Tumor-specifically activated protein drug and use thereof

Abstract

Provided is a cleavable sequence (CS) that can be recognized and cleaved by a protease specifically expressed in the tumor microenvironment. The CS sequence can be recognized and cleaved by a protease specifically expressed in the tumor microenvironment. Further provided is a single-domain antibody capable of neutralizing the activity of an SP34 antibody. Further provided is an isolated polypeptide, which can be a tumor-specifically activated protein drug, and is a prodrug structure having no or extremely low biological activity. The isolated polypeptide comprises 1) a polypeptide (aCD3) that targets and binds to a CD3 molecule on the surface of a T cell; 2) domain 1 (BD1) that blocks the binding of aCD3 to a CD3 molecule on the surface of a T cell; 3) an antibody fragment (aTA) that recognizes and binds to a tumor antigen; and 4) a cleavable linker peptide (CL) that contains a cleavable sequence (CS) and / or 5) domain 2 (BD2) that blocks the binding of aTA to a target antigen thereof.

Description

Tumor-specifically activated protein drugs and applications thereof TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to a tumor-specifically activated protein drug and applications thereof. BACKGROUND

[0002] T-cell engager (TCE) is a kind of bispecific antibody targeting tumor antigen and T cell surface CD3 molecule, which activates T cells by forming an artificial immune synapse between target cells and T cells, releases perforin and granzyme to exert cytotoxic activity, and kills cancer cells. TCE directly targets CD3, bypasses the dependence of TCR recognition on specific peptide-major histocompatibility complex (pMHC), and is not limited by the down-regulation of MHC antigen presentation in cancer cells. Therefore, TCE has always been a hot spot in the research and development of tumor drugs.

[0003] So far, there are only a few TCEs approved for marketing, far fewer than monoclonal antibodies. In 2009, Catumaxomab (mouse-rat hybrid IgG antibody targeting EpCAM / CD3) became the first bispecific TCE approved for marketing, with a clinical indication for malignant ascites. Although Catumaxomab generated cancer-specific T cell responses, the Fc domain of the antibody also interacted with other immune cells. Patients showed hepatotoxicity from cytokine release syndrome (CRS) and off-target FcγR interactions, and Catumaxomab was eventually withdrawn from the market in 2017. In 2014, Blinatumomab became the second bispecific TCE approved for marketing. Unlike Catumaxomab, Blinatumomab is a CD19 x CD3 single-chain antibody (scFv) structure for the treatment of acute lymphoblastic leukemia. In recent years, the number of TCEs approved has begun to increase, but it is still concentrated in hematological tumors, and the progress of TCEs in solid tumors is very slow. This is mainly because, although TCEs have significant tumor cell killing activity, they are also accompanied by significant side effects, the most notable of which are CRS and immune effector cell-associated neurotoxicity syndrome (ICANS). The strong activity of TCEs triggers a large amount of cytokine release before reaching the tumor site, leading to severe clinical TEAE (treatment emergent adverse event) events. Grade 3 or higher TEAEs are often an important reason for the clinical failure of TCEs. In addition, too strong activity can also cause damage to normal tissues and organs, such as the TCE bispecific antibody Solitomab targeting EpCAM / CD3, which was terminated in phase 1 clinical trials due to gastrointestinal toxicity (Oncoimmunology. 2018 Apr 18; 7(8): e1450710.).

[0004] Therefore, how to better utilize the strong killing activity of TCEs while avoiding their side effects, and provide safer and more effective treatment drugs for clinical cancer patients, has become a major problem that needs to be solved. SUMMARY

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a protein drug specifically activated in the tumor microenvironment, for solving the problems in the prior art.

[0006] To achieve the above object and other related objects, the present application first provides an isolated polypeptide comprising a cleavable sequence (CS) that can be recognized and cleaved by a proteinase specifically expressed in a tumor microenvironment. The CS sequence is obtained by screening and comprises an amino acid sequence as shown in SEQ ID NO. 1 (LSGRSDRG, designated as CS1). The CS sequence can be recognized and cleaved by a proteinase specifically expressed in a tumor microenvironment, including but not limited to matrix metalloproteinase (MMP), serine protease, cysteine protease, etc. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13 or MMP14. In some embodiments, the serine protease includes urokinase-type plasminogen activator (uPA), Matriptase / MT-SP1 / ST14 (the three can be used interchangeably), Hepsin. In some embodiments, the cysteine protease includes legumain.

[0007] Another aspect of the present application provides a single domain antibody that can neutralize the activity of human CD3 antibody-SP34 clone (PNAS USA. 1990 Sep; 87(18):7220-4). The single domain antibody that can neutralize the activity of SP34 antibody is of a llama origin.

[0008] The single domain antibody that can neutralize the activity of SP34 antibody can be further humanized to become a humanized antibody.

[0009] The present application further provides an isolated polypeptide, which in some embodiments can be a single-chain or multi-chain polypeptide structure. In some specific embodiments, the isolated polypeptide is a tumor-specific activated protein drug.

[0010] The isolated polypeptide comprises, in structure, 1) a polypeptide targeting binding to a T cell surface CD3 molecule (aCD3); 2) a domain 1 (BD1) blocking the binding of aCD3 to a T cell surface CD3 molecule; 3) an antibody fragment recognizing and binding to a tumor antigen (aTA); 4) a cleavable linker peptide (CL) containing a cleavable sequence (CS); and or 5) a domain 2 (BD2) blocking the binding of aTA to its target antigen. The aTA and aCD3 constitute an active molecule TCE (T cell engager) that is not cleavable. There is a cleavable linker peptide (CL) between BD1 and the active molecule TCE, and when BD2 is present, there is also a cleavable linker peptide (CL) between BD2 and the active molecule TCE. Exemplary structures of the isolated polypeptide are shown in FIG. 5, FIG. 7 or FIG. 14.

[0011] In some embodiments of the present application, the aCD3 is selected from a single chain antibody (scFv). In some embodiments, the amino acid sequence of the aCD3 single chain antibody (scFv) is in the order of N to C terminal: VH-VL; in other embodiments, the amino acid sequence of the single chain antibody (scFv) is in the order of N to C terminal: VL-VH. In some embodiments, the aCD3 single chain antibody is derived from SP34, which has the CDRs of the SP34 antibody. In some embodiments, the aCD3 has the CDRs of the antibody shown in SEQ ID NO. 40 or 41, such as the underlined portion of SEQ ID NO. 40 or 41 in the sequence listing.

[0012] In the present application, the antibody fragment (aTA) that recognizes and binds to tumor antigen is selected from an antibody or an antibody fragment; in some embodiments of the present application, the aTA is selected from a Fab fragment of IgG antibody of mammalian origin; in other preferred embodiments of the present application, the aTA is selected from a single domain antibody. The tumor antigen includes but is not limited to EpCAM, Trop-2, HER-2, HER3, EGFR, PSMA, Claudin 6, Claudin 18.2, MUC-1, MUC-6, MUC-17, CEA, MSLN, FRa, etc.

[0013] Wherein the aTA and the aCD3 form an active molecule TCE, which can crosslink tumor target cells and T cells, form an immunological synapse, activate T cells and induce T cells to release perforin and granzyme, and kill tumor cells. Those skilled in the art know that the TCE provided by the present application can be used alone or in combination with other structures. When BD1 exists and is non-covalently bound to aCD3, it will shield the activity of the TCE active molecule, so as to ensure that no toxicity is generated to normal tissues in the peripheral circulation before entering the tumor tissue. When entering the tumor tissue, specific proteases in the tumor microenvironment will cut at the CS sequence, so as to degrade BD1, lose the shielding effect on the TCE active molecule, and restore the activity of the TCE molecule to kill tumor cells.

[0014] In some embodiments of the present application, the BD1 is selected from a single domain antibody (sdAb), a single chain antibody (scFv), an antibody fragment or a shielding peptide (such as xten). In a preferred embodiment of the present application, the BD1 is selected from a single domain antibody, which has an amino acid sequence as shown in any one of SEQ ID NO. 19-21.

[0015] In some embodiments of the present application, a domain 2 (BD2) that blocks the binding of the aTA to its target antigen can be further included. The BD2 can be an antibody, an antigen or an antigen fragment recognized by the aTA, or a masking peptide (e.g., xten). In a preferred embodiment of the present application, the BD2 is selected from the extracellular domain region of EpCAM, and the amino acid sequence is shown in SEQ ID NO. 35.

[0016] In some embodiments of the present application, when the aTA is in the Fab structure, the binding of the BD1 to the aCD3 can change the conformation of the aTA, significantly reducing its affinity to the antigen, that is, the BD1, by binding to the aCD3, also blocks the binding of the aTA to the tumor target antigen; in this case, the BD1 is also the BD2, and additional BD2 can no longer be needed. Exemplary examples are shown in the structures in FIG. 5 and FIG. 26.

[0017] In some embodiments of the present application, a long-acting unit (LA) is further included, and the long-acting unit is selected from a mammalian IgG Fc fragment, serum albumin, a single-domain antibody (ABD) that binds to serum albumin, a single-chain antibody (scFv) that binds to serum albumin, etc. In a preferred embodiment of the present application, the long-acting unit is selected from a single-domain antibody, and the amino acid sequence is shown in SEQ ID NO. 37. The long-acting unit can be connected to the BD1 or the BD2. When the tumor-specific protease recognizes and cleaves the CS sequence, the BD1 and the BD2 (if any) are dissociated from the TCE along with the long-acting unit, so that the activated TCE molecule is quickly metabolized and cleared from the circulation system, reducing the toxicity to normal tissues. BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is the ELISA result of the preferred BD1 clone 20D1 binding to CD3.

[0019] FIG. 2 is the ELISA result of the preferred BD1 clone 20D1 antagonizing the binding of CD3 to aCD3.

[0020] FIG. 3 is the affinity (left) and antagonistic activity (right) of different humanized versions of the preferred BD1 clone 20D1 to aCD3.

[0021] FIG. 4 is a schematic diagram of the structure of Trop2-TCE Ab-1 and DR50726, and HC and LC represent the heavy chain and the light chain of the Fab fragment of the antibody, respectively.

[0022] Figure 5 is a schematic diagram of the structure of proTrop2-TCE DR50722, DR50724, DR50725 and DR50728, and proTrop2-TCE-NC DR50722NC, DR50724NC, DR50725NC and DR50727NC, wherein the red crosses represent the CS sequence, and HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0023] Figure 6 is a schematic diagram of the structure of Trop2-TCE DR50721, wherein HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0024] Figure 7 is a schematic diagram of the structure of proTrop2-TCE DR50723 and proTrop2-TCE-NC DR50723NC, wherein the red crosses represent the CS sequence, and HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0025] Figure 8 is a schematic diagram of the structure of EpCAM-TCE DR50808, wherein HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0026] Figure 9 is a schematic diagram of the structure of proEpCAM-TCE DR50809 and proEpCAM-TCE-NC DR50809NC, wherein the red crosses represent the CS sequence, and HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0027] Figure 10 is a schematic diagram of the structure of EpCAM-TCE DR50812 and DR50814, wherein HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0028] Figure 11 is a schematic diagram of the structure of proEpCAM-TCE-NC DR50813NC and DR50815NC, wherein HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0029] Figure 12A is the in vitro cell killing result of Anti-EpCAM-1H5V4-Fc.

[0030] Figure 12B is the anti-tumor effect of aEpcam-1H5V4-Fc-CPD3-D4 in a HT1376 mouse model.

[0031] Figure 12C is the effect of aEpcam-1H5V4-Fc-CPD3-D4 on the body weight of a HT1376 mouse model.

[0032] Figure 13 is a schematic representation of the structure of EpCAM-TCE DR50818, CH1 and CL represent the heavy chain constant region 1 and the light chain constant region in the CH1-CL domain of the antibody, respectively.

[0033] Figure 14 is a schematic representation of the structure of proEpCAM-TCE DR50819, DR50824, DR50825, DR50828, DR50831, DR50833 and DR50837 and proEpCAM-TCE-NC DR50819NC, wherein the red crosses represent the CS sequence, CH1 and CL represent the heavy chain constant region 1 and the light chain constant region in the CH1-CL domain of the antibody, respectively.

[0034] Figure 15A is the in vitro cell activity of Ab-1 and DR50722NC (left: BxPC3 cells; right: MDA-MB-231 cells).

[0035] Figure 15B is the in vitro cell activity of DR50722 after uPA cleavage.

[0036] Figure 15C is the in vitro cell activity of DR50721 and DR50723NC.

[0037] Figure 15D is the in vitro cell activity of DR50721 and DR50723 after uPA cleavage.

[0038] Figure 15E is the in vitro cell activity of Ab-1, DR50724 and DR50724NC.

[0039] Figure 15F is the in vitro cell activity of Ab-1, DR50725NC (left) and DR50725 after uPA cleavage (right).

[0040] Figure 15G is the in vitro cell activity of Ab-1, DR50726 and DR50727NC.

[0041] Figure 16A is the in vitro cell activity of DR50808 and DR50809.

[0042] Figure 16B is the in vitro cell activity of DR50808, DR50812 and DR50813NC (left: BxPC3 cells; right: MDA-MB-231 cells).

[0043] Figure 16C is the in vitro cell activity of DR50808, DR50814 and DR50815NC (left: BxPC3 cells; right: MDA-MB-231 cells).

[0044] Figure 16D is the in vitro cell activity of DR50818, DR50819 and DR50819NC (left) and DR50819 after ST14 cleavage (right).

[0045] Figure 16E is the in vitro cell activity of DR50818, DR50824 and DR50828 (BxPC3 cells).

[0046] Figure 16F is the in vitro cell activity of DR50818, DR50819, DR50825 and DR50825 after ST14 digestion.

[0047] Figure 16G is the in vitro cell activity of DR50818, DR50824 and DR50828 (BxPC3 cells).

[0048] Figure 16H is the in vitro cell activity of DR50808, DR50831 and DR50831 after MMP9 digestion.

[0049] Figure 16I is the in vitro cell activity of DR50818, DR50832 and DR50832 after MMP9 digestion.

[0050] Figure 17 is the in vitro PBMC killing assay of DR50818, DR50819 and DR50819NC (left: HCT116 cells; right: Colo205 cells).

[0051] Figure 18 is the efficacy results of DR50818, DR50819 and DR50819NC in HT29 mouse model.

[0052] Figure 19 is the serum stability results of DR50819, DR50833 and DR50819PC1.

[0053] Figure 20 is the in vitro digestion results of DR50833 and DR50819PC1.

[0054] Figure 21 is the in vitro cell activity of DR50818, DR50833 and DR50833 after uPA digestion.

[0055] Figure 22 is the in vitro cell activity of DR50818, DR50837 and DR50837 after ST14 (left) and MMP9 (right) digestion.

[0056] Figure 23 is the anti-tumor effect of DR50837 in HT29 mouse model.

[0057] Figure 24 is the effect of DR50837 on body weight of HT29 mouse model.

[0058] Figure 25 is a structural diagram of EGFR-TCE DR51001, HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0059] Figure 26 is a structural schematic of proEGFR-TCE-NC DR51002NC and proEGFR-TCE DR51002 and DR51003, wherein the red cross represents the CS sequence, and HC and LC represent the heavy chain and light chain of the antibody Fab fragment, respectively.

[0060] Figure 27A is the in vitro cell activity of DR51001, DR51002 and DR51002NC.

[0061] Figure 27B is the in vitro cell activity of DR51001, DR51002 and DR51002 after cleavage by ST14 protease.

[0062] Figure 27C is the in vitro cell activity of DR51001, DR51003 and DR51002NC (left: N87 cells; right: NCI-H82 cells).

[0063] Figure 27D is the in vitro cell activity of DR51001, DR51003 and DR51002NC and DR51003 after cleavage by ST14 protease (HT29 cells).

[0064] Figure 28 is the flow binding of DR51001, DR51002, DR51003 and DR51002NC on hCD3 high expression cells.

[0065] Figure 29 is the flow binding of DR51001, DR51002 and DR51003 on hEGFR high expression cells.

[0066] Figure 30 is the anti-tumor effect of DR51003 in HT29 mouse model.

[0067] Figure 31 is the effect of DR51003 on the body weight of HT29 mouse model.

[0068] Figure 32 is the anti-tumor effect of DR51003 in PC-9 mouse model.

[0069] Figure 33 is the effect of DR51003 on the body weight of PC-9 mouse model.

