi):

[0022] (i) a multispecific antibody, a recombinant protein and an immunoconjugate containing the Nanobody or the antigen-binding fragment thereof as set forth in any one of the preceding.

[0023] (ii) a Chimeric Antigen Receptor (CAR) comprising an extracellular region, a transmembrane region and an intracellular region; wherein the extracellular region comprises the Nanobody or the antigen-binding fragment thereof of the first aspect.

[0024] (iii) a nucleic acid molecule encoding the Nanobody or antigen-binding fragment thereof of the first aspect, the multispecific antibody, recombinant protein and immunoconjugate of (i), or the chimeric antigen receptor of (ii).

[0025] (iv) a vector carrying the nucleic acid molecule of (iii).

[0026] (v) a recombinant cell carrying the nucleic acid molecule of (iii), or the vector of (iv); or, the recombinant cell expresses the Nanobody or antigen-binding fragment thereof of the first aspect, the multispecific antibody, recombinant protein and immunoconjugate of (i), or the chimeric antigen receptor of (ii).

[0027] (vi) an engineered immune effector cell expressing the chimeric antigen receptor of (ii) or containing a nucleic acid molecule encoding the chimeric antigen receptor of (iii).

[0028] In a third aspect, the present application provides a reagent or kit for detecting the CD3 / TCR complex, comprising the Nanobody or antigen-binding fragment thereof of the first aspect.

[0029] In a fourth aspect, the present application provides the Nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the reagent or kit of the eighth aspect, for use in detecting CD3 or a cell expressing CD3, or has an application in:

[0030] (I) detecting CD3 or a cell expressing CD3;

[0031] (II) preparing a product for detecting CD3 or a cell expressing CD3.

[0032] In a fifth aspect, the present application further provides a pharmaceutical composition comprising the Nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect.

[0033] In a sixth aspect, the present application further provides the Nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the pharmaceutical composition of the fifth aspect, for use in treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, an autoimmune disease, an infection, immune rejection of organ transplantation, or has an application in:

[0034] (I) preparing a medicament for treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, an autoimmune disease, an infection, immune rejection of organ transplantation;

[0035] (II) treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation.

[0036] In a seventh aspect, there is further provided a method for detecting a CD3 / TCR complex or a cell expressing a CD3 / TCR complex, comprising detecting a sample to be tested using the nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the reagent or kit of the third aspect.

[0037] In an eighth aspect, there is further provided a method for treating, preventing or alleviating a disease, disorder or condition, including a tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation, comprising administering to a subject a therapeutically effective amount of the nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the pharmaceutical composition of the fifth aspect. Advantages:

[0038] The nanobody or antigen-binding fragment thereof of the present application specifically binding to a CD3 / TCR complex can specifically bind to a CD3 / TCR complex, and has a human monkey cross-reactivity; and the nanobody or antigen-binding fragment thereof of the present application specifically binding to a CD3 / TCR complex has a small cytotoxicity. Thus, the multispecific antibody, recombinant protein, immunoconjugate, chimeric antigen receptor, engineered immune effector cell, pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof of the present application specifically binding to a CD3 / TCR complex can effectively prevent and treat a disease, disorder or condition associated with a tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation.

[0039] The amino acid sequence table A1 of the present application is shown as follows:

[0040] Additional aspects and advantages of the present application will be in part apparent and in part pointed out below in the description of embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0041] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0042] FIG. 1 is a result of the binding activity of each antibody to overexpressing strain hCD3-293T or to 293T in Example 2 of the present application.

[0043] Figure 2 is the binding activity result of each antibody to natural tumor cell line Jurkat in Example 3 of the present application;

[0044] Figure 3 is the binding activity result of each antibody to human PBMC in Example 4 of the present application;

[0045] Figure 4 is the binding activity result of each antibody to cyno PBMC in Example 5 of the present application;

[0046] Figure 5 is the binding activity result of each antibody to mouse PBMC in Example 6 of the present application;

[0047] Figure 6 is the reporter gene activation activity result of each antibody in Example 7 of the present application;

[0048] Figure 7 is the binding activity result of each humanized nanobody and corresponding parent nanobody to human PBMC in Example 9 of the present application;

[0049] Figure 8 is the binding activity result of each humanized nanobody and corresponding parent nanobody to cyno PBMC in Example 10 of the present application;

[0050] Figure 9 is the reporter gene activation activity result of each humanized nanobody and corresponding parent nanobody in Example 11 of the present application. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts are within the protection scope of the present application.

[0052] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are contained in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0053] The articles "a", "an", and "the" in this application include the plural, unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or to more than one antibody.

[0054] In this application, the terms "first", "second", etc. are used only to describe particular elements, but do not imply or suggest relative importance or a quantity of the specified technical features. Thus, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise explicitly and specifically limited.

[0055] In this context, the term "optionally", "optional" or "optional" generally means that the event or condition subsequently described can or can not occur, and the description includes situations where the event or condition occurs, as well as situations where it does not occur.

[0056] In this context, the term "antibody" generally refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal, polyclonal, multimeric antibody and CDR grafted antibody.

[0057] In the present application, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The term "antibody" is used in the broadest sense, covering various antibody structures including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, polyspecific antibodies, full-length antibodies, nanobodies, antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Generally, a natural intact antibody contains two heavy (H) chains and two light (L) chains. According to whether it contains α, δ, ε, γ and μ heavy chains, the antibody can be divided into five main categories or isotypes: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain), etc. Each heavy chain is composed of a variable region (VH) and a first, second, third and fourth (optionally) constant region (CH1, CH2, CH3, CH4, respectively). The variable region of the heavy chain determines the binding of the antigen. The variable region of each chain usually contains three hypervariable regions, called "complementarity determining regions (CDRs)", in which the heavy chain CDRs include HCDR1, HCDR2 and HCDR3. Each variable region (VH and VL) is composed of three complementarity determining regions connected by four framework regions (FR). Usually, the variable region VHand the FR of the heavy chain can be connected by the following combination of CDRs and FRs: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0058] In the present application, the term "nanobody" is also referred to as "single domain antibody", which refers to a heavy chain antibody (such as: derived from Camelidae or shark) lacking light chain, a single domain antibody cloned from the variable region thereof, which is the smallest functional antigen-binding fragment, with a relative molecular mass (Mr) of only about 15000. Nanobodies have the characteristics of small molecular mass, strong stability, good solubility, easy expression, low immunogenicity, etc.

[0059] In the present application, the term "antigen-binding fragment" is a substance comprising all or part of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies, single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies). The above antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0060] In the present application, the term "antigen-binding fragment" is a substance comprising all or part of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies, single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies). The above antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0061] The CDR boundaries of the antibodies or antigen-binding fragments thereof in the present application can be defined or identified according to IMGT, Kabat, Chothia, AbM, Contact definition, or CDR defined in other ways acceptable in the art, and CDR defined in other ways acceptable in the art also belong to the protection scope of the present application (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); R. M. MacCallum et al.,. Antibody-antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, A. C. R. Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Developmental and Comparative Immunology 27 (2003) 55-77).

[0062] In the present application, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0063] In the present application, the terms "variant Fc region", "Fc mutant", "Fc region carrying mutation", "mutated Fc region", "Fc region variant", "Fc variant", "variant Fc region", and "mutated Fc region" and the like can be used interchangeably, which refer to an Fc region comprising at least one amino acid modification that distinguishes it from a native sequence Fc region / wild-type Fc region.

[0064] In the present application, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Common naturally occurring amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G); histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). An amino acid analog refers to a compound having the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. An amino acid analog typically has a modified R group (e.g., norleucine) or a modified peptide backbone, but retains the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound having a structure different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.

[0065] In the present application, the terms "identity," "homology," or "similarity" are used in the context of comparing amino acid sequences or nucleic acid sequences to a reference sequence to refer to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as determined by conventional methods, which compare the best alignment of two sequences, and the percent of amino acids in the two polypeptides that are the same at equivalent positions. Alignment of amino acid sequence identity percentages can be performed using a variety of ways known in the art, such as the software programs BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, and the like, which are well known in the art. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximum alignment of the entire length of the compared sequences.

[0066] Without materially affecting the activity of the antibody (retaining at least 80% activity), one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be substituted, added, and / or deleted from the sequences of the present application to obtain variants of the antibody sequences by those skilled in the art. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences described in the present application can have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequences. The sequence identity described in the present application can be measured using sequence analysis software. For example, using the computer program BLAST, especially BLASTP or TBLASTN with default parameters. The amino acid sequences described in the present application are shown in the order of N-terminal to C-terminal.

[0067] It should be noted that in the claims and specification herein, the variants of the antibody sequences are obtained by adding and / or deleting one or more amino acids based on the heavy chain or the variable region of the heavy chain, and the position or number of the mutation sites defined in the specification and claims of the present application also need to be adjusted according to the number and position of the added and / or deleted amino acids.

[0068] In this document, the term "at least 80% homology" means at least 80% homology with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 85% homology" means at least 85% homology with each reference sequence, which can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 90% homology" means at least 90% homology with each reference sequence, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology.

[0069] In the present application, the term "specifically binds" or "binds specifically" refers to a non-random binding reaction between two molecules, for example, the reaction between an antibody and an antigen. In some specific embodiments, for example, as determined by flow cytometric fluorescence sorting technology.

[0070] In the present application, the term "CD3 / TCR complex" refers to a functional transmembrane protein complex located on the surface of T cells, which is composed of T cell receptor (TCR) and CD3 molecules. The TCR contains an a / b chain or g / d chain heterodimer connected by disulfide bonds, which is responsible for specific recognition of antigen peptide-MHC complex; the CD3 molecule is composed of g (CD3g), d (CD3d), e (CD3e) three subunits of heterodimer (ge and de) and z z homodimer (CD247), which binds to TCR to form a complete complex through non-covalent bonds. The complex triggers antigen-specific recognition through the antigen binding domain of TCR, and relies on the immunoreceptor tyrosine activation motif (ITAM) of the intracellular region of CD3 subunit to transmit activation signals and initiate downstream signaling pathways (such as ZAP-70 phosphorylation cascade), inducing T cell immune response. In some embodiments, in the present application, it includes naturally occurring human or non-human CD3 / TCR complex, its conservative structural variants (such as homologues or functionally equivalent bodies with > 80% sequence identity), and pathological or engineered state complex forms (such as abnormal assemblers in tumor microenvironment or genetically edited modified chimeric receptors).

[0071] In the present application, the term "affinity" or "avidity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen. The strength or affinity of the immunological binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. KD can be determined by using any conventional method known in the art, including but not limited to Biacore assay, Octet method, microthermal phoresis method, HPLC-MS method and flow cytometric fluorescence sorting technique.

