WO2024240064A1 - Antibody-drug conjugate targeting antigenic epitope polypeptide, and use thereof - Google Patents

Abstract

Provided is a use of an antibody targeting an antigenic epitope polypeptide or an antigen-binding fragment thereof in the preparation of an antibody-drug conjugate. An antigenic epitope polypeptide is differentially expressed on tumor cells/cancer cells and normal human cells. The antibody or the antigen-binding fragment thereof can target and bind to the antigenic epitope polypeptide to show an antibody internalization function, and thus can be applied to the preparation of an antibody-drug conjugate and play a tumor treatment effect.

Description

Antibody-drug conjugates targeting antigen epitope peptides and their applications Technical Field

The present invention relates to the field of medicine, specifically to the field of antibody-drug conjugates, and in particular to antibody-drug conjugates targeting antigen epitope polypeptides and applications thereof.

Background Art

Antibody-drug conjugate (ADC) is a type of biological drug that connects a biologically active cytotoxic drug (payload) to an antibody through a chemical linker. In recent years, multiple antibody-drug conjugates have made breakthrough progress in tumor treatment, making it a major emerging treatment method after surgery, chemotherapy, and radiotherapy. ADC drugs combine the highly specific targeting ability of antibodies with the efficient killing effect of cytotoxic drugs, and can achieve accurate and efficient removal of cancer cells. It has become one of the hot spots in the field of cancer research and development. As of March 2023, 16 antibody-drug conjugates have been approved worldwide. At present, the research field of ADC is still expanding, and it is mainly used in the research of hematological malignancies and solid tumors.

Although ADC drugs have been favored by many medical workers and developed rapidly, their toxicity is still a very important challenge faced in clinical practice. For example, the uptake of ADC in non-targeted normal cells will transmit cytotoxicity. For example, the expression of ADC-targeted antigens in normal tissues (although the expression level is low) may lead to ADC target-dependent uptake and secondary toxicity. The ideal ADC drug aims to improve the therapeutic index of cytotoxic drugs and reduce exposure in normal cells by more selectively delivering cytotoxic drugs to tumor cells. How to avoid or reduce drug toxicity during the development of ADC drugs is a problem that R&D personnel need to think about during development.

Summary of the invention

The present invention solves at least one of the technical problems mentioned to a certain extent. The selection of antigens that are low-expressed or not expressed in normal tissues for specific targeting can undoubtedly effectively reduce the toxicity of ADC. The targets or antigens targeted by ADC drugs that have been marketed or are under research are all antigens that are highly expressed in tumor cells and low-expressed in normal cells. Even so, cytotoxicity will inevitably be produced. The present invention can be used to distinguish tumor cells and/or cancer cells from normal cells by modifying the surface of tumor cells and/or cancer cells with a unique amino acid sequence (for example, an amino acid sequence (antigenic epitope polypeptide) that is not contained in the amino acid sequence of mammalian cell membrane proteins or secretory proteins in the natural state), and to bring tumor cells and/or cancer cells to "target". Then, by targeting the amino acid sequence modified on the surface of tumor cells and/or cancer cells with antibodies, specific targeting of tumor cells and/or cancer cells can be achieved; by killing tumor cells or cancer cells with cytotoxic drugs mediated by antibody endocytosis, specific killing of tumors can be achieved. Taking TT3 as an example, the TT3 sequence contains an antigen epitope polypeptide. By comparing it with the human genome database, it was found that the antigen epitope polypeptide is not expressed in the human genome, so it can be used to modify the surface of tumor cells and/or cancer cells. Antibodies against TT3 can specifically recognize and bind to TT3, achieve specific targeting of tumor cells and/or cancer cells, and kill tumor cells or cancer cells through antibody-mediated cytotoxic drugs, thereby improving the anti-tumor killing effect.

Specifically, the present invention provides the following technical solutions:

In the first aspect of the present invention, there is provided a use of an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide in the preparation of an antibody conjugate; wherein in a natural state, the amino acid sequence of a mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen-binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells, and exhibits endocytosis with EC50≤100nM. For example, endocytosis can be exhibited with EC50≤90nM, EC50≤80nM, EC50≤70nM, EC50≤60nM, EC50≤50nM, EC50≤40nM, EC50≤30nM, EC50≤20nM, EC50≤10nM, EC50≤5nM, EC50≤4nM, EC50≤3nM, EC50≤2nM, EC50≤1nM.

In a second aspect of the present invention, there is provided a use of an antibody-drug conjugate in the preparation of a drug for treating cancer and/or tumors, wherein the antibody-drug conjugate is an antibody or antigen-binding fragment targeting an antigen epitope polypeptide coupled to one or more therapeutic agents; wherein in a natural state, the amino acid sequence of a mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen-binding fragment binds to the tumor epitope polypeptide. It specifically binds to antigen epitope peptides on tumor cells/cancer cells and exhibits endocytosis with EC50 ≤ 100nM.

In the third aspect of the present invention, an antibody-drug conjugate targeting an antigen epitope polypeptide is provided, comprising an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide coupled to one or more therapeutic agents; wherein in a natural state, the amino acid sequence of a mammalian cell membrane protein or a secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen-binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells and exhibits endocytosis with an EC50 ≤ 100 nM.

In a fourth aspect, the present invention provides a pharmaceutical composition, which comprises the antibody-drug conjugate according to the third aspect of the present invention.

In a fifth aspect, the present invention provides a kit, comprising the antibody-drug conjugate according to the third aspect of the present invention.

The sixth aspect of the present invention provides a method for preventing and/or treating a disease, the method comprising administering an effective amount of the antibody-drug conjugate of the third aspect, or the pharmaceutical composition of the fourth aspect to a subject in need.

The seventh aspect of the present invention provides a use of an antibody-drug conjugate in the preparation of a drug or a kit, wherein the antibody-drug conjugate is the antibody-drug conjugate described in the third aspect.

The eighth aspect of the present invention provides a use of an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide in the preparation of an antibody-drug conjugate;

Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide;

Compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells;

The amino acid sequence of the antigenic epitope polypeptide has one or more sequences derived from Strep tag I or Strep tag II.

The ninth aspect of the present invention provides an antibody-drug conjugate targeting an antigen epitope polypeptide, comprising an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide coupled to one or more therapeutic agents;

Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide;

Compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells;

The amino acid sequence of the antigenic epitope polypeptide has one or more sequences derived from Strep tag I or Strep tag II.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the ELISA test results of the antibody targeting TT3 and its humanized antibody provided according to an embodiment of the present invention, as well as the FACs test results of HCT116-TT3 cells, SK-HEP-1-TT3 cells and MC38-TT3 cells.

FIG. 2 is a diagram showing the SDS-PAGE results of the antibody-drug conjugate TT3-MMAE provided in Example 2 of the present invention.

FIG3 is a graph showing the SEC results of the antibody drug conjugate TT3-MMAE provided in Example 2 of the present invention.

FIG. 4 is a UV-Vis result diagram of the antibody drug conjugate TT3-MMAE provided according to Example 2 of the present invention.

FIG5 is a graph showing the cell binding activity results of the antibody drug conjugate TT3-MMAE provided in Example 3 of the present invention.

FIG6 is a diagram showing the endocytosis results of the antibody-drug conjugate TT3-MMAE provided in Example 3 of the present invention.

FIG. 7 is a graph showing the cell killing results of the antibody-drug conjugate TT3-MMAE provided in Example 3 of the present invention.

Specific implementation plan

The technical solution of the present invention is described in detail below with reference to specific examples. At the same time, in order to facilitate the understanding of those skilled in the art, some terms of the present invention are explained and described. It should be noted that these explanations and descriptions are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention.

As used in this specification and claims, "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. The words "tumor", "cancer", "tumor cell", and "cancer cell" have the meanings commonly recognized in the art. The term "plurality" includes two.

The term "effective amount" herein refers to the amount of a drug, preparation or active ingredient that can show a detectable therapeutic effect, inhibitory effect or preventive effect. The detectable therapeutic effect, inhibitory effect or preventive effect mentioned can be detected by any detection method known in the art.

As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in tumor cells and lyse tumor cells.

As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide comprising an antigen binding site, an antigen binding portion or an antigen binding fragment, covering natural antibodies and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen binding fragments of antibodies.

Herein, "complete antibody" or "complete antibody" etc. are used to express the same meaning, indicating a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (also referred to as a heavy chain constant domain, abbreviated as CH). The heavy chain constant region includes a heavy chain constant domain CH1, a heavy chain constant domain CH2, and a heavy chain constant domain CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (also referred to as a light chain constant domain, abbreviated as CL). VH and VL can be further divided into complementary determining regions (also referred to as hypervariable regions or hypervariable regions, abbreviated as CDR or HVR), with conserved framework regions (FR) inserted therebetween. Each VH and VL comprises three CDRs and four FRs, arranged in the following sequence from the amino terminus (N terminus) to the carboxyl terminus (C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively, starting from the amino terminus, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively, starting from the amino terminus.