[0070] Figure 34 is the anti-tumor effect of DR51003 in PDX HNSC mouse model.

[0071] Figure 35 is the effect of DR51003 on the body weight of PDX HNSC mouse model.

[0072] Figure 36 is the blood concentration-time curve of DR51003 in cynomolgus monkey. DETAILED DESCRIPTION

[0073] The inventors of the present application have invented, through a large amount of exploratory research, a cleavable sequence (CS) that can be recognized and enzymatically cleaved by a proteinase specifically expressed in a tumor microenvironment. On this basis, the inventors have invented a tumor-specific activated protein drug. The tumor-specific activated protein drug has no activity or very low activity in normal tissues, but when it enters a tumor tissue, it is cleaved by a tumor-specific proteinase to release activity, thereby producing an antitumor activity. On this basis, the present application is completed.

[0074] In the present text, the terms "antigen", "target antigen", "targeted antigen" or "tumor antigen" are used interchangeably and refer to polypeptide or protein molecules, etc. that can be specifically recognized and bound by an antibody.

[0075] The term "antibody" or "immunoglobulin" is used in the present text in accordance with the well-known understanding in the art. With the progress of scientific discoveries, the definition of "antibody" has been continuously expanded. In addition to the classic antibody / immunoglobulin (IgG, IgA, IgD, IgM and IgE) form comprising two identical light chains and two identical heavy chains, single-domain antibodies containing only heavy chains have also been discovered. They are all active molecules with the function of "specifically recognizing and binding to an antigen". Among them, IgG is the most commonly used form of biopharmaceuticals (especially therapeutic monoclonal antibodies) and consists of two Fab fragments, a hinge region and an Fc fragment. Each Fab fragment consists of a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1), the core of which is the "antigen binding site" (containing 6 CDRs) formed by VH and VL. CH1 and CL can be combined into a heterodimer through non-covalent and or disulfide bonds, which are represented by "CHI-CL" in the present text.

[0076] The term "sequence" (for example, in the terms "immunoglobulin sequence", "antibody sequence", "VHH sequence" or "protein sequence", etc.) used in the present text should be generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited interpretation is required in the present text.

[0077] The term "monoclonal antibody" refers to a preparation of antibody molecules consisting of single molecules. Monoclonal antibodies exhibit unique affinity and specificity for a specific epitope.

[0078] The term "domain" refers to a specific three-dimensional structural unit of a polypeptide chain of a protein that is formed by folding and that can maintain its tertiary structure independently of the rest of the protein. Generally, a single domain carries out a particular function of the protein; often, the domain can be removed from the original protein molecule without significantly affecting the function of the rest of the original protein molecule; or the domain, when transferred to another protein molecule, does not significantly affect the function of the rest of the protein molecule and of the domain itself.

[0079] In the present text, "Fab", "Fab fragment", "antibody Fab fragment", "antibody Fab domain" are used interchangeably.

[0080] In the present text, "CH1-CL", "CH1-CL fragment", "antibody CH1-CL fragment", "antibody CH1-CL domain" are used interchangeably.

[0081] In the present application, the terms "single domain antibody (sdAb)" and "VHH (variable heavy domain of heavy chain)" have the same meaning and refer to the variable region of the heavy chain of an antibody, which is the smallest antigen-binding fragment with full function. Usually, the antibody gene naturally lacking the light chain and the constant region 1 (CH1) of the heavy chain is first obtained from the immunized serum of a llama, and then the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region. The single domain antibody comprises four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, which are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of a single domain antibody can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The single domain antibody confers its specificity for an antigen due to the presence of the antigen-binding site.

[0082] The term "IMGT numbering system" is an integrated information system for immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of human and other vertebrates, i.e. THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM, which is available at http: / / imgt.cines.fr / textes / numbering / . The IMGT numbering system is based on the Kabat numbering system, but it is more accurate and more consistent. (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). Antibody light and heavy chain genes are analyzed for the framework and CDR regions of the variable region by accessing the IMGT® website (http: / / www.imgt.org / IMGT_vquest). The "location" of CDRs within the immunoglobulin variable region is conserved between species and occurs in a structure called a "loop," so CDRs and framework regions are readily identified by using a numbering system in which the variable region sequences are aligned using the structural features. This information can be used to graft and substitute CDR residues from an immunoglobulin of one species into an acceptor framework from a human antibody. Unless otherwise indicated, in the specification, claims and appended drawings, antibodies are numbered using the IMGT® numbering method to identify CDR regions and framework regions.

[0083] The term "single-chain antibody (scFv)" generally refers to an antibody formed by the connection of an antibody heavy chain variable region (VH) and a light chain variable region (VL) through a connecting peptide. In sequence, the C-terminus of VH can be connected to the N-terminus of VL through a connecting peptide, or the C-terminus of VL can be connected to the N-terminus of VH through a connecting peptide. Preferably, the connecting peptide is selected from a flexible polypeptide chain composed of alanine and / or serine and / or glycine, and the length of the connecting peptide can be 3-40 amino acids.

[0084] The term "humanized" refers herein to a key technology that reduces the immunogenicity of antibodies of non-human origin (such as murine, rabbit, or llama origin) in humans by genetically engineering the antigen-binding site of the antibody to fuse or reengineer the framework structure of a human antibody, while retaining the ability of the original antibody to specifically bind to the antigen. The humanized antibody molecule has an antigen-binding site from a non-human immunoglobulin, while the rest of the sequence is from a human immunoglobulin sequence. The antigen-binding site can include the entire variable region, or only the CDRs. The antigen-binding site can be wild-type, or modified by one or more amino acid substitutions to be more similar to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., humanized single-domain antibodies containing all three CDRs from a llama). Other forms have one or more CDRs that have been altered with respect to the original antibody.

[0085] The term "pharmaceutical composition" refers to a preparation that is in a form suitable for delivery of an active agent to a subject in need of treatment, and that does not contain amounts of other ingredients that would render it unacceptable for use.

[0086] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredients that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0087] Cleavable Sequence (CS) and Cleavable Linker (CL)

[0088] A large number of studies have shown that in solid tumor tissues, in addition to tumor cells, there are also extracellular matrix (ECM) and basement membrane. ECM is an extracellular scaffold composed of a complex mixture of proteins, which plays a key role in tumor progression. ECM remodeling is a key process in tumor migration and invasion during metastasis. ECM can be remodeled through several processes, including synthesis, contraction, and proteolytic degradation. In order to cross the ECM barrier, tumor cells produce a series of extracellular proteases, including matrix metalloproteinases (MMPs), serine proteases, and cysteine proteases, to degrade ECM components. In normal tissues, however, these proteases are present in very small amounts or are inactivated due to the presence of inhibitors.

[0089] The first aspect of the present application provides an isolated polypeptide that can be recognized and cleaved by a protease specifically expressed in the tumor microenvironment, referred to as a cleavable sequence (CS). The CS sequence can be recognized and cleaved by matrix metalloproteinases (MMPs), serine proteases, cysteine proteases, threonine proteases, or aspartic proteases, etc. Metalloproteinases include but are not limited to MMP-2, MMP-7, MMP-9, MMP-11, MMP-13, MMP-14, etc. In some embodiments, serine proteases include but are not limited to urokinase-type plasminogen activator (uPA), Matriptase (also known as MT-SP1 or ST14), Hepsin, etc. In some embodiments, cysteine proteases include but are not limited to Legumain, etc.

[0090] Due to the heterogeneity of the tumor microenvironment of different solid tumor patients, different CS sequence combinations can increase the cleavage efficiency and improve the treatment effect. In a preferred embodiment of the present application, the CS1 sequence can also be combined with the cleavable sequence shown in SEQ ID NO. 2 (ISSGL, CS7) to form a CS2 sequence (SEQ ID NO. 4); in another preferred embodiment of the present application, the CS1 sequence is combined with the cleavable sequence shown in SEQ ID NO. 3 (PLGLAG, CS8) to form a CS3 sequence (SEQ ID NO. 5). Since different CS sequences are recognized by different proteases, there should be no order restriction for the connection of different CS sequences, for example, CS1-CS7 or CS7-CS1 should have similar cleavage effect, CS1-CS8 or CS8-CS1 should have similar cleavage effect, CS1-CS7-CS8 or CS1-CS8-CS7 or CS7-CS1-CS8 or CS7-CS8-CS1 or CS8-CS1-CS7 or CS8-CS7-CS1 should have similar cleavage effect.

[0091] In some specific embodiments, the CS sequence has a sequence selected from any one of SEQ ID NO. 1-7, SEQ ID NO. 76-80, or a combination thereof.

[0092] On one side or both sides of the CS sequence, a flexible amino acid with increased freedom can be provided to form a cleavable linker (CL). The flexible amino acid with increased freedom can be alanine (A) and / or serine (S) and / or glycine (G), and the number of flexible amino acids can be 1-20 amino acids, preferably 2-18, more preferably 3-15, more preferably 3-10, and most preferably 3-8. It should be emphasized that when the CL sequence does not contain flexible amino acids, the CS sequence is equivalent to the CL sequence. Therefore, it can be said that the CL has a sequence selected from any one of SEQ ID NO. 1-7, SEQ ID NO. 76-95, or a combination thereof.

[0093] In some specific embodiments, the CL sequence containing CS1 has a sequence selected from any one of SEQ ID NO. 1, 4-5, SEQ ID NO. 92-95, or a combination thereof.

[0094] Single-domain antibody capable of neutralizing the activity of SP34 antibody

[0095] The second aspect of the present application provides a single-domain antibody capable of neutralizing the activity of SP34 antibody (PNAS USA. 1990 Sep; 87(18): 7220-4), wherein the complementarity determining regions (CDRs) of the single-domain antibody contain CDR1 to CDR3 with the amino acid sequences shown as follows, and the SP34 antibody refers to an antibody derived from the SP34 clone capable of binding to CD3 molecules on human T cells:

[0096] CDR1 shown in SEQ ID NO: 8,

[0097] CDR2 shown in SEQ ID NO: 9,

[0098] CDR3 shown in SEQ ID NO: 10.

[0099] The single-domain antibody capable of neutralizing the activity of the SP34 antibody is derived from a llama, and the framework regions (FR1 to FR4) thereof contain the amino acid sequences shown as follows:

[0100] FR1 shown in SEQ ID NO: 11,

[0101] FR2 shown in SEQ ID NO: 12,

[0102] FR3 shown in SEQ ID NO: 13,

[0103] FR4 shown in SEQ ID NO: 14.

[0104] The single-domain antibody capable of neutralizing the activity of the SP34 antibody can be further humanized into a humanized antibody, and the framework regions (FR1 to FR4) of the humanized antibody contain the amino acid sequences shown as follows:

[0105] FR1 shown in SEQ ID NO: 15 or 16;

[0106] FR2 shown in SEQ ID NO: 12 or 17;

[0107] FR3 shown in SEQ ID NO: 18;

[0108] FR4 shown in SEQ ID NO: 14.

[0109] Further, the humanized antibody has the amino acid sequence shown in any one of:

[0110] (1) SEQ ID NO: 19 to 21; or

[0111] (2) a sequence having 80% or more identity to any one of the sequences represented by SEQ ID NOs: 19 to 21, and having the function represented by (1). Preferably, a sequence having 85% or more identity to any one of the sequences represented by SEQ ID NOs: 19 to 21, and having the function represented by (1); more preferably, having 90% or more identity; still more preferably, having 95% or more identity; yet still more preferably, having 98% or more identity.

[0112] Tumor-specifically activated protein drugs

[0113] The third aspect of the present application provides an isolated polypeptide comprising 1) a polypeptide targeting binding to a T cell surface CD3 molecule (aCD3); 2) a domain 1 (BD1) blocking the binding of aCD3 to a T cell surface CD3 molecule; 3) an antibody fragment recognizing and binding to a tumor antigen (aTA); 4) a cleavable linker peptide (CL) containing a CS sequence; and / or 5) a domain 2 (BD2) blocking the binding of aTA to its target antigen.

[0114] The aTA and aCD3 constitute an uncleavable active molecule TCE, and there is a cleavable linker peptide (CL) between BD1 and TCE, and when BD2 is present, there is also a cleavable linker peptide (CL) between BD2 and TCE.

[0115] In some embodiments, the isolated polypeptide is a tumor-specifically activated protein drug. The tumor-specifically activated protein drug is a prodrug structure with no or very low biological activity.

[0116] It is well known that T cells recognize exogenous polypeptides on infected or mutated cells through T cell receptors (TCRs). The TCR does not directly recognize the exogenous peptide, but binds to the exogenous peptide / MHC complex with a low affinity of 1-100 uM. Therefore, the T cell response is the result of the avidity effect generated by the joint action of multiple low-affinity TCRs (Omid Vafa and Nathan D. Trinklein, Front Oncol. 2020; 10: 446.). The TCR is a complex of multiple proteins, and the CD3 subunit in it converts the signal of cell surface antigen binding into an intracellular phosphorylation signal cascade. These phosphorylation reactions ultimately activate transcription factors such as NFAT and NFKB, and ultimately trigger the expression and release of cytokines and effector proteins such as granzymes and perforins. The signal strength of the TCR complex ultimately determines the fate of the T cell, including cytotoxic activity, proliferation, exhaustion, and apoptosis.

[0117] In this invention, aCD3 is selected from anti-CD3 antibodies. These anti-CD3 antibodies are derived from the SP34 clone. When the SP34 antibody is used alone, it activates T cells by binding to CD3 on the surface of T cells. Furthermore, when the anti-CD3 antibody is combined with an antibody targeting a tumor antigen to form a bispecific TCE (T cell engager) antibody, an immune synapse is formed between T cells and tumor cells without the need for the binding reaction of the TCR and the exogenous peptide / MHC complex. Classical TCE forms include... TandAb diabody, CrossMAb 2+1 structure (Roche), and IgG heterodimer, etc. In these TCE forms, anti-CD3 antibodies can exist in Fab form or as single-chain antibodies (scFv). In a preferred embodiment of the invention, aCD3 is in the form of a single-chain antibody (scFv). Preferably, the amino acid sequence of aCD3 is as shown in SEQ ID NO. 40; more preferably, the amino acid sequence of aCD3 is as shown in SEQ ID NO. 41.

[0118] Tumor antigens are targets for anticancer drugs and are mainly divided into two categories: tumor-specific antigens (TSA) and tumor-associated antigens (TAA). Tumor-specific antigens (TSA) are endogenously expressed antigens specific to tumor cells. Tumor-associated antigens (TAA) are antigens not specific to tumor cells, present in both tumor and normal tissues, but present in much higher amounts in tumor tissues than in normal tissues; these are known as "overexpressed antigens." The first widely known TAA is HER2 / neu, a well-known target in breast cancer and a member of the epidermal growth factor receptor family. In this invention, the tumor antigens recognized by the aTA include, but are not limited to, EpCAM, Trop-2, HER-2, HER3, EGFR, PSMA, Claudin 6, Claudin 18.2, MUC-1, MUC-6, MUC-17, CEA, MSLN, and FRα.

[0119] The aTA and aCD3 form the active molecule TCE. Therefore, the present invention also provides a TCE that can be used alone as an active molecule or in combination with other structures.

[0120] In some embodiments, the aTA is an antibody Fab domain, and one chain of the antibody Fab domain is linked to CD3 to form the TCE.

[0121] In some embodiments, the aTA is a single domain antibody, the aTA is connected to CD3 via the antibody CH1-CL domain, constituting the TCE. The aTA is located at the N-terminus of the antibody CH1-CL domain and the aCD3 is located at the C-terminus of the antibody CH1-CL domain, or the aTA is located at the C-terminus of the antibody CH1-CL domain and the aCD3 is located at the N-terminus of the antibody CH1-CL domain.