[0072] The binding of the nanobody, antibody or any antigen-binding fragment provided in the present application to the CD3 / TCR complex can also be expressed as "half maximal effective concentration (EC50)", which refers to the concentration of the drug or antibody that can achieve 50% of the maximum biological effect after a certain exposure time. Generally, the smaller the EC50, the better the affinity, indicating that the target protein can be bound at a lower concentration. The EC50 value can be determined by binding detection methods known in the art, such as direct or indirect binding detection methods (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technique and other binding detection methods).

[0073] In the present text, the terms "polyantibody" and "multispecific antibody" are synonymous and refer to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or to multiple (two or more) different antigens, for example an antibody that recognizes two antigenic epitopes (a bispecific antibody), an antibody that recognizes three antigenic epitopes or an antibody that recognizes four antigenic epitopes, which is to be understood in a broad sense and is not limited in its structure, as long as it recognizes multiple antigenic epitopes.

[0074] A "chimeric antigen receptor", CAR, in the present application is an artificial receptor that mimics the function of TCR, which is composed of an extracellular domain, a transmembrane domain and an intracellular signaling domain connected in turn. When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal can be transmitted to the intracellular through the hinge region and the transmembrane region. The intracellular signaling domain then converts the signal into an activation signal, activates the effector cell, and the effector cell kills the tumor cell by secreting perforin or producing cytokines, while the effector cell itself also undergoes expansion, further expanding the immune killing effect. The extracellular domain is generally composed of a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to an antigen and a hinge region (Hinge) that serves as a connecting function. The single-chain variable fragment is the antigen-binding domain of CAR, which determines the specificity and function of CAR- immune cells. The hinge region is the extracellular structure region of CAR connecting the single-chain variable fragment and the transmembrane domain, which usually maintains the stability required for robust CAR expression and activity in effector cells. The hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8a / CD28. The type and length of the hinge region have an important influence on the functional activity of CAR. The transmembrane domain connects the extracellular domain of CAR with the intracellular signal transduction domain. The commonly used transmembrane domain is derived from CD4, CD8, CD28 and CD3 zeta or their derivatives. The choice of transmembrane domain affects the degree of activation of CAR structure in cell function. The intracellular domain is composed of a costimulatory domain and a signal transduction domain. The costimulatory domain is usually from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). The signal transduction domain is usually the T cell receptor TCR / CD3 zeta chain or the immunoglobulin Fc receptor Fc epsilon RI gamma chain.

[0075] The CARs of the present application (including functional parts and functional variants thereof) can be obtained by methods known in the art, for example, by any suitable method for preparing polypeptides or proteins.

[0076] In the present application, the term "engineered immune effector cell" refers to an immune effector cell expressing a CAR or an immune effector cell modified with a CAR. Among others, an "immune effector cell" generally refers to a cell involved in an immune response, e.g., that promotes an immune effector response. Exemplary immune effector cells include, but are not limited to, T cells (e.g., alpha / beta T cells and gamma / delta T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells (Natural killer T cell), dendritic cells, granulocytes, B cells, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0077] In the present application, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. When the nucleic acid molecule codes for a protein or polypeptide, the coding is optionally for the sense or antisense strand. A nucleic acid molecule can be natural or synthetic, recombinant, or any combination thereof. "Nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably.

[0078] In alternative embodiments, the nucleic acid molecule is RNA or DNA, which can be single- or double-stranded, with the latter preferred. A nucleic acid molecule is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. When a nucleic acid molecule is incorporated into a vector, it is preferably DNA.

[0079] In the present application, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and from which the genetic element is obtained expression, e.g., production of a protein, RNA or DNA encoded by the genetic element, or replication of the genetic element. The vector can be used to transform, transduce or transfect a host cell, so that the genetic element carried by the vector is expressed in the host cell. For example, the vector includes: a plasmid, a phagemid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1 -derived artificial chromosome (PAC), a bacteriophage such as a lambda phage or a M13 phage, and an animal virus, etc. The vector can contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector can also contain a replication initiation site. The vector can also include components that assist its entry into a cell, including but not limited to a viral particle, a liposome, or a protein coat. The vector can be an expression vector or a cloning vector. In some embodiments, the vector (e.g., expression vector) provided in the present application contains a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof described in the present application, at least one promoter (e.g., SV40, CMV, EF-1 a) operably linked to the nucleic acid sequence, and at least one selection marker.

[0080] In the present application, the term "purified" or "isolated" in association with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, e.g., if it is naturally occurring, from the natural environment. In association with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0081] In the present application, the term "recombinant cell" refers to a cell into which a foreign polynucleotide and / or vector can be or has been introduced. The foreign polynucleotide can or can not be integrated into the genome of the "recombinant cell". When the vector is contained in the recombinant cell, the vector can be introduced into a mammalian cell to construct a recombinant cell, and then the recombinant cell is used to express the antibody or antigen-binding fragment provided in the present application. The recombinant cell is cultured, and the corresponding antibody can be obtained. The available mammalian cell can be a CHO cell, etc.

[0082] In the present application, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective, and does not contain additional components that are unacceptable toxic to the subject to which the composition will be administered. In some specific embodiments, the antibody or expressed antibody contained in the above-mentioned pharmaceutical composition can specifically target the binding of the CD3 / TCR complex.

[0083] In the present application, "pharmaceutically acceptable carrier" can include any solvent, carrier, excipient, diluent or other liquid excipient, and the like, suitable for the particular target dosage form. Except insofar as any conventional carrier is incompatible with the nanobodies or antigen-binding fragments thereof of the present application, for example, as it is produced any adverse biological effects or interacts in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, their use is contemplated to be within the scope of this application.

[0084] In the present application, the term "subject" or "patient" refers to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, llamas, avians, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having a cancer, an autoimmune disease or condition, and / or an infection.

[0085] In the present application, the term "diagnosing" refers to the identification of a pathological state, disease or condition, for example, the identification of a CD3- or TCR-related disease, or to the identification of a subject who has a CD3- or TCR-related disease who can benefit from a particular treatment regimen. In some embodiments, diagnosing comprises identifying an abnormal amount or activity of CD3 or TCR. In some embodiments, diagnosing refers to identifying a cancer or an autoimmune disease in a subject.

[0086] In the present application, the term "effective amount" refers to a therapeutic amount, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof; prevent the advancement of a disease; cause regression of a disease; prevent the recurrence, development or progression of one or more symptoms associated with a disease; detect a disease; or enhance or improve the prophylactic or therapeutic efficacy of another therapy, such as a prophylactic or therapeutic agent. The therapeutically effective dose of the antibody or antigen-binding fragment thereof described herein depends on a variety of factors well known in the art, such as body weight, age, past medical history, concurrent treatments, the subject's health status and potential for cross-infection, allergies, hypersensitivity, and side effects, as well as the route of administration and the extent of tumor development. A person skilled in the art, such as a physician or veterinarian, can reduce or increase the dose proportionally according to these or other conditions or requirements.

[0087] In the present context, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present application can be administered by any common route, so long as it can reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, and the like, although the present application is not limited to these exemplified modes of administration. Preferably, the composition of the present application is administered by intravenous or subcutaneous injection.

[0088] In the present context, the term "treatment" refers to obtaining a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any use of a drug or a Nanobody or antigen binding fragment thereof specifically binding to a CD3 / TCR complex to treat, cure, relieve, ameliorate, lessen, or inhibit a disease in an individual, including, but not limited to, administering a drug comprising a Nanobody or antigen binding fragment thereof specifically binding to a CD3 / TCR complex as described herein to an individual in need thereof.

[0089] Detailed description of the Nanobody or antigen binding fragment thereof specifically binding to a CD3 / TCR complex of the present application and its use

[0090] The present application provides a Nanobody or antigen binding fragment thereof specifically binding to a CD3 / TCR complex, a multispecific antibody, an antigen chimeric receptor, a nucleic acid molecule, a vector, a recombinant cell, an engineered immune effector cell, a kit for detecting a CD3 / TCR complex, a pharmaceutical composition, and their uses, methods of detecting a CD3 / TCR complex or of detecting a cell expressing a CD3 / TCR complex, methods of treating or preventing or alleviating a disease or condition or a disorder, which will be described in detail below, respectively.

[0091] Nanobody or antigen binding fragment thereof specifically binding to a CD3 / TCR complex

[0092] In a first aspect, the present application provides a Nanobody or antigen binding fragment thereof specifically binding to a CD3 / TCR complex.

[0093] In particular as follows:

[0094] In one aspect, there is provided a Nanobody or an antigen binding fragment thereof that specifically binds to a CD3 / TCR complex, comprising HCDRs as defined in a heavy chain variable region set forth in any one of SEQ ID NOs: 1-15.

[0095] In some alternative embodiments of the present application, the HCDRs are determined by Kabat, Chothia, AbM, Contact or IMGT definition from a heavy chain variable region set forth in any one of SEQ ID NOs: 1-15.

[0096] In the present disclosure, the term "complementarity determining region", "CDR" or "CDRs" refers to the hypervariable region of the heavy chain of an immunoglobulin, which refers to the region comprising one or more, or even all, of the amino acid residues that contribute primarily to the binding affinity for the antigen or epitope recognized. In the detailed description of the present disclosure, CDRs refer to the hypervariable region of the heavy chain of the antibody.

[0097] In the present disclosure, the term "complementarity determining region", "CDR" or "CDRs" refers to the hypervariable region of the heavy chain of an immunoglobulin, which refers to the region comprising one or more, or even all, of the amino acid residues that contribute primarily to the binding affinity for the antigen or epitope recognized. In the detailed description of the present disclosure, CDRs refer to the hypervariable region of the heavy chain of the antibody.

[0098] Kabat et al. also defined a numbering system for variable region sequences of any antibody. One of ordinary skill in the art can unambiguously apply this Kabat numbering system to any variable region sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0099] According to embodiments of the present application, the HCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT.

[0100] In an alternative embodiment of the present application, the CDRs of the amino acid sequence set forth in SEQ ID NO: 1 are defined based on different definitions as follows:

[0101] In an alternative embodiment of the present application, the CDRs of the amino acid sequence set forth in SEQ ID NO: 3 are defined based on different definitions as follows:

[0102] In an alternative embodiment of the present application, the CDRs of the amino acid sequence set forth in SEQ ID NO: 4 are defined based on different definitions as follows:

[0103] In an alternative embodiment of the present application, the CDRs of the amino acid sequence set forth in SEQ ID NO: 5 are defined based on different definitions as follows:

[0104] In another aspect, there is provided a Nanobody or an antigen binding fragment thereof that specifically binds to a CD3 / TCR complex, comprising the complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region;

[0105] The complementarity determining region HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58;

[0106] The complementarity determining region HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59;

[0107] The complementarity determining region HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60.

[0108] In some embodiments of the application, the complementarity determining region has an amino acid sequence obtained using the IMGT definition based on the amino acid sequence as set forth in any one of SEQ ID NOs: 16-60.

[0109] In some embodiments of the application, the complementarity determining region has an amino acid sequence obtained using the Kabat, Chothia, AbM, or Contact definition based on the amino acid sequence as set forth in any one of SEQ ID NOs: 16-60.