"Antigen binding fragment", "antigen binding portion", or "antigen binding site" refers to a portion of a full-length antibody that exhibits specific binding to an antigen. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen binding fragments or antigen binding portions include, but are not limited to, Fab, Fab', F(ab') ₂ , bispecific Fab' and Fv fragments, linear antibodies, single-chain antibodies, single-domain antibodies, bispecific antibodies, and multispecific antibodies. Papain digestion of an intact antibody produces two identical antigen binding fragments, called Fab fragments, each of which contains a heavy and light chain variable region and a light chain constant domain and a heavy chain constant domain CH1. The Fab' fragment differs from the Fab fragment by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the hinge region of the antibody. Pepsin digestion of an intact antibody yields a F(ab') ₂ fragment. The F(ab') ₂ fragment has two antigen-binding F(ab) parts linked together by a disulfide bond, and the F(ab') ₂ fragment is a bivalent antibody. A single-chain antibody is a fusion protein formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a flexible short peptide consisting of about 10-25 amino acids. A single-domain antibody is an antibody fragment composed of the variable region of a single monomer. Since single-domain antibodies are usually derived from the variable region of the heavy chain of camel antibodies or shark antibodies, they are often called nanobodies. "Multispecific antibodies" (also called multi-antibodies) refer to antigen-binding portions that specifically bind to epitopes of at least two different biological molecules or at least two different epitopes of the same biological molecule. For example, a "multispecific antibody can be a bispecific antibody (also called a bispecific antibody), or a trispecific antibody, etc. "Multi" means two or more.

When the term "and/or" or "/" is used to connect two or more optional items, it should be understood to mean any one of the optional items or any two or more of the optional items.

The term "comprising" or "including" means including the mentioned elements or steps, but not excluding other elements or steps. Of course, unless otherwise stated, comprising or including also covers the situation consisting of the mentioned elements or steps. For example, when referring to an antibody variable region comprising a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and/or stability of the binding sites between an antigen and an antibody or an antigen-binding fragment.

"Specific recognition", "specific recognition of", "specific binding" or "specific binding to", "binding to", "specific targeting" a specific antigen or epitope, or "having specificity" or "capable of binding" to a specific antigen or epitope means to distinguish from non-specific interactions, and this specific binding or specific recognition effect can be measured by some methods commonly used in the art. The ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying antigens can be detected by flow cytometry, and the competitive binding between the antibody to be tested and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation. In conjunction with the specific embodiments of the present invention, the provided antibodies have endocytosis and exhibit endocytosis with an EC50 value of ≤10nM, ≤9nM, ≤8nM, ≤7nM, ≤6nM, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.9nM, ≤0.8nM, ≤0.7nM, ≤0.6nM, ≤0.5nM, ≤0.4nM, ≤0.3nM, ≤0.2nM, ≤0.1nM, ≤0.05nM, ≤0.01nM, ≤0.005nM, ≤0.001nM. In addition, the binding activity of the antibody to the antigen can also be determined by surface isoparticle resonance technology (SPR) or biofilm interferometry technology (BLI).

"Humanized" antibodies generally refer to antibodies based on antigen-binding portions derived from non-human species and based on partial structures and sequences of human immunoglobulin molecules. For example, in humanized antibodies, the entire antibody except for CDR is encoded by a polynucleotide of human origin, which retains the antigen-binding activity while reducing immunogenicity.

The term "therapeutic agent" refers to any substance or entity that can play a therapeutic role (e.g., treat, prevent, alleviate or inhibit any disease and/or cancer), including but not limited to: chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, thermal therapeutic agents, etc.

Epitope peptide

Select the amino acid sequence that the amino acid sequence of mammalian cell membrane protein or secretory protein does not contain as antigen epitope polypeptide under natural state, modify tumor cell and/or cancer cell surface, make antigen epitope polypeptide be modified on the surface of tumor cell and/or cancer cell through expression, thereby can distinguish tumor cell and/or cancer cell from normal cell.According to specific embodiment, in order to modify antigen epitope polypeptide on tumor cell and/or cancer cell surface, extracellular antigen determining region, spacer part and transmembrane part can be operably connected as needed, and together constitute marker polypeptide.Antigenic epitope polypeptide can be set as one or more as needed as part of forming extracellular antigen determining region.By expressing marker polypeptide on tumor cell and/or cancer cell, thereby modifying antigen epitope polypeptide on tumor cell and/or cancer cell surface.Herein, when describing protein or polypeptide expression on tumor cell or cancer cell or normal cell, or expression in tumor cell or cancer cell or normal cell, the position of protein or polypeptide on cell is not specifically limited (i.e., not specifically limited to expression in cell or on cell or cell surface). Those skilled in the art can determine the expression location of a protein or polypeptide based on common technical knowledge, for example, the extracellular antigenic determining region is expressed on the cell surface, and the transmembrane portion is expressed on the cell membrane. The mentioned marker polypeptide and its contained extracellular antigenic determining region, spacer portion and transmembrane portion and other polypeptide sequences are all recorded in the PCT patent application with international application number PCT/CN2019/102480 and international publication number WO2020038490A, which can be cited or partially cited in this article as needed. The term "extracellular antigenic determining region" refers to the portion of the marker polypeptide containing the antigenic epitope polypeptide located outside the cell membrane when it is expressed on the cell surface.

According to a specific embodiment, the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of a protein existing in nature, or is an artificially synthesized amino acid sequence that does not exist in nature. Wherein, the protein existing in nature includes proteins in mammalian cells and proteins of other organisms except mammals. Proteins of other organisms except mammals include viral proteins, bacterial proteins, fungal proteins, protozoan proteins, plant proteins, and other animal proteins except mammals. In some embodiments, the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of the following tags: Myc tag, HA tag, Strep tag I, Strep tag II, Flag tag, HAT tag, S tag, Sl tag, protein C tag, tag-100 tag, E2 tag, TAP tag, HSV tag, KT3 tag, V5 tag, VSV-G tag, His tag or RFP tag, etc. The amino acid sequence and nucleotide sequence of these tags are all known and can be queried by the public database commonly used in the art.

In some preferred embodiments, the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of the following tags: human Myc tag (the corresponding marker polypeptide is recorded as TT1), influenza virus HA tag (the corresponding marker polypeptide is recorded as TT2), Strep tag II (the corresponding marker polypeptide is recorded as TT3) or Strep tag I. The amino acid sequence of the Strep tag I or Strep tag II mentioned is at least 3 amino acids long, such as 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11, 12, 13, 14 or 15 amino acids, and may contain amino acids derived from the sequence shown in NWSHPQFEK (SEQ ID NO: 26) or AWRHPQFGG (SEQ ID NO: 27), and may also be a partial sequence or a combination of partial sequences of these sequences. More specifically, the amino acid sequence of Strep tag II may be, for example, WSHPQFEK (SEQ ID NO: 28), NWSHPQFEK (SEQ ID NO: 26). The amino acid sequence of Strep tag I mentioned may be, for example, AWRHPQFGG (SEQ ID NO: 27). Also preferably, the amino acid sequence of the antigenic epitope polypeptide is consistent with the 410th to 419th amino acid sequence of human Myc protein; or the amino acid sequence of the antigenic epitope polypeptide is consistent with the 119th to 127th amino acid sequence of influenza virus HA protein; or the amino acid sequence of the antigenic epitope polypeptide is consistent with Strep tag II (for a detailed description of Strep tag II, see the document "Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin, Thomas G.M.Schmidt, Jurgen Koepke, Ronald Frank and Arne Skerra"). In other embodiments of the present invention, the amino acid sequence of the antigenic epitope polypeptides of TT1 and TT2 may be extended to no more than 10 amino acids upstream and downstream of the above sequence. Among them, the amino acid sequence of human Myc protein may be the amino acid sequence numbered P01106 isoform1 in UniProtKB. The amino acid sequence of the influenza virus HA protein may be the amino acid sequence numbered Q03909 in UniProtKB.

In some specific embodiments, the amino acid sequence of the extracellular antigenic determinant region of the marker polypeptide includes the amino acid sequence of one or more epitope polypeptides, wherein when the amino acid sequence of the extracellular antigenic determinant region of the marker polypeptide includes the amino acid sequence of multiple epitope polypeptides, each two adjacent epitope polypeptides are operably connected, for example, they can be connected by a linker, or they can be directly connected without a linker. The amino acid sequence of the linker can be (for example): G (as used in marker polypeptides C1&2a, C1&2b), GGS (as used in C1&2a, C1&2b), GGGGSGGGGS (as used in TT1-TT3).

In order to enhance the immunogenicity of the marker polypeptide, the extracellular antigenic determinant region of the marker polypeptide preferably comprises n antigenic epitope polypeptides, wherein n is an integer greater than or equal to 1, for example, n=1, 2, 3, 4... Preferably, n is an integer between 1 and 10; more preferably, n is an integer between 2 and 5; more preferably, n=2 or 3.