[0122] In some embodiments, the aTA is a Fab structure, such as Trop2 Fab, EpCAM Fab, EGFR Fab, Her2 Fab, etc. In some embodiments, the aTA is a Fab fragment targeting EGFR, preferably the Fab fragment is derived from Zalutumumab, comprising a VH as set forth in SEQ ID NO. 111 and a VL as set forth in SEQ ID NO. 112. In some embodiments, the Fab fragment targeting EGFR has light and heavy chain sequences as set forth in SEQ ID NO. 34, SEQ ID NO. 110. In some embodiments, the aTA is a Fab fragment targeting Trop2, preferably the Fab fragment is derived from hRS7 (Sacituzumab), comprising a VH as set forth in SEQ ID NO. 114 and a VL as set forth in SEQ ID NO. 115. In some embodiments, the Fab fragment targeting Trop2 has light and heavy chain sequences as set forth in SEQ ID NO. 23 and SEQ ID NO. 113. In some embodiments, the aTA is a Fab fragment targeting EpCAM, preferably the Fab fragment is derived from 3622W94, comprising a VH as set forth in SEQ ID NO. 108 and a VL as set forth in SEQ ID NO. 109. In some embodiments, the Fab fragment targeting EpCAM has light and heavy chain sequences as set forth in SEQ ID NO. 27, SEQ ID NO. 107.

[0123] In some embodiments, the aTA is a single domain antibody (VHH or sdAb), such as Trop2 VHH, EpCAM VHH, EGFR VHH, Her2 VHH, etc. In some embodiments, the aTA is an EpCAM VHH, thus the present application also provides an EpCAM VHH having a CDR1 as set forth in SEQ ID NO. 104, a CDR2 as set forth in SEQ ID NO. 105, and a CDR3 as set forth in SEQ ID NO. 106. In some embodiments, the EpCAM VHH has a full length sequence as set forth in SEQ ID NO. 36.

[0124] The aTA and aCD3 constitute an active molecule TCE, and preferred exemplary active molecule TCE is shown in Table 1.

[0125] Table 1 Preferred exemplary TCE active molecule

[0126] Wherein Ab-1 is derived from patent WO2022046658.

[0127] In the present application, aCD3 and aTA constitute a classic TCE structure. TCE can effectively direct T cells and trigger strong tumor killing effect, but also has the risk of uncontrollable and even fatal CRS or ICANS. Especially when the antigen is widely expressed in normal tissues, the risk is particularly significant. So far, CRS has been observed in almost all TCE drugs in clinical trials, such as Blinatumomab, which can only control CRS by adjusting the dose and dexamethasone treatment. The purpose of the present application is to provide a protein drug that has no or very low activity in normal tissues and is specifically activated in tumor microenvironment. To achieve this purpose, the present application designs and provides a domain 1, BD1, which can block the binding of aCD3 to CD3 molecules on the surface of T cells. BD1 effectively shields the activity of active molecule TCE by non-covalent binding with aCD3, to ensure that there is no toxicity to normal tissues in the peripheral circulation before entering the tumor tissue. When entering the tumor tissue, the specific protease in the tumor microenvironment will be cleaved at the CS sequence, so that BD1 is degraded, losing the shielding effect of active molecule TCE, and the activity of TCE molecule is restored, killing tumor cells again.

[0128] In the present application, the preferred BD1 domain is selected from single domain antibody (sdAb). The single domain antibody is obtained from immunized vicuna (Vicugna pacos), and the preferred sequence is selected from clone 20D1, the CDR1 sequence of which is shown as SEQ ID NO. 8; the CDR2 sequence is shown as SEQ ID NO. 9; and the CDR3 sequence is shown as SEQ ID NO. 10. Clone 20D1 can specifically antagonize or neutralize the activity of anti-CD3 antibody derived from SP34. At the same time, to avoid the risk of immunogenicity, the 20D1 clone can be further humanized (hu20D1). In an embodiment of the present application, the sequence after humanization is shown as SEQ ID NO. 20, named hu20D1V3. Other humanized sequences are shown as SEQ ID NO. 19 (hu20D1V1) and SEQ ID NO. 21 (hu20D1V4), and different humanized sequences have no obvious effect on affinity.

[0129] Since the target antigen is not absolutely only expressed in tumor tissue, but also expressed at low or even moderate levels in normal tissues, further shielding the binding of aTA can further improve the safety of the drug in normal tissues, while reducing the impact of target-mediated drug disposition (TMDD) on drug exposure and improving the pharmacokinetic properties of the drug. For this purpose, in some embodiments of the present application, a domain 2 (BD2) that blocks the binding of aTA to its target antigen can be further included. The BD2 can be an antibody, or an antigen or antigen fragment recognized by the aTA. The antigen fragment can be the extracellular domain of the antigen. For example, in a preferred embodiment of the present application, when the tumor target antigen is EpCAM, the BD2 is selected from the extracellular domain region of EpCAM, and the amino acid sequence thereof is shown in SEQ ID NO. 35.

[0130] In some embodiments, the aTA is an antibody Fab domain, one chain of which is connected to CD3 to form the TCE. The BD1 is connected to the TCE through a cleavable linker peptide (CL), and is located in a different chain from aCD3. Preferably, the aCD3 and BD1 are both located at the N-terminus of the aTA, or the aCD3 and BD1 are both located at the C-terminus of the aTA.

[0131] When further including BD2, the BD2 is connected to one chain of the aTA through a cleavable linker peptide (CL).

[0132] In some embodiments, the aTA is a single-domain antibody, which is connected to CD3 through an antibody CH1-CL domain to form the TCE. The aTA is located at the N-terminus of the antibody CH1-CL domain and the aCD3 is located at the C-terminus of the antibody CH1-CL domain, or the aTA is located at the C-terminus of the antibody CH1-CL domain and the aCD3 is located at the N-terminus of the antibody CH1-CL domain. The BD1 is connected to the TCE through a cleavable linker peptide (CL), and is located in a different chain from aCD3. Preferably, the aCD3 and BD1 are both located at the N-terminus of the antibody CH1-CL domain, or the aCD3 and BD1 are both located at the C-terminus of the antibody CH1-CL domain.

[0133] When further including BD2, the BD2 is connected to the TCE through a cleavable linker peptide (CL), and is located in a different chain from the aTA.

[0134] In some embodiments of the present application, when aTA is in the structure of Fab, the binding of BD1 to aCD3 can change the conformation of aTA, significantly reducing its affinity for antigen binding, that is, BD1, by binding to aCD3, also blocks the binding of aTA to tumor target antigen; in this case, BD1 is also BD2, and additional BD2 may not be needed. Exemplary examples are shown in the structures of FIG. 5 and FIG. 26, specifically DR50722 or DR51003.

[0135] Due to the heterogeneity of the tumor microenvironment of different solid tumor patients, different CS sequence combinations can increase the cutting efficiency and improve the therapeutic effect. In a preferred embodiment of the present application, the CS1 sequence can also be combined with the cleavable sequence shown in SEQ ID NO. 2 (ISSGL, CS7) to form a CS2 sequence; in another preferred embodiment of the present application, the CS1 sequence is combined with the cleavable sequence shown in SEQ ID NO. 3 (PLGLAG, CS8) to form a CS3 sequence.

[0136] In some specific embodiments, the cleavable sequence (CS) comprises an amino acid sequence selected from any one of SEQ ID NO. 1-7, SEQ ID NO. 76-80.

[0137] On one side or both sides of the CS sequence, flexible amino acids that increase the degree of freedom can be provided to form a cleavable linker (CL). In some specific embodiments, the cleavable linker (CL) is selected from an amino acid sequence shown in any one of SEQ ID NO. 81-95. The CL can also not contain flexible amino acids. Therefore, the CL can comprise an amino acid sequence selected from any one of SEQ ID NO. 1-7, SEQ ID NO. 76-80.

[0138] In some embodiments of the present application, a long-acting unit (LA) is also included, which is selected from a mammalian IgG Fc fragment, serum albumin, a single-domain antibody that binds serum albumin, a single-chain antibody (scFv) that binds serum albumin, etc. In a preferred embodiment of the present application, the long-acting unit is selected from a single-domain antibody, and the amino acid sequence is shown in SEQ ID NO. 37.

[0139] In some embodiments of the present application, the long-acting unit is connected to BD1, or the long-acting unit is connected to BD2. When the tumor-specific protease recognizes and cuts the CS sequence, BD1 and BD2 (if any) will be dissociated from the TCE along with the long-acting unit, so that the activated TCE molecule is quickly metabolized and cleared from the circulatory system, reducing the toxicity to normal tissues.

[0140] In some embodiments of the present application, the aCD3 and aTA, BD1 and TCE, long acting unit (LA) and BD1, long acting unit (LA) and BD2 can or can not comprise a linker peptide. The linker peptide can be a flexible polypeptide chain consisting of alanine (A) and / or serine (S) and / or glycine (G), and the length of the linker peptide can be 3-40 amino acids, preferably 3-9, 9-12, 12-16, 16-20, 20-25, 25-30, 30-35, 35-40, and in another specific embodiment of the present application, the length of the linker peptide can be 5 or 8 or 15 or 18.

[0141] In the tumor-specifically activated protein drugs of the present application, the protein targeting tumor surface antigen Trop2 is named proTrop2-TCE, and its active molecule is named Trop2-TCE; the protein targeting tumor surface antigen EpCAM is named proEpCAM-TCE, and its active molecule is named EpCAM-TCE; the protein targeting tumor surface antigen EGFR is named proEGFR-TCE, and its active molecule is named EGFR-TCE; and so on. If the code name is followed by "NC", it means "non-cleavable" sequence, and the cleavable linker peptide (CL) is replaced by a flexible linker peptide. The amino acid sequences of the preferred structures are shown in Table 2:

[0142] Table 2 Preferred tumor-specifically activated protein drugs

[0143] Isolated polynucleotide

[0144] The fourth aspect of the present application provides an isolated polynucleotide encoding the single-domain antibody capable of neutralizing the SP34 antibody, the cleavable sequence (CS) or the tumor-specifically activated protein drug of the preceding aspects of the present application, which can be RNA, DNA or cDNA, etc. The method for providing the isolated polynucleotide should be known to those skilled in the art, for example, it can be prepared by automatic DNA synthesis and / or recombinant DNA technology, etc., or it can be isolated from suitable natural sources.

[0145] Expression vector

[0146] The fifth aspect of the present application provides an expression vector comprising the isolated polynucleotide according to the fourth aspect of the present application. The method for constructing the expression vector is known to those skilled in the art, for example, the expression vector can be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like, more specifically, the expression vector can be constructed by inserting the isolated polynucleotide into a multiple cloning site of the expression vector. The expression vector in the present application generally refers to various commercially available expression vectors known in the art, for example, it can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus or other vectors. The vector can further comprise one or more regulatory sequences operably linked to the polynucleotide sequence, which can comprise a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the amino acid sequence to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutated, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. The regulatory sequence can further comprise a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator functional in the selected host cell can be used in the present application.

[0147] Generally, a suitable vector can comprise an origin of replication functional in at least one organism, a promoter sequence, convenient restriction sites, and one or more selectable markers. For example, the promoters can be, but are not limited to, the lac or trp promoter of E. coli; the PL promoter of bacteriophage lambda; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter from Pichia pastoris and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, for example, can be, but are not limited to, dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP) for E. coli, or tetracycline or ampicillin resistance for E. coli, etc. When the polynucleotide is expressed, an enhancer sequence can also be included in the expression vector, which will enhance transcription if inserted into the vector, the enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, which acts on the promoter to enhance transcription of the gene.

[0148] Expression system

[0149] In a sixth aspect, the present application provides an expression system comprising the polynucleotide, the expression vector, or the genome of the fourth aspect of the present application, or the recombinant cell of the fifth aspect of the present application. In particular, the expression system can be a recombinant cell. Any cell suitable for expression of the expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, in particular, the host cell can be one or more of a combination of a bacterial cell, such as Escherichia coli, Streptomyces; Salmonella typhimurium; a fungal cell, such as yeast, filamentous fungi, a plant cell; a fruit fly S2 or Sf9 insect cell; a CHO, COS, HEK293 cell, or a Bowes melanoma cell, and the like. Methods for constructing the expression system are known to one skilled in the art, for example, the method can be one or more of a combination of a microinjection, a biolistic method, electroporation, a virus-mediated transformation, an electron bombardment, a calcium phosphate precipitation, and the like.

[0150] Pharmaceutical composition

[0151] In a seventh aspect, the present application provides a pharmaceutical composition comprising the polynucleotide, the expression vector, the recombinant cell, the single-domain antibody capable of neutralizing the SP34 antibody, or the isolated polypeptide of the preceding aspects of the present application. The isolated polypeptide can be a cleavable sequence (CS), and / or a tumor-specific activated protein drug.

[0152] The pharmaceutical composition can further comprise various pharmaceutically acceptable carriers in the art. The pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, in particular, the pharmaceutically acceptable carriers can be one or more of a combination of a buffer, such as acetate, Tris, phosphate, citrate, and other organic acids; an antioxidant, including ascorbic acid and methionine; a preservative (such as octadecyldimethylbenzyl ammonium chloride; chlorhexidine; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); a protein, such as serum albumin, gelatin, or immunoglobulin; a hydrophilic polymer, such as polyvinylpyrrolidone; an amino acid, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, a disaccharide, and other carbohydrates, including glucose, mannose, or dextrins; a chelating agent, such as EDTA; a tonicity adjusting agent, such as trehalose and sodium chloride; a sugar, such as sucrose, mannitol, trehalose, or sorbitol; a surfactant, such as polysorbate; a salt-forming counterion, such as sodium; a metal complex (such as a Zn-protein complex); and / or a non-ionic surfactant, such as or polyethylene glycol (PEG). The pharmaceutical preparation for in vivo administration is generally sterile, and methods for achieving the sterility of the pharmaceutical preparation should be known to those skilled in the art, for example, the pharmaceutical preparation can be filtered through a sterile filter membrane, etc. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the desired dosage form of the pharmaceutical composition to prepare it into different dosage forms, for example, the pharmaceutical composition of the present application can be various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0153] In the pharmaceutical composition, the content of the tumor-specifically activated protein drug is generally an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the subject to be treated and the specific administration method. For example, the content of the tumor-specifically activated protein drug can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%, based on the total mass of the pharmaceutical composition.

[0154] The tumor-specifically activated protein drug and the pharmaceutical composition of the present application can be administered as a single effective ingredient, or can be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can be the combination of the fusion protein, the immunoconjugate, the pharmaceutical composition with at least one other anti-tumor drug. For another example, the combination therapy can be the combination of the fusion protein, the immunoconjugate, the pharmaceutical composition with an immune checkpoint inhibitor, which includes but is not limited to a combination of one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor, and the inhibitor can preferably be a monoclonal antibody.

[0155] Use

[0156] The eighth aspect of the present application provides the use of the aforementioned polynucleotide, expression vector, recombinant cell, single-domain antibody capable of neutralizing the SP34 antibody, isolated polypeptide, tumor-specifically activated protein drug, or pharmaceutical composition of the seventh aspect of the present application in the preparation of a medicament for the diagnosis, treatment or prevention of a disease associated with cells expressing tumor surface antigens.

[0157] A "therapeutically effective amount" of the tumor-specifically activated protein drugs, pharmaceutical compositions provided herein preferably results in a decrease in severity of a disease's symptoms, an increase in frequency and duration of disease symptom-free periods, or an amelioration of a disease's painful or debilitating effects. For example, for the treatment of a tumor associated with a tumor surface antigen, a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to a subject who has not received treatment. The ability to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, the ability to inhibit cell growth can also be evaluated, which can be determined in vitro by assays known to the skilled artisan. A therapeutically effective amount of a nanobody, fusion protein, immunoconjugate, pharmaceutical composition is generally capable of reducing the size of a tumor, or otherwise ameliorating the symptoms of a subject. The skilled artisan can select an appropriate therapeutically effective amount depending on the actual circumstances, e.g., the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen. Prescription of treatment (e.g., the decision to dose, etc.) can be made by a physician, taking into account factors such as the disease to be treated, the individual case of the patient, the delivery site, the method of administration, and other factors. A prophylactically effective amount refers to an amount effective to prevent a desired prophylactic effect at a necessary dosage, and for a necessary period of time. Generally, but not necessarily, a "prophylactically effective amount" is less than a "therapeutically effective amount" since a prophylactic dose is used before or at an early stage of disease onset, in a subject who is not yet suffering from a disease.

[0158] Examples of diseases associated with cells expressing tumor-associated antigens that can be diagnosed, treated and / or prevented by the present application can include all hematological and solid tumors expressing the respective tumor surface antigen. The solid tumors can in particular be gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, melanoma, head and neck cancer, neuroendocrine cancer, adrenal cancer, gallbladder cancer, mesothelioma, and the like, which can be in an early, intermediate or advanced stage, e.g., metastatic cancer. In some embodiments of the present application, the disease associated with cells expressing tumor surface antigens is selected from the group consisting of a tumor selected from one or more of a hematological tumor, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, melanoma, head and neck cancer, neuroendocrine cancer, adrenal cancer, gallbladder cancer, and mesothelioma.