[0110] In some embodiments of the application, the complementarity determining region has an amino acid sequence obtained using the Kabat, Chothia, AbM, or Contact definition based on the amino acid sequence as set forth in any one of SEQ ID NOs: 1-15.

[0111] In some embodiments of the application, the complementarity determining region HCDR1, HCDR2 and HCDR3 of the Nanobody or antigen-binding fragment thereof is selected from any one of the following, according to the IMGT definition:

[0112] 1) has an HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 16, has an HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 17, and has an HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 18;

[0113] 2) has an HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 19, has an HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 20, and has an HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 21;

[0114] 3) has an HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 22, has an HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 23, and has an HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 24;

[0115] 4) has an HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 25, has an HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 26, and has an HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 27;

[0116] 5) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 28, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 29, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 30;

[0117] 6) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 31, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 32, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 33;

[0118] 7) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 34, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 35, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 36;

[0119] 8) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 37, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 38, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 39;

[0120] 9) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 40, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 41, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 42;

[0121] 10) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 43, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 44, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 45;

[0122] 11) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 46, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 47, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 48;

[0123] 12) HCDR1 as set forth in the amino acid sequence of SEQ ID NO: 49, HCDR2 as set forth in the amino acid sequence of SEQ ID NO: 50, HCDR3 as set forth in the amino acid sequence of SEQ ID NO: 51;

[0124] 13) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 52, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 53, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 54;

[0125] 14) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 55, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 56, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 57;

[0126] 15) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 58, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 59, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 60.

[0127] In another aspect, there is provided a Nanobody or an antigen binding fragment thereof that specifically binds to a CD3 / TCR complex, characterized in that it comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region;

[0128] The amino acid sequence of the complementarity determining region HCDR1 comprises D or N;

[0129] The amino acid sequence of the complementarity determining region HCDR2 comprises: S-X1-X2-G-X3, wherein X1 is T, I or Y, X2 is S, G or D, and X3 is S, A or T;

[0130] The amino acid sequence of the complementarity determining region HCDR3 comprises: L-X4, or AGFTG, wherein X4 is G or H.

[0131] In some optional embodiments of the present application, X1 is T.

[0132] In some optional embodiments of the present application, X1 is I.

[0133] In some optional embodiments of the present application, X1 is Y.

[0134] In some optional embodiments of the present application, X2 is S.

[0135] In some optional embodiments of the present application, X2 is G.

[0136] In some optional embodiments of the present application, X2 is D.

[0137] In some optional embodiments of the present application, X3 is S.

[0138] In some optional embodiments of the present application, X3 is A.

[0139] In some embodiments of the application, X3 is T.

[0140] In some embodiments of the application, X4 is G.

[0141] In some embodiments of the application, X4 is H.

[0142] In some embodiments of the application, X1 is T, X2 is S, X3 is S.

[0143] In some embodiments of the application, X1 is T, X2 is S, X3 is S, X4 is G.

[0144] In some embodiments of the application, X1 is I, X2 is G, X3 is A.

[0145] In some embodiments of the application, X1 is I, X2 is G, X3 is A, X4 is H.

[0146] In some embodiments of the application, X1 is Y, X2 is S, X3 is A.

[0147] In some embodiments of the application, X1 is Y, X2 is S, X3 is A, X4 is G.

[0148] In some embodiments of the application, X1 is T, X2 is D, X3 is T.

[0149] In some embodiments of the application, X1 is T, X2 is D, X3 is T, X4 is G.

[0150] In some embodiments of the application, X1 is T, X2 is D, X3 is T, the amino acid sequence of the complementarity determining region HCDR3 comprises AGFTG.

[0151] In some embodiments of the application, the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the nanobody or antigen binding fragment thereof are selected from any one of:

[0152] 1) the amino acid sequence of the complementarity determining region HCDR1 comprises D, the amino acid sequence of the complementarity determining region HCDR2 comprises S-X1-S-G-X3, the amino acid sequence of the complementarity determining region HCDR3 comprises L-G, wherein X1 is T or Y, X3 is S or A;

[0153] 2) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-I-G-G-A, the amino acid sequence of the complementarity determining region HCDR3 comprises L-H;

[0154] 3) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-D-G-T, and the amino acid sequence of the complementarity determining region HCDR3 comprises L-G or AGFTG.

[0155] In some alternative embodiments of the application, the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the Nanobody or antigen-binding fragment thereof are selected from any one of:

[0156] 1) the amino acid sequence of the complementarity determining region HCDR1 comprises D, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-S-G-S, and the amino acid sequence of the complementarity determining region HCDR3 comprises L-G;

[0157] 2) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-I-G-G-A, and the amino acid sequence of the complementarity determining region HCDR3 comprises L-H;

[0158] 3) the amino acid sequence of the complementarity determining region HCDR1 comprises D, the amino acid sequence of the complementarity determining region HCDR2 comprises S-Y-S-G-A, and the amino acid sequence of the complementarity determining region HCDR3 comprises L-G;

[0159] 4) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-D-G-T, and the amino acid sequence of the complementarity determining region HCDR3 comprises L-G;

[0160] 5) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-D-G-T, and the amino acid sequence of the complementarity determining region HCDR3 comprises AGFTG.

[0161] In some alternative embodiments of the application, the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the Nanobody or antigen-binding fragment thereof are selected from any one of:

[0162] 1) has a HCDR1 as shown in the amino acid sequence of SEQ ID NO: 16, has a HCDR2 as shown in the amino acid sequence of SEQ ID NO: 17, and has a HCDR3 as shown in the amino acid sequence of SEQ ID NO: 18;

[0163] 2) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 19, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 20, and has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 21;

[0164] 3) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 22, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 23, and has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 24;

[0165] 4) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 25, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 26, and has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 27;

[0166] 5) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 28, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 29, and has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 30.

[0167] According to an embodiment of the present application, the above-mentioned nanobody or antigen binding fragment thereof can further have at least one of the following technical features:

[0168] In some optional embodiments of the present application, the nanobody or antigen binding fragment thereof is used for specifically binding to CD3 / TCR complex.

[0169] In some optional embodiments of the present application, the heavy chain variable region contains a heavy chain framework region from at least one of a camelid-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof.

[0170] In some optional embodiments of the present application, the heavy chain framework region comprises HFR1, HFR2, HFR3, and HFR4.

[0171] In some optional embodiments of the present application, the HFR1 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117, or an amino acid sequence with at least 85% identity thereto, or a humanized modification thereof.

[0172] In the present disclosure, the term "humanization modification" refers to modification of amino acid sites of a target region of a non-human antibody to eliminate or reduce immunogenicity of the antibody, and to improve safety and therapeutic effect of the antibody. Exemplarily, the humanization modification of the framework region can be mutation of amino acid sites of the framework region to achieve the purpose of reducing immunogenicity, and the specific mutation position and number are not limited and are within the protection scope of the present disclosure.

[0173] In some optional embodiments of the present disclosure, the HFR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, and 118, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof.

[0174] In some optional embodiments of the present disclosure, the HFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, and 119, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof.

[0175] In some optional embodiments of the present disclosure, the HFR4 comprises an amino acid sequence as shown in any one of SEQ ID NO: 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, and 120, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof.

[0176] In some optional embodiments of the present disclosure, the heavy chain framework region is selected from any one of the following groups:

[0177] 1) HFR1 as shown in an amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as shown in an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as shown in an amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as shown in an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0178] 2) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0179] 3) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0180] 4) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 76;

[0181] 5) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0182] 6) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0183] 7) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 86, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0184] 8) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 91, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0185] 9) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 95, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 96, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0186] 10) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 98, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 99, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof;

[0187] 11) HFR1 having an amino acid sequence as set forth in SEQ ID NO: 101, or an amino acid sequence having at least 85% identity thereto, or a humanized modification of the amino acid sequence, HFR2 having an amino acid sequence as set forth in SEQ ID NO: 102, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence, HFR3 having an amino acid sequence as set forth in SEQ ID NO: 103, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence, HFR4 having an amino acid sequence as set forth in SEQ ID NO: 104, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence;

[0188] 12) HFR1 having an amino acid sequence as set forth in SEQ ID NO: 105, or an amino acid sequence having at least 85% identity thereto, or a humanized modification of the amino acid sequence, HFR2 having an amino acid sequence as set forth in SEQ ID NO: 106, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence, HFR3 having an amino acid sequence as set forth in SEQ ID NO: 107, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence, HFR4 having an amino acid sequence as set forth in SEQ ID NO: 108, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence;

[0189] 13) HFR1 having an amino acid sequence as set forth in SEQ ID NO: 109, or an amino acid sequence having at least 85% identity thereto, or a humanized modification of the amino acid sequence, HFR2 having an amino acid sequence as set forth in SEQ ID NO: 110, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence, HFR3 having an amino acid sequence as set forth in SEQ ID NO: 111, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence, HFR4 having an amino acid sequence as set forth in SEQ ID NO: 112, or an amino acid sequence having at least 90% identity thereto, or a humanized modification of the amino acid sequence;

[0190] 14) HFR1 as shown in the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence having at least 85% identity thereto, or an amino acid sequence of humanized modification thereof, HFR2 as shown in the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence of humanized modification thereof, HFR3 as shown in the amino acid sequence of SEQ ID NO: 115, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence of humanized modification thereof, HFR4 as shown in the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence of humanized modification thereof;

[0191] 15) HFR1 as shown in the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence having at least 85% identity thereto, or an amino acid sequence of humanized modification thereof, HFR2 as shown in the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence of humanized modification thereof, HFR3 as shown in the amino acid sequence of SEQ ID NO: 119, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence of humanized modification thereof, HFR4 as shown in the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence of humanized modification thereof.

[0192] In some optional embodiments of the present application, the heavy chain framework region is selected from any one of the following groups:

[0193] HFR1: X5VQX6VESGGGLVQX7GGSLRLSCVAS, X5 is Q or E, X6 is V or L, X7 is V or P;

[0194] HFR2: MAWYRQAPGKQRELVAR;

[0195] HFR3: YYADSVKGRFTISRGNAKNX8X9YLRMNX 10 X 11 RX 12 EDTAVYYC, X8 is I or S, X9 is V or L, X 10 is I or S, X 11 is V or L, X 12 is P or A;

[0196] HFR4: WGQGTX 13 VTVSS, X 13 is Q or L.

[0197] In some alternative embodiments of the application, the heavy chain framework regions are selected from:

[0198] HFR1 : EVQLVESGGGLVQVGGSLRLSCAAS,

[0199] HFR2: MAWYRQAPGKQRELVAR,

[0200] HFR3: YYADSVKGRFTISRGNAKNIVYLRMNILRAEDTAVYYC,

[0201] HFR4: WGQGTLVTVSS. See, for example, the R4792 antibody in the Examples.