According to a specific embodiment, the extracellular antigenic determinant region of the marker polypeptide may contain 1, 2, 3, 4 or 5 The invention relates to a polypeptide having an antigen epitope derived from Strep tag I or Strep tag II.

According to a specific embodiment, the sequence of the antigen epitope polypeptide is shown in SEQ ID NO:24 or SEQ ID NO:25.

According to a specific embodiment, the extracellular antigen determining region and the transmembrane portion are connected by a spacer. The structure of this region should be flexible so that the extracellular antigen determining region can adapt to different directions to promote the recognition and binding of the corresponding multispecific antibodies. The simplest form of the spacer is the hinge region of the immunoglobulin IgG1, or it can be a part of the immunoglobulin _{CH2CH3 region} . Through research and experimental exploration, it is found that the spacer is preferably from the hinge region of CD8α, and the transmembrane region is preferably from the transmembrane region of CD8α. Preferably, the spacer and the transmembrane portion constitute the spacer transmembrane portion, and wherein the amino acid sequence of the spacer transmembrane portion is consistent with the amino acid sequence of the Y-210th position of CD8α, and 118≤Y≤128, and Y is an integer. Among them, the UniProtKB number of the amino acid sequence of CD8α can be P01732. That is, the amino acid sequence of the spacer transmembrane portion is preferably selected from the 118th-210th amino acids of CD8α and contains the 128th-210th amino acids. For example, the amino acid sequence of the spacer transmembrane portion is as shown in any one of the following amino acid sequence groups: amino acids 118-210, 119-210, 120-210, 121-210, 122-210, 123-210, 124-210, 125-210, 126-210, 127-210, or 128-210 of CD8α.

The transmembrane portion can be any type I transmembrane region sequence, and the type I transmembrane region sequence mentioned refers to a single transmembrane sequence, and the N-terminus of the peptide chain is located outside the cell membrane, and the C-terminus of the peptide chain is located inside the cell membrane. According to a specific embodiment, the transmembrane portion is derived from the transmembrane region of CD8, CD3ζ, CD4 or CD28, and its full-length amino acid sequence and nucleotide sequence are known and can be queried from public databases commonly used in the art. It is also preferred that the transmembrane portion is derived from the transmembrane region of human CD8α. CD8 is a transmembrane glycosylated membrane protein composed of two subunits, α and β, which works together with T cell surface receptors to bind T cells to specific antigens. CD8 specifically binds to MHC I and mediates the killing effect of cytotoxic T cells. The transmembrane region is usually a hydrophobic α helix that spans the cell membrane.

The definitions of the terms "spacer portion" and "transmembrane portion" used in this article are known in the art. For details, please refer to "Introduction to Immunology, Yu Shanqian, Higher Education Press, 2008"; and "Immunobiology, 7th edition, Kenneth Murphy, Paul Travers, Mark Walport, etc."

The present invention further discovered that the marker polypeptide can express two antigenic polypeptides at the same time. Specifically, the present invention designed a marker polypeptide Cl&2a, whose antigenic determining region contains three repeats of the Myc tag and the HA tag. The present invention also designed a marker polypeptide Cl&2b, whose antigenic determining region contains two repeats of the Myc tag and the HA tag; in order to further stabilize the expression of the marker polypeptide on the cell membrane surface, a TIGIT spacer was added to the intergenic part of Cl&2b, wherein the amino acid sequence of the added spacer is consistent with the amino acid sequence of TIGIT at positions 24 to 140. Among them, the amino acid sequence of TIGIT comes from UniProt KB-Q495A1.

According to a specific embodiment, the amino acid sequence of the marker polypeptide is as shown in SEQ ID NO: 18 (corresponding to TT1), 19 (corresponding to TT2), 20 (corresponding to TT3), 21 (corresponding to Cl&2a) or 22 (corresponding to Cl&2b).

In some embodiments, the marker polypeptide can be expressed on tumor cells/cancer cells by recombinant viruses, and the genome of the recombinant virus has a marker polypeptide coding sequence. In the nucleic acid having a marker polypeptide coding sequence of the present invention, a signal peptide coding sequence may also be preferably included before the 5' end of the marker polypeptide coding sequence, and the signal peptide has the function of guiding the secretion of the target protein to the cell surface. The present invention has found that the combination of the extracellular antigenic determinant region and the signal peptide from the GM-CSFα chain can make the marker polypeptide expressed on the surface of tumor cells. The GM-CSFα chain signal peptide is a leader sequence that targets the marker polypeptide of the present invention to the secretory pathway. Its coding sequence is first translated into protein in the cell together with the coding sequence of the marker polypeptide, guiding the synthesized protein into the intracellular secretory pathway. Before the marker polypeptide is expressed on the cell surface, the signal peptide is removed. The full-length amino acid sequence and nucleotide sequence of the GM-CSFα chain are both known and can be queried from the public database commonly used in the art. Preferably, the amino acid sequence of the signal peptide is selected from the 1st to 22nd amino acids of the human GM-CSFα chain. More preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 23. Wherein, the amino acid sequence of the GM-CSF α chain is from UniProtKB-P15509.

The recombinant virus mentioned includes a selection of replicative repeat oncolytic viruses or replication-deficient recombinant viruses. According to the embodiment, the recombinant oncolytic virus is derived from a genetically mutated virus with oncolytic effect and a wild-type virus with oncolytic effect; preferably, the recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus with oncolytic effect. The oncolytic virus can selectively replicate in tumor cells, thereby expressing the marker polypeptide in tumor cells or cancer cells, so that the oncolytic virus can significantly enhance the expression of antigen epitope polypeptides on the surface of tumor cells while killing tumor cells or cancer cells, and then combine with antibody-bridged immune cells to show a synergistic therapeutic effect.

According to a specific embodiment, the nucleic acid of the above-mentioned marker polypeptide coding sequence can be inserted into the genome of the recombinant virus. According to a specific embodiment, the recombinant oncolytic virus is a recombinant oncolytic poxvirus with TK gene and VGF gene functional defects. The TK gene can make the TK gene functionally defective by inserting an exogenous nucleotide sequence. The VGF gene can make the VGF gene functionally defective by gene knockout or insertion of an exogenous nucleotide sequence, but the VGF gene is preferably knocked out. According to a specific embodiment, the recombinant virus is configured for intratumoral injection, intravenous administration, intraperitoneal injection, or intracranial injection.

According to specific embodiments, the coding sequence is DNA or RNA, including mRNA transcribed from DNA.

According to a specific embodiment, the DNA is formulated for intratumoral injection, intravenous injection, intraperitoneal injection or intracranial injection; the mRNA The drug is formulated for administration by intratumoral, intravenous, intraperitoneal or intracranial injection.

Antibody drug conjugates and their uses

The present invention provides a use of an antibody or antigen-binding fragment thereof targeting an antigen epitope polypeptide in the preparation of an antibody-drug conjugate for treating cancer and/or tumors. The present invention also provides an antibody-drug conjugate targeting an antigen epitope polypeptide, comprising an antibody or antigen-binding fragment thereof targeting an antigen epitope polypeptide coupled to one or more therapeutic agents. The antigen epitope polypeptide is the antigen epitope polypeptide mentioned above. According to a specific embodiment, in a natural state, the amino acid sequence of a mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigenic epitope polypeptide; compared with normal cells, the antigenic epitope polypeptide is differentially highly expressed on tumor cells/cancer cells, and the antibody or antigen-binding fragment specifically binds to the antigenic epitope polypeptide on the tumor cells/cancer cells, and exhibits endocytosis with EC50≤100nM, for example, EC50≤90nM, EC50≤80nM, EC50≤70nM, EC50≤60nM, EC50≤50nM, EC50≤40nM, EC50≤30nM, EC50≤20nM, EC50≤10nM, EC50≤5nM, EC50≤1nM, EC50≤0.5nM, EC50≤0.1nM, etc. The antigenic epitope polypeptide mentioned is the antigenic epitope polypeptide mentioned above. The "differential high expression" mentioned here means that the antigen epitope polypeptide is highly expressed on tumor cells/cancer cells, is not expressed or is lowly expressed in normal human cells, and the expression in tumor cells/cancer cells is significantly different from that in normal human cells. The significant difference mentioned here means that the statistical p value detected is less than or equal to 0.05.

According to a specific embodiment, the antigenic epitope polypeptide has one or more sequences derived from Strep tag I or Strep tag II. The "plurality" mentioned includes two, for example, it can be two, three, four, five, six, seven, etc. It can be multiple repeated Strep tag I, or multiple repeated Strep tag II, or any combination of strep tag I and strep tag II. It can be the entire sequence of multiple repeated Strep tag I or Strep tag II, or a partial sequence. According to a specific embodiment, the antigenic epitope polypeptide has one or more repeated sequences derived from Strep tag I or Strep tag II. The "repeat" mentioned refers to multiple repetitions of Strep tag I, or multiple repetitions of Strep tag II. According to a specific embodiment, the antigenic epitope polypeptide has the sequence shown in SEQ ID NO:24 or SEQ ID NO:25.