[0159] In some embodiments of the present application, the tumor-specifically activated protein drugs targeting the tumor surface antigen Trop2 can be used for the diagnosis, treatment and / or prevention of Trop2-expressing tumors, including but not limited to gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, melanoma, head and neck cancer, neuroendocrine cancer, adrenal cancer, gallbladder cancer, and mesothelioma, etc.

[0160] In some embodiments of the present application, the tumor-specifically activated protein drugs targeting the tumor surface antigen EpCAM can be used for the diagnosis, treatment and / or prevention of EpCAM-expressing tumors, including but not limited to gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, melanoma, head and neck cancer, neuroendocrine cancer, adrenal cancer, gallbladder cancer, and mesothelioma, etc.

[0161] In some embodiments of the present application, the tumor-specifically activated protein drugs targeting the tumor surface antigen EGFR can be used for the diagnosis, treatment and / or prevention of EGFR-expressing tumors, including but not limited to gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, melanoma, head and neck cancer, neuroendocrine cancer, adrenal cancer, gallbladder cancer, and mesothelioma, etc.

[0162] The specific embodiments of the present application are described herein by way of specific examples. Other advantages and benefits of the present application will be readily appreciated by those skilled in the art in view of the disclosure herein. The present application can also be carried out in ways not specifically described herein without departing from the spirit and scope of the application. Numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the present application, the application can be practiced otherwise than as specifically described herein.

[0163] Before further description of the present application, it is understood that the present application is not limited in scope to the specific embodiments described herein; and that the terms used in the present application's examples are for the purpose of describing particular embodiments and are not intended to limit the present application's scope.

[0164] When a numerical range is given in the examples, it is understood that, unless otherwise stated by the present application, each numerical range's two endpoints and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to specific methods, devices, materials, etc. described in the examples, any methods, devices, and materials similar or equivalent to those described in the present application's examples can be used in the practice of the present application, based on the knowledge in the art and the teachings of the present application.

[0165] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all employ conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields. These techniques are well described in the existing literature, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P. B. Becker, ed.) Humana Press, Totowa, 1999, and the like.

[0166] Example 1, Screening of CS sequences

[0167] 1. Construction of CS phage display library

[0168] The various partial elements of the gene-synthetic phage surface display (SEQ ID NO. 96) were inserted between the EcoRI and NotI sites of the pcomb3xss vector to obtain the plasmid pcomb3gfpGAG-PB-GS-TS.

[0169] The pcomb3gfpGAG-PB-GS-TS plasmid was used as a template for PCR amplification with primers pcomb3gfp-uPA2-R (SEQ ID NO. 97) and pcomb3gfp-uPA2-F (SEQ ID NO. 98) using high-fidelity enzyme Phanta Max Master (Novozyme) to cut the gel and recover as a carrier fragment. Primers pcomb3gfp-uPA-F1 (SEQ ID NO. 99) and pcomb3gfp-uPA-R1 (SEQ ID NO. 100) were annealed without a template, and the annealing product was recovered and extended with high-fidelity enzyme Phanta Max Master (Novozyme) to complete the extension. The recovered annealing product was ligated with the prepared carrier fragment using Uniclone One Step Seamless Cloning Kit (Jinsa) to recombine and transform competent cells XL1-Blue (Nanjing Humagen Edium Biotechnology Co., Ltd.) to obtain a CS sequence library containing the XSGRSXXX coding sequence, wherein X is any amino acid. The library capacity was determined by gradient dilution plating to be 3 x 10 6 / ml. The constructed CS library was packaged with helper phage M13KO7 (NEB) to obtain a recombinant phage display library with a titer of 1 x 10 13 pfu / ml.

[0170] 2. Phage library panning

[0171] In the first round of panning, 100 uL of the CS phage display library was diluted to 2 mL with 50 mM Tris-HCl + 0.01% Tween pH 8.5 buffer, filtered to remove bacteria, and placed in a sterile EP tube for 37°C enzyme digestion for 24 h. Then, the enzyme-digested library supernatant was combined with an immunotube coated with recombinant Strep-Tactin (r-STc, Hangzhou Nuolong Biotechnology Co., Ltd.) and blocked with 3% BSA (Shanghai Yovgen Biotech Co., Ltd.), and incubated at 37°C for 1 h. The phage supernatant in the immunotube was recovered. This process was repeated three times to remove the recombinant phage that was not digested in the library. The phage library titer in the recovered supernatant was determined, and the phage library was used to infect ER2738 and amplified. The library amplified in the first round of panning was used for the second round of panning, with the same steps as the first round of panning. The phage library titer in the recovered supernatant was determined.

[0172] 3. Identification of phage monoclonal

[0173] The monoclonal culture was picked from the titer plate, and a negative control pcomb3gfpGAG-PB-GS-TS was added, and cultured at 37°C for about 4h, and then infected and packaged with M13KO7 helper phage to obtain the culture supernatant containing recombinant phage. After dilution with 50mM Tris-HCl+0.01% Tween (pH8.5) at 1:50, 75μL was transferred to a new 96-well plate A, and 75μL of 0.5μg / ml uPA (Suzhou Coastal Protein Technology Co., Ltd.) was added for enzyme cutting; 75μL of the diluted phage was taken to a new 96-well plate B, and mixed with 75μL of 50mM Tris-HCl+0.01% Tween (pH8.5) as a negative control without enzyme cutting. The two groups of experimental 96-well plates were incubated in a 37°C incubator overnight.

[0174] Two ELISA plates were coated with rSTc (100ng / well) and blocked with 5% skim milk powder, and after PBST washing, the phage supernatant on plates A and B incubated overnight was added, and incubated at 37°C for 1 hour. After 5 times of PBST washing, 100μl of 0.1μg / mL Anti-M13 Antibody (HRP), Mouse Monoclonal (SinoBiological) was added to each well, and incubated at 37°C for 1 hour. After 5 times of PBST washing, TMB color developing working solution (Huzhou Yingchuang Biotechnology Co., Ltd.) was added, and after color development at 37°C for 5 minutes, 1M sulfuric acid was added to terminate the reaction, and OD450nm was read.

[0175] According to the ELISA results, the clones with no color or weak color on plate A and corresponding color on plate B were selected as candidate clones, and part of the clones are shown in Table 3, the coding genes were obtained by sequencing, and the repeated sequence clones were removed.

[0176] Table 3

[0177] 4. Identification of CS sequence based on recombinant protein

[0178] In this embodiment, the candidate CS sequence obtained by screening was connected with a single-domain antibody (ABD) binding to serum albumin and human IgG1 Fc, and a Twin Strep tag label was introduced at the N-terminus to construct a fusion protein in the form of TS-ABD-GS-CS-Fc structure, and an exemplary sequence is shown in SEQ ID NO. 101.

[0179] The above sequence was synthesized and inserted into the pcDNA3.4 expression vector, and the expression vector and transfection reagent PEI (Yixing Biotech (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3, and the cells were cultured in a 37°C, 5% CO2 incubator for 7 days. After centrifugal recovery of the expression supernatant, the antibody was purified by a Protein A affinity chromatography column (Bogu Biotech Co., Ltd.), and after purity detection and quantitative analysis, the fusion protein was obtained.

[0180] 4 μg of the obtained fusion protein was subjected to enzyme digestion with 0.6 μg of uPA protease (Suzhou Jinan Protein Technology Co., Ltd.) or ST14 protease (RD systems) in a system of 40 μL of 50 mM Tris-HCl+0.01% Tween (pH 8.5) solution, incubated at 37°C for 24 h, and subjected to SDS-PAGE electrophoresis to detect the enzyme digestion effect. The preferred sequence of this embodiment is named CS1 (SEQ ID NO. 1).

[0181] Example 2, Construction of BD1 Single Domain Antibody Library

[0182] The nucleic acid sequence of the aCD3 scFv derived from SP34 was connected to the nucleic acid sequence encoding the llama Fc, the gene was synthesized and inserted into the pcDNA3.4 expression vector, and the expression vector and transfection reagent PEI (Yixing Biotech (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3, and the cells were cultured in a 37°C, 5% CO2 incubator for 7 days. After centrifugal recovery of the supernatant, the antibody was purified by a Protein A affinity chromatography column (Bogu Biotech Co., Ltd.), and the fusion protein Anti-CD3 Scfv-Llama Fc (SEQ NO. 38) was obtained.

[0183] One healthy vicugna (Vicugna pacos) was immunized with 1 mg Anti-CD3 Scfv-Llama Fc fusion protein and 1 mL Freund's complete adjuvant (Sigma) after emulsification, and then immunized again after 21 days, a total of 3 times. One week after the third immunization, 30 mL of vicugna blood was collected with a vacuum blood collection tube, and lymphocytes were separated with lymphocyte separation medium (Tianjin Haoyang Huake Biological Technology Co., Ltd.). Total RNA was extracted by Trizol method. 3 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Invitrogen), and VHH was amplified using nested PCR and the following primers: the upstream primer 5'-CTTGGTGGTCCTGGCTGC-3'(SEQ ID NO. 71) and the downstream primer 5'-GGTACGTGCTGTTGAACTGTTCC-3'(SEQ ID NO. 72) for the first round of PCR; the second round of PCR used the first round of PCR as a template, and the upstream primer 5'-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC-3'(SEQ ID NO. 73) and the downstream primer 5'-CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG-3'(SEQ ID NO. 74) or 5'-CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG-3'(SEQ ID NO. 75) were used for amplification. The target VHH nucleic acid fragment was recovered, and restriction endonuclease Sfil (NEB) was used for enzyme digestion, and inserted into the same enzyme-digested phage display vector pcomb3xss (Addgene plasmid #63890; RRID: Addgene_63890) and connected by T4 ligase (Takara). The ligation product was transformed into XL1-Blue (Nanjing Heming Yinggu Biotechnology Co., Ltd.) to construct the CD3 anti-antibody library.

[0184] The constructed CD3 anti-antibody library was packaged with helper phage M13KO7 (NEB), and the recombinant phage titer of the display library was measured to be 2.0 x 10 14 PFU / mL. The Anti-CD3 scFv-OVAp-TS-His fusion protein (SEQ ID NO. 39) was diluted to 10 μg / mL with coating solution, 100 μL per well, and coated at 4°C overnight. Washed with PBST for three times, and added 3% BSA-PBST at 37°C for 2 hours. Washed with PBST for three times, and added about 2.0 x 10 11The recombinant phage library containing PFU was incubated at 37°C for 2 hours. Unbound phages were aspirated, and the cells were washed 5 times with PBST and 10 times with PBS. 100 μL of 1 mg / mL BSA (0.1 M Gly-HCl buffer, pH 2.2) was added to each well, and the cells were incubated for 10 minutes to elute. The phages were then neutralized with 1 M pH 8.0 Tris-HCl and filtered for sterilization. The phage titer was determined to be approximately 1.15 × 10⁻⁶. 4 PFU / mL. The above phage elution buffer was amplified, and the titer was determined to be approximately 2 × 10⁻⁶. 13 PFU / mL.

[0185] Example 3: Screening and Identification of BD1 Single-Domain Antibodies

[0186] Dilute the Anti-CD3 Scfv-Llama Fc fusion protein to 5 μg / mL using coating buffer, add 100 μL to each well of an ELISA plate, and coat overnight at 4°C. Wash three times with PBST, add 3% OVA-PBST, and block at 37°C for 2 hours. Wash three times with PBST, and add approximately 2 × 10⁻⁶ of the amplified library from the first round of panning. 11 PFU was incubated at 37°C for 1 hour. Unbound phages were aspirated, and the cells were washed 10 times with PBST and then 10 times with PBS. 100 μL of 1 mg / ml BSA (0.1 mol / L Gly-HCl buffer, pH 2.2) was added to each well, and the cells were incubated for 10 minutes to elute. The phages were then neutralized with 1 mol / L pH 8.0 Tris-HCl and filtered for sterilization. The phage titer was determined to be approximately 3.6 × 10⁻⁶. 4 PFU / mL.

[0187] 188 single clones were picked from the phage titer assay plate after the second round of washing and elution, and cultured in 96-well plates. M13KO7 helper phage was used for infection and packaging to obtain the accumulation of recombinant phage in the supernatant. The Anti-CD3 Scfv-His fusion protein was diluted to 1 μg / mL with coating buffer, and 100 μL was added to each well of the ELISA plate. The plate was incubated overnight at 4°C. After washing three times with PBST, 5% skim milk powder-PBST was added, and the plate was blocked at 37°C for 2 hours. 100 μL of phage supernatant was diluted 1:1 with 5% skim milk powder-PBST, mixed well, and 100 μL was added to the blocked ELISA plate. The plate was incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of 0.1 μg / mL Anti-M13 Antibody (HRP) and Mouse Monoclonal (SinoBiological) was added to each well, and the plate was incubated at 37°C for 1 hour. After washing 6 times with PBST, TMB substrate was added and incubated at 37°C. After 5 minutes of incubation for color development, the reaction was stopped by adding 1M sulfuric acid. The result was measured at OD. 450nmThe light absorption value was measured. When the OD value of the sample hole was more than twice the OD value of the control hole, it was determined to be a positive clone, and Sange sequencing was performed.

[0188] The candidate VHH sequence gene was synthesized into a pCDNA3.4 vector containing a signal peptide and a human IgG1 Fc sequence (SEQ ID NO. 42), and the expression plasmid and transfection reagent PEI (Yixing Biotech (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days. After the expressed protein or antibody was recovered by centrifugation, antibody purification was performed by a Protein A affinity chromatography column (Bogu Biotech Co., Ltd.), and an Anti-aCD3 scFv VHH-Fc fusion protein was obtained.

[0189] The Anti-CD3 scfv-His fusion protein was diluted to 1 pg / mL with the coating solution, 100 pL was added to each well of the enzyme-labeled plate, and it was coated at 4°C overnight. After washing three times with PBST, 5% skimmed milk powder-PBST was added for blocking at 37°C for 2 hours. The Anti-aCD3 scFv VHH-Fc fusion protein was gradiently diluted with 1% BSA-PBST, added to the blocked enzyme-labeled plate, and incubated at 37°C for 1 hour. After 5 times of PBST washing, 100 pL of 1:10,000 diluted Goat Anti-Human IgG, Monkey ads-HRP (SouthernBiotech) was added to each well, and incubated at 37°C for 1 hour. After 6 times of PBST washing, TMB substrate was added, and color development was performed after incubation at 37°C for 5 minutes. The reaction was terminated by adding 1M sulfuric acid, and the OD450nm was read. The experimental results of the preferred sequence 20D1 clone are shown in Figure 1.

[0190] The CD3ed-Fc fusion protein was diluted to 1 pg / mL with the coating solution, 100 pL was added to each well of the enzyme-labeled plate, and it was coated at 4°C overnight. After washing three times with PBST, 5% skimmed milk powder-PBST was added for blocking at 37°C for 2 hours. The Anti-aCD3 scFv VHH-Fc fusion protein was gradiently diluted with 1% BSA-PBST and pre-incubated with Bio-aCD3 scfv-Llama Fc (final concentration 0.02 pg / mL) at 37°C for 1 hour, and then added to the blocked enzyme-labeled plate, and incubated at 37°C for 1 hour. After 5 times of PBST washing, 100 pL of 1:2,500 diluted HRP-Strep Avidin was added to each well, and incubated at 37°C for 1 hour. After 6 times of PBST washing, TMB substrate was added, and incubated at 37°C. Color development was performed after incubation for 5 minutes, the reaction was terminated by adding 1M sulfuric acid, and the OD450nm was read. 450nm The light absorption value was measured. The experimental results are shown in Figure 2.

[0191] The sequence 20D1 was preferably humanized, and the humanization method used the VHH humanization universal framework grafting method established by Vincke C et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. J Biol Chem. 2009; 284(5): 3273-3284). According to the sequence homology design, the universal humanized VHH framework h-NbBcII10FGLA (PDB code: 3EAK) was completed, the corresponding CDR region was replaced with the CDR region of Anti-aCD3 scFv VHH, and the back mutation adjustment of individual amino acids in the FR region was adjusted as different humanized variants, such as "hu1A11V3" representing the humanized V3 variant of the 1A11 clone sequence.