[0202] In some alternative embodiments of the application, the heavy chain framework regions are selected from:

[0203] HFR1 : EVQVVESGGGLVQVGGSLRLSCVAS,

[0204] HFR2: MAWYRQAPGKQRELVAR,

[0205] HFR3: YYADSVKGRFTISRGNAKNIVYLRMNIVRPEDTAVYYC,

[0206] HFR4: WGQGTLVTVSS. See, for example, the R4380 antibody in the Examples.

[0207] In some alternative embodiments of the application, the heavy chain framework regions are selected from:

[0208] HFR1 : EVQLVESGGGLVQPGGSLRLSCAAS,

[0209] HFR2: MAWYRQAPGKQRELVAR,

[0210] HFR3: YYADSVKGRFTISRGNAKNIVYLRMNSLRAEDTAVYYC,

[0211] HFR4: WGQGTLVTVSS. See, for example, the R4466 antibody in the Examples.

[0212] In some alternative embodiments of the application, the heavy chain framework regions are selected from:

[0213] HFR1 : EVQLVESGGGLVQPGGSLRLSCAAS,

[0214] HFR2: MAWYRQAPGKQRELVAR,

[0215] HFR3: YYADSVKGRFTISRGNAKNILYLRMNILRAEDTAVYYC,

[0216] HFR4: WGQGTLVTVSS. Exemplarily, refer to R4789 antibody in the embodiments.

[0217] In some optional embodiments of the present application, the heavy chain framework region is selected from:

[0218] HFR1: EVQLVESGGGLVQPGGSLRLSCAAS,

[0219] HFR2: MAWYRQAPGKQRELVAR,

[0220] HFR3: YYADSVKGRFTISRGNAKNILYLRMNILRAEDTAVYYC,

[0221] HFR4: WGQGTLVTVSS. Exemplarily, refer to R4790 antibody in the embodiments.

[0222] In some optional embodiments of the present application, the heavy chain framework region is selected from:

[0223] HFR1: EVQLVESGGGLVQPGGSLRLSCAAS,

[0224] HFR2: MAWYRQAPGKQRELVAR,

[0225] HFR3: YYADSVKGRFTISRGNAKNILYLRMNILRAEDTAVYYC,

[0226] HFR4: WGQGTLVTVSS. Exemplarily, refer to R4791 antibody in the embodiments.

[0227] In some optional embodiments of the present application, the nanobody or the antigen binding fragment thereof has a heavy chain variable region as shown in any one of SEQ ID NOs: 1-15, 138-143 or a heavy chain variable region having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% sequence identity thereto.

[0228] Exemplarily, with reference to the heavy chain variable region shown in SEQ ID NO: 1, SEQ ID NOs: 138-143 have at least 90% identity with SEQ ID NO: 1, provided that the CDRs are unchanged.

[0229] In some embodiments of the present application, the nanobody or antigen-binding fragment thereof has a heavy chain variable region as set forth in any one of SEQ ID NOs: 1-15, 138-143.

[0230] In some embodiments of the present application, the nanobody or antigen-binding fragment thereof further comprises a constant region of a heavy chain.

[0231] In some embodiments of the present application, the constant region of the heavy chain is connected to the C-terminus of the heavy chain variable region at the N-terminus.

[0232] In some embodiments of the present application, the heavy chain constant region is from a sequence of at least one of a portion or all of a camelid-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof.

[0233] In some embodiments of the present application, the heavy chain constant region comprises a sequence of a partial constant region or a complete constant region of at least one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a mutant thereof.

[0234] In some embodiments of the present application, the heavy chain constant region is a sequence of a partial or complete constant region of IgG1.

[0235] In some embodiments of the present application, the heavy chain constant region is a sequence of a partial or complete constant region of human-derived IgG1.

[0236] In some embodiments of the present application, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO: 121 or an amino acid sequence having at least 80% sequence identity thereto.

[0237] In some embodiments of the present application, the nanobody antigen-binding fragment comprises one or more of F(ab’)2, Fab’, Fab, Fv, scFv, dsFv, and a minimal recognition unit of an antibody.

[0238] In some alternative embodiments of the present application, the nanobody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 122-136 or an amino acid sequence having at least 80% sequence identity thereto, or has an amino acid sequence after splicing of an amino acid sequence as shown in any one of SEQ ID NOs: 138-143 with an amino acid sequence as shown in SEQ ID NO: 121 or an amino acid sequence having at least 80% sequence identity thereto. Exemplarily, the "amino acid sequence after splicing of an amino acid sequence as shown in SEQ ID NO: 138 with an amino acid sequence as shown in SEQ ID NO: 121" refers to an amino acid sequence after splicing of the C-terminus of SEQ ID NO: 138 with the N-terminus of SEQ ID NO: 121.

[0239] biological material

[0240] In a second aspect, the present application provides a biological material comprising any one of (i) to (vi) below:

[0241] (i) a multispecific antibody, a recombinant protein and an immunoconjugate containing the nanobody or antigen-binding fragment thereof as shown in any one of the preceding embodiments.

[0242] (ii) a chimeric antigen receptor (CAR) comprising an extracellular region, a transmembrane region and an intracellular region; wherein the extracellular region comprises the nanobody or antigen-binding fragment thereof of the first aspect.

[0243] (iii) a nucleic acid molecule encoding the nanobody or antigen-binding fragment thereof of the first aspect, the multispecific antibody, recombinant protein and immunoconjugate of (i), or the chimeric antigen receptor of (ii).

[0244] (iv) a vector carrying the nucleic acid molecule of (iii).

[0245] (v) a recombinant cell carrying the nucleic acid molecule of (iii) or the vector of (iv); or the recombinant cell expresses the nanobody or antigen-binding fragment thereof of the first aspect, the multispecific antibody, recombinant protein and immunoconjugate of (i), or the chimeric antigen receptor of (ii).

[0246] (vi) an engineered immune effector cell expressing the chimeric antigen receptor of (ii) or containing a nucleic acid molecule encoding the chimeric antigen receptor of (iii).

[0247] In some embodiments of the present application, the multispecific antibody, the recombinant protein and the immunoconjugate comprise the first binding region, which comprises the nanobody or the antigen-binding fragment thereof as shown in any of the preceding embodiments.

[0248] In some embodiments of the present application, the multispecific antibody is a bispecific antibody, which further comprises a second binding region having a specific binding to a target molecule activity.

[0249] In some embodiments of the present application, the target molecule is selected from a tumor surface antigen or an immune checkpoint.

[0250] In some embodiments of the present application, the tumor surface antigen includes, but is not limited to, MUC16, VEGFR-2 (KDR / FIK-1), K-RAS, CD2, CD3, CD19, CD19, CD20, CD21, CD22, CD30, CD32B, CD33, CD38, CD39, CD40, CD45, CD52, CD70, CD80, CD60, CD62, CD72, CD79a, CD79B, CD123, EGFR, HER2, HER3, HER4, GPC3, CLL1, TGF-β, TGF-beta RII, VEGF, GD3, CCR4, CCR5, BCMA, CTLA4, mesothelin, OX40.

[0251] In some embodiments of the present application, the immune checkpoint includes, but is not limited to, PD-1, PD-L1, PD-L2, TIGIT, CD47, CD27, CD28, CD40, CD122, CD137, CD94 / NKG2A, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, LAG3, TIM-3, VISTA, GARP, PS, CSF1R, TDO, CTLA-4, IDO, KIR, GITR, TNFR, FasR / DcR and CEACAM1.

[0252] According to embodiments of the present application, the recombinant protein or the immunoconjugate further comprises at least one of a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

[0253] According to embodiments of the present application, the biologically active protein or the fragment thereof includes at least one selected from a protein tag, a protein toxin or a fragment thereof, a tumor necrosis factor or a fragment thereof, an interferon or a fragment thereof, a biological response modifier or a fragment thereof, and an Fc fragment.

[0254] In the present disclosure, "protein tag" generally refers to a polypeptide or protein fused with a target protein (antibody or antigen binding fragment) for expression, detection, tracking or purification of the target protein. It includes but is not limited to His tag (also known as His-Tag, sequence HHHHHH), Flag tag (also known as Flag-Tag, sequence DYKDDDDK), GST tag (also known as GST-Tag, glutathione S-transferase tag), MBP tag (also known as MBP-Tag, maltose binding protein tag), SUMO tag and C-Myc tag, etc.

[0255] In the present disclosure, "toxin" generally refers to a substance toxic to a host, including protein toxins and non-protein toxins. The protein toxins include but are not limited to abrin, ricin A, pseudomonas exotoxin and diphtheria toxin, etc. In the present disclosure, the protein toxin is preferably a protein toxin with enzymatic activity.

[0256] In the present disclosure, "tumor necrosis factor" generally refers to a substance capable of causing hemorrhagic necrosis of various tumors, including but not limited to TNF-α and TNF-β.

[0257] In the present disclosure, "interferon" generally refers to a glycoprotein with direct killing or inhibiting virus. It includes but is not limited to IFN-α, INF-β and IFN-γ.

[0258] In the present disclosure, "biological response modifier" generally refers to a class of protein substances that directly or indirectly enhance the anti-tumor effect of the immune system. It includes but is not limited to lymphokines, IL-2, IL-6, IL-10 and GM-CSF, etc.

[0259] In the present disclosure, "Fc fragment" generally refers to the Fc region from IgG (such as IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM, including CH2, CH3 region and optionally hinge region. Preferably, IgG, IgA1, IgA2, IgD, IgE or IgM is derived from murine, human, primate or camelid.

[0260] The present disclosure provides a chimeric antigen receptor (CAR), comprising an extracellular region, a transmembrane region and an intracellular region; wherein the extracellular region comprises the nanobody or antigen binding fragment thereof of the first aspect.

[0261] In some optional embodiments of the present disclosure, the antigen binding domain contains a single-chain antibody scFv, wherein the heavy chain variable region has the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the nanobody or antigen binding fragment thereof of any one of the preceding aspects.

[0262] In some embodiments of the application, the antigen binding domain comprises a single chain antibody (scFv), wherein the heavy chain variable region has the heavy chain variable region of any one of the afore-mentioned Nanobodies or antigen binding fragments thereof.

[0263] In the process of preparing or obtaining the Nanobodies or antigen binding fragments thereof, the multispecific antibodies, the recombinant proteins and immunoconjugates, the chimeric antigen receptors of the first aspect, the nucleic acids encoding these Nanobodies or antigen binding fragments thereof, the multispecific antibodies, the chimeric antigen receptors, the recombinant proteins can be ligated to different vectors and then expressed in different cells to obtain the corresponding Nanobodies or antigen binding fragments thereof, the multispecific antibodies, the chimeric antigen receptors, the recombinant proteins.

[0264] The present application provides a nucleic acid molecule encoding the Nanobodies or antigen binding fragments thereof, the multispecific antibodies, the chimeric antigen receptors, or the recombinant proteins of the first aspect. The nucleic acid according to the embodiments of the present application can encode the Nanobodies or antigen binding fragments thereof, the multispecific antibodies, the chimeric antigen receptors, the recombinant proteins and the immunoconjugates.