According to a specific embodiment, the form of the antibody or its antigen-binding fragment mentioned is not particularly required, including but not limited to monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, recombinant proteins, bispecific antibodies, multispecific antibodies, monovalent antibodies, multivalent antibodies, complete antibodies, Fab, Fd, scFv, F(ab') ₂ fragments, Fab' fragments, Fv fragments, single-chain antibodies. According to a specific embodiment, the antibody or its antigen-binding fragment also includes a constant region of an immunoglobulin, and the constant region of the immunoglobulin is IgG1, IgG2, IgG3, IgG4, or a variant thereof. For example, the constant region of an immunoglobulin includes Fc or a variant thereof. The Fc mentioned can be derived from a natural Fc or a modified Fc. The natural Fc can be of any origin, including but not limited to mouse, primate, human, preferably human Fc regions. The human Fc region mentioned can be obtained in a public database. The mutated Fc includes but is not limited to L234A/L235A, L234F/L235E/P331S, G236R/L328R, L234A/L245A/P329G, etc. By mutating Fc, the binding of Fc to related receptors can be regulated, thereby regulating the function of ADCC.

According to a specific embodiment, the amino acid sequence of the light chain of the antigen binding domain targeting TT3 is consistent with the light chain of the anti-Strep tag II antibody (see (for example) patent document EP2871189A1), and the amino acid sequence of the heavy chain is consistent with the heavy chain of the anti-Strep tag II antibody (see (for example) patent document EP2871189A1). In conjunction with the specific embodiment of patent document EP2871189A1, the disclosed antibody capable of specifically targeting TT3 has a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:8. The antibody can specifically recognize and bind to TT3. In order to reduce immunogenicity, the antibody having the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:8 can be humanized. The humanized antibody maintains a high affinity for TT3. According to a specific embodiment, the humanized antibody includes a heavy chain variable region shown in SEQ ID NO: 1, 2 or 3 and a light chain variable region shown in SEQ ID NO: 4, 5 or 6. The sequences of the heavy chain variable region and the light chain variable region mentioned can be freely combined, and the antibodies formed have been shown to be able to show high affinity activity with TT3. Moreover, any humanized heavy chain variable region can be combined with the light chain variable region shown in SEQ ID NO: 8, and any humanized light chain variable region can be combined with the heavy chain variable region shown in SEQ ID NO: 7 to specifically target TT3. The antibody or antigen-binding fragment thereof specifically targeting TT3 mentioned in this article refers to an antigen epitope polypeptide or an extracellular antigen-determining region that specifically targets TT3. According to a specific embodiment, the antibody or antigen-binding fragment thereof specifically targeting TT3 specifically targets the sequence shown in SEQ ID NO: 25.

According to a specific embodiment, an antibody or antigen-binding fragment thereof targeting TT3 antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: the amino acid sequences of HCDR1, HCDR2 and HCDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:8. According to a specific embodiment, the CDR sequences of the antibody or antigen-binding fragment thereof targeting TT3 are not the same according to different definition schemes. For example, it is defined according to the IMGT definition scheme, as shown in SEQ ID NO:12-17. In addition, it can also be defined according to definition schemes such as Kabat, Chothia, AbM, Contact, etc. Different CDR sequences obtained according to different definition schemes are all included in the protection scope of the present invention.

According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain can be The variable region is selected from the sequence shown in SEQ ID NO: 1, 2, 3 or 7, and the light chain variable region is selected from the sequence shown in SEQ ID NO: 4, 5, 6 or 8. The heavy chain variable region mentioned can be connected to the heavy chain constant domain, and the light chain variable region can be connected to the light chain constant domain to form an IgG type antibody. According to a specific embodiment, the sequence of the heavy chain constant domain is shown in SEQ ID NO: 9 or 10. According to a specific embodiment, the sequence of the light chain constant domain is shown in SEQ ID NO: 11.

According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:4; or has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:5; or has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:6; or has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:8; or has a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:4; or has a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:5; or has a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:6; or has a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:8 NO:8; or having the heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:4; or having the heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:5; or having the heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:6; or having the heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:8; or having the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:8; or having the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:4; or having the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:5; or having the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:6. The heavy chain variable region mentioned can be connected to the heavy chain constant domain shown in SEQ ID NO:9 or 10, and the light chain variable region mentioned can be connected to the light chain constant domain shown in SEQ ID NO:11.

The amino acid sequence mentioned is as follows:

According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered as Ab-1hu, which has a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 4, the heavy chain variable region is connected to the heavy chain constant domain as shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain as shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered as Ab-2hu, which has a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 5, the heavy chain variable region is connected to the heavy chain constant domain as shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain as shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered Ab-3hu, which has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 6, the heavy chain variable region is connected to the heavy chain constant domain shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, and in Fc L234F/L235E/P331S mutations occurred in the region, numbered as Ab-3hu-mu, and the amino acid sequence is a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 6, the heavy chain variable region is connected to the heavy chain constant domain shown in SEQ ID NO: 10, and the light chain variable region is connected to the light chain constant domain shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered as Ab-4hu, which has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO: 5, the heavy chain variable region is connected to the heavy chain constant domain shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 antigen, Ab-5hu, has a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 6, the heavy chain variable region is connected to the heavy chain constant domain as shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain as shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered Ab-6hu, which has a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 4, the heavy chain variable region is connected to the heavy chain constant domain as shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain as shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered as Ab-7hu, which has a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 6, the heavy chain variable region is connected to the heavy chain constant domain as shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain as shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or antigen-binding fragment thereof targeting TT3 is an IgG1 antibody, numbered as Ab-8hu, which has a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 4, the heavy chain variable region is connected to the heavy chain constant domain as shown in SEQ ID NO: 9, and the light chain variable region is connected to the light chain constant domain as shown in SEQ ID NO: 11. According to a specific embodiment, the antibody or its antigen-binding fragment targeting TT3 is an IgG1 antibody, numbered Ab-9hu, which has a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:5, the heavy chain variable region is connected to the heavy chain constant domain shown in SEQ ID NO:9, and the light chain variable region is connected to the light chain constant domain shown in SEQ ID NO:11.

The antibodies or antigen binding fragments thereof mentioned herein are usually separable or recombinant. "Separable" means that it can be identified and separated and/or recovered from cells or cell cultures expressing polypeptides or proteins. Usually, the separated polypeptide will be prepared by at least one purification step. "Separated antibody" means that it is substantially free of other antibodies or antigen binding fragments with different antigen specificities. "Recombinant" means that antibodies can be produced in exogenous host cells using genetic recombination techniques.

The antibody or antigen-binding fragment mentioned above can be inserted into a replicable expression vector by the nucleotide encoding it, and expressed in a host cell or a cell-free expression system. The construct can be obtained by inserting the nucleotide encoding the antibody or antigen-binding fragment into a replicable expression vector. A variety of methods commonly used in the art can be used to obtain the construct, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. For example, a polynucleotide can be inserted into the multiple cloning site of the expression vector to form a construct. The construct can contain a variety of operating factors such as promoters, terminators, marker genes, etc. as needed, and these operating factors can be operably connected to the polynucleotide. The promoter is usually used to provide a signal to start transcription. The promoter can select lactose promoter (Lac), Trp promoter, Tac promoter, PL and PR promoters of bacteriophage as needed; the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening. Of course, there can also be enhancers as needed to enhance protein expression. The expression vector is not particularly limited, and can be some commercially available expression vectors, or it can be an expression vector artificially modified as needed, such as a plasmid, a bacteriophage, a virus, etc. The virus may be a plant cell virus, a mammalian cell virus, etc. The construct may express antibodies or proteins in vitro, or may be transferred into cells to express antibodies or proteins.

Any cell suitable for expressing antibodies or proteins with polynucleotides or constructs can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a yeast cell, a mammalian cell, etc. Commonly used host cells can be yeast cells, CHO, HEK-293 cells, COS cells, insect cells of Drosophila S2 or Sf9. Host cells containing polynucleotides or constructs can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc. Antibodies or antigen-binding fragments thereof targeting TT3 antigens can be obtained by culturing the above host cells and collecting from the culture. The antibodies or antigen-binding fragments collected from the culture can be purified to obtain substantially pure products. "Substantially pure" means that the purity of the antibody or antigen-binding fragment reaches more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or even more than 99.5%, 99.6%, 99.7%, or 99.8%.