[0192] The binding activity and blocking activity of the humanized Anti-aCD3 scFv VHH fusion protein were detected by the above-mentioned ELISA method, and the results are shown in Figure 3. There was no significant difference in the affinity and antagonistic activity of different humanized versions (hu20D1v1 and hu20D1v3) to aCD3.

[0193] Example 4, Preparation of Trop2-TCE

[0194] 1. Construction and expression

[0195] The structures of Trop2-TCE Ab-1 and DR50726 are shown in Figure 4. According to the related sequences shown in Table 1, the DNA sequence corresponding to Ab-1 was optimized and synthesized by Jiangsu Huakang Biotechnology Co., Ltd., and the heavy chain and light chain were inserted into the pcDNA3.4 vector containing the signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids.

[0196] Trop2-TCE Ab-1 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.), until the cell density reached 2-4 x 10 6 Live cells / mL, and the viability was ≥95%. The light chain and heavy chain expression plasmids were extracted using a large extraction kit for endotoxin-free plasmids, filtered through a 0.22 μm filter to remove bacteria, mixed at a ratio of 1:1, and the expression plasmid and transfection reagent PEI (Yixing Biotechnology (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and the culture supernatant was harvested.

[0197] 2. Purification

[0198] The cell expression suspension of Ab-1 was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto a protein G affinity chromatography column (GE Healthcare Life Sciences). The equilibration liquid was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution liquid was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning liquid was a 0.2 mol / L HAC solution; and the neutralization liquid was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration liquid, and the sample was loaded at a loading amount of not more than 10 mg of target protein per ml of filler at a retention time of 1-2 min. After loading, the chromatography column was equilibrated with the equilibration liquid, and then the target peak was eluted with the elution liquid. The target peak was adjusted to pH 6.0 with the neutralization liquid, and the precipitate was removed by centrifugation. The centrifugal supernatant was adjusted to a conductivity of less than 5 mS / cm and then loaded onto a Diamond SP cation exchange chromatography column (Borgolon (Shanghai) Biotechnology Co., Ltd.). Elution was performed at a linear gradient of 0-100% B (0-500 mmol / L NaCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV), and a sample with a SDS-PAGE electrophoretic purity of ≥95% and a SEC purity of ≥90% was obtained.

[0199] DR50726 was prepared and purified in the same manner as Ab-1.

[0200] Example 5, Preparation of proTrop2-TCE and proTrop2-TCE-NC

[0201] DR50722, DR50722NC, DR50724, DR50724NC, DR50725, DR50725NC, DR50728, and DR50727NC1, Construction and Expression

[0202] The structure is shown in FIG. 5. According to the relevant sequences shown in Table 2, the DNA sequence corresponding to DR50722 was optimized and synthesized by Jiangsu Huagong Biotechnology Co., Ltd., and the heavy chain and the light chain were inserted into the pcDNA3.4 vector containing a signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50722-HC and pcDNA3.4-DR50722-LC.

[0203] According to the relevant sequences shown in Table 2, the DNA sequence corresponding to DR50722NC was optimized and synthesized by Jiangsu Huagong Biotechnology Co., Ltd., and the heavy chain and the light chain were inserted into the pcDNA3.4 vector containing a signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50722NC-HC and pcDNA3.4-DR50722NC-LC.

[0204] DR50722 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2-4 x 10 6 Live cells / mL, viability ≥ 95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR50722-HC and pcDNA3.4-DR50722-LC were extracted using an endotoxin-free plasmid maxi extraction kit, filtered through a 0.22 μm filter to remove bacteria, and mixed at a ratio of 1:1. The expression plasmids and transfection reagent PEI (Yixing Biotechnology (Shanghai) Co., Ltd.) were mixed at a ratio of 1:3 to transfect Expi293F cells (Thermo). The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and the culture supernatant was harvested.

[0205] DR50722NC expression preparation: The two plasmids pDR01-DR50722-HC and pDR01-DR50722NC-LC were extracted using an endotoxin-free plasmid maxi extraction kit, filtered through a 0.22 μm filter to remove bacteria, and mixed at a ratio of 1:1. The mixed plasmids and transfection reagent PEI were mixed at a ratio of 1:3 and allowed to stand for 30 minutes, then added to Expi293F cells. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and the supernatant was collected by centrifugation.

[0206] 2. Purification of DR50722 and DR50722NC

[0207] The cell expression suspension was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane. The protein G affinity chromatography column (GE Healthcare Life Sciences) was equilibrated with 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the eluent was 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning solution was 0.2 mol / L HAC solution; and the neutralizing solution was 1 mol / L Tris-HCl, pH 8.0. The column was equilibrated with the equilibration buffer, and the sample was loaded at a loading capacity of not more than 10 mg target protein / ml packing with a retention time of 1 min. After loading, the column was equilibrated with the equilibration buffer, and the target peak was eluted with the eluent. The target peak was adjusted to pH 6.0 with the neutralizing solution, centrifuged to remove the precipitate, and the supernatant was collected. The centrifugal supernatant was adjusted to a conductivity of less than 5 mS / cm and loaded onto a Diamond SP cation exchange chromatography column (Borgolon (Shanghai) Biotechnology Co., Ltd.). Elution was performed with a linear gradient of 0-30% B (0-500 mmol / L NaCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV), and a sample with a SDS-PAGE electrophoretic purity of ≥ 95% and SEC purity of ≥ 90% was obtained.

[0208] Since DR50722, DR50724, DR50725 and DR50728 have the same structure, the difference is only in the length of the connecting peptide between aCD3 scFv and aTA (anti-Trop-2 Fab) or the length of the connecting peptide between BD1 and aTA (anti-Trop-2 Fab), therefore, DR50724, DR50725 and DR50728 are constructed to express the same as DR50722, and DR50724NC, DR50725NC and DR50727NC are constructed to express the same as DR50722NC.

[0209] Example 6, Preparation of Trop2-TCE DR50721

[0210] 1. Construction and expression

[0211] The structure of DR50721 is shown in Figure 6. According to the relevant sequences shown in Table 1, the corresponding DNA sequence of DR50721 was entrusted to Jiangsu Huakang Biotechnology Co., Ltd. for optimization and synthesis. The heavy chain and light chain were inserted into the pcDNA3.4 vector containing the signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50721-HC and pcDNA3.4-DR50721-LC.

[0212] 2. Preparation of DR50721 expression

[0213] Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biological Technology Co., Ltd.) until the cell density reached 2-4 x 10 6 Live cells / mL, and the viability was ≥95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR50721-HC and pcDNA3.4-DR50721-LC were extracted using a large-endotoxin-free plasmid extraction kit, filtered through a 0.22 μm filter to remove bacteria, and then mixed at a ratio of 1:1. The expression plasmid and transfection reagent PEI (Yixing Biological Technology (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and then the culture supernatant was harvested.

[0214] 3. Purification of DR50721

[0215] The same as Ab-1 and DR50726.

[0216] Example 7, Preparation of proTrop2-TCE and proTrop2-TCE-NC: DR50723 and DR50723NC

[0217] 1. Construction and expression

[0218] The structure is shown in Figure 7. According to the related sequence shown in Table 2, the DNA sequence corresponding to DR50723 is entrusted to Jiangsu Huakang Biotechnology Co., Ltd. for optimization and synthesis, and the heavy chain and light chain are inserted into the pcDNA3.4 vector containing the signal peptide respectively to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50723-HC and pcDNA3.4-DR50723-LC.

[0219] 2. Preparation of DR50723 expression

[0220] Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2-4 x 10 6 Live cells / mL, and the viability was ≥95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR50723-HC and pcDNA3.4-DR50723-LC were extracted using a large-endotoxin-free plasmid extraction kit, filtered through a 0.22 μm filter to remove bacteria, and then mixed at a ratio of 1:1. The expression plasmid and transfection reagent PEI (Yixing Biosciences (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and then the culture supernatant was harvested.

[0221] DR50723NC was constructed and expressed in the same way as DR50723.

[0222] 2. Purification of DR50723 and DR50723NC

[0223] The cell expression suspension was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto a protein G affinity chromatography column (GE Healthcare Life Sciences). The equilibration liquid was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution liquid was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning liquid was a 0.2 mol / L HAC solution; and the neutralization liquid was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration liquid, and the sample was loaded at a loading amount of not more than 10 mg of target protein per ml of filler at a retention time of 1 min. After loading, the chromatography column was equilibrated with the equilibration liquid, and then the target peak was eluted with the elution liquid. The target peak was adjusted to pH 6.0 with the neutralization liquid, and the precipitate was removed by centrifugation. The centrifugal supernatant was adjusted to a conductivity of less than 5 mS / cm and then loaded onto a Diamond SP cation exchange chromatography column (Borgolon (Shanghai) Biotechnology Co., Ltd.). Elution was performed at a linear gradient of 0-30% B (0-500 mmol / L NaCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV), and a sample with a SDS-PAGE electrophoretic purity of ≥95% and a SEC purity of ≥90% was obtained.

[0224] Example 8, Preparation of EpCAM-TCE DR50808

[0225] 1. Construction and expression

[0226] The structure is shown in FIG. 8. According to the relevant sequences shown in Table 1, the DNA sequence corresponding to DR50808 was entrusted to Jiangsu Huakang Biotechnology Co., Ltd. for optimization and synthesis. The heavy chain and the light chain were respectively inserted into a pcDNA3.4 vector containing a signal peptide to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50808-HC and pcDNA3.4-DR50808-LC.

[0227] DR50808 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2-4 x 10 6 The light chain and heavy chain expression plasmids pcDNA3.4-DR50808-HC and pcDNA3.4-DR50808-LC were extracted using a large extraction kit for endotoxin-free plasmids, filtered through a 0.22 μm filter to remove bacteria, mixed at a ratio of 1:1, and then transfected into Expi293F cells (Thermo) at a ratio of 1:3 with the expression plasmid and transfection reagent PEI (Yixing Biotechnology Co., Ltd., Shanghai). The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and then the culture supernatant was harvested.

[0228] 2. Purification

[0229] The cell expression suspension was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 pm membrane and then loaded onto a protein G affinity chromatography column (GE Healthcare Life Sciences). The equilibration liquid was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution liquid was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning liquid was a 0.2 mol / L HAC solution; and the neutralization liquid was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration liquid, and the sample was loaded at a loading amount of not more than 10 mg of target protein per ml of filler at a retention time of 1 min. After loading, the chromatography column was equilibrated with the equilibration liquid, and then the target peak was eluted with the elution liquid. The target peak was adjusted to pH 6.0 with the neutralization liquid, the precipitate was removed by centrifugation, and the supernatant was taken. The sample obtained had a SDS-PAGE electrophoretic purity and SEC purity of ≥95%.

[0230] Example 9, Preparation of proEpCAM-TCE and proEpCAM-TCE-NC: DR50809 and DR50809NC

[0231] 1. Construction and expression

[0232] The structure is shown in FIG. 9. According to the relevant sequences shown in Table 2, the DNA sequence corresponding to DR50809 was entrusted to Jiangsu Huakang Biotechnology Co., Ltd. for optimization and synthesis, and the heavy chain and the light chain were respectively inserted into a pcDNA3.4 vector containing a signal peptide to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50809-HC and pcDNA3.4-DR50809-LC.

[0233] DR50809 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.), until the cell density reached 2-4 x 10 6 The light chain and heavy chain expression plasmids pcDNA3.4-DR50809-HC and pcDNA3.4-DR50809-LC were extracted using a large extraction kit for endotoxin-free plasmids, filtered through a 0.22 pm filter to remove bacteria, mixed at a ratio of 1:1, and then the expression plasmids and transfection reagent PEI (Yixing Biotechnology Co., Ltd.) were used to transfect Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and then the culture supernatant was harvested.

[0234] The construction and expression of DR50809NC were the same as those of DR50809.

[0235] 2. Purification of DR50809 and DR50809NC

[0236] The cell expression suspension was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto a protein G affinity chromatography column (GE Healthcare Life Sciences). The equilibration liquid was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution liquid was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning liquid was a 0.2 mol / L HAC solution; and the neutralization liquid was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration liquid, and the sample was loaded at a loading amount of not more than 10 mg of target protein per ml of filler at a retention time of 1 min. After loading, the chromatography column was equilibrated with the equilibration liquid, and then the target peak was eluted with the elution liquid. The target peak was adjusted to pH 6.0 with the neutralization liquid, and the precipitate was removed by centrifugation, and the supernatant was taken. The centrifugal supernatant was adjusted to a conductivity of less than 5 mS / cm and then loaded onto a Diamond SP cation exchange chromatography column (Borglun (Shanghai) Biotechnology Co., Ltd.). Elution was performed at a linear gradient of 0-30% B (0-500 mmol / L NaCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 30 CV), and a sample with a SDS-PAGE electrophoretic purity of ≥95% and a SEC purity of ≥90% was obtained.

[0237] Example 10. Preparation of EpCAM-TCE DR50812 and DR50814

[0238] The structure is shown in FIG. 10. The EpCAM-TCE structures DR50812 and DR50814 were prepared by expression as for DR50808.

[0239] Example 11. Preparation of proEpCAM-TCE-NC: Preparation of DR50813NC and DR50815NC

[0240] The structures of DR50813NC and DR50815NC are shown in FIG. 11, and the preparation was as for DR50809NC.

[0241] Example 12. Preparation of EpCAM-TCE DR50818

[0242] In this example, aEpCAM VHH obtained by screening was used as aTA to construct EpCAM-TCE.

[0243] 1. Screening, expression and purification of aEpCAM VHH

[0244] The healthy alpaca is immunized with the Human EpCAM extracellular domain multiple times to stimulate B cells to express antigen-specific single-domain antibodies. The alpaca blood is collected, lymphocytes are separated, total RNA is extracted, cDNA is reverse transcribed, and VHH is amplified by nested PCR. The target VHH nucleic acid fragment is recovered and recombined into the phage display vector pcomb3xss. The recombinant vector is transformed into ER2738 electrocompetent cells to construct an Anti-EpCAM VHH library. After multiple rounds of screening and enrichment, the Anti-EpCAM single-domain antibody clone 1H5 that binds to the EpCAM-His (ACRO) fusion protein is selected.

[0245] The selected 1H5 gene fragment is subjected to PCR amplification, and the PCR product is recovered and inserted into a suitable expression vector such as HEK293F cells. After 7 days of culture at 37°C in a 5% CO2 incubator, the supernatant is centrifuged and purified by chromatography column to obtain the target single-domain antibody Anti-EpCAM-1H5.

[0246] 2. Affinity of Anti-EpCAM-1H5V4

[0247] The Anti-EpCAM-1H5 is humanized using the VHH humanization universal framework grafting method established by Vincke C et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. J Biol Chem. 2009; 284(5): 3273-3284), obtaining Anti-EpCAM-1H5V4. The binding force of Anti-EpCAM-1H5V4 to human EpCAM-His fusion protein is detected by ELISA, and the EC50 is 3.555 nM. Anti-EpCAM-1H5V4 is fused with human IgG1 Fc to obtain Anti-EpCAM-1H5V4-Fc fusion protein, and the binding force of Anti-EpCAM-1H5V4-Fc fusion protein to human EpCAM-His fusion protein is detected by ELISA, and the EC50 is 0.046 nM.

[0248] ELISA detection steps are as follows: the EpCAM-His fusion protein is diluted to 1 μg / mL with coating solution, 100 μL is added to each well of the enzyme-labeled plate, and it is coated at 4°C overnight. Wash with PBST three times, add 5% skim milk-PBST to block at 37°C for 2 hours. The sample to be tested is diluted with 1% BSA-PBST, added to the blocked enzyme-labeled plate, and incubated at 37°C for 1 hour. After 5 times of PBST washing, 100 μL of 1:10000 diluted HRP-Goat anti-human IgG Fc antibody (Thermo Scientific) is added to each well, incubated at 37°C for 1 hour. After 6 times of PBST washing, TMB substrate is added, and incubated at 37°C. After 5 minutes of color development, 1M sulfuric acid is added to terminate the reaction, and the OD 450nm The absorbance is measured. The data is processed and plotted using software GraphPad Prism v5.0, and the EC 50 value is obtained to reflect the affinity binding ability of the antibody to EpCAM.