[0265] According to the embodiments of the present application, the nucleic acid comprises DNA or RNA.

[0266] It should be noted that for the nucleic acid mentioned herein, a person skilled in the art should understand that either one of the complementary double strands or both are actually included. For the convenience, in the present application, although only one strand is given in most cases, the other complementary strand is also disclosed. In addition, the sequence of the molecule in the present application comprises DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0267] The present application provides a vector carrying the above-mentioned nucleic acid molecule. In the process of ligating the above-mentioned nucleic acid molecule to the above-mentioned vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control elements.

[0268] "Operably linked" herein refers to the linkage of an exogenous gene to a vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the vector according to some embodiments of the present application is introduced into a suitable recipient cell, the expression of the aforementioned nanobodies or antigen-binding fragments thereof, multispecific antibodies, chimeric antigen receptors, recombinant proteins can be effectively realized under the mediation of the regulation system, thereby realizing the in vitro mass acquisition of nanobodies or antigen-binding fragments thereof, multispecific antibodies, chimeric antigen receptors, recombinant proteins.

[0269] According to embodiments of the present application, the vector can refer to a cloning vector, which can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present application is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0270] In some optional embodiments of the present application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0271] In some optional embodiments of the present application, the expression vector is a plasmid expression vector or a lentivirus expression vector.

[0272] The present application provides a recombinant cell carrying the aforementioned nucleic acid molecule, vector; or the recombinant cell expresses the nanobodies or antigen-binding fragments thereof of the first aspect, the multispecific antibodies, chimeric antigen receptors, recombinant proteins or immunoconjugates described above. Using the cell or host under suitable conditions, the aforementioned nanobodies or antigen-binding fragments thereof, multispecific antibodies, chimeric antigen receptors, recombinant proteins and immunoconjugates can be effectively expressed in the cell or host.

[0273] According to embodiments of the present application, the cell is obtained by introducing the aforementioned vector into the cell or host.

[0274] It should be noted that the cell or host of the present application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cell includes fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, etc., insect cells such as Spodoptera exigua, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0275] In an alternative embodiment of the application, the cell is a mammalian cell, including a BHK cell, a CHO cell, an NSO cell or a COS cell, and excluding an animal reproductive cell, a zygote or an embryonic stem cell.

[0276] It is to be understood that the "suitable conditions" as described in the present application refer to the conditions suitable for the expression of the nanobodies or antigen binding fragments thereof, multispecific antibodies, chimeric antigen receptors, recombinant proteins and immunoconjugates as described in the present application. It is readily understood by the skilled person that the conditions suitable for the expression of the nanobodies or antigen binding fragments thereof, multispecific antibodies, chimeric antigen receptors, recombinant proteins include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy cell status, suitable cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited and the skilled person can optimize the most suitable conditions for the expression of the nanobodies or antigen binding fragments thereof, multispecific antibodies, chimeric antigen receptors, recombinant proteins and immunoconjugates according to the specific environment of the laboratory.

[0277] The present application provides an engineered immune effector cell expressing the aforementioned chimeric antigen receptor or containing a nucleic acid molecule encoding the aforementioned chimeric antigen receptor.

[0278] Reagent or kit

[0279] In a third aspect, the present application provides a reagent or kit for detecting the CD3 / TCR complex, comprising the nanobodies or antigen binding fragments thereof of the first aspect.

[0280] In some alternative embodiments of the present application, the product of any of the above aspects, for example, but not limited to, the antibody or antigen binding fragment thereof in the reagent, kit, blocking agent and pharmaceutical composition, is coupled with at least one diagnostic agent and / or therapeutic agent to form an immunoconjugate.

[0281] The diagnostic agent is selected from one or more of a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer;

[0282] The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulatory agent, an anti-apoptotic agent, a photosensitive therapeutic agent, an immunoconjugate and an oligonucleotide.

[0283] The radionuclide includes, but is not limited to 110 In、 111 In、 177 Lu、 18 F、 52 Fe、 62 Cu、 64 Cu、 67Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb and 83 Sr.

[0284] Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0285] Fluorescent labels include, but are not limited to, one or more of Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, Aminocoumarin, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 5-Carboxy-2',4',5',7'-tetrachlorofluorescein, 5-Carboxyfluorescein, 5-Carboxyrhodamine, 6-Carboxyrhodamine, 6-Carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-Nitrobenzo-2-oxa-l,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresyl Fast Violet, Cresyl Blue Violet, Brilliant Cresyl Blue, p-Aminobenzoic acid, Erythrosin, Phthalocyanine, Azomethine, Cyanine, Xanthine, Succinylfluorescein, Rare earth cryptate, Terbium cryptate or chelate, Europium cryptate or chelate, Diamine, Bis-cyanin, La Jolla Blue dye, Allophycocyanin B, Allophycocyanin C, Allophycocyanin R, Thioamine, Phycoerythrin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), Tetramethylrhodamine, and Texas Red.

[0286] Oligonucleotides include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

[0287] Drugs include, but are not limited to, methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, mechlorethamine, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, dactinomycin, doxorubicin, daunorubicin, vinblastine, paclitaxel, harringtonine, and L-asparaginase.

[0288] Immunomodulators include, but are not limited to, one or more of a cytokine, a chemokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon, an erythropoietin, a thrombopoietin, a tumor necrosis factor (TNF), an interleukin (IL), a granulocyte-colony stimulating factor (G-CSF), a granulocyte macrophage-colony stimulating factor (GM-CSF), and a stem cell growth factor.

[0289] Radionuclides include, but are not limited to 111 In、 111 At、 177 Lu、 211 Bi、 212 Bi、 213 Bi、 211 At、 62 Cu、 67 Cu、 90 Y、 125 I、 131 I、 133 I、 32 P、 33 P、 47 Sc、 111 Ag、 67 Ga、 153 Sm、 161 Tb、 152 Dy、 166 Dy、 161 Ho、 166 Ho、 186 Re、 188 Re、 189 Re、 211 Pb、 212 Pb、 223 Ra、 225 Ac、 77 As、 89 Sr、 99 Mo、 105 Rh、 149 Pm、 169 Er、 194 Ir、 58 Co、 80 mBr、 99 mTc、 103 mRh、 109 Pt、 119 Sb、 189 mOs、 192 Ir、 219 Rn、 215 Po、 221 Fr、 255 Fm、 11 C、 13 N、 15 O、 75 Br、 198 Au、 199 Au、 224 Ac、 77 Br、 113 mIn、 95 Ru、 97 Ru、103 Ru, 105 Ru, 107 Hg, 203 Hg, 121 mTe, 122 mTe, 125 mTe, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 142 Pr, 143 Pr, 161 Tb, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, 76 Br and 169 Yb.

[0290] Applications

[0291] In a fourth aspect, the present application provides an application of the nanobody or the antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the reagent or the kit of the third aspect in:

[0292] (I) detecting CD3 / TCR complex or cells expressing CD3 / TCR complex;

[0293] (II) preparing a product for detecting CD3 / TCR complex or cells expressing CD3 / TCR complex.

[0294] Pharmaceutical composition

[0295] In a fifth aspect, the present application further provides a pharmaceutical composition comprising the nanobody or the antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect.

[0296] In some optional embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0297] According to embodiments of the present application, the excipient includes but is not limited to one or more diluents, stabilizers, or pH adjusters, etc. which are pharmaceutically acceptable.

[0298] According to embodiments of the present application, the pharmaceutical composition is an injection.

[0299] It is to be noted that the pharmaceutical composition includes a combination separated in time and / or space as long as it can act together to achieve the purpose of the present application. For example, the components contained in the pharmaceutical composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the pharmaceutical composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0300] The pharmaceutical composition of the present application contains a safe and effective amount of the active ingredient of the present application and a pharmaceutically acceptable adjuvant. Such adjuvants include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical composition of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, sustained-release preparation. For example, it is prepared by conventional methods with normal saline or aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0301] The effective amount of the active ingredient described in the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be given daily, or the dose can be proportionally reduced, according to the exigencies of the therapeutic situation.

[0302] The pharmaceutically acceptable adjuvant described in the present application includes, but is not limited to, water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combinations thereof. The selection of the carrier should be matched with the administration mode, which is well known to a person of ordinary skill in the art.

[0303] Use

[0304] The sixth aspect also provides the use of the nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the pharmaceutical composition of the fifth aspect in:

[0305] (I) preparing a medicament for treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation;

[0306] (II) treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation.

[0307] The terms "treatment" and "prevention" and words stemming therefrom as used herein do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as having a potential benefit or therapeutic effect. Also, the treatment or prevention provided herein can include the treatment or prevention of one or more of the conditions or symptoms of the disease being treated or prevented, e.g., cancer. In addition, "prevention" for the purposes herein can encompass delaying the onset of a disease or its symptoms or conditions.

[0308] Methods

[0309] In a seventh aspect, there is further provided a method for detecting a CD3 / TCR complex or a cell expressing a CD3 / TCR complex, which comprises detecting a sample to be tested using the nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the reagent or kit for detecting a CD3 / TCR complex of the third aspect.

[0310] In an eighth aspect, there is further provided a method for treating, preventing or alleviating a disease, disorder or condition, including a tumor, inflammation, autoimmune disease, infection, immune rejection resulting from organ transplantation; which comprises administering to a subject a therapeutically effective amount of the nanobody or antigen-binding fragment thereof of the first aspect, the biomaterial of the second aspect, or the pharmaceutical composition of the fifth aspect.

[0311] It is noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal to be evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, an individual having cancer, an individual having an autoimmune disease, an individual having a pathogen infection, and the like. The subject can be a human, but also includes other mammals, particularly mammals useful as laboratory models for human diseases, e.g., mice, rats, and the like.

[0312] The effective amount of the nanobody or antigen-binding fragment thereof, multispecific antibody, chimeric antigen receptor, recombinant protein, nucleic acid, vector, cell or host, or pharmaceutical composition described herein can vary depending on the mode of administration, the severity of the disease to be treated, and the like. The selection of an optimal effective amount can be made by one of ordinary skill in the art (e.g., by clinical trial). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the weight and immune status of the patient, the route of administration, and the like. For example, the dosage can be administered several times a day, or the dose can be proportionally reduced as appropriate, by the exigencies of the therapeutic situation.

[0313] In the present disclosure, the tumor, inflammation, autoimmune disease, infection, and immune rejection caused by organ transplantation include, but are not limited to, one or more of the following:

[0314] gastric cancer, esophageal cancer, head and neck cancer, bladder cancer, cervical cancer, sarcoma, cell tumor, lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, squamous cell carcinoma, urogenital cancer, rectal cancer, sarcoma, testicular cancer, cervical cancer, mastocytoma, hemangioma, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, rectal cancer, uterine cancer, brain cancer, and glioma.