The antibody-drug conjugate provided also includes one or more therapeutic agents coupled to the antibody or antigen-binding fragment through a linker. The linker used when the therapeutic agent is coupled to the antibody or antigen-binding fragment may be a linker commonly used in the art. It can be connected to the antibody by any means known in the art, preferably connected by a sulfhydryl group and/or an amino group. In some specific embodiments, the antibody of the present invention is connected to the linker through a sulfhydryl group. The linker used in the present invention may be a cleavable linker (i.e., a linker that can be broken in the in vivo environment) or a non-cleavable linker. The cleavable linker can be broken in or on the target cell to release the drug. In some embodiments, the linker of the present invention has good stability, greatly reducing the release of the drug during delivery to the target (e.g., in the blood), thereby reducing side effects and toxicity. In some embodiments, the linker of the present invention is selected from a cleavable linker, such as a disulfide-based linker (which selectively breaks in tumor cells with a higher concentration of sulfhydryl groups), a peptide linker (which is cut by enzymes in tumor cells), and a hydrazone linker. In some embodiments, the linker used is a non-cleavable linker, such as a thioether linker. For example, the linker used is a cleavable mc-vc-PAB linker, valine-citrulline (vc) or valine-alanine (va) and a non-cleavable mc linker (maleimidoacetyl). For example, examples of linker therapeutic agents formed by a linker and a therapeutic agent may include mc-vc-PAB-MMAE (which may be abbreviated as mc-vc-MMAE and vc-MMAE), mc- MMAF and mc-vc-MMAF.

The therapeutic agent mentioned is selected from the group consisting of cytotoxic molecules, immunopotentiators and radioisotopes. According to a specific embodiment, the cytotoxic molecule is selected from the group consisting of tubulin inhibitors or DNA damaging agents.

Tubulin inhibitors can disrupt microtubule assembly and affect mitosis. Microtubules are an important component of the cytoskeleton and play an important role in the process of cell division. Since tumor cells maintain rapid proliferation, tubulin inhibitors that interfere with tumor cell mitosis have become one of the directions of tumor drug research and development. The tubulin inhibitors mentioned are selected from dolastatin or auristatin cytotoxic molecules, maytansine cytotoxic molecules; the auristatin cytotoxic molecules mentioned are selected from MMAE (monomethyl auristation E) or MMAF (monomethyl auristation F) or their derivatives, and the maytansine cytotoxic molecules are selected from DM1, DM4 or their derivatives. MMAE or MMAF is a typical promoter of tubulin polymerization, which acts on the β-subunit of the α-β tubulin dimer, making microtubule growth unregulated, and is an effective payload. The DNA damaging agent mentioned is selected from calicheamicins, duocarmycins, anthramycin derivatives PBD, camptothecins and camptothecin derivatives, SN-38, and Dxd.

According to a specific embodiment, the present invention provides an antibody drug conjugate, the general structural formula of which is Ab-(L-U)n, wherein:

The Ab represents an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide, the U is an active drug unit, the L is any linking group, and the L is covalently connected to the Ab and the U respectively; n is an integer selected from 1, 2, 3, 4, 5, 6, 7 or 8; 1, 2, 3, 4, 5, 6, 7 or 8 U are connected to the Ab through one or more L.

According to a specific embodiment, the L is covalently linked to an amino residue or a thiol residue on the Ab; preferably, the L is covalently linked to a thiol residue on the Ab; more preferably, the L is covalently linked to a thiol residue formed after the interchain disulfide bond on the Ab is opened.

According to a specific embodiment, L includes a cleavable linker and a non-cleavable linker. According to a specific embodiment, the cleavable linker comprises 2 to 20 amino acid units, preferably, the amino acid units are selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) or a combination thereof.

According to a specific embodiment, U is selected from the general formula (I) or a pharmaceutically acceptable salt or solvate thereof:

Among them, in the general formula (I)

_R1 and _R2 are each independently selected from H or C1-C6 alkyl;

_R3 is selected from C1-C8 alkyl;

_R4 and _R5 are each independently selected from H, C1-C6 alkyl, aryl, or _R4 and _R5 form a C3-C8 membered ring;

_R6 is selected from C1-C8 alkyl;

_R7 is selected from C1-C8 alkyl;

R ₈ is selected from H or C1-C8 alkyl;

R ₉ is selected from H, C1-C8 alkyl, -OH, -COOH, -COO(C1-C6 alkyl), -CONH(C1-C6 alkyl), C5-C7 heterocycle;

R ₁₀ is selected from H, -OH, or a bond, and when R ₁₀ is a bond, the resulting OR10 is =O;

R ₁₁ is selected from H, C1-C6 alkyl, or -OH.

According to a preferred embodiment, R1 is independently selected from H or methyl, ethyl. According to a specific embodiment, R3 is methyl, ethyl, isopropyl. According to a specific embodiment, _R4 and _R5 are independently selected from H, methyl or ethyl. According to a specific embodiment, _R6 is H, methyl or ethyl. _R7 is methyl, ethyl, isopropyl, n-butyl, isobutyl. According to a specific embodiment, _R8 is H, methyl or ethyl. According to a specific embodiment, _R9 is methyl, -COOH, -CONH- _CH3 .

According to a specific embodiment, R ₁₀ is H or -OH.

According to a specific embodiment, R ₁₁ is H or -OH.

According to a specific embodiment, said U is selected from the following structures:

According to a specific embodiment, said L is selected from the following structures:

According to a specific embodiment, the Ab includes a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: the amino acid sequences of HCDR1, HCDR2 and HCDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:8.

According to a specific embodiment, the Ab includes a heavy chain variable region and a light chain variable region, the heavy chain variable region is selected from the sequence shown in SEQ ID NO: 1, 2, 3 or 7, and the light chain variable region is selected from the sequence shown in SEQ ID NO: 4, 5, 6 or 8.

According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:4. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:5. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:6. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:4. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:5. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:6. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:4. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:5. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:6. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:4. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:5. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:6. According to a specific embodiment, the Ab has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:8. According to a specific embodiment, the Ab has a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO: 8. According to a specific embodiment, the Ab has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 8.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising the above antibody-drug conjugate and a pharmaceutically acceptable carrier.

According to a specific embodiment, the pharmaceutically acceptable carrier can be a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (for example, as described in Berge, S.M.etal (1977) J.Pharma.Sci.66:1-19). The pharmaceutically acceptable carrier mentioned is acceptable to the subject at the dose or concentration used. Pharmaceutically acceptable carriers include, but are not limited to, buffers or salts, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; sugars, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtering with a sterile filter membrane. Those skilled in the art can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed to prepare different dosage forms, such as freeze-dried dosage forms, injections, etc. The prepared different pharmaceutical dosage forms can be formulated into any suitable administration route for administration to the subject, including but not limited to intravenous, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.

Test kit or medicine box

The present invention also provides a kit, the kit includes the above-mentioned antibody drug conjugate. The kit may also include a container, a buffer reagent, a control such as a positive control and a negative control as needed. Those skilled in the art can make corresponding selections as needed. Accordingly, the kit may also include instructions for use to facilitate the operation and use of those skilled in the art. In some embodiments, the present invention also provides a medicine box, the medicine box includes the above-mentioned antibody drug conjugate.

Methods of preventing and/or treating disease

In addition, the present invention provides a method for preventing and/or treating a disease, comprising: administering an effective amount of the above-mentioned antibody-drug conjugate drug or the above-mentioned pharmaceutical composition to a subject in need.

The "treatment" mentioned here means that it can lead to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or a reduction in the pain caused by the disease. The effective amount of antibodies used to "prevent" the disease will usually be lower than the effective amount of antibodies used to treat the disease. "Effective amount" is an amount sufficient to achieve or at least partially achieve the desired effect. After treatment with antibody drug conjugates, the inhibition rate of tumor cells in the subject is more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or even more than 90% or more than 95%. The subject mentioned here can be an animal or a human. For example, it can be a mammal, including cattle, sheep, mice, horses, etc.

The antibody-drug conjugate provided can be used to treat a variety of solid tumors and hematological malignancies in the human body, especially breast cancer, gastric cancer, colorectal cancer, urothelial carcinoma, ovarian cancer, uterine cancer, lung cancer (especially non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), mesothelioma, liver cancer, thyroid cancer, melanoma, pancreatic cancer, bile duct cancer, esophageal cancer, head and neck cancer, prostate cancer, leukemia, synovial cancer, kidney cancer, connective tissue cancer, melanoma, esophageal cancer, colon cancer, rectal cancer, brain cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome Syndrome, anal cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, skin squamous cell carcinoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, peritoneal cancer, pleural cancer and blood cancer, etc.

The antibody-drug conjugate mentioned above can also be combined with other treatment methods to treat diseases. The other treatment methods mentioned above can be chemotherapy or radiotherapy. By combining chemotherapy or radiotherapy and other treatment methods clinically, the therapeutic effect of the drug can be further improved, and the treatment effect of the patient can be improved.

The present invention also provides the use of antibody-drug conjugates in preparing a kit. The use is for treating cancer. The provided antibodies or antigen-binding fragments can be used to prepare drugs for treating various diseases. They can also be used to prepare kits for use as immunodiagnostic reagents.

The technical scheme of the present invention is described in detail with reference to the embodiments below. It should be noted that these embodiments are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. The embodiments of the present invention are described in detail below, and the described embodiments are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

HCT116 is a human colorectal cancer cell line. SK-HEP-1 is a human liver cancer cell line and MC38 is a mouse colon cancer cell line. The HCT116-TT3 cell line mentioned is a HCT116 cell line stably expressing TT3, which is constructed by lentiviral transfection. Other cell lines, such as the SK-HEP-1 cell line stably expressing TT3 (SK-HEP-1-TT3) and the MC38 cell line stably expressing TT3 (MC38-TT3), are constructed by similar methods.