[0249] 3. Anti-tumor effect of Anti-EpCAM-1H5V4-Fc-CPD3 (DAR4) in human bladder cancer HT1376 xenograft model

[0250] To verify the anti-tumor activity of Anti-EpCAM-1H5V4 VHH, the fusion protein of Anti-EpCAM-1H5V4 VHH and Fc (Anti-EpCAM-1H5V4-Fc) and its antibody drug conjugate (Anti-EpCAM-1H5V4-Fc-CPD3) were prepared in this embodiment.

[0251] The antibody drug conjugate Anti-EpCAM-1H5V4-Fc-CPD3 has the following structure:

[0252] wherein Ab is Anti-EpCAM-1H5V4-Fc. n represents the average DAR value, in this example n is 4. The preparation steps are as follows: take 8 mL aEpcam-1H5V4-Fc, the concentration is 1.38 mg / mL, ultrafiltration to PBS (pH 7.4) solution, the exchange ratio is more than 100 times, the concentration of the obtained sample is 1.05 mg / ml, and the volume is 9.1 ml. 100 eq. (molar times relative to the antibody) of TCEP (tris (2-carboxyethyl) phosphine, 10 mM TCEP solution, 1.153 ml) is added to the above solution system, mixed, incubated at 37°C for 2h. 0.777 ml DMSO is added to the above solution system, mixed, and then 10 eq. of CPD3 (10 mM CPD3 stock solution, DMSO solution system, 102.8 μl) is added, mixed, and shaken at 25 degrees for 1h. Finally, 20 eq. of cysteine (10 mM cysteine solution stock, 0.205 ml) is added to terminate the reaction. Finally, the final sample is obtained by affinity purification, which is aEpcam-1H5V4-Fc-pym-PEG8-VA-PAB-Exatecan-D4 (abbreviated as aEpcam-1H5V4-Fc-CPD3-D4, solvent is 100 mM Gly + 100 mM NaAC, 8% trehalose, pH 5.0) 9.6 mg. Electrophoresis, SEC and RP are sent for inspection. SEC shows no obvious aggregate, and the average DAR value measured by RP method is 3.7. The structure of CPD3 is as follows:

[0253] 3.1 Anti-EpCAM-1H5V4-Fc in vitro cell killing (DTZ method)

[0254] This example uses a method based on diphtheria toxin (DT) and domain Z combined with IgG Fc to prepare a method for evaluating endocytosis function based on killing (DTZ method).

[0255] Take MDA-MB-468, N87 cells in logarithmic growth phase, trypsinize, resuspend in complete culture medium, adjust the cell density to 30,000 cells / mL, 100 μL / well into a 96-well white plate, incubate in a 37°C incubator overnight. The next day, discard 25 μL of culture medium, and prepare the sample to be tested with a 1:2 molar ratio of antibody and DTZ, incubate at room temperature for 30 min, then dilute with complete culture medium to the specified concentration, take 75 μL of the gradient-diluted sample to be tested and add it to the 96-well white plate, incubate for 120 h, and then use the CellTiter-Glo luminescent live cell detection kit (Promega) to detect cell viability. Figure 12A shows the in vitro cell killing results of Anti-EpCAM-1H5V4-Fc. The results show that Anti-EpCAM-1H5V4-Fc has high cell killing activity, i.e. high endocytosis activity.

[0256] 3.2 In vivo antitumor activity of antibody drug conjugates

[0257] Human bladder cancer HT1376 cells were mixed with Matrigel at a ratio of 1:1 and subcutaneously inoculated into the right flank of SPF grade B-NDG mice to establish a subcutaneous tumor model. The tumor volume and body weight of all mice were measured, and when the average tumor volume reached 171 mm 3 , the mice were randomly divided into groups according to the tumor volume and body weight, with 5 mice in each group and 2 groups in total. On the day of grouping (D0), the mice were administered according to the group design in Table 4. The tumor volume and body weight of the mice were monitored 2-3 times per week after administration, and the monitoring was continued until D21.

[0258] Table 4 Test design and results summary Note: The statistical data of each index are represented as Mean ± SEM. *** p < 0.001 compared with the Vehicle group. i.v.: intravenous injection.

[0259] The tumor volume and tumor inhibition rate (TGI) of the mice are shown in Figure 12B and Table 4. On D21 after grouping and administration, the aEpcam-1H5V4-Fc-CPD3-D4 group showed a significant antitumor effect compared with the Vehicle group, with a TGI of 62.86%. During the test period, no drug-related decrease in body weight was observed in the mice, as shown in Figure 12C and Table 4.

[0260] In summary, in the human bladder cancer HT1376 xenograft model, the aEpcam-1H5V4-Fc-CPD3-D4 group showed a better antitumor effect, which was significantly different from the Vehicle group (p < 0.001).

[0261] 4. Construction and expression of EpCAM-TCE DR50818

[0262] The structure is shown in Figure 13. According to the relevant sequences shown in Table 1, the DNA sequence corresponding to DR50818 was synthesized by Jiangsu Huakang Biotechnology Co., Ltd. The heavy chain and light chain were inserted into the pcDNA3.4 vector containing the signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50818HC and pcDNA3.4-DR50818LC.

[0263] DR50818 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2-4 x 10 6Live cell count: 1.0 x 107cells / mL, viability ≥ 95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR50809-HC and pcDNA3.4-DR50809-LC were extracted using a Endo-Free Plasmid Miniprep Kit, filtered through a 0.22 μm filter to remove bacteria, and mixed at a ratio of 1:1. The expression plasmids and transfection reagent PEI (Yixing Biotech (Shanghai) Co., Ltd.) were used to transfect Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and the culture supernatant was harvested.

[0264] 5. Purification of EpCAM-TCE DR50818

[0265] The cell expression suspension was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto an Amsphere A3 affinity chromatography column (JSR Corporation). The equilibration solution was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution solution was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning solution was a 0.1 mol / L NaOH solution; and the neutralization solution was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration solution, and the sample was loaded at a loading amount of no more than 10 mg target protein / ml filler at a retention time of 1 min. After loading, the chromatography column was equilibrated with the equilibration solution, and then the target peak was eluted with the elution solution. The target peak was adjusted to pH 6.0 with the neutralization solution, and the precipitate was removed by centrifugation. The centrifugal supernatant was adjusted to a conductivity of less than 5 mS / cm and then loaded onto a Capto SP impres cation exchange chromatography column (GE Healthcare Life Sciences). Elution was performed at a linear gradient of 30%-50% B (0-500 mmol / L NaCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV), and a sample with a SDS-PAGE electrophoretic purity of ≥ 95% and a SEC purity of ≥ 90% was obtained.

[0266] Example 13. Preparation of proEpCAM-TCE and proEpCAM-TCE-NC: DR50819NC, DR50819, DR50824, DR50825, DR50828, DR50831, DR50833, DR50837

[0267] 1. Construction and expression

[0268] The structure is shown in Figure 14. According to the relevant sequence shown in Table 2, the DNA sequence corresponding to DR50819 is optimized and synthesized by Jiangsu Huakang Biotechnology Co., Ltd., and the heavy chain and light chain are inserted into the pcDNA3.4 vector containing the signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR50819HC and pcDNA3.4-DR50819LC.

[0269] DR50819 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2-4 x 10 6 Live cells / mL, viability ≥95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR50819HC and pcDNA3.4-DR50819LC were extracted using a large-endotoxin-free plasmid extraction kit, filtered through a 0.22 μm filter to remove bacteria, and then mixed at a ratio of 1:1. The expression plasmid and transfection reagent PEI (Yixing Biotechnology (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and then the culture supernatant was harvested.

[0270] DR50819NC was constructed and expressed as DR50819.

[0271] Since DR50819, DR50824, DR50825, DR50828, DR50831, DR50833, and DR50837 have the same structure, the only difference is the sequence of the cleavable linker peptide between EpCAM ECD and the light chain constant region (CL), hu20D1V3 and the light chain constant region (CL), therefore the construction and expression of DR50824, DR50825, DR50828, DR50831, DR50833, and DR50837 are the same as DR50819.

[0272] 2. Purification of DR50819NC, DR50819, DR50824, DR50825, DR50828, DR50831, DR50833, and DR50837

[0273] The cell expression suspension of DR50819 was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto an Amsphere A3 affinity chromatography column (JSR Corporation). The equilibration liquid was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution liquid was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning liquid was a 0.1 mol / L NaOH solution; and the neutralization liquid was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration liquid, and the sample was loaded at a loading amount of not more than 10 mg of target protein per ml of filler at a retention time of 1 min. After loading, the chromatography column was equilibrated with the equilibration liquid, and then the target peak was eluted with the elution liquid. The target peak was adjusted to pH 6.0 with the neutralization liquid, the precipitate was removed by centrifugation, and the supernatant was taken. After the conductivity of the centrifugal supernatant was adjusted to be lower than 4 mS / cm, the sample was loaded onto a Capto SP impres cation exchange chromatography column (GE Healthcare Life Sciences), and eluted in a linear gradient of 0-40% B (25-250 mmol / L Arg-HCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV) to obtain a sample with a SDS-PAGE electrophoretic purity of ≥95% and a SEC purity of ≥90%.

[0274] The purification of DR50819 NC, DR50824, DR50825, DR50828, DR50831, DR50833 and DR50837 was the same as that of DR50819.

[0275] Example 14: Construction of cells for in vitro activity measurement

[0276] 1. Effector cells: Construction of Jurkat-NFAT-Luc2p activity measurement cell strain

[0277] Jurkat cells were transfected with a plasmid containing an NFAT-RE-Luc2p luciferase reporter gene, cultured in a complete RPMI1640 culture medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator under the condition of containing puromycin (4 μg / ml), adjusted for 96-well plate, and after the cells grew stably, the single clone cell line with positive luciferase signal was picked and cultured.

[0278] 2. Target cells: Human pancreatic cancer cells BxPC3 and human breast cancer cells MDA-MB-231 which simultaneously express Trop2 and EpCAM were used as activity measurement target cells.

[0279] Example 15: Recombinant protease cleavage experiment

[0280] The protein with CL sequence was digested with recombinant protease before activity detection to evaluate the efficiency of activity recovery.

[0281] 1. Digestion Buffer preparation:

[0282] 50mM Tris-HCl + 0.01% Tween20 Buffer (pH 8.5): Take 0.157g Tris-HCl, make up to 20ml with ddH2O, add 2μl Tween20, adjust pH to 8.5.

[0283] 2. Digestion with recombinant uPA protease:

[0284] Take 20μg of sample to be digested, add 3μg of uPA protease, add the same volume of digestion Buffer as the sample to be digested, mix well, and incubate in a 37°C oven for 40h.

[0285] 3. Digestion with recombinant ST14 protease:

[0286] Dilute ST14 protease to 0.1μg / ml with digestion Buffer, take 20μg of sample to be digested, add the same volume of ST14 protease at a concentration of 0.1μg / ml, mix well, and incubate in a 37°C oven for 40h.

[0287] 4. Digestion with MMP9 metalloprotease:

[0288] MMP9 digestion Buffer preparation: components are 50mM Tris, 5mM CaCL2, 150mM NaCL, and 0.05% Brij35; preparation process: weigh 0.302g Tris, 0.028g CaCL2, 0.438g NaCL, and 0.025g Brij35, make up to 50ml with pure water.

[0289] Table 5 Digestion system

[0290] Some substrates (i.e. cleavable sequences CS) of the three enzymes reported in the prior art are listed below, as shown in Table 6.

[0291] Table 6 Substrates (cleavable sequences CS) of different proteases

[0292] Example 16 In vitro cell activity assay of proTROP2-TCE and proTrop2-TCE-NC

[0293] Target cell plating: The day before protein sample addition, take appropriate amount of recombinant cells or tumor cells as target cells, digest, centrifuge at 1000 rpm for 5 min, discard supernatant, resuspend with medium containing 10% FBS, count, adjust cell density to 1.5x10 5 cells / ml, plate 96-well luminescence plate, 100 μl per well, incubate overnight at 37°C in a 5% CO2 incubator. In this example, the target cells are BxPC3 cells with high expression of TROP-2 and MDA-MB-231 cells with low expression of TROP-2.

[0294] Protein dilution: the next day, dilute the protein to be tested with RPMI1640 medium containing 1% FBS to the specified concentration.

[0295] Effector cell processing: take appropriate amount of Jurkat-NFAT-Luc2p 10F2 cells, centrifuge at 1000 rpm for 5 min, discard supernatant, resuspend with RPMI1640 medium containing 1% FBS, count, adjust cell density to 1.2x10 6 cells / ml.

[0296] Incubation: take out the target cells plated the day before, add 50 μl of diluted protein after aspirating the supernatant, and add 50 μl of effector cells with adjusted cell density, incubate in a 37°C, 5% CO2 incubator for 6 hours.

[0297] Detection: add luciferase detection solution 10 μl / well, shake for 1 min 30 s, set full wavelength, integration time 500 ms, and detect in a microplate reader.

[0298] Exemplary cell viability assay results are shown in FIGS. 15A-15G (samples tested on the same plate are shown in the same figure). FIG. 15A shows the in vitro cell viability of TCE active molecules Ab-1 and DR50722NC (left: BxPC3 cells; right: MDA-MB-231 cells). FIG. 15B shows the in vitro cell viability of DR50722 after uPA cleavage. FIG. 15C shows the in vitro cell viability of DR50721 and DR50723NC. FIG. 15D shows the in vitro cell viability of DR50721 and DR50723 after uPA cleavage. FIG. 15E shows the in vitro cell viability of Ab-1, DR50724 and DR50724NC. FIG. 15F shows the in vitro cell viability of Ab-1, DR50725NC (left) and DR50725 after uPA cleavage (right). FIG. 15G shows the in vitro cell viability of Ab-1, DR50726 and DR50727NC. All Trop-2-TCEs in Table 1 exhibited high TDCC activity in both target cells, while all proTrop2-TCE-NCs and proTrop2-TCEs in Table 2 showed little or no activity in the cell viability assay, indicating that the activity was almost completely shielded. The proTrop2-TCEs were able to restore activity close to that of the TCE in the active state after cleavage by the proteases (uPA, ST14 or MMP9).

[0299] The proTrop2-TCE results show that the BD1 (20D1 clone) screened by the present application can efficiently shield the activity of the aCD3 antibody derived from SP34, and the tumor-specific activated protein drugs constructed on this basis have little or very low activity before being cleaved by proteases. To verify whether the combination of BD1 (20D1 clone) and the aCD3 derived from SP34 can form different tumor-specific activated protein drugs in combination with any antibody or antibody fragment, the present inventors further constructed tumor-specific activated protein drugs for EpCAM and EGFR targets. In addition, the present inventors also applied the cleavable sequence (CS1, SEQ ID NO. 1) screened to the tumor-specific activated protein drugs for EpCAM and EGFR targets.

[0300] Example 17, in vitro cell viability verification of proEpCAM-TCE and proEpCAM-TCE-NC

[0301] 1. Fluorescent signal method

[0302] The procedure of this example is the same as that of Example 16, and the target cells are also BxPC3 and MDA-MB-231 cells. Exemplary results of the activity assay are shown in Figures 16A-16H (samples detected in the same plate are shown in the same figure). Figure 16A is the in vitro cell activity of DR50808 and DR50809. Figure 16B is the in vitro cell activity of DR50808, DR50812 and DR50813 NC (left: BxPC3 cells; right: MDA-MB-231 cells). Figure 16C is the in vitro cell activity of DR50808, DR50814 and DR50815 NC (left: BxPC3 cells; right: MDA-MB-231 cells). Figure 16D is the in vitro cell activity of DR50818, DR50819 and DR50819 NC (left) and DR50819 after ST14 enzyme digestion (right). Figure 16E is the in vitro cell activity of DR50818, DR50824 and DR50828. Figure 16F is the in vitro cell activity of DR50818, DR50819, DR50825 and DR50825 after ST14 enzyme digestion. Figure 16G is the in vitro cell activity of DR50818, DR50824 and DR50828. Figure 16H is the in vitro cell activity of DR50818, DR50831 and DR50831 after MMP9 enzyme digestion. Figure 16I is the in vitro cell activity of DR50818, DR50832 and DR50832 after MMP9 enzyme digestion.