[0315] In the present disclosure, the tumor includes, but is not limited to, a hematological tumor, and exemplary hematological tumors include, but are not limited to, one or more of the following: hematological tumor chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML), B-cell malignancies, lymphoma (Hodgkins Lymphoma, non-Hodgkins lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, myeloma), and myeloproliferative disorders.

[0316] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be considered as limiting the scope of the present application. If a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained commercially.

[0317] Example 1. Production of Nanobodies Specifically Binding to CD3 / TCR Complex

[0318] The alpaca is immunized by subcutaneous multiple point injection using a fusion protein of recombinant human CD3, Jurkat cells, etc. as immunogen to produce antibodies against human CD3 / TCR complex. After completion of immunization, the peripheral blood of the animal is collected and PBMC is separated to construct a phage library or a mammalian cell display library. After multiple rounds of panning or enrichment using the antigen, the single clone is selected for detection. The clone specifically binding to human CD3 / TCR complex antigen is selected for sequencing, recombinant expression, and purification. In the present embodiment, 15 nanobodies are selected, the amino acid sequences of the heavy chain variable regions of the 15 nanobodies are shown in SEQ ID NO: 1-15, and the amino acid sequences of the heavy chains of the 15 nanobodies are shown in SEQ ID NO: 122-136.

[0319] Example 2. Binding activity of nanobodies specifically binding to CD3 / TCR complex to hCD3-293T cells and 293T cells

[0320] Sufficient hCD3-293T cells and 293T cells overexpressing human CD3 were cultured and collected by centrifugation. Meanwhile, the candidate nanobodies (see Example 1 for details) specifically binding to CD3 / TCR complex, the corresponding positive antibody R3772 (see WO2016180982 for its preparation method, the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 137), the corresponding positive antibody R3632 (i.e. the SP34 antibody known in the art), the negative control R0861 were diluted with PBS+3% FBS, starting from 33.33 nM, 3-fold gradient dilution, 11 concentration gradients were obtained, ready for use. After washing the collected cells with PBS+3% FBS once, the cells were resuspended with PBS+3% FBS to 1×10 6 cells / mL, and the cells were plated in a 96-well plate at 100 μL (1×10 5 cells) per well, 100 μL of diluted antibody was added, and the cells were incubated at 4°C for 30 minutes; centrifuged to remove the supernatant, and the cells were washed with PBS+3% FBS for 1-2 times, and then resuspended with 100 μL of diluted 647 fluorescently labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) per well, and the cells were incubated at 4°C in the dark for 30 minutes, washed with PBS for 1-2 times, resuspended with 100 μL of PBS per well, and then detected on the machine. The median fluorescence intensity was used to analyze and calculate the binding affinity EC 50 values of the antibodies to hCD3-293T and 293T by using the software GraphPad Prism 7.0.

[0321] The results are shown in Figure 1. Among the 15 candidate nanobodies specifically binding to CD3 / TCR complex, 11 molecules of R4068, R4098, R4099, R4100, R4101, R4102, R4103, R4104, R4106, R4110, and R4111 had better binding activity to the overexpressing strain hCD3-293T, and the activity was better than or equivalent to the positive nanobody R3772.

[0322] The results are shown in Figure 1. Among the 15 candidate nanobodies, 14 molecules of R4098, R4099, R4100, R4101, R4102, R4103, R4104, R4105, R4106, R4107, R4108, R4109, R4110, and R4111 had very weak binding activity to 293T, and the candidate R4068 nanobody did not bind to 293T.

[0323] Example 3. Binding activity of Nanobodies specifically binding to CD3 / TCR complex to native Jurkat cells

[0324] Sufficient native tumor cell Jurkat cells were cultured and collected by centrifugation. Meanwhile, the candidate Nanobodies and the corresponding positive antibody were diluted with PBS+3% FBS, starting from a concentration of 33.33 nM, 3-fold gradient dilution, to obtain 11 concentration gradients, ready for use. After washing the collected cells with PBS+3% FBS once, the cells were resuspended with PBS+3% FBS to 1 x 10 6 cells / mL, and the cells were plated in a 96-well plate, 100 μL (1 x 10 5 cells) per well. 100 μL of the diluted antibody was added, and the cells were incubated at 4°C for 30 minutes. After centrifugation to remove the supernatant, the cells were washed with PBS+3% FBS for 1-2 times, and then resuspended with 100 μL of diluted 647 fluorescently labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) per well. The cells were incubated at 4°C in the dark for 30 minutes, washed with PBS for 1-2 times, resuspended with 100 μL of PBS per well, and then detected on the machine. The median fluorescence intensity was used to analyze and calculate the binding affinity EC 50 value of the antibody to Jurkat cells by using the software GraphPad Prism 7.0.

[0325] The results are shown in Figure 2. Among the 15 candidate Nanobodies, R4068, R4101, R4102, R4103, and R4104, a total of 5 molecules, had better binding activity to the native tumor cell line Jurkat, and the activity was better than that of the positive Nanobody R3772.

[0326] Example 4. Binding activity of Nanobodies specifically binding to CD3 / TCR complex to human PBMC

[0327] Sufficient human PBMC cells were cultured and collected by centrifugation. Meanwhile, the candidate Nanobodies and the corresponding positive antibody were diluted with PBS+3% FBS, starting from a concentration of 33.33 nM, 3-fold gradient dilution, to obtain 11 concentration gradients, ready for use. After washing the collected cells with PBS+3% FBS once, the cells were resuspended with PBS+3% FBS to 1 x 10 6 cells / mL, and the cells were plated in a 96-well plate, 100 μL (1 x 10 5PBMCs, centrifuge to remove supernatant, wash cells with PBS+3%FBS for 1-2 times, then resuspend cells with 100 μL diluted 647 fluorescent labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) per well, incubate at 4°C for 30 minutes in the dark, wash with PBS for 1-2 times, then resuspend cells with 100 μL PBS per well, detect on the machine, and calculate the binding affinity EC 50 values of the antibody and cyno PBMCs by using software GraphPad Prism 7.0.

[0328] The results are shown in Figure 3. Of the 15 candidate nanobodies, R4068, R4101, R4102, R4103, and R4104 had better binding activity with human PBMCs, and the activity was better than that of the positive nanobody R3772.

[0329] Example 5. Binding activity of nanobodies specifically binding to CD3 / TCR complex with cyno PBMCs

[0330] Sufficient cyno PBMC cells were cultured, and the cells were collected by centrifugation. Meanwhile, the candidate nanobodies and the corresponding positive antibodies were diluted with PBS+3%FBS, and the concentration was from 33.33nM, 3-fold gradient dilution, to obtain 11 concentration gradients, for standby use. The collected cells were washed with PBS+3%FBS for 1 time, then resuspended with PBS+3%FBS to 1×10 6 cells / mL, and the cells were plated in a 96-well plate, 100 μL (1×10 5 cells) per well, 100 μL of diluted antibody was added, and incubated at 4°C for 30 minutes; centrifuged to remove supernatant, washed cells with PBS+3%FBS for 1-2 times, then resuspended cells with 100 μL diluted 647 fluorescent labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) per well, incubated at 4°C for 30 minutes in the dark, washed with PBS for 1-2 times, then resuspended cells with 100 μL PBS per well, detected on the machine, and calculated the binding affinity EC 50 values of the antibody and cyno PBMCs by using software GraphPad Prism 7.0.

[0331] Results are shown in Figure 4. Of the 15 candidate nanobodies, 5 molecules, R4068, R4101, R4102, R4103, and R4104, had better binding activity with cyno PBMC. Nanobody R3772 did not bind to cyno PBMC.

[0332] Example 6. Binding activity of nanobodies specifically binding to CD3 / TCR complex with mouse PBMC

[0333] Sufficient mouse PBMC cells were cultured, and the cells were collected by centrifugation. Meanwhile, the candidate nanobodies and the corresponding positive antibodies were diluted with PBS+3% FBS, starting from a concentration of 33.33 nM, and 3-fold gradient dilution was performed to obtain 11 concentration gradients for standby. After the collected cells were washed once with PBS+3% FBS, the cells were resuspended in PBS+3% FBS to 1 x 10 6 cells / mL, and the cells were plated in a 96-well plate at 100 μL (1 x 10 5 cells) per well. Then, 100 μL of the diluted antibodies were added, and the cells were incubated at 4°C for 30 minutes. After centrifugation to remove the supernatant, the cells were washed once or twice with PBS+3% FBS, and then 100 μL of diluted 647 fluorescently labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) was added to resuspend the cells. The cells were incubated at 4°C in the dark for 30 minutes, washed once or twice with PBS, resuspended in 100 μL PBS per well, and then subjected to machine detection. The median fluorescence intensity was used to analyze and calculate the binding affinity EC 50 values of the antibodies with mouse PBMC by using software GraphPad Prism 7.0.

[0334] Results are shown in Figure 5. Of the 5 candidate nanobodies, 5 molecules, R4068, R4101, R4102, R4103, and R4104, did not have binding activity with mouse PBMC.

[0335] Example 7. Reporter gene activation activity of nanobodies specifically binding to CD3 / TCR complex

[0336] Sufficient CHO-CD32b cross-linked cells and Jurkat NFAT Luciferase Reporter Gene Cells were cultured and collected by centrifugation. Simultaneously, candidate nanobodies and corresponding positive antibodies were diluted with 1640 complete medium (containing 10% FBS) at 100 nM, with 4-fold serial dilutions to obtain 12 concentration gradients for later use. The collected CHO-CD32b cross-linked cells and Jurkat NFAT Luciferase Reporter Gene Cells were resuspended in 1640 complete medium (containing 10% FBS) to 8 × 10⁻⁶ cells / mL. 5 cells / mL and 4×10 6 Cells / mL were seeded at a rate of 25 μL / well into 96-well flat-bottomed white plates. 50 μL of diluted antibody was added, and the plates were incubated at 37°C and 5% CO2 for 4 hours. The plates were then removed and allowed to equilibrate at room temperature for 10 min. Next, 100 μL of equilibrated Bright-Lumi™ firefly luciferase reporter gene assay reagent was added to each well. The plates were gently shaken on a horizontal shaker for 5 min in the dark. The Lum value was then read using a microplate reader. The reporter gene activation activity (EC5) of the antibody was calculated using the Lum value and analyzed with GraphPadPrism 7.0 software. 50 value.

[0337] As shown in Figure 6, five molecules—R4068, R4101, R4102, R4103, and R4104—among the 15 candidate nanobodies exhibited superior reporter gene activation activity.