For details, please refer to the following methods:

After the wild-type HCT116 was digested into single cells with 0.25% trypsin, it was inoculated into a 48-well plate at 1×10 ⁵ /500μL/well, and 300μL of lentivirus LV-TT3-GFP and polybrene (commercially available from: Shanghai Yishen Biotechnology Co., Ltd., final concentration 8μg/mL) were added, and placed in an incubator for culture and passage expansion. One week later, the infected cells were digested and inoculated into a 96-well plate at 2 cells/well, 100μL per well, and placed in an incubator for continued culture. After about 10 days of continuous culture to grow monoclonal cells, all cells were GFP-positive and there was only one group of cell clones under the microscope, and passaged and expanded. The expression of GFP and TT3 was detected by flow cytometry, and the optimal monoclonal clone was finally obtained according to the expression ratio and intensity of GFP and TT3, and a stable cell line expressing TT3 (HCT116-TT3) was obtained.

The antibody (numbered Ab-TT3, its heavy chain variable region is SEQ ID NO:7, and its light chain variable region is SEQ ID NO:8) was humanized. Specifically, the humanized sequence template was retrieved in the database based on the Ab-TT3 antibody sequence. In order to maintain the conformation of the CDR region, three types of residues were focused on, including residues located on the VL and VH binding interface, residues close to the CDR region and embedded in the protein, and residues that directly interacted with the CDR region. The interactions included: hydrophobic interactions, hydrogen bonds, and salt bridges. The importance of different amino acids in the 3D structure was analyzed, and the key amino acids that affected the structure were reverse mutated when necessary to determine the corresponding humanized antibody.

The humanized antibody was fused with the constant region sequence of human origin and constructed into the PCDNA3.1 mammalian expression vector to form a chimeric antibody. Then, the obtained different antibodies were expressed and purified using a mammalian cell system to obtain different antibodies with a purity of at least 90%. The expressed antibodies were Ab-1hu, Ab-2hu, Ab-3hu, Ab-4hu, Ab-5hu, Ab-6hu, Ab-7hu, Ab-8hu, and Ab-9hu. At the same time, the Fc region of the antibody numbered Ab-3hu was mutated to have L234F/L235E/P331S mutations, and the mutated antibody was numbered Ab-3hu-mu. When the activity of these antibodies was detected by the ELISA method, the TT3 antigen used was obtained by preparation, and the polypeptide shown in SEQ ID NO:24 was fused with the GST protein sequence, and the TT3 antigen was obtained by expression and purification for the activity detection of specific antibodies.

The activity of the prepared humanized antibody was tested by ELISA. The experimental process is as follows: dilute the TT3 antigen to 0.5ug/mL with coating solution, add 100uL/well to a 96-well plate, and incubate overnight at 4℃. Wash with PBST, then add 200uL antibody blocking solution to each well, and block at 37℃ for 2h. Wash with PBST, add different antibodies (starting concentration 100nM, gradient dilution according to 1:6) at 100uL/well, and incubate at 37℃ for 2h. Wash with PBST, add HRP-conjugated goat anti-human/mouse IgG antibody at 100uL/well, and incubate at 37℃ for 1h. Wash with PBST, add TMB at 100uL/well, and color at 37℃ for 10-20min; add 50ul 2M sulfuric acid to each well to terminate the reaction. Read the OD value at a wavelength of 450nm. The EC50 values of antibodies Ab-1hu, Ab-2hu, Ab-3hu, and Ab-TT3 were 0.0104 nM, 0.0105 nM, 0.0140 nM, and 0.0878 nM, respectively, as shown in FIG1 .

The activity of the humanized antibody prepared in Example 1 was detected by the FACs method. The experimental process was as follows: different cells (HCT116-TT3, SK-HEP-1-TT3, MC38-TT3) were plated, then washed with FACS buffer, centrifuged at 1200rpm for 3-5min, and different concentrations of diluted antibodies were added (the initial concentration was 100nM, and the gradient dilution was performed according to 1:5), incubated at 4°C for 0.5h, centrifuged at 1200rpm for 3-5min. Washed with FACS buffer, then diluted goat anti-human IgG secondary antibody (Abcam, ab98596) at 1:250, added to the plate at 100uL/well, incubated at 4 degrees for 0.5h, centrifuged at 1200rpm for 3-5min, washed with FACs buffer, and then 50uL of FACs buffer was added to resuspend the cells and detected by flow cytometry. Among them, the EC50 results of the FACs affinity activity of antibodies Ab-1hu, Ab-2hu, and Ab-3hu with HCT116-TT3 cells were 0.568nM, 0.958nM, and 0.692nM, respectively; the EC50 results of the FACs affinity activity with SK-HEP-1-TT3 cells were 0.546nM, 1.527nM, and 0.738nM, respectively; the EC50 results of the FACs affinity activity with MC38-TT3 cells were 1.201nM, 2.327nM, and 0.588nM, respectively; as shown in Figure 1.

The same method was used to detect the antibody after Fc mutation (Ab-3hu-mu), and it was found that both before and after Fc mutation could bind to TT3 antigen and had biological activity, and the binding activity of the two was not much different.

Using these antibodies as examples to prepare antibody-drug conjugates, antibody-drug conjugates with similar functions and activities were obtained. The following examples record the preparation of antibody-drug conjugates using antibody Ab-3hu-mu as an example.

Example 1 TT3-MMAE conjugate

Example 1 TT3-MMAE conjugate was prepared by the following method, comprising:

(1) Antibody reduction

TT3-SH was obtained by reducing the disulfide bond of anti-TT3 antibody (the antibody used was numbered Ab-3hu-mu) with tri(2-carboxyethyl)phosphine (TCEP). The process is as follows: first, tri(2-carboxyethyl)phosphine (TCEP, 2 mg/mL) was dissolved in a solution containing 0.025 M sodium borate, 0.025 M NaCl, and 5 mM EDTA (pH 7.5), and then anti-TT3 antibody was added, wherein the molar ratio of TCEP to anti-TT3 antibody was set to 5:1, and the reaction was placed at room temperature and 115 rpm for 2 hours to obtain TT3-SH. After the reaction, the content of thiol groups was analyzed using Ellman’s reagent (DTNB).

DTNB is a widely used reagent for quantitative determination of thiol groups. The product of its reaction with thiol groups has an absorption peak at 412nm. Therefore, DTNB can be used to determine the thiol content on TT3-SH. Take 90μL TT3-SH and 10μL DTNB (3.96mg/mL) and incubate them at room temperature for 15min. Then measure the absorbance at 412nm by UV-Vis spectrophotometer. The standard curve should be configured to calculate the content of thiol groups.

The molar ratio of antibody to thiol was calculated to be 1:9.4.

(2) Preparation of Antibody Drug Conjugate TT3-MMAE

Freshly prepared VC-MMAE DMSO solution (10 mg/mL) was added to TT3-SH (TT3 antibody concentration was 2.5 mg/mL), and the molar ratio of TT3-SH to VC-MMAE was set to 1:10. After reacting on a shaker at room temperature and 115 rpm for 2 h, free monoclonal antibody, TCEP and VC-MMAE were purified using a HIC column to obtain TT3-MMAE.

Example 2 Physical and chemical analysis of TT3-MMAE

Example 2 The antibody conjugate prepared in Example 1 was analyzed by SEC and SDS-PAGE. The absorbance of TT3-MMAE at 248 nm (MMAE absorbs at 248 nm) was measured by ultraviolet spectrophotometry (UV-Vis), and the DAR value was calculated. The results are shown below:

(I) SDS-PAGE

The TT3-MMAE conjugate was analyzed by SDS-PAGE. During the purification process using the HIC column, the stronger the hydrophobicity of the molecule, the later the peak time.

The results are shown in Figure 2. The numbers 1-9 represent samples collected at different peak times, and the larger the number, the later the sample collection time. From the SDS-PAGE graph, it can be seen that except for tube 1, the remaining samples all have multiple bands. When the disulfide bond is reduced, there is still a non-covalent bond between H and H, H-L, but when SDS exists, the non-covalent bond is destroyed, so multiple bands such as H, L, and HL are observed in SDS-PAGE, but no small molecule fragments are detected on the SEC spectrum, indicating that the antibody is intact.

(II) SEC

The purity was determined by size exclusion chromatography (SEC, Waters ACQUITY Arc chromatograph). The XBridge Premier Protein SEC 250A column (7.8 mm x 300 mm, 2.5 μm) was used for analysis. The mobile phase was 100 mM phosphate buffer (pH 7.2-7.6) containing 200 mM arginine hydrochloride and 5% isopropanol, the flow rate was 0.5 mL/min, the column temperature was 30 ° C, and the detection wavelength was 280 nm. The SEC results are shown in Figure 3. From the SEC spectrum, it can be seen that the elution time of the TT3-MMAE conjugate is consistent with that of the anti-TT3 antibody, the purification process of the TT3-MMAE conjugate is good, the main peak accounts for a high proportion, and the main peak of the product has a significant tailing phenomenon compared with the anti-TT3 antibody, indicating that the hydrophobic small molecule MMAE is successfully coupled to the anti-TT3 antibody.