[0303] As with proTrop2-TCE-NC and proTrop2-TCE, all EpCAM-TCEs in Table 1 exhibited high TDCC activity in both target cells, while all proEpCAM-TCE-NCs and proEpCAM-TCEs in Table 2 had almost no activity or very low activity in the activity assay system, indicating that the activity was almost completely shielded. After enzyme digestion by proteases (uPA, ST14 or MMP9), the proEpCAM-TCEs all restored activity close to that of TCE in the active state.

[0304] 2. In vitro PBMC killing

[0305] In vitro PBMC (peripheral blood mononuclear cell) killing used HCT116 and Colo205 as target cells, and the effector-to-target ratio was 10:1. After incubation for 48 h, cell survival rate was detected. As shown in Figure 17, the activity of TCE active molecule DR50818 was thousands of times different from that of DR50819 and DR50819NC.

[0306] Example 18, in vivo efficacy test of proEpCAM-TCE in mice

[0307] This example tests the in vivo pharmacodynamics of the sample on a PBMC immune system humanized HT29 human colon cancer subcutaneous xenograft mouse model. SPF level, body weight 22-26 g, 6-8 weeks old, male Mus Musculus NCG mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd.) are used. The dosing regimen shown in Table 7 is used as a reference.

[0308] Table 7 Experimental groups Note: The dosing volume is 10 μl / g; i.p. intraperitoneal injection; QD: once a day, Q3D: once every 3 days; 2W: 2 weeks.

[0309] The results are shown in Figure 18. The TCE active molecule DR50818 completely inhibited tumor growth at a dose of 0.3 mg / kg, with a TGI of 106%. Compared with R50819NC, the TGI of DR50819 at a low dose was 49%, and at a high dose, the TGI of DR50819 was 106%, consistent with DR50818. This indicates that DR50819 has a tumor-specific activated antitumor effect.

[0310] The results of Examples 17 and 18 show that the combination of aCD3 antibodies derived from BD1 (20D1 clone) and SP34 is also successfully applied to two different structures of EpCAM target antibodies (e.g. DR50809 and DR50819), and the tumor-specific activated protein drugs prepared therefrom release activity after treatment with tumor-specific expression proteases, significantly killing tumor cells. And in the HT29 colorectal cancer model, they exhibit excellent antitumor activity.

[0311] Example 19, verification of CS1 sequence (SEQ ID NO. 1)

[0312] The cleavable sequence (CS1, SEQ ID NO. 1) obtained by screening was applied to the proEpCAM-TCE with the same structure as DR50819, and DR50833 (CS2, ISSGLLSGRSDRG), DR50837 (CS3, LSGRSDRGSPLGLAG) and DR50819PC1 (PC1, ISSGLLSGRSDNH, SEQ ID NO. 7) were prepared. The cleavable sequence (CS1, SEQ ID NO. 1) can be recognized and cleaved by serine proteases, and CS2 and CS3 can be further recognized by metalloproteinases. DR50819 and DR50833 were incubated in mouse serum and human serum for 1 day and 3 days, and the serum stability was observed by non-reducing SDS-PAGE WB experiment, and it was found that the stability of DR50833 was significantly better than that of DR50819 and DR50819PC1 (Figure 19). At the same time, the results of in vitro recombinant protease cleavage (Figure 20) showed that the cleavage efficiency of DR50833 by uPA (incubated for 40h) and ST14 protease (incubated for 40h) was significantly higher than that of DR50819PC1. Since DR50833 and DR50819PC1 contain the same metalloproteinase recognition sequence (ISSGL), the difference affecting the serum stability and the cleavage efficiency of serine proteases is only in CS1 (LSGRSDRG, SEQ ID NO. 1) and CS4 (LSGRSDNH, SEQ ID NO. 6), and these results show that the cleavage efficiency of the cleavable sequence (CS1, SEQ ID NO. 1) by serine proteases is higher than that of CS4, and the serum stability is better.

[0313] Example 20, Preparation and in vitro identification of DR50837

[0314] 1. Preparation and purification of DR50837 (containing CS3 sequence) is the same as DR50833

[0315] 2. In vitro cell activity test

[0316] In the in vitro cell activity test, DR50833 (containing CS2 sequence) showed very low activity, and the activity was completely restored after uPA enzyme digestion, as shown in Figure 21.

[0317] In the in vitro cell activity test, DR50837 also showed very low activity, which can be efficiently cleaved by ST14 and MMP9 and the activity is restored, as shown in Figure 22.

[0318] 3. ELISA method for detecting in vitro serum stability

[0319] The degradation rate of DR50837 was less than 0.1% in human and mouse serum after 3 days of incubation, as shown in Table 8 below, using 100 ng / well CD3ed-Fc plate coating capture, EpCAM-his (1.0 μg / ml) detection, His tag antibody (1:5000) method to detect DR50837 degradation products (the "DR508 Active" in the table below refers to the active molecule TCE).

[0320] Table 8

[0321] Example 21, Anti-tumor effect of proEpCAM-TCE DR50837 in co-inoculated PBMC and human colon cancer HT29 xenograft tumor model

[0322] Human PBMC and human colon cancer HT29 cells were mixed at a ratio of 1:1 and inoculated subcutaneously into the right flank of SPF-grade B-NDG mice to establish a subcutaneous tumor model. The tumor volume and body weight of all mice were measured, and when the average tumor volume reached 150 mm 3 left, the mice were randomly divided into groups according to tumor volume and body weight, with 5 mice in each group and 3 groups in total. On the day of grouping (D0), the mice were administered according to the group design in Table 9. The tumor volume and body weight of the mice were monitored 2-3 times per week after administration, and the experiment was terminated on D24.

[0323] Table 9 Experimental design and results summary table Note: The statistical data of each index is represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with the Vehicle group. QD: once a day; i.p.: intraperitoneal injection; TGI: tumor inhibition rate.

[0324] The tumor volume and tumor inhibition rate (TGI) of the mice are shown in Figure 23 and Table 9. Compared with the Vehicle group, the DR50837 group had a strong tumor inhibition effect, with a TGI of 107.87% on D24 after grouping. The DR50819NC group had no tumor inhibition effect, with a TGI of -22.83%. During the experiment, the body weight of the mice was monitored, as shown in Figure 24 and Table 9, and no drug-related decrease in body weight was observed in the mice of each group.

[0325] In summary, in this co-inoculated PBMC and human colon cancer HT29 model, DR50837 had a strong tumor inhibition effect. The control DR50819NC without a cleavable sequence did not have a tumor inhibition effect at the same molar dose as DR50837, indicating that DR50837 had a good tumor-specific activated tumor inhibition effect.

[0326] Example 22, Preparation of EGFR-TCE DR51001

[0327] 1. Construction and expression

[0328] The structure is shown in Figure 25. According to the relevant sequences shown in Table 1, the DNA sequence corresponding to DR51001 was entrusted to Jiangsu Huakang Biotechnology Co., Ltd. for optimization and synthesis, and the heavy chain and light chain were inserted into the pcDNA3.4 vector containing the signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR51001-HC and pcDNA3.4-DR51001-LC.

[0329] DR51001 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2-4 x 10 6 Live cells / mL, and the viability was ≥95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR51001-HC and pcDNA3.4-DR51001-LC were extracted using a large-endotoxin-free plasmid extraction kit, filtered through a 0.22 μm filter to remove bacteria, and then mixed at a ratio of 1:1. The expression plasmid and transfection reagent PEI (Yixing Biotechnology Co., Ltd.) were used to transfect Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and then the culture supernatant was harvested.

[0330] 2. Sample purification

[0331] The cell expression suspension of DR51001 was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto a Preasto 70CH1 affinity chromatography column (Purolite Corporation). The equilibration solution was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution solution was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning solution was a 0.2 mol / L HAC solution; and the neutralization solution was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration solution, and the sample was loaded at a loading capacity of not more than 10 mg target protein / ml packing with a retention time of 1-2 min. After loading, the chromatography column was equilibrated with the equilibration solution, and then the target peak was eluted with the elution solution. The target peak was adjusted to pH 6.0 with the neutralization solution, the precipitate was removed by centrifugation, and the supernatant was taken. The centrifugal supernatant was adjusted to a conductivity of less than 5 mS / cm and then loaded onto a Diamond SP cation exchange chromatography column (Borgolon (Shanghai) Biotechnology Co., Ltd.). Elution was performed with a linear gradient of 0-100% B (0-500 mmol / L NaCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV), and a sample with a SDS-PAGE electrophoretic purity of ≥95% and a SEC purity of ≥90% was obtained.

[0332] Example 23, Preparation of proEGFR-TCE and proEGFR-TCE-NC: DR51002, DR51002NC and DR51003

[0333] 1. Construction and expression

[0334] The structure is shown in Figure 26. According to the relevant sequences shown in Table 2, the DNA sequence corresponding to DR51002 was optimized and synthesized by Jiangsu Huakang Biotechnology Co., Ltd., and the heavy chain and light chain were inserted into the pcDNA3.4 vector containing the signal peptide, respectively, to obtain the light chain and heavy chain expression plasmids pcDNA3.4-DR51002-HC and pcDNA3.4-DR51002-LC.

[0335] DR51002 expression preparation: Expi293F cells were subcultured and expanded using CD01 medium (Hangzhou Peiding Biological Technology Co., Ltd.) until the cell density reached 2-4 x 10 6 live cells / mL, and the viability was ≥95%. The light chain and heavy chain expression plasmids pcDNA3.4-DR51002-HC and pcDNA3.4-DR51002-LC were extracted using a large-endotoxin-free plasmid extraction kit, filtered through a 0.22 μm filter to remove bacteria, mixed at a ratio of 1:1, and the expression plasmid and transfection reagent PEI (Yixing Biological Technology (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, and the culture supernatant was harvested.

[0336] DR51002 and DR51003 contain the same CS2 sequence, and the only difference is the length of the flexible linker peptide on both sides of the CS sequence.

[0337] DR51002NC and DR51003 were constructed and expressed in the same way as DR51002.

[0338] 2. Sample purification

[0339] The cell expression suspension of DR51002 was centrifuged at high speed, and the centrifugal supernatant was filtered through a 1.2 μm membrane and then loaded onto an Amsphere A3 affinity chromatography column (JSR Corporation). The equilibration liquid was a 20 mmol / L Na2HPO4-NaH2PO4, 150 mmol / L NaCl, pH 7.0 solution; the elution liquid was a 100 mmol / L Gly-HCl, pH 3.0 solution; the cleaning liquid was a 0.1 mol / L NaOH solution; and the neutralization liquid was a 1 mol / L Tris-HCl, pH 8.0 solution. The chromatography column was equilibrated with the equilibration liquid, and the sample was loaded at a loading amount of not more than 10 mg of target protein per ml of filler at a retention time of 1 min. After loading, the chromatography column was equilibrated with the equilibration liquid, and then the target peak was eluted with the elution liquid. The target peak was adjusted to pH 6.0 with the neutralization liquid, and the precipitate was removed by centrifugation, and the supernatant was taken. The centrifugal supernatant was adjusted to a conductivity of less than 4 mS / cm and then loaded onto a Capto SP impres cation exchange chromatography column (GE Healthcare Life Sciences), and eluted with a linear gradient of 0-60% B (25-250 mmol / L Arg-HCl, 20 mmol / L HAC-NaAC, pH 6.0 solution, 20 CV), to obtain a sample with a SDS-PAGE electrophoretic purity of ≥95% and a SEC purity of ≥90%.

[0340] DR51002NC and DR51003 were purified in the same manner as DR51002.

[0341] Example 24, in vitro cell activity

[0342] The steps of this example were the same as those of Example 16, and the target cells were various EGFR-positive tumor cells. FIG. 27A shows the in vitro cell activity of DR51001, DR51002, and DR51002NC. FIG. 27B shows the in vitro cell activity of DR51001, DR51002, and DR51002 after being cleaved by the ST14 protease. FIG. 27C shows the in vitro cell activity of DR51001, DR51003, and DR51002NC (left: N87 cells; right: NCI-H82 cells). FIG. 27D shows the in vitro cell activity of DR51001, DR51003, and DR51002NC (left) and DR51003 after being cleaved by the ST14 protease. The TCE active molecule DR51001 showed high activity on various cells, while the proEGFR-TCE structure and the proEGFR-TCE-NC structure showed almost no activity or very low activity. The activity of DR51002 and DR51003 was completely restored after being cleaved by the ST14 protease, proving that the conformation was correct.

[0343] Example 25, flow binding with CD3

[0344] Positive control JANX analog (SEQ ID NO. 102, SEQ ID NO. 103) was prepared according to patent WO2022240637. In this example, the flow binding affinity of DR51001, DR51002, DR51002NC and DR51003 to Jurkat cells (high expression of human CD3) was detected. As shown in Figure 28 below, the affinity of DR51002 and DR51003 to CD3 decreased significantly, equivalent to the double shielding effect of JANX analog, proving that the binding of BD1 (hu20D1V3) to aCD3 shields the activity of aCD3.

[0345] Example 26, Flow binding with hEGFR

[0346] In this example, the flow binding affinity of DR51001, DR51002 and DR51003 to human EGFR (hEGFR) positive cells was detected. As shown in Figure 29 below, the affinity of DR51002 and DR51003 to hEGFR decreased significantly, equivalent to the double shielding effect of JANX analog, proving that the binding of BD1 (hu20D1V3) to aCD3 also affects the conformation of EGFR Fab (aTA), therefore, BD1 also plays the role of BD2 at the same time, and only one BD is needed to achieve the double shielding effect.

[0347] Example 27, Anti-tumor effect of proEGFR-TCE DR51003 in co-inoculated PBMC and human colon cancer HT29 xenograft model

[0348] Human PBMC and human colon cancer HT29 cells were mixed at a ratio of 1:1 and subcutaneously inoculated into the right flank of SPF grade B-NDG mice to establish a subcutaneous tumor model. The tumor volume and body weight of all mice were measured, and when the average tumor volume reached 130 mm 3 left, they were randomly divided into 7 groups according to tumor volume and body weight, 5 mice per group. On the day of grouping (D0), the mice were administered according to the group design in Table 10. After administration, the tumor volume and body weight of the mice were monitored 2-3 times per week, and the experiment was terminated on D24.

[0349] Table 10 Test design and result summary table Note: The statistical data of each index is represented by Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with the Vehicle group. QD: once a day; i.p.: intraperitoneal injection.

[0350] The tumor accumulation and tumor inhibition rate (TGI) of mice are shown in Figure 30 and Table 10. On D24 after grouping, the DR51001 group, the DR51003-M group (JANX Analog equimolar dose group), and the DR51003-H group all had strong tumor inhibition effects compared with the Vehicle group, with TGIs of 112.17%, 110.66%, and 111.51%, respectively. The DR51003-L group and the JANX Analog group had limited tumor inhibition effects, with TGIs of 22.87% and 66.46%, respectively. The DR51002NC group did not produce a tumor inhibition effect, with a TGI of -6.12%. During the experiment, the body weight of the mice was monitored, as shown in Figure 31 and Table 10. No drug-related decrease in the body weight of the mice in each group was observed.

[0351] In summary, in the PBMC human colon cancer HT29 model in this example, DR51003 had a strong tumor inhibition effect in a dose-dependent manner, and was equivalent to the equimolar dose of DR51001, while the DR51002NC control without the enzyme cleavage site had no obvious tumor inhibition effect. This indicates that DR51003 has a good tumor-specific activated tumor inhibition effect, and its tumor inhibition efficacy is better than that of the equimolar dose of JANX Analog.

[0352] Example 28, Anti-tumor effect of proEGFR-TCE DR51003 in a human lung adenocarcinoma PC-9 xenograft model with human PBMC immune system humanization

[0353] Human PBMCs were inoculated into the tail vein of SPF-level NCG mice, and human lung adenocarcinoma PC-9 cells were inoculated subcutaneously into the right flank of the mice to establish a subcutaneous tumor model. The tumor volume and body weight of all mice were measured, and when the average tumor volume reached 92 mm 3 left and right, the mice were randomly grouped according to the tumor volume, body weight, and immune reconstruction level, with 5 mice in each group, and a total of 5 groups. On the grouping day (D0), the mice were administered according to the group design in Table 11. The tumor volume and body weight of the mice were monitored 2-3 times per week after administration, and the monitoring was continued until D20, when the experiment was ended.

[0354] Table 11 Test design and result summary table Note: The statistical data of each index are represented as Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 compared with the Vehicle group. QD: once a day; i.p.: intraperitoneal injection.