[0338] Example 8. Humanization process of nanobodies that specifically bind to the CD3 / TCR complex

[0339] The humanization process is described by taking the humanization of R4068 as an example. Homology modeling is performed on R4068, and the humanization design is performed by the "CDR grafting" method. Based on the modeling results, the matching germline gene is selected as the template, and the CDR of the nanobody is grafted into the framework of the full human antibody VH. The sequence of IGHV3-7*01 germline gene is used as the template in the humanization design process of R4068, and the sequence of hJH1 J region is directly replaced. After generating the corresponding humanized sequence, the key amino acids are checked one by one, and the important difference residues are subjected to back mutation to generate the final humanized sequence. After sequence confirmation, gene synthesis, recombinant expression and purification are performed to obtain the target protein for subsequent activity detection. The humanized molecules corresponding to R4068 are: R4380 (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 143), R4466 (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 139), R4789 (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 140), R4790 (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 141), R4791 (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 142), and R4792 (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 138).

[0340] Example 9. Binding activity of humanized nanobody to human PBMC

[0341] Sufficient human PBMC cells are cultured, and the cells are collected by centrifugation. At the same time, the candidate humanized nanobody (see Example 8 for details) and the corresponding parent nanobody R4068 (see Example 1 for details) are diluted with PBS+3% FBS, and the concentration is started from 30nM, 4-fold gradient dilution, to obtain 8 concentration gradients, ready for use. After washing the collected cells with PBS+3% FBS once, resuspend the cells with PBS+3% FBS to 1x10 6 cells / mL, plate the cells in a 96-well plate, 100μL (1x10 5 cells) per well, add 100μL of diluted antibody, incubate at 4°C for 30 minutes; centrifuge to remove the supernatant, wash the cells with PBS+3% FBS for 1-2 times, then resuspend the cells with 100μL of diluted 647 fluorescent labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) per well, incubate at 4°C in the dark for 30 minutes, wash 1-2 times with PBS, resuspend the cells with 100μL PBS per well, and detect on the machine. The median fluorescence intensity is used to calculate the EC 50 value of the binding affinity of the antibody to human PBMC by software GraphPad Prism 7.0.

[0342] The results show that the six candidate humanized nanobodies all have better binding activity with human PBMC, and the activity is better than or equivalent to the parent nanobody R4068. This application exemplarily displays the results of part of the humanized nanobodies, see FIG. 7.

[0343] Example 10. Binding activity of humanized nanobodies with cyno PBMC

[0344] Sufficient cyno PBMC cells were cultured, and the cells were collected by centrifugation. Meanwhile, the candidate nanobodies (see Example 8) and the corresponding positive antibody R4068 (see Example 1) were diluted with PBS+3% FBS, and the concentration was 30nM, 4-fold gradient dilution, to obtain 8 concentration gradients, for standby. The collected cells were washed once with PBS+3% FBS, and then resuspended in PBS+3% FBS to 1x10 6 cells / mL, and the cells were plated in a 96-well plate, 100μL (1x10 5 cells) per well. 100μL of diluted antibody was added, and incubated at 4°C for 30 minutes. The supernatant was removed by centrifugation, and the cells were washed 1-2 times with PBS+3% FBS. Then 100μL of diluted 647 fluorescent labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) was added to resuspend the cells, and incubated at 4°C in the dark for 30 minutes. The cells were washed 1-2 times with PBS, and then resuspended in 100μL PBS per well. The cells were detected by machine, and the median fluorescence intensity was used to analyze and calculate the binding affinity EC 50 value of the antibody with cyno PBMC by using software GraphPad Prism 7.0.

[0345] The results show that the six candidate humanized nanobodies all have better binding activity with cyno PBMC, and the activity is better than or equivalent to the parent nanobody R4068. This application exemplarily displays the results of part of the humanized nanobodies, see FIG. 8.

[0346] Example 11. Reporter gene activation activity of humanized nanobodies

[0347] Sufficient cross-linking cell CHO-CD32b cells and effector cell Jurkat NFAT Luciferase Reporter Gene Cell cells were cultured and centrifuged to collect the cells. At the same time, the candidate humanized nanobodies (see Example 8 for details) and the corresponding parent nanobody R4068 (see Example 1 for details) were diluted with 1640 complete culture medium (containing 10% FBS) at a concentration of 100 nM, 4-fold gradient dilution, 10 concentration gradients were obtained, and were ready for use. The collected cross-linking cell CHO-CD32b and effector cell Jurkat NFAT Luciferase Reporter Gene Cell were resuspended in 1640 complete culture medium (containing 10% FBS) to 8 x 10 5 6 cells / mL and 4 x 10 6 cells / mL, respectively, and plated in a 96-well flat-bottom full-white plate at 25 μL / well. 50 μL of diluted antibody was added, and incubated at 37°C, 5% CO2 for 4 hours. The 96-well flat-bottom full-white plate was removed, equilibrated at room temperature for 10 min, and then 100 μL / well of equilibrated Bright-LumiTM Firefly Luciferase Reporter Gene Detection Reagent was added. The plate was placed on a horizontal shaker for 5 min, and then placed in an enzyme-labeled instrument to read the Lum value. The Lum value was used to analyze and calculate the EC 50 value of the reporter gene activation activity of the antibody by using software GraphPad Prism 7.0.

[0348] The results showed that the six candidate humanized nanobodies all had relatively optimal reporter gene activation activity, and the activity was equivalent to that of the parent nanobody R4068. This application exemplarily displays the results of some humanized nanobodies, see Figure 9 for details.

[0349] Example 12. Binding of humanized nanobodies to CD3 / TCR complex

[0350] Sufficient empty cell CHO-FP1, 293T, Expi293 cells and cell strains transfected with CD3-TCR complex CHO-hCD3-hTCR (de V region), 293T-hCD3-hTCR (de C and V region), Expi293-mCD3-hTCR cells were collected by centrifugation. At the same time, the candidate humanized nanobodies (see Example 8 for details) and the corresponding positive TCR antibody hTRAC mAb (TRAC Monoclonal Antibody (3A8), ThermoFisher, TCR1145) and positive CD3 antibodies R3772 and R3632 (see Example 2 for details), negative control R0861 (see Example 2 for details) were diluted with PBS+3%FBS, starting from 100nM, 4-fold gradient dilution, 7 concentration gradients were obtained, ready for use. The collected cells were washed once with PBS+3%FBS, then resuspended with PBS+3%FBS to 1×10 6 cells / mL, and plated in a 96-well plate, 100μL (1×10 5 cells) per well, 100μL of diluted antibody was added, and incubated at 4°C for 30 minutes; centrifuged to remove the supernatant, and the cells were washed 1-2 times with PBS+3%FBS, then resuspended with 100μL of diluted 647 fluorescently labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, 109-605-098) or 647 fluorescently labeled anti-mouse IgG Fc antibody (Jackson ImmunoResearch, 115-605-008) per well, and incubated at 4°C in the dark for 30 minutes. Washed 1-2 times with PBS, resuspended the cells with 100μL PBS per well, and detected by machine, then calculated the binding affinity EC 50 values of the antibodies to each empty cell strain and each transfected CD3 / TCR complex cell strain by software GraphPad Prism 7.0.

[0351] The results are shown in Table 1. The candidate humanized nanobody R4792 only has binding activity with the CHO-hCD3-hTCR (de V region) cell strain, indicating that the candidate humanized nanobody R4792 does not bind to the CD3 region or the TCR region alone, but binds to the junction region of the CD3-TCR complex.

[0352] Table 1

[0353] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A Nanobody or antigen-binding fragment thereof that specifically binds to the CD3 / TCR complex, characterized in that, HCDRs defined in a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-15; the HCDRs are determined by Kabat, Chothia, AbM, Contact, or IMGT definition.

2. A Nanobody or antigen-binding fragment thereof that specifically binds to the CD3 / TCR complex, characterized in that, complementarity determining regions HCDR1, HCDR2, and HCDR3 of a heavy chain variable region; the complementarity determining region HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58; the complementarity determining region HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59; the complementarity determining region HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60.

3. The Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that, According to IMGT definition, the complementarity determining regions HCDR1, HCDR2, and HCDR3 of the Nanobody or antigen-binding fragment thereof are selected from any one of: 1) HCDR1 as set forth in an amino acid sequence of SEQ ID NO: 16, HCDR2 as set forth in an amino acid sequence of SEQ ID NO: 17, HCDR3 as set forth in an amino acid sequence of SEQ ID NO: 18; 2) HCDR1 as set forth in an amino acid sequence of SEQ ID NO: 19, HCDR2 as set forth in an amino acid sequence of SEQ ID NO: 20, HCDR3 as set forth in an amino acid sequence of SEQ ID NO: 21; 3) HCDR1 as set forth in an amino acid sequence of SEQ ID NO: 22, HCDR2 as set forth in an amino acid sequence of SEQ ID NO: 23, HCDR3 as set forth in an amino acid sequence of SEQ ID NO: 24; 4) HCDR1 as set forth in an amino acid sequence of SEQ ID NO: 25, HCDR2 as set forth in an amino acid sequence of SEQ ID NO: 26, HCDR3 as set forth in an amino acid sequence of SEQ ID NO: 27; 5) HCDR1 as set forth in an amino acid sequence of SEQ ID NO: 28, HCDR2 as set forth in an amino acid sequence of SEQ ID NO: 29, HCDR3 as set forth in an amino acid sequence of SEQ ID NO: 30; 6) HCDR1 as set forth in an amino acid sequence of SEQ ID NO: 31, HCDR2 as set forth in an amino acid sequence of SEQ ID NO: 32, HCDR3 as set forth in an amino acid sequence of SEQ ID NO: 33; 7) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 34, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 35, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 36; 8) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 37, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 38, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 39; 9) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 40, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 41, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 42; 10) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 43, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 44, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 45; 11) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 46, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 47, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 48; 12) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 49, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 50, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 51; 13) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 52, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 53, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 54; 14) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 55, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 56, has HCDR3 as shown in the amino acid sequence of SEQ ID NO: 57; 15) has HCDR1 as shown in the amino acid sequence of SEQ ID NO: 58, has HCDR2 as shown in the amino acid sequence of SEQ ID NO: 59, has HCDR3 as shown in the amino acid sequence of SEQ ID NO:

60.

4. A Nanobody or antigen-binding fragment thereof that specifically binds to the CD3 / TCR complex, characterized in that, a complementarity determining region HCDR1, a complementarity determining region HCDR2, and a complementarity determining region HCDR3 of a heavy chain variable region; the amino acid sequence of the complementarity determining region HCDR1 comprises D or N; the amino acid sequence of the complementarity determining region HCDR2 comprises: S-X1-X2-G-X3, wherein X1 is T, I or Y, X2 is S, G or D, X3 is S, A or T; the amino acid sequence of the complementarity determining region HCDR3 comprises: L-X4, or AGFTG, wherein X4 is G or H.