(III) UV-Vis

The absorbance was measured at 248 nm by UV-vis method. The results are shown in FIG4 .

A standard curve was prepared based on the ultraviolet absorption value of MMAE (0.195-100ug/mL) at 248nm; secondly, using the anti-TT3 antibody solution with the same antibody concentration as the baseline, the ultraviolet absorption value of the antibody-drug conjugate TT3-MMAE at 248nm was 0.1746, and the concentration of TT3-MMAE was 14.7ug/mL when substituting it into the standard curve. After calculation, DAR=8.38≈8.

Example 3 Functional Analysis

Example 3 The cell binding activity and antibody endocytosis of the prepared antibody drug conjugate TT3-MMAE were studied.

The experiment is as follows:

(I) Cell binding activity

In order to study the binding of TT3-MMAE to cells overexpressing TT3, flow cytometry was used to detect the binding of TT3-MMAE and anti-TT3 antibodies to HCT116-TT3 cells overexpressing TT3 and HCT116 cells not expressing TT3.

First, HCT116-TT3 and HCT116 cells were digested with trypsin, resuspended and counted, and plated in a 96-well round-bottom plate at 1E5 cells/well. The cells were washed twice with FACs buffer (2% FBS+PBS) (1000rpm, 5min) to remove the supernatant. Then, TT3-MMAE and anti-TT3 antibodies were gradiently diluted with FACs buffer (initial concentration was 100nM, gradient dilution was performed at 1:5, and a total of 8 dilution points were diluted), added to the 96-well round-bottom plate at 100uL/well, and incubated at 4℃ for 1h. After the incubation, the cells were washed with FACs buffer. Anti-Human IgG (PE) secondary antibody was diluted at 1:250 with FACs buffer, and added to the 96-well round-bottom plate at 100uL/well, and incubated at 4℃ for 1h. Finally, the plate was washed twice with FACs buffer to remove unbound secondary antibodies, 50uL FACs buffer was added to resuspend the cells, and flow cytometry was used for analysis. The results are shown in Figure 5 and Table 1 below.

Table 1 FACs binding activity

ND stands for not detected.

The experimental results showed that: 1) compared with the anti-TT3 antibody, the binding activity of the antibody-drug conjugate TT3-MMAE and HCT116-TT3 was not much different, indicating that the conjugation of MMAE with the anti-TT3 antibody did not affect the binding activity of the anti-TT3 antibody with the TT3 antigen;

2) The antibody drug conjugate TT3-MMAE binds to HCT116-TT3 cells but not to HCT116 cells, indicating that the binding of the antibody drug conjugate TT3-MMAE to the TT3 antigen is specific.

(II) Antibody internalization

Flow cytometry was used to study the internalization of antibody-drug conjugate TT3-MMAE by HCT116-TT3 cells. First, HCT116-TT3 and HCT116 cells were digested with trypsin, resuspended and counted, and plated in a 96-well round-bottom plate at 1E5 cells/well, washed with FACs buffer (2% FBS+PBS), and centrifuged at 1000rpm for 5 minutes to remove the supernatant. Then, TT3-MMAE and TT3 were gradiently diluted with FACs buffer (initial concentration was 100nM, gradient dilution was performed in 5 times, and a total of 8 dilution points were diluted), added to a 96-well round-bottom plate at 100uL/well, and incubated at 4℃ for 1h. After the incubation, the cells were washed with FACs buffer and 100uL of culture medium was added to each well to resuspend the cells. The 0h sample was placed in a 4℃ refrigerator for 2h, and the remaining samples were placed in a 37℃ incubation for 2h. Then, all samples were centrifuged and washed with FACs buffer; then, Anti-Human IgG (Dylight 650) secondary antibody was diluted 1:250 with FACs buffer and added to the 96-well plate containing the samples at 100uL/well and incubated at 4°C for 1h. Finally, the plate was washed twice with FACs buffer to remove the unbound secondary antibody, and the cells were resuspended in FACs buffer and analyzed using a flow cytometer. Endocytosis ratio = MFI (0h) - MFI (2h) / MFI (0h).

As shown in Figure 6 and Table 2 below.

Table 2

The experimental results show that:

1) Compared with the anti-TT3 mAb, the internalization degree of TT3-MMAE by HCT116-TT3 was not much different, indicating that the conjugation of MMAE did not affect the internalization of the antibody;

2) TT3-MMAE and anti-TT3 antibodies are internalized through TT3 receptor.

(III) Cell Killing

In order to study the cytotoxicity and targeting ability of the antibody-drug conjugate TT3-MMAE, the growth inhibition of TT3-positive HCT116-TT3 cells and TT3-negative HCT116 cells was determined by CCK8 method. HCT116-TT3 and HCT116 cells were plated in a 96-well culture plate at a density of 1E4 cells/well and incubated in a 37°C, 5% CO ₂ incubator for 24 hours. Then, 100 μL of culture medium solutions containing different concentrations of TT3-MMAE and TT3 monoclonal antibody were added to replace the old culture medium. They were placed in a 37°C, 5% CO ₂ incubator for 72 hours, and then 10 μL of CCK8 solution was added to each well, incubated for 2 hours, and the ultraviolet absorbance of each well at 450 nm was measured by an enzyme reader. The cell viability was calculated by comparing the absorbance of each well at 450 nm (minus the absorbance of the culture medium) with that of the PBS control group (minus the absorbance of the culture medium), and the IC50 was calculated by Prism nonlinear regression (sigmoidal).

The results are shown in Figure 7. The experimental results show that:

1) As shown in Figure 7A, after TT3-MMAE was co-incubated with HCT116-TT3 cells for 72 h, TT3-MMAE showed a significant killing effect in the cells with an IC50 of 1.02 nM, while TT3 monoclonal antibody had no killing effect.

2) As shown in Figure 7B, after TT3-MMAE and HCT116 were co-incubated for 72 hours, neither anti-TT3 antibody nor TT3-MMAE had killing effect, indicating that TT3-MMAE was specific to TT3-HCT116. 3) TT3 monoclonal antibody had no killing effect on both cells, indicating that the specific killing effect mainly came from the small molecule MMAE.

In the description of this specification, reference to terms such as "one embodiment", "some embodiments", "implementation", "specific implementation", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (37)