[0355] The tumor accumulation and tumor inhibition rate (TGI) of mice are shown in Figure 32 and Table 11. On D20 after grouping, the JANX Analog group, the DR51003-L group, the DR51003-M group (JANX Analog equimolar dose group), and the DR51003-H group all had strong tumor inhibition effects compared with the Vehicle group, with TGI of 94.42%, 110.31%, 110.94%, and 110.91%, respectively, and 4 cases (80%), 3 cases (60%), 5 cases (100%), and 5 cases (100%) of complete remission (CR) were observed in the four groups, respectively. During the experiment, the body weight of mice in each group was monitored, as shown in Figure 33 and Table 11. No drug-related decrease in body weight was observed in mice in each group. One mouse in the Vehicle group and one mouse in the JANX Analog group were close to death or died due to model graft versus host disease (GVHD) in the late stage of the experiment.

[0356] In summary, in the human lung adenocarcinoma PC-9 model of PBMC immune system humanization, both JANX Analog and DR51003 have strong tumor inhibition effects, and the tumor inhibition efficacy of DR51003 in all dose groups is better than that of JANX Analog.

[0357] Example 29, Anti-tumor effect of proEGFR-TCE DR51003 in a PBMC immune system humanized human head and neck cancer PDX model

[0358] Human PBMCs were inoculated into the tail vein of SPF-level NCG mice, and the human head and neck cancer PDX model tumor was cut into small pieces and inoculated into the right rib of the mice to establish the PDX model. The tumor volume and body weight of all mice were measured, and when the average tumor volume reached 190 mm 3 The mice were randomly divided into 4 groups of 5 according to the tumor volume, body weight, and immune reconstruction level when the average tumor volume reached 190 mm

[0359] Table 12 Test design and results summary table Note: The statistical data of each index are represented as Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 compared with the Vehicle group. # p < 0.05; ## p < 0.01; ### p < 0.001; ####p<0.0001 vs. JANX Analog group. QD: once daily; i.p.: intraperitoneal injection.

[0360] Tumor volume and tumor inhibition rate (TGI) of mice are shown in Figure 34 and Table 12. On D18 after grouping, DR51003-L group (JANX Analog equimolar dose group) and DR51003-H group had stronger tumor inhibition effect compared with the Vehicle group, and the TGI was 96.20% and 106.14%, respectively. The tumor inhibition effect of JANX Analog group was limited, and the TGI was 35.65%. During the experiment, the body weight of mice was monitored, and no drug-related decrease in body weight was observed in each group, as shown in Figure 35 and Table 12.

[0361] In summary, in the humanized head and neck cancer PDX model of PBMC immune system in this example, DR51003 has a strong tumor inhibition effect, and its tumor inhibition efficacy is significantly better than that of JANX Analog at the equimolar dose.

[0362] Example 30. Pharmacokinetic study of proEGFR-TCE DR51003 in cynomolgus monkeys

[0363] Two healthy cynomolgus monkeys (Wuyin monkeys, half male and half female, 3-4 years old, 2.6 kg and 3.1 kg) were each single intravenously infused with 0.066 mg / kg of DR51003, and the administration time was 1 h per monkey, and the administration volume was 5 mL / kg. Serum samples were collected before administration and at 0.5, 1, 2, 6, 24, 48, 96, 120, and 168 h after the start of administration (with the start of intravenous infusion as the starting time) for detection of the concentrations of DR51003 intact structure (Intact) and TCE active structure (Active), to draw the blood concentration-time curve and calculate the pharmacokinetic parameters, and to evaluate the pharmacokinetic characteristics and degradation rate in cynomolgus monkeys.

[0364] During the experiment, no abnormalities were observed in animal body weight, food intake, body temperature, and clinical general observation. The drug was stable within 168 h after administration of DR51003, and the blood concentration-time curve is shown in Figure 36. The t 1 / 2 was 133.72 ± 6.89 h, and other pharmacokinetic parameters are shown in Table 13. Active was detected only at 1-6 h after the start of administration, and the detection at other time points was below the detection limit. The degradation rate calculation formula was: C active / (C active +C Intact ) × 100%, and C: the concentration of the corresponding structure of the drug.

[0365] In summary, DR51003 has good stability in cynomolgus monkeys.

[0366] Table 13 Pharmacokinetic parameters of DR51003 in cynomolgus monkeys

[0367] Example 31 Toxicology dose exploration study of proEGFR-TCE DR51003 in cynomolgus monkeys

[0368] Four weeks of intravenous administration of DR51003 to cynomolgus monkeys once a week, a total of 4 times, to observe the possible toxic reactions caused by DR51003. Two healthy cynomolgus monkeys (Wuyin monkeys, half male and half female, 3-4 years old, 2.5 kg and 3.0 kg) were intravenously infused with DR51003 on D1, D8, D15 and D22, respectively, with a dose of 0.65 mg / kg (dose volume of 5.4 mL / kg) on D1 and D8, and a dose of 1.3 mg / kg (dose volume of 10.8 mL / kg) on D15 and D22, with a dose time of 1 h per animal. The observation indicators include death / near-death observation, general observation, body weight, food intake, body temperature, electrocardiogram (lead II), blood pressure, hematology and coagulation, serum biochemistry and immune function, urine test, cytokine detection, and gross necropsy and major organ weight at the end of the observation period (D29) for all animals.

[0369] During the experiment, all animals survived to the planned dissection. No abnormalities were observed in general observation, body weight, body temperature, blood pressure, electrocardiogram (lead II), hematology, coagulation, serum biochemistry, urine test, gross necropsy and organ weight. The main abnormalities related to the test product were: a decrease in food intake (25%-50%) was observed in female and male animals from D13, and the animals ate the auxiliary food given from D18-D28, and food intake was restored on D27; IL-6 increased slightly (≤160.25 pg / mL) 6 hours after administration of D1, and returned to normal 24 hours after administration. Other cytokine tests were below the detection limit. See Table 14. No obvious abnormalities were observed in the general observation of animals and other indicators, and this change had no toxicological significance.

[0370] In summary, DR51003 has good safety in cynomolgus monkeys.

[0371] Table 14 Cytokine detection results Note:BLQ:Below the Lower Limit of Quantification(19.53pg / mL).Quantitative Range of Cytokine Detection:19.53~5000.00pg / mL.

[0372] SEQUENCE LISTING

Claims

1. A single domain antibody, which has complementarity determining regions (CDRs) comprising CDR1 to CDR3 with amino acid sequences as shown below: CDR1 as shown in SEQ ID NO: 8, CDR2 as shown in SEQ ID NO: 9, and CDR3 as shown in SEQ ID NO:

10. Also comprising framework regions (FRs) comprising FR1 to FR4 with amino acid sequences as shown below: FR1 as shown in SEQ ID NO: 11, FR2 as shown in SEQ ID NO: 12, FR3 as shown in SEQ ID NO: 13, and FR4 as shown in SEQ ID NO:

14. The single domain antibody is a humanized antibody.

4. The single domain antibody of claim 3, which humanized antibody comprises FR1 to FR4 selected from the group consisting of: FR1 as shown in SEQ ID NO: 15 or 16, FR2 as shown in SEQ ID NO: 12 or 17, FR3 as shown in SEQ ID NO: 18, and FR4 as shown in SEQ ID NO:

14.

2. The single domain antibody of claim 2, wherein, The humanized antibody has: (1) an amino acid sequence as shown in any one of SEQ ID NOs: 19 to 21; or (2) a sequence having 80% or more identity to the sequence as shown in SEQ ID NOs: 19 to 21 and having the function as shown in (1). CDR1 as shown in SEQ ID NO. 104, CDR2 as shown in SEQ ID NO. 105, and CDR3 as shown in SEQ ID NO.

106. has an amino acid sequence as shown in SEQ ID NO. 36 or a sequence having 80% or more identity to the sequence as shown in SEQ ID NO.

36. comprises a cleavable sequence (CS) as shown in SEQ ID NO.

1. The cleavable sequence (CS) is cleavable by a tumor-specific protease; 3. The single domain antibody of claim 1, wherein Optionally, the tumor-specific protease comprises a metalloprotease, a serine protease, or a cysteine protease. The metalloprotease comprises MMP-2, MMP-7, MMP-9, MMP-11, MMP-13, MMP-14; the serine protease comprises urokinase-type plasminogen activator, Matriptase, or Hepsin; and the cysteine protease comprises Legumain. The isolated polypeptide comprises: 1) a polypeptide that targets binding to a T cell surface CD3 molecule (aCD3); 2) a polypeptide 1 (BD1) that blocks the binding of aCD3 to a T cell surface CD3 molecule; 3) an antibody fragment that recognizes and binds to a tumor antigen (aTA); 4) a cleavable linker peptide (CL) comprising a cleavable sequence (CS) sequence, and / or 5) a polypeptide 2 (BD2) that blocks the binding of aTA to its target antigen; the aCD3 and aTA together form an active molecule T cell engager (TCE). ​ ​ ​ 5. The single domain antibody according to any one of claims 3 or 4, wherein ​ ​ ​ 6. A single domain antibody, characterized in that, ​ ​ 7. The single domain antibody of claim 6, wherein ​ 8. An isolated polypeptide, comprising, ​ 9. The isolated polypeptide of claim 8, wherein, ​ ​ 10. The isolated polypeptide of claim 9, wherein, ​ ​ 11. An isolated polypeptide, comprising, ​ ​ 12. The isolated polypeptide of claim 11, characterized by, The aTA is an antibody Fab domain, one chain of the antibody Fab domain is linked to aCD3 to form the TCE; the BD1 is linked to the TCE through CL, and is located in a different chain from aCD3.

13. The isolated polypeptide of claim 11, characterized by, The aTA is a single domain antibody, the aTA and aCD3 are linked through an antibody CH1-CL domain to form the TCE; the aTA is located at the N-terminal of the antibody CH1-CL domain and the aCD3 is located at the C-terminal of the antibody CH1-CL domain, or the aTA is located at the C-terminal of the antibody CH1-CL domain and the aCD3 is located at the N-terminal of the antibody CH1-CL domain; the BD1 is linked to the TCE through CL, and is located in a different chain from aCD3.

14. The isolated polypeptide of claim 12, further comprising a polypeptide 2 (BD2) that blocks the binding of the aTA to its target antigen, the BD2 being linked to one chain of the aTA through CL when the BD2 is present.

15. The isolated polypeptide of claim 13, wherein the polypeptide is characterized by, Further comprising a polypeptide 2 (BD2) that blocks the binding of the aTA to its target antigen, the BD2 being linked to the TCE through CL when the BD2 is present, and being located in a different chain from the aTA.

16. The isolated polypeptide of claim 14 or 15, wherein the BD2 is selected from the group consisting of an antibody, an antibody fragment, a masking peptide, an antigen recognized by the aTA, or an antigen fragment; optionally, the antigen fragment is selected from the extracellular domain of an antigen.

17. The isolated polypeptide of claim 11, wherein, The aCD3 is derived from SP34 antibody; preferably, the aCD3 derived from SP34 antibody is a single chain antibody; more preferably, the aCD3 is selected from the sequences shown in SEQ ID NO. 40 or 41; the BD1 is an antibody, an antibody fragment, or a masking peptide; preferably, the BD1 is a single domain antibody according to any one of claims 1-5; preferably, the BD1 is selected from the sequences shown in any one of SEQ ID NO: 19-21 or a sequence having more than 80% identity to the sequences shown in SEQ ID NO: 19-21.

18. The isolated polypeptide of claim 11, wherein, The CL sequence consists of a CS sequence, or further comprises alanine (A) and / or serine (S) and / or glycine (G); optionally, the CL has a sequence selected from any one of SEQ ID NO. 1-7, SEQ ID NO. 76-95.

19. The isolated polypeptide of claim 11, wherein, Further comprising a long-acting unit, optionally, the long-acting unit is selected from a single domain antibody that binds serum albumin; optionally, the long-acting unit comprises a sequence shown in SEQ ID NO.

37.

20. The isolated polypeptide of claim 19, wherein, The long-acting unit is linked to BD1 or BD2; optionally, the long-acting unit is linked to BD1; optionally, the long-acting unit is linked to BD2.

21. The isolated polypeptide of any of claims 11-20, wherein, There can be a connecting peptide between the aCD3 and the antibody Fab domain, between the aCD3 and the antibody CH1-CL domain, between the aTA and the antibody CH1-CL domain, between the BD1 and the long-acting unit, and between the BD2 and the long-acting unit, which can be a flexible polypeptide chain composed of alanine (A) and / or serine (S) and / or glycine (G), and the length of the connecting peptide can be 3-40 amino acids, preferably 3-9, 9-12, 12-16, 16-20, 20-25, 25-30, 30-35, 35-40, more preferably 5 or 8 or 15 or 18.

22. The isolated polypeptide of any of claims 11-20, wherein, The tumor antigen includes, but is not limited to, at least one of EpCAM, Trop-2, HER-2, HER3, EGFR, PSMA, Claudin 6, Claudin 18.2, MUC-1, MUC-6, MUC-17, CEA, MSLN, and FRa, etc.; optionally, the tumor antigen is Trop-2; optionally, the tumor antigen is EpCAM; optionally, the tumor antigen is EGFR.

23. The isolated polypeptide of any of claims 11-22, wherein, The isolated polypeptide includes the sequence shown in SEQ ID NO. 22 and SEQ ID NO. 43, the sequence shown in SEQ ID NO. 24 and SEQ ID NO. 45, the sequence shown in SEQ ID NO. 47 and SEQ ID NO. 43, the sequence shown in SEQ ID NO. 48 and SEQ ID NO. 43, the sequence shown in SEQ ID NO. 22 and SEQ ID NO. 49, the sequence shown in SEQ ID NO. 26 and SEQ ID NO. 50, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 56, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 57, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 58, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 59, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 60, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 61, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 62, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 64, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 65, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 66, the sequence shown in SEQ ID NO. 54 and SEQ ID NO. 67, the sequence shown in SEQ ID NO. 33 and SEQ ID NO. 69, and the sequence shown in SEQ ID NO. 33 and SEQ ID NO.

70.

24. An isolated polynucleotide encoding the single-domain antibody of any one of claims 1 to 7, the isolated polypeptide of any one of claims 8 to 23.

25. An expression vector comprising the isolated polynucleotide of claim 24.

26. A recombinant cell comprising the expression vector of claim 25 or the polynucleotide of claim 24 integrated into the genome of the recombinant cell.

27. The single-domain antibody according to any one of claims 1 to 7, the method of producing an isolated polypeptide according to any one of claims 8 to 23, comprising the step of: culturing the recombinant cell according to claim 26 under conditions suitable for protein expression to obtain the single-domain antibody or the isolated polypeptide; wherein, The recombinant cell comprises the polynucleotide of claim 24 or the expression vector of claim 25, the polynucleotide encoding the single-domain antibody of any one of claims 1 to 7 or the isolated polypeptide of any one of claims 8 to 23; the expression vector comprising the polynucleotide encoding the single-domain antibody of any one of claims 1 to 7 or the isolated polypeptide of any one of claims 8 to 23.

28. A pharmaceutical composition comprising the polynucleotide of claim 24, the expression vector of claim 25, the recombinant cell of claim 26, the single-domain antibody of any one of claims 1 to 7, or the isolated polypeptide of any one of claims 8 to 23.

29. The pharmaceutical composition of claim 28, wherein, Also included are pharmaceutically acceptable carriers.

30. Use of the polynucleotide of claim 24, the expression vector of claim 25, the recombinant cell of claim 26, the single-domain antibody of any one of claims 1 to 7, the isolated polypeptide of any one of claims 8 to 23, or the pharmaceutical composition of claim 28 in the manufacture of a medicament for the diagnosis, treatment, or prevention of a disease associated with cells expressing a tumor antigen.

31. The use of claim 30, wherein, The disease associated with cells expressing a tumor antigen is selected from the group of one or more of a hematological tumor, a gastric cancer, an esophageal cancer, a pancreatic cancer, a breast cancer, a lung cancer, an ovarian cancer, a cervical cancer, a colorectal cancer, a liver cancer, a kidney cancer, a bladder cancer, a prostate cancer, a melanoma, a head and neck cancer, a neuroendocrine cancer, an adrenal cancer, a gallbladder cancer, and a mesothelioma.

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