5. The Nanobody or antigen-binding fragment thereof according to claim 4, characterized in that, The nanobody or antigen-binding fragment thereof satisfies one or more of the following conditions: a) X1 is T; and / or, X1 is I; and / or, X1 is Y; and / or, X2 is S; and / or, X2 is G; and / or, X2 is D; and / or, X3 is S; and / or, X3 is A; and / or, X3 is T; and / or, X4 is G; and / or, X4 is H; and / or, X1 is T, X2 is S, X3 is S; and / or, X1 is T, X2 is S, X3 is S, X4 is G; and / or, X1 is I, X2 is G, X3 is A; and / or, X1 is I, X2 is G, X3 is A, X4 is H; and / or, X1 is Y, X2 is S, X3 is A; and / or, X1 is Y, X2 is S, X3 is A, X4 is G; and / or, X1 is T, X2 is D, X3 is T; and / or, X1 is T, X2 is D, X3 is T, X4 is G; and / or, X1 is T, X2 is D, X3 is T, the amino acid sequence of the complementarity determining region HCDR3 comprises AGFTG; b) the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the nanobody or antigen-binding fragment thereof are selected from any one of the following: 1) the amino acid sequence of the complementarity determining region HCDR1 comprises D, the amino acid sequence of the complementarity determining region HCDR2 comprises S-X1-S-G-X3, the amino acid sequence of the complementarity determining region HCDR3 comprises L-G, wherein X1 is T or Y, and X3 is S or A; 2) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-I-G-G-A, the amino acid sequence of the complementarity determining region HCDR3 comprises L-H; 3) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-D-G-T, the amino acid sequence of the complementarity determining region HCDR3 comprises L-G or AGFTG; c) the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the nanobody or antigen-binding fragment thereof are selected from any one of the following: 1) the amino acid sequence of the complementarity determining region HCDR1 comprises D, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-S-G-S, the amino acid sequence of the complementarity determining region HCDR3 comprises L-G; 2) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-I-G-G-A, the amino acid sequence of the complementarity determining region HCDR3 comprises L-H; 3) the amino acid sequence of the complementarity determining region HCDR1 comprises D, the amino acid sequence of the complementarity determining region HCDR2 comprises S-Y-S-G-A, the amino acid sequence of the complementarity determining region HCDR3 comprises L-G; 4) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-D-G-T, and the amino acid sequence of the complementarity determining region HCDR3 comprises L-G; 5) the amino acid sequence of the complementarity determining region HCDR1 comprises N, the amino acid sequence of the complementarity determining region HCDR2 comprises S-T-D-G-T, and the amino acid sequence of the complementarity determining region HCDR3 comprises AGFTG; d) the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the Nanobody or antigen binding fragment thereof are selected from any one of: 1) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; 2) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 20, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 21; 3) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 22, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 23, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 24; 4) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 25, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 26, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 27; 5) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 28, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 29, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO:

30.

6. The Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4 to 5, characterized in that, the heavy chain variable region contains a heavy chain framework region from at least one of a camelid-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof; optionally, the heavy chain framework region comprises HFR1, HFR2, HFR3, and HFR4; and / or, the HFR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117, or an amino acid sequence that is at least 85% identical thereto, or a humanized modification thereof; and / or, the HFR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, and 118, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; and / or, the HFR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, and 119, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; and / or, the HFR4 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, and 120, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; and / or, the heavy chain framework region is selected from any one of the following groups: 1) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 2) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 3) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 4) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 5) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 6) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 7) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 86, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 8) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 91, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 9) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 95, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 96, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 10) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 98, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 99, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 11) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 101, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 102, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 103, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 12) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 105, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 106, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 107, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 13) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 109, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 110, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 111, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 112, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 14) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 113, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 114, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 115, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 116, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; 15) HFR1 as set forth in an amino acid sequence of SEQ ID NO: 117, or an amino acid sequence having at least 85% identity thereto, or a humanized modification thereof, HFR2 as set forth in an amino acid sequence of SEQ ID NO: 118, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR3 as set forth in an amino acid sequence of SEQ ID NO: 119, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof, HFR4 as set forth in an amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 90% identity thereto, or a humanized modification thereof; and / or, the heavy chain framework regions are selected from any one of the following groups: HFR1: X5VQX6VESGGGLVQX7GGSLRLSCVAS, X5 is Q or E, X6 is V or L, X7 is V or P; HFR2: MAWYRQAPGKQRELVAR; HFR3: YYADSVKGRFTISR GNAKX8X9YLRMNX 10 X 11 RX 12 EDTAVYYC, X8 is I or S, X9 is V or L, X 10 is I or S, X 11 is V or L, X 12 is P or A; HFR4: WGQGTX 13 VTVSS, X 13 is Q or L; and / or, the heavy chain framework regions are selected from any one of the following groups: 1) HFR1: EVQLVESGGGLVQVGGSLRLSCAAS, HFR2: MAWYRQAPGKQRELVAR, HFR3: YYADSVKGRFTISRGNAKNIVYLRMNILRAEDTAVYYC, HFR4: WGQGTLVTVSS; 2) HFR1: EVQVVESGGGLVQVGGSLRLSCVAS, HFR2: MAWYRQAPGKQRELVAR, HFR3: YYADSVKGRFTISRGNAKNIVYLRMNIVRPEDTAVYYC, HFR4: WGQGTLVTVSS; 3) HFR1: EVQLVESGGGLVQPGGSLRLSCAAS, HFR2: MAWYRQAPGKQRELVAR, HFR3: YYADSVKGRFTISRGNAKNIVYLRMNSLRAEDTAVYYC, HFR4: WGQGTLVTVSS; 4) HFR1: EVQLVESGGGLVQPGGSLRLSCAAS, HFR2: MAWYRQAPGKQRELVAR, HFR3: YYADSVKGRFTISRGNAKNSLYLRMNILRAEDTAVYYC, HFR4: WGQGTLVTVSS; 5) HFR1: EVQLVESGGGLVQPGGSLRLSCAAS, HFR2: MAWYRQAPGKQRELVAR, HFR3: YYADSVKGRFTISRGNAKNILYLRMNILRAEDTAVYYC, HFR4: WGQGTLVTVSS; 6) HFR1: EVQLVESGGGLVQVGGSLRLSCAAS, HFR2: MAWYRQAPGKQRELVAR, HFR3: YYADSVKGRFTISRGNAKNSVYLRMNILRAEDTAVYYC, HFR4: WGQGTLVTVSS.

7. The Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4 to 5, characterized in that, the nanobody or the antigen-binding fragment thereof has a heavy chain variable region as set forth in any one of SEQ ID NOs: 1-15, 138-143 or a sequence having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% sequence identity thereto; optionally, further comprising a constant region of a heavy chain; optionally, the constant region of the heavy chain is connected to the C-terminus of the heavy chain variable region at the N-terminus; optionally, the constant region of the heavy chain is from at least one of a sequence of a part or all of a camelid-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof; and / or, the constant region of the heavy chain comprises a sequence of a part or all of a constant region of at least one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a mutant thereof; and / or, the constant region of the heavy chain is a sequence of a part or all of a constant region of IgGl; and / or, the constant region of the heavy chain is a sequence of a part or all of a constant region of a human-derived IgGl; and / or, the constant region of the heavy chain has an amino acid sequence as set forth in SEQ ID NO: 121 or an amino acid sequence having at least 80% sequence identity thereto; optionally, the nanobody or the antigen-binding fragment thereof has a human monkey cross-reactivity; and / or, the nanobody comprises one or more of a full-length monoclonal antibody, a nanobody, a multispecific antibody; and / or, the antigen-binding fragment of the nanobody comprises one or more of F(ab’)2, Fab’, Fab, Fv, scFv, dsFv, and a minimal recognition unit of an antibody.

8. A biomaterial, characterized by, the biomaterial comprises any one of (i) to (vi): (i) a multispecific antibody, a recombinant protein, and an immunoconjugate, comprising the nanobody or the antigen-binding fragment thereof of any one of claims 1-7; preferably, the multispecific antibody, the recombinant protein, and the immunoconjugate comprise a first binding region, the first binding region comprising the nanobody or the antigen-binding fragment thereof of any one of claims 1-7; preferably, the multispecific antibody is a bispecific antibody, the bispecific antibody further comprising a second binding region, the second binding region having a specific binding activity to a target molecule; preferably, the target molecule is selected from a tumor surface antigen or an immune checkpoint; (ii) a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular region, a transmembrane region, and an intracellular region; wherein the extracellular region comprises the nanobody or the antigen-binding fragment thereof of any one of claims 1-7; (iii) a nucleic acid molecule, the nucleic acid molecule encoding the nanobody or the antigen-binding fragment thereof of any one of claims 1-7, the multispecific antibody, the recombinant protein, and the immunoconjugate of (i), or the chimeric antigen receptor of (i); (iv) a vector carrying the nucleic acid molecule of (iii); (v) a recombinant cell characterized in that it comprises the nucleic acid molecule of (iii), or the vector of (iv), or expresses the Nanobody or antigen-binding fragment thereof of any one of claims 1-7, or expresses the multispecific antibody, recombinant protein and immunoconjugate of (i), or expresses the chimeric antigen receptor of (ii); (vi) an engineered immune effector cell expressing the chimeric antigen receptor of (ii) or containing a nucleic acid molecule encoding the chimeric antigen receptor of (ii).

9. A reagent or kit characterized in that, comprising the Nanobody or antigen-binding fragment thereof of any one of claims 1-7.

10. The Nanobody or antigen-binding fragment thereof of any one of claims 1-7, the biomaterial of claim 8, or the reagent or kit of claim 9, for use in detecting a CD3 / TCR complex or a cell expressing a CD3 / TCR complex, or having the following among: (I) detecting a CD3 / TCR complex or a cell expressing a CD3 / TCR complex; (II) preparing a product for detecting a CD3 / TCR complex or a cell expressing a CD3 / TCR complex.

11. A pharmaceutical composition, characterized by, the pharmaceutical composition comprising the Nanobody or antigen-binding fragment thereof of any one of claims 1-7, the biomaterial of claim 8; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

12. The Nanobody or antigen-binding fragment thereof of any one of claims 1-7, the biomaterial of claim 8, or the pharmaceutical composition of claim 11, for use in treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, an autoimmune disease, an infection, immune rejection of organ transplantation, or having the following among: (I) preparing a medicament for treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, an autoimmune disease, an infection, immune rejection of organ transplantation; (II) treating, preventing or alleviating a disease, disorder or condition associated with a tumor, inflammation, an autoimmune disease, an infection, immune rejection of organ transplantation.

13. A method of detecting a CD3 / TCR complex or detecting a cell expressing a CD3 / TCR complex, characterized in that, The method comprises: detecting a test sample using the Nanobody or antigen-binding fragment thereof of any one of claims 1-7, the biomaterial of claim 8, or the reagent or kit of claim 9.

14. A method of treating, preventing or alleviating a disease, disorder, or condition, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-13. The method comprises: administering to a subject a therapeutically effective amount of the Nanobody or antigen-binding fragment thereof of any one of claims 1-7, the biomaterial of claim 8, or the pharmaceutical composition of claim 11; Preferably, the disease, disorder or condition comprises a tumor, inflammation, an autoimmune disease, an infection, immune rejection of organ transplantation.

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