Use of antibodies or antigen-binding fragments thereof targeting antigen epitope polypeptides in the preparation of antibody-drug conjugates; Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; Compared with normal human cells, the antigen epitope polypeptide is differentially highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells and exhibits endocytosis with EC50≤100nM. The use according to claim 1 is characterized in that the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of a protein existing in nature, or is an artificially synthesized amino acid sequence that does not exist in nature. The use according to claim 2 is characterized in that the proteins existing in nature include mammalian cell proteins, viral proteins, and other non-mammalian proteins. The use according to claim 1 is characterized in that the amino acid sequence of the antigen epitope polypeptide includes the amino acid sequence of the following tags: Myc tag, HA tag, Strep tag II, Strep tag I, Flag tag, HAT tag, S tag, S1 tag, protein C tag, tag-100 tag, E2 tag, TAP tag, HSV tag, KT3 tag, V5 tag, VSV-G tag, His tag or RFP tag. The use according to claim 1, characterized in that the amino acid sequence of the antigenic epitope polypeptide has one or more sequences derived from Strep tag I or Strep tag II; Optionally, the amino acid sequence of the antigenic epitope polypeptide has one or more repeated sequences derived from Strep tag I or Strep tag II; The use according to claim 1 is characterized in that the antigen epitope polypeptide has a sequence as shown in SEQ ID NO:24 or SEQ ID NO:25. The use according to claim 1 is characterized in that the extracellular antigen determining region is formed by the amino acid sequence of one or more of the antigenic epitope polypeptides, and the extracellular antigen determining region is operably connected to the spacer part and the transmembrane part to form a marker polypeptide, and the marker polypeptide is expressed on tumor cells/cancer cells. The use according to claim 7, characterized in that the spacer portion is derived from the hinge region of CD8α, the hinge region of IgG or the hinge region of IgD; wherein the transmembrane portion is derived from any type I transmembrane region sequence; Optionally, the transmembrane portion is derived from the transmembrane region of CD8, CD3ζ, CD4 or CD28. The use according to claim 7 is characterized in that the amino acid sequence of the marker polypeptide is as shown in SEQ ID NO: 18, 19, 20, 21 or 22. The use according to claim 7 is characterized in that the marker polypeptide is expressed on tumor cells/cancer cells through a recombinant virus, the genome of the recombinant virus has a marker polypeptide coding sequence, and the recombinant virus includes a selective replication-type recombinant oncolytic virus or a replication-deficient recombinant virus. The use according to claim 10, characterized in that the recombinant oncolytic virus is derived from a genetically mutated virus with oncolytic effect and a wild-type virus with oncolytic effect; preferably, the recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus with oncolytic effect. The use according to claim 10 is characterized in that the recombinant virus is configured for intratumoral injection, intravenous administration, intraperitoneal injection or intracranial injection. The use according to claim 10, characterized in that the coding sequence is DNA or RNA, and the RNA includes mRNA transcribed from DNA; Optionally, the DNA is formulated for administration by intratumoral injection, intravenous injection, intraperitoneal injection or intracranial injection; the mRNA is formulated for administration by intratumoral injection, intravenous injection, intraperitoneal injection or intracranial injection. The use according to claim 1, characterized in that the antibody or its antigen-binding fragment is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant protein or a Fab fragment, a F(ab') fragment, a F(ab') ₂ fragment, a Fv fragment, a dAb, a Fd or a single-chain antibody. The use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: The amino acid sequences of HCDR1, HCDR2 and HCDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, The amino acid sequences of LCDR1, LCDR2 and LCDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:8. CDR3; Optionally, the CDR sequence of the antibody or its antigen-binding fragment is defined according to the IMGT definition scheme, as shown in SEQ ID NO: 12 to 17. The use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof is selected from: Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:6; or Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:8; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:6; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:8; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:6; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:8; or Having a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:6; or It has a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:8. The use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof further comprises a constant region of an immunoglobulin, and the immunoglobulin is selected from IgG1, IgG2, IgG3, IgG4, or a variant thereof. A use of an antibody-drug conjugate in the preparation of a drug for treating cancer and/or tumors, characterized in that the antibody-drug conjugate is an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide coupled to one or more therapeutic agents; Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; Compared with normal human cells, the antigen epitope polypeptide is differentially highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells and exhibits endocytosis with EC50≤100nM. The use according to claim 18, characterized in that the antigen epitope polypeptide is the antigen epitope polypeptide according to any one of claims 1 to 17. The use according to claim 18, characterized in that the therapeutic agent is selected from cytotoxic molecules, immunopotentiators and radioactive isotopes, Optionally, the cytotoxic molecule is selected from a tubulin inhibitor or a DNA damaging agent; Preferably, the tubulin inhibitor is selected from dolastatin or auristatin cytotoxic molecules, maytansine cytotoxic molecules; the DNA damaging agent is selected from calicheamicin, duocarmycin, anthramycin derivative PBD, camptothecins and camptothecin derivatives, SN-38, Dxd; The auristatin cytotoxic molecule is selected from MMAE or MMAF or their derivatives, and the maytansine cytotoxic molecule is selected from DM1, DM4 or their derivatives; Optionally, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: The amino acid sequences of HCDR1, HCDR2 and HCDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, The amino acid sequences of LCDR1, LCDR2 and LCDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:8; Optionally, the CDR sequence of the antibody or its antigen-binding fragment is defined according to the IMGT definition scheme, as shown in SEQ ID NO: 12 to 17. An antibody-drug conjugate targeting an antigen epitope polypeptide, characterized in that it comprises an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide coupled to one or more therapeutic agents; Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; Compared with normal human cells, the antigen epitope polypeptide is differentially highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells and exhibits endocytosis with EC50≤100nM. The antibody-drug conjugate according to claim 21, characterized in that the antigen epitope polypeptide is any of claims 1 to 17 The antigen epitope polypeptide. The antibody-drug conjugate according to claim 21, characterized in that the therapeutic agent is selected from a cytotoxic molecule, an immunopotentiator and a radioisotope, Optionally, the cytotoxic molecule is selected from a tubulin inhibitor or a DNA damaging agent; Preferably, the tubulin inhibitor is selected from dolastatin or auristatin cytotoxic molecules, maytansine cytotoxic molecules; the DNA damaging agent is selected from calicheamicin, duocarmycin, anthramycin derivative PBD, camptothecins and camptothecin derivatives, SN-38, Dxd; The auristatin-type cytotoxic molecule is selected from MMAE or MMAF or derivatives thereof, and the maytansine-type cytotoxic molecule is selected from DM1, DM4 or derivatives thereof. The antibody-drug conjugate according to claim 21, characterized in that the general structural formula of the antibody-drug conjugate is Ab-(L-U)n, wherein: The Ab represents an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide, The U is an active drug unit, The L is any linking group, and the L is covalently linked to the Ab and the U respectively; n is an integer selected from 1, 2, 3, 4, 5, 6, 7 or 8; One, two, three, four, five, six, seven, eight, nine or ten U's are connected to the Ab via one or more L's. The antibody-drug conjugate according to claim 24, characterized in that the L is covalently linked to an amino residue or a thiol residue on the Ab; Preferably, said L is covalently linked to a thiol residue on said Ab; More preferably, the L is covalently linked to the sulfhydryl residue formed after the interchain disulfide bonds on the Ab are opened. The antibody-drug conjugate according to claim 24, characterized in that L comprises a cleavable linker and a non-cleavable linker; The cleavable linker comprises 2 to 20 amino acid units, preferably, the amino acid units are selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) or a combination thereof. The antibody drug conjugate according to claim 24, characterized in that L is selected from the following structures:







The antibody drug conjugate according to claim 24, characterized in that U is selected from the following structures:
Among them, in the general formula (I) _R1 and _R2 are each independently selected from H or C1-C6 alkyl; _R3 is selected from C1-C8 alkyl; _R4 and _R5 are each independently selected from H, C1-C6 alkyl, aryl, or _R4 and _R5 form a C3-C8 membered ring; _R6 is selected from C1-C8 alkyl; _R7 is selected from C1-C8 alkyl; R ₈ is selected from H or C1-C8 alkyl; R ₉ is selected from H, C1-C8 alkyl, -OH, -COOH, -COO(C1-C6 alkyl), -CONH(C1-C6 alkyl), C5-C7 heterocycle; R ₁₀ is selected from H, -OH, or a bond, and when R ₁₀ is a bond, the resulting OR ₁₀ is =O; R ₁₁ is selected from H, C1-C6 alkyl, or -OH; Preferably, R ₁ is independently selected from H or methyl, ethyl; Preferably, R ₃ is methyl, ethyl, isopropyl; Preferably, _R4 and _R5 are each independently H, methyl or ethyl; Preferably, R ₆ is H, methyl or ethyl; Preferably, R ₇ is methyl, ethyl, isopropyl, n-butyl, isobutyl; Preferably, R ₈ is H, methyl or ethyl; Preferably, R ₉ is methyl, -COOH, -CONH-CH 3 . Preferably, R ₁₀ is H or -OH; Preferably, R ₁₁ is H or -OH; Preferably, U is selected from the following structures:





The antibody-drug conjugate according to claim 24, characterized in that the Ab comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: The amino acid sequences of HCDR1, HCDR2 and HCDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, The amino acid sequences of LCDR1, LCDR2 and LCDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:8; Optionally, the CDR sequence of the Ab is defined according to the IMGT definition scheme, as shown in SEQ ID NO:12~17. The antibody-drug conjugate according to claim 24, characterized in that the Ab is selected from: Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:6; or Having a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:8; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:6; or Having a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:8; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:6; or Having a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:8; or Having a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:4; or Having a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:5; or Having a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:6; or It has a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:8. A pharmaceutical composition, characterized in that it comprises the antibody-drug conjugate according to any one of claims 21 to 30, and a pharmaceutically acceptable carrier. A kit, characterized in that it comprises the antibody-drug conjugate according to any one of claims 21 to 30. A method for preventing and/or treating a disease, characterized in that it comprises: administering an effective amount of the antibody-drug conjugate according to any one of claims 21 to 30, or the pharmaceutical composition according to claim 31, to a subject in need; Optionally, the disease is a tumor and/or cancer. The method according to claim 33 is characterized in that the tumors and/or cancers include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, cartilage At least one of sarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multiapical myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, peritoneal cancer, pleural cancer, and blood cancer. Use of the antibody-drug conjugate according to any one of claims 21 to 30 in the preparation of a drug or a kit. Use of antibodies or antigen-binding fragments thereof targeting antigen epitope polypeptides in the preparation of antibody-drug conjugates; Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; Compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells; The amino acid sequence of the antigenic epitope polypeptide has one or more sequences derived from Strep tag I or Strep tag II. An antibody-drug conjugate targeting an antigen epitope polypeptide, characterized in that it comprises an antibody or an antigen-binding fragment thereof targeting an antigen epitope polypeptide coupled to one or more therapeutic agents; Wherein, in the natural state, the amino acid sequence of the mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigen epitope polypeptide; Compared with normal human cells, the antigen epitope polypeptide is differentially and highly expressed on tumor cells/cancer cells, and the antibody or antigen binding fragment specifically binds to the antigen epitope polypeptide on the tumor cells/cancer cells; The amino acid sequence of the antigenic epitope polypeptide has one or more sequences derived from Strep tag I or Strep tag